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The Prostate 38:317–360 (1999)
Abstracts From The 1998 International
Symposium on Biology of Prostate Growth
National Institutes of Health
Natcher Conference Center
Bethesda, Maryland
March 15–18, 1998
INVESTIGATION OF MOLECULAR MECHANISMS OF p75NTR
REGULATION AND EXPRESSION IN THE HUMAN PROSTATE
Scott Krygier, Beth Pflug, and Daniel Djakiew
Department of Cell Biology, Georgetown University,
Washington, DC 20007
The low affinity nerve growth factor receptor is a 75 kD
peptide (p75NTR) that binds NGF and other members of the
neurotrophin gene family. The complete function of p75NTR
still remains to be fully evaluated, although in some model
systems it plays a role as a negative regulator of growth. Part
of this role appears to occur through the apoptotic pathway
causing cell death, although other pathways may be important for cell quiescence. In normal prostate epithelium it
appears that p75NTR functions as a negative regulator of
growth. Transfections of p75NTR into a p75NTR deficient tumor cell line (TSU-pr1) induced these cells to undergo apoptosis depending on the level of p75NTR expression. Examination of p75NTR expression in the pathological human prostate by immunohistochemistry and Western blots shows that
p75NTR is present in normal prostate epithelium; its expression is partially lost in organ-confined adenocarcinoma tissue and expression is nonexistent in four tumor cell lines
derived from metastases. This loss of expression can occur at
several levels which include loss of the gene, transcriptional
control, and posttranscriptional control. Since all four tumor
lines do not express p75NTR, it is possible that there may be
common elements of downregulation. The mRNA of p75NTR
is about 3.8 kb, with the ORF of only 1500 bp. This leaves
about 2.3 kb in the 3⬘ UTR, which may be playing a role in
post-transcriptional regulation of this gene. With this in
mind we investigated the possibility that there is some posttranscriptional regulation of p75NTR. RT-PCR utilizing primers that will PCR out the first half of the ORF shows that all
four tumor cell lines express mRNA that contains this region, although the levels are extremely low relative to the
A875 cell line that expresses the protein. RNase protection
further supports the RT-PCR results that all four cell lines
contain mRNA for p75NTR. This shows that transcription is
occurring, at least at a low level. If control was purely transcriptional, then we would not expect any message to be
seen in both the RT-PCR and RNase protection assay. This
lends credence to the hypothesis that there is some alteration
© 1999 Wiley-Liss, Inc.
in the post-transcriptional regulation of p75NTR within the
tumor cells that contributes to the loss of expression during
malignant transformation.
LOSS OF EXPRESSION OF INHIBIN ALPHA SUBUNIT RESULTS IN
AN INTERPLAY BETWEEN ACTIVIN AND FOLLISTATINS IN HIGH
GRADE PROSTATE CANCER
G.P. Risbridger,1 S. Mellor,1 T. Thomas,1 M. Richards,1 D.M.
Robertson,2 N. Groome,3 W. Lee3 and J. Pedersen4
1
Institute of Reproduction and Development, Level 3, Block E,
Monash Medical Centre, Clayton, Victoria 3168, Australia
2
Prince Henry’s Institute of Medical Research, Level 4, Block E,
Monash Medical Centre, Clayton, Victoria 3168, Australia
3
Oxford Brookes University, Headington, Oxford OX4 3DH,
United Kingdom
4
Melbourne Pathology, Collingwood, Victoria 3066, Australia
Inhibins and activins are dimeric proteins, composed of ␣
and ␤ subunits, which are members of the TGF␤ superfamily. In human ovarian cancer, serum levels of inhibin are
elevated but the deletion of inhibin ␣ in mice results in tumour formation which implies that inhibin has tumour suppressive actions. The aim of this study was to determine if
inhibin ␣ and activin ␤A and ␤B subunit gene expression
was altered in men with high grade prostate cancer. The
expression and localisation of ␣ and ␤ subunit mRNAs and
proteins was examined in tissue sections from men with
high grade prostate cancer using in situ hybridisation and
immunostaining with specific antibodies to the subunits. A
comparison was made between malignant and non malignant regions of tissue. Needle biopsies were obtained from a
total of 46 men who were grouped according to histological
diagnosis of BPH or prostate cancer (Gleason grade 4 or 5).
The results demonstrate that the ␣ subunit is down regulated and there is no detectable expression or localisation of
protein in poorly differentiated tumours. In contrast there
was no corresponding loss in the expression or localisation
of the ␤A or ␤B subunits; therefore the epithelial tumour
tissue retains the capacity to synthesise activins, but not inhibins. Activins are known to inhibit IL-6 induced activities
and suppress inflammatory responses which would effect
the host defense; but some human prostate tumour cell lines
are growth inhibited by activin. The latter effect is blocked
by activin binding proteins, follistatins. We have previously
reported that follistatin mRNA and protein production is
318
Bethesda Abstracts
altered in tumour tissue from patients with high grade cancer, and suggested that resistance to activin by the cells
themselves is conferred through the expression of specific
forms of FS on the tumour cells which block the autocrine,
but not systemic action of activins. Thus it is concluded that
the regulation of epithelial tumour cell growth involves autocrine regulation of activin action by follistatins and that
this contributes to malignant progression.
NEONATAL ESTROGENIZATION STIMULATES PROLIFERATION OF
A ZONE OF PERIDUCTAL FIBROBLASTS THAT IMPEDE DUCTAL
BRANCHING AND CELL-CELL INTERACTIONS IN THE
DEVELOPING RAT VENTRAL PROSTATE
William Y. Chang, Lynn Birch, and Gail S. Prins
Department of Urology, University of Illinois at Chicago,
Chicago, IL 60612
Neonatal estrogenization of male rats leads to growth
and developmental retardation of the prostate gland characterized by epithelial differentiation blockade, decreased
epithelial proliferation and reduced branching morphogenesis during the first 25 days of life. The role of the extracellular matrix (ECM), which can regulate branching morphogenesis and differentiation, has not been characterized in
this model. This study set out to determine whether neonatal
estrogen exposure alters ECM components in the developing rat ventral prostate. S-D rat pups were given 25 ␮g estradiol benzoate or oil on days 1, 3 and 5 (neoEB) and were
sacrificed on days 6, 10, 15 or 30. Control pups were also
sacrificed on days 2–5. Prostatic complexes were microdissected at 4°C and frozen in OCT. Immunocytochemistry was
performed on frozen sections along the longitudinal axis of
the tissues. Laminin and collagen IV were immunolocalized
to the basement membrane (BM) and basal lamina surrounding differentiating periductal smooth muscle and endothelial cells of the control developing prostates while fibronectin and collagen III were localized only to the periductal ECM. While neoEB did not alter laminin or collagen
IV in the BM, a clear layer (zone) of cells free of laminin and
collagen IV was evident immediately adjacent to the BM.
Outside of this clear zone, actin-positive smooth muscle cells
immunostained for laminin and collagen IV which created a
halo appearance around each duct. The ECM in the zone
was positive for fibronectin and the zone cells were negative
for actin but immunopositive for vimentin indicating that
the cells were of mesenchymal origin. Evaluation by electron
microscopy positively identified the zone cells as fibroblasts.
EM revealed that the control prostates possessed an extremely flattened single cell layer of fibroblasts outside the
BM in the proximal ducts while in the neoEB prostates, the
fibroblast layer was several cells thick. The fibroblast zone
was most prominent in neoEB day 10 rat ventral prostates,
was thickest in the proximal ducts and diminished distally.
Immunostaining for PCNA revealed that the periductal fibroblasts in neoEB rats were actively proliferating to a
greater extent than observed in controls. Some, but not all, of
the fibroblasts in the zone stained for ER␣ indicating that the
effect of estradiol may be direct. The periductal fibroblast
zone was devoid of urokinase- and tissue-plasminogen activator (PA) immunostain. Since PAs activate ECM proteases, we propose that the fibroblast zone may act as a physical constraint against branching morphogenesis. Furthermore, the immediate periductal location of the fibroblast
zone also presents as a potential physical barrier for paracrine factors which mediate cell-cell interactions between the
smooth muscle and epithelium in the rat ventral prostate. In
conclusion, our data demonstrate an altered cell and ECM
composition adjacent to the BM of epithelial ducts in neoEB
prostates which may mediate some of the events of developmental estrogenization. (supported by NIH DK-40890)
ESTRADIOL CAUSES A DOSE DEPENDENT INCREASE IN
PROSTATE SIZE IN CASTRATE BEAGLE DOGS
W.P. Feeney,1 V.D.H. Ding,1 H.M. Saunders,2 D.F. Hora Jr.,1
P.K. Cunningham,1 L.A. Balogh,1 R.K. Kemp,1 J. Pivnichny,1 V.
Pikounis,1 L. Gantert,1 W. Rosner,3 D.E. Moller,1 R.G. Smith,1
and L. Rhodes1
1
Merck Research Laboratories, Rahway, NJ
University of Pennsylvania School of Veterinary Medicine,
Philadelphia, PA
3
St. Luke’s/Roosevelt Hospital, New York, NY
2
Previous studies have shown that giving castrate dogs
androgen does not cause enlargement of the prostate, but
adding a small amount of estrogen results in significant
prostate growth. However, the effects of estrogen alone have
not been well studied. The purpose of this experiment was
to evaluate the effect of estradiol on prostate growth in dogs.
Dogs (n = 25) were randomly divided into groups (n = 5)
and treated as follows: castration alone (CC), castration +
low dose estradiol (E low), castration + high dose estradiol
(E high), castration + estradiol + androstandiol (AE), or no
steroid, no castration (NC). Silastic implants containing 5␣androstan-3␣-17␤-diol (3␣diol), and/or 17␤-estradiol were
used for continuous delivery of steroids. E low and E high
treatments were estimated to result in estrogen release rates
of 0.22 and 0.45 mg/week/dog, respectively. Prostate volume was measured by transrectal ultrasonography, and
blood was drawn for estradiol and SHBG determinations at
−1, 1, 3, 5, 7, 9 and 12 weeks following castration and silastic
implant placement. Results show that serum estradiol and
SHBG levels were fairly constant over 12 weeks in all
groups. In the NC and CC groups, mean serum estradiol
was approximately 25 pg/ml, while the E low, AE and E
high groups had mean serum estradiol values of approximately 40, 40 and 60 pg/ml respectively, showing treatment
with estradiol from silastic implants caused a sustained
dose-dependent rise in serum estradiol. Prostate volume
measurement showed that at initiation of the study, all
groups had similar prostate volumes (12.6 ± 5.0 cm3, mean ±
SEM). Over 12 wks, the CC group had a decline in prostate
volume, whereas the NC and AE groups maintained a constant prostate volume. The E low and E high groups had a
large, late onset (wk 7), dose-dependent increase in prostate
volume relative to the NC group (P < 0.01). At 12 wks,
animals were euthanized and prostates weighed. Mean
prostate weight correlated with mean prostate volume measured by ultrasonography. The mean prostate weights in
each group were: NC 9.5 ± 2.0, CC 2.6 ± 0.4, AE 7.9 ± 1.4, E
low 15.2 ± 7.8, and E high 41.1 ± 15.2 gms (mean ± SEM).
Histologically, the prostates of estrogen treated dogs
showed metaplastic, squamous cells, with the E high group
showing marked glandular dilation. The AE group showed
a more normal prostate histology. These results demonstrate
that estrogen causes marked stimulation of prostate growth
in the dog. This growth may be due to an estrogen receptor
Bethesda Abstracts
319
mediated effect or estradiol’s interaction with SHBG and its
receptor.
TELOMERASE ACTIVITY IN THE BROWN NORWAY RAT
PROSTATE: LOBE-SPECIFIC REGIONAL DISTRIBUTION AND
AGE-DEPENDENT CHANGES
Partha P. Banerjee, Subhadra Banerjee, Barry R. Zirkin, and
Terry R. Brown
Division of Reproductive Biology, Johns Hopkins University,
School of Hygiene and Public Health, Baltimore, MD 21205
It is generally established that telomerase activity is present in most cancer cells, germ line cells, and established cell
lines, but typically is not present in normal somatic cells.
Among the few exceptions, telomerase activity has been detected in hematopoietic stem cells and in physiologically
renewable epithelial cell populations (e.g., skin, small intestine). The adult rat prostate gland is a tissue that has the
ability to regenerate in response to androgen. We hypothesized, therefore, that telomerase activity should be present
in the normal adult rat prostate. In this study, using a highly
sensitive PCR-based telomerase assay [the telomeric repeat
amplification protocol (TRAP) assay], we tested this hypothesis by measuring telomerase activity in the ventral, dorsal,
lateral and anterior lobes of the adult Brown Norway rat
prostate. Telomerase activity indeed is present in whole tissue extracts of the ventral, dorsal and anterior lobes, though
at differing levels, but not the lateral lobe. Quantitative
analyses revealed that compared to the activity present in
lung (100%), the relative telomerase activities in the ventral,
dorsal and anterior prostatic lobes were 6%, 13%, and 20%,
respectively. Lateral lobe prostatic fluid was found to contain a factor(s) that is inhibitory for the TRAP reaction, and
hence telomerase activity was detected only after removal of
this fluid. Telomerase activities were not uniform within the
prostatic ducts, but rather telomerase positive cells were regionally distributed within the distal, intermediate and
proximal ducts of the microdissected segments of each of the
lobes. In the ventral, dorsal and lateral lobes, telomerase
activity was highest in the proximal segment (∼20% of lung),
activity was less in the distal tips, and the least activity was
seen in the intermediate segment. Telomerase activity in the
anterior lobe was highest in the proximal segment, lower in
the intermediate segment and lowest in the distal segment.
We discovered that lobe-specific overgrowth of the prostate
also occurred spontaneously in Brown Norway rats as a
function of age, which led us to investigate whether telomerase activity changed with age in a lobe-specific manner.
Compared to the activity present in lung (100%), the relative
telomerase activity in the ventral prostatic lobes of young(4month-old) and old (24-month-old) rats was in the range of
5–7%. In the dorsal lobe, telomerase activity was agedependent, being 1.4-fold higher in old rats than in young
rats. In the anterior lobe of old rats, telomerase activity was
2.6-fold higher than in the anterior lobe of young rats. We
speculate from these results that androgen-stimulated regeneration of the rat prostate following androgen ablation
may depend upon the telomerase-positive pool of cells present within specific regions of the ductal network. Moreover,
the age-dependent and lobe-specific increases in expression
of telomerase activity in the Brown Norway rat prostate may
be indicative of potential sites for the development of hyperplastic changes during aging. (Supported by NIA P01AG08321)
CONTROL OF uPA/uPAR EXPRESSION AND MAPK PATHWAYS IN
PROSTATE CELLS
D. Gavrilov,1 O. Kenzior,1 R. Callaluce,2 M. Evans,2 and W.
Folk2
1
Department of Biochemistry, University of Missouri School of
Medicine, Columbia, MO
2
Department of Pathology, University of Missouri School of
Medicine, Columbia, MO
Expression of urokinase plasminogen activator (uPA)
and its receptor (uPAR) are required for tissue remodeling
during development and are highly correlated with prostate
cancer metastasis. These proteins are regulated by ets and
AP1 families of transcription factors whose synthesis and
phosphorylation are determined by signal transduction
pathways acting through mitogen activated protein kinases
(MAPK). In the present study we evaluated the expression
of uPA and uPAR in primary high-grade prostate adenocarcinoma tissue sections, and in established human prostate
cancer cell lines. Elevated levels of uPA and uPAR in prostate adenocarcinoma epithelial cells co-localized with three
ets-proteins—Elf-1, Erg 1, 2 and Fli-1, but not with PEA3,
Ets-1, Ets-2 and PU.1. We also detected c-Jun and c-Fos proteins (components of AP1) in epithelial cells and the surrounding stroma, but no apparent association with tumor
grade was noticed. We detected expression of Erg 1, 2, Elf-1
and Fli-1 transcription factors in PC3 cells, avid expressors
of uPA/uPAR, but not in LNCaP cells, which do not express
uPA/uPAR. Members of the AP1 family were detected in
both cell lines but levels of c-Jun and c-Fos were significantly
higher in PC3 cells. We characterized different branches of
the MAPK pathway, which activate AP1 and ets-proteins.
The MAPK pathway leading to activation of c-Jun is fully
active in PC3 cells. Both MEK1 and SEK1 dominant negative
mutants and the MEK1 inhibitor PD 098059 inhibit the expression of the reporter genes dependent upon Elk and AP1
and significantly inhibit the expression of uPA. In LNCaP
cells, the MAPK pathway is not activated, but can be stimulated by constitutively active MEK1 and MEKK; however
uPA expression still remains low, suggesting additional elements are required for uPA expression.
PRIMARY CULTURE AND IMMORTALIZATION OF EPITHELIAL
AND MESENCHYMAL CELLS FROM HUMAN BENIGN PROSTATIC
HYPERPLASIA
Patricia M. Fry, John R.W. Masters, and Michael J. O’Hare
Institute of Urology and Department of Surgery, University
College London, London W1P 7PN, UK
To produce primary cultures of epithelial cells from biopsies of benign prostatic hyperplasia obtained by transurethral resection, the tissue was minced using crossed scalpels
to a size of approximately 1 mm3 in size. This was incubated
overnight in 0.5 mg/ml collagenase type 1A, washed and
centrifuged at 800 rpm for 20 seconds. The pellet was plated
in serum-free medium. Primary cultures were established in
19/20 cases in WAJC 404 (Life Technologies), mammary epithelial, keratinocyte and endothelial cell growth media
(Clonetics). In WAJC 404 medium the cells became heavily
vacuolated within 3–4 weeks in culture, whereas in the other
320
Bethesda Abstracts
media there was less vacuolation, but the cells became squamous. However, in prostate epithelial cell growth medium
(Clonetics), there was little vacuolation and cell proliferation
was maintained. Primary cultures of mesenchymal cells
were obtained by incubating minced prostate tissue in 5
mg/ml collagenase type 1A for 2 h followed by filtration
down to 35 ␮m. The filtrate was plated in DMEM:F12 + 20%
FCS and produced primary cultures in 20/25 attempts.
Both epithelial and mesenchymal cells were successfully
transduced using a temperature-sensitive mutant of the
SV40 large T-antigen gene (tsA58-U19), as previously described (Stamps et al., Int J Cancer 57:865–874, 1994). Primary cultures were transduced with the tsT gene using an
amphotropic retroviral vector, selected in G418, and have
been maintained for several months in continuous culture.
The epithelial cells retain their typical phenotype when
grown on a 3T3 feeder layer at low density. The mesenchymal cultures have been separated into three morphological
types: 1) regular growth pattern and classical orthogonal
overlaying typical of fibroblasts, 2) ‘‘hill and valley’’ phenotype typical of smooth muscle cultures and 3) a chunky cell
type which we have not yet identified.
This work was supported by The Wellcome Trust.
SYNERGISTICAL INTERACTION OF THEARUBIGINS AND
GENEISTEIN ON PC-3 CELL GROWTH AND CELL
CYCLE PERTURBATION
K. Sakamoto,1 Y. Hara,2 and J.A. Milner1
1
Pennsylvania State University, University Park, PA 16802
Food Research Laboratories, Mitsui Norin Co. Ltd., Fujieda,
Japan
2
Epidemiological evidence has shown that habitual consumption of higher amounts of green tea (Camellia sinensis)
than the average Japanese population intakes is correlated
with a reduction in cancer risk at various sites in both sexes.
In addition, another epidemiological study suggests that
low mortality in prostate cancer of Japanese men consuming
a traditional diet may be due to the high intake of soy bean
products. In general, the traditional Oriental diet includes
soy foods and is consumed daily with tea at the meal. The
combination of genistein from soy beans and polyphenols
from green or black tea may significantly influence cancer
prevention more than either provided alone.
Therefore, the present study examined the interaction of
thearubigins (TR) from black tea (Camellia sinensis) and
genistein from soy bean isoflavone in human prostate PC-3
tumor cell growth. Black tea extract contains approximately
20–30% polyphenols. Among them, thearubigains (TR) are
the most abundant (containing 10–15%) and highly oxidized
polymer forms of polyphenols.
Supplementing increased doses of TR or genistein, provided either alone (at 0.5, 1, 5, 10, 20 ␮g/ml), for 60 h did not
alter the growth of PC-3 cells, except for higher doses of
genistein at 10 and 20 g/ml. However, combined low dosage
of TR with genistein at certain ratios altered the cell proliferation. A low dose (0.75 ␮g/ml) of TR alone that did not
decrease the growth of PC-3 tumor cells did enhance the
effect of genistein(30 ␮g/ml) on altering cell growth and also
perturbing the cell cycle regulation at the 2nd check point.
Furthermore, the increasing low doses of TR at 0.125, 0.25 or
0.5 ␮g/ml that did not alter PC-3 cell growth, suppressed
tumor cell growth and disrupted cell cycle at G2/M phase at
dose dependent when combined with genistein (a ratio, 1:40,
TR to genistein) at 5, 10, or 20 ␮g/ml. Even though a low
dose of either TR (0.0625 ␮g/ml) or genistein (2.5 ␮g/ml)
provided alone did not inhibit the growth of tumor cells, the
combined treatment of these compounds significantly altered the tumor cell (PC-3) proliferation (30% control inhibition). These findings indicate that the potential of using
combined phytochemicals may achieve cancer prevention.
LOSS OF CASPASE-3-LIKE ACTIVITIES IN HUMAN PROSTATE
CANCER CELLS: IMPLICATION IN DRUG RESISTANCE
Sandhya Mandlekar,1 Wei Lei,1 Rong Yu,1 Adedigbo
Fasanmade,1 Mark Heller,2 and A.-N. Tony Kong1
1
Center for Pharmaceutical Biotechnology, Department of
Pharmaceutics and Pharmacodynamics, University of Illinois,
Chicago, IL
2
Division of Hematology/Oncology, Department of Medicine,
University of Illinois, Chicago, IL
Advanced prostate cancer cells are extremely insensitive
to a variety of anticancer drugs. However, the mechanism
underlying such drug-resistance remains unknown. Recent
advances in our understanding of cellular signal transduction mechanisms in response to many cytotoxic agents provided valuable insights. There is growing evidence that signals leading to the activation of intracellular ICE/Ced-3 proteases (caspases) or c-Jun N-terminal kinases (JNKs) play a
pivotal role in apoptosis induced by various stimuli. We
compared the activation of JNKs and caspases in human
prostate cancer cells, including androgen-independent PC-3,
and MH-PR1, and androgen-dependent LNCaP, and in
other non-prostate cancer cells, in response to different
apoptotic agents. Treatment with tumor necrosis factoralpha (TNF-␣), diethyl stilbesterol (DES), phenethyl isothiocyanate (PEITC), and quinacrine caused 10–20 fold induction of caspase-3-like activities in human cervical carcinoma
HeLa cells or human breast cancer BT-20 cells. However, no
activation of caspase-3-like proteases activity was observed
in the three human prostate cancer cells. Unlike caspases,
JNK activation was intact in both prostate and non-prostate
cancer cell lines. Thus, a defect in caspase signaling may
contribute to the resistance of advanced prostate cancer cells
to apoptosis-inducing agents in human.
HIGH FREQUENCY REGRESSION OF ESTABLISHED HUMAN
PROSTATE CARCINOMA PC3 XENOGRAFTS BY SYSTEMIC
TREATMENT WITH ANTISENSE JUN KINASE
Frédéric Bost,1 Olga Potapova,1, Chaoting Liu,1 Yong-Min
Yang,1 W. Charbono,1 Nicholas Dean,2 Robert McKay,2
Xian-Ping Lu,4 Magnus Pfahl,1,3 and Dan Mercola1,5
1
Sidney Kimmel Cancer Center, San Diego, CA 92121
Isis Pharmaceutical, Inc., Carlsbad, CA 92008
3
Maxia Pharmaceuticals, Inc., San Diego, CA 92121
4
Galderma Research, Inc., San Diego, CA 92121
5
Center for Molecular Genetics, University of California at San
Diego, La Jolla, CA 92093
Previous studies have shown that N-terminal Jun Kinase
(JNK) cooperates in the transformation of primary fibroblasts and plays a role in human tumor cell growth. To test
this further, we used previously characterized high affinity
2
Bethesda Abstracts
phosphorothioate oligonucleotides that are complementary
to two major forms of JNK in systemic treatment of established PC3 xenografts with and without cisplatin. All combinations lead to decreased tumor JNK activity and to tumor
arrest with up to 89% inhibition for the best combination. In
addition antisense treatment alone preferentially caused ulceration and complete regression leading to flat sores in
100% of cases, significantly greater (P = 0.022) than the second best combination with cisplatin, 45%. Conversely, PC3
cells that express a dominant negative inhibitor of c-Jun (cJun(S63A,S73A)) were unable to develop any tumors
whereas empty-vector control cells were 100% tumorigenic.
To test the generality of these results we examined the in
vitro growth properties of PC3 cells and 8 additional established human prostate carcinoma cells. Lipofection with 0.2
␮M antisense JNK eliminated steady state JNK protein by at
least 70% in all cases and inhibited serum-supported proliferation up to 80% in direct proportion to the level of seruminducible JNK activity (2-to-9 fold over basal activity) with
an overall Pearson correlation coefficient of 0.65 when all
cell lines were included whereas the one normal prostate cell
line available exhibited high serum-stimulated JNK activity
(10.6-fold) but little (1.5-fold) increased growth compared to
basal (0.5% serum) growth. These results indicate that JNK is
essential for the development and growth of human prostate
PC3 carcinoma xenografts, that the JNK signal transduction
pathway is a common serum-dependent growth pathway
among human prostate carcinoma cells and that the JNK
signal transduction pathway may be an important new target for intervention.
THE ANTISENSE DOWNREGULATION OF bcl-xL PROTEIN IN
PROSTATE CANCER CELL LINES
I.V. Lebedeva, A.L. Weitzman, and C.A. Stein
Columbia University, New York, NY 10032
Prostate cancer is a major cause of deaths and currently
one of the most commonly diagnosed cancer in the U.S. Data
suggest that the overexpression of the antiapoptotic protein
bcl-xL is correlated with resistance to cytotoxic chemotherapeutic agents in hormone refractory prostate cancer. We
have screened forty 18mer and 20mer phosphorothioate oligonucleotides randomly targeting different regions of the
bcl-xL mRNA. These molecules were tested in LNCaP, DU145 and PC3 prostate cancer cell lines. Two novel agents for
the intracellular delivery of oligonucleotides were used:
tetra meso-(4-methylpyridyl)porphine (TMP) is a cation
which forms 2:1 complexes; tetra meso-(trimethylammonium)porphine (TAP) is also a cation, but forms 3:1 complexes
with oligonucleotides. The concentration of oligonucleotides
used was 1 ␮M. By Western blot analysis we have demonstrated effective down-regulation of bcl-xL with five of the
oligonucleotides tested. Control oligonucleotides did not effect expression of bcl-xL and viability of the cells. According
to DAPI stain analysis data these five active oligonucleotides
used alone did not induce apoptosis in prostate cancer cell
lines. Northern blot analysis shows decrease in bcl-xL
mRNA signal after antisense treatment. Appropriate controls have been used to demonstrate specificity. The data
suggest that these novel porphine delivery agents can be
successfully used in antisense technology with diverse cell
types. Furthermore, we propose using an antisense for bclxL in combination with cytotoxic chemotherapy as a strategy of overcoming drug resistance in prostate cancer.
321
LNCaP CELLS OVEREXPRESSING bcl-xL AS A MODEL FOR
STUDYING OF APOPTOSIS IN PROSTATE ADENOCARCINOMAS
I.V. Lebedeva, A.L. Weitzman, and C.A. Stein
Columbia University, New York, NY 10032
A recent immunohistochemical survey of neoplastic human prostate cancer tissues showed that expression of bclxL protein increases during progression of prostate cancer.
Overexpression of this protein might be a factor enabling
prostate cancer cells to survive in an androgen-deprived environment. To mimic the hormone-insensitive, metastatic
adenocarcinoma phenotype and to determine the degree to
which overexpression of bcl-xL can protect human prostate
cancer cells from apoptosis, we transfected human prostate
cancer cells (LNCaP) with a neomycin-selectable eukaryotic
expression vector containing cDNA encoding human bcl-xL.
Two transfected clonal variants that overexpress bcl-xL (LNCaP/bcl-xL) have been selected. These clones were unaltered with regard to their growth rate in 10% serumcontaining medium and the level of expression of other bclfamily proteins such as bax and bak. Taxanes induce a
downregulation of bcl-xL in prostate cancer cells. In contrast
to the parental or mock-transfected cells, LNCaP/bcl-xL
cells were highly resistant to the taxol treatment as determined by MTT assay. These bcl-xL-overexpressing LNCaP
cell lines may serve as a good model for developing further
strategies for prostate cancer treatment and represent additional evidence of the important role that bcl-xL plays in
prostate cancer resistance to these chemotherapy.
POTENTIAL ROLE OF FER TYROSINE KINASES IN
PROSTATE CANCER
P. Allard, P. Beaulieu, S. Tessier, F. Landry, A. Aprikian, L.R.
Begin, and S. Chevalier
Urologic Oncology Research Group, Montreal General Hospital
Research Institute, McGill University, and Department of
Biochemistry and Medicine, University of Montreal, Montreal,
Quebec, Canada
Androgens play important regulatory roles in prostate
functions and for this reason, endocrine therapies are commonly used to treat prostate cancer (PCa). However, the
relapse observed in a majority of patients, along with numerous studies on peptide growth factors, indicates that androgen-independent pathways are also up-regulated in PCa.
GFs transduce mitogenic signals from the membrane to the
nucleus via tyrosine kinases (TKs), which are often powerful
oncogene products. Since TKs are implicated in signaling
pathways regulating cell adhesion/migration, we undertook the cloning of prostatic TKs from an expression library
derived from GF-responsive but steroid-unresponsive canine prostatic epithelial (basal) cells in primary culture.
Among others, we sequenced the fer (fps/fes related) cDNA,
encoding a 94 kDa non-receptor TK with a putative nuclear
localization site. To evaluate its potential role in the prostate,
we raised polyclonal antibodies against an FER peptide and
studied its expression as well as distribution in prostate epithelial cells, normal (canine) and malignant (human cell
lines). The level of p94Fer was highly increased in proliferating canine prostatic epithelial cells once seeded in culture
when compared to freshly dispersed resting cells. The protein was absent from the nuclear fraction but present in the
cytoplasm (78%) and membrane (20%) fractions. Also, a 110
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kDa Fer-like protein (p110Fer) specifically recognized by our
antibody was detected only in dividing cells and its distribution was exclusively nuclear. Interestingly, p94Fer was
found in nuclei (12–31%) of PC-3, DU145 and LNCaP cells,
along with p110Fer which was again strictly nuclear. The
levels of both proteins were negligible or low in normal (n =
4) and hyperplastic (n = 7) human prostates, but high in
prostate extracts from PCa patients (n = 11) where the
p94Fer protein was detected in 90% of specimens and
p110Fer in 30%. In vivo in the dog, the expression of both
proteins was related to basal cell metaplasia, as induced by
estrogen in castrated animals, but not to the differentiated
luminal phenotype (normal and hyperplastic prostates and
from androgen-treated castrated dogs). To get insights into
the functional role of Fer proteins, we examined associated
signaling molecules following bombesin stimulation of PC-3
cells, knowing that this neuropeptide stimulates PC-3 motility and invasiveness (Aprikian et al., Int J Cancer 1997). By
co-immunoprecipitation, we observed that p110Fer but not
p94Fer was associated with p60Src, a finding suggesting that
the two proteins are not involved in the same signaling pathways and biological functions. However, using an antisense
strategy (Fer constructs) in PC-3 cells, we observed that
clones with the lowest expression of both proteins (50–75%
reduction) had the lowest growth rate on a solid support
and were unable to form colonies in soft agar. Collectively,
our findings suggest that expression of Fer proteins in prostate epithelial cells and tissues correlates with phenotypes
where growth and adhesion/migration are activated. Also,
knowing that protein segregation in cytoplasmic vs. nuclear
compartments is an important regulatory mechanism in signaling pathways, an abnormal localization of p94Fer in PCa
cells could also affect positively prostate cell growth. (Supported by grants from MRCC).
DISTINCT EFFECT BETWEEN HYDROXYFLUTAMIDE AND
BICALUTAMIDE ON ANDROGEN RECEPTOR MEDIATED
SIGNAL TRANSDUCTION
L.G. Wang, X.M. Liu, W. Kreis, and D.R. Budman
Department of Medicine, New York University School of
Medicine, Don Monti Research Laboratory, North Shore
University Hospital, Manhasset, New York 11030
The androgen receptor (AR) is a ligand-dependent transcriptional regulator required for the differentiation, development, and maintenance of normal male reproductive
functions. Androgen binding initiates the process of receptor
transformation that eventually results in specific binding of
the receptor complex to steroid receptor binding consensus
(SRBC) of AR target-genes and thus transcriptional regulation. Antiandrogens exert their effects on prostate cancer
cells through blocking androgen binding to the AR leading
to interruption of AR mediated signal transduction.
Flutamide (Euletin威) and bicalutamide (Casodex威) are
widely used antiandrogens for the treatment of prostate cancer. Recently the action of these two androgen antagonists
has been re-evaluated. In this report, we show the distinct
effects between hydroxylutamide, the active form of flutamide, and bicalutamide on androgen receptor signal
transduction. Bicalutamide exhibits an inhibitory effect on
LNCaP cell growth in both serum and serum free RPMI 1640
medium, and down-regulates the expression of prostate specific antigen (PSA). Also, no transactivation of luciferase activity is observed in HeLa cells co-transfected with wild type
AR expression plasmid pAR0 and AR driven reporter plasmid GRE-tk-LUC in the presence of different concentrations
of bicalutamide (1.0 to 10.0 ␮M). Most importantly, bicalutamide significantly inhibits androgen receptor phosphorylation in a concentration-dependent manner (1.0 to 10.0 ␮M),
followed by a decrease of binding of AR SRBC in the promoter region of PSA. In contrast, no inhibitory effect on AR
phosphorylation, and no changes in the levels of binding
complex between AR and SRBC are observed after LNCaP
cells are treated with different concentrations (7.5 to 58 ␮M)
of hydroxyflutamide. Moreover, hydroxyflutamide shows
slight, but significant stimulation of LNCaP cell growth in
the serum-free medium and moderately up-regulates PSA
expression. The approximately 10-fold induction of luciferase activity is also observed in gene transactivation assay
using pAR0 and GRE-tk-LUC in the presence of hydroxyflutamide. Our data indicate that bicalutamide is a pure androgen antagonist, whereas hydroxyflutamide acts as a partial agonist, and that androgen receptor phosphorylation is a
determining factor for AR function.
Supported by Don Monti Research Memorial Foundation.
LOSS OF THE CYCLIN-DEPENDENT KINASE INHIBITOR p27Kip1
PROTEIN IN HUMAN PROSTATE CANCER CORRELATES WITH
TUMOR GRADE
Yanping Guo, Geoffrey N. Sklar, Andrew Borkowski, and
Natasha Kyprianou
Department of Biochemistry and Molecular Biology, and
Division of Urology, University of Maryland School of
Medicine, Baltimore, MD 21201
TGF-␤1 acts as a physiological regulator of prostate epithelial cell growth by arresting activity proliferating cells
and inducing apoptosis in both normal and malignant prostate cells. Cyclin-dependent kinase inhibitors have been implicated as postreceptor effectors for the intracellular transduction of TGF-␤1 signal. Since the cdk inhibitor,
p27Kip1plays a central role in the basic machinery controlling
TGF-␤-mediated negative effects, we hypothesized that potential defects in this downstream effector may be involved
in abrogating TGF-␤ signal transduction during prostate carcinogenesis. In this study, we investigated the relationship
between p27Kip1 protein expression and tumor grade in human prostate cancer, by conducting an immunohistochemical analysis in a series of normal, benign and malignant
prostate specimens. The proliferative activity of prostatic tumors was determined on the basis of the Ki-67 nuclear antigen staining. A uniformly intense immunoreactivity for
p27Kip1 was localized to the nuclei of glandular epithelial
cells of normal prostates. The benign glandular epithelia exhibited moderate immunostaining. In the malignant prostate
tissue, however, a heterogenous pattern of substantially reduced p27Kip1 immunoreactivity was found among the glandular epithelial cells. The majority of primary prostate cancer specimens (68%) were totally negative for p27Kip1 immunoreactivity, while the rest exhibited a significantly
decreased p27Kip1 expression, compared with the normal
prostate (P < 0.01). Moreover, there was progressively diminished p27Kip1 immunostaining with increased tumor
grade. Significantly enough, this loss of p27Kip1 was associated with an increase in the proliferative index of prostatic
tumors (r = 0.08). There was no significant relationship between p27Kip1 loss and the TGF-␤ status of prostatic adeno-
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carcinomas. These results indicate that frequent loss of the
cdk-inhibitor p27Kip1 in human prostate cancer cells, correlates with advancing histological aggressiveness, implicating deregulation of p27Kip1 in prostate tumor progression.
Our findings are in accord with recent evidence suggesting
that loss of expression or mutational deletion of cdk inhibitor, p27Kip1, is involved in development of other human malignancies. Overall, the present study strongly implicates
that loss of p27Kip1 may have a prognostic value as a marker
for prostate cancer progression.
INDICATIONS OF LIVING CIRCULATING PROSTATIC CANCER
CELLS IN THE PROSTATIC CANCER PATIENTS’ BLOOD
Z.P. Wang,1 S.J. Di,1 M.A. Eisenberger,2 A.W. Partin,3 and
P.O.P. Ts’o1
1
Cell Works, Inc., University of Maryland Baltimore County,
Baltimore, Maryland
2
Department of Oncology, Johns Hopkins University, Baltimore,
Maryland
3
Department of Urology, Johns Hopkins University, Baltimore,
Maryland
The morphological observation of circulating cancer cells
in our more than 200 prostatic cancer patients’ blood tests
demonstrated that some characteristics of the circulating
prostate cancer cells might indicate potential growth ability.
The indications of potential growth ability of circulating cancer cells included the following aspects: 1) stem-like cancer
cells: some cancer cells possess stem-cell characteristics enabling them to exhibit very ‘‘young’’ morphological features. The size of the cancer cells was comparable to monocytes, they were present in high density, possessed very
developed cytokeratin systems in the cytoplasm, and exhibited aneuploidy of chromosome 18. They were distinguishable from ‘‘typical’’ circulating cancer cells which possess a
large cell body and nuclei; 2) dividing circulating cancer
cells: most of the circulating cancer cells were in interphase
of the cell cycle. Some circulating cancer cells in our tests
were in the ‘‘M’’ phase. The occurrence of circulating cancer
cells in M phase might indicate that some cancer cells were
growing in the patient’s circulatory system; 3) cloning circulating cancer cell cluster: the distribution of circulating
cancer cells isolated from patients’ blood on the slides
showed us two distributed patterns. First, the cancer cells
were in single distribution on the slides. Second, the cancer
cells formed a cell cluster. The formation of a cancer cell
cluster might occur in the patients’ circulation and/or be
formed during the sample processing in vitro due to the
sticky nature of the cancer cells’ surfaces. The possibility of
cloning growth of circulating cancer cells in the patients’
circulatory systems may be considered as a primary means
to form a circulating cancer cell cluster through cytomorphological analyses. The appearance of stem-like circulating cancer cells, dividing circulating cancer cells, and
cloning circulating cancer cell clusters in the cancer patients’
circulation may indicate that some circulating cancer cells
are living and growing in the patients’ blood.
INTERLEUKIN-8 (IL-8) SERUM PROTEIN IN PROSTATE DISEASE:
CLINICAL SIGNIFICANCE FOR THE DIFFERENTIAL DIAGNOSIS OF
LOCALIZED PROSTATE CANCER
323
Robert W. Veltri, M. Craig Miller, Angela Ng, Hitendra Patel,
Gang Zhao, and David A. Ralph
Oklahoma City, Oklahoma
INTRODUCTION AND OBJECTIVES: The literature
supports the contention that the immune system plays a key
role in prostate cancer progression and that some cytokines
may regulate key physiological events in this process. We
surveyed the immune systems (nucleated cells in the peripheral blood circulation) of metastatic prostate cancer patients
and healthy controls using an unbiased method that identifies differentially expressed mRNAs. We identified that IL-8
was overexpressed in the prostate cancer (CaP) patients.
METHODS: Based upon the demonstration of significantly elevated IL-8 mRNA levels in nucleated peripheral
blood cells of patients with metastatic prostate cancer, we
investigated circulating levels of the IL-8 protein in patients
with prostate disease. Using an ELISA kit from R&D Systems, Inc. (Rochester, MN), we examined the IL-8 protein
levels in sera taken from 193 prostate disease patients and
controls.
RESULTS: Serologic examination of patients’ bloods revealed that IL-8 was a sensitive biomarker for the detection
of localized CaP in patients with an elevated prostate specific antigen level (艌2.5 ng/ml). Sera from normal controls
(n = 37) and patients with localized CaP (N = 87); stages A,
B and C1), metastatic (n = 14) CaP, or BPH (n = 55) were
examined for IL-8 protein levels, total PSA concentrations,
and their free and total (f/t) PSA ratios. The mean concentration (pg/ml) of IL-8 observed in the sera from these
groups were: normals = 6.7; BPH = 6.8; stage A and B CaP =
15.4; stage C CaP = 19.1; and metastatic CaP = 78.9. IL-8
outperformed total PSA and yielded similar sensitivity and
specificity results as those of the f/t PSA ratio at a 14% cutoff
for the differentiation of CaP from BPH. The IL-8 levels,
when combined with the f/t PSA ratio using logistic regression, outperformed both tests alone with differentiating CaP
from BPH (sensitivity = 80.0%, specificity = 80.0%, area under ROC = 88.6%). The combination of the IL-8 serum protein results and the f/t PSA ratio improved the specificity for
the detection of BPH by approximately 13% when compared
to the results using the two tests alone.
CONCLUSION: IL-8 protein concentrations are increased in blood of CaP patients. The IL-8 serum protein
level, in conjunction with the total PSA concentration and
the f/t PSA ratio, may serve to improve the diagnosis of
localized vs. metastatic CaP and more clearly distinguish
CaP from BPH. The IL-8 serum protein levels improve the
specificity for the differential diagnosis of BPH in patients
with prostate disease.
BENIGN PROSTATIC HYPERPLASTIC STROMAL CELLS EXPRESS
BOTH l- AND h-CALDESMON
Victor K. Lin, Dolores J. Vazquez, John D. McConnell, and R.
Ann Word
Departments of Urology and Obstetrics-Gynecology, University
of Texas Southwestern Medical Center, Dallas, TX 75235
INTRODUCTION: Smooth muscle (SM) is a major component of prostatic stroma and plays an important role in
the pathogenesis of benign prostatic hyperplasia (BPH). The
enlargement of the gland has been characterized as a stromal
process. Caldesmon (CD) is a thin filament associated protein and is suggested to play a regulatory role in SM contractility. High molecular weight caldesmon isoform (h-CD,
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150 kDa) is only expressed in SM tissues while the low molecular weight isoform (l-CD, 75 kDa) was detected in some
non-muscle cells and SM cells in a rapid proliferation state.
CD expression, therefore could be used as a molecular
marker for SM phenotypic modulation.
METHODS: We utilized immuno-blot and immunohistochemistry to investigate the expression of h- and l-CD in
prostate from normal young adults (n = 6) and BPH patients
(n = 8).
RESULTS: Immuno-blot analysis indicates that h-CD is
expressed in both normal and BPH specimens. l-CD is not
expressed in normal prostate, as in other normal SM tissues
such as bladder. However, BPH tissue expresses l-CD at the
same level as that of h-CD suggesting that SM in BPH tissue
undergoes a de-differentiation process. Immunohistochemistry results indicate that antibody recognizing both h- and
l-CD localizes to the prostatic stroma but not to the glandular epithelial cells on both normal and BPH tissues suggesting that the expression of l-CD shown in immunoblot is from
prostatic stroma. Furthermore, antibody specific for h-CD
co-localizes with the antibody recognizing both h- and l-CD
in stroma suggesting that h- and l-CD are co-expressed in
BPH stroma.
CONCLUSIONS: These data suggest that a de-differentiation process of prostatic stroma SM is associated with
BPH pathogenesis.
STUDIES ON THE ROLE OF THE HYDROPHILIC REGION OF THE
PHOSPHOLIPID ENVIRONMENT IN THE MODULATION OF
5␣-REDUCTASE ACTIVITY IN EPITHELIUM AND STROMA OF
HUMAN BENIGN PROSTATIC HYPERPLASIA (BPH)
Heike Weisser, Tjalf Ziemssen, and Michael Krieg
Institute of Clinical Chemistry, Transfusion and Laboratory
Medicine, University Clinic Bergmannsheil, Bochum, Germany
Obviously membrane components such as phospholipids
play an important role in the regulatory process of 5␣reductase activity. In this context, our own recent studies
demonstrated that 5␣-reductase activity can be inhibited by
phospholipases (PL) altering either the hydrophobic or the
hydrophilic region of phospholipids. In order to elucidate
whether any alteration of the hydrophilic membrane environment results in an uniform modulation of 5␣-reductase
activity, the effect of phosphatidylinositol-specific PLC (PIPLC), PLC type XIV, PLC type III, and PLD was investigated. These phospholipases, altering the polar, hydrophilic
region of phospholipids, differ with respect to their mode of
phospholipid hydrolysis as well as to their substrate specificity. BPH tissue was separated mechanically in epithelium
and stroma. 5␣-reductase activity was determined using testosterone (585 nM) and various phospholipase concentrations (0.01–100 U/ml). The androgen metabolites were separated by HPLC. The significance of the differences between
the means was calculated by Student’s t-test (P < 0.05). Results: Using generally a phospholipase concentration of 10
U/ml, as compared to controls, in epithelium the mean relative 5␣-reductase activities (±SEM) were 82% ± 2 (PI-PLC),
85% ± 8 (PLC type XIV), 42% ± 4 (PLC type III), and 92% ±
3 (PLD). In stroma, the mean 5␣-reductase activities were
86% ± 6 (PI-PLC), 73% ± 5 (PLC type XIV), 34% ± 4 (PLC
type III), and 105% ± 2 (PLD). Among all phospholipases
modifying the hydrophilic milieu, PLC type III was the most
potent inhibitor of epithelial and stromal 5␣-reductase ac-
tivity. In contrast to PLC type III, the slight inhibition of
epithelial 5␣-reductase activity by PI-PLC, PLC type XIV,
and PLD was not dose-dependent. In stroma, PLC type XIV
and PLC type III inhibited 5␣-reductase activity dosedependently, whereas PI-PLC and PLD did not exhibit such
a dose-dependent 5␣-reductase inhibition. The effects of
PLD, PI-PLC, PLC type XIV were not significantly different
between the epithelial and stromal 5␣-reductase, whereas
PLC type III inhibited the 5␣-reductase activity significantly
stronger in stroma than in epithelium. The present data
demonstrate that phospholipases with various substrate
specificities and modes of phospholipid hydrolysis exhibit
significantly different effects on 5␣-reductase activity. Thus,
prostatic 5␣-reductase activity is not unspecifically affected
by any alteration of the hydrophilic phospholipid milieu.
Rather, the various phospholipids act specifically. The significanty lower inhibition of 5␣-reductase by PLC type XIV
as compared to PLC type III suggests that phosphatidylcholine is not absolutely necessary for 5␣-reductase activity.
Phospholipids such as phosphatidylethanolamine, serine,
and inositol which are not hydrolyzed by PLC type XIV
seem to maintain the 5␣-reductase activity. Among those
phospholipids, phosphatidylinositol seems not to be an important modulator as incubation with PI-PLC did not inhibit
significantly the 5␣-reductase activity. In addition, the
present study demonstrated that the effect of phospholipases on 5␣-reductase activity depends not only on the substrate specificity but also on the mode of hydrolysis. In contrast to PLC type III, PLD exhibited no or merely a slight
inhibition of 5␣-reductase activity. Both PLC type III and
PLD are characterized by a broad substrate affinity. Thus,
the significant differences between the PLC type III and PLD
mediated effects on 5␣-reductase must be due to different
hydrolysis products generated by both phospholipases. In
summary, our data point towards the role of specific phospholipids and phospholipolysis mediated degradation
products as regulators of prostatic 5␣-reductase.
FREQUENT ABNORMALITIES OF TSG101 TRANSCRIPTS IN HUMAN
PROSTATE CANCER
Zijie Sun, Jing Pan, Glenn Bubley, and Steven P. Balk
Cancer Biology Program, Division of Hematology-Oncology,
Department of Medicine, Beth Israel Deaconess Medical Center
and Harvard Medical School, Boston, MA 02215
TSG101 has been identified as a candidate tumor suppressor gene and abnormal transcripts have been identified
in a substantial fraction of breast cancers. To determine
whether TSG101 expression is commonly altered in other
tumors, a series of 15 primary and metastatic prostate cancers were analyzed by reverse transcriptase-PCR amplification. Abnormal transcripts with extensive deletions in the
coding region were found in 9 of these tumors, while only
the normal transcript was found in control and benign prostatic hypertrophy tissues. More than one abnormal transcript was found in 4 of these 9 cases and distinct abnormal
TSG101 transcripts were found in separate biopsies taken
from one tumor. Importantly, the normal TSG101 transcript
was undetectable in two metastatic prostate cancers, indicating the absence of tsg101 protein. Sequence analysis demonstrated that there were at least 6 distinct deletions, with
four of these deletions found in more than one tumor
sample. The most commonly identified deletion, from bp
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153 to 1055, was identical to a deletion reported previously
in breast cancer. These results demonstrate that TSG101
transcripts are frequently abnormal in prostate cancer.
To further determine the biological roles of TSG101, we
tested its transcription activity in vitro. The N-terminal 230
AA’s, when fused to the Gal4 DNA binding domain, can
activate transcription; this function is inhibited by the Cterminal region, which binds to the transcription co-factor
CBP/p300. The full-length protein or its separate halves can
suppress ligand-dependent transcriptional activation by the
androgen receptor (AR) or estrogen receptor (ER), which
have central roles in prostate and breast cancers, respectively. Our results suggest that the anti-tumor effects of
TSG101 on prostate and breast cancers may be through
modulation of AR and ER-mediated transcriptional activation.
THE IMPORTANCE OF CYTOCHROME P450c17 POLYMORPHISMS
IN MEDIATING PROSTATE CANCER RISK
Abhijit A. Shinde,1,2 Stewart L. MacLeod,2,4 Anjali Shinde,1,3
Jason R. Plaxco,4 Bobby Lawson,4 Roger C. Stotts,4 Don E.
Johnson,5 and Nicholas P. Lang 1,2,4,6
1
Department of Physiology and Biophysics, University of
Arkansas for Medical Sciences, Little Rock, AR 72205
2
Department of Surgery, University of Arkansas for Medical
Sciences, Little Rock, AR 72205
3
Department of Pathology, University of Arkansas for Medical
Sciences, Little Rock, AR 72205
4
Arkansas Cancer Research Center, Little Rock, AR, 72205
5
Urology Service, John McLellan VA Hospital, Little Rock, AR
72205
6
Surgical Service, John McLellan VA Hospital, Little Rock, AR
72205
Cytochrome P-450c17 (CYP17) catalyzes the rate-limiting
step in testosterone biosynthesis, thereby influencing androgen levels. Higher androgen levels may be an important risk
factor for developing prostate cancer. A single T→C substitution in the 5⬘ promoter region of the CYP17 gene creates an
additional SP-1 type (CCACC box) promoter site that may
cause increased gene expression. The T→C substitution also
creates an MspA1 recognition site detected by PCR-RFLP
analysis. The CYP17 promoter region was amplified from
lymphocyte DNA, digested with MspA1 and analyzed by
4% agarose gel electrophoresis. Representative genotypes
were confirmed by fluorescent cycle sequencing. We report
here genotyping results from 61 prostate cancer patients and
138 frequency-matched disease-free controls. Heterozygous
individuals (with the polymorphism on one CYP17 allele)
were 1.4 times more likely to have prostate cancer (O.R. =
1.38, P = 0.29). However, men homozygous for the polymorphism (on both alleles) had an elevated risk of developing
prostate cancer (O.R. = 3.45, P = 0.03). The increase in risk
rises in a dose-dependent manner with the number of polymorphic alleles present, possibly corresponding to higher
transcriptional activity. Higher CYP17 activity would increase androgen levels, contributing to an elevated lifetime
exposure to androgens. The higher prostate cancer risk observed in individuals with the CYP17 polymorphisms is
probably due to higher androgen levels which have been
implicated as a significant risk factor for developing the disease.
325
HERITABILITY OF THE SYMPTOMS OF BENIGN PROSTATIC
HYPERPLASIA AND THE ROLES OF AGE AND ZONAL PROSTATE
VOLUMES IN TWINS
A. Wayne Meikle, Aruna Bansal, Darrell K. Murray, Robert A.
Stephenson, and Richard G. Middleton
Departments of Medicine, Urology, and Medical Informatics,
University of Utah School of Medicine, Salt Lake City, Utah
84132
OBJECTIVE: Both benign prostatic hyperplasia and
lower urinary tract symptoms (LUTS) have been shown to
increase with age in men, but a causal relationship between
prostate volume and symptoms has not been established.
This study has two aims, firstly to investigate the interrelationships of age, symptoms, and various zonal measurements in the prostate, and secondly, to assess the impact of
heritable influences on symptom score.
METHODS: Eighty-three monozygotic twin pairs, and
83 dizygotic twin pairs were studied to determine age and
lower urinary tract symptoms (LUTS) as assessed by the
American Urological Association (AUA) symptom score.
Their prostate volumes (total, transition zone, and peripheral zone) were measured by transrectal ultrasound (TRUS).
RESULTS: There was significant evidence of pairwise
correlation between TZ and symptom score (P = 0.04), and
between age and symptom score (P = 0.03). Age also showed
significant correlation with all volume measurements. Heritability appears to account for 82.6% of the variability in
symptom score in men older than 50 years.
CONCLUSION: This study shows evidence that age and
transition zone volume play a role in LUTS, but also that
their influence is not strong. Estimates of heritability suggest
that hereditary factory factors contribute substantially to
LUTS.
Supported in Part by Grants DK-45760, DK-43344 and
RR-00064 from the National Institute of Health USPHS.
TRANSURETHRAL BALLOON LASER THERMOTHERAPY INDUCES
LONG-LASTING APOPTOSIS OF PROSTATIC SMOOTH MUSCLE
CELLS IN HUMAN BENIGN PROSTATIC HYPERPLASIA
T. Tsukamoto, N. Itoh, N. Masumori, and S. Furuya
Department of Urology, Sapporo Medical University, Sapporo,
Japan
Transurethral balloon laser thermotherapy (TUBAL-T)
has been developed recently as one of the noninvasive treatments for benign prostatic hyperplasia (BPH). However,
previous studies have not fully revealed cellular events in
prostatic tissue after thermotherapy. In the previous study,
we reported that significant decrease in noradrenalininduced peristalsis of the prostate occurred after TUBAL-T.
Since a decrease in prostate volume as great as 20% was
achieved after TUBAL-T, it may be valid to speculate about
some other mechanism to explain reduced adrenergic activity in the prostatic urethra than the volume reduction by
TUBAL-T-induced necrosis. Prostatic smooth muscle cells
(PSMCs), the cells most responsible for dynamic urinary obstruction, have been reported to be more heat sensitive than
prostatic gland cells. Thus, we examined apoptosis of PSMCs in the prostate 2–4 weeks after the treatment by electronmicroscopy (EM) and the DNA nick end-labeling
(TUNEL) method. The EM study revealed condensation of
chromatin at the nuclear margin in PSMCs located in the
area adjacent to periurethral coagulative necrosis. PSMCs
with condensed chromatin were often associated with cyto-
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Bethesda Abstracts
plasmic vacuoles in the perinuclear area. Adjacent to the
areas of necrosis, a zone of loose stromal tissue containing
considerable numbers of spindle cells with acidophilic cytoplasm was found. These cells were immunoreactive for alpha-smooth muscle actin as well as the heat-shock protein,
but it cannot be determined whether they were smooth
muscle cells or myofibroblasts. When the prostatic tissues
were stained by the TUNEL method, nuclei of most spindle
cells in the loose stromal zone showed strong positive reaction. The current results suggest that long-standing apoptosis was induced in PSMCs by TUBAL-T and that apoptosis
may contribute to clinical improvement of outlet obstruction
through its influence on PSMC function.
INHIBITION OF APOPTOSIS IN THE VENTRAL PROSTATE
FOLLOWING CASTRATION BY ANDROGENS IS ASSOCIATED
WITH ALTERATION OF THE IGF SYSTEM OF MITOGENS
Hung Huynh and Tara Nickerson
Lady Davis Research Institute of the Jewish General Hospital
and the Department of Medicine, McGill University, Montreal,
Quebec H3T 1E2, Canada
Insulin-like growth factor (IGF) has been shown to protect cells from apoptosis and plays an essential role in normal prostate physiology. We have previously shown that
apoptosis is induced in the ventral prostate within 6 hours
following castration and is temporally associated with increased gene expression of IGF binding proteins (IGFBPs) -2,
-3, -4, and -5. Because IGFBPs modulate IGF action and are
potential mediators of apoptosis and involution of the prostate gland, we have studied the effects of androgenreplacement on apoptosis and the expression of IGFs and
IGFBPs in the ventral prostate following castration. Shortterm administration of 5-␣-dihydrotestosterone (DHT) by
daily injections (3 ␮g/kg BW) slightly delayed, but was not
able to overcome, castration-induced IGFBP gene expression
and apoptosis in the ventral prostate. Induction of TRPM-2
gene expression, a gene known to be associated with involution in the prostate, following castration was also slightly
delayed by injections of DHT and was observed to lag behind expression of IGFBPs and induction of apoptosis. In
contrast, constant release of either testosterone or DHT from
implants for 3 weeks effectively prevented castrationinduced IGFBP gene expression and apoptosis. Our data
suggest that prostatic testosterone or DHT levels must stay
above a critical threshold in order to inhibit IGFBP gene
expression and to maintain healthy prostate cells.
CHARACTERIZATION OF AN INHIBITOR OF PROSTATE CANCER
CELL GROWTH
Mohsen Abolhassani1,2 and J.W. Chiao2
1
Department of Immunology, Pasteur Institute of Iran, Tehran
13164, Iran
2
Department of Medicine, New York Medical College, Valhalla,
New York 10595
Prostate cancer is currently the most common malignancy and is the second leading cause of cancer death
among males in the United States. For more than 50 years,
hormonal therapy has been the major therapeutic procedure
for metastatic prostate cancer. After initial response, patients
inevitably develop a tumor that is hormonally unresponsive
and resistant to the current therapeutic procedures. Other
available treatments such as radiation, surgery and chemotherapy have not prolonged survival of patients with metastatic carcinoma. We have identified a new antiproliferative
protein (APP) from serum-free conditioned medium of two
androgen-independent prostatic carcinoma cell lines (PC3
and DU-145). This protein selectively inhibits cell proliferation of an androgen-dependent prostatic carcinoma cell line
(LNCaP), but not proliferation of other cell lines tested or
normal human peripheral lymphocytes. APP does not induce apoptosis, but it prevents the LNCaP cells from entering into the S phase of the cell cycle. The antiproliferative
activity is reversible and is not affected by neutralizing antibodies against the well known cytokines. APP was partially purified with an apparent molecular weight of 50 kDa
and is sensitive to protease digestion but not to reducing
agent, DNase or RNase digestion. APP is immunogenic and
can induce anti-APP antibody in mice. Further purification
and characterization of APP is under way.
TYROSINE KINASE EXPRESSION BY BONE MARROW METASTASES
FROM HUMAN ANDROGEN INDEPENDENT PROSTATE CANCERS
Andreas Chott, Zijie Sun, Glenn Bubley, and Steven Balk
Hematology-Oncology Division, Beth Israel Deaconess Medical
Center and Harvard Medical School, Boston, MA
Prostate stroma-epithelial cell interactions play a critical
role in regulating normal prostate growth. An important
aspect of human prostate cancer biology is preferential
metastatic growth in bone marrow and this likely also reflects the need for specific interactions between tumor cells
and bone marrow stroma or other myeloid elements. The
lack of cell lines representative of human bone marrow metastases has hindered efforts to identify biochemically the
relevant growth factors and/or stromal elements. Therefore,
an alternative approach we have taken is to identify growth
factor receptors expressed by freshly isolated metastatic
prostate cancer cells from bone marrow biopsies. Initial efforts have focused on receptor tyrosine kinases as they represent a large family and can be cloned from the small quantities of available tissue by degenerate oligonucleotide RTPCR methods. This approach was applied to frozen sections
from seven fresh bone marrow biopsies that were largely
replaced by androgen independent prostate cancers. The ␣
platelet derived growth factor (␣PDGF-R) was consistently
identified in these biopsies. Expression of the ␣PDGF-R by
bone marrow metastases as well as primary prostate cancers, but not normal prostate, was confirmed by immunohistochemistry. Non-receptor tyrosine kinases were also
identified in this screen, the most common being Jak 1.
Immunohistochemistry confirmed Jak 1 protein expression in bone marrow metastases as well as in primary prostate cancers. Finally, direct analyses demonstrated that the
type I insulin like growth factor and the fibroblast growth
factor receptors, important tyrosine kinase receptors in normal prostate and primary prostate cancer, were not expressed by metastatic prostate cancers in bone marrow.
These results indicate that metastatic prostate cancers in
bone marrow may rely on a particular spectrum of growth
factors, including PDGF and possibly others which signal
through as yet unidentified receptors upstream of JAK 1.
Further tyrosine kinases identified in these studies will also
be discussed.
Bethesda Abstracts
Based upon these studies, a phase II clinical trial of a
PDGF-R antagonist in androgen independent prostate cancer is now underway. This work was supported by NIH
grant CA-65647 to S.B. and by the Hershey Family Prostate
Cancer Research Fund.
327
THE EVALUATION OF SERUM AND TISSUE ZINC LEVELS IN SEX
HORMONE-INDUCED PROSTATIC CARCINOGENESIS IN THE
NOBLE (Nb) RAT
Y.C. Wong,1 Y.Z. Wang,1 J.S.K. Lee,2 Neville N.C. Tam,1 and
Davy Lee1
1
POSSIBLE ROLE OF MATRIX MOLECULES IN BPH
L. True, T. Wight, and T. Norwood
University of Washington, Seattle, WA
BACKGROUND: Benign prostatic hypertrophy (BPH) is
characterized by the formation of discrete nodules of hyperplastic parenchyma in the transition zone (TZ) of the prostate. There is a relative increase in the ratio of stromal to
epithelial cells in the average nodule. This, and other evidence, suggest a primary role for stromal cells in nodule
formation. A variety of specific growth factors, many the
products of stromal cells, are overexpressed in BPH, or in
cells from nodules—basic FGF, TGF ␤2, TGF ␣, insulin-like
growth factors I and II, keratinocyte growth factor, and EGF.
We might better understand the pathogenesis of BPH by
comparing the disease with another chronic disease of stromal/smooth muscle cells, atherosclerosis. In atherosclerosis,
the cytokines PDGF and bFGF stimulate synthesis of the
proteoglycans versican and hyaluronan by vascular smooth
muscle cells. These matrix molecules increase total tissue
volume, which plays at least a permissive, if not a stimulatory, role in smooth muscle cell proliferation. A similar role
for these matrix molecules may occur in BPH. However,
specific questions and mechanisms concerning BPH have
not been answered, including: what is the relative role of
epithelial and stromal cells in BPH, why does nodule formation occur primarily in the TZ and not in other zones of
the prostate, and, is there a role for matrix molecules in
BPH?
INVESTIGATIONS: Samples of nodular and nonnodular TZ parenchyma from 3 prostates were analyzed for
cell composition by morphometry, for overexpression of
versican mRNA by Northern blots and for versican and hyaluronan by affinity histochemistry using a versican-specific
antibody and a hyaluronan-specific probe in an indirect avidin-biotin-peroxidase assay, and for the proliferative state of
cell compartments by immunostaining with the cell cycle
marker Ki67. The ratio of epithelial:stromal cells in nodules
(from 2:1 to 1:5) overlapped with that in control, nonnodular TZ tissue (1.5:1 to 1:4). Versican mRNA levels were
1.5 to 2.5-fold greater in RNA preparations from nodules
compared with control non-nodular samples from the same
patient’s prostate. Immunoreactive versican and labeled hyaluronan were more intense in the stroma of nodules than in
non-nodular stroma. And, there was a tendency for the fraction of Ki67-positive stromal cells to be highest in regions of
nodules with intense versican and hyaluronan reactivity
than in less intensely stained regions of nodules, or in nonnodular tissue.
CONCLUSION: Our investigations (1) more precisely
define the cell composition of nodules, demonstrating more
marked heterogeneity in the cell composition of nodules
than has been previously recognized, (2) demonstrate overexpression of the matrix molecules versican and hyaluronan,
suggesting that they may play a role in BPH that is similar
to their role in atherosclerosis, and (3) provide preliminary
evidence that these matrix molecules are associated with a
relative state of stromal cell hyperproliferation.
Department of Anatomy, Faculty of Medicine, The University
of Hong Kong, Hong Kong, SAR, People’s Republic of China
2
Department of Clinical Biochemistry, Queen Mary Hospital,
Hong Kong, SAR, People’s Republic of China
In an effort to develop new prostatic tumor markers that
may be useful to the diagnosis and prognosis of this disease
we investigated the changes of serum and tissue zinc levels
in the Noble rat prostate gland under different pathological
conditions induced by administration of a combination of
testosterone (T) and 17␤-estradiol (E2). The results showed
that there were significant differences in serum zinc values
between the normal and hormone-treated rats with prostatic
hyperplasia, dysplasia and prostatic carcinoma (P < 0.05),
although there was no significant difference among rats
with different forms of prostatic lesions (i.e., hyperplasia,
dysplasia and prostatic adenocarcinoma). There was also a
difference in zinc content among LP, VP and DP in normal.
The zinc levels of LP were several fold greater than that of
either VP or DP (P < 0.01). There was also a great difference
in the zinc levels between the normal and hyperplastic/
dysplastic and carcinomatous LP and VP (P < 0.05). The
levels of zinc in both LP and VP were increased in hyperplasia/dysplasia and carcinoma. On the other hand, the zinc
levels of LP were much higher than that of VP in hyperplasia/dysplasia and carcinoma (P < 0.01), which may be correlated with the incidence of prostate cancers in these lobes
(i.e., being higher in LP and much lower in VP). In contrast,
in DP which was resistant to carcinogenesis, the levels of
zinc were not affected by this hormonal regime. Our data
suggest that 1) the differences in Zn levels between these
lobes reflect the intraprostatic heterogeneity in biological
function within the rat prostate; 2) the growth and development of the prostatic lesions in LP and VP may be positively
correlated with their significant increase in tissue zinc levels
while the lack of response of DP to carcinogenesis may be
due to its relatively stable low zinc levels. It is suggestive
that the tissue zinc content may be used as a marker for
prostatic lesions including hyperplasia, dysplasia and carcinoma while the serum zinc levels may be a useful indicator
for abnormal prostatic growth.
PSA DETECTION OF PROSTATE CANCER IN WHITE AMERICAN,
BLACK AMERICAN AND ASIAN MEN
Hee Sun Jeon, M.D., Ph.D., Jerome Kowal, M.D., and Yun Sik
Kwak, M.D., Ph.D.
Division of Geriatric Medicine, Department of Medicine, Case
Western Reserve University School of Medicine, Cleveland, OH,
and Department of Laboratory Medicine, Ajou University
School of Medicine, Suwon, Korea
There are large racial and geographical variations in
prostate cancer (PC) prevalence and aggressiveness, ranging
from 0.082% in black Americans to 0.001–0.006% in Asian
men. Prostate specific antigen (PSA) is considered to be a
useful screening tool for PC because of its organ specificity,
but serum PSA measurement has several problems, includ-
328
Bethesda Abstracts
ing diagnostic overlap between PC and benign prostatic hyperplasia (BPH), resulting in unnecessary invasive biopsy
procedures. To determine the effectiveness of PSA in diagnosing PC in populations with wide variations in PC prevalence, we retrospectively analyzed serum PSA levels in 302
black Americans (BA) and 522 white Americans (WA) at the
Cleveland VAMC, and 134 Koreans at Ajou University Hospital, Suwon, Korea. These patients had been subjected to
prostate biopsy as a result of serum PSA levels greater than
4 ng/ml and/or abnormal digital rectal examination. The
distribution in BA was 51.0% PC, 31.8% BPH and 17.2%
normal prostate histology; WA 41.6% PC, 39.3% BPH and
19.1% normal; and Koreans 18.7% PC, 79.8% BPH and 1.5%
normal. The mean age of Koreans with PC (75.0) was significantly higher than BA (68.5) and WA (67.7) with PC.
The serum PSA levels (ng/ml) of BA with PC (mean: 69.0,
median: 21.3) were significantly higher than WA with PC
(41.0, 10.9), but Koreans with PC (88.9, 62.3) were not different from BA or WA with PC; 23.1% BA, 31.8% WA and
17.2% Koreans fell into the diagnostic gray zone (PSA 4.0–10
ng/ml). Pathologic distribution was 35.7% PC, 50.0% BPH
and 14.2% normal histology among BA, 44.0% PC, 32.5%
BPH and 22.9% normal among WA, and 9.8% PC and 90.2%
BPH among Koreans. Using a serum PSA cutoff of 4 ng/ml,
the specificity of the PSA assay was low in all three groups,
48% in BA, 57% in WA and 47% in Koreans. Sensitivity was
relatively low but slightly higher in Koreans (92%) than in
BA (83%) and WA (84%). The positive predictive value was
much lower in Koreans (28%) than in BA (62%) and WA
(58%), due to the lower prevalence in Koreans. The negative
predictive value was higher in Koreans (96%) than in BA
(74%) and WA (83%). The false positive rate was 52% in BA,
43% in WA and 54% in Koreans. Conversely, the false negative rate was 16% in BA and WA, and 8% in Koreans.
These data indicated that the serum PSA levels of Korean
men with PC or BPH, while they had the lowest incidence
rate of PC, more closely resembled those of BA men who
had the highest incidence rate of PC than WA. Using the
generally accepted serum PSA cutoff of 4 ng/ml, specificity
and predictive values of PSA assay in differentiating PC
from benign conditions were low among all three groups.
This initial study was limited by the number of cases eligible
for inclusion and the dependence on retrospective data of
biopsy-proven prostatic pathology. However, these data
clearly demonstrate the limitations of elevated PSA measurements alone in accurately detecting PC, particularly in
Asians. More definitive diagnostic studies to reduce invasive prostate biopsy procedures are required in all three
populations.
HETEROCYCLIC AMINE ADDUCT FORMATION AND
N-ACETYL-TRANSFERASE EXPRESSION IN HUMAN PROSTATE
Richard F. Jones,1 Maria Debiec-Rychter,1 Herman J. Schut,2
Philo P. Morse,1 Gabriel P. Haas,1 and Ching Y. Wang1
1
Department of Urology, SUNY Health Science Center and VA
Medical Center, Syracuse, NY
2
Department of Pathology, Medical College of Ohio, Toledo, OH
INTRODUCTION AND OBJECTIVES: Environmental
agents including heterocyclic amines (HAs) are implicated
in the etiology of prostate cancer. In the present study, we
analyzed the carcinogenic potential for human prostate of
two common HAs found in overcooked meat and cigarette
smoke: N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (N-OH-PhIP) and N-hydroxy-2-amino-3,8dimethylimidazo[4,5-f]quinoxaline (N-OH-MelQx).
METHODS: Prostate epithelial cells (PECs) were isolated
from fresh prostates from 2 individuals. The PECs were incubated in vitro with 10−5 M N-OH-PhIP or N-OH-MelQx for
either 2 hr for DNA adduct formation, determined by 32Ppostlabeling analysis; or 18 hr for unscheduled (repair) DNA
synthesis, determined by incorporation of 3H-thymidine
into DNA in the presence of hydroxyurea (to block DNA
replication). The expression of N-acetyltransferases (NATs)
was determined by in situ hybridization to RNA in prostate
sections with probes specific for either NAT1 or NAT2 transcripts. NATs are enzymes capable of metabolically activating aromatic and heterocyclic amines to DNA reactive
forms.
RESULTS: 32P-Postlabeling analysis detected DNA adducts in both HA-treated PEC samples, but not in controls.
Adduct levels were ∼100× greater in N-OH-PhIP- vs. N-OHMelQx-exposed cells. Unscheduled (repair) DNA synthesis
was induced in the PECs exposed to either HA. In situ hybridization showed that mRNAs for both NAT- I and NAT-2
were present in glandular prostate cells, but absent in stromal tissue.
CONCLUSIONS: Both N-OH-PhIP and N-OH-MelQx
induce DNA adduct formation and unscheduled (repair)
DNA synthesis in prostate epithelial cells. Thus the prostate
expresses NATs that are capable of metabolically activating
procarcinogenic N-hydroxy derivatives of these heterocyclic
amines to 0-acetoxyamines which are both DNA-reactive
and mutagenic genotoxins. N-OH-PhIP and N-OH-MelQx
are therefore both potential prostate carcinogens. (Source of
funding: PHS grants CA23386 and CA23800)
ANTIPROLIFERATIVE EFFECT OF THE POLYSACCHARIDE
FRACTION OF 20% METHANOLIC STINGING NETTLE ROOT
EXTRACT IN VIVO (MOUSE) AND IN VITRO (LNCaP)
J.J. Lichius, C. Muth, C. Lenz, and L. Konrad
Department of Pharmacological Biology and Department of
Anatomy and Cell Biology, University of Marburg, D-35033
Marburg, Germany
Methanolic and aqueous extracts of stinging nettle roots
(Urtica dioica L., Urticaceae) are used in Germany (and Europe) in the treatment of benign prostatic hyperplasia (BPH).
We used the BPH-model of Chung in our laboratories by
directly implanting an intact fetal urogenital sinus (UGS)
into the ventral prostate gland of an adult mouse. The fetuses of 16 day-old Balb/c mice were harvested and the UGS
with connecting tissues were microdissected. The UGS was
directly transplanted into one lobe of the prostate of an adult
male mouse. The contralateral lobe of each prostate was
simply pierced with a fine forceps and served as a control
experiment (sham-operation). The implants were allowed to
grow and develop for 28 days. We tested in this model the
effect of the polysaccharide fraction of 20% methanolic stinging nettle root extract.
In addition we investigated the antiproliferative effect of
this extract on LNCaP cells. The proliferation was measured
by determining the total amount of genomic DNA during
one week. Antiproliferative effects were found in the mouse
model and LNCaP growth (40–50% suppression).
The results of our experiments show that the polysaccha-
Bethesda Abstracts
ride fraction of stinging nettle root extract may play an active role in antiproliferative activity, especially on epithelial
cells.
(Supported in parts by Kanoldt Arzneimittel Höchstädt/
Donau Germany)
NEUROTROPHIC FACTORS CNTF AND GDNF IN PROSTATE AND
PROSTATIC CANCER
Albert A. Geldof,1,2 Ernst P. van Haarst,1 and Don W.W.
Newling1
1
Department of Urology, University Hospital Vrije Universiteit,
Amsterdam, The Netherlands
2
Department of Nuclear Medicine, University Hospital Vrije
Universiteit, Amsterdam, The Netherlands
Recent progress in growth factor research has led to a
reexamination of the involvement of neurotrophic factors
outside their classical domain of the nervous system. These
last few years have seen a substantial accumulation of data
concerning nerve growth factor (NGF)’s prevalence within
the prostate. NGF and its receptors were reported from normal prostatic tissue, benign hyperplasia and prostatic cancer.
Using a panel of 5 specific antibodies, directed against
various neurotrophic factors (CNTF: ciliary neurotrophic
factor; BDNF: brain derived neurotrophic factor; GDNF:
glial-cell derived neurotrophic factor; NT#: neurotrophin-3
and NT-4: neurotrophin-4), we have made an estimate of the
prevalence and distribution of these factors within prostatic
tissue. Immunohistochemistry using a two-step staining
technique was performed on preparations of human prostatic cell lines LNCaP-FGC, PC-3 and DU-145 and rat anaplastic prostate cell line R3327-MATLyLu and cryostat sections of tissue biopsy samples from eleven patients suffering
from carcinoma of the prostate.
In eight out of nine evaluable clinical carcinoma samples,
specific staining for CNTF and GDNF was detected. A role
for such factors, their specific receptors and the relation with
processes of malignant transformation require further investigation.
CASTRATION INDUCES A RAPID AND SIGNIFICANT REDUCTION
OF BLOOD FLOW TO THE RAT PROSTATE GLAND THAT
PRECEDES THE APPEARANCE OF APOPTOTIC EPITHELIAL CELLS
Ahmad Shabsigh, David Chang, Peter Puchner, Carl A. Olsson,
and Ralph Buttyan
Columbia University, New York, NY
Androgenic steroids regulate prostate size by repressing
prostate cell apoptosis. Classically, this effect has been attributed to direct androgenic stimulation of the prostatic epithelium. Yet for other tissues of the body, local blood flow
regulation can be one of the simplest means to control tissue
size. To determine if androgen action on the prostate might
involve blood flow regulation, we used a microsphere perfusion technique to measure the acute effects of castration on
blood flow to the rat ventral prostate gland.
Four groups of adult male Sprague Dawley rats were
studied: 1) intact controls; 2) sham-operated controls; 3) castrated controls receiving simultaneous testosterone replacement; and 4) castrated rats with no androgen replacement.
Catheters were placed in anesthetized rats through the
femoral artery and through the carotid artery to the aorta.
Fluorescent microspheres (4 × 105) (15 ␮M diameter) were
329
injected into the peripheral circulation through the carotid
catheter. These microspheres lodge in the capillary bed of
the tissue to which they perfuse. A specimen of blood, obtained through the femoral catheter simultaneous with microsphere infusion is used as a reference to calculate tissue
blood flow. Two minutes after infusion, rats were sacrificed
and prostates, bladders and spleens were recovered,
weighed and digested in an alkaline solution to recover microspheres. Microspheres were quantified by flow cytometry and relative blood flow (RBF) was calculated based on
the number of microspheres in the reference blood and tissue samples.
The results of this study demonstrated a significant decrease in prostate blood flow at both 18 and 24 hrs after
castration when compared to control groups. RBF to the
ventral prostate was reduced by 38% at 18 hrs after castration and by 52% at 24 hrs after castration (P < 0.05). RBF to
the bladder and spleen was unchanged by castration compared to all control groups. The drastic reduction in prostate
blood flow observed in our studies precedes the onset of
epithelial cell apoptosis that begins at 24 hrs after castration.
We propose that this early and acute reduction in prostate
blood flow might creates a hypoxic condition conducive to
the onset of massive cellular apoptosis leading to prostate
regression.
From: Biology of Prostate Growth, March 1998, Bethesda
MD.
MOLECULAR CLONING, GENOME STRUCTURE AND GENE
EXPRESSION ANALYSIS OF PSP94 (PROSTATE SECRETORY
PROTEIN OF 94 AMINO ACIDS)
Jim W. Xuan,1 Sean Jiang,1 Sue Guo,2 Joseph L. Chin,1 Joseph
Kwong,3 and Franky L. Chan3
1
Department of Surgery, University of Western Ontario,
Ontario, Canada
2
Procyon Biopharma, Inc.
3
Department of Anatomy, The Chinese University of Hong
Kong, Hong Kong
PSP94 is one of the major proteins secreted by the human
prostate . It is a small, non-glycosylated protein rich in cysteine residues with a molecular weight of 10.7 kDa. Its potential use as a marker and a therapeutic agent for prostate
cancer has raised much interest.
In order to establish a mouse animal model to further
elucidate its biological role, we cloned by RT-PCR and sequenced mouse PSP94 cDNA (∼500 bp) and disclosed genomic structure of PSP94. We have studied gene expression
and distribution of PSP94 mRNA in the rat prostate and its
regulation by androgen by in-situ hybridization (ISH). The
whole mouse PSP94 gene (>18 kb) was amplified by long
and accurate-PCR and also by screening of a mouse embryo
stem cell genomic library. ISH showed that the hybridization signal was strongly positive in the cytoplasm of the
secretory epithelial cells in lateral prostate and also weakly
expressed in the nuclei of the epithelial cells in dorsal prostate, but negative in ventral prostate, coagulating gland and
seminal vesicle, indicating a specific pattern of secretion
from the prostate.
PSP94 gene expression appears to be androgen-regulated
as it can be removed by long-term castration. The hybridization signal in the lateral prostate was weakened significantly after 30 days castration and became completely lost
after castration for 60 days.
330
Bethesda Abstracts
Computational and statistical analyses indicate several
highly conserved characteristics of PSP94. Comparison
PSP94 from human, monkey, dog and rodents revealed that
their cysteine content comprises up to 10% of whole coding
sequence (94 amino acids) and most are highly conserved.
The three intron-four exon structure, the consensus sequence ( . . . . GT- intron-AG . . . ), i.e., mRNA splicing of
PSP94, is also strikingly conserved. High divergence in the
genomic DNA sequence and the protein coding region,
however, was observed among these species. For example,
overall identity of mouse and rat coding sequences is only
78.6%. Comparing ␣-globin, a typical evolutionarily conserved gene, with the PSP94 gene, the rate of synonymous
and nonsynonymous changes per site per year is about two
times higher. Therefore PSP94 gene has been under far less
evolutionary constraints than other genes, This may indicate
a potential role as a species barrier in reproductive biology.
ROLE OF GROWTH FACTORS AND SEX STEROIDS ON
PROLIFERATION OF ADULT RODENT PROSTATE
S. Yoshida,1 Y. Sugimura,1 T. Hioki,1 Y. Yamada,1 M.
Tatematsu,1 K. Arima,2 and J. Kawamura2
Gridley,2 Cory Abate-Shen,1 and Michael M. Shen1
1
Center for Advanced Biotechnology and Medicine and
UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ
2
Jackson Laboratories, Bar Harbor, ME
Homeobox genes encode transcriptional regulatory proteins that play an important role during development. We
have identified a novel homeobox gene, termed Nkx3.1,
whose molecular properties signify its essential role in prostate development and a possible involvement in prostate
carcinoma. Human Nkx3.1 is localized to chromosome region 8p21, which is deleted in 80% of prostate carcinomas.
We have now shown that Nkx3.1 is one of the earliest markers of the prospective prostate epithelium during embryogenesis. In adult male mice, Nkx3.1 expression is androgen
dependent and is primarily restricted to the prostate. We
have examined the function of Nkx3.1 during mouse development using targeted gene disruption. Homozygous
(Nkx3.1−/−) male and female mice are viable and fertile. Our
analysis of the homozygous male mice indicates a defect in
the prostatic ductal branching and secretory protein production by the prostate. Moreover, we find that forced expression of Nkx3.1 in a prostatic carcinoma cell line reverts its
transformed phenotype in culture. Further work will help us
understand the role played by this homeobox gene in prostate development and oncogenesis.
1
Department of Urology and Pathology, Aichi Cancer Center,
Nagoya, Japan
2
Department of Urology, Mie University School of Medicine,
Mie, Japan
It is well known that the normal adult prostatic cells
show minimal DNA synthesis; however, little is known
about the hemostatic mechanism in the proliferative activity
in the adult prostate. To determine the role of growth factors
and sex steroids on the adult mouse prostate, microdissected prostate were grown in a serum-free organ culture
system. Micro-dissected ducts from adult mouse vental and
dorsolateral prostate were incubated on the Millipore filters
(Millipore, USA) for 6 days under serum and growth factorfree conditions. The basal media contains DMEM-Ham’s
F-12 (1:1 vol/vol) supplemented with insulin, transferrin,
gantamycin. Media was changed every 3 days. Direct effects
of TGF-␣ and estradiol were histologically studied by adding them into testosterone deficient media. Proliferative activity was also histologically analyzed by BrdU incorporation. Both TGF-␣ (10 ng/ml) and estradiol (10−9 M) induced
epithelial hyperplasia, resulting in squamous morphology
with extensive epithelial proliferation into the luminal space
in the proximal ducts of dorsolateral lobe. On the other
hand, atypical hyperplastic changes were observed in the
distal ducts of ventral lobe. BrdU labeling indicated excessive proliferation of basal cells in both treatment. These
hyperplastic changes were partially inhibited by co-administration of physiological dose of testosterone (10−8 M).
These results suggest that prostatic epithelial growth is
regulated by interaction between growth factors and sex steroid hormone. Testosterone can maintain ductal morphology by suppressing the aberrant growth stimulation, which
is locally induced by growth factors or cytokines.
THE Nkx3.1 HOMEOBOX GENE IS REQUIRED FOR NORMAL
PROSTATE DEVELOPMENT
Rajula Bhatia-Gaur,1 Peter Sciavolino,1 Nishita Desai,1 Tom
EFFECTS OF ANDROGEN ABLATION ON ANGIOGENISIS IN
BENIGN PROSTATIC HYPERPLASIA
Durwood E. Neal Jr., Rama Gangula, William Elfarr, Andi
Arnautovic, Nilson Salas, and Massoud Motamedi
Galveston, TX
INTRODUCTION AND OBJECTIVES: We previously
reported in a canine model that prostatic blood flow was
dramatically reduced either by administration of finasteride
or by bilateral orchiectomy. This was observed at both 45
and 90 days of treatment. These changes occurred along
with and prior to prostatic size reduction. These were expected findings based on theoretical and anecdotal information. The mechanisms by which this takes place are as yet
undefined.
METHODS: In the second phase of this experiment, we
examined twelve dog prostate glands by immunohistochemical staining for factor VIII, to identify the vascular
endothelium. This was accomplished on paraffin fixed sections of whole mount prostate glands. Also, we performed
reverse transcriptase polymerase chain reaction on fresh
prostatic tissue, to test for interleukin 8 (IL8) activity, a
known angiogenesis factor.
RESULTS: In both the orchiectomized and finasteride
treated dogs, the factor VIII staining revealed a markedly
reduced blood vessel density of 6.8 + 4 and 9.2 + blood
vessels per square centimeter, compared with control prostates that had 29 + .3, P = .0007 and .0004, respectively. There
were fewer vessels in the orchiectomized animals, but the
difference was not statistically significant, p = 0.27. The IL8
levels were not detectable in either the control or the orchiectomized groups, whereas there were high levels in the
finasteride group.
CONCLUSIONS: In conclusion, the prostatic blood flow
does indeed diminish with androgen ablation, manifested
by a decrease in blood vessels per unit area in the prostate.
The IL8 gene is upregulated in the finasteride treated ani-
Bethesda Abstracts
mals, but not in the orchiectomized ones. This implies a
different mechanism of reduction in blood flow between the
finasteride and orchiectomized animals. Further studies are
ongoing to elucidate this.
GENISTEIN INHIBITS HYPOXIA-INDUCED VEGF EXPRESSION IN
PROSTATE CANCER CELLS
Alexander Kirschenbaum, Xin-Hua Liu, and Alice C. Levine
Departments of Medicine and Urology, Mount Sinai School of
Medicine, New York, NY
INTRODUCTION AND OBJECTIVES: We have previously demonstrated that human prostate fetal fibroblasts
(FF) and LNCaP prostate cancer cells express vascular endothelial cell growth factor (VEGF), a potent angiogenic and
permeability factor. We compared VEGF expression in response to hypoxia in FF, LNCaP, and the less differentiated
PC3 prostate cancer cell line and its metastatic subline,
PC3ML. We further tested the effects of an isoflavinoid,
genistein, on hypoxia-induced VEGF expression.
METHODS: A hypoxic state was induced with 100 ␮M
cobalt chloride for 2 days in FF, LNCaP, PC3 and PC3ML
cells. Genistein (100 ␮M) was added for 2 days. VEGF secretion was measured by ELISA.
RESULTS: Baseline VEGF levels (ng/106 cells/24 h) were
as follows: FF = 4.8, LNCaP = 4.8, PC3 = 2.5, PC-3ML =
2.1. Hypoxia had a modest effect on VEGF secretion by FF,
LNCaP and PC3 cells, but had a marked effect (8-fold increase in VEGF secretion) on PC3-ML cells (hypoxiainduced VEGF secretion by FF = 6.8, LNCaP = 7, PC-3 = 4.7,
PC3-ML = 15.9). Genistein treatment had no effect on hypoxia-induced VEGF secretion in LNCaP cells, but inhibited
the hypoxia-induced increase in VEGF secretion in the PC3ML cells (control = 1.78, genistein alone 2.4, hypoxia = 20.9,
genistein + hypoxia = 9.1).
CONCLUSIONS: Although baseline VEGF secretion is
highest in non-cancerous stroma and the well-differentiated
prostate cancer cell line LNCaP, the more aggressive and
metastatic cell line, PC3-ML, can respond to hypoxic conditions by increasing VEGF secretion 8-fold, thereby increasing tumor angiogenesis and metastatic potential. This hypoxia-inducible VEGF secretion is partially blocked by treatment with genistein which may be a potential antiangiogenic therapy for advanced prostate cancer.
SOURCE OF FUNDING: T.J. Martell Foundation for
Leukemia, Cancer and AIDS Research.
ANDROGENS INCREASE THE PRODUCTION AND SECRETION OF
VASCULAR ENDOTHELIAL CELL GROWTH FACTOR BY HUMAN
PROSTATE FETAL FIBROBLASTS
Alexander Kirschenbaum, Xin-Hua Liu, Pietra D Greenberg,
Stuart A. Aaronson, James F. Holland, Alice C. Levine
Departments of Medicine (Endocrinology), Urology, and Derald
H. Ruttenberg Cancer Center, Mount Sinai School of Medicine,
New York, N.Y. 10029
INTRODUCTION AND OBJECTIVES: Human fetal
prostate development is known to be dependent upon androgens and stromal-epithelial interactions. Vascular endothelial cell growth factor (VEGF) is an endothelial cellspecific mitogen which is critical to normal vasculogenesis.
331
We determined the cell-specific expression of VEGF in the
human fetal prostate and studied its androgenic regulation
in prostate fetal fibroblasts.
METHODS: Fetal prostates were derived from 17–22
week gestation fetuses. Immunohistochemical staining for
VEGF expression was carried out on formalin fixed tissues.
Human prostate fetal fibroblasts were grown in culture ± the
addition of dihydrotestosterone ( DHT 10−10-10−6M) and
VEGF mRNA and protein levels determined by Northern
blot and Western blot, respectively. VEGF mRNA transcription rates in the fibroblasts ± DHT were determined by
Nuclear Run-On Assay.
RESULTS: Immunohistochemical studies localized
VEGF protein expression to the stromal cells of the developing prostate. DHT upregulated VEGF mRNA and both
the cell-associated and secreted isoforms of VEGF within
12–24 hours. Maximal upregulation (2-fold) of VEGF expression was demonstrated with DHT 10−8 M. Nuclear run-on
assay demonstrated that the increase in VEGF expression
was at least partially due to increased VEGF mRNA transcription which was maximal 2 hrs after DHT addition.
CONCLUSIONS: VEGF is expressed by the stromal cells
of the human fetal prostate. VEGF expression by human
prostate fetal fibroblasts in under androgenic regulation.
Androgens influence prostate development via effects on
vasculogenesis.
SOURCE OF FUNDING: T.J. Martell Foundation for
Leukemia, Cancer and AIDS Research.
REGULATION OF PRIMARY HUMAN PROSTATE EPITHELIAL
CELL APOPTOSIS
Michael Morgan, Michael Lloyd, Jacqueline Thorburn, and
Andrew Thorburn
Huntsman Cancer Institute, University of Utah, Eccles
Institute of Human Genetics, Salt Lake City, Utah 84112
In the most general sense, cancer occurs when cell growth
and survival are misregulated and this fact lies behind the
enormous effort that is expended upon understanding the
molecular mechanisms through which mammalian cell
growth and survival/apoptosis are regulated. While significant progress has been made in identifying and understanding signaling pathways that may be altered in cancer cells,
this information comes primarily from studies in immortal
cell lines that can be easily maintained in culture and experimentally manipulated. However, most primary human
cells, even from tumor tissue, do not survive and grow well
in culture implying that the cell lines must be inherently
different in some respects from cells in tissues. Ironically,
such differences are most likely to be manifested for responses such as survival and growth that are under selective
pressure in culture, the phenotypes that we are most interested in understanding from the perspective of diseases
such as cancer. This fact raises a potential problem in that
conclusions regarding regulatory mechanisms that are based
only on studies in cell lines may not apply to human cells in
vivo. We may avoid some aspects of this problem by performing mechanistic studies in both standard prostate cell
lines and primary human prostate epithelial cells from normal and tumor tissue which are more likely to maintain the
regulatory mechanisms that apply in vivo. To begin this effort, we have developed methods to study molecular mechanisms in primary human prostate epithelial cells focusing on
332
Bethesda Abstracts
understanding the regulation of an aspect of prostate cell
biology that is altered in tumor cells—the control of adhesion-dependent survival and apoptosis. Our data suggest
that stress-induced signaling pathways that are activated by
the protein kinase MEKK1 regulate apoptosis when prostate
epithelial cells are detached from the extracellular matrix.
Recent data describing the molecular mechanisms that mediate this response will be presented. Our approaches to
extend molecular analysis to primary human prostate cells
will be generally applicable to other systems and may represent a way to extend our understanding of the mechanisms that control important cellular phenotypes to an experimental system that more closely reflects the situation in
vivo.
PAR, A NOVEL GENE DIFFERENTIALLY REGULATED BY
ANDROGENS IN HORMONE SENSITIVE AND RESISTANT
PROSTATIC CARCINOMA CELLS
O. Platica, M. Lopingo, J.F. Holland, and M. Platica
VA Medical Center, Brooklyn, NY 11209, and Mount Sinai
Medical Center, New York, NY 10029
During our previous work on the mechanism of hormone
resistance of prostatic carcinomas a gene (PAR) was isolated
from an LNCaP androgen resistant subline (LNCaP-r) using
a modified representational difference analysis (RDA). The
gene cDNA is 1.3 kb in size from which 650 bp have been
sequenced so far. The search in GenBank database using
BLAST program revealed no significant homology with any
other sequences already described except the EST with the
accession #RO1857.
The cDNA PAR partial sequence includes a poly A tail at
3⬘ extremity and a continuous ORF beginning at 5⬘ extremity
of the clone and going up to nucleotide 530. They deduced
PAR amino acid sequence has functional motifs for protein
kinase C and cyclic AMP phosphorylases.
The PAR gene is expressed in various tissues. The white
blood and prostate cells express the highest levels, while
thymus has the lowest level. The gene is expressed at higher
levels in androgen resistant prostatic carcinoma cell lines
PC3, DU145 and LNCaPr in comparison with LNCaP androgen sensitive cells (LNCaPs). In LNCaPs cells PAR expression is down regulated by androgens, while no androgen regulatory effect has been observed in hormone resistant prostatic carcinoma cells.
Further work is carried out in this laboratory to determine the biological function of this gene in prostatic cells
and whether PAR has any role in the mechanism of hormone resistance.
EXPRESSION PATTERNS OF THE MYC/MAX/MAD NETWORK IN
DEVELOPING MURINEPROSTATE
Barry G. Timms,1 Leo Kretzner,2 Erin Harmon,2 and Qi Luo2
1
Department of Anatomy and Structural Biology, University of
South Dakota School of Medicine, Vermillion, SD
2
Department of Biochemistry and Molecular Biology, University
of South Dakota School of Medicine, Vermillion, SD
The prostate develops into a gland of complex ductal
architecture through finely orchestrated growth control
mechanisms. Though some of the mechanisms have been
elucidated, it remains unclear how similar yet aberrant processes lead to inappropriate new ductal branching, as in
benign prostatic hypertropy, or completely override normal
growth controls, as in cancer. Because many growth control
genes are expressed during fetal and perinatal development,
we are directing our atten tion to this period of growth and
to patterns of gene expression during the switch from proliferation to differentiation. The Myc family of protooncogenes (c-, L-, and N-myc) and their antagonists, the
Mad genes (Mxi1 and Mads 1, 3, and 4), are known to be
differentially expressed during the development of many
tissues. Expression of c-myc in proliferating prostate cells is
well established, but the activity of other members of the
Myc/Max/Mad network of transcription factors in prostate
is completely uncharacterized. This is of interest for several
reasons. Mxi1 displays allelic loss and mutation in some
cases of prostate cancer, though others have questioned how
general this finding may be. Furthermore, N-myc is involved in the branching morphogenesis of lung tissue—
functionally similar to prostate ductal formation—but Nmyc expression in the prostate has not yet been described.
We hypothesize that differential expression of the Myc/
Max/Mad gene network will coincide with the switch from
proliferation to differentiation in developing prostate, and
we have used in situ hybridization and RT-PCR to characterize the patterns of expression of these genes in this organ.
Initial studies have been performed with ventral prostate
from mouse pups at postnatal days three and twenty-four.
We find high expression of c- and N-myc genes at day three.
Myc expression is localized to the proliferating epithelium of
ductal branches, while message from the ubiquitously expressed Max is present in both the epithelial and stromal
compartments. Positive results have also been obtained for
the Mad1 and Mad4 genes, and their upregulation in ductal
epithelial cells by day twenty-four suggests that these cells
are committed to differentiation by this time point. These
and other data will be presented.
MOLECULAR AND GENETIC MECHANISMS OF
PROSTATE CANCER
J.S. Rhim
National Cancer Institute, Frederick, MD 21702
Prostate cancer is one of the most prevalent diseases and
the second leading cause of cancer death of older American
men. The incidence of prostate cancer is still rising. The
genetic and environmental factors responsible for the high
incidence of prostate cancers are largely unknown. In addition, unlike other major cancers such as breast, lung and
colon cancer, little is known at the molecular and genetic
levels about prostate cancer. In vitro models of human prostate epithelial (HPE) cells provide a practical approach to
analyze the molecular and genetic mechanisms underlying
prostate carcinogenesis. We reported the immortalization of
normal adult HPE cells by DNA transfection of the HPV-18
DNA and the subsequent conversion of such nontumorigenic but immortalized cells (HPV-18 C-1) into tumorigenic
cells by the introduction of an activated Ki-ras oncogene.
Recently, we have demonstrated the malignant transformation of HPV-18 C-1 cells after multiple exposure to the
chemical carcinogen N-nitroso-N-methylurea. Such transformants showed morphological alterations and anchorageindependent growth in soft agar and induced carcinomas
Bethesda Abstracts
when transplanted into nude mice. No p53 and ras mutations were observed. Stepwise chromosomal changes in the
progression to tumorigenicity were observed. Loss of the p
arms of chromosome 8 (p10>pter) and chromosome 10
(p10>pter) and gain of the q arm of chromosome 8 (q10>qtr)
(the most frequently observed cytogenetic changes observed
directly from prostate cancer patients) were only observed
in the tumor outgrowths. These findings provide the first
evidence of malignant transformation of HPE exposed to a
chemical carcinogen. Molecular analysis of these cells will be
needed to determine the specific events that are responsible
for chemical-carcinogen-induced malignant transformation.
INHIBITION OF PROSTATE CELL PROLIFERATION BY THE
␤1C INTEGRIN
Mara Fornaro,1 Michela Manzotti,1 Duo-Qi Zheng,1 Amy E.
Slear,1 Harris Foster Jr.,2 Giovanni Tallini,1 and Lucia R.
Languino1
1
2
Department of Pathology, Yale University, New Haven, CT
Department of Surgery, Yale University, New Haven, CT
The interactions between cells and extracellular matrix
proteins control cell proliferation. These interactions are mediated by integrins, cell surface receptors composed of one ␣
and one ␤ subunit. The ␤1C integrin is an alternatively
spliced variant of the ␤1A subunit, that, in contrast to ␤1A,
inhibits fibroblast cell proliferation.
The ␤1C integrin is expressed in benign prostate epithelium and is downregulated in prostate cancer as well as in
hyperplastic prostate glands which exhibit regenerative features. To investigate whether ␤1C plays a role in prostate
epithelial cell proliferation, stable PC3 prostate cell transfectants, expressing either ␤1C or ␤1A cytoplasmic tails under
the control of an inducible promoter, were generated and
tested in proliferation assays. In contrast to ␤1A, the expression of ␤1C completely inhibited thymidine incorporation by
serum-stimulated prostate cells.
Analysis of in vivo expression of ␤1C and of the cyclindependent kinase inhibitor p27kip1, another inhibitor of cell
cycle progression, shows that ␤1C and p27kip1 are coexpressed in non proliferating human benign prostate epithelial cells, whereas both are downregulated in prostate
adenocarcinoma. In vitro, p27kip1 protein levels were found
to be increased in response to ␤1C expression, suggesting
that p27kip1 is a downstream target of ␤1C-regulated signaling pathways. In contrast, transient activation of the mitogen
activated protein kinase, Erk2, in response to ␤1 integrin
engagement, was unaffected in ␤1C-transfected cells.
These results suggest a novel pathophysiological role for
the ␤1C integrin variant in normal and aberrant prostate cell
proliferation.
RELATIONSHIP OF CATHEPSIN B AND STEFIN A mRNA
LOCALIZATION TO GLEASON GRADES OF HUMAN PROSTATE
CANCER: AN IN SITU HYBRIDIZATION AND IMAGE
ANALYSIS STUDY
Akhouri A. Sinha, Michael J. Wilson, Donald F. Gleason, and
Bonnie F. Sloane
333
Departments of Genetics and Cell Biology, Urologic Surgery,
Laboratory Medicine, and Pathology, University of Minnesota
and Research Service, Veterans Affairs Medical Center,
Minneapolis, MN, and Department of Pharmacology, Wayne
State University, Detroit, MI
Increased expressions of cathepsin B (CB) message, protein, and membrane association have been linked to malignancy in animal and human solid organ tumors, including
human prostate cancer. Stefins/cystatins regulate CB activities in normal and tumor tissues. Proteins of CB and its
inhibitors (stefins A, B and cystatin C) have been demonstrated in prostate cancer, but there are few studies of their
mRNAs in human prostate cancer using in situ hybridization
techniques. Our objective was to determine the relationship
of CB and stefin A (or cystatin A) mRNA localization to the
Gleason grades (histologic scores) in the hope of distinguishing human prostate cancers with aggressive and less aggressive phenotypes. We used a 25-base biotinylated oligonucleotide CB cDNA antisense probe to localize CB message
and a 27-base biotinylated oligonucleotide stefin A/cystatin
A cDNA antisense probe to localize stefin A message. Prostate samples from 29 patients were frozen in liquid nitrogen
or on dry ice and sectioned at 4 to 6 ␮m with a cryostat.
Sections were hybridized with probes using techniques reported by Sinha et al. (Anat. Rec., 235:233–240, 1993). In
control studies, sections hybridized by pBR 322 probe or
after pretreatment with RNAse showed little to no reaction
products. Distribution of CB and stefin A mRNA localization in relation to each Gleason grade was quantitated by an
image analysis system. Although reaction products for CB
and stefin A probes varied within and between Gleason
grades, they consistently localized in neoplastic epithelial
and invasive cells and occasionally in stromal cells. Our data
indicated that localization patterns could be categorized in
three patterns, namely, prostate cancers with higher levels of
CB than stefin A, similar levels of CB and stefin A, and lower
levels of CB than stefin A. Prostate cancers representing
Gleason grades 3, 4, 5, 6, 9 and 10 showed higher levels of
CB than stefin A. Similar levels of CB and stefin A staining
were observed in Gleason grades 3, 4, 6, and 7 and lower
levels of CB than stefin A in Gleason grades 3, 6 and 7. In 22
prostatectomy samples showing Gleason grade 6 tumors,
the three CB and stefin A localization patterns were observed, suggesting heterogeneity in this Gleason grade.
Based on analysis of our data, we hypothesize that cancer
cases showing higher levels CB to stefin A represent an aggressive variant of prostate cancer within a Gleason grade. If
this is the case, aggressive variants of prostate cancer would
occur within Gleason grades 3 to 10 even though higher
grades of prostate cancers are usually considered more aggressive. As the Gleason grading system is based on analyses of several thousand cases and survival data of patients
and our study is based upon a limited number of prostatectomy samples, we, therefore, emphasize that a larger series
of prostate cancer cases needs to be examined before our
hypothesis can be confirmed. This research was supported
by USPHS grant DK-51348 to A.A.S. and in part by USPHS
grant CA-36481 to B.F.S., and in part by the Research Service, Minneapolis VA Medical Center.
OPTIMIZATION OF EXTRACTION AND PCR OF DNA FROM
FROZEN SERUM
S.C. Dixon, Y. Guo, J. Horti, E. Reed, and W.D. Figg
334
Bethesda Abstracts
Medicine Branch, Division of Clinical Sciences, NCI, NIH,
Bethesda, MD 20852
A variety of biological materials have been used as a
DNA source for PCR. DNA extracted from buffy coats is
most commonly used for genetic studies. However, it is not
always possible to obtain a buffy coat from a subject for
DNA extraction. In addition, many laboratories have large
banks of frozen sera stored on specific patient populations
that could be used for genetics studies. These observations
prompted us to optimize DNA extraction and amplification
from frozen serum. Thirteen extraction methods were evaluated initially. From these methods, six yielded sufficient
DNA of good quality based on spectrophotometric measurements. Four of these six methods were chosen for further
evaluation based on their ease of use. Frozen sera from 110
prostate cancer patients were extracted by each method and
DNA recovery was determined spectrophotometrically and
by fluorescence assays. Modifications of two commercially
available DNA extraction kits, the Qiagen QIAamp blood kit
and the InVitrogen Easy-DNA kit, produced the most consistent results. The polymorphic trinucleotide (CAG) repeat
length in the first exon of the androgen receptor was amplified for each DNA extracted by these two methods. This
region has been associated with prostate cancer risk. Individuals with a shorter repeat region may have a higher risk
of prostate cancer than those individuals with a longer repeat region. Using each method, we successfully amplified
56–70% of the extracted DNA. Since the Qiagen procedure is
less labor-intensive and does not require the use of organic
solvents, we have chosen it as our primary method. If this
amplification fails, the Easy-DNA method is used. In this
manner, 88.5% of the DNAs could be amplified. The percentage of amplifiable DNAs as well as the sensitivity of the
assay was increased by reamplification of the primary PCR
product with a nested set of PCR primers. Using the product
from either the primary or the nested amplifications, we
were able to successfully sequence the region to accurately
determine the number of repeated trinucleotides.
AN INVESTIGATION OF THE INFLUENCE OF PLANT EXTRACTS
ON THE IN VITRO GROWTH AND ANCHORAGE-INDEPENDENCE
OF RAT PROSTATE CARCINOMA CELLS
Mark S. Condon,1,2 Andrew Seal,3 and Maarten C. Bosland1,4
1
Department of Environmental Medicine, New York University
School of Medicine, Tuxedo, NY
2
Department of Biological Sciences, Dutchess Community
College, Poughkeepsie, NY
3
Department of Environmental Science, New England College,
Henniker, NH
4
Department of Urology, New York University School of
Medicine, New York, NY
Experimental evidence suggests that extracts of saw palmetto (Serona repens) berries, Pygeum africanum bark, and
nettle (Urtica dioica) leaves can inhibit the growth of benign
hyperplastic cells of the prostate. In the present study, the
potential of these extracts to modulate the growth and tumorigenic potential of malignant prostate cells was investigated. The cell lines CKB-H7 and CKB-I8 were established
from rat (Fisher 344 and Wistar Furth, respectively) dorsolateral prostate adenocarcinomas. The cells are of epithelial
in origin, divide rapidly in vitro (50 hour and 15 hour doubling times, respectively), and readily form moderately-
differentiated, metastasizing tumors when injected subcutaneously into syngeneic hosts. CKB-H7 cells were grown in
the presence of three different (non-cytotoxic) concentrations of each aforementioned plant extract. Cell counts were
performed at appropriate intervals during these exposures
and these data were plotted to establish ‘‘growth curves.’’
Results indicate that the highest concentration of each extract significantly inhibited the mitotic activity of the CKBH7 cells as compared to vehicle-exposed controls. In a related study, CKB-H7 and CKB-I8 cells were used in standard
anchorage-independence (‘‘soft agar’’) assays in order to investigate the possible usefulness of saw palmetto and
Pygeum africanum extracts as anti-tumorigenic agents. Cells
were suspended in culture medium containing 0.36% agar
and a plant extract (concentrations = 75% of the highest
concentrations used in the aforementioned study). Three
weeks after plating, the colony-forming efficiencies were determined and compared to controls. Saw palmetto significantly inhibited the colony-forming efficiency of both the
CKB-H7 and CKB-I8 cells (P < 0.0001). However, the Pygeum
africanum extract had no significant effect on the anchorageindependent growth of either cell line tested. These results
suggest that certain extracts used in this study might be
useful in modulating the growth and tumorigenicity of malignant prostate epithelial cells. Further investigations are
necessary to elucidate their mechanism(s) of action and in
vivo usefulness.
GATA TRANSCRIPTION FACTORS IN PROSTATE CANCER CELLS
Carlos Perez-Stable, Alicia De Las Pozas, Edward L. Oates,
and Bernard A. Roos
Miami VA Medical Center, University of Miami School of
Medicine, Miami, FL
Androgen-independent metastatic prostate cancer unexpectedly develops in a transgenic mouse line containing the
human fetal globin promoter linked to the SV40 T antigen
oncogene. We hypothesize that the fetal globin promoter
contains DNA elements active in prostate cells, which results in the activation of T antigen and the eventual development of prostate tumors in transgenic mice. Our previous
experiments have shown that the fetal globin promoter between -140 and -201 is active in aggressive androgenindependent human prostate cell lines like DU-145 and inactive in the less aggressive androgen-responsive prostate
cell line LNCaP. This DNA region contains protein binding
sites for GATA, Oct, and Sp1 transcription factors. Identification of the prostate factors which bind to the -140 to -201
region may help to better identify aggressive androgenindependent prostate cancer cells and provide insight into
the progression of prostate cancer. Here, we focus on the
GATA transcription factor family.
The GATA transcription factor family consists of six
members important in the regulation of numerous genes
and expressed in a variety of tissues during development
and in the adult. GATA-1, -2, and -3 consist of one subfamily
predominantly expressed in blood and endothelial cells;
GATA-4, -5, and -6 consist of another subfamily predominantly expressed in heart, lung, smooth muscle, and bladder. Gel shift competition assays uncovered subtle differences in the GATA transcription factors from aggressive and
Bethesda Abstracts
less aggressive prostate cancer cells. We used degenerate
oligonucleotide primers specific for the highly conserved
zinc finger DNA binding domain of the GATA family and
reverse transcription polymerase chain reaction (RT-PCR) to
identify the expression of GATA-2, -3, and -6 in mouse and
human prostate cancer cells. RNase protection assay confirmed that GATA-2 is highly expressed in LNCaP but not in
DU-145 or PC3 cells. In contrast, GATA-3 appears to be
expressed in DU-145 and PC3 but not in LNCaP. GATA-6
appears to be expressed in human prostate cancer cells but
not in normal mouse prostate and prostate tumor.
We are currently investigating whether the GATA family
of transcription factors plays a role in the regulation of prostate-specific genes and in the progression of prostate cancer
cells. We speculate that differential expression of GATA
transcription factors may reflect androgen-responsive and
androgen-independent prostate cell phenotypes and this expression pattern is instrumental in the development of prostate cancer in the transgenic mice.
DEREGULATION OF FGF-7/KGF SIGNALING IN PROSTATIC
EPITHELIUM DISRUPTS STROMAL-EPITHELIAL INTERACTIONS
WHILE CHANGES IN THE FGF AXIS ARE ASSOCIATED WITH
PROSTATE CANCER PROGRESSION
Barbara A. Foster, Jeffrey Gingrich, and Norman M. Greenberg
Department of Cell Biology, Baylor College of Medicine,
Houston, TX 77030
The probasin (PB) promoter was used to target expression of human FGF-7 (KGF) directly to prostatic epithelium
of transgenic mice (PKS) converting KGF from a paracrine to
an autocrine factor. Several PKS lines express the transgene
in the dorsolateral prostate (DLP). PKS DLP epithelium is
hyperplastic by histological analysis. Many of the prostatic
ducts are filled with epithelial cells. However, BrdU incorporation indicates the rate of proliferation in PKS DLP is not
different from nontransgenic littermates. There are areas of
stromal thickening and distortion of the ductal smooth
muscle layer. This suggests that ectopic expression of KGF in
prostatic epithelium disrupts epithelial-mesenchymal interactions resulting in epithelial hyperplasia.
To functionally abrogate endogenous KGF signaling, the
PB promoter was used to target a dominant negative KGF
receptor (KDNR) to prostatic epithelium. The KDNR construct contains exon iiib conferring KGF ligand specificity,
but lacks the internal signaling domain. Dissection and gross
examination of the reproductive tracts of KDNR mice indicate all four lobes of the prostate are present but the DLP is
smaller in size. Histological analysis indicates that in many
of the ducts the DLP epithelium is disorganized and not
associated with the basement membrane. The stroma is also
disorganized and does not form a tight layer of smooth
muscle around the epithelial ducts. This suggests expression
of a dominant KGF receptor in prostatic epithelium disrupts
epithelial-mesenchymal interactions resulting in disorganization of both the epithelium and stroma.
Reverse transcriptase-polymerase chain reaction analysis
was used to test the hypothesis that changes in the expression of fibroblast growth factor ligands (FGF) and receptors
(FGFR) correlate with tumor progression in prostatic adenocarcinoma. In these studies, prostate tumor samples and epithelial cell lines derived from the autochthonous TRAMP
mouse model were analyzed. FGF-1 (acidic FGF) appears to
335
decrease during TRAMP tumor progression and is barely
detectable in the TRAMP cell lines. Expression of FGF-2 (basic FGF), FGF-7 (KGF) and FGF-10 is detectable in all of the
samples. Since FGF-7 is thought to be exclusively expressed
by the stroma and acts as an andromedin through the epithelial FGFR2iiib receptor, it was surprising to find FGF-7
expressed in all of the epithelial TRAMP cell lines. This suggests that activation of FGF-7 is an early event in the transformation of prostate epithelium. Expression of FGFR1iiib,
FGFR1iiic and FGFR2iiic is detectable in all samples. Variant
forms of FGFR1iiic are detectable in late stage tumors and in
the cell lines. Multiple forms of FGFR2iiib and FGFR3iiib are
detectable in normal prostate and TRAMP tumors; however,
they are undetectable or barely detectable in the cell lines.
FGFR3iiic and FGFR4 are undetectable in all samples. The
multiple splice forms of the receptors are currently being
isolated to determine ligand specificity and functional role
in prostate cancer progression. These observations support
the hypothesis that changes in the FGF axis correlate with
prostatic cancer progression and thus might form the basis
of improved diagnostic and therapeutic strategies for the
treatment of prostate disease.
INHIBITION OF PROSTATE CANCER CELL GROWTH BY
RIBOZYMES TARGETED TO INSULIN-LIKE GROWTH
FACTOR (IGF)-II
Yoko Fujita-Yamaguchi,1 Zhao-Dong Xu,1 Lily Oey,1,2 Nan
Sook Lee,1 Mark H. Kawachi,2 Timothy G. Wilson,2 and John J.
Rossi1
1
Department of Molecular Biology, Beckman Research Institute
of the City of Hope, City of Hope Medical Center, Duarte, CA
91010
2
Department of Urologic Oncology, City of Hope Medical
Center, Duarte, CA 91010
IGFs have previously been reported to promote prostate
cancer cell growth via IGF-I receptor in an autocrine/
paracrine manner. We have shown by ISH and IHC that
IGF-II is expressed in over 50% of prostate, breast, and bladder cancer tissues, and in 9/9 of paragangliomas. In all positive cases, the expression is localized to malignant cells (Li et
al., Cell Tissue Res 291, 469–479, 1998). These data are consistent with the hypothesis that cancer cell growth may be
regulated by IGF-II in an autocrine manner. To test this hypothesis, we have constructed catalytically active IGF-II
ribozymes, and intracellularly expressed them in human
prostate cancer PC-3 cells. Single- and double-hammerhead
ribozymes that are complementary to +16 to 31 (R) and +16
to 46 (RR) downstream of AUG of human IGF-II mRNA,
respectively, were synthesized and cloned into the
pTZU6+27 or pcDNA vectors. A point mutation of G to A
was introduced at a highly conserved base required for
cleavage (M, MM, RM, MR). All the functional ribozymes (R,
RR, RM, and MR) transcribed in vitro cleaved ∼82 base- and/
or ∼1 kilobase-IGF-II substrates examined, while M or MM
did not. Kcat/Km for the shorter IGF-II substrate by RR and
R was 4772 vs. 1546 M−1s−1, respectively, suggesting that RR
was ∼3-fold more efficient in cleaving the IGF-II substrate
than R in vitro. PC-3 stable transfectants expressing R or RR,
driven by U6 promoter, reduced the endogenous IGF-II
mRNA level similarly by 60% compared to those expressing
336
Bethesda Abstracts
M. In parallel experiments, expression of CMV promoterdriven R in PC-3 cells reduced the levels of endogenous
IGF-II mRNA and IGF-II protein secretion compared to vector-control cells. Furthermore, PC-3 cells expressing R grew
poorly under serum-free or 2% FCS conditions as judged by
growth curves, supporting our hypothesis that IGF-II plays
a critical role in prostate cancer cell growth.
THE BLADDER IS A PRIMARY TARGET FOR DYSFUNCTIONS
INDUCED BY BPH
Robert M. Levin, Ph.D.,1 and Ralph Buttyan, Ph.D.2
1
2
Albany College of Pharmacy, Albany, NY
Columbia University, New York, NY
Benign prostatic hypertrophy (BPH) is a disease caused
by an abnormal age-associated growth of the prostate gland,
but it is a disease whose symptomatic presentation is determined by the development of major dysfunctions in the urinary bladder resulting from the bladder outlet obstruction
associated with BPH. Since animals other than humans
rarely get BPH, bladder dysfunctions must be studied in
experimental animal models in which the bladder is partially obstructed through a reversible surgical procedure
wherein a ligature is placed around the bladder outlet. This
presentation will discuss our extensive experience with a
rabbit model of partial bladder outlet obstruction and the
accumulating physiological, cellular and molecular evidence
that bladder dysfunction associated with bladder outlet obstruction is caused by a progressive, cyclical hypoxia within
the bladder proper.
The progressive response of the rabbit bladder to partial
outlet obstruction can be divided into three discrete temporal phases referred to as hypertrophy, compensation and
decompensation. The hypertrophy phase is the most studied
bladder response. The onset of hypertrophy can be characterized by an ‘‘early molecular response’’ associated with
induced proto-oncogene and heat-shock gene expression
that can also be initiated by bladder overdistension or by
partial bladder ischemia in a manner that describes a mechanistic pathway in which bladder ischemia appears to be the
most pertinent event. This phase is also accompanied by
distinct changes in growth factor expression (increased basic
fibroblast growth factor and decreased transforming growth
factor-␤ expression), replication of urothelial and serosal fibroblast cells and hypertrophy of the smooth muscle cells.
The bladder hypertrophy phase is ended coincident with the
induced expression of tumor suppressor gene products (p53
and WAF-1) and the bladder now enters a transient stage in
which normal function has been regained. Current studies
are ongoing to determine what molecular events drive the
bladder to enter the final stage, decompensation, wherein
bladder function is severely compromised, but preliminary
data strongly suggest that a continuing cyclic ischemia associated with bladder filling is important for this latter transition.
In summary, we hope to emphasize the importance of the
urinary bladder to the disease process underlying BPH and
promote the dissemination of our experimental results to
research scientists more focused on studying the prostate
aspects of BPH. We believe that increased investigations on
the bladder as a target organ of BPH will lead to more rapid
progress in identifying successful treatments for this extremely common human affliction.
A NEW MODEL SYSTEM FOR STUDYING SEX-HORMONE
INDUCED PROSTATIC LESIONS
Y.Z. Wang, Simon W. Hayward, Anne A. Donjacour, Peter
Young, and Gerald R. Cunha
Department of Anatomy, University of California at San
Francisco, San Francisco, CA 94143-0540
Benign prostatic hyperplasia and prostate cancer are
among the most commonly diagnosed neoplastic conditions
in men. The etiologies of these diseases are not well understood. Research has been hampered by a lack of suitable
experimentally manipulable models. In the present study,
both rat (Noble, Fischer 344, Wistar, Sprague-Dawley) and
mouse (Balb/c, C57 BC/L) prostatic embryonic rudiments
and/or neonatal prostates were dissected and grafted under
the renal capsule of nude mice. The experimental hosts were
implanted with silastic tubing containing testosterone propionate (T) and 17-␤-estradiol (E2), while the control group
was implanted with empty tubing. The grafted prostatic tissues were harvested after treatment with T + E2 for 3 to 6
weeks. The control grafts showed normal prostatic morphology. In prostatic grafts of the T + E2 treated group, the vast
majority of ventral prostatic grafts showed hyperplastic
and/or dysplastic changes. In dorsolateral prostatic grafts,
these changes were more sporadic with localized regions of
hyperplasia and/or dysplasia, and many unaffected ducts
which remained normal. Moreover, the sex hormoneinduced prostatic lesions were present in all strains of mice
and rats tested. The results indicated that: 1) Rodent prostatic rudiments and neonatal rodent prostates are sensitive
to T + E2 treatment; 2) The T + E2 treatment can induce
hyperplastic and/or dysplastic lesions in as early as 3 weeks
with no apparent strain-specificity; 3) The model system
may supply the possibility to dissect and analyze the hormones and growth factors pathways involved in the development of prostatic lesions by separating and recombining
targeted prostatic epithelium and/or mesenchyme. Future
studies will focus on each sex hormone and growth factor
pathway alone as well as their cross talk to provide data on
both normal and abnormal prostatic development.
ATYPICAL ADENOMATOUS HYPERPLASIA OF THE PROSTATE: A
PREMALIGNANT LESION?
L. Cheng, M.D., A. Shan, M.D., J.C. Cheville, M.D., J. Qian,
M.D., and D.G. Bostwick, M.D.
Mayo Clinic, Rochester, MN
Atypical adenomatous hyperplasia (AAH) of the prostate
is a small acinar proliferation which mimics carcinoma and
has been proposed to be a possible precursor lesion of prostate cancer. Our understanding of genetic alterations of
AAH is limited. In this study, we examined the prevalence
of allelic imbalance (AI) at 5 microsatellite polymorphic
markers on chromosome 7q31–q35, 8p12–21, 8p22, 8q22.2,
and 18q12.2 from 15 patients with AAH. DNA samples were
obtained from formalin-fixed, paraffin-embedded sections
using tissue microdissection. Polymerase chain reaction and
gel electrophoresis were performed. Autoradiographs of
paired normal and AAH were analyzed by densitometry
using NIH Image 1.47 software. The criterion for AI was a
paired AAH allele:control (normal) allele intensity ratio of
Bethesda Abstracts
艌1.5. We found AI in 7 (47%) of 15 cases of AAH. Genetic
changes that commonly occur in early prostatic carcinogenesis and prostate carcinoma are found in AAH. Current data
provide evidence of a genetic link between some cases of
AAH and carcinoma. Further investigation is warranted to
establish the putative preinvasive nature of this lesion.
MECHANISM OF ANDROGEN AND GROWTH FACTOR MEDIATED
REGULATION OF PROTEIN KINASE CK2 SIGNAL IN PROSTATE
CANCER CELLS
K. Ahmed, A. Davis, and C. Guo
Cellular and Molecular Biochemical Research Laboratory, VA
Medical Center, and Department of Laboratory Medicine and
Pathology, University of Minnesota, Minneapolis, MN 55417
Protein kinase CK2 is a multi-functional ser/thr kinase
that has been implicated in cell growth and proliferation.
Studies on CK2 regulation in the rat prostate have demonstrated profound regulation of the nuclear kinase by androgenic stimulus. Various data have implicated CK2 in the
neoplastic process, and dysregulation of CK2 has been observed in neoplasias, including prostate cancer. A significant
amount of nuclear CK2 is associated with the nuclear matrix
(NM), a subnuclear structure believed to play a fundamental
role in regulation of gene activity and cell proliferation, suggesting that NM is a differential locus of CK2 signaling in
the nucleus (Tawfic, Faust, Gapany, Ahmed: J. Cell. Biochem. 62, 165–171, 1996). We have therefore investigated the
mechanism of regulation of CK2 signal in the prostate cancer
cells LNCaP and PC3 in response to androgenic and growth
factor stimuli. Androgen withdrawal from the culture medium results in loss of nuclear CK2 while addition of androgen results in translocation of the kinase from the cytoplasm
to the nucleus. Growth factors (EGF, serum) induce similar
effects in LNCaP. On the other hand, androgen had no effect
on the CK2 in the PC3 cells. However, it was influenced by
the growth factors. The nuclear modulation of CK2 by these
factors is preferentially apparent in the NM fraction, as determined by measurement of CK2 activity and immunoreactive protein, and correlates with the status of cell proliferation. These results indicate that, depending on the nature
of the prostate cancer cell type, the CK2 signal is affected by
both the androgenic and growth factor stimuli. This may be
pertinent to the role of CK2 in growth of these cells in response to these stimuli. [Supported by research grant CA15062 awarded by N.C.I., D.H.H.S., and the Medical Research Fund of the Department of Veterans Affairs.]
DIHYDROTESTOSTERONE AS A SELECTIVE CELLULAR/NUCLEAR
LOCALIZATION VECTOR FOR ANTI-GENE PEPTIDE NUCLEIC
ACID IN PROSTATIC CARCINOMA CELLS
P.L. Morris,1,2 L.C. Boffa,3 M.R. Mariani,3 U. Benatti,4 S. Scarfi,4
A. Gasparini,4 L. Mitchell,1 and G. Damonte4
1
Population Council, New York, NY
Rockefeller University, New York, NY
3
Department of Experimental Oncology, National Cancer
Institute, IST, Genoa, Italy
2
4
337
Institute of Biochemistry, University of Genoa Medical School,
Genoa, Italy
Peptide nucleic acids (PNAs) represent a recent development in the field of oligonucleotide analogues, in which the
natural phosphate ribose backbone has been substituted by
a synthetic polyaminic structure. There are numerous advantages of using PNA oligomers over conventional antisense oligonucleotides, which are used to try to downregulate target gene expression. These advantages include: (1)
their artificial backbone makes them resistant to proteases
and nucleases, (2) they can invade duplex DNA and hybridize with complementary sequences with superior thermal
stability to successfully compete and eventually displace the
natural complementary strand, and (3) on entering the cell
nuclei, PNAs bind to the complementary DNA strand and
selectively block gene transcription. All of these features
make PNAs effective anti-gene candidates in vitro and potentially therapeutic agents in vivo. However, significant issues remain that currently limit their practical use, viz. PNAs
enter the cells by pinocytosis and become trapped in cytoplasmic vesicles without the opportunity to enter the
nucleus and affect gene expression.
Recently we obtained evidence that any anti-gene PNA
covalently linked to a nuclear localization signal (nls) peptide penetrates into the nuclei of living cells, effectively inhibiting targeted gene expression. Prostatic carcinoma cell
lines provide an important model to test if the biologically
active form of testosterone could function as a cell specific
nls in a cell line (LNCaP) that expresses the nuclear androgen receptor (AR) gene. As an appropriate negative control,
we used a second prostate cell line, DU145, in which the AR
gene is functionally silent. Dihydrotestosterone (DHT) was
covalently linked to the N-terminal position of a 14-mer
PNA complementary to a unique sequence located at the
beginning of the second exon of the c-myc oncogene expressed in both cell lines. In order to detect the cellular localization of the PNA construct, a rhodamine (R) group was
attached at the C-terminal position of the PNA (PNA-RT*).
A matching PNA that was rhodaminated but not linked to
androgen (PNA-R) was also synthesized.
Using 2 ␮M concentrations of either PNA-RT or PNA-R
for up to 24 hours in vitro, the kinetics of cellular and nuclear
PNA uptake were monitored by fluorescence and phase contrast confocal microscopy. PNA-R localized in cytoplasmic
vesicles of both cell lines without nuclear uptake. Under the
same experimental conditions, PNA-RT* clearly localized
not only in the cytoplasm, but also in LNCaP nuclei, while
the uptake of this construct was quite low and solely cytoplasmic in DU145 cells. Using Western immunoblot analysis
with total cellular protein from treated LNCaP cells, MYC
remains unchanged by exposure to PNA-R while a significant decrease of ∼30% was induced by PNA-RT*. In contrast,
MYC expression in treated DU145 cells was not affected
either by PNA-R or PNA-RT*. We show that DHT can indeed act as a specific active nls in prostatic cells expressing
AR gene; DHT allows myc anti-gene PNA to enter the
nucleus followed by effective inhibition of c-myc expression.
These results demonstrate the feasibility of this approach;
our data suggest that development of specific anti-gene
therapy for androgen-dependent prostatic carcinomas is
possible using this strategy.
Studies supported by grants from NIH HD16149 (PLM),
Associazione Italiana per la Ricerca sul Cancro, AIRC (LCB),
Consiglio Nazionale delle Ricerche, CNR, ‘‘Short Term Mobility Fellowship’’ (SS and AG).
338
Bethesda Abstracts
DOES THE APPARENT ABSENCE OF BCL-2 IN PC-3 CELLS
CONTRIBUTE TO THE ATYPICAL PROGRAMMED CELL DEATH
INDUCED BY SELECTIVE INHIBITORS OF 5-LIPOXYGENASE?
A.M. Anderson, W.A. Alrefai, D. Ou, and J.E. Harris
Departments of Medicine and of Biochemistry, Rush Medical
College, Chicago, IL, and Department of Pathology, University
of Illinois at Chicago, Chicago, IL 60612
The selective inhibitors of 5-lipoxygenase, SC412661A
and MK886, both inhibit proliferation and with continued
culture, induce ‘‘classic’’ type 1 programmed cell death in
U937 monoblastoid cells. These inhibitors reduced PC-3
prostate cell proliferation and induced cellular suicide, as
judged from results with treated cells by flow cytometry,
laddering of DNA after electrophoresis on agarose gels and
by light and electron microscopy. SC41661A, an oxidation/
reduction agent, induced a morphologically incomplete type
1 cellular suicide. MK886, an indirect inhibitor of 5-LPOX
which inhibits the association between arachidonic acid, 5-lipoxygenase activating protein and 5-LPOX., induced nonnecrotic changes largely confined to the cytoplasm, probably
most consistent with a form of type 2 ‘‘autophagic’’ cell
death.
The antioxidant and precursor to glutathione, N-acetyl-lcysteine but not butylated hydroxy toluene or a -tocopherol,
partially reduced inhibition of proliferation due to
SC41661A. When SC45662, a less effective inhibitor of
5-LPOX, was compared with SC41661A, the inability of the
former compound to inhibit cell proliferation was associated
with the absence of DNA laddering; inhibition of proliferation must proceed or accompany the induction of programmed cell death. MK886 rapidly increased the cytoplasmic concentration of Ca2+ in U937 cells, mostly from internal
stores, but did not raise that of PC-3 cells studied in suspension. Finally, we were unable to detect Bcl-2 by Western
blotting in PC-3 cells, although high concentrations were
present in U937 cells exhibiting classic type 1 PCD. To the
extent that Bcl-2 may be associated with mitochondria in the
implementation of PCD, its apparent absence, or at least
very low concentration in PC-3 cells could suggest that these
cells utilize alternate or incomplete pathways for cellular
suicide.
Since PC-3 cells have been shown to express mRNA for
both 5-LPOX and FLAP (Prostate, in press, and AACR, 1998)
and can synthesize 5-HETE (Gosh, J, Myers, CE. Biochem
Biophys Res Commun 235, 418, 1997), they represent a suitable model for studying the mechanism by which PCD is
induced by these inhibitors. (Supported by the Weinberg
Foundation, the Seidel Family Trust and the Wadsworth
Memorial Trust.)
An update. Bcl-2 mRNA, assessed by RT-PCR, was present in U937 cells and absent from our PC-3 cell clone. Bcl-x,
Bax, Bak, Mcl-1 and Bik were present in both cell lines, as
determined by a commercial RNase protection assay.
In comparing PC-3 and U937 cells: (a) Is Bcl-2 expression
required for ‘‘classic’’ type 1 PCD, and are DNA ‘‘laddering’’
and 5-LPOX activity associated? (b) Why is there no Ca2+
response in PC-3 cells and is Bcl-2 and P53 expression inversely related? Is the relative lack of response to therapy of
many solid cancers due to inherently atypical forms of PCD
in pre-malignant ‘‘stem’’ cells coupled with defective expression from karyotypic changes as tumors progress?
LOSS OF HETEROZYGOSITY IN PROSTATIC ATYPICAL
ADENOMATOUS HYPERPLASIA (AAH) AND CARCINOMAS
Jennifer A. Doll,1 Xiaopei Zhu,2 Peter A. Humphrey,2 John
Meyer,3 Jaime Furman,2 Zahid Kaleem,2 Carlos Torres,2 and
Helen Donis-Keller1
1
Division of Human Molecular Genetics, Department of
Surgery, Washington University School of Medicine, St. Louis,
MO
2
Department of Anatomical Pathology, Washington University
School of Medicine, St. Louis, MO
3
Department of Pathology, St. Luke’s Hospital, Chesterfield,
MO
Atypical adenomatous hyperplasia (AAH) is a microglandular proliferation of the prostate gland arising most
often in the transition zone of the prostate. This zone is also
the site of ∼20% of the cancer lesions, the majority of which
are well-differentiated carcinomas (Gleason score [GS] 艋4).
AAH and well-differentiated carcinomas are similar histologically. This observation as well as other characteristics of
AAH has led to the hypothesis that AAH is the precursor
lesion to the subset of well-differentiated cancers arising in
the transition zone. The identification of genetic alterations
is one way to provide direct evidence for or against this
hypothesis. Therefore, we used loss of heterozygosity (LOH)
assays to screen DNA from 25 microdissected AAH lesions
for deletions in regions of the genome frequently reported to
be deleted in prostate cancer samples, using one marker
each on chromosome arms 1q, 6q, 7q, 10q, 13q, 16q, 17p, 17q
and 18q and 19 markers on chromosome arm 8p. Patient
matched normal control DNA, used to identify heterozygosity, was isolated from benign prostate tissue. In this study,
we observed 12% (3/25) LOH in our AAH samples. Loss
was only observed on chromosome 8p at markers D8S1104
and D8S268 which span a region of approximately 2–4 Mb.
In prostate cancer, any loss observed at a frequency of 20%
or greater is considered significant due to the low level of
background loss in this cancer (<9%). In addition, Qian et al.
(1995) reported that transition zone cancers have fewer genetic alterations than do peripheral zone cancers (17% versus 64%, P = 0.04) which may indicate that the 12% LOH
observed in our AAH samples is significant. In a concurrent
study of DNA from 113 microdissected cancer samples, 25%
LOH was observed in this region, with markers D8S1104
and D8S1817 defining the minimal region of deletion, a region within the D8S1104 to D8S268 region. An increase in
LOH frequency was observed in moderately- to poorlydifferentiated (GS 艌5) tumors versus well-differentiated (GS
艋4) tumors; however, the zone of origin of these tumors is
unknown. These observations indicate the need for further
characterization of genetic alterations specifically in welldifferentiated transition zone cancers and subsequently in
AAH lesions to determine if AAH is a precursor lesion to
this subset of cancers.
THE EFFECT OF ET-1 ON PROLIFERATION AND APOPTOSIS OF
HUMAN PROSTATIC SMOOTH MUSCLE CELLS
Jinshyun R. Wu-Wong, William J. Chiou, Jiahong Wang,
Cathleen E. Berg, and Terry J. Opgenorth
Abbott Laboratories, Abbott Park, IL
Bethesda Abstracts
339
An imbalance between proliferation and apoptosis is an
important causal factor for disorders involving abnormal
cell accumulation. Endothelin-1 (ET-1) is a 21-amino acid
peptide with mitogenic and vasoconstricting activities. In
this report, we characterize ET receptors in primary culture
of human prostatic smooth muscle cells, and study the effects of ET-1 on proliferation and apoptosis in these cells.
[125I]ET-1 binding was of high affinity and the Bmax and Kd
values were 870 fmol/1 × 106 cells and 0.37 nM, respectively.
ET-1 and ET-3 inhibited [125I]ET-1 binding to these cells with
IC50 values of 0.1 and 0.5 nM, respectively. Reverse transcription-polymerase chain reaction confirmed that these
cells expressed both ETA and ETB receptors. When cells were
deprived of serum, addition of ET-1 did not stimulate the
proliferation of these cells as determined by thymidine incorporation, while 10% fetal bovine serum stimulated DNA
synthesis by more than 10-fold. Serum withdrawal for 48 h
caused DNA fragmentation, a characteristic of apoptosis, in
these cells. Addition of ET-1 attenuated DNA fragmentation
caused by serum withdrawal in a dose-dependent manner
with an EC50 at ∼0.1 nM. The attenuating effect of ET-1 on
DNA fragmentation was not affected by wortmannin (1
␮M), an inhibitor of phosphatidylinositol 3-kinase (PI3⬘K),
and bisindolylmaleimide I (2 ␮M), a protein kinase C inhibitor, but was partially blocked by tyrphostin B42 (1–20 ␮M),
a JAK-2 inhibitor, in a dose-dependent manner. These results suggest that ET is a potential survival factor for the
human prostatic smooth muscle cells, and may be involved
in aberrant cell growth in the prostate.
cultured for at least 20 days with 1200 U/ml rhIL-2, and
tested for their ability to kill prostate carcinoma (DU145),
ovarian carcinoma (AD10), and renal carcinoma (R11) cell
lines. PTIL from patient 1 exhibited little or no killing of
DU145, either in the presence or absence of the Ca++ chelator, EGTA/MgCl2, which blocks the degranulation cytotoxic
pathway but not the Fas/FasL pathway. However, both
CDDP and VP-16 (subtoxic concentrations) sensitized
DU145 cells to PTIL in a Ca++-independent manner. In contrast, PTIL from patient 2 killed DU145 cells and cytotoxicity
was blocked by EGTA/MgCl2. PTIL from patient 2 also
killed AD10 and R11 cells. As with patient 1, PTIL from
patient 2 were able to kill drug-sensitized DU145 cells via
the Fas/FasL pathway. This study demonstrates that prostate-derived TIL cells cannot kill Fas+ DU145 cells via the
Fas/FasL cytotoxic pathway unless the tumor cells are sensitized with drugs. Furthermore, some PTIL preparations
may not exert any detectable cytotoxic activity unless the
tumor cells are sensitized. These findings suggest that drugmediated sensitization of tumor cells may potentiate the effectiveness of immunotherapy. We are currently examining
both the mechanism of immunosensitization of tumor cells,
as well as the mechanism of TIL-mediated recognition and
killing of target cells.
IMMUNOSENSITIZATION OF PROSTATE CANCER CELLS TO Fas
LIGAND-MEDIATED KILLING BY PROSTATE-DERIVED HUMAN
TUMOR-INFILTRATING LYMPHOCYTES
P. Frost,1 R. Kaboo,2 A. Belldegrun,2 and B. Bonavida1
1
1
Department of Microbiology and Immunology, UCLA School
of Medicine and Jonsson Comprehensive Cancer Center,
University of California, Los Angeles, CA 90095-1747
2
Department of Urology, UCLA School of Medicine and Jonsson
Comprehensive Cancer Center, University of California, Los
Angeles, CA 90095-1747
The primary treatments of prostate cancer are surgery,
hormone ablation, and chemotherapy. In many cases, the
initial effectiveness of these treatments is high, but tumors
frequently recur that are resistant to subsequent therapies.
Therefore, the development of resistant tumor cells has led
to the exploration of new treatments, such as gene- and immunotherapy. However, these therapies may also select resistant tumor cells and/or are not effective with acquired
immunoresistance, thereby limiting their success. Most immunotherapy strategies have been directed toward eliciting
a specific anti-tumor response, with few studies on overcoming immunoresistance. Cytotoxic cells have been shown
to kill via both the degranulation (perforin/granzyme) and
Fas/FasL cytotoxic pathways. Recent studies in our laboratory have shown that the Fas- and drug-resistant human
prostate tumor cell lines, PC-3 and DU145, can be sensitized
to killing by Fas-ligand expressing CTL, LAK and renal derived TIL (RTIL) cells following treatment with subtoxic
concentrations of chemotherapeutic drugs (ADR, CDDP,
VP-16) (Frost et al., 1997. Cell. Immunol. 180:70). This study
examined the cytotoxicity of prostate-derived TIL (PTIL)
from two patients with hormone refractory prostate cancer,
EXTRACELLULAR MATRIX AND METHYLATION DEPENDENT
REGULATION OF METALLOTHIONEIN GENE EXPRESSION IN
HUMAN PROSTATE CANCER CELL LINES
Seshadri Tekur,1 Kin Mang Lau,1 Brian C.S. Liu,2 and Shuk-Mei
Ho1
Department of Biology, Tufts University, Medford, MA 02155
Department of Urology, Mount Sinai School of Medicine, New
York, NY 10029
2
Resistance to anti-neoplastic agents is a major obstacle to
cancer treatment. Overexpression of metallothioneins (MT)
represents one mechanism of resistance to a subset of clinically important anti-cancer drugs. Most neoplastic cells with
acquired resistance to the anti-neoplastic agent cisplatin
overexpressed MT and demonstrated cross-resistance to alkylating agents such as chlorambucil and melphalan. Human carcinoma cells that maintained high levels of MT due
to chronic exposure to heavy metals were resistant to cisplatin, chlorambucil and melphalan. Thus, overexpression
of MT represents one mechanism of resistance to a subset of
clinically important anti-cancer drugs.
The long-term goal of our research is to understand the
regulation of metallothionein gene expression in human
prostate cancer (PCa) progression. As a first step towards
this goal we have characterized the expression of MT isoforms in several human PCa cell lines. There are 10 different
known isoforms of metallothioneins expressed in humans.
These include IA, IB, IE, IF, IG, IH, IX, MTIIA, MT3 and MT4.
Using a semi-quantitative RT-PCR we looked at the levels of
these MTs in DU145, PC-3, LNCaP and TSU-Pr1 cell lines.
Our results indicate that in the hormone-dependent LNCaP
cells all isoforms are expressed except IB, MT3 and MT4. In
another hormone-dependent PCa cell line, TSU-Pr1, all isoforms are expressed except IB, MT3 and MT4. Coincidentally both LNCaP and TSU-Pr1 were derived from lymph
node metastasis. On the contrary, in hormone-independent
cell lines, DU145 cells, which were derived from brain metastasis, express IA, IE, IF, IX and MTIIA while PC-3 cells,
340
Bethesda Abstracts
which were derived from bone marrow metastasis, express
only IF, IX and MTIIA and do not express IA and IE isoforms. We therefore speculate that in PCa selective isoforms
of MT may be down-regulated as the cells progress from a
hormone-dependent state to hormone-independent stage.
Since methylation of genes is often implicated in cancer
progression, our next objective was to investigate if methylation was involved in the selective down-regulation of MT
gene expression. Treatment of PC-3 and DU145 cells with
the demethylating agent 5-azacytidine restored expression
of IA and IE isoforms in PC-3 cells and MT3 isoform in
DU145 cells indicating that methylation of these genes is
involved in their down-regulation in the respective cell
types. Interestingly, LNCaP cells grown on extracellular matrix (ECM) of bone marrow and prostatic fibroblasts show
down-regulation of metallothionein IA and IE gene expression and these cells show a similar MT expression pattern as
PC-3. These observations illustrate the importance of stromal-epithelial interaction and suggest that extracellular matrix may play a crucial role in regulating metallothionein
gene expression during growth and progression of human
prostate cancer and metastasis. These studies may help us
design strategies to down-regulate specific MT isoform expression which would form the basis for designing a supportive therapeutic tool for the anti-neoplastic treatment of
human prostate cancer.
DOES 5 ␣-REDUCTASE INHIBITION ENHANCE
IMMUNE FUNCTIONS?
A.G. Alias, M.D.
Chester Mental Health Center, Chester, IL 62233
5␣-reductase (5␣R) converts testosterone (T) to dihydrotestosterone (DHT). DHT is needed for the development
of external genitalia of male fetus, and later, for body hair
growth. 5␣R 2 deficiency 46 XY pseudohermaphrodites,
who are born with female or ambiguous genitalia and are
often raised as girls, tend to dramatically turn around towards puberty to become muscular heterosexual men with
small genitalia and negligible tendency for body hair growth
due to lack of DHT. By contrast, the author noted, which has
been strengthened if not confirmed by several published
studies during the past 80 years, that gay men while much
less muscular are much more hirsute. Thus, T to DHT ratio
is likely to be lower in gay men as Doerr et al. noted, who
wrote, ‘‘the elevated plasma DHT [in gay men] and the lack
of correlation between DHT and T cannot be explained.’’
DHT has specific immune suppressing properties, inhibiting the production of interleukin4 (IL-4), IL-5, and gamma
interferon, but not of IL-2 in activated murine cells, while
predominantly anabolic androgens possess immune enhancing properties. Tolentino writes, ‘‘Ghione was the first to
demonstrate an ‘anti-infective’ action of 4-chlorotestosterone
[an ‘anabolic’ derivative of T] in experimental infection . . . in
rabbits and mice.’’ Araneo et al. wrote, ‘‘the inhibition of
IL-4 production [by T] was abrogated if 4MA, a specific 5␣R
inhibitor, was added to macrophage cultures containing T.’’
Further, a ‘‘novel, potent and selective dual 5␣R inhibitor [of
both 5␣R 1 and 2], Pnu 157706,’’ reduced a prostatic carcinoma growth in the rat by more than 50%.
Hirsutism, or hypertrichosis, is a common, dose-related
side effect of cyclosporin A (CSA), and it is given to organ
transplant patients as an immunosuppressant. CSA stimu-
lates 5␣R, but suppresses plasma T, while finasteride, a 5␣R
inhibitor, substantially raises T, and lowers DHT, etc., in
human benign prostatic hyperplasia tissue, for example.
CSA exerts immune suppression partly at least through enhanced DHT formation by stimulating 5␣R.
Thus, it is plausible that finasteride, and other 5␣R inhibitors, perhaps by inhibiting DHT formation, and/or by
raising tissue levels of T, or of other predominantly ‘‘anabolic T metabolites,’’ possess immune enhancing properties.
Although anabolic steroids such as oxandrolone (11) have
been effectively been used to prevent wasting in AIDS patients, their immune enhancing properties have not been
appreciated. Futher, an immune enhancing role for leptin, in
addition to its role in obesity, and malnutrition, has been
demonstrated.
TUMORIGENESIS OF RAT PROSTATE EPITHELIAL CELLS IS
ASSOCIATED WITH AN ALTERED RETINOIC ACID/RAR PATHWAY
F. Richter,1 H.F.S. Huang,1 M.T. Li,1 D. Danielpour,2 and R.J.
Irwin1
1
VA Medical Center, East Orange, UMD-New Jersey Medical
School, Newark, NJ
2
NCI, Bethesda, MD
INTRODUCTION: Our recent observations of high
abundance of mRNA transcripts for RAR-␣ and -␥ in rat
prostate, and that expression of these transcripts can be
modulated by testosterone (T), suggest that retinoic acid/
RAR mediated processes may be involved in androgen regulation of prostate physiology. In this study, we compared the
growth responses of carcinoma (NRP-154) and noncarcinoma (NRP-152) rat prostate epithelial cells to androgen and retinoic acid, as well as T regulation of mRNAs for
RARs in these cell lines.
METHODS: NRP-154 and NRP-152 cells were propagated in DMEM/F12 medium supplemented with 10% FCS,
2 mM glutamine, penicillin/streptomycin (20 ml/l), fungizone (4 ml/l), EGF (20 ␮g/l), insulin (5 mg/l), dexamethasone (0.1 ␮M) and cholera toxin (10 ␮g/l) in a humidified
37°C CO2 incubator. The effects of retinoic acid and androgen were examined after the cells were cultured in medium
in which FCS was replaced by charcoal stripped FCS for 48
h. Northern blot analysis was used to determine the level of
androgen receptor (AR) and RAR mRNA transcripts.
RESULTS: In the presence of 10% FCS, cell number of
both NRP-152 and NRP-154 cells increased linearly for at
least 6 days without medium change. At that time, the number of NRP-154 cells was 4-fold higher than that of the NRP152 cells (P < 0.01). Treatment of these cells with T for 4 days
resulted in a dose-dependent increase in cell number with a
4-fold greater effect in NRP-152 than in NRP-154 cells.
Northern blot analysis of poly (A)+ enriched RNA isolated
from untreated cells revealed the presence of the 9.4 kb androgen receptor (AR) mRNA in both cell lines, but the level
was at least 10-fold higher in NRP-152 cells. Treatment of
NRP-152 cells with retinoic acid stimulated cell growth in a
dose-dependent, biphasic manner. Identical retinoic acid
regimens failed to induce significant cell growth in NRP-154
cells. The steady levels of the 2.7 and 3.4 kb RAR-␣ mRNA
transcripts in untreated NRP-154 cells were higher than
those of NRP-152 cells. On the other hand, the 3.4 kb RAR-␥
mRNA was lower in NRP-154 cells. Treatment of NRP-152
cells with increasing concentrations of T resulted in a bipha-
Bethesda Abstracts
sic decrease in RAR-␣ and -␥ mRNA levels, whereas the
identical treatments resulted in a biphasic increase in RAR-␣
and -␥ mRNA levels in NRP-154 cells.
CONCLUSION: Current results demonstrate that carcinogenesis of rat prostate epithelial cells is associated with an
increase in baseline cell proliferation but a decrease in androgen-stimulated cell growth. The latter is attributable to a
lower AR expression. Futhermore, an over-expression of
RAR-␣ mRNA and an under-expression of RAR-␥ mRNA,
and a change in the responses of RAR genes to T in NRP-154
cells, indicate an altered retinoic acid/RAR signal transduction pathway in these cells. (Supported by C.R. Bard Endowed Fund and a grant from VA Rahab R & D; F. Richter
is a recipient of a training grant from the Deutsche Forschungsgemeinschaft, Germany.)
341
mary (66%) and metastatic tissues (50%) of these patients
which needs to be further characterized.
CONCLUSIONS: This study revealed a very high rate of
6q alterations in specimens of CaP patients including homozygous deletions (62.5%) in primary and metastatic CaP.
Taken together, this study and our previous observations
support the involvement of a tumor suppressor gene on
chromosome 6q16.3–21 in prostate tumorigenesis.
CLINICALLY-RELEVANT ORTHOTOPIC-METASTATIC AND
HISTOCULTURE MODELS OF HUMAN PROSTATE CANCER
J. Geller,1 Z. An,1 X. Wang,1 G. Olbina,1 A.R. Moossa,2 and
R.M. Hoffman1,2
1
AntiCancer, Inc., San Diego, CA
Department of Surgery, University of California, San Diego
School of Medicine, San Diego, CA
2
HIGH FREQUENCY OF CHROMOSOME 6q16.3–21 ALTERATIONS
IN HUMAN METASTATIC PROSTATE CANCER
Vasantha Srikantan,1 Leland D. Davis,1 Robert C. Dean,2 Judd
W. Moul,1,2 Shiv Srivastava,1 F. Kash Mostofi,3 David G.
McLeod,2 and Isabell A. Sesterhenn3
1
Center for Prostate Disease Research, Uniformed Services
University of the Health Sciences, Bethesda, MD
2
Urology Service, Walter Reed Army Medical Center,
Washington, DC
3
Department of GU Pathology, Armed Forces Institute of
Pathology, Washington, DC
INTRODUCTION AND OBJECTIVES: Chromosomal
allelic losses suggesting tumor suppressor gene inactivation
have been noted for chromosomes 2q , 5q, 6q, 8p, 10q, 13q,
16q, 18q in prostate cancer (CaP) as evidenced by loss of
heterozygosity (LOH) or comparative genomic hybridization (CGH) studies. We recently reported LOH on chromosome 6q16.3–21 loci in 27% of primary prostate cancers with
an increased trend of this alteration in stage T3 disease. This
report compares our observations of 6q alterations in primary and metastatic CaP.
METHODS: Genomic DNA from paraffin embedded,
microdissected, normal, primary and metastatic CaP tissues
were analyzed for 6q16.3–21 alterations by using microsatellite markers (D6S300, D6S283, and D6S314) that exhibited
the highest frequency of LOH deletions in our previous
study. Autopsy derived multiple metastatic cancer tissues
from each patient were analyzed. Sixteen archival paraffin
embedded specimens representing lymph node, bone, lung,
and liver metastasis, eight primary cancer specimens, and
nine normal tissues from eight patients with metastatic CaP
were subjected to a comprehensive analysis of 6q microsatellite markers.
RESULTS: Using the D6S300 marker, we have found either LOH/homozygous deletion or band shifts suggestive of
microsatellite instability (MSI) in one or more specimens
from eight of eight (100%) CaP patients with metastatic disease. Further, we have found an increased rate of 6q deletions in metastatic CaP (62.5%) as compared to the primary
CaP (33%) in this study. Multiple metastatic specimens from
the individual patients exhibited considerable heterogeneity
with respect to 6q status, e.g., DNA from different metastatic
sites of individual patients exhibited heterogeneity with respect to 6q alterations. An intriguing observation included
the high incidence of band shifts suggestive of MSI in pri-
In order to develop a clinically-relevant animal model of
human prostate cancer, we have developed microsurgical
techniques, termed surgical orthotopic implantation (SOI),
to implant histologically intact tumor tissues orthotopically
in immunodeficient mice. In this study intact tissue of the
human prostate cancer cell line PC-3, harvested from a subcutaneous PC-3 tumor in a nude mouse, was implanted to
the ventral lateral lobes of the prostate gland in a series of
nude mice. Extensive and widespread lymph-node and lung
metastases following orthotopic implantation of PC-3 were
found. In contrast to orthotopic injection of cell suspensions,
no multiple metastatic cell selection was necessary after SOI
for significant expression of the metastatic potential of PC-3.
In order to develop a clinically-relevant in vitro model of
human prostate cancer, we have used collagen-sponge-gelsupported three-dimensional histoculture to measure androgen-independent and androgen-dependent growth in vitro.
Paired specimens of BPH and prostate cancer from 23 radical prostatectomies were compared. Both androgenindependent growth and androgen-dependent growth are
measures of important biological characteristics of benign
and malignant prostate tissue. The effect of hydroxyflutamide and antiandrogens on dihydrotestosterone (DHT)
stimulated incorporation of 3H-thymidine into both paired
specimens of BPH and cancer was utilized to measure androgen-independent and androgen-dependent growth. The
percentage decrease in 3H-thymidine incorporation/␮g protein in the hydroxyflutamide-treated specimen compared to
the DHT-treated specimen represented androgen-dependent growth. Residual 3H-thymidine incorporation/␮g protein during hydroxyflutamide administration represented
androgen-independent growth.
Androgen-independent growth was significantly greater
(P = 0.015) in the BPH compared to the cancer paired tissue.
Androgen-dependent growth was significantly higher in 23
paired specimens of cancer compared to BPH (P = <0.03).
There was no correlation of either growth parameter with
Gleason tumor grade. Future clinical correlations will indicate whether either growth parameter represents an important prognostic factor for prostate cancer aggressiveness.
These in vitro and in vivo models should prove useful for the
study of and individualized therapy and drug discovery for
androgen-dependent and -independent prostate cancer
growth and metastasis.
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Bethesda Abstracts
PREGNENOLONE STIMULATES GROWTH OF THE LNCaP
PROSTATE CANCER CELL LINE BY INTERACTING WITH A
SPECIFIC BINDING SITE
D.N. Grigoryev, B.J. Long, and A.M.H. Brodie
Department of Pharmacology and Experimental Therapeutics,
University of Maryland School of Medicine, Baltimore, MD
21201
We have determined the effects of various steroids on the
growth of the LNCaP prostate cancer cells over a nine day
period (changing the media and adding fresh steroid every
3 days). Pregnenolone (P5), a common precursor of many
steroids, is present in the blood of normal adult males at a
concentration 1–3 nM and was found to stimulate LNCaP
cell proliferation 7–8-fold at physiological (2 nM) and 3–
4-fold at subphysiological concentration (0.2 nM). The
stimulation at 2 nM was comparable to that of the androgen,
dihydrotestosterone (DHT) at its physiological concentration (0.5 nM; 9–10-fold increase in cell number). To determine whether it was P5 itself or metabolites of this steroid
that were mediating this growth response, we performed
high performance liquid chromatography (HPLC) with
[3H]-P5 that had been exposed to LNCaP cells. Following
an 8 hr exposure to 5 × 104 cells, one minor P5 metabolite was detected. The amount of this metabolite was increased following 24 hr and 48 hr exposure and by 24 hr
a second metabolite was also detected at low levels. By 48
hr the levels of these metabolites were 14% and 9% of
the starting amount of P5 added. To eliminate the possible
growth stimulatory effects of these metabolites, growth
studies were repeated changing the media every 48 hr and
cells were also treated with P5 that had been pre-exposed
to high density LNCaP so that it contained an increased
proportion of metabolites (P5:metabolites = 0.02 nM:0.27
nM as determined by HPLC). LNCaP cells proliferated at
a faster rate when they were exposed to fresh P5 every 2
rather than 3 days; however, this difference did not gain
statistical significance. The P5 metabolites also increased
LNCaP cell growth (1.5-fold vs. vehicle), but this was significantly less than treatment with P5 alone (3-fold vs.
vehicle), suggesting that these growth effects were being
mediated by P5 itself. In order to determine whether P5
was binding to a specific steroid receptor in LNCaP cells,
we performed receptor binding analysis and displacement
studies. Following treatment of cells with various concentrations of [3H]-P5 (0.1 nM–20 nM) in the presence and
absence of a 200-fold excess of cold P5, Scatchard analysis
predicted for a single binding site with an apparent Kd = 2.4
nM. LNCaP cells were then incubated with 2.4 nM of [3H]-P5
in the presence of different concentrations of unlabeled P5,
progesterone (P4), 17␤-estradiol (E2), and DHT. [3H]-P5
was displaced by 96%, 22%, 0% and 0% by 10 nM of cold
P5, P4, E2, and DHT respectively, and 100%, 61%, 76%, and
0%, by 100 nM of cold P5, P4, E2, and DHT respectively.
These results show that P5 stimulates the growth of LNCaP
prostate cancer cells and that this effect is likely to be mediated by a specific high affinity binding site for this steroid.
These results suggest that the growth of hormonedependent prostate tumors is being mediated by other steroids in addition to androgens and may have important implications in the design of novel drugs for the treatment of
prostate cancer.
EXPRESSION AND FUNCTION OF ps20 IN PROSTATE SMOOTH
MUSCLE: A NOVEL GROWTH INHIBITORY PROTEIN IN THE
WAP-TYPE ‘‘FOUR DISULFIDE CORE DOMAIN’’ PROTEASE
INHIBITOR FAMILY
S.J. Ressler, M. Larsen, B. Lu, T.D. Dang, J.A. Tuxhorn, and
D.R. Rowley
Baylor College of Medicine, Houston, TX 77030
Our previous studies have purified and characterized a
novel growth inhibitory protein termed ps20, which is secreted by rat prostate stromal cell lines ( J. Biol. Chem. 270:
22058–22065). We have subsequently reported the cloning of
rat ps20 ( J. Biol. Chem. 273:4574–4584), a 20–22 kDa protein
in the WAP-type ‘‘four-disulfide core domain’’ family. To
extend these studies, we have cloned the human ps20 homolog, analyzed cell-specific expression patterns, and addressed mechanisms of action. Human ps20 shares a 95%
similarity at the amino acid level and conservation of the
WAP domain. WAP family proteins include secreted serine
protease inhibitors which exhibit growth, differentiation,
and tissue remodeling functions. Expression analysis in rat
tissues demonstrated moderate to high expression of ps20 in
the dorsal-lateral prostate, vas deferens, testis, uterus, ovary,
and particularly high expression in the heart. Immunolocalization revealed expression was specific to the smooth
muscle cell type. In the prostate, expression was specific to
the periacinar smooth muscle, whereas stromal fibroblasts
were negative. In the heart, expression was specific to the
tunica media smooth muscle cells in coronary arteries. Gut
(stomach and intestine) smooth muscle exhibited low ps20
immunoreactivity. Human normal prostate and BPH specimens exhibited strong staining in smooth muscle cells. In
contrast, a variable pattern of expression was observed in
the smooth muscle of prostatic carcinoma specimens. Prostate stromal (smooth muscle) cell lines generated by our
laboratory were used to assess ps20 expression in vitro. Both
the rat (U4F and PS-1) and human (HPS-3 and HTS-1) prostate stromal cell lines exhibited ps20 expression by Northern
(U4F, PS-1), Western and immunoprecipitation analysis.
Each cell line stained positive for smothe muscle markers
including smooth muscle ␣-actin, desmin, smooth muscle
myosin heavy chain, and calponin. In addition, these cell
lines were androgen receptor positive, androgen responsive
(Endocrinology 137:864–872, PS-1 cells), and, typical of
smooth muscle cells, were able to form spheroids in culture.
To address ps20 mechanisms of action, we expressed ps20 as
a GST fusion protein in bacteria. Recombinant ps20 was
growth inhibitory to human prostate carcinoma PC-3 cells
and other cell lines, similar to what was reported with the
native protein. Stable transfected COS cells secreted ps20
into the media, were also growth inhibited, and exhibited
typical ps20-induced phenotypic alterations (including filopodia and pseudopodia extensions). Reverse zymography
demonstrated that ps20 inhibited elastase and trypsin-like
proteolytic activities which is consistent with the function of
other WAP family serine protease inhibitors. Since elastase
has been shown to activate and/or release several growth
factors, we propose that the growth inhibitor activity of ps20
is mediated through its protease inhibitor function. These
studies suggest that ps20 is a new smooth muscle-specific,
secreted protein, which may function as a protease inhibitor
in tissue remodeling and/or regulation of growth factor bioavailability. Future studies will address the biological sig-
Bethesda Abstracts
nificance of ps20 to cell growth and differentiation control,
and the stromal cell responses in prostate development, the
genesis of BPH, and prostate carcinoma progression. Supported by DK45909, CA58093, and SPORE CA58204.
AN ANALYSIS COMPARING MICROVESSEL DENSITY, ANDROGEN
RECEPTORS, p53 AND HER-2/neu EXPRESSION AND GLEASON
SCORE IN PROSTATE CANCER: THERAPEUTIC IMPLICATIONS
J.H. Mydlo, J.G. Kral, M. Volpe, C. Axiotis, R.J. Macchia, and
L.P. Pertschuk
SUNY Health Science Center at Brooklyn, Brooklyn, NY
We wanted to investigate whether there are any interrelationships between microvessel density (MVD), androgen
receptor (AR), p53, HER-2/neu expression and Gleason
score (GS) in prostate cancer (CAP) in an attempt to further
characterize markers of malignancy and have a greater understanding of prostate tumor biology.
Slides of prostate cancer specimens from radical surgery
and channel transurethral resection of the prostate (TURPs)
were tested for AR by immunocytochemical assay, for MVD
using antibodies to the endothelial cell membrane molecule
PECAM-1/CD-31, for p53 using the monoclonal antibody
D07, and for HER-2/neu using the HER-2 9G6 mouse monoclonal antibody. They were compared to GS and clinical
stage at the time of operation.
We found a fourfold increase in MVD expression in CAP
compared to neighboring benign tissue. Gleason score correlated with mutant p53 expression (r = 0.57, P < 0.05) and
with MVD (r = 0.40, P = 0.06), but not with AR or HER-2/
neu. Furthermore, more than half of the specimens that had
a 50% or greater expression of mutant p53 were in the D2
stage. There were no correlations with AR and HER-2/neu.
In conclusion, we found correlations only between MVD
and p53 to both GS and stage of CAP. This suggests that
increased neovascularization may reflect increased potential
for metastasis, while mutant p53 expression may promote
cell division, potentially influencing the radiosensitivity of
the tumor. Furthermore, since the neovascularity of prostate
tumors can be attenuated by radiation and hormones, while
mutant p53 expression may confer resistance to apoptosis
from such treatment, it appears that p53 may play a more
important role than angiogenesis in the virulence of prostate
cancer. These data may aid in allocating patients to different
treatment modalities.
ESTABLISHMENT OF NORMAL NOVEL MOUSE EPITHELIAL AND
SMOOTH MUSCLE PROSTATE CELL LINES
Yasuhiro Koikawa,1 Varrie Ogilvie,1 Emma Moore,2 Ellen
Shapiro,3 Herbert Lepor,3 Tung-Tien Sun,3 David Moscatelli,3
and Lynette Wilson3
1
Department of Cell Biology, NYU Medical Center, New York,
NY 10016
2
Zymogenetics, Inc., Seattle, WA 98102
3
Departments of Cell Biology and Urology, NYU Medical
Center, New York, NY 10016
In order to develop models for understanding the interactions that occur between prostatic epithelial and stromal
cells, we have developed 14 murine prostatic cell lines.
These were derived from both the ventral (VP) and dorso-
343
lateral (DLP) prostate tissue from p53 knock out mice. p53
knock out mice were chosen as it has previously been shown
that normal cell lines can be obtained from these animals.
The cell lines were established in both serum-free and serum-containing media. The cell lines have been passaged
32–45 times and are of both epithelial and smooth muscle
origin. We have 8 cell lines from the DLP and 6 from the VP.
The cell lines were stained with antibodies to cytokeratins
and smooth muscle actin to determine whether they were of
epithelial or smooth muscle origin. One of the cell lines is
purely epithelial, two of the lines are of smooth muscle origin and the remaining 11 lines have both epithelial and stromal components in varying amounts. The cell lines have
generation times varying from 15–43 hours. None of the
lines forms tumors when inoculated subcutaneously at
doses up to 5 × 106 cells per mouse. TGF-␤ was significantly
inhibitory for epithelial growth but only modestly inhibited
the growth of the smooth muscle cells. TGF-␤ is produced
by many of the lines but all in the latent or inactive form. We
have therefore developed a panel of mouse prostate cell
lines of both epithelial and smooth muscle origin. These
lines are currently being used to study prostatic epithelial/
smooth muscle interactions.
IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES USING
PROSTATE CANCER XENOGRAFT MODELS
R.S. Hubert, E. Chen, E. Bezema, S. Rastegar, I. Vivanco, R.
Harrell, A. Raitano, D. Saffran, and D. Afar
UroGenesys, Inc., Santa Monica, CA
A complete understanding of the molecular basis for
prostate cancer (CaP) has been hindered by the paucity of
cell-lines and in vivo animal models for CaP. The recently
developed LAPC (Klein et al., 1997) xenograft mouse models
overcome the limitations of existing cell-lines and can recapitulate the transition of androgen dependence to androgen
independence. Using a subtractive hybridization method
with cDNAs derived from these LAPC xenografts and normal prostates, five cDNA fragments were identified that are
differentially expressed. Sequencing of approximately 3500
cDNA fragments gave 70% known genes, 25% ESTs, and 5%
novel genes. Fifty-five of these cDNA fragments were initially evaluated by RT-PCR on a panel consisting of normalized first strand cDNAs from normal brain and prostate,
androgen dependent and independent xenograft tumors,
and non-prostate tumor cell-lines. Five of these cDNA fragments showed differential expression in CaP. BLAST homology searches of these cDNA fragments revealed no homology to known genes in GenBank or ESTs in dBEST. One
cDNA fragment, UG01, is up-regulated in the androgen independent prostate cancer xenograft. The two cDNA fragments, UG02 and UG03, are up-regulated in both androgen
dependent and independent CaP xenografts, and UG04 is
down-regulated. UG05 shows restricted expression in prostate tissues. Northern blot analysis confirmed these expression profiles. Additional RT-PCR analysis on normal tissues
demonstrated restricted expression patterns with UG01 being expressed at low levels only in placenta, ovary and normal prostate while UG04 showed very high expression in
normal prostate and lower expression in lung. In summary,
xenograft models have been used successfully to identify
five genes that are differentially expressed in CaP and may
represent novel markers and/or therapeutic targets.
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Bethesda Abstracts
EXPRESSION OF HOX GENES IN BENIGN PROSTATIC TISSUES
AND CELLS
Gary J. Miller, Heidi L. Miller, and Robert E. Donohue
The homeodomain proteins are a very large family of
transcription factors characterized by the presence of a
highly conserved homeobox. The HOX genes represent a
distinct subfamily of these genes. This subfamily is located
in clusters on 4 separate chromosomes that are the result of
tandem reduplication of a primitive set of genes present in
invertebrates. These genes are well recognized for their roles
in development including spatial organization of the musculoskeletal and central nervous systems. Comparatively
little is known about their expression in post-gestational
cells or tissues. We have used a degenerate RT-PCR technique to examine the range of HOX genes that are transcribed in the adult prostate. Whole RNA from histologically-normal adult prostate, a highly epithelial benign hyperplastic nodule and primary cultures of normal,
postpubertal prostatic epithelial cells with or without treatment with vitamin D was reverse transcribed to cDNA. PCR
was carried out using degenerate primers representing the
conserved peptide motifs, ELEKEF and KIWFQN, from the
5⬘ and 3⬘ ends of the homeobox, respectively. PCR products
were cloned into pCR2.1-TOPO vectors for sequencing. A
total of 116 inserts were examined. Overall, transcripts from
18/38 of the known HOX genes were detected. The expression of 9 different homeobox genes was found in adult human prostate tissue. These included 5 from chromosome 7
(A3, A6, A7, A9 and A10) one from chromosome 17 (B3) and
two from chromosome 2 (D1 and D4). Over 30% of the inserts were from A9 and A10. In addition, the androgen responsive homeobox gene NKX3.1 was also expressed. The
transcripts from untreated primary epithelial cells were enriched for A9 and A10 (16/32). Other notable differences
were the presence of A1(7/32), B7, D9, D10 and the absence
of A7. When these cells were treated with 10−9 M 1␣,25dihydroxyvitamin D3, there was a relative increase in the
3⬘-most members of the A cluster (A1, A3, A4, A6 and A7)
balanced by a decrease in A9 (8/33) and an absence of A10.
Similar HOX genes were expressed in BPH. A3, A4 and A5
accounted for 14% of the BPH derived inserts. A9 and A10
were again prominent (15/29) and B3, C6, D1, D8, D9, and
D12 were detected. NKX3.1 was also found (3/29). In summary, while their precise role in the prostate remains unknown, it is clear that several of the HOX genes on chromosomes 2 and 7 are expressed in adult prostatic epithelial
cells. Further elucidation of their functions should provide
valuable insight into regulation of prostatic differentiation in
normal and neoplastic cells.
EXPRESSION OF IGFBP-7/mac25 IN PROSTATE CANCER
A. Degeorges, F.A. Wang, H.F. Frierson Jr., A. Seth, and R.A.
Sikes
Department of Urology and Pathology, University of Virginia,
Charlottesville, VA 22908, and Laboratory of Molecular
Pathology, Women’s College Hospital, University of Toronto,
Toronto ON M5S 1B3, Canada
The Insulin-like growth factor (IGF) axis is composed
of IGF-I and -II, receptors I and II, and binding proteins
(IGFBPs) responsible for the bioavailability of the IGFs. Recently a new IGFBP, IGFBP-7, has been characterized. Since
the IGF axis is modified in benign and malignant human
prostate tumors, we evaluated IGFBP-7 expression in prostate specimens. Immunohistochemistry was performed using a polyclonal rabbit antibody directed against T1A12, a
protein shown to be IGFBP-7. A preliminary study on normal human tissues showed that IGFBP-7 stained nerves
strongly in all tissues examined. Cytoplasmic staining was
observed in epithelium in various tissues and smooth
muscle cells. A normal prostate specimen showed no staining except for the nerves. We have stained 64 specimens of
paraffin embedded prostate adenocarcinoma. Nerves were
always strongly positive, smooth muscle cells moderately
positive and lymphocytes infiltrating the tumor always
negative. When normal prostate secretory cells were present
on a section, their staining was usually negative; when positive, the staining was weak and mostly focal, localized to the
basal cell layer. Tumor cells were often more positive than
normal secretory cells and when normal epithelium was
weakly positive, intensity in tumor cells was superior. Fiftyeight of 64 (91%) tumors were positive, with a positivity
ranging from 25 to 100% of the tumor cells. When high grade
PIN was present, the immunohistochemical pattern was
same as in the invasive tumor. One metastasis stained
strongly. Unlike prostate adenocarcinoma, IGFBP-7 staining
in normal breast epithelium is intensely positive and subsequently lost in adenocarcinoma cells. By Northern blot, we
have shown that none of the human prostate cancer cell lines
express IGFBP-7 RNA, nor can androgen induce its expression in LNCaP cells. The role of IGFBP-7 in prostate and
breast biology and tumorigenicity remains to be elucidated;
however, it may be a novel prostate cancer progression
marker.
DEVELOPMENT OF A REPORTER-TAGGED HUMAN PROSTATE
LINE AND ITS USE AS A METASTATIC MODEL
Mark Heller,1 Karen Ferrer,2 Bao-Hua Xue,1 David Peace,1 and
Carol Westbrook1
1
Division of Hematology/Oncology, Department of Medicine,
University of Illinois at Chicago, Chicago, Illinois
2
Department of Pathology, University of Illinois at Chicago
Hospital, Chicago, Illinois
Progression of localized prostate cancer to metastatic and
invasive disease is a key issue of prostate biology and clinical management. As a step toward improved in vitro and
preclinical xenograft models of prostate metastasis, we have
stably transduced a new hormone-independent human
prostate line with the reporter gene tag ␤-galactosidase and
designate these cells MH-PR1. MH-PR1 reliably metastasizes to retroperitoneal/paraaortic lymph nodes and other
distant sites when orthotopically implanted in the prostate
of athymic nude mice. Preliminary characterization of MHPR1 shows it does not express androgen receptor or PSA but
does express prostate specific membrane antigen (PSMA).
To establish a metastatic-progressed lineage from a progenitor ␤-gal tagged prostate cell clone, we have recovered
tagged cells from distal metastatic sites such as lymph node,
soft tissue mets, and bone marrow, and metastasis of these
derivatives from the orthotopic prostate site in nude mice
was repeated. Efforts are underway to develop sublines
Bethesda Abstracts
which have defined affinity in the nude mouse model for
metastatic sites such as skeletal bone, marrow, etc. We are
also using microcell mediated transfer of individual human
chromosomes into MH-PR1 for suppression of metastasis as
an approach to identify prostate metastasis suppressor
genes.
NOVEL FETAL UROGENITAL SINUS GENE EXPRESSION IN
PROSTATE CANCER PROGRESSION
Robert A. Sikes,1 Jin-Hee Kim,1 Fuan A. Wang,1 Armelle
Degeorges,1 Leland W.K. Chung,1 Claudia Fasciana,2 and Jan
Trapman2
1
2
Charlottesville, VA
Rotterdam, The Netherlands
Fetal markers such as ␣-fetoprotein and carcinoembryonic antigen have been widely used in the diagnosis, treatment and prognosis of gastrointestinal and testicluar cancers. To date, no such fetal markers have been identified for
prostate cancer (PCa). We chose to isolate clones from the
mouse fetal urogenital sinus (UGS), the prostatic anlage, in
order to identify new markers associated with PCa progression from androgen responsive to androgen insensitive as an
aid in the diagnosis and treatment of human PCa. Seven
hundred eighty-seven clones were selected randomly from a
mouse UGS plasmid library and subjected to chain terminiation sequencing. These cDNA fragments were then analyzed by FASTA (GCG/Wisconsin) against the GenBank
plus (known genes plus expressed sequence tag (EST) database) to search for homology to known genes. Two hundred
and six out of 787 (26.2%) were found to match known gene
sequences; 59/787 (7.5%) had moderate homologies to
known genes; 497/787 (63.2%) of sequences matched ESTs
or were unique to UGS; and 25/787 (3.2%) contained only
vector sequences. To efficiently screen the 787 ESTs, we developed a method to systematically pool clones in dot matrix
format in combination cDNA hybridization. Duplicate
membranes were hybridized using 32-P radiolabeled cDNA
pools synthesized from total RNAs isolated from LNCaP,
human androgen-responsive prostate cancer cell line, and
C4-2, a LNCaP-derived cell subline that has acquired androgen insensitivity thereby mimicking PCa progression. The
exposed films were then compared for differential expression of the UGS clones between these two human PCa cell
lines. Thirty-five of the 787 (4.4%) clones showed differential
expression in LNCaP versus C4-2 cell lines on the dot matrix
blots. Expression of 28 clones decreased with progression
from LNCaP to C4-2 while 7 clones had increased expression. For further confirmation, RNA blot analysis using
30 ␮g of total RNA from the LNCaP progression model
(LNCaP, C4, C4-2, C4-2B4 each ± 1 nM R1881 for 48 hr) were
probed with the EST clones demonstrating differential expression. To date, one clone (ug311) that encodes a novel
octamer-binding protein sequence has shown loss of expression from LNCaP to C4-2 while a second unique clone
(ug494) demonstrates up-regulation in C4-2 and limited homology to an mRNA splicing factor. Therefore, we have
demonstrated for the first time the re-expression of fetal
UGS-derived genes in a progression model of human prostate cancer. Two fetal UGS-derived ESTs have been found in
the LNCaP PCa progression model. One gene loses its expression in the aggressive, androgen independent PCa cell
345
line, C4-2, while a second gene shows increased expression
in the C4-2 cell line as compared to LNCaP. The remaining
33 differentially-expressed ESTs are now being characterized and may hold promise for further development into
diagnostic and prognostic markers for human PCa.
TGF-␤1 INDUCED PROLIFERATION IS MEDIATED THROUGH PDGF
IN THE PROSTATE CANCER CELL LINE, TSU-Pr1
S.M. Sintich, M. Lamm, J.A. Sensibar, and C. Lee
Department of Urology, Northwestern University, Chicago, IL
60611
Previous studies showed that TGF-␤1 stimulated the proliferation of the prostate cancer cell line, TSU-Pr1. This observation is unexpected, for TGF-␤ usually inhibits prostate
cancer cell proliferation. The present study examines the
mechanism through which TGF-␤1 induces this proliferation. We have hypothesized that TGF-␤1 action is through
an indirect mechanism by inducing PDGF expression which
in turn stimulates proliferation. Cells were treated with
TGF-␤1 with or without PDGF neutralizing antibodies in
serum-free media. TGF-␤1 stimulated proliferation of TSUPr1 cells in a dose-dependent manner (0.1 ng/ml 87%, 1
ng/ml 132%, 10 ng/ml 135% increase). The TGF-␤1 induced
proliferation can be abrogated by treatment with a PDGF
neutralizing antibody. The use of goat IgG in place of the
neutralizing antibody was ineffective in inhibiting TGF-␤
induced proliferation in these cells. An ELISA assay determined that treatment with 10 ng/ml TGF-␤1 increased TSU
secretion of PDGF into the conditioned media. This increase
was evident after 3 h of treatment and remained elevated for
at least 24 h in the presence of TGF-␤1. In addition, TSU-Pr1
proliferation was increased by 80% in the presence of 1 ng/
ml PDGF. This observation indicates that TGF-␤1 induced
expression of the mitogenic growth factor PDGF which in
turn stimulated proliferation in TSU-Pr1 cells. These results
provide a mechanism for a proliferative role of TGF-␤ in
prostate cancer cells. [Supported in part by CA09560 and
CA69851]
EXPRESSION OF WINGED HELIX TRANSCRIPTION FACTORS IN
PROSTATE, SEMINAL VESICLE, BLADDER, AND PROSTATIC CELL
LINES
Will Kopachik,1 Simon W. Hayward,2 and Gerald R. Cunha2
1
Department of Zoology and Genetics Program, Michigan State
University, Ann Arbor, Michigan
2
Department of Anatomy, University of California at San
Francisco, San Francisco, California
The hepatocyte-forkhead-homolog (HFH) transcription
factors are a large class which are similar in the winged helix
DNA-binding domain. In mammals the prototype HFH
genes, hepatocyte nuclear factor-3 ␣, ␤ and ␥ play a role in
activation of liver and lung gene expression. In order to
determine if HFH genes are expressed in male sex accessory
organs, RT-PCR of mRNA from rats was performed using
degenerate primers flanking the conserved winged helix domain sequence. Fourteen distinct sequences (four known
and 10 novel) were cloned and analyzed from the 20 day old
rat ventral prostate cDNA. The expression of HNF-3␣ in
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Bethesda Abstracts
male accessory sex organs was determined by Northern
blotting, in situ hybridization and electrophoretic mobility
shift analysis (EMSA). RNA from the dorsolateral prostate
(DP), ventral prostate (VP), anterior prostate (AP), seminal
vesicle (SV) and bladder (BL) was compared with RNA from
the positive control, liver, and negative control, spleen.
HNF-3␣ mRNA levels in the DP,VP, AP and BL were 20, 14,
5 and 6 times higher than in the SV which was equivalent to
that of the liver. Among 10 cell lines tested, HNF-3␣ mRNA
was found in 8 (rat NRP 152 and 154, mouse Pr14, human
DU-145, LNCaP, ND-1 and BPH-1) but not in rat Dunning
epithelial, mouse Pr12 cells or in one stromal cell line, DT3.
In the VP, HNF-3␣ mRNA levels drastically drop after castration but do not in castrates given testosterone; thus the
expression of HNF-3␣ is at least indirectly regulated by androgen. In situ hybridization analysis revealed that HNF-3␣
mRNA is expressed in epithelial cells of the urogenital sinus
and its derivatives (VP, AP, DP, BL) and the Wolffian duct
derivative, the SV. EMSA using VP cell extracts confirmed
that protein with HNF-3␣ activity and inhibited by specific
antibody was present in the VP. The results suggest that
HFH genes may play a role in development and maintenance of urogenital tract epithelial cells.
didate cDNAs that were up- or down-regulated during progression of tumours to androgen-independent growth. One
of these genes, IK factor, was detected by dd-PCR to be
upregulated in LNCaP tumours growing in castrated as
compared to intact mice. Expression of IK factor, which
downregulates interferon-␥ induced HLA class II antigen
expression has not been reported previously in human prostate cancers. IK factor mRNA expression is detectable in
benign human prostate cancer tissues and in primary human prostate tumours; however, its function as a regulator
of HLA class II expression or as a growth regulatory cytokine in normal or malignant prostate is not yet characterised.
In LNCaP cells, expression of IK factor may contribute to the
resistance of cells to the growth inhibitory effects of interferon-␥. Identification of cytokines facilitating the growth of
cancer cells constitutes an important strategy towards development of successful adjuvant therapies for advanced
disease.
DEVELOPMENTAL AND HORMONAL REGULATION OF PEPTIDE
GROWTH FACTOR GENE EXPRESSION IN RAT VENTRAL
PROSTATE
N. Itoh,1 T. Tsukamoto,1 A.S. Cupp,2 and M.K. Skinner2
1
DIFFERENTIAL EXPRESSION OF GENES DURING
ANDROGEN-INDEPENDENT PROGRESSION OF PROSTATE
CANCERS
Jacqueline Bentel, Nicole Moore, Hugh Dawkins, and Wayne
Tilley
Urological Research Centre, Level 2, M Block, QEII Medical
Centre, Nedlands, WA, 6009, and Flinders Cancer Centre,
Flinders University of SA, Adelaide, SA, 5042, Australia
Androgen ablation therapies are initially very successful
in the treatment of advanced prostate cancer; however, development of androgen-independent tumours occurs rapidly, resulting in resumption of tumour growth, tumour metastasis and death of the patient, despite maintenance of
circulating testosterone at castrate levels. The failure of androgen ablation therapies to provide sustained control of
prostate cancer growth is likely to result from multiple genetic and epigenetic events. Several studies have demonstrated that androgen independent progression of prostate
cancers is not associated with loss of expression of androgen
receptors, which mediate androgen action in prostate cancer
and other androgen target cells. However, a significant
number of advanced prostate cancers contain mutations in
the androgen receptor gene which may contribute to the
development of androgen independent tumours. Identification of additional genes with altered expression and/or mutations will lead to increased understanding of this disease
process and the development of new treatments for hormone refractory tumours based on these findings. Investigation of genetic alterations associated with androgenindependent progression of prostate cancer has been carried
out using the human prostate cancer cell line, LNCaP, which
contains a point mutation in the androgen receptor gene.
Identification of genes contributing to the androgenindependent progression of prostate cancers has been determined by comparison of gene expression in subcutaneous
xenografts of LNCaP cells growing in intact and castrated
nude mice. Differential display PCR (dd-PCR) performed on
these LNCaP tumours has resulted in the isolation of can-
Department of Urology, Sapporo Medical University, Sapporo,
Japan
2
Center for Reproductive Biology, Washington State University,
Pullman, WA
OBJECTIVE: Androgen has an important role in growth
and development of the prostate, and its actions are mediated by locally produced growth factors through stromalepithelial interactions. To maintain normal prostatic physiology, homeostasis requires a balance between the biological
effects of growth stimulatory (i.e., TGF-␣) and inhibitory
(i.e., TGF-␤s) factors. The hypothesis tested in the present
study is that these growth factors are regulated by not only
androgens but other locally produced growth factors to
equilibrate their balance.
METHODS: Male SD rats aged 1 to 100 days were used
for the study of prostatic development. Organ culture of
ventral prostate used 0-day-old rat. Epithelial and stromal
cells were isolated from 20-day-old rat ventral prostate and
cultured separately. The expression of mRNA for TGF-␤1, 2,
3, and TGF-␣ by testosterone and a variety of growth factors
(EGF, KGF, TGF-␤) was analyzed by quantitative reverse
transcription polymerase chain reaction.
RESULTS: The patterns of TGF-␤ and TGF-␣ expression
during prostatic development were different. After castration of a 60-day-old rat, all growth factors except for TGF-␤3
were enhanced. However, testosterone did not show any
effect on TGF-␤ and TGF-␣ expression by cultured prostatic
cells. EGF and TGF-␤ inhibited prostatic ductal morphogenesis and growth in organ culture. EGF stimulated TGF-␤1
and TGF-␣ mRNA expression in stromal cells (6-fold and
4.5-fold, respectively), while KGF stimulated TGF-␤2 mRNA
(2.8-fold) in epithelial cells.
CONCLUSIONS: The in vivo effects of castration on
growth factors are regulated indirectly through a complex
network of growth factors, not simply direct androgen
depletion. The ability of EGF to inhibit prostatic ductal morphogenesis and growth in organ culture is postulated to be
in part mediated by the increase of TGF-␤1 expression.
Regulatory mechanism between growth factors is specu-
Bethesda Abstracts
lated to maintain the balance between growth stimulation
and inhibition during prostate development.
ANALYSIS OF THE FUNCTION OF HUMAN TR3 ORPHAN
RECEPTOR GENE IN PROSTATE CANCER
Hiroji Uemura, Yoshinobu Kubota, Kazunori Takase, Yasuhide
Miyoshi, Shinsuke Ohta, and Masahiko Hosaka
Department of Urology, Yokohama City University, School of
Medicine, Yokohama, 236, Japan
In a previous meeting, we demonstrated that human TR3
orphan receptor, a member of the steroid receptor superfamily, can be rapidly induced by stimulation of growth factors
and androgen in prostate cancer cells. Furthermore, this receptor can be rapidly induced with apoptosis-inducing reagents, e.g., calcium ionophore or etoposide in LNCaP or
PC-3 cells.
We then performed in situ hybridization using TR3 cDNA
probe on human prostate cancer samples, benign prostatic hyperplasia (BPH) and normal prostate tissues. These data
showed that TR3 mRNA was highly expressed in prostate cancer region compared with BPH or normal prostate regions. To
investigate whether the expression of TR3 mRNA can be
quantitated in prostate cancer, RT-PCR was performed using total RNAs extracted from human prostate tissues. It
was demonstrated that there was significant difference of
TR3 expression between prostate cancer, and BPH, normal
prostate. However, no relationship was found between TR3
expression and pathological differentiation of prostate cancer.
These results indicate that human TR3 orphan receptor is
thought to be one of the early response genes which play
important roles in mitogenesis and apoptosis in prostate
cancer cells, and suggest that this gene may play a key role
in the development of prostate cancer.
APOPTOSIS OF PROSTATE FIBROBLASTS AND VASCULAR
ENDOTHELIAL CELLS DURING CASTRATION-INDUCED
REGRESSION OF THE RAT VENTRAL PROSTATE GLAND
Ahmad Shabsigh, Erik T. Goluboff, Aaron E. Katz, Carl A.
Olsson, and Ralph Buttyan
Columbia University, New York, NY
The rat ventral prostate gland is a good in vivo model
system to study the means by which castration induces apoptosis of prostate cells. Castration of an adult male rat
causes regression of the ventral prostate by inducing apoptosis of prostate epithelial cells. Although a previous report
described the loss of stromal cells as well (English et al.,
1985, Prostate, 7:41), this aspect is usually ignored in contemporary studies of the model. Using an in situ immunohistochemical technique that identifies apoptotic cells by enzymatic labeling of fragmented nuclear DNA (TUNEL assay), we reexamined regressing rat ventral prostates to
characterize apoptosis in the stroma.
Ventral prostates were obtained from unoperated or
sham-operated rats and from rats at daily intervals after
castration. Tissues were fixed, embedded and sectioned
prior to staining for apoptosis using a TUNEL assay kit.
TUNEL-positive nuclei were counted by microscopy.
Smooth muscle cells were identified by staining with antismooth muscle actin antibody; vascular endothelial cells
347
were identified by anti-CD31 antibody staining. No TUNEL
staining (apoptotic) stromal cells were observed in sections
from control rat prostate tissues. Eight percent of prostate
stromal cells were TUNEL-positive at 12 hrs after castration
and 15% of stromal cells at 24 hrs after castration, decreasing
thereafter to less than 10% of the stromal cells at 48 hrs after
castration. At 72 hrs and after, TUNEL-positive nuclei were
no longer found in the stroma of the regressing rat prostate
gland. TUNEL-positive cells in the stroma were restricted to
the vascular endothelial and non-specific fibroblast population with no labeling of smooth muscle cells.
The stromal cell deletion previously described during rat
ventral prostate regression occurs by means of apoptosis.
Stromal cell apoptosis precedes and is coincident with the
early onset of prostate epithelial cell apoptosis but was
greatly diminished during the later period of regression.
Non-specific fibroblasts as well as vascular endothelial cells
were involved in this apoptosis. Study of the mechanism by
which androgen withdrawal leads to prostate regression
must account for the induced apoptosis of stromal cells as
well as epithelial cells following castration.
INDUCED FAS ANTIGEN EXPRESSION AND ACTIVATION DURING
REGRESSION OF THE RAT VENTRAL PROSTATE GLAND:
MOLECULAR EVIDENCE FOR HYPOXIC INSULT ASSOCIATED
WITH CASTRATION-INDUCED PROSTATE REGRESSION
Min-Wei Chen, Ahmad Shabsigh, Carl A. Olsson, and Ralph
Buttyan
Columbia University, New York NY
Androgens regulate prostate growth to the adult size,
acting through a mechanism that is not yet well defined. To
study this mechanism, we use an experimental animal
model, the rat ventral prostate gland, that enables us to determine the effects of androgen-withdrawal/androgen
supplementation on the prostate. Previously, we found that
castration of a rat significantly reduced blood flow to the
ventral prostate gland and we proposed that the reduced
blood flow results in a hypoxic state sufficient to induce
apoptosis of prostatic cells. To find further molecular evidence that reduced blood flow might be a factor in the onset
of apoptosis associated with rat prostate regression, we surveyed regressing rat prostate tissues for two prominent
markers of a cellular response to hypoxia/ischemia; namely,
upregulation of cell surface fas antigen expression and activation of the intracellular fas-signaling complex (identified
by binding of RIP and FADD proteins to the fas antigen).
Ventral prostate glands were obtained from unoperated
adult rats, sham-operated rats and rats at daily intervals
after castration. The tissues were surveyed for fas antigen
expression by RNase protection assays using a rat fas antisense RNA probe and by Western blot analysis of prostate
protein extracts using anti-murine fas antibodies. Fas activation was analyzed by immunoprecipitation of fas antigen
complexes from prostate membrane fractions and subsequent analysis for co-precipitation of RIP and FADD. At 72
hrs after castration, rat ventral prostate fas antigen mRNA
was found to be induced >5-fold compared to levels in control prostates. Coordinate upregulation of fas protein expression was confirmed by Western blots. Immunoprecipitation
of fas antigen from control prostate membranes did not coprecipitate RIP or FADD whereas these two proteins were
abundant in fas immunoprecipitates made from the membranes of 66 hr castrated ventral prostate glands.
348
Bethesda Abstracts
In conclusion, we have found that fas antigen expression
is upregulated early during castration-induced expression of
the rat ventral prostate. Coordinate with fas upregulation,
the prostate cell fas signaling pathway is activated as shown
by complexes of fas-FADD-RIP that are found on prostate
membranes only after castration. Based on these and other
results, we propose that a significant portion of prostate cell
apoptosis that occurs following castration is due to hypoxic/
ischemic-induced apoptotic cell deaths.
potential of this new strategy will be realized if a larger
number of samples are analyzed.
PSP94 (PROSTATE SECRETORY PROTEIN OF 94 AMINO ACIDS)
EXPRESSION IN PROSTATE CANCER: CORRELATION WITH
TUMOR PROGRESSION AND DIFFERENTIATION
Y. Imasato,1,3 M. Moussa,2 P. Cordeiro,2 J. Izawa,1 H. Sakai,3 J.
Xuan,1 and J. Chin1
1
SERUM BOUND FORM AND DIFFERENTIAL TEST OF PSP94
(PROSTATE SECRETORY PROTEIN OF 94 AMINO ACIDS) AS A
PROGNOSTIC MARKER FOR PROSTATE CANCER
Jim W. Xuan,1 Joseph L. Chin,1 Varagur M. Venkatesan,1 Glenn
S. Bauman,1 Susan Guo,2 and Hideki Sakai3
1
University of Western Ontario, London, ON, Canada
Procyon Biopharma Inc., London, ON, Canada
3
Nagasaki University, Nagasaki, Japan
BACKGROUND: Both prostate specific antigen (PSA)
and PSP94 are regarded as possible prostate cancer markers,
however, the role of PSP94 has been controversial. All prior
studies had been designed to test only the free form of PSP94
in serum. We have identified serum bound form of PSP94
and performed a differential test between serum free and
bound forms of PSP94 on the sera of prostate cancer patients
prior to radiotherapy.
METHODS AND RESULTS: Both native and SDSPAGE (polyacrylamide gel electrophoresis) were tested by
Western blot experiments. There was no PSP94 immunoactive signal in native gel, while strong signal were obtained in
SDS-PAGE treated by SDS-␤-mercaptoethanol. Molecular
sieve chromatography of serum sample showed one major
peak of PSP94-bound complexes (>100 kDa) located near
IgG and albumin peaks. Biotinylated PSP94 was used to
monitor the free form (<16 kDa) in the column isolation.
Two rounds of protein A column purification were used for
further purification. The bound complexes were not recognized by any polyclonal or monoclonal antibody. In 58 of 70
sera from Pca patients, bound PSP94 complexes were identified and comprise the majority of serum total PSP94. PSP94
bound forms were very stable, resistant to the treatments of
SDS, urea, guanidine hydrochloride and NaCl. We have performed a preliminary clinical test of bound form of PSP94
(bPSP94) pretreatment in the archived sera taken from 42
prostate cancer (Pca) patients with non metastatic Pca who
received radical radiotherapy. In the survival curves, we
have found serum bound (less than 0.35 ␮g/ml) and free
form (less than 8 ng/ml) of PSP94 have positive and inverse
association with the outcome separately. Patients with high
b/fPSP94 (6 or more than 6) had a poorer prognosis than
those with low b/fPSP94 (logrank test: P = 0.045; generalized Wilcoxon test: P = 0.030). The analysis was also repeated for the subgroups of 27 patients with a favorable
prostate specific antigen (PSA, <10 ng/ml) pretreatment;
higher ratios of bound to free form of serum PSP94 (b/
fPSP94) were a stronger predictor (P = 0.0074) of the relapse
post radiotherapy as compared with PSA (P = 0.070).
CONCLUSIONS: Clinical value of serum PSP94 as a
prostate marker must be re-evaluated. The ratio of b/fPSP94
may be of additional prognostic value, particularly for prostate cancer patients with lower PSA. We expect that the real
2
Department of Surgery and 2Pathology, University of Western
Ontario, Ontario, Canada
3
Department of Urology, Nagasaki University School of
Medicine, Nagasaki, Japan
INTRODUCTION: PSP94 and PSA are the two most
abundant proteins secreted by human prostate gland. Elevated serum levels of both proteins have been considered
as an indicator of prostate cancer (Pca). Tissue PSA expression, however, has shown an inverse correlation to Pca progression. Similarly, an inverse correlation of tissue PSP94
expression to Pca has been reported, but the observation was
limited only to advanced Pca patients. Thus, we examined
tissue PSP94 expression in patients with early stages of Pca
and evaluated the correlation with other pathological variables.
METERIALS AND METHODS: Thirty-three patients
who underwent prostatectomy without neoadjuvant hormonal therapy between January 1994 and December 1997
were studied. All formalin-fixed, paraffin-embedded tissues
were stained by ABC technique using a primary rabbit antibody against human PSP94 purified from semen. Tumor
foci on each slide were identified and were graded according to the scheme of Gleason. The intensity of the staining
was graded on a scale of 0 to 2+, and the extension of the
staining was classified as 0, 25, 50, 75, 100% on each focus.
Other clinical data of pretreatment serum PSA, pathological
stage and Gleason score were collected and analyzed by
chi-square test.
RESULTS: Pathological stages of 33 patients were determined: 5 stage B1, 12 stage B2, 9 stage C1 and 7 stage C2. A
total number of 56 tumor foci were identified, 10 of which
were grade 2, 31 were grade 3, 11 were grade 4 and 4 were
grade 5, respectively. Normal and hyperplastic tissues surrounding the cancer tissue showed mostly intense (2+) staining on the epithelial lines of the glands. Staining in cancer
tissues was generally heterogeneous. Positive staining was
seen in 100% of grade 2, 77% of grade 3, 18% of grade 4 and
0% of grade 5, respectively. Positive staining was seen in
79% of cases (26 of 33) and in 66% of foci (37 of 56) respectively, and was significantly higher than previously reported
in advanced Pca cases (7.7–38.5%). Intensity of tissue PSP94
expression showed positive correlation with its extension (P
< 0.001). Intensity and extension of the staining on tumor
foci all showed an inverse correlation with Gleason grade (P
< 0.001). All grade 2 tumors showed strongly positive (2+)
and the grade 5 tumors were negative. However intensity of
the staining did not show significant correlation with stage
(P = 0.199) and serum PSA (P = 0.490). Pretreatment serum
PSA did not show any significant correlation (P > 0.40) with
the other variables.
CONCLUSIONS: PSP94 expression in prostate tissue has
an inverse correlation with the tumor grade. Tissue PSP94
may be a useful marker to assess the biological potential of
the tumor.
Bethesda Abstracts
EXPRESSION OF AMINOPEPTIDASE N IN HUMAN PROSTATE
CANCER
Y. Sugimura,1 T. Hioki,1 Y. Yamada,1 M. Fumino,1 S. Yoshida,1
M. Tatematsu,1 K. Ishii,2 and K. Hirano2
1
Department of Urology and Pathology, Aichi Cancer Center,
Nagoya, Japan
2
Laboratory of Pharmaceutics, Gifu Pharmaceutical University,
Gifu, Japan
The novel peptide hydrolysis activity was found in human prostate tissues. This hydrolysis activity was identified
to aminopeptidase N (CD13). The expression of aminopeptidase N in normal and abnormal prostate gland was immunohistochemically studied. Human prostate tissues obtained
chromatographies, and digested by V8 protease to determine the N-terminal amino acid sequence. The polyclonal
antibody was made to use immunohistochemistry. The expression of aminopeptidase N was compared with that of
prostate specific antigen (PSA) using immunoperoxidase assay on formalin-fixed, paraffin-embedded tissue sections of
normal and cancerous prostate specimens, which were obtained from radical prostatectomy of 5 patients.
Aminopeptidase N was expressed in the normal or hyperplastic cells, but more restricted to carcinoma cells. PSA
expression was predominantly cytoplasmic domain of epithelial cells, and aminopeptidase N showed luminal membrane staining of prostate gland epithelia. Aminopeptidase
N (AP-N) is not prostate specific and localized in small intestine, renal tubules, membranes of central nervous system,
and on the surface of monocytes and gulanurocytes. Recent
papers indicate that aminopeptidase N activity may possibly
contribute to the tumor cell growth and tumor metastasis.
The staining pattern of aminopeptidase N seems close to
that of prostate-specific membrane antigen (PSMA). Further
study is required to investigate the biological role of aminopeptidase N and the correlation between the expression of
aminopeptidase N and PSMA in prostate cancer.
349
the status of NKX3.1 expression in primary prostate cancer
specimens.
METHODS: NKX3.1 expression was analyzed by RTPCR in carefully microdissected primary tumors and normal
tissues derived from radical prostatectomy from 53 prostate
cancer patients. The expression of cytokeratin 18 as a marker
for prostatic epithelial cells and androgen receptor (AR) was
similarly analyzed by RT-PCR. Identity of AR and NKX3.1
was confirmed by DNA sequencing of randomly selected
PCR products.
RESULTS: A complex pattern of NKX3.1 expression was
noted with 94% of normal and 96% of tumor samples exhibiting some level of expression. Comparison of NKX3.1 expression between microdissected normal and tumor tissues
revealed an increased expression in 32% tumor specimens
(17 of 53), decreased expression in 21% specimens (11 of 53)
and no change in 47% specimens (25 of 53). When these
expression patterns were stratified by pathologic stage T2
and stage T3 disease, a higher percentage of patients exhibited NKX3.1 overexpression in stage T3 (40%) vs. T2 (22%)
disease (P < 0.05). Tumor associated decrease in expression
of NKX3.1 was notably similar in stage T2 and stage T3
disease. The NKX3.1 expression was observed correlating
with the AR expression (P < 0.01).
CONCLUSIONS: Increased levels of the androgen regulated NKX3.1 transcripts are associated with non-organ confined (Stage T3) disease and may serve as an indicator for a
more aggressive CaP phenotype. Further molecular characterization of NKX3.1 transcripts in CaP is warranted.
DETECTION OF PSA-EXPRESSING PROSTATIC CELLS IN BONE
MARROW OF RADICAL PROSTATECTOMY PATIENTS BY
SENSITIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN
REACTION (RT-PCR)
Chun-Ling Gao,1 Robert Dean,2 Angela Pinto,2 Renee
Mooneyhan,2 David G. McLeod,2 Shiv Srivastava,1 and Judd W.
Moul1,2
1
EVALUATION OF EXPRESSION OF AN ANDROGEN REGULATED
PROSTATE-SPECIFIC HOMEBOX GENE, NKX3.1, IN HUMAN
PROSTATE CANCER
Linda L. Xu,1 Vasantha Srikantan,1 Isabell A. Sesterhenn,2
Meena Augustus,1 David Sui,1 Judd W. Moul,1,3 Kenneth C.
Carter,4 and Shiv Srivastava1
1
Center for Prostate Disease Research, Department of Surgery,
Uniformed Services University of Health Sciences,
Bethedsa, MD
2
Department of GU Pathology, Armed Forces Institute of
Pathology, Washington DC
3
Urology Service, Walter Reed Army Medical Center,
Washington, DC
4
Human Genome Sciences, Inc., Rockville, MD
BACKGROUND: NKX3.1 is a newly discovered prostate
specific androgen regulated gene which belongs to homeobox gene family. Chromosomal localization of NKX3.1
on 8p21, a region frequently deleted in prostate cancer (CaP)
and suspected to harbor a tumor suppressor gene has
prompted further investigation to evaluate the possibility of
NKX3.1 being a candidate tumor suppressor gene involved
in prostate tumorigenesis. In this report we have evaluated
Center for Prostate Disease Research, Department of Surgery,
USUHS, Bethesda, MD
2
Urology Service, Walter Reed Army Medical Center,
Washington, DC
INTRODUCTION AND OBJECTIVES: The RT-PCR assay for prostate-specific antigen (PSA) expressing cells in the
blood circulation has been under intense investigation since
1992. Although early reports suggested that this technology
could be used as ‘‘molecular staging’’ for occult prostatic
hematogenous metastases, later studies, including an investigation from our group, were unable to confirm RT-PCRPSA of peripheral blood to predict stage or recurrence in
radical prostatectomy (RP) patients. To date, only two studies have been performed with bone marrow and both suggested good correlation with stage. The goal of this study
was to perform bone marrow RT-PCR-PSA on a large cohort
of RP patients with pathologic stage correlation.
MATERIALS AND METHODS: Unilateral anterior iliac
crest bone marrow aspirates were performed on 106 patients
immediately prior to incision for RP. RP specimens were
processed as whole-mounts. A sensitive nested RT-PCR assay with specific primers derived from the prostate specific
350
Bethesda Abstracts
antigen (PSA) sequence was used. This method enabled us
to detect PSA-expressing prostate LNCaP cells as low as one
cancer cell from ten million lymphocytes (1/107). Three RTPCR-PSA reactions were performed on all patients and at
least two positive tests were required to define positivity.
The results were also evaluated on the basis of any positivity.
RESULTS: RT-PCR-PSA assay detected PSA-expressing
cells in 50 of 106 (47.2%). In 10 randomly selected cases, the
RT-PCR product was confirmed as PSA by DNA sequencing. Twenty-six of 55 (47.3%) patients with non-organ confined disease were positive in BM RT-PCR-PSA as compared
to 24 of 51 (49.0%) patients with organ confined disease.
Furthermore, BM-RT-PCR-PSA was not associated age,
grade, pre-treatment PSA value, clinical stage, or margin
studies.
CONCLUSION: BM-RT-PCR-PSA of 106 prostate cancer
patients did not correlate to pathological stage, age, serum
PSA or margin positivity. Further studies are needed to
evaluate the prognostic value of RT-PCR-PSA assay in bone
marrow of patients undergoing radical prostatectomy.
EVALUATION OF THE MECHANISMS OF ANTI-TUMORIGENIC
EFFECTS OF THE ADENOVIRUS wt p53 ON DU145 CELLS DERIVED
TUMORS IN NUDE MICE
Kekule Asgari,1 Linda Xu,1 Shanmugam Naga,1 Isabell A.
Sesterhenn,3 David G. McLeod,2 Prem Seth,4 Judd W. Moul,1,2
and Shiv Srivastava1
1
Center for Prostate Disease Research, Department of Surgery,
Uniformed Services University of the Health Sciences,
Bethesda, MD
2
Urology Service, Walter Reed Army Medical Center,
Washington, DC
3
Armed Forces Institute of Pathology, Washington, DC
4
Medical Breast Section, Medical Branch, National Cancer
Institute, National Institutes of Health, Bethesda, MD
OBJECTIVE: Three independent experiments involving
6–7 months follow up following single intratumoral injections of the AdWTp53 have yielded similar tumor growth
inhibitory effects on prostate cancer cell derived tumors in
nude mice. In an attempt to better understand the long-term
anti-tumorigenic effects of the AdWTp53 vector, we are
carefully evaluating (1) the efficiencies of adenovirus infection by intratumoral injections of the adenovirusbetagalactosidase (Ad-␤gal) vector, (2) in vivo expression of
wt p53 and its effect of cell proliferation, (3) p53 expression
regulated genes that may have field effects, e.g., thrombospondin and (4) activation of immune functions, e.g., NK cell
activation.
METHODS: Three weeks after cell inoculation, tumors
were 4–5 mm in diameter and twenty mice in each group
were treated with only one intratumoral injection of AdWTp53 (2.0 × 109 pfu) or control virus (d1312) (2.0 × 109 pfu).
Tumor volumes were measured once a week for twenty-five
weeks. In separate experiments, 4–5 mm tumor nodules
were injected with AdWTp53, d1312 or Ad-␤gal vectors and
tissues were harvested at 48 and 144 hours post injections.
The tissues were embedded in OCT and ten of 8 ␮ thick
serial sections from each specimen were analyzed for in situ
␤gal activity, immunostaining of p53 and cell proliferation
by bromodeoxyuridine staining.
RESULTS: After twenty-five weeks follow up, significant
tumor suppressing effects (70–80%) via single injections of
the ADWTp53 vector into the tumors have been confirmed.
A quantitative evaluation of the in situ ␤gal staining revealed an average of 3–5% ␤gal positive cells per tissue section. Detectable p53 expression was noted in injected area
around needle tracks which correlated with DNA synthesis
inhibition as measured by in situ BrdU assay. Higher percentage ␤gal or p53 positive cells were noted at 48 hours
post injection as compared to 144 hours post injection. Additionally, when compared to d1312 treated tumors, TGFbeta, thrombospondin expression and lymphocytic infiltration. NK cell activation was not altered in AdWTp53 treated
tumors.
CONCLUSIONS: AdWTp53 infected cells exhibited inhibition of DNA synthesis. Expression of AdWTp53 only in
a small fraction of tumor cells in vivo warrants further investigation to understand field/bystander effects of the
AdWTp53 vector.
CHARACTERIZATION OF PROSTATE CANCER CELLS
IMMORTALIZED WITH E6/E7 GENES
Leland D. Davis,1,2 Isabell Sesterhenn,3 Judd Moul,1,4 and Shiv
Srivastava1
1
Center for Prostate Disease Research, Depart. of Surgery,
USUHS, Bethesda, MD
2
Molecular and Cell Biology Program, USUHS, Bethesda, MD
3
Genitourinary Pathology, Armed Forces Institute of Pathology,
Washington, DC
4
Urology Service, Walter Reed Army Medical Center,
Washington, DC
RATIONALE: There are only a few well-characterized
prostate cancer cell lines, most of which are from metastatic
tumors. Metastatic tumors typically have many complex
biological and molecular alterations resulting from in vivo
selection pressures imposed in the evolution of the metastatic phenotype. The role of multiple genetic alterations associated with metastatic cancer cells makes it difficult to
establish the causal relationship of these gene alterations to
prostate tumor development. To study early genetic lesions
of prostate cancer, cell lines derived from primary tumors
are needed. These cell lines should have fewer, more informative alterations. Androgen regulation of prostate growth
as well as the widely used androgen deprivation therapy for
prostate cancer treatment necessitates a better understanding of the role of androgen in prostate cancer biology. Only
one well-characterized prostate cancer cell line (LNCaP) is
known to express androgen receptor (AR), but it is a mutant
receptor. Therefore, development of primary prostate tumor
derived cell lines will have significant impact in studying
both early genetic alterations and in evaluating the role of
androgen signaling pathways in prostate cancer.
METHODS: Biopsies from palpable tumors of radical
prostatectomy specimens were used for primary culture development. Minced pieces of tissue were distributed to several collagen coated cell culture dishes with MEM based
media supplemented with 10% fetal bovine serum and
growth factors, or keratinocyte serum free medium (Life
Technologies) supplemented with 5% serum. Tissue explants were grown for three or four weeks and passaged at
a split ratio of 1:3. Primary prostate cultures were infected
with a replication deficient retroviral construct,
LXSN16E6E7 (kindly provided by Denise A. Galloway),
Bethesda Abstracts
which contains the genes for human papilloma virus 16 E6
and E7 and a neomycin resistance gene. Infected cells were
selected in the media containing geneticin for four weeks
and further passaged in the same media.
RESULTS: We obtained four immortalized cultures,
CPDR1 through 4, from four different patients out of twelve
immortalization experiments. These cultures exhibit continued growth, in one case beyond ten passages. Primary cultures generally became senescent, and went in crisis beyond
the third passage. All of these immortalized cultures show
an epithelial morphology in culture. To further confirm the
epithelial nature of these cells, RT-PCR was done on these
cells showing a consistent expression of cytokeratin 18 and
19, which are characteristic of prostatic epithelial cells. AR
expression was also evaluated by RT-PCR. The primary cultures giving rise to CPDR2 and 3 expressed AR. However, it
was CPDR2 that continued to express androgen receptor
even after the immortalization. CPDR1 and 4 did not express
AR. The immortalized cells are further being analyzed for
expression of PSA, NKX3.1, V5 and HPRAS, and for 8p21
lesions.
CONCLUSIONS: Immortalization with the E6/E7 retroviral construct constitutes an efficient means of producing
immortal cell cultures of primary prostate cancer which
should aid the study of early stage cancer. We have obtained
a cell line, CPDR2, which expresses AR. The androgen response properties of this cell line should help approach
questions related to androgen regulation of prostate cells.
LANDMARKING THE PROSTATE GENOME BY SPECTRAL
KARYOTYPING (SKY) AND COMPARATIVE GENOMIC
HYBRIDIZATION (CGH)
Meena Augustus,1,2 Evelin Schröck,2 Leland Davis,1 Kerstin
Heselmeyer,2 Judd Moul,1 Shiv Srivastava,1 and Thomas Ried2
1
Center for Prostate Disease Research, Dept. of Surgery,
USUHS, and 2Genome Technology Branch, National Human
Genome Research Institute-NIH, Bethesda, MD
INTRODUCTION AND OBJECTIVES: Altered cell
growth and differentiation and a highly abnormal karyotype
are accepted hallmarks of tumorigenic conversion and progression of several human cancers. However, genetic
changes and master control genes, crucial for the initiation
and progression of human prostate cancer (CaP), need to be
better defined. Spectral karyotyping (SKY) and comparative
genomic hybridization (CGH) are two very powerful molecular cytogenetic methodologies that have independently
proven to be successful as whole-genome screening tools.
Recent CGH studies in prostate cancer have helped identify
some chromosomal loci relevant to prostate tumorigenesis.
A direct visualization of these genetic changes by karyotyping has unfortunately been compounded by pitfalls in tumor
cell-culture and limitations imposed by conventional Gband-based cytogenetic analysis. SKY, on the other hand,
uses combinatorial multi-color fluorescence in-situ hybridization (FISH) to classify nearly every chromosomal abnormality, with accuracy. To date, there are no SKY analyses of
prostate cancer cells. Used alone and in combination with
CGH, this technique has tremendous potential to identify
novel prostate specific genetic alterations with enhanced
resolution and precision. As a first step in this direction, SKY
and CGH have been applied to characterize the three most
commonly studied CaP cell-lines, LNCaP, PC3 and DU145,
and an immortalized primary CaP cell-line, CPDR-1.
351
METHODS: For the CGH analysis, genomic DNAs extracted from cultures of LNCaP, PC3, DU 145 and CPDR-1
were used as the biotin-labeled ‘‘test’’ DNA and cohybridized with digoxygenin-labeled normal ‘‘reference’’
male DNA on target, normal, male, metaphase chromosomes. Fluorescein iso-thiocyanate (FITC) and tetra-methyl
rhodamine iso-thiocyanate (TRITC) were used for detection
of the test and reference DNAs, respectively. Ratio profiles
of fluorescent intensities along all the chromosomes were
obtained using digital imaging and analysis (Leica Q-FISH
image acquisition system and Applied Imaging Analysis
software). For SKY, metaphase chromosome preparations
from the same cultures were used as the target chromosomes that were hybridized with a kit containing 24 differentially labeled fluorescent whole human chromosome paint
probes. Metaphases were counter-stained with 4⬘, 6-diamidine-2⬘-phenylindole dihydrochloride (DAPI). Image acquisition and analysis were done using the Spectral Imaging
and SkyView software (Applied Spectral Imaging, Carlsbad,
CA). SKY-karyotypes were analyzed using the spectral/
classification colors and the inverted DAPI images (that resemble the conventional G-banding pattern of human chromosomes).
RESULTS: SKY analysis allowed the direct visualization
of gross and subtle chromosome alterations and aberrations
with exquisite cytogenetic detail hitherto not revealed by
conventional G-banding techniques. CGH helped confirm
the involvement of some specific chromosomes/regions/
aberrations in the relatively straightforward and less complex primary cell-line CPDR-1, but could barely reflect the
karyotypic complexity of the grossly rearranged metastatic
cells of PC3, DU145 and LNCaP (in that order of reducing
complexity).
CONCLUSIONS: With the advent and widespread application of SKY as a novel and successful molecular cytogenetic FISH technology, it is inevitable and more than
likely that tumor cytogenetic data, as it exists today, will
have to be re-evaluated. The point in case is amply confirmed by our preliminary prostate cancer cytogenetic
analyses. Needless to say that the amazing array of chromosome hot-spots and underlying prospect of candidate cancer
genes revealed by SKY will not only help in the molecular
definition of prostate tumorigenesis, but will lend that critical burst of speed needed in the search for genes for cancer
prevention, early detection, rationalized, well-tolerated
treatment and successful cures.
Fgf10 EXPRESSION IN PROSTATIC DEVELOPMENT AND DISEASE
Axel A. Thomson and Gerald R. Cunha
Department of Anatomy, University of California San
Francisco, San Francisco, CA 94143-0452
Development of the prostate and other male sex accessory organs is dependent upon androgens and mesenchymal/epithelial interactions. We have identified fibroblast
growth factor 10 (Fgf10) as a likely paracrine regulator of the
urogenital epithelia during the development of the prostate
and seminal vesicle. Also, Fgf10 mRNA was expressed in
benign prostatic hyperplasia (BPH) surgical specimens. In
situ hybridization of embryonic urogenital sinus showed
that Fgf10 mRNA was present in a subset of mesenchymal
cells overlying the areas where the prostate and the seminal
vesicle form, but was not expressed in epithelial cells. In the
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Bethesda Abstracts
neonatal rat ventral prostate (VP) Fgf10 mRNA was expressed in mesenchyme surrounding distal ductal tips undergoing branching morphogenesis, and was absent in mesenchyme surrounding proximal ducts where ductal growth
was minimal. In the neonatal rat seminal vesicle (SV) Fgf10
mRNA was highest in mesenchyme subadjacent to the serosa (the periphery of the organ), the zone into which the
epithelium grows.
RNase protection assays were used to quantitate levels of
Fgf10 transcripts. Fgf10 mRNA levels were high in both SV
and VP during neonatal development, declined around puberty, and were very low or absent in adult tissues (where
growth is minimal). To determine if Fgf10 was regulated by
androgens Fgf10 transcripts were meaured in neonatal SVs
and VPs grown in vitro. Fgf10 mRNA levels showed a fourfold increase in Sv and twofold increase in VP, when grown
in the presence of testosterone for five days, compared to
untreated organs. However, if organs were grown with testosterone for four days and then treated with anti-androgen
for one day there was no decrease in Fgf10 mRNA levels.
Therefore, we concluded that Fgf10 mRNA was not directly
regulated by androgens.
To determine if Fgf10 might be involved in human BPH,
RNA from transurethral prostatic resection (TURP) samples
was analyzed by RNase protection assay. Fgf10 mRNA was
present in 60% (n = 10) of BPH samples. When a number of
individual TURP chips from the same patient were analyzed, different levels of Fgf10 mRNA were observed. However, since the samples may have contained areas of normal
prostate and may have had different stromal/epithelial ratios it was difficult to interpret differences in Fgf10 levels
from the BPH samples.
A PHASE II TRIAL OF GALLIUM NITRATE IN PATIENTS WITH
ANDROGEN-INDEPENDENT METASTATIC PROSTATE CANCER
J. Horti, A.J. Senderowicz, S.C. Dixon, R. Reid, D. Headlee,
W.D. Figg, and E. Sausville
Medicine Branch, Division of Clinical Sciences, NCI, NIH,
Bethesda, MD 20852
Due to in vitro and clinical data suggesting antitumor
activity, we sought to evaluate the safety and activity of
gallium nitrate in patients with androgen-independent prostate cancer.
METHOD: Eligibility criteria included the presence of
either measurable metastatic lesions or evaluable disease (elevation of PSA), disease progression following combined
androgen ablation and failed antiandrogen withdrawal, an
ECOG performance status of >2, and adequate bone marrow
and renal function. Therapy consisted of gallium nitrate 200
mg/m2/day as a continuous infusion for 7 days, administered every 21 days, with hydration (100 ml/m2/hr). Individuals that had previously received suramin were treated
at a dose of 150 mg/m2/day of gallium nitrate.
RESULTS: A total of eight patients were enrolled: four
patients at 200 mg/m2/day and four patients at the lower
dosage. One out of eight patients had a greater than 75%
decline in PSA that lasted for four months. Three patients
were noted to have stable disease with a median duration of
5 months (range 3–5 months). In two cases, we observed
grade 4 toxicities. One patient developed a sudden vision
loss and another had pulmonary interstitial infiltrates. The
patient with optic neuropathy had a lower serum gallium
nitrate level compared to those individuals that had their
pharmacokinetics determined. Anemia was the most common side effect; all patients had blood transfusions and five
of eight patients developed grade 3 anemia.
CONCLUSION: This trial was prematurely terminated
because repeat administration of gallium nitrate was poorly
tolerated in an elderly population with androgenindependent prostate cancer. In addition, gallium nitrate
may have limited clinical activity in this disease. In vitro
data suggest that PSA is down-regulated following gallium
nitrate exposure.
ANDROGENS INCREASE THE PRODUCTION AND SECRETION OF
VASCULAR ENDOTHELIAL CELL GROWTH FACTOR BY HUMAN
PROSTATE FETAL FIBROBLASTS
Alexander Kirschenbaum, Xin-Hua Liu, Pietra D. Greenberg,
Stuart A. Aaronson, James F. Holland, and Alice C. Levine
Departments of Medicine (Endocrinology) and Urology, and
Derald H. Ruttenberg Cancer Center, Mount Sinai School of
Medicine, New York, NY 10029
Introduction and Objectives: Human fetal prostate development is known to be dependent upon androgens and
stromal-epithelial interactions. Vascular endothelial cell
growth factor (VEGF) is an endothelial cell-specific mitogen
which is critical to normal vasculogenesis. We determined
that cell-specific expression of VEGF in the human fetal
prostate and studied its androgenic regulation in prostate
fetal fibroblasts.
METHODS: Fetal prostates were derived from 17–22
week gestation fetuses. Immunohistochemical staining for
VEGF expression was carried out on formalin fixed tissues.
Human prostate fetal fibroblasts were grown in culture ± the
addition of dihydrotestosterone (DHT 10−10–10−6 M) and
VEGF mRNA and protein levels determined by Northern
blot and Western blot, respectively. VEGF mRNA transcription rates in the fibroblasts ± DHT were determined by
nuclear run-on assay.
RESULTS: Immunohistochemical studies localized
VEGF protein expression to the stromal cells of the developing prostate. DHT upregulated VEGF mRNA and both
the cell-associated and secreted isoforms of VEGF within
12–24 hours. Maximal upregulation (>2-fold) of VEGF expression was demonstrated with DHT 10−8 M. Nuclear runon assay demonstrated that the increase in VEGF expression
was at least partially due to increased VEGF mRNA transcription which was maximal 2 hrs after DHT addition.
CONCLUSIONS: VEGF is expressed by the stromal cells
of the human fetal prostate VEGF expression by human
prostate fetal fibroblasts is under androgenic regulation. Androgens influence prostate development via effects on vasculogenesis.
SOURCE OF FUNDING: T.J. Martell Foundation for
Leukemia, Cancer and AIDS Research.
IGFBP-3 MEDIATES TNF-␣ INDUCED APOPTOSIS IN THE
PROSTATE CANCER CELL LINE, PC-3: ROLE OF bcl-2
PHOSPHORYLATION
Roopmathy Rajah and Pinchas Cohen
Bethesda Abstracts
Department of Pediatrics, University of Pennsylvania,
Philadelphia, PA 19104
Insulin-like growth factor (IGF) binding protein-3
(IGFBP-3) is known to block IGF action and inhibit cell
growth. IGFBP-3 is thought to act by sequestering free IGFs
or, possibly, via a novel IGF-independent mechanism. Recently, we demonstrated the apoptosis inducing effects of
IGFBP-3 in the prostate cancer cell line, PC-3 and showed
that this effect is, in part, IGf dependent. We also showed
that IGFBP-3 induces apoptosis through a caspase cascade.
Supporting its role as a primary growth inhibitor, IGFBP-3
production has been shown to be increased by cell growthinhibitory agents, such as transforming growth factor-beta
(TGF-␤), tumor necrosis factor-alpha (TNF-␣), and the tumor suppressor gene p53. We have previously shown that
TGF-␤ induces apoptosis in the p53-negative cell line, PC-3,
and that this effect is mediated through IGFBP-3. In this
communication, we demonstrate the role of IGFBP-3 as a
mediator of apoptosis induced by TNF-␣, and show that this
process involves bcl-2 phosphorylation. Addition of recombinant IGFBP-3 to PC-3 cells resulted in a dose dependent
induction of apoptosis. Treatment with TNF-␣, a known apoptosis-inducing agent, resulted in the induction of IGFBP-3
mRNA and peptide expression followed by induction of apoptosis. The apoptosis inducing effect of TNF-␣ was prevented by co-treatment with IGFBP-3 neutralizing antibodies or IGFBP-3 specific antisense thiolated-oligonucleotides.
Both IGFBP-3 and TNF-␣ treatment increased the inactive
phosphorylated form of bcl-2 (a well recognized inhibitor of
apoptotic cell death) in PC-3 cell lysates. In addition, immunodetection with anti-phosphoserine antibodies using bcl-2
immunoprecipitated cell lysates confirmed that bcl-2 is specifically phosphorylated at the serine residues after IGFBP-3
treatment. The effect of TNF-␣ on bcl-2 serine-phosphorylation was blocked by IGFBP-3 neutralizing antibodies as
well as by IGFBP-3 antisense oligomer. These findings confirm that IGFBP-3 not only induces apoptosis through a
novel pathway independent of p53 but is also necessary for
TNF-␣ induced apoptosis in PC-3 cells and this effect may be
mediated by the inactivation of bcl-2 through serine phosphorylation.
INDUCTION OF HUMAN PROSTATE CANCER DIFFERENTIATION
BY VITAMIN D ANALOGUES IS ATTENUATED BY BONE STROMAL
AND EXTRACELLULAR MATRIX CELLS
Mitchell H. Sokoloff,1 Thomas A. Gardner,1 Timothy H.
Caven,1 Alex Wood,2 and Leland W.K. Chung1
1
Department of Urology, University of Virginia, Charlottesville,
Virginia
2
Hoffman-La Roche, Inc. Nutley, NJ
Most men diagnosed with prostate cancer will eventually
develop osteoblastic metastases. Such lesions cause severe
pain, bone fractures, and spinal cord compression, all of
which result in significant disability, discomfort, and distress. Unfortunately, few treatment options exist for men
with advanced disease, and none offer more than palliation.
Vitamin-D is intimately associated with skeletal growth and
development. Furthermore, population-based studies suggest that increased vitamin-D levels may prevent prostate
cancer development. Due to its ability to inhibit prostate
tumor cell growth and predispose prostate cells towards
terminal differentiation, vitamin-D is currently under investigation as a putative anti-prostate cancer therapy.
353
The effects of prostate cancer cell proliferation in vitro by
vitamin-D and twelve functional analogues have been assessed in our laboratory. Each of the vitamin-D analogues
demonstrated dose-dependent inhibition of LNCaP and lineage-derived C4-2 prostate cancer cell growth, although differences in the potency of growth inhibition between the
analogues were noted. Vitamin-D analogues also promoted
differentiation of the prostate cancer cells, and increased
PSA synthesis and secretion. The increases in cellular PSA
production were similar in all twelve treatment groups, indicating that anti-proliferative and cell differentiation functions of the analogues can be successfully separated.
We have also investigated whether the presence of bone
stromal elements can influence the activity of vitamin-D on
prostate cancer cell lines in vitro by co-culturing LNCaP or
lineage-derived C4-2 cells with and without bone stromal
cells derived from an osteosarcoma patient. Compared to
controls, the presence of stromal cells enhanced the susceptibility of prostate cancer cells to the growth inhibiting effect
of vitamin-D when regular uncoated plastic plates were
used. When these chimeric tumor cells were cultured on
collagen gel, the growth inhibitory effect of vitamin-D was
still enhanced by the presence of stromal cells, albeit less
than when on plastic plates alone. However, when the chimeric prostate cancer and bone stromal cells were cultured
together in collagen gel, the presence of the stromal cells
inhibited the actions of vitamin-D. This was demonstrated
by using the cluster-forming ability of prostate tumor cells
on collagen as a measure of tumorigenicity. When C4-2 cells
were grown alone, cluster-formation (which correlates
closely with tumorigenicity) decreased upon the addition to
increasing concentrations of vitamin-D. However, when
C4-2 cells were grown in the presence of bone stroma, their
cluster forming-response to vitamin-D was only marginally
affected, suggesting that bone stromal cells attenuated the
therapeutic responses of androgen-independent prostate
cancer cells toward vitamin-D in vitro.
These results demonstrated that the in vitro effects of vitamin-D analogues need to be carefully evaluated as such
effects may be influenced greatly by the cell culture conditions. Interestingly, co-culture of prostate cancer with bone
stromal cells in collagen gels appears to be least sensitive to
vitamin-D analogue-induced growth inhibition and differentiation. This underscores the importance of establishing
relevant in vivo models for evaluating the efficacy of vitamin-D analogues on prostate cancer growth and progression. Such in vivo studies have recently been initiated by us
in both a subcutaneous C4-2 prostate cancer model and a
novel subcutaneous osteoblastic prostate tumor model. Finally, our results substantiate that PSA is a poor surrogate
marker to measure responsiveness to vitamin-D, and that
novel biomarkers are needed to monitor therapeutic outcomes.
IDENTIFICATION OF POSITIVE AND NEGATIVE REGULATOR
ELEMENTS FOR THE TISSUE INHIBITOR OF METALLOPROTEINASE
1 GENE
Min Wang, Youji Hu, Ishiro Shima, and Mark E. Stearns
Department of Pathology, Allegheny University,
Philadelphia, PA
We have identified an IL-10 inducible enhancer (HTE)
(5⬘-CACGATGACTCATCACTGTTGAAAGACA-3⬘) (−864 to
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Bethesda Abstracts
−836 bp) and associated silencer element (HTS) (5⬘-CCACTGGCCCATCGTATAT-3⬘) (−1284 to −1266 bp) in the 5⬘ promoter region of the human tissue inhibitor of metalloproteinase-1 (TIMP-1) gene. Chloramphenicol acetyl transferase
(CAT), electrophoretic migration shift assays (EMSAs) and
DNase footprinting revealed that IL-10 (15 ng/ml for 1–2 hr)
induced the HTE enhancer. In comparison, 100 nM phorbol
ester stimulated the HTS silencer and blocked IL-10’s effects
in a dose-dependent, orientation- and position-independent fashion, suggesting that HTS is a true silencer element.
EMSAs combined with deletion and mutation analysis of the
THE and HTS elements confirmed these observation. Finally, the Northern blot, Western blot, immunoprecipitation
and ELISA analysis showed that IL-10 (15 ng/ml) induced
TIMP-1 expression (∼10 fold by 18 hr), whereas PMA (100
ng/ml) inhibited the stimulatory effects of IL-10 on TIMP-1
expression. Invasion assays with Matrigel coated Boyden
chambers revealed that IL-10 blocked PC-3 ML tumor cell
invasion by up regulating TIMP-1. Ten nM PMA also inhibited invasion, whereas 100 nM PMA stimulated invasion
and blocked IL-10 stimulated increases in TIMP-1 expression. Tumor metastasis studies in SCID mice further showed
that IL-10 blocked metastasis of the PC-3 ML cells, whereas
high PMA stimulated metastasis in the presence of IL-10.
The data indicate that HTE and HTS function as positive and
negative regulatory elements which control human TIMP-1
expression to directly influence the invasive and metastatic
behavior of PC-3 ml tumor cells.
Supported by grant no. CA 75122 to MES.
EXPRESSION OF ANDROGEN METABOLIZING ENZYMES IN
BENIGN PROSTATIC HYPERPLASIA (BPH) AND IN PRIMARY
CULTURES OF EPITHELIAL AND STROMAL BPH CELLS
P. Negri-Cesi,1 A. Colciago,1 A. Poletti,1 M. Andres,2 V.
Franchini,2 and M. Motta1
1
Center for Endocrinological Oncology, Department of
Endocrinology, University of Milan, and 2Division of Urology,
Hospital S. Pio X, Milan, Italy
To better understand the etiology and the maintenance of
human prostatic benign hyperplasia (BPH), increasing attention has been focused on the cellular distribution of the enzymes which form active metabolites from testosterone (T),
and on the interactions between stroma and epithelium in
the control of their expression. T might be transformed either to 5␣-dihydrotestosterone (DHT) or to estradiol
through the action of the 5␣-reductase (5␣-R) and the aromatase (Aro) respectively. Two isoforms of the human 5␣-R
(type 1 and type 2) with different biochemical properties,
tissue localization and probably different physiological
functions have been identified. In particular, the 5␣-R1, less
specific for androgen-dependent structures and present
mainly in liver, has been demonstrated in our laboratory to
be also present in BPH, where it seems to be confined to the
epithelial compartment. On the contrary, the highly specific,
5␣-R2 isozyme, typical of the androgen-dependent structures, is particularly concentrated in BPH stroma. The possible differential expression of the two isoforms in relation to
BPH is not well clarified. It is known that estrogens might be
involved in the pathogenesis and progression of BPH, since
estrogen receptors are expressed in this disease; however,
their intracellular formation from T via Aro, and its relative
expression in the stroma and/or in the epithelium of BPH,
are still controversial. Primary cultures of either epithelial or
stromal cells derived from BPH specimens were prepared by
centrifugation on Ficoll gradient and maintained in cellspecific growth media. The cultures were evaluated by
phase contrast microscopy and characterized by immunocytochemical staining with monoclonal antibodies specific either for epithelial cells (cytokeratine) or fibroblasts (␣-actin).
Both cell components had very little or no contaminants,
indicating a high level of purity. Total RNA extracted from
cultured epithelial or stromal cells was then used to evaluate, by RT-PCR and Southern analysis, the expression of the
5␣-R isoforms and of Aro; RNA obtained from whole BPH
tissue was also used. 5␣-R1 is present in BPH tissue as well
as in both prostatic cell cultures; however, in contrast with
the epithelial localization in the whole tissue, its expression
seems to be higher in the BPH stromal then in the epithelial
cultured cells. The 5␣-R2, which is highly present, in the
stromal compartment of BPH tissue, is poorly expressed in
cultured stromal cells, and it is absent in the epithelium.
Finally Aro, which is absent in the whole BPH, and in cultured epithelial cells, appears to be highly expressed in the
cultured BPH stromal cells. Altogether, these results indicate
a difference in the expression of the androgen metabolizing
enzymes between BPH tissue and primary cultures of BPH
cells. It is possible to hypothesize that the expression of 5␣Rs and Aro in one compartment is controlled by paracrine
factors produced by the other compartment; however, one
cannot disregard the possibility that the observed differences in the expression of these enzymes might be related to
a phenomenon of dedifferentiation which seems to occur in
cultured prostatic cells. Supported by AIRC, MURST and
CNR (ARCO #96.00594.PF39).
AGONISTIC ACTION OF BICALUTAMIDE (CASODEX) IN A NEW
LNCaP SUBLINE OBTAINED AFTER LONG-TERM ANDROGEN
ABLATION
Zoran Culig,1 Jans Hoffmann,2 Iris E. Eder,1 Claudia Nessler,1
Martin Erdel,2 Alfred Hobisch,1 Gerd Utermann,2 Georg
Bartsch,2 Karsten Parczyk,3 and Helmut Klocker
1
Departments of 2Urology and 2Medical Biology and Human
Genetics, University of Innsbruck, Austria
3
Schering AG, Experimental Oncology, Berlin, Germany
Prostate cancer cells may adapt to an androgen-depleted
environment by utilizing a hyperreactive androgen receptor
(AR). We report on a new in vitro model which closely mimics the situation in patients who receive androgen ablation
therapy for carcinoma of the prostate. In order to simulate
long-term androgen ablation, LNCaP cells were grown in
steroid-depleted medium for one and a half years. This selection process established a subline LNCaP-hr (hyperreactive) which shows changes in the androgen response pattern
and the transcriptional AR activity. These cells were compared to parental LNCaP cells which were maintained in
unstripped serum. In passages around 70, LNCaP-hr cells
were stimulated by very low concentrations (0.001 nM) of
the synthetic androgens methyltrienolone and mibolerone.
In latter passages (ⱖ100), however, androgens did not exert
any proliferative effect. Surprisingly, in contrast to the inhibitory effects in parental LNCaP cells, the nonsteroidal
antiandrogen bicalutamide stimulated the proliferation of
LNCaP-hr cells both in the absence or presence of androgen.
For measurement of AR transcriptional activity, the cells
Bethesda Abstracts
were transiently transfected with an androgen-responsive
reporter gene. Reporter gene (CAT) activity was measured
after 24 hours of incubation with androgens and/or AR antagonists. In the absence of any hormone, the reporter gene
activity in LNCaP-hr cells was 10-fold higher than the reporter gene activity measured in parental control cells. The
androgen dose-response curve of AR activation was shifted
to lower concentrations in LNCaP-hr cells with a maximal
induction at 0.1 nM of methyltrienolone as compared to
maximal induction in LNCaP cells at 1 nM. The nonsteroidal
antiandrogen hydroxyflutamide stimulated the AR 4 times
stronger in LNCaP-hr cells than in control cells. Most interestingly, bicalutamide also displayed agonistic effects on
CAT activity in long-term androgen-ablated cells. Maximal
stimulation of reporter gene activity by bicalutamide was
2-fold. The ability of bicalutamide to antagonize androgeninduced increase in reporter gene activity as seen in parental
LNCaP cells was reduced or lost in the LNCaP-hr subline.
The changes in AR transcriptional activity were not associated with amplification of the AR gene.
In summary, our study reveals new mechanisms by
which prostate cancer cells adapt to an androgen-depleted
environment.
ANDROGEN RECEPTOR DOWNREGULATION BY
ANTISENSE-PHOSPHOROTHIOATE OLIGONUCLEOTIDES IN
LNCaP CELLS
Iris E. Eder, Zoran Culig, Georg Bartsch, and Helmut Klocker
Department of Urology, University of Innsbruck, Innsbruck,
Austria
Endocrine treatment of metastatic prostate cancer is only
palliative. In therapy-refractory primary prostate tumors
and in their metastases the presence of androgen receptor
(AR) has been demonstrated by immunohistochemistry suggesting a role of the AR in an advanced-stage disease. By
using antisense technology we downregulated AR expression and investigated the effects on androgen signal transduction and cell growth in LNCaP cells.
We used electroporation to ensure the uptake of various
phosphorothioate oligodeoxynucleotides (PS-ODNs) by
LNCaP cells as incubation of cells with PS-ODNsupplemented culture medium was not effective. PS-ODNs
directed against the poly Gln and the poly Gly regions were
chosen. AR expression was evaluated by Western blotting
and radioligand binding assay. Proliferation was determined by MTT assay. Secretion of prostate-specific antigen
(PSA) into cell culture supernatant was measured by an enzyme immunoassay.
Treatment with either antisense PS-ODNs as 750/15 and
as 1944/15 alone or with a mixture of both resulted in a
downregulation of AR to less than 10% of an untreated control within 24 hours after electroporation. After 72 hours AR
content was reduced by 60% in cells treated with as 1944/15.
AR downregulation persisted up to 120 hours. In contrast,
AR protein expression was not affected by treatment with
missense (ms) control ODNs, ms 750/15 and ms 1944/15,
respectively. Proliferative response to androgen stimulation
was reduced in antisense-treated cells whereas untreated as
well as missense controls showed the typical biphasic
growth curve. The synthetic androgen mibolerone stimulated PSA secretion in control cells 10-fold. On the other
hand, only a 3-fold increase in PSA secretion was measured
in antisense-treated cells.
355
These results demonstrate that application of AR antisense PS-ODNs may be a promising new approach in prostate cancer treatment.
STIMULATION OF ANDROGEN RECEPTOR TRANSCRIPTIONAL
ACTIVITY BY INTERLEUKIN-6 IN PROSTATE CANCER CELLS
Alfred Hobisch, Iris E. Eder, Thomas Putz, Georg Bartsch,
Helmut Klocker, and Zoran Culig
Department of Urology, University of Innsbruck, Innsbruck,
Austria
The human androgen receptor (AR) can be activated by a
variety of nonsteroidal substances such as cyclic adenosine
monophosphate analogues, growth factors and vitamin D in
a ligand-independent and ligand-dependent (synergistic)
manner. It was recently shown that interleukin-6 (IL-6) levels are elevated in sera of patients with metastatic carcinoma
of the prostate. Therefore we asked whether IL-6 is able to
stimulate AR activity in the absence of ligand or in the presence of very low concentrations of androgen. An androgenresponsive reporter gene and an AR expression vector were
transfected into DU-145 cells and reporter gene activity was
measured after treatment with androgen and/or IL-6. Dosedependent stimulatory effects of IL-6 on AR activity were
observed. The reporter gene activity induced by IL-6 varied
in individual experiments between 30 and 90% of the maximal activity stimulated by the synthetic androgen methyltrienolone. Low doses of androgen (5–10 pM of R1881) in
combination with IL-6 (10–20 ng/ml) induced strong synergistic activation of the AR. Both ligand-independent and
synergistic activation of AR by IL-6 were antagonized by
the nonsteroidal antiandrogen bicalutamide (Casodex). In
LNCaP cells, which express endogenous AR, IL-6 induced a
8-fold increase in the levels of prostate-specific antigen
(PSA) whereas PSA induction by androgens was 25-fold.
Again, IL-6 effects on PSA protein were blocked by bicalutamide. Then we investigated the role of protein kinases in
AR activation by IL-6. An inhibitor of protein kinase A
(PKA), PKI, nearly completely abolished ligand-independent and ligand-dependent IL-6 effects on AR. This compound also partially antagonized the effects of androgen on
AR activation (by 60%) thus indicating that protein kinase A
is involved in both ligand-dependent and ligand-independent AR activation. IL-6 effects on reporter gene activity
were also blocked by the mitogen-activated protein kinase
kinase (MEK) inhibitor PD 98059. These results suggest an
involvement of a mitogen-activated protein kinase pathway
(MAPK) in the process controlling AR activation.
In summary, our results show that IL-6 is able to stimulate androgen signaling pathways and that PKA and MAPK
pathways are needed for this activation.
VIDEOIMAGING OF PROSTATIC STROMAL CELL CONTRACTION:
AN IN VITRO MODEL FOR STUDYING DRUG EFFECTS
Stefan Corvin, Simone Bösch, Iris Eder, Martin Thurnher, Ju
Zhang, Georg Bartsch, and Helmut Klocker
Department of Urology, University of Innsbruck, Innsbruck,
Austria
INTRODUCTION AND OBJECTIVES: Stromal cell
contractility is known to play an important role in the de-
356
Bethesda Abstracts
velopment of bladder outlet obstruction caused by benign
prostate hyperplasia (BPH). Prostatic stromal cell contractions are mainly mediated by ␣1-adrenergic stimulation. We
established an in vitro model for videomicroscopy of single
cell contractions and used it to investigate the effect of the ␣1
adrenoreceptor antagonist doxazosin.
MATERIALS AND METHODS: Human prostatic stromal cells were isolated from prostatectomy- and cystoprostatectomy-specimens by means of an explant culture technique. The cells were cultured in a selective medium supplemented with growth factors and steroid hormones.
Immunohistochemistry with various smooth-muscle-cell
markers and an antifibroblast antibody was used for characterization of the cells. For videomicroscopy the culture
flasks were coated with viscous agents to allow cell contraction. Contractions were visualized with a cell culture microscope with a time-lapse system. For quantification the percentage of contracting cells was evaluated.
RESULTS: Immunohistochemistry showed these cells to
exhibit smooth-muscle-cell markers like ␣-actin, myosin,
desmin, vimentin, smoothelin and caldesmon. However, a
weak staining could also be seen with an antifibroblast antibody revealing these cells to be myofibroblasts. Without
stimulation 19% of the cells were seen to contract. The number of spontaneously contracting cells showed a dose dependent but not significant decrease after preincubation with
doxazosin (10 nM, 100 nM, 1 ␮M). Adrenergic stimulation
with phenyleprine (1 ␮M, 10 ␮M, 100 ␮M) led to a significant increase of contracting cells. A concentration of 10 ␮M
was seen to be most effective with a contraction rate of 55%.
This pehylephrine-induced effect was significantly reduced
by preincubation with doxazosin.
CONCLUSION: With this model it is possible to culture
prostatic stromal cells. Videomicroscopy offers the possibility to study stromal cell contractility. Drug effects on prostatic stromal cells can be investigated with this simple in
vitro model.
MODULATION OF SMOOTH MUSCLE PHENOTYPE BY
TRANSFORMING GROWTH FACTOR ␤-1 AND ANDROGENS
Michael J. Gerdes, Truong Dang, and David R. Rowley
stromal cells in culture, the PS-1 cell line was established and
characterized as smooth muscle based on expression of
smooth muscle ␣-actin, smooth muscle myosin heavy chain,
calponin, and desmin. Further, these cells express androgen
receptor, consistent with prostate smooth muscle in vivo.
PS-1 cells were cultured in chemically defined media ± TGF␤1 (25 nM) and ± DHT (10 nM). Under these conditions,
TGF-␤1 is not growth inhibitory to the PS-1 cells; however,
it was found to be a potent inhibitor of androgen induced
cell proliferation, based on change in cell numbers. To address the specific mechanisms of this modulation in cell proliferation, 3 cell cycle regulatory proteins were examined,
including PCNA and the cyclin dependent kinase inhibitors
p16 and p27. In the presence of androgens, PCNA expression was found to be unchanged through 4 days in culture,
while both p16 and p27 expression was reduced by DHT. In
the presence of TGF-␤1, however, PCNA expression was
dramatically reduced, coincident with a marked increase in
p16 and p27. Further, TGF-␤1 was found to override the
pattern seen in the presence of androgens. To characterize
the potential mechanism for this inhibition, cells were examined for androgen receptor alterations. In defined media
alone, AR is found in the nucleus of the cells independent of
hormone through 7 days in culture. In the presence of TGF␤1, however, AR was found exclusively in the cytoplasm
with a filamentous-like distribution after 24 hrs in culture.
The process was rapid, reversible, and did not require new
protein synthesis. Analysis of smooth muscle markers indicates that TGF-␤1 promotes smooth muscle differentiation
and acts cooperatively with androgens to induce smooth
muscle phenotype as determined by induction of smooth
muscle ␣-actin and calponin. The induction of these smooth
muscle proteins was most pronounced after 96 hrs. Finally,
a novel putative smooth muscle marker, KE4, has been identified, cloned, and is androgen regulated both in vitro and in
vivo. Preliminary in situ hybridization data indicate expression of KE4 is restricted to smooth muscle. TGF-␤1 acted
coperatively with androgens to stimulate KE4 expression,
consistent with other markers examined to date. These studies implicate TGF-␤1 as a potential paracrine mediator of
stromal-epithelial interactions in prostate carcinoma and development. Supported by NIH grants DK-45909, CA-58093
and CA-58204.
Department of Cell Biology, Baylor College of Medicine,
Houston, TX 77030
Transforming growth factor-␤1 (TGF ␤-1) has been implicated in prostate development, and elevated expression
of TGF ␤1 has been correlated with prostate carcinogenesis.
In this study, cell type specificity of TGF-␤1 and TGF-␤ receptor type II (RcII) protein expression was determined by
immunohistochemistry in human normal prostate and compared to prostate carcinoma tissues. Localization of LAPTGF ␤1 (TGF-␤1 precursor) and RcII was observed at moderate levels in both epithelial and mesenchymal cells in fetal
prostate. Intensity and homogeneity of LAP-TGF ␤1 staining
was increased in neonatal, prepubertal, and adult prostate.
In stromal tissues, RcII localization was correlated with
staining for smooth muscle ␣-actin. In prostatic carcinoma,
LAP-TGF ␤1 increased staining intensity localized to carcinoma cells. Carcinoma epithelia, however, exhibited low to
nondetectable RcII staining, while adjacent smooth muscle
maintained expression of RcII, implicating this cell type as
the primary target for TGF-␤1 action in carcinoma. To examine the actions of TGF-␤1 and androgens on prostatic
LNCaP CELLS OVEREXPRESSING bcl-xL AS A MODEL FOR
STUDYING OF APOPTOSIS IN PROSTATE ADENOCARCINOMAS
I.V. Lebedeva, A.L. Weitzman, and C.A. Stein
Columbia University, New York, NY 10032
A recent immunohistochemical survey of neoplastic human prostate cancer tissues showed that expression of bclxL protein increases during progression of prostate cancer.
Overexpression of this protein might be a factor enabling
prostate cancer cells to survive in an androgen-deprived environment. To mimic the hormone-insensitive, metastatic
adenocarcinoma phenotype and to determine the degree to
which overexpression of bcl-xL can protect human prostate
cancer cells from apoptosis, we transfected human prostate
cancer cells (LNCaP) with a neomycin-selectable eukaryotic
expression vector containing cDNA encoding human bcl-xL.
Two transfected clonal variants that overexpress bcl-xL
(LNCaP/bcl-xL) have been selected. These clones were unaltered with regard to their growth rate in 10% serum-
Bethesda Abstracts
containing medium and the level of expression of other bclfamily proteins such as bax and bak. Taxanes induce a
downregulation of bcl-xL in prostate cancer cells. In contrast
to the parental or mock-transfected cells, LNCaP/bcl-xL
cells were highly resistant to the taxol treatment as determined by MTT assay. These bcl-xL-overexpressing LNCaP
cell lines may serve as a good model for developing further
strategies for prostate cancer treatment and represent additional evidence of the important role that bcl-xL plays in
prostate cancer resistance to chemotherapy.
INDICATIONS OF LIVING CIRCULATING PROSTATIC CANCER
CELLS IN THE PROSTATIC CANCER PATIENTS’ BLOOD
Z.P. Wang,1 S.J. Di,1 M.A. Eisenbergerand,2 A.W. Partin,3 and
P.O.P. Ts’o1
1
Cells Works, Inc., University of Maryland Baltimore County,
Department of Oncology, 3Department of Urology, Johns
Hopkins University, Baltimore, Maryland
The morphological observation of circulating cancer cells
in our more than 200 prostatic cancer patients’ blood tests
showed that some characteristics of the circulating prostate
cancer cells might indicate potential growth ability. The indications of potential growth ability of circulating cancer
cells included the following aspects: 1) Stem-like cancer
cells: some cancer cells possess stem-like cellular morphological characteristics that appear as very ‘‘young’’ morphological features. The size of the cancer cells was like monocytes with high density and very developed cytokeratin systems in the cytoplasm and aneuploidy of chromosome 18.
They were distinguished from ‘‘typical’’ circulating cancer
cells which had very large cell bodies and nuclei. 2) Dividing
circulating cancer cells: most circulating cancer cells were in
interphase of the cell cycle. Some circulating cancer cells in
our tests were in the ‘‘M’’ phase. The occurrence of M phase
circulating cancer cells might indicate that some cancer cells
were growing in the patient’s circulation system. 3) Cloning
circulating cancer cell cluster: the distribution of circulating
cancer cells isolated from patients’ blood on the slides
showed us two distributed patterns. One was that the cancer
cells showed a single distribution on the slides. The other
pattern was that the cancer cells formed cell clusters. The
formation of cancer cell clusters might occur in the patient’s
circulation and/or be formed in the sample process in vitro
because of the sticky cancer cell surface. The possibility of
cloning growth of circulating cancer cells in the patient’s
circulation system may be considered as a primary manner
to form circulating cancer cell clusters through cytomorphological analyses. The appearance of stem-like circulating cancer cells, dividing circulating cancer cells, and
cloning circulating cancer cell clusters in the cancer patient’s
circulation may indicate that some circulating cancer cells
are living and growing in the patient’s blood
2
DIFFERENTIAL EXPRESSION OF GENES DURING
ANDROGEN-INDEPENDENT PROGRESSION OF PROSTATE
CANCERS
Jacqueline Bentel, Nicole Moore, Hugh Dawkins, and Wayne
Tilley
Urological Research Centre, Level 2, M Block, OEII Medical
Centre, Nedlands, WA, 6009, Flinders Cancer Centre, Flinders
University of SA, Adelaide, SA, 5042, Australia
357
Androgen ablation therapies are initially very successful
in the treatment of advanced prostate cancer; however, development of androgen-independent tumours occurs rapidly resulting in resumption of tumour growth, tumour metastasis and death of the patient, despite maintenance of
circulating testosterone at castrate levels. The failure of androgen ablation therapies to provide sustained control of
prostate cancer growth is likely to result from multiple genetic and epigenetic events. Several studies have demonstrated that androgen independent progression of prostate
cancers is not associated with loss of expression of androgen
receptors, which mediate androgen action in prostate cancer
and other androgen target cells. However, a significant
number of advanced prostate cancers contain mutations in
the androgen receptor gene which may contribute to the
development of androgen independent tumours. Identification of additional genes with altered expression and/or mutations will lead to increased understanding of this disease
process and the development of new treatments for hormone refractory tumours based on these findings. Investigation of genetic alterations associated with androgenindependent progression of prostate cancer has been carried
out using the human prostate cancer cell line, LNCaP, which
contains a point mutation in the androgen receptor gene.
Identification of genes contributing to the androgenindependent progression of prostate cancers has been determined by comparison of gene expression in subcutaneous
xenografts of LNCaP cells growing in intact and castrated
nude mice. Differential display PCR (dd-PCR) performed on
these LNCaP tumours has resulted in the isolation of candidate cDNA’s that were up- or down-regulated during progression of tumours to androgen-independent growth. One
of these genes, IK factor, was detected by dd-PCR to be
upregulated in LNCaP tumours growing in castrated as
compared to intact mice. Expression of IK factor, which
downregulates interferon-␥ induced HLA class II antigen
expression has not been reported previously in human prostate cancers. IK factor mRNA expression is detectable in
benign human prostate cancer tissues and in primary human prostate tumours; however, its function as a regulator
of HLA class II expression or as a growth regulatory cytokine in normal or malignant prostate is not yet characterised.
In LNCaP cells, expression of IK factor may contribute to the
resistance of cells to the growth inhibitory effects of interferon-␥. Identification of cytokines facilitating the growth of
cancer cells constitutes an important strategy towards development of successful adjuvant therapies for advanced
disease.
STAUROSPORINE-INDUCED APOPTOSIS OF THE PROSTATE
CANCER CELL LINE LNCaP IS MEDIATED BY CASPASE-3 AND -7,
AND IS INHIBITED BY OVEREXPRESSION OF THE ONCOPROTEIN
BCL-2
Marco Marcelli,1 Glenn R. Cunningham,1 Margaret Walkup,1
Lydia Sturgis,2 Carolina Kagan,2 Larry Denner,2
1
Departments of Medicine and Cell Biology, Baylor College of
Medicine-VA Medical Center,
2
Department of Cell Biology, Texas Biotechnology Corporation,
Houston, TX 77030
Understanding the molecular events associated with apoptosis cell lines derived from patients with prostate cancer
358
Bethesda Abstracts
may help in developing new effective medical treatments for
this disease. In this work we show that apoptosis of the
prostate cancer cell line LNCaP was induced after 6 hours of
treatment with the protein kinase inhibitor, staurosporine
(STS). Caspase-3 and -7 were proteolytically activated during STS-induced apoptosis. Their activation was followed by
cleavage of the downstream targets of apoptosis, poly(ADPribose) polymerase (PARP) and DNA fragmentation factor
(DFF). The pancaspase inhibitor z-VAD-CH2F was used to
demonstrate the central role played by caspase-3 and -7 in
mediating STS-induced apoptosis. This inhibitor prevented
caspase-3 and -7 activation, apoptosis, and cleavage of the
downstream targets PARP and DFF.
Overexpression of the oncoprotein BCL-2 is associated
with the development of androgen-independent prostate
cancer. We utilized a LNCaP cell line overexpressing BCL-2
(LNCaP-BCL-2). This cell line was resistant to STS-induced
apoptosis. This was associated with lack of caspase-3 and -7
activation, and of PARP and DFF cleavage.
These experiments have shown that: 1. STS-induced apoptosis of LNCaP cells was mediated by activation of
caspase-3 and -7. 2. Prevention of caspase-3 and -7 activation
prevented apoptosis and cleavage of the two downstream
targets of activated caspases, PARP and DFF. 3. Overexpression of BCL-2 prevented apoptosis of LNCaP cells by blocking caspase-3 and -7 activation.
Since caspase-3 and -7 are downstream of BCL-2 in the
executioner phase of apoptosis, manipulation of their expression/activation may be a way to force prostate cancer
cells to undergo apoptosis.
MOLECULAR MECHANISMS OF THE VARIABLE
ANTIPROLIFERATIVE EFFECT OF 1␣,25-(OH)2 VITAMIN D3 IN
HUMAN PROSTATE CANCER CELL LINES
Sen-Hong Zhuang and Kerry L. Burnstein
Department of Molecular and Cellular Pharmacology,
University of Miami School of Medicine, Miami, FL 33136
1␣,25-dihydroxyvitamin D3 (1,25 D), the most active metabolite of vitamin D, inhibits the growth of human prostate
cancer cells in vitro and in vivo. However, the magnitude of
growth inhibition is variable. The majority of 1,25 D effects
are mediated by the vitamin D receptor (VDR), a ligandactivated transcription factor and member of the steroid/
thyroid hormone receptor superfamily. To understand the
different sensitivities of human prostate cancer cell lines to
growth inhibition by 1,25 D, we assayed VDR content and
VDR transcriptional activity in four human prostate cancer
cell lines: LNCaP, PC3, DU145, and ALVA 31. PC3 and
DU145 prostate cells, which contain the lowest VDR content/transcriptional activity, were relatively insensitive to
1,25 D-mediated growth inhibition. Stable transfection of
PC3 with a VDR cDNA (PC3/VDR) resulted in modest 1,25
D-mediated growth inhibition. However, in LNCaP and
ALVA 31 cells, VDR content and antiproliferative effects of
1,25 D did not correlate. LNCaP cells express about half the
number of functional VDR as ALVA 31 cells yet LNCaP
were more profoundly growth inhibited than ALVA 31 cells.
VDRs in both LNCaP and ALVA 31 cells were transcriptionally active as determined by reporter gene assays. Thus,
VDR is necessary but not sufficient for maximal antiproliferative effects of 1,25 D. 1,25 D did not promote DNA fragmentation in either LNCaP or ALVA 31 cells suggesting that
apoptosis is not involved in the differential 1,25 D growth
sensitivity of these cell lines. 1,25 D caused LNCaP cells to
accumulate in the G1/G0 phase of the cell cycle, but had no
effect on cell cycle distribution of ALVA 31 or PC3/VDR
cells. 1,25 D treatment of LNCaP cells resulted in decreased
retinoblastoma (Rb) protein phosphorylation, repressed E2F
transcriptional activity, increased levels of the cyclindependent kinase inhibitors p21WAF1, CIP1 and p27KIP1 and a
profound reduction in cyclin dependent kinase 2 (CDK2)
activity. Moreover, 1,25 D increased the amount of p21 and
p27 associated with CDK2. In contrast, neither p21 nor p27
were detected or 1,25 D-inducible in ALVA 31 or PC3/VDR
cells. These results suggest that maximal antiproliferative
effects of 1,25 D require VDR and a functional RB pathway,
including p21 and p27 expression and 1,25 D regulation of
CDK2 activity.
DIPEPTIDYLPEPTIDASE IV (CD26) ACTIVITIES ARE ELEVATED IN
PROSTATE CANCERS AND ADJACENT BENIGN HYPERPLASTIC
(BPH) GLANDS VERSUS BPH ALONE
M.J. Wilson,1,2 A.R. Ruhland, B.J. Quast, P.K. Reddy,2 S.L.
Ewing,1 and A.A. Sinha3
1
VA Medical Center and Departments of Laboratory Medicine
and Pathology, 2Urologic Surgery, and Genetics and Cell
Biology, 3University of Minnesota, Minneapolis, MN 55417
Dipeptidylpeptidase IV (DPP IV) is a serine-type exopeptidase which cleaves N-terminal dipeptides from polypeptides with a proline, and to a limited extent, alanine, at the
penultimate position. DPP IV has been identified as the cluster differentiation antigen CD26. It has also been implicated
in peptide transport, cell adhesion to extracellular matrix
proteins, and metabolism of hormones, cytokines, and
growth factors. We have shown that DPP IV is secreted by
the prostate and the DPP IV activity in semen is derived
exclusively from this gland. Because of the interaction of this
peptidase with extracellular matrix and its metabolism of
bioactive peptides, we undertook this study to determine
whether DPP IV activities changed with carcinogenesis in
the prostate gland. The histology of human prostatectomy
tissue specimens was examined in hematoxylin and eosinstained frozen sections. Samples that histologically were
BPH or cancer (>65% cancer cells) were homogenized in
0.1% Triton X-100 (pH 6.5) and the DPP IV activity of the
extracts was measured with glycylprolyl-p-nitroanaline as
substrate in a microtiter plate assay. The DPP IV activity of
cancer tissues (336 ± 143 mU/mg protein [±SEM], N = 4,
Gleason grade 6) was 2 fold greater than that of BPH (172 ±
70, N = 6). DPP IV activities in human prostate tissues were
examined further by histochemistry in frozen sections (prefixed for 1 min with formalin:acetone:PBS [1:40:10] at 4°C)
using glycylprolyl-4-methoxyl-2-naphtylamide as substrate
coupled with Fast Blue B. No counter stain was used. DPP
IV activity was localized in the epithelium of BPH and cancer tissues. The enzyme activity was quantitated by the relative intensity of reaction products determined by threshold
mapping of gray values using Metamorph software and
computer driven image analysis. The activity in cancer tissues (N = 13); 1 sample of Gleason grade 4, 10 or Gleason
grade 6, 2 of Gleason grade 8) was statistically greater than
that of BPH alone (N = 9). There were 6 cancer specimens
which also contained BPH glands; in these tissue sections
the DPP IV activity in BPH glands was elevated and was
Bethesda Abstracts
similar to that of the cancers. Thus, DPP IV activities were
increased in BPH glands associated with cancers, and were
statistically greater than activities in BPH glands ot tissue
sections containing no cancer. The relative activity of DPP IV
was similar in the Gleason grade 4 and 8 cancers. These data
indicate that DPP IV activity is increased not only in prostatic cancers but also in associated BPH glands, suggesting
that there may be some local factor(s) produced by cancer
cells that influences BPH epithelial cells. The increased DPP
IV activity may result in metabolism of some peptide(s) that
changes the tissue environment to one favorable for cancer
growth. The cancer induced increase in DPP IV activity in
BPH glands may also be reflected in seminal plasma where
DPP IV measurement may serve as a marker for prostate
disease. Supported in part by the Department of Veterans
Affairs, the Medical School Grant Program of Merck & Co.,
Inc. and USPHS grant DK-51348 to A.A.S.
CHARACTERIZATION OF AN INHIBITOR OF PROSTATE CANCER
CELL GROWTH
Mohsen Abolhassani1 and J.W. Chiao2
1
Department of Immunology, Pasteur Institute of Iran, Tehran
13164, Iran
2
Department of Medicine, New York Medical College, Valhalla,
New York 10595
Prostate cancer is currently the most common malignancy and is the second leading cause of cancer death
among males in the United States. For more than 50 years,
hormonal therapy has been the major therapeutic procedure
for metastatic prostate cancer. After initial response, patients
inevitably develop a tumor that is hormonally unresponsive
and resistant to the current therapeutic procedures. Other
available treatments such as radiation, surgery and chemotherapy have not prolonged survival of patients with metastatic carcinoma. We have identified a new antiproliferative
protein (APP) from serum-free conditioned medium of two
androgen-independent prostatic carcinoma cell lines (PC3
and DU-145). This protein selectively inhibits cell proliferation of an androgen-dependent prostatic carcinoma cell line
(LNCaP), but not proliferation of other cell lines tested or
normal human peripheral lymphocytes. APP does not induce apoptosis, but it prevents the LNCaP cells from entering into the S phase of the cell cycle. The antiproliferative
activity in reversible and is not affected by neutralizing antibodies against the well known cytokines. APP was partially purified with an apparent molecular weight of 50 kDa
and is sensitive to protease digestion but not to reducing
agent, DNase or RNase digestion. APP is immunogenic and
can induce anti-APP antibody in mice. Further purification
and characterization of APP are underway.
EFFECTS OF ANDROGEN ABLATION ON ANGIOGENISIS IN
BENIGN PROSTATIC HYPERPLASIA
Durwood E. Neal, Jr., Rama Gangula, William Elfarr, Andi
Arnautovic, Nilson Salas, and Massoud Motamedi
Galveston, TX
INTRODUCTION AND OBJECTIVES: We previously
reported in a canine model that prostatic blood flow was
dramatically reduced by either administration of finasteride
359
or by bilateral orchiectomy. This was observed at both 45
and 90 days of treatment. These changes occurred along
with and prior to prostatic size reduction. These were expected findings based on theoretical and anecdotal information. The mechanisms by which this takes place is as yet
undefined.
METHODS: In the second phase of this experiment, we
examined twelve dog prostate glands by immunohistochemical staining for factor VIII, to identify the vascular
endothelium. This was accomplished on paraffin fixed sections of whole mount prostate glands. Also, we performed
reverse transcriptase polymerase chain reaction on fresh
prostatic tissue, to test for interleukin 8 (IL8) activity, a
known angiogenesis factor.
RESULTS: In both the orchiectomized and finasteride
treated dogs, the factor VIII staining revealed a markedly
reduced blood vessel density of 6.8 + 4 and 9.2 + blood
vessels per square centimeter, compared with control prostates that had 29 + 3, P = .0007 and .0004, respectively. There
were fewer vessels in the orchiectomized animals, but the
difference was not statistically significant, P = 0.27. The IL8
levels were not detectable in either the control or the orchiectomized groups, whereas there were high levels in the
finasteride group.
CONCLUSIONS: In conclusion, the prostatic blood flow
does indeed diminish with androgen ablation, manifested
by a decrease in blood vessels per unit area in the prostate.
The IL8 gene is up-regulated in the finasteride treated animals, but not in the orchiectomized ones. This implies a
different mechanism of reduction in blood flow between the
finasteride and orchiectomized animals. Further studies are
ongoing to elucidate this.
SYNERGISTIC ACTION OF STEROIDS AND SPERMATOCELE FLUID
ON PROLIFERATION OF HUMAN PROSTATE STROMA IN
CULTURE IS ABOLISHED BY OH-FLUTAMIDE
Kenneth Y. Ilio, Julia A. Sensibar, Richard Kasjanski, Roxanne
White, James Kozlowski, Chung Lee, and John T. Grayhack
Department of Urology, Northwestern University Medical
School, Chicago, IL 60611
INTRODUCTION: Previous studies in our laboratory
have demonstrated the presence of prostate growthpromoting substances in human spermatocele fluid that act
synergistically with androgens on proliferation of prostate
stromal cells in culture. In this study, we further characterize
this synergy by adding an anti-androgen into the incubation
media.
METHODS: Spermatocele fluid, a rete testis-derived
fluid, was tested for proliferative activity on primary culture
of human prostate stroma routinely grown in RPMI-1640
with 10% fetal bovine serum. Cultures were treated with
basal media (RPMI-1640 and ITS+) containing spermatocele
fluid or ether-stripped spermatocele fluid, testosterone and
hydroxy-flutamide added alone or in combination. Results
of the treatments were assessed by cell counts. Immunocytochemical localization of androgen receptors was also undertaken.
RESULTS: Whole spermatocele fluid added to basal media significantly stimulated proliferation of prostatic stromal
cells. Ether-stripping of the spermatocele fluid did not result
in significant reduction of cell counts. Combination of testosterone and ether-stripped spermatocele resulted in sig-
360
Bethesda Abstracts
nificant increase in cell counts over that of ether-stripped
fluid. The addition of testosterone alone did not change cell
number significantly over that of basal media. When hydroxy-flutamide was added to the media containing testosterone and ether-stripped spermatocele fluid, the synergistic
proliferative effect of the steroid and fluid was abolished.
Addition of hydroxy-flutamide to basal media alone did not
alter cell count significantly. Phenotypic evaluation of stromal cultures indicated the presence of a mixture of fibro-
blasts, myofibroblasts and smooth muscles which are positive for androgen receptors.
CONCLUSIONS: Taken together, the results indicate
that testicular/epididymal secretions contain prostate stromal growth-promoting factors acting synergistically with
androgens that could be mediated through androgen receptors.
This study was supported in part by NIH/NIDDK (grant
DK 39250).
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