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Int. J. Cancer: 67, 785-790 (1996)
0 1996 Wiley-Liss, Inc.
Puolcatlon 01 tne Internal onal h o n Agamst Cancer
Publcation ae ‘Union Internal onale Contre le Cancer
Franck BLADOU‘,’,Robert L. VESSELLA’,
Kent R. BUHLER’,William J. ELLIS’,Lawrence D. TRUE’and Paul H. LANCE’
Departments of I Urology and ‘Pathology, University of Washington Medical School, Seattle, WA, USA.
The biological mechanisms involved in androgen-dependent
and -independent prostate cancer growth after castration were
analyzed in the LuCaP 23. I human prostate cancer xenograft
model. Athymic mice (n = 82) bearing LuCaP 23.1 xenograft
were castrated and tumors were harvested at different time
points from day 0 to day I I 2 post castration. In each group of
mice, tumor growth rate (TGR), serum PSA concentration,
percentage of tumor cells incorporating bromodeoxyuridine
(BUdR index), percentage of apoptotic tumor cells assessed by
morphological analysis (apoptotic index), and presence of apoptosis-related DNA “ladder” were analyzed. Castration induced
a significant decrease in TGR and serum PSA from day I to day
7, and a progressive increase in the 2 parametersfrom day 14 to
day I 12, heralding androgen-independenttumor relapse. Meanwhile the BUdR and apoptotic indexes varied as follows after
castration: an increase was noted for both at day 3, a significant
increase in apoptotic index with a decrease in BUdR index from
day 5 to day 14, and a progressive decrease in apoptotic index
while BUdR index remained at 50% of the pre-castration value
from day 28 to day 112. DNA ladder was present sparsely in
tumors grown in non-castrated hosts, universally present in
tumors from day I to day 28 post castration, and frequent in
tumors from day 56 to I 12. Castration-induced effects in LuCaP
23.1 tumors were characterized by an increase in number of
apoptotic cells and a decrease in proliferative activity. The
androgen-independenttumor relapse after castration was associated with a low apoptotic index with no increase in proliferative activity.
o 1996 Wilqv-Liss,Inc.
Advanced prostate cancer is at present not curable because
androgen-independent (AI) tumor relapse occurs following
medical or surgical castration, and eventually causes the death
of the patient. The effects of androgen ablation and the
mechanisms involved in A1 tumor growth are poorly understood. Indeed, prostate-cancer studies are limited because
animal models are either animal tumors (i.e., Dunning and
Noble rat models) or inadequate human prostate cancer
models (since prostate cancer is a slow-growing tumor, most of
the in vira and in vitro human models were established from
explants of metastatic loci and do not mimic the clinical
Androgen ablation induces genetically programmed cell
death, or apoptosis, in the epithelial cells of the rat ventral
prostate and in the androgen-dependent human prostate
cancer xenograft PC82 within a few hours following castration
(Kyprianou and Isaacs, 1988; Kyprianou et al., 1990). Within
the first week post castration, apoptosis is responsible for the
death of 80% of the epithelial cells in the rat ventral prostate
(English et a/., 1989), and for a 9-fold increase in the percentage of apoptotic cells in the regressing PC82 tumor model
(Kyprianou et al., 1990). However, 2 other androgen-sensitive
prostate cancer models do not undergo apoptosis in response
to castration. Gleave et al. (1992) in the LNCaP tumor model
as well as Westin et al. (1993) and Brandstrom et al. (1994) in
the Dunning R3327-PAP rat tumor model found no evidence
of castration-induced apoptosis as assessed by the absence of
DNA fragmentation, no change in the number of apoptotic
cells, and no induction of the TRPM-2 apoptosis-related gene.
Moreover, Westin et al. (1995) showed that the apoptotic index
was increased in only a minority of prostate-cancer patients at
day 7 following castration.
The question of whether A1 growth of prostate cancer is due
to adaptation of androgen-dependent cells or to clonal selection of A1 cells has been debated for years, and remains
controversial (Isaacs and Coffey, 1981). A1 tumor relapse is
also associated with abnormal regulation of autocrine and
paracrine growth factors (Davies and Eaton, 1991; Ware,
1993). Finally, the equation: “cell number = cell proliferation - cell death” had led to the conclusion that uncontrolled
tumor growth is caused by cell populations increasing their
rate of proliferation, decreasing their rate of death, or both
(Barr and Tomei, 1994). Recently. studies on oncogenesis have
focused not only on the regulation of cell proliferation, but also
on negative growth control such as apoptosis. In A1 prostatecancer relapse, the proliferation rate is low; thus, a logical
conclusion is that apoptosis may be down-regulated in these
tumors, although this hypothesis remains controversial (Westin et al., 1995).
We have characterized a new prostate-cancer model, the
LuCaP 23.x series of xenografts (Liu et al., 1996) that exhibits
some of the salient properties of clinical prostate cancer, such
as androgen sensitivity, PSA production and A1 tumor relapse
following a period of regression after hormonal ablation. The
aim of this study was to analyze the role of cell proliferation
and apoptosis during tumor involution following androgen
ablation and A1 prostate-cancer relapse in this promising
animal model.
LuCaP 23.1 human prostate cancerxenograft
LuCaP 23.1 is a subline of the established human prostate
cancer xenograft LuCaP 23.x. The donor was a 63-year-old
white male diagnosed with a stage-D3 prostate adenocarcinoma. He had previously received androgen ablation therapy,
radiotherapy and chemotherapy. Different metastatic foci
were harvested within 2 hr post mortem for the generation of
xenografts in athymic mice. Three sublines have been developed in our laboratory. Two of the xenograft sublines were
established from lymph nodes (LuCaP 23.1 and LuCaP 23.8)
and one from liver metastasis (LuCaP 23.12). The LuCaP 23.1
xenograft was chosen for this study because it produces high
levels of PSA, shows androgen sensitivity and has the potential
to undergo A1 growth post castration.
All animal uses and procedures were performed in compliance with the recommendations of the University of Washington Animal Care Committee. Tumor implantation and castration were performed under 130 mg ketamine/8.8 mg xylazineikg
anesthesia. Male BALB/c nu/nu mice aged 6-8 weeks (Simonsen, Gilroy, CA) were implanted subcutaneously with 20-25
3To whom correspondence and reprint requests should be sent, at
the Department of Surgical Oncology, Paoli-Calmettes Cancer Institute, 232 Bd Ste Marguerite, 13273 Marseille Cedex 9, France. Fax:
011 33 91 22 35 50.
Received: December 19,1995 and in revised form April 25,1996.
mm3 LuCaP 23.1 (passage 10). Tumor volume in individual
animals was measured weekly with micro-calipers and calculated as: V (mm’) = (L x W x H) x 0.5236 (Janek et al.,
1975). The percentage of tumor growth rate (TGR) was
calculated by the formula: TGR = (tumor volume at end of the
observation period)/(tumor volume at time of castration)
(Landstrom et al., 1994). When the tumor volume reached 200
to 500 mm7, the mice were killed and LuCaP 23.1 tumors were
harvested from 10 animals. Seventy-two mice were castrated
transabdominally and tumors were harvested from randomized groups of 8 mice at day 1, 3, 5, 7, 14, 28, 56 and 112
post-castration. Following euthanasia, LuCaP 23.1 tumor volumes were measured and the xenografts were excised, weighed,
and cut into 3 or 4 pieces. Tumor pieces were fixed in different
histological media or flash-frozen in liquid nitrogen (see
Measurement of serum PSA concentration
Sequential serum PSA concentrations were measured weekly
from samples obtained by capillary prick of the tail vein in all
animals. Approximately 40 pl of blood were collected. Serum
was separated by centrifugation and stored at -20°C until
assay. A 1 : l O dilution in Hanks’ BSS was made and 100 pl of
diluted serum were used for PSA determination by the
automated IMx PSA immunoassay system (Abbott, Chicago,
IL). The percentage of serum PSA rate was also calculated by
the formula: PSA rate = (PSA at end of the observation
period)/(PSA at time of castration).
Extraction and analysis of DNA
LuCaP 23.1 tumors were flash-frozen and powdered under
liquid nitrogen. DNA was extracted from SO mg powdered
tumor using the A.S.A.P. Genomic DNA Isolation Kit (Boehringer Mannheim, Indianapolis, IN). Briefly, powdered tumors
were incubated in lysis buffer with RNase DNase-free solution
(10 mg/ml) at 37°C for 30 min. Proteinase K (20 mg/ml) and
guanidine hydrochloride (5M solution) were added and samples
were incubated at 55°C for 3 hr. DNA was eluted in a
chromatography column, precipitated with isopropanol and
rinsed in 70% ethanol. DNA was then resuspended in TE
buffer and quantified by spectrophotometry at 260 nm, after
which 10-pg aliquots of DNA were electrophoresed on a 1.5%
agarose gel. Gels were stained with ethidium bromide ( 5
pg/lOO PI), and DNA was visualized by UV fluorescence.
Morphological analysis and electron microscopy
Tissue samples (30-40 mm3) were fixed in a mixture of 2.5%
glutaraldehyde and 2% paraformaldehyde in cacodylate buffer
at -4°C overnight. The tissue was post-fixed in osmium
tetroxide, dehydrated in graded ethanol solutions and propylene oxide, infiltrated with a mixture of epoxy and propylene
oxide, and finally embedded in cpoxy (Medcast, BALTEC
Product, Middlebury, CT). Then, 1-pm sections were stained
with Azure IIiMethylene blue. Morphological analysis to
estimate the percentage of apoptotic cells per ~ 4 0 field
performed with an Olympus BH-2 microscope in a 10 X 10
grid. An average of 3,000 cells in regions of tumor which were
free of necrosis has counted per tumor and 3 tumors were
counted per group (27 tumors).
For electron microscopy, selected areas were thin-sectioned,
stained with uranyl acetatellead citrate, and examined under a
Philips (Eindhoven, The Netherlands) 410 electron microscope.
Proliferative activig
One hour before tumor harvest, mice were injected intraperitoneally with 80 mg/kg BUdR (Sigma, St. Louis, MO). BUdR
incorporated into S-phase cell DNA was visualized in paraffinembedded tissue by incubation of 5-pm tissue slices with a
monoclonal anti-BUdR antibody (Zymed, South San Francisco, CA). Incorporated BUdR was detected with an indirect
streptavidin-biotin-peroxidase system. Quantificative estimates of proliferative activity were assessed under 1OOx
magnification (Olympus BH-2 microscope) and were expressed as a percentage of positively staining cells per x 100
field on a 10 x 10 grid. An average of 3,000 cells was counted
per tumor and 3 tumors were counted per group (27 tumors).
Values are expressed as the mean and standard error of the
mean. Statistical analysis was performed by Student’s t-test for
unpaired comparison.
Tumor growth and PSA production
In non-castrated hosts, serum PSA concentration and tumor
volume increased proportionally with a correlation factor r2 =
0.56. Although castration induced a retardation of the TGR in
most animals, tumor volume did not significantly decrease
during the first few weeks post castration in all animals. As
shown in Table I, TGR was significantly lower in the groups of
animals killed between day 1 and day 28 following castration
when compared to the group of non-castrated mice. After
castration, serum PSA concentration decreased significantly
during the same period (until 4 weeks post castration) in the
majority of animals (mean serum PSA decrease = 54%,
p < 0.001). Following a 2-week period, an increase in serum
PSA rate and TGR was observed in almost all castrated
animals, heralding A1 tumor growth (Table I ; Fig. 1).
Castration-induced changes in cell proliferation
The average percentage of BUdR-stained epithelial cells, or
BUdR index, in LuCaP 23.1 tumors grown in intact male mice
was 8.6%. Within 3 days post castration, the BUdR index
increased 1.25- to 1.5-fold, and then decreased progressively to
a level of 5% 2 weeks after castration ( p < 0.01). This BUdR
index remained low and stable up to 4 months post-castration,
even in groups of relapsing tumors (Table I).
Castration-induced changes in programmed cell death
An early event in castration-induced apoptosis is the activation of a Ca++/Mg++-dependentendonuclease which causes a
typical DNA fragmentation into multiples of a 180 nucleotide
base-pair fragment, or “DNA ladder” (Kyprianou et al., 1988).
Some low-molecular-weight nucleosomal fragments were detectable in a few LuCaP 23.1 grown in non-castrated hosts and
in the TK-177 renal-cell carcinoma xenograft used as a tumor
control. However, typical DNA ladder was universally present
in tumors examined from day 1 to day 28 post castration and
was frequent in the tumors harvested at days 56 and 112 post
castration (Fig. 2).
Programmed cell death was also noted in LuCaP 23.1 grown
in intact mice, as indicated by the presence of apoptotic cells
(less than 1% of the tumor cells). Apoptotic cells appeared as
single rounded cells with condensed chromatin in semilunar
nuclear caps surrounded by viable tumoral cells (Fig. 3).
Apoptotic cells were often fragmented into apoptotic bodies
presented as heterophagic vacuoles in neighboring tumor cells.
When several apoptotic bodies were present at the same
location, they were counted as pieces of the same cell.
Castration induced a 2- to 3-fold increase in the percentage of
LuCaP cells undergoing apoptosis 1 and 2 weeks post castration ( p < 0.05). The percentage of LuCaP apoptotic cells
decreased progressively from day 14, to reach the precastration value 16 weeks post castration, as shown in Table I.
The androgen-sensitive period after castration (1 week after
castration) was characterized by a 50% decrease in prolifera-
TGR (%)
Mean 2 SEM
PSA Rate (%)
Mean & SEM
Apoptotic index (%)
Mean & SEM
1 day
3 days
5 days
7 days
14 days
28 days
56 days
112 davs
291.1 f 114.7
318.6 f 113.1
0.66 f 0.13
87 f 13.7**
115 2 44’
116.5 ? 14.5*
98.8 f 18.8**
146.9 f 28.6*
150.2 f 57.8*
362.6 f 117.9
475 2 225.3
88.7 f 12.3**
71.8 f 16.4**
36.3 2 26.4**
36.5 2 22.5**
67.3 f 20.5**
120.2 f 86.2*
353.2 f 75
508.3 2 117.6
1.13 f 0.25
1.29 f 0.14*
1.69 f 0.41*
1.30 f 0.18
1.29 f 0.17
0.89 f 0.37
0.83 f 0.11
0.54 f 0.08
BUdR index (9%)
Mean ? SEM
8.6 2 0.6
10.7 f 1.1*
8.3 f 1.7
6.6 f 0.9*
5.0 f 0.6*
4.5 f 0.6**
5.1 f 1.1**
4.4 +. 0.9**
* , p < 0.05; **, p < 0.01: Student’s t-test, unpaired comparisons of non-castrated group versus
castrated groups.
Castration induced a 2- to 3-fold increase in the apoptotic index within the first week, then the
apoptotic index decreased steadily to reach the pre-castration value 4 months post-castration. The
BUdR index (percentage of S-phase cells) increased during the first 3 days post-castration and
decreased linearly during the next 3 weeks. The BUdR index remained at a low level (half of the
pre-castration value) 4 months post-castration.
Time post Castration
FIGURE 1 - Evolution of average tumor growth rate (TGR
(0-0) and PSA rate (B-B) in non-castrated hosts (controls1
and at different time points after castration. The decrease in PSA
rate was higher than the decrease in TGR during the 4 weeks post
tive activity and a 2- to 3-fold increase in apoptotic cells. A1
tumor relapse was characterized by a progressive decrease in
the apoptotic index with no increase in the proliferative
activity (Fig. 4).
Electron niicroscopic analysis
Electron microscopic analysis showed the presence of clusters of round to oval osmiophilic “pseudo-granules” within the
cytoplasm of tumor cells. These inclusions ranged in diameter
from 0.5 to 5 km, their electron density varied from cell to cell,
and they did not have a polar distribution within the tumor
cells. The electron-dense inclusions were interpreted as lipid
droplets and/or secretory granules. They were markedly increased in the tumor cells of castrated hosts, and particularly
in the cells surrounding necrotic areas (Fig. 5).
In this study, androgen ablation induced both an early
increase in apoptosis and a decrease in cell proliferation in the
FIGURE 2 - Electrophoretic analysis of DNA isolated from
LuCaP 23.1 prostate cancer xenografts from intact and castrated
hosts, and from TK 177 renal cell carcinoma xenograft. Lane 1,
100-bp DNA ladder as a molecular weight marker; Lanes 2 and 3,
control LuCaP 23.1 tumor DNA from non-castrated mice; Lanes
4 , 5 , 6 , 7 and 8, DNA isolated from LuCaP 23.1 tumors I, 7, 14,56
and 112 days post-castration; lane 9, DNA isolated from TK 177
xenograft. There is no evidence of DNA fragmentation in the first
LuCaP tumor in a non-castrated host but some low-molecularweight nucleosomal fragments are visible in lanes 2 and 9 due to
spontaneous apoptosis occurring in non-treated growing tumors.
A typical DNA ladder is visible in LuCaP 23.1 tumors following
castration (lanes 4-8).
LuCaP 23.1 human prostate-cancer xenograft. The progression to androgen independence was concordant with a progressive decrease in apoptosis without an increase in cell proliferation. LuCaP 23.1 is a unique and promising model for in vivo
prostate cancer studies. This model exhibits many of the
defining characteristics of clinical prostate cancer, for example, heterogeneous growth kinetics, PSA production, castration-induced apoptosis, and A1 tumor relapse. Tumor growth
and PSA production are heterogeneous within the same
subline LuCaP 23.1 xenograft grown in non-castrated athymic
male mice. The castration-induced response is variable as well.
Castration induced a moderate regression in tumor volume
FIGURE3 - Apoptotic cells and apoptotic bodies (arrows) shown in a 1-km section of plastic-embedded LuCaP 23.1 tumor on day 7
post-castration. Scale bar: 5 Krn.
and a significant decline in serum PSA concentration during
the first few weeks. We speculate that there is a population of
androgen-dependent cells in such tumors, although the initial
tumor was from a stage-D3 prostate cancer patient treated
with androgen ablation. However, some tumors exhibited a
relatively androgen-insensitive status. This heterogeneity in
tumor growth and response to castration in LuCaP 23.1 is
similar to that seen in patients treated with androgen ablation
for advanced prostate cancer (Westin et al., 1995).
Apoptosis is the most important contributory factor to the
continuous cell loss from most growing tumors (Green et al.,
1994; Wyllie, 1992). This process occurs in small as well as
large tumors and may be caused by numerous factors such as
hypoxia. inadequate levels of growth factors or hormones, or
any sublethal injury (Cotter e f al., 1990). In LuCaP 23.1
xenografts in intact hosts, apoptosis occurs spontaneously, as
evidenced by the presence of some degree of DNA fragmentation and a 0.5 to l%level of apoptotic cells. There are varying
opinions regarding the best method of measuring apoptosis.
Certain authors (Green et al., 1994; Collins et al., 1992) report
that apoptosis can occur in the absence of DNA ladder, and
thus DNA fragmentation should not be considered as the
definitive feature of apoptosis. According to Green et al.
(1994), cell morphology analysis is the most important parameter defining apoptosis. For these reasons, we employed a
combination of 2 parameters (DNA analysis and cell morphol-
ogy) to identify and quantify apoptosis in the LuCaP 23.1
Castration induced a 3-step response in the LuCaP 23.1
model. During the first 3 days post-castration, an increase was
noted in BUdR index and apoptotic index. Two different
hypotheses have been suggested to explain the increase in the
BUdR index during the first few days post castration in the rat
ventral prostate model (Colombel et al., 1992; Berges et al.,
1993). The first hypothesis is that BUdR is incorporated into
the DNA of GO epithelial cells as they enter an abortive cell
cycle which ends in apoptosis (Colombel et al., 1992). The
second hypothesis is that BUdR is incorporated during the
cellular repair of damaged DNA, called “futile DNA repair,”
which proceeds independently of the programmed cell death
(Berges et al., 1993). However, the increase in the BUdR index
noted on day 3 in this study was only approximately 20%. In
contrast, within 1 day following castration there was more than
a doubling of the apoptotic index and by day 5 there was a
nearly 3-fold increase in this apoptotic index. These data
suggest that androgen-dependent cells undergo programmed
cell death rapidly following androgen ablation.
During the second week after castration, the tumor response
was characterized by a significant decrease in serum PSA
concentration, a stable decrease in BUdR index, and a
progressive decrease in the apoptotic index. As previously
described in clinical studies and the LNCaP model (Leo et al.,
- ._- . . _ _ _- -. ..-_
. .I
0 1
I n P z % I -n - ,
Time post Castmtion (days)
4 - Relative percentage of BUdR index (0-0),
apoptotic index (-),
TGR (-)
and PSA (----)rate plotted against
time post-castration in the LuCaP 23.1 xenograft model. The
decrease in the BUdR index, TGR and PSA rate was concomitant
with an increase in the apoptotic index during the first week
following castration. The androgen-independent tumor relapse
heralded by an increase in TGR and PSA rate was characterized
by a progressive decrease in apoptosis while proliferative activity
remained at a low level.
FIGURE5 - Electron-micrograph of LuCaP 23.1 tumoral cells
showing round to oval, osmiophilic, intracytoplasmic, electrondense inclusions ranging in diameter from 0.5 to 5 km in the cells
surrounding necrotic areas. Scale bar: 3 km.
1991; Gleave et al., 1992), we observed a loss of correlation
between tumor volume and serum PSA post-castration in the
LuCaP 23.1 model: the PSA rate decrease was more important
than the T G R decrease during the 4 weeks following castration (see Fig. 1). Androgen ablation results in the reduction of
PSA production on a per-cell basis and the enrichment of
undifferentiated, non-secreting cells. In our model, tumor
volume and morphology did not vary markedly after castration.
Thus, the major effect of androgen withdrawal appears to be a
tumor-cell repression revealed by a significant decrease in
TGR, PSA production and proliferative activity. This observation is concordant with the experimental (Gleave et al., 1992;
Westin et al., 1993; Brandstrom et al., 1994) and clinical
findings (Murphy et al., 1991; Westin et al., 1995) reported in
the literature. The mechanism of androgen withdrawal must
be considered as suppression rather than as effective ablation
in prostate cancer cells, and androgen withdrawal may induce
a cell-cycle repression in prostate cancer cells for a limited
period of time (Isaacs, 1994).
Electron micrographic analysis showed an increase in cytoplasmic electron-dense inclusions in tumor cells after castration, and particularly around necrotic areas. Further studies
are ongoing in our laboratory to determine whether these
electron-dense inclusions are secretory granules containing
PSA protein.
Post-castration tumor relapse, the third step in the tumor
response, occurred in almost all the LuCaP 23.1 xenografts.
Tumor relapse was heralded by an increase in serum PSA from
a nadir within 2 weeks post-castration, in much the same way
as PSA rises in advanced prostate cancer patients who have
undergone androgen ablation. In this study, tumors growing in
an A1 manner had a decreased proliferative activity compared
to the tumors before castration. This finding is concordant with
the fact that the overall proliferation rate of recurrent prostate
tumors is low, as shown by Landstrom et a1. (1994) in rat
Dunning R3327-PAP tumors and that anti-proliferative chemotherapeutic agents are not effective in prostate cancer (Murphy, 1988). The significance of a decreased proliferative
activity in an A1 relapsing tumor is not clear, and requires
further investigation. Interestingly, tumor relapse after castration seemed to be related to a decrease in apoptosis rather
than an increase in proliferative activity, as allready reported
in a study on the Dunning R3327-PAP rat prostatic tumor
model (Landstrom et al., 1994). A new concept in the mechanism of prostate cancer relapse (as well as chemotherapeutic
resistance, for example) focuses on the role of new factors, ie.,
anti-apoptosis oncoproteins such as mutant p53 and bcl-2
proteins (Westin et al., 1995). Indeed bcl-2 is overexpressed in
A1 prostate cancer (McDonnell et al.. 1992; Colombel et al.,
1993) and in a recent report, we showed that hormoneindependent LuCaP 23.1 tumors expressed bcl-2 as well (Liuet
al., 1996). Castration-induced apoptosis could be blocked (by
bcl-2 or mutant p53) and induce A1 prostate-cancer relapse. If
this hypothesis is confirmed, activation of programmed cell
death may be a new therapeutic strategy for advanced prostate
cancer, either by induction of apoptosis-activating genes or by
inhibition of apoptosis-inhibiting genes, in conjunction with
androgen withdrawal. Further studies are ongoing on bcl-2 and
mutant p53 expressions in androgen-dependent and -independent LuCaP xenografts, as well as on induction of apoptosis in
A1 tumors.
In conclusion, the LuCaP 23.1 prostate cancer xenograft
appears to be a promising in vivo model for the study of PSA
production, effects of androgen ablation and mechanisms
involved in A1 tumor relapse. Castration-induced effects observed in the LuCaP 23.1 model may be explained by both the
decrease in proliferative activity and the increase in apoptotic
cells. Tumor relapse after castration seemed to be related to a
progressive decrease in apoptosis rather than an increase in
proliferative activity.
This work was supported in part by a Veterans Administration RAGS grant, the Lucas Foundation and the O’Brien
Center grant. F.B. was supported by a grant from the French
Ligue Nationale contre le Cancer.
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