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The Prostate 41:49–57 (1999)
Isochromosome 8q Formation Is Associated
With 8p Loss of Heterozygosity in a Prostate
Cancer Cell Line
Jeffrey B. Virgin,1,2,3* Patrick M. Hurley,4 Fatimah A. Nahhas,3
Karen G. Bebchuk,2 Anwar N. Mohamed,1,2 Wael A. Sakr,1,2
Robert K. Bright,1,5 and Michael L. Cher1,2,3,4
1
Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, Michigan
2
Department of Pathology, Wayne State University, Detroit, Michigan
3
Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan
4
Department of Urology, Wayne State University, Detroit, Michigan
5
Department of Surgery, Wayne State University, Detroit, Michigan
BACKGROUND. In advanced prostate cancer, loss of chromosomal regions on 8p is frequently associated with gain of 8q. We studied the gross chromosomal abnormalities associated with 8p loss of heterozygosity (LOH) in the prostate tumor cell line 1542 CP3Tx. The cell
line was previously established from a primary prostatic adenocarcinoma by immortalization
with a recombinant retrovirus carrying the E6 and E7 genes of human papilloma virus type
16. Allelotyping studies demonstrated LOH at multiple markers on 8p.
METHODS. To investigate the relationship of 8p LOH to gross chromosomal rearrangements, and to screen for other genetic abnormalities in 1542 CP3Tx, we used comparative
genomic hybridization (CGH), conventional karyotyping, fluorescence in situ hybridization
(FISH), and allelotyping.
RESULTS. CGH revealed loss of the entire 8p arm, associated with gain of the entire 8q arm.
Other abnormalities included chromosome 4 loss and chromosome 11 gain. The karyotype
showed an isochromosome (8q), monosomy 4, and trisomy 11. FISH and allelotyping confirmed and extended these results.
CONCLUSIONS. These results demonstrate that i(8q) formation is a mechanism for associated 8p loss and 8q gain in prostate cancer. Furthermore, the small number of chromosomal
abnormalities in this cell line indicates that immortalization of low-passage cultures with viral
oncogenes provides a method for obtaining cell lines for studying genetic abnormalities in
prostate cancer. Prostate 41:49–57, 1999. © 1999 Wiley-Liss, Inc.
KEY WORDS:
prostate cancer; isochromosome; cell line; immortalization; chromosomal instability
INTRODUCTION
Loss of heterozygosity (LOH) is a frequent finding
in many human tumors, but the underlying mechanisms for this common genetic abnormality are not
well-understood. Several possible mechanisms include whole chromosome loss, subchromosomal deletion, unbalanced translocation, mitotic recombination,
and isochromosome formation. The distinction between these different mechanisms may be important,
© 1999 Wiley-Liss, Inc.
since they involve different forms of chromosomal instability. For example, unbalanced translocation, miGrant sponsor: Barbara Ann Karmanos Cancer Institute; Grant
sponsor: Fund for Medical Research and Education of Wayne State
University School of Medicine.
*Correspondence to: Jeffrey B. Virgin, Department of Pathology,
Wayne State University, 540 East Canfield Ave., Detroit, MI 48201.
E-mail: jvirgin@med.wayne.edu
Received 12 February 1999; Accepted 31 March 1999
50
Virgin et al.
totic recombination, and isochromosome formation all
involve DNA recombination, whereas whole chromosome loss results from aberrant segregation. Understanding the types of abnormalities leading to LOH
will improve our understanding of the underlying
mechanisms of chromosomal instability in human
cancer.
Two very common genetic abnormalities observed
in prostate cancer are 8p LOH and 8q gain (summarized by Isaacs and Bova [1]). Deletions of 8p were first
described in association with development of androgen resistance in the prostate carcinoma cell line
LNCaP [2]. Subsequently, many allelotyping studies
have shown 8p LOH in the majority of tumors analyzed. LOH on 8p is found at a high frequency in both
early lesions (prostatic intraepithelial neoplasia) [3],
and advanced tumors and metastatic deposits [4,5],
suggesting that it is an early event and that it could
play a role in tumor progression. In addition to 8p
LOH, 8q gains have been identified in advanced prostate tumors in studies using Southern blotting [6],
comparative genomic hybridization (CGH) [4,7–9],
and fluorescence in situ hybridization (FISH) [10]. The
prostate cancer cell lines DU-145 and PC-3, both established from metastatic deposits of prostate cancer,
show 8q gains [11], as do two prostate cancer xenografts [12]. Since 8q gains have been identified at a
higher frequency in advanced and metastatic lesions
than in primary tumors, amplification of genes on 8q
may play a role in prostate tumor progression.
The mechanisms of chromosomal abnormalities
have been difficult to characterize in prostate cancer
due to the difficulty in obtaining metaphases representative of tumor cells. The application of molecular
cytogenetics has made it evident that the extent of
chromosomal abnormalities is underestimated by conventional karyotyping of cultured cells from prostate
tumors [13–16]. By CGH, chromosomal losses and
gains frequently cover large regions, many including
the entire arm [4,7,9]. Furthermore, there is a frequent
association of loss of the entire 8p arm and gain of the
entire 8q arm in the same tumor, suggesting the possibility of an isochromosome 8q [8]. Cytogenetic
analysis has identified i(8q) in several metastatic lesions from prostate carcinomas [15,17]. However, to
our knowledge, definitive identification of i(8q) by
conventional karyotyping has not been reported in
cells from a primary prostate tumor. This could be due
to a lack of representative metaphases as noted above,
or i(8q) may be uncommon in primary tumors if it
arises late in tumor progression.
The goal of this study was to characterize the chromosomal abnormalities associated with 8p LOH in a
primary prostate tumor cell line. We studied a lowpassage cell line, 1542 CP3Tx [18], established from a
primary tumor by immortalization with a recombinant retrovirus encoding the human papilloma virus
type 16 (HPV16) E6 and E7 genes [19]. LOH for multiple 8p markers was previously demonstrated in this
cell line [18]. In this study, we used CGH, conventional karyotyping, FISH, and allelotyping to more
fully characterize the genetic abnormalities in this cell
line. The results show that 8p loss and 8q gain resulted
from i(8q) formation. In addition, the genomic analysis with multiple methods revealed few other abnormalities, suggesting that immortalization with HPV16
E6/E7 did not result in major chromosomal instability.
MATERIALS AND METHODS
Cell Lines
Establishment of the cell line 1542 CP3Tx by HPV16
E6/E7 immortalization of cells grown from explants of
a human prostatic adenocarcinoma was reported previously [18]. A subclone (8.4) of this cell line was used
in the present study. A corresponding cell line (1542
NPTx), established from histologically normal prostate from the same patient [18], was used for comparison in the allelotyping studies. Immortalization of
both cell lines was at passage 3, and the clonal line
CP3Tx.8.4 was obtained by dilution cloning at passage
8 [18]. Cells at passage 22 (CP3Tx) or passage 27
(NPTx) were split into three flasks and used for CGH,
karyotyping, and FISH. Allelotyping was performed
on cells from passages 18 and 21 (CP3Tx) or passages
27 and 34 (NPTx). Cell culture conditions were as previously described [18].
CGH
Cells were trypsinized, placed in digestion buffer
(0.5% SDS, 25 mM EDTA, 100 mM NaCl, 10 mM TRIS,
pH 8.0, and 1 mg/ml proteinase K) and incubated at
55°C overnight. DNA was purified by phenol/
chloroform extraction and ethanol precipitation. DNA
yields were quantified by fluorometry. Nick translation of tumor DNA with FITC-12-dUTP and normal
DNA with Texas Red-5-dUTP (NEN Research Products, Boston, MA), and hybridization to peripheral
blood metaphases (Vysis, Inc., Downer’s Grove, IL),
were performed as previously described [4]. After
washing, images were analyzed on the QUIPS Image
Analysis System (Vysis, Inc.).
Fluorescence intensity green:red ratio distributions
were generated from nine of the best metaphase images from the hybridization. These ratio distributions
were organized into 1,747 data channels along the entire genome. In this system, each channel corresponds
to roughly 1.9 Mb, and channel-by-channel t-statistics
Isochromosome 8q in Prostate Cancer
51
for each tumor-normal hybridization were calculated
as described previously [4,20]. Positive values of t indicated gains; negative values indicated deletions. The
magnitude of the absolute value of t gave an indication of the relative confidence of the true presence of a
gain or deletion. Previously, this analysis was demonstrated to have several advantages over standard interpretations of fluorescence ratio tracings [4,20].
Cytogenetic Analysis
Cell cultures were harvested as previously described [21]. Briefly, freshly fed near-confluent cultures were exposed to colcemid (0.05 ␮g/ml) for 1 hr
at 37°C. Cells were dislodged with trypsin-EDTA,
transferred to centrifuge tubes, and treated with 0.075
M KCl for 30 min at 37°C. Cells were fixed with
methanol:acetic acid (3:1). G- and Q-banding were
used for chromosome analysis. Interpretation was
based on the International System for Human Cytogenetic Nomenclature [22].
FISH and Chromosome Painting
Cells were harvested as for cytogenetic analysis.
Dual-color FISH or chromosome 8 painting was performed with DAPI counterstaining, as previously described [23]. Probes CEP 8, c-myc, and chromosome 8
paint were obtained from Vysis, Inc. RMC08P020
(“p20”) and RMC08P027 (“p27”) are P1 probes specific
for 8p11 and 8q11, respectively (generously supplied
by Dr. Joe Gray, University of California at San Francisco).
Allelotyping
In order to detect regions of allelic gain as well as
loss, we used a nonradioactive semiquantitative
method of allelotyping. Primers for two different microsatellite markers were included in each reaction to
control for variability in amplification efficiency [24],
and reactions were limited to 22 cycles, which was
within the range of exponential amplification (data
not shown). Each PCR reaction contained 100 ng
DNA, 1.2 ␮M each oligodeoxynucleotide primer (Research Genetics, Huntsville, AL), 0.2 mM each dATP,
dCTP, dGTP, and TTP, 1 unit Taq DNA Polymerase
(Fisher Scientific, Itasca, IL), 10 mM Tris-HCl, pH
8.3, 50 mM KCl, and 1.5 mM MgCl2. Reactions were
run for 22 cycles at 95°C for 30 sec, 55–57°C for 60 sec,
and 72°C for 60 sec on a GeneAmp 9600 thermal cycler
(Perkin Elmer, Foster City, CA). Products were run on
a nondenaturing 6% polyacrylamide gel (19:1 acrylamide:2% bis-acrylamide). The gel was stained with
Fig. 1. CGH abnormalities in cell line 1542 CP3Tx. DNA from
1542 CP3Tx was labeled with FITC-dUTP, and peripheral blood
DNA from a normal volunteer was labeled with Texas Red-dUTP.
Ideograms showing regions of chromosomal loss (bars to left of
chromosomes) and gain (bars to right of chromosomes) are presented with the t-statistic plots, which are derived from the tumor:normal ratio distribution compared with normal:normal ratio
distribution data (see Materials and Methods). Horizontal axis is
t-statistic value. Negative values indicate regions of loss, and positive values indicate regions of gain.
SYBR Green I (Molecular Probes, Eugene, OR) and
scanned on the blue fluorescence setting at 900 V with
the STORM 960 laser fluorescence scanner (Molecular
Dynamics, Sunnyvale, CA). Quantitation of band intensities was performed using ImageQuant software
(Molecular Dynamics).
52
Virgin et al.
Fig. 2. G-banded karyotype of 1542 CP3Tx, i.e., 46, XY, −4, i(8)(q10), +11.
RESULTS
CGH
To determine if 8p LOH in 1542 CP3Tx resulted
from a subchromosomal deletion, we screened for regions of chromosomal gains and losses by CGH. On
chromosome 8 there was loss of the entire p arm and
gain of the entire q arm (Fig. 1). These results suggested the possibility of i(8q) formation as a mechanism for 8p LOH, as observed previously in tumor
tissue [4,8,9]. The only other abnormalities observed
were loss of chromosome 4 and gain of chromosome
11, suggesting a low level of chromosomal instability
in this cell line. These abnormalities were evident by
visual examination of the images (data not shown)
and were confirmed by t-statistics (Fig. 1).
Cytogenetic Studies
To determine if the 8p loss and 8q gain observed by
CGH were due to an isochromosome, we determined
the karyotype by G-banding. Of the 19 metaphases
analyzed in detail, 15 were 46 XY, -4, i(8)(q10), +11
(Fig. 2). The other four metaphases had some or all of
the same abnormalities, with additional nonclonal abnormalities. Seventeen metaphases showed i(8q), and
in the two remaining metaphases i(8q) was replaced
by nonclonal translocations involving chromosomes 8,
11, and 19. Thus, the 8p LOH in this cell line was
associated with formation of i(8q). All the abnormalities observed in the karyotype were consistent with
the CGH results.
FISH and Chromosome Painting
We wanted to determine if the relatively uniform
abnormal karyotype obtained with this cloned immortalized cell line was representative of the entire population of cells, or if other cells were present that were
not identified in the analyzed metaphases. Therefore,
we analyzed both metaphase and interphase cells by
dual-color FISH (Fig. 3 and Table I). In most metaphases the asymmetric p and q arms of the normal
chromosome 8 were distinguishable from the symmetric q arms of i(8q) by DAPI staining. More definite
identification of the normal chromosome 8 and i(8q)
Isochromosome 8q in Prostate Cancer
53
Fig. 3. Dual-color FISH and chromosome paint on 1542 CP3Tx. Metaphases were simultaneously hybridized with a centromere 8 probe
(red) and a pericentromeric probe (green) specific for either 8p (A) or 8q (B). Isochromosome 8q shows no 8p signal and duplicated 8q
signals. C: Metaphases were simultaneously hybridized with a centromere 8 probe (red) and a chromosome 8 paint (green). A normal
chromosome 8 and an isochromosome 8 are apparent. No other chromosome 8 material is present in the genome.
was achieved by hybridizing slides with a combination of a centromere 8 probe (CEP 8) and a P1 probe
specific for a region of either 8p (p20) or 8q (p27 or
c-myc). When a CEP 8 probe was hybridized in combination with a proximal 8p probe, the normal chromosome 8 showed the CEP 8 signal adjacent to the
p-arm signal, whereas i(8q) showed only the CEP 8
signal (Fig. 3A). When a CEP 8 probe was used in
combination with a proximal 8q probe, the normal
chromosome 8 showed the CEP 8 signal, with one
signal adjacent on the q arm (Fig. 3B). i(8q) showed the
CEP 8 signal flanked by signals on both arms of the
isochromosome. With a combination of CEP 8 and cmyc probes, the pattern was similar to that in Figure
3B, with two q-arm signals flanking the CEP 8 signal,
except that the signals were farther apart (data not
shown). In interphase cells, two CEP 8 signals and
three c-myc signals were found.
For each combination of probes, 100 metaphases
were analyzed (Table I). Based on the pattern of hybridization signals and the DAPI-stained chromosomes, the cells were assigned as containing i(8q), two
normal chromosomes 8, or other (if the pattern did not
fit either normal or i(8q)). With the p- and q-arm
probes, 86–90% of metaphases contained i(8q), and
4–6% of metaphases showed two apparently normal
copies of chromosome 8. With the c-myc probe, 80% of
metaphases showed the pattern of i(8q), and approximately 20% of cells showed two apparently normal
chromosomes 8. The small difference between the results with the p20 and p27 probes and the c-myc probe
in the proportion of cells interpreted as i(8q) was likely
54
Virgin et al.
TABLE I. Quantitation of FISH Results
Probea
c-myc
p20
metaphase
p27
metaphase
Metaphase
Interphase
89
4
7
100
86
6
8
100
80
20
0
100
81
19
0
100
b
Analysis
i(8q) pattern
Normal pattern
Other pattern
Total
a
P1 probes were pericentromeric on 8p (p20) or 8q (p27). The c-myc probe localizes to 8q24.2 (see
Materials and Methods).
b
Number of metaphases or interphases with the indicated FISH results for chromosome 8. For
scoring of i(8q), Normal, or Other pattern, see text and Figure 3.
due to sampling or technical differences. With the cmyc probe, a similar proportion of interphase nuclei
(81%) showed evidence of i(8q), indicating that the
interphase and metaphase cells were similar populations with regard to i(8q). These results demonstrate
that the karyotype was representative of the cell population.
Finally, to determine if chromosome 8 material
could be deposited elsewhere in the genome by an
occult rearrangement, we hybridized slides with CEP
8 combined with chromosome 8 paint (Fig. 3C). No
chromosome 8 material was found attached to other
chromosomes. i(8q) was evident by the similar length
of the arms flanking CEP 8.
mortalized with HPV16 E6 and E7. CGH showed no
abnormalities in this cell line (data not shown).
The allelotyping patterns were consistent with the
results from the CGH, karyotype, and FISH studies.
There was LOH on 4p, 4q, and 8p (Table II and Fig. 4).
No other LOH events were detected. In cases of LOH
in this clonal cell line, the missing allele was undetectable above background (Fig. 4). With markers on 8q,
the intensities of the two alleles were unbalanced according to both visual inspection (Fig. 4) and band
quantitation (data not shown), consistent with allelic
gain. There was no evidence of mitotic recombination
with the markers studied.
DISCUSSION
Allelotyping
In addition to determining the underlying chromosome abnormality associated with 8p LOH, we
wanted to evaluate the level of global chromosomal
instability in 1542 CP3Tx. CGH and karyotyping suggested a low level of chromosomal instability. However, these methods lack sufficient resolution to detect
losses or gains less than approximately 10 megabase
pairs. Also, CGH and karyotyping do not detect LOH
events that are due to mitotic recombination, because
there is no cytogenetically detectable loss or rearrangement of chromosomal material. Therefore, we
used PCR-based allelotyping to screen for submicroscopic genomic alterations. In all, 42 markers distributed over 24 chromosome arms were studied (Table
II). We chose markers that mapped to the distal regions of the chromosome to maximize the sensitivity
for LOH resulting from chromosomal rearrangements
or mitotic recombination events that included a continuous region from a proximal breakpoint to the telomere. As a source of normal DNA for comparison, we
used the cell line 1542 NPTx, derived from histologically normal prostate from the same patient, and im-
Chromosomal instability, involving loss, gain, and
rearrangement of chromosomes, is a prominent feature of most human cancers. Different types of chromosomal abnormalities may reflect different underlying defects in DNA repair and recombination pathways, or chromosome segregation, in different
tumors. In prostate cancer, numerous chromosomal
abnormalities have been found using molecular genetic techniques such as CGH, FISH, and allelotyping.
However, the chromosomal events underlying these
abnormalities have not been well-documented in
prostate cancer because of difficulties in obtaining
metaphases representative of tumor cells for cytogenetic studies. In this study, we showed that 8p loss
and 8q gain were associated with formation of i(8q) in
a low-passage cell line from a primary prostate tumor.
Several studies using CGH suggested that i(8q) is a
frequent event in prostate carcinogenesis [4,8,9], but
this abnormality had been previously documented by
karyotype only in metastatic deposits [15,17].
Many studies suggest that chromosome 8 instability is important in prostate carcinogenesis. Gains of
regions of 8q are frequently associated with advanced
Isochromosome 8q in Prostate Cancer
55
TABLE II. Allelotyping Results With
Heterozygous Markers*
Marker
1S534
1S1590
1S1609
2S172
3S1530
4S2375
4S2390
5S1492
5S117
5S408
5S498
7S481
7S2208
7S676
8S264
8S136
8S1104
8S587
8S522
8S263
8S373
9S281
9S925
9S934
10S591
10S1435
10S1212
12S372
12S373
12S1027
13S802
13S796
13S766
14S582
17S849
17S513
CHRNB1
18S59
19S246
20S431
21S415
22S450
Locus
Result
1p12
1q
1q43
2q33–37
3q27–qter
4p
4q
5p
5p15.3–15.1
5qter
5q35.2
7pter–15
7q
7q33–35
8p
8p
8pcen
8q
8q24.12–24.1
8q24.13–qter
8q
9p
9p
9q31–33
10p15.3–15.2
10p
10p
12p
12p
12p
13q12.1
13q32–34
13q32–34
14p
17pter
17p13
17p12–11
18pter–11.22
19q13.3–13.4
20p
21q11.2–21
22q13–ter
ROH
ROH
ROH
ROH
ROH
LOH-L
LOH-U
ROH
ROH
ROH
ROH
ROH
ROH
ROH
LOH-U
LOH-U
LOH-L
AI
AI
AI
AI
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
ROH
*ROH, retention of heterozygosity; LOH-U, loss of upper allele;
LOH-L, loss of lower allele; AI, allelic imbalance. Abnormalities
are shown in bold type.
prostate tumors [4,6,9], and gains of 8q24, where the
c-myc gene is located, have been associated with poor
prognosis [25]. Also, chromosome 8 gains detected by
pericentromeric FISH probes have been associated
with a poor prognosis [26]. Because the 8q gains frequently cover large regions of the chromosome arm,
and because in one study the region of most common
gain was 8q21.3 [4], proximal to the c-myc locus, there
Fig. 4. Allelotyping with 1542 CP3Tx. Primers for two different
short tandem repeat markers were included in each reaction (see
Materials and Methods). T, template DNA from 1542 CP3Tx; N,
template DNA from 1542 NPTx (see Materials and Methods).
Lanes 1, 2: below, 8S264 (8p); above, 7S481 (7p); lanes 3, 4:
below, 4S2390 (4q); above, 8S587 (8q); lanes 5, 6: below, 12S372
(12p); above, 8S373 (8q). Arrowheads, loss of heterozygosity
(LOH); arrows, allelic imbalance (AI).
may be multiple genes on 8q important in tumorigenesis. The association of 8q gains and i(8q) with advanced and metastatic prostate cancer (see Introduction) suggests that quantitative alterations in a gene or
genes on 8q may play a role in prostate tumor progression.
The finding of i(8q) in 1542 CP3Tx, derived from a
primary prostate carcinoma, provides evidence for
one mechanism by which concomitant 8p losses and
8q gains occur during prostate carcinogenesis. The
correspondence between the allelotyping results with
four different markers from 8p12–p22 from the cell
line and tumor tissue [18] is consistent with origin of
i(8q) in vivo. Due to a lack of available tumor tissue,
we were unable to obtain further support for the presence of i(8q) in the tumor from which this cell line was
derived. It has been suggested that chromosome 8 abnormalities in prostate cancer may result from multiple sequential events within a single tumor, beginning with localized 8p losses in early lesions and followed by chromosome 8 loss, duplication, and
56
Virgin et al.
isochromosome formation [10]. This suggestion is supported by the finding that localized 8p losses are frequently detected in early lesions, including prostatic
intraepithelial neoplasia (PIN) [3], whereas whole-arm
8p losses and 8q gains are more commonly detected in
advanced tumors [4,9]. However, chromosome 8 gains
can occur early in prostate cancer, since numerical abnormalities of chromosome 8 have been detected by
FISH in clinically localized cancers and PIN [27].
Whether the apparent differences in the timing of appearance of 8p losses and 8q gains truly reflect prostate tumor biology, or are merely due to the different
applications and sensitivities of detection methods for
losses and gains, remains to be determined. In any
case, the frequent concomitant appearance of 8p losses
and 8q gains in advanced prostate tumors suggests a
role for quantitative alterations on both arms of chromosome 8 in prostate carcinogenesis. Therefore, i(8q)
formation may be an important component of chromosome 8 instability.
Isochromosomes have been associated with gains
of subchromosomal regions in a variety of tumor
types (summarized by Mitelman et al. [28]). i(8q) has
been reported most commonly in adenocarcinomas of
the colon, breast, kidney, lung, and stomach, melanomas of the eye, and leukemias and lymphomas. i(1q)
and i(17q) are also common abnormalities in tumors of
different lineages. In testicular germ-cell tumors
(TGCT), i(12p) is present in >80% of cases [29,30]. In
i(12p)-negative cases of TGCT, FISH studies revealed
amplified 12p material elsewhere in the genome
[31,32]. The minimum amplified segment has been
narrowed to 12p11.1–p12.1 [33]. Similarly, in breast
carcinoma, in which i(8q) is frequent, two different
regions of 8q are amplified in some tumor cell lines by
mechanisms other than isochromosome formation
[34]. One of these regions includes the oncogene c-myc
at 8q23–q24, which is frequently amplified in breast
and other cancers [35]. Mechanisms other than isochromosome formation have also been suggested for
8q gains in prostate cancer [8,10,12]. Taken together,
these studies suggest that isochromosome formation is
one of several mechanisms for low-copy amplification
of subchromosomal regions, and that alterations in
gene dosage within these regions play a role in tumorigenesis.
It is notable that very few chromosomal abnormalities were observed in this low-passage cell line immortalized by retroviral transduction of HPV16 E6 and E7
genes. In addition to i(8q), monosomy 4 and trisomy
11 were present (Figs. 1, 2). Many cell divisions occurred between the immortalization and genome
analysis, with viral transduction and immortalization
at passage 2, and genomic analysis at passages 18–22.
Furthermore, repeat CGH analysis at passage 32
showed the same abnormalities as the earlier passage
with one additional abnormality, trisomy 20 (data not
shown). These results suggest that immortalization
with the HPV16 E6 and E7 oncoproteins did not induce a high degree of chromosomal instability. Another study showed a moderate degree of chromosomal instability associated with HPV16 E6/E7 immortalization of epithelial cells from human bladder
[36]. This contrasts with the markedly aneuploid
karyotypes of two cell lines derived by immortalization of benign and malignant prostate cells with the
entire HPV18 genome [37]. Since those cell lines were
analyzed at higher passages, it is unclear if the differences between that study and our study are due to the
different immortalization procedures or the increased
time in culture. Our results suggest that HPV16 E6/E7
immortalization can be used to capture chromosomal
abnormalities in prostate cancer cells and analyze
them in detail to better understand the mechanisms of
chromosomal instability.
ACKNOWLEDGMENTS
We thank Dr. Joe Gray (University of California at
San Francisco) for P1 probes. We thank Jeffrey Bailey
and Fred Koppitch for technical assistance and Jerry
Turner and Shijie Sheng for helpful comments on the
manuscript.
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