Cell Cycle Checkpoint Function in Bladder Cancer
Sharon C. Doherty, Stephanie R. McKeown, Valerie McKelvey-Martin, C. Stephen Downes,
Anthony Atala, James J. Yoo, Dennis A. Simpson, William K. Kaufmann
Background: Cell cycle checkpoints function to maintain
genetic stability by providing additional time for repair of
DNA damage and completion of events that are necessary
for accurate cell division. Some checkpoints, such as the
DNA damage G1 checkpoint, are dependent on p53, whereas
other checkpoints, such as the decatenation G2 checkpoint,
are not. Because bladder transitional cell carcinomas (TCCs)
often contain numerous chromosomal aberrations and appear to have highly unstable genomes, we analyzed cell cycle
checkpoint functions in a panel of TCC lines. Methods: Cell
cycle arrest was induced in normal human fibroblasts
(NHF1-hTERT) and normal human uroepithelial cells
(HUCs), and TCC lines and checkpoint functions were quantified using flow cytometry and fluorescence microscopy. The
inducers and checkpoints were ionizing radiation (i.e., DNA
damage) (G1 and G2 checkpoints), the mitotic inhibitor colcemid (polyploidy checkpoint), or the topoisomerase II catalytic inhibitor ICRF-193 (decatenation G2 checkpoint).
Four of the five TCC lines expressed mutant p53. Results:
HUCs had an effective G1 checkpoint response to ionizing
radiation, with 68% of cells inhibited from moving from G1
into S phase. By contrast, G1 checkpoint function was severely attenuated (<15% inhibition) in three of the five TCC
lines and moderately attenuated (<50% inhibition) in the
other two lines. NHF1-hTERT had an effective polyploidy
checkpoint response, but three of five TCC lines were defective in this checkpoint. HUCs had effective ionizing radiation
and decatenation G2 checkpoint responses. All TCC lines
had a relatively effective G2 checkpoint response to DNA
damage, although the responses of two of the TCC lines were
moderately attenuated relative to HUCs. All TCC lines had
a severe defect in the decatenation G2 checkpoint response.
Conclusion: Bladder TCC lines have defective cell cycle
checkpoint functions, suggesting that the p53-independent
decatenation G2 checkpoint may cooperate with the p53dependent G1 checkpoints to preserve chromosomal stability
and suppress bladder carcinogenesis. [J Natl Cancer Inst
2003;95:1859 – 68]
Transitional cell carcinomas (TCCs) account for more than
90% of bladder cancers (1). The molecular, genetic, and cellular
changes that occur in TCC of the bladder are numerous and
include chromosomal alterations, loss of heterozygosity, and
loss of cell growth regulation. Loss of heterozygosity has been
reported for many chromosomal regions in TCCs, including sites
Affiliations of authors: Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland (SCD,
VMM, CSD); Radiation Science Research Group, School of Applied Medical
Science and Sports Studies, University of Ulster, Jordanstown, Northern Ireland
(SCD, SRM); Laboratory for Tissue Engineering and Cellular Therapeutics,
Children’s Hospital and Harvard Medical School, Boston, MA (AA, JJY);
Department of Pathology and Laboratory Medicine, Lineberger Comprehensive
Cancer Center and Center for Environmental Health and Susceptibility, University of North Carolina at Chapel Hill, Chapel Hill (DAS, WKK).
Correspondence to: William K. Kaufmann, PhD, Department of Pathology
and Laboratory Medicine, Lineberger Comprehensive Cancer Center and Center
for Environmental Health and Susceptibility, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599-7295 (e-mail: [email protected]).
See “Notes” following “References.”
DOI: 10.1093/jnci/djg120
Journal of the National Cancer Institute, Vol. 95, No. 24, © Oxford University
Press 2003, all rights reserved.
Journal of the National Cancer Institute, Vol. 95, No. 24, December 17, 2003
ARTICLES 1859
on 3p, 4p, 8p, 9p, 9q, 11p, 13q, 14q, 17p, and 18q (2–5). Several
of these genetic alterations have been linked with inactivation of
tumor suppressor genes. In bladder TCC, two of the most
frequently inactivated tumor suppressor genes are the retinoblastoma (RB) and p53 genes. Genetic alterations in RB and p53
occur in approximately 35% and 50% of bladder cancers, respectively, and are associated with high-stage and high-grade
TCCs (6 –9). The RB and p53 genes encode key proteins of the
cell cycle checkpoints that arrest cells in G1. Deletion of 9p21 is
also a common and early event in the development of TCC (10);
this region encodes three distinct proteins (p16, ARF, and p15 in
humans) that are negative regulators of RB and p53 function.
Thus, bladder cancers display mutations in cell cycle checkpoint
genes that are associated with malignant growth and genetic
instability.
There are two major classes of cell cycle checkpoints: DNA
damage checkpoints and dependency checkpoints. DNA damage
checkpoints delay cell cycle transitions from G1 to S and from
G2 to M, thereby providing more time for DNA repair. Essential
components of the G1 checkpoint include ATM, p53, RB, Chk2,
and p21Waf1 (11,12). DNA damage activates ATM kinase, which
phosphorylates p53 and Chk2, leading to the induction and
activation of p53. In turn, p53 transactivates p21Waf1, which
inhibits the G1 cyclin– dependent kinases that normally inactivate RB, and thereby represses the E2F transcription factors that
initiate S phase (13,14). Key protein regulators of the G2 checkpoint include ATM, ATR, NBS1, Plk1, BRCA1, Chk1, Cdc25C,
Wee1, Myt1, and 14-3-3␴/⑀ (15–18). G2 arrest or block that
occurs after DNA damage is achieved by three mechanisms; two
act immediately and simultaneously, and the third acts after a
delay of several hours. The first mechanism is an ATM- and
BRCA1-dependent phosphorylation of Cdc25C by the Chk1
protein kinase (19). This phosphorylation event promotes an
interaction between Cdc25C and 14-3-3 proteins that sequesters
Cdc25C into the cytosol and prevents activation of the nuclear
pool of Cdk1/cyclin B1, thereby delaying mitosis (20,21). The
second mechanism is the exclusion of cyclin B1 from the nucleus (22) by the Crm1 nuclear exporter protein (23). In the
absence of DNA damage, Plk1 phosphorylates cyclin B1 within
the nuclear export sequence, blocking interaction with Crm1 and
promoting nuclear accumulation of cyclin B1 (24). In the presence of DNA damage, ATM- and ATR-dependent inhibition of
Plk1 sustains nuclear exclusion of cyclin B1, thereby delaying
mitosis. These immediate ATM-dependent G2 checkpoint responses to DNA damage are independent of p53 (25,26). The
delayed third mechanism involves the p53-dependent induction
of 14-3-3␴ and p21Waf1 and may serve to sustain mitotic delay
after high doses of ionizing radiation (26,27).
Dependency checkpoints, such as the spindle damage,
polyploidy, and decatenation checkpoints, are those that delay
cell cycle phase transitions when essential cell cycle events have
not been completed. The spindle damage checkpoint delays
anaphase and chromosome segregation until metaphase has been
completed. p53 enforces a polyploidy checkpoint that prevents
initiation of DNA synthesis pending completion of mitosis and
cytokinesis (28). The decatenation checkpoint delays entry into
mitosis until newly synthesized daughter DNA molecules are
sufficiently decatenated by DNA topoisomerase II to permit
chromosome condensation and segregation (29,30). ICRF-193 is
a topoisomerase II catalytic inhibitor that keeps the enzyme in
the form of a closed clamp so that it cannot form a covalent
1860 ARTICLES
complex with DNA. When cells in G2 phase are incubated
briefly with ICRF-193, decatenation is blocked and entry into
mitosis is delayed (29,31,32). The mitotic delay in response to
ICRF-193 is not a DNA damage checkpoint because it is independent of ATM and p53 activities and does not involve activation of Chk1 or Chk2 (30,33). Genetic evidence for the
decatenation checkpoint function was demonstrated by the expression of a kinase-inactive ATR allele that ablated the mitotic
delay induced by ICRF-193 (30). A requirement for the breast
and ovarian cancer tumor suppressor gene BRCA1 in the decatenation checkpoint was also indicated, suggesting that malignant
progression might include inactivation of the decatenation
checkpoint function (30). The decatenation checkpoint delays
mitosis by sustaining cytosolic sequestration of Cdk1– cyclin B1
complexes through the inhibition of Plk1 activity (16,30).
Ablation or attenuation of cell cycle checkpoint function is
associated with increased growth and genetic instability, two
hallmarks of cancer. In this study, we hypothesized that the
inactivation of checkpoint functions may contribute to the genetic instability in bladder cancer. We therefore evaluated
whether there were differences in the function of DNA damage
and dependency checkpoints in human uroepithelial cells
(HUCs) and a series of bladder cancer cell lines.
MATERIALS
AND
METHODS
Cell Lines and Cell Culture
Normal HUCs were obtained from biopsy tissue from pediatric patients at Children’s Hospital in Boston, MA. The primary
cultures were established at Harvard Medical School and
shipped to the University of North Carolina at Chapel Hill
Lineberger Comprehensive Cancer Center at passages 3–5. The
cells were confirmed to be of bladder epithelial origin as described (34) and were grown using serum-free keratinocyte
medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with bovine pituitary extract at 50 ␮g/mL and epidermal
growth factor at 10 ng/mL. Checkpoint function was analyzed in
HUCs within two passages of their receipt at the University of
North Carolina at Chapel Hill.
The following bladder cancer lines were obtained from
American Type Culture Collection (Manassas, VA): T24, J82,
TCC-SUP, RT4, and UM-UC-3. The T24 and RT4 lines were
maintained in McCoy’s 5A medium supplemented with 10%
fetal bovine serum (FBS). The TCC-SUP and UM-UC-3 lines
were maintained in minimum essential medium (MEM) supplemented with 1 mM sodium pyruvate and 10% FBS. The J82 line
was maintained in MEM with nonessential amino acids and
Earle’s basic salt solution supplemented with 10% FBS. Diploid
human fibroblasts, obtained from neonatal foreskin at the University of North Carolina at Chapel Hill, were engineered to
express the catalytic subunit of telomerase (NHF1-hTERT) and
maintained in MEM supplemented with 10% FBS and 2 mM
glutamine as checkpoint-proficient controls (16,30). All cell
lines were cultured at 37 °C in a humidified atmosphere of 5%
CO2. Four of the five TCC lines have mutant p53 (Table 1).
G1 Checkpoint Function
G1 checkpoint function was quantified using a flow cytometric assay, as described (35–37). HUC cells, NHF1-hTERT, and
the bladder TCC lines, when growing logarithmically, were
Journal of the National Cancer Institute, Vol. 95, No. 24, December 17, 2003
Table 1. Cell cycle checkpoint function, ploidy, and tumor suppressor gene status in normal human uroepithelial cells (HUCs), normal human fibroblasts
(NHF1-hTERT), and bladder cancer cell lines*
Cell cycle checkpoints†
DNA damage
Cell line
HUCs
RT4
UM-UC-3
TCC-SUP
J82
T24
NHF1-hTERT
G1
⫹
⫹/⫺
⫺
⫺
⫺
⫹/⫺
⫹
Dependency
Gene status§
G2
DC
PP
Cell ploidy‡
p53
RB㛳
ARF/p16
⫹
⫹
⫹/⫺
⫹/⫺
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫹
ND
⫹
⫺
⫹
⫺
⫺
⫹
Diploid
Near-tetraploid
Hypertriploid
Hypertriploid
Triploid
Hypotetraploid
Diploid
(wt)
wt
mt
mt
mt
mt
(wt)
(⫹)
⫺
⫹
⫺
⫺
⫹
(⫹)
(wt/wt)
hd/hd
hd/hd
wt/wt
wt/wt
wt/mt
(wt/wt)
*NHF1-hTERT ⫽ normal human fibroblasts expressing the human catalytic subunit of telomerase; DC ⫽ decatenation; PP ⫽ polyploidy; RB ⫽ retinoblastoma;
ND ⫽ not determined; wt ⫽ wild-type allele; mt ⫽ mutant allele; hd ⫽ homozygous deletion (ARF and p16).
†Cell cycle checkpoint scoring: ⫹ ⫽ effective or modestly attenuated (although the DNA damage G2 checkpoint response in J82 and T24 lines was significantly
altered in comparison to that of HUCs, ⬎90% of G2 cells in these cancer cell lines delayed entry into mitosis); ⫹/⫺ ⫽ moderately attenuated (TCC lines with reduced
DNA damage G1 and G2 checkpoint function relative to HUCs, but with an intermediate level of response between HUCs and severely attenuated TCC lines;
⫺ ⫽ severely attenuated (cells with ⬍15% G1 arrest after irradiation, with more than an eightfold increase in the number of polyploid nuclei after incubation with
colcemid, or ⬍62% G2 delay during incubation with ICRF-193).
‡Cytogenetic analysis of NHF1-hTERT revealed a diploid 46 XY karyotype with no chromosomal aberrations (56). The ploidy of HUCs was estimated from DNA
content as determined by flow cytometry (37,41). The ploidy of cancer cell lines was obtained from information provided by American Type Culture Collection
(Manassas, VA).
§Tumor suppressor gene status from Sanchez-Carbayo et al. (53). Because gene status was not determined directly in HUCs and fibroblasts, the inferred status
is shown in parentheses.
㛳Retinoblastoma gene status implied from the expression of protein localized to the nucleus. ⫹ ⫽ presence of nuclear expression; ⫺ ⫽ absence of nuclear
expression.
␥-irradiated with 1.5 Gy from a 137Cs source (Gammacell 40;
MDS Nordion, Ottawa, Ontario, Canada) at a dose rate of 86
cGy/min and then returned to the incubator. Control cultures
were sham-treated, with the same movements to and from the
irradiation source. At 6, 9, or 12 hours after irradiation or sham
treatment, 10 ␮M bromodeoxyuridine (BrdU) was added to the
media for 2 hours to label cells actively synthesizing DNA. Cells
were released from dishes with trypsin, washed once with
phosphate-buffered saline (PBS), and fixed in 70% ethanol in
PBS overnight. Cells were incubated with 0.08% pepsin in 0.1 M
HCl for 20 minutes at 37 °C to yield a crude nuclear preparation.
These nuclei were partially denatured by an additional incubation in 2 N HCl for 20 minutes at 37 °C before the solution was
neutralized with 3 M sodium borate. The nuclei were washed in
Buffer A containing 4% FBS, 10 mM HEPES (pH 7.4), 150 mM
NaCl, 0.1% sodium azide, and 0.5% Tween 20 before an incubation for 30 minutes on ice in 80 ␮L of Buffer A plus 20 ␮L
of fluorescein isothiocyanate (FITC)-labeled anti-BrdU antibody
(BD Biosciences, San Jose, CA). The nuclei were then rinsed in
Buffer A and counterstained with propidium iodide at 50 ␮g/mL
in Buffer A, and RNase A (5 mg/mL) was added. A FACScan
flow cytometer (BD Biosciences) was used to quantify the
percentage of cells in the first quarter of S phase (BrdU-labeled
nuclei with 2–2.5 N DNA content). Analysis of cell cycle
distribution was performed using the Summit Program (DakoCytomation, Carpinteria, CA). A total of 3 ⫻ 104 nuclei were
analyzed per sample. Independent determinations of radiationinduced G1 arrest were conducted three times with bladder
cancer and fibroblast cell lines. Three independent HUC preparations were analyzed twice, and a fourth preparation was analyzed once. The percentage of cells in early S 6 –9 hours after
irradiation was expressed as a fraction of the sham-treated control to provide an index of the proportion of G1 cells evading the
DNA damage checkpoint. G1 checkpoint function in bladder
cancer lines was arbitrarily judged to be severely attenuated
when less than 15% of G1 cells arrested entry to S and moderately attenuated when more than 15% but less than 50% of G1
cells arrested entry to S.
Polyploidy Checkpoint Function
Cells growing logarithmically were incubated with colcemid
to depolymerize microtubules and arrest cells in metaphase.
BrdU was added to a final concentration of 10 ␮M 2 hours
before harvesting for flow cytometric analysis (described
above). Because the initiation of DNA synthesis depends on the
completion of mitosis and cytokinesis, polyploidy checkpoint
function was evaluated as the colcemid-induced increase in the
percentage of nuclei with DNA content 2.2 to 4 times (i.e.,
polyploid nuclei in S, G2, and M) the DNA content of the control
cells in G1. NHF1-hTERT cells displayed a normal karyotype
(37) and were used as a control for diploid (2N) DNA content.
Because the HUC nuclei were predominately diploid, with a
minority of tetraploid nuclei, and the bladder cancer lines all
predominately displayed a G1 nuclear DNA content of 3N or
greater, appropriate adjustments were carried out during flow
cytometry to capture profiles of BrdU labeling against DNA
content. Polyploidy checkpoint function in bladder cancer and
fibroblast lines was analyzed once. Cell lines were judged to
have a severely attenuated polyploidy checkpoint if the fraction
of polyploid nuclei was increased by more than eightfold over
the control after a 48-hour incubation with colcemid.
G2 Checkpoint Function
Mitotic delay to assess the G2 checkpoint function was determined by quantifying mitotic cells as previously described
(36,37). Cells growing logarithmically were ␥-irradiated with
1.5 Gy or were sham-treated. After incubation for 2 hours at
Journal of the National Cancer Institute, Vol. 95, No. 24, December 17, 2003
ARTICLES 1861
37 °C, cells were fixed on dishes with a solution of methanol:
glacial acetic acid (3:1), rinsed with water, and then stained with
DAPI (4⬘,5⬘-diamidino-2-phenylindole dihydrochloride, 1 ␮g/
mL) in Antifade (Molecular Probes, Eugene, OR). The number
of mitotic cells in a minimum of 2000 cells per sample was
counted by fluorescence microscopy.
Mitotic cells were also quantified by a second method that
used flow cytometry to measure the expression of a mitosisspecific form of phosphohistone H3 (38). Cells were harvested by release from dishes with trypsin 2 hours after
irradiation and, after one wash with PBS, fixed with 1%
formaldehyde in PBS for 20 minutes at room temperature.
The cells were stored overnight in 70% ethanol at 4 °C and
then incubated with 0.5 ␮g of a rabbit anti-phosphohistone
H3 antibody (Upstate Biotechnology, Lake Placid, NY) in
100 ␮L of Buffer A, which was detected with an FITClabeled goat anti-rabbit antibody (20 ␮g/mL in Buffer A;
Santa Cruz Biotechnology, Santa Cruz, CA). DNA was
stained with propidium iodide (50 ␮g/mL in Buffer A). For
each sample, 3 ⫻ 104 cells were evaluated by two-parameter
(DNA content versus H3 expression) analysis using a BD
Biosciences FACScan flow cytometer (16,38). DNA damage
G2 checkpoint function was calculated as the fraction of
mitotic cells in irradiated cultures and expressed as a percentage of the fraction of mitotic cells in sham-treated control
cultures (mitotic index treated/mitotic index control ⫻ 100%).
To quantify decatenation checkpoint function, cells were
incubated with 2 ␮M ICRF-193 (provided by Dr. Andrew
Creighton, St. Bartholomew’s Hospital Medical College, London, U.K.), or control solvent (0.5% dimethyl sulfoxide) in
medium. After 2 hours, the mitotic index was determined, as
described above.
Each bladder TCC line and the fibroblast line were evaluated for both G2 checkpoint responses three times by the
microscopic method and up to three times using the cytometric method. Each of three independent HUC preparations was
evaluated once for each G2 checkpoint by the microscopic
method. Cells were judged to have effective or modestly
attenuated G2 checkpoint functions if less than 10% of irradiated or ICRF-193-treated G2 cells failed to delay entry to
mitosis. Lines with greater than 10% but less than 35% of
cells evading the checkpoint were judged to have a moderately attenuated checkpoint response, whereas lines with
greater than 35% of cells evading the checkpoint were judged
to have a severely attenuated checkpoint response.
Statistical Analysis
Irradiation- and ICRF-193-induced G1 and G2 checkpoint
functions were expressed as the ratio of the index of DNA
synthesis or mitosis, respectively, in irradiation- or ICRF193-treated cells divided by the same index in sham- or
solvent-treated control cells. Mean ratios from multiple independent experiments were compared by Student’s t test on
log-transformed data using Statistical Analysis Software, version 8.2 (SAS Institute, Cary, NC). Tukey’s method was used
to adjust for multiple comparisons between HUCs and bladder TCC lines.
1862 ARTICLES
RESULTS
DNA Damage G1 Checkpoint Function
DNA damage G1 checkpoint function can be measured by
the selective emptying of the early S-phase compartment
6 –12 hours after exposure to irradiation. This selective emptying of early S is a consequence of p53- and ATM-dependent
induction of p21Waf1, which inhibits G1 cyclin– dependent
kinase complexes and arrests the progression of cells from G1
phase into S phase (13,39). Because the G1 checkpoint response of normal HUCs was unknown, we determined the
percentage of cells in S phase 6, 9, and 12 hours after
radiation or sham treatments. At 6 hours after irradiation,
there was a selective 73% reduction in the percentage of cells
in the first quarter of S, with little change in the fractions of
cells in other quarters of S, compared with sham-treated
control cells (Fig. 1, A and B). By 9 hours post-radiation,
there was a reduction in the percentages of cells in the first
half of S phase and, by 12 hours post-radiation, the percentages of cells in each of the first three quarters of the S phase
compartment were reduced by 50%– 60% relative to the
sham-treated control (Fig. 1, B). The normal HUC thus displayed a radiation-induced emptying of S phase that spread
with time from the beginning (i.e., front) to the end (i.e.,
back) of the compartment. This selective emptying of the S
phase compartment from front to back is characteristic of a
p53-dependent G1 checkpoint response (40,41). In a total of
four experiments using different normal HUC strains that
were tested 6 –9 hours after irradiation, the percentage of
irradiated cells in the first quarter of S was, on average, 32%
(95% confidence interval [CI] ⫽ 14% to 50%) of the percentage of sham-treated control for a 68% inhibition of progression of G1 cells into S (Fig. 1, D). In comparable experiments
in which NHF1-hTERT cells were tested 6 hours after irradiation (Fig. 1, C and D), the percentage of irradiated cells in
early S was 5% (95% CI ⫽ 2% to 7%) of the sham control,
representing a 95% inhibition of progression from G1 into S.
Compared with the G1 checkpoint response in normal HUCs,
the G1 checkpoint response to irradiation was statistically
significantly reduced in all of the bladder cancer lines
(P⬍.001) and, after adjustment for multiple comparisons,
four of five TCC lines were statistically significantly different
from HUCs (Fig. 1, C and D). Little or no reduction in the
percentage of S phase cells was detected among the TCCSUP (104%, 95% CI ⫽ 86% to 129%), UM-UC-3 (86%, 95%
CI ⫽ 82% to 89%), or J82 (93%, 95% CI ⫽ 85% to 100%)
lines when tested 6 hours after irradiation, whereas a partial
reduction in the percentage of S phase cells was detected
among the RT4 (57%, 95% CI ⫽ 51% to 62%) and T24 (51%,
95% CI ⫽ 46% to 56%) lines. Thus, in comparison with
HUCs, three of five bladder cancer lines displayed a severe
attenuation of DNA damage G1 checkpoint function, whereas
two others displayed a moderate attenuation of G1 checkpoint
function.
Polyploidy Checkpoint Function
Flow cytometry was used to assess growth arrest during
incubation with colcemid as a measure of polyploidy checkpoint
function in the bladder TCC lines. Colcemid depolymerizes
microtubules and arrests mitotic cells in metaphase. After a
Journal of the National Cancer Institute, Vol. 95, No. 24, December 17, 2003
Fig. 1. G1 checkpoint function in normal human uroepithelial cells (HUCs), five
bladder cancer cell lines, and normal human fibroblasts expressing the human
catalytic subunit of telomerase (NHF1-hTERT), which serves as a positive
control. A) HUCs were ␥-irradiated with 1.5 Gy (gamma) or were sham-treated
(sham). After 6, 9, or 12 hours, bromodeoxyuridine (BrdU) was added to the
culture medium for 2 hours. Cells were harvested and stained for flow cytometric
analysis with a fluorescein isothiocyanate–labeled anti-BrdU antibody (to identify S-phase nuclei) and propidium iodide (to label DNA). The three panels show
the time-dependent emptying of the early S-phase compartment after irradiation
of HUCs. The early S-phase compartment, or first quartile, is identified by the
inset box. The percentage near the inset box refers to the fraction of events
within the early S-phase compartment. For the irradiated cells, the percentage of
cells in the early S-phase compartment is also expressed as a percentage of early
S-phase compartment nuclei from sham-treated control cells. Data shown at 6
and 9 hours are representative of results from several preparations of HUCs.
B) Percentage of irradiated HUCs in each quartile of S-phase over time expressed as a percentage of the sham-treated control cells. C) Comparison of
BrdU incorporation by NHF1-hTERT, T24, and J82 cell lines 6 hours after
irradiation (1.5 Gy) or sham treatment. Representative data are shown. D) G1
checkpoint function in NHF1-hTERT, HUCs, and bladder cancer cell lines was
determined as the mean fraction of early S-phase compartment cells in irradiated
cells expressed as a percentage of the sham-treated control cells. Error bars
enclose the upper 95% confidence interval above the mean of multiple experiments (n ⫽ 3– 4). Values statistically significantly different from those for HUCs
are marked with * (P⬍.001) and # (P ⫽ .004) and are not adjusted for multiple
comparisons. When adjusting for all pairwise comparisons using Tukey’s
method, all comparisons between bladder transitional cell carcinoma cell lines
and HUCs remained statistically significant at the .05 level.
variable interval of metaphase arrest during incubation with
colcemid, mitosis collapses and cells reform interphase nuclei
with decondensed chromatin surrounded by nuclear envelope
(i.e, restitution nuclei). p53 signals to prevent these restitution
nuclei from initiating DNA synthesis (28,37,41). Cells with
wild-type p53 do not initiate DNA synthesis or enter S phase
from the 4N compartment during incubation with colcemid,
whereas cells with mutant or inactive p53 do. The standard
Journal of the National Cancer Institute, Vol. 95, No. 24, December 17, 2003
ARTICLES 1863
protocol to assay polyploidy checkpoint function involved adding colcemid 24 hours after seeding cells onto culture dishes and
monitoring BrdU incorporation into polyploid nuclei 48 hours
after the addition of colcemid. Insufficient numbers of HUCs
were available in these studies to permit quantitation of
polyploidy checkpoint function.
To provide a comparison for analysis of polyploidy checkpoint function in the bladder TCC lines, NHF1-hTERT fibroblasts, which have an effective checkpoint function, were used
as the positive control. After a total of 48 hours of incubation in
colcemid, which culminated with a 2-hour BrdU pulse, NHF1hTERT fibroblasts displayed an accumulation of unlabeled nuclei with 4N and less DNA content representing restitution
nuclei, i.e., those nuclei that had collected a variable fraction of
the nuclear DNA within a single nucleus (Fig. 2) (41,42). The
tetraploid nuclei normally present in the culture also arrested
with 8N and less DNA content. BrdU-labeled tetraploid S phase
nuclei were detected infrequently (⬍1% of the total nuclei) after
48 hours in colcemid, demonstrating the efficiency of this
polyploidy checkpoint.
After incubation with colcemid, the bladder TCC lines also
accumulated unlabeled restitution nuclei with DNA content
similar to that found in G2 nuclei (4N) or less. Three of the
five bladder TCC lines reinitiated DNA synthesis, as detected
by BrdU incorporation, from the restitution compartment
(Fig. 2). A representative example (Fig. 2) shows that at least
58% of nuclei from T24 cells had polyploid DNA content
after incubation with colcemid. Although the T24 line displayed a moderately defective DNA damage G1 checkpoint response, this line displayed severely defective p53-dependent
polyploidy checkpoint function. This polyploidization was
not a universal feature of all bladder cancer lines, however,
because the RT4 (Fig. 2) and TCC-SUP lines with moderately
and severely defective DNA damage G1 checkpoint function,
respectively, did not display the trait. All of the bladder
cancer lines displayed a substantial defect in at least one of
the p53-dependent G1 checkpoints (DNA damage and
polyploidy), and two lines (J82 and UM-UC-3) were severely
defective in both.
DNA Damage G2 Checkpoint Function in Normal HUCs
and Bladder Cancer Lines
Irradiation was also used to assess the G2 checkpoint response to
DNA damage. Cells were treated with 1.5 Gy and then incubated
for 2 hours to allow cells that were in mitosis at the time of
treatment to finish mitosis and move into G1. When the G2 checkpoint is intact, few cells progress into M phase. When the G2
checkpoint is not intact, irradiated cells progress into M phase. Cells
in M phase were quantified by fluorescence microscopy and by the
expression of phosphohistone H3 (Fig. 3, A). The two methods
for enumeration of mitotic cells yielded equivalent results (Fig.
3, B). G2 checkpoint function was quantified as the percentage of
G2 cells that evaded radiation-induced mitotic delay.
In NHF1-hTERT cells and HUCs, the G2 checkpoint response to irradiation was effective, with not more than 5%
(95% CI ⫽ 1% to 4% for NHF1-hTERT and 95% CI ⫽ 2%
to 9% for HUCs) of cells evading the checkpoint. The RT4
cancer line had an effective G2 checkpoint response to DNA
damage, with 4% (95% CI ⫽ 3% to 6%) of cells evading the
checkpoint. Compared with the DNA damage G2 checkpoint
response in normal HUCs, the T24 and J82 cell lines had a
modest but statistically significant attenuation of response,
with 9% (95% CI ⫽ 5% to 12%; P ⫽ .03) of J82 cells and
9% (95% CI ⫽ 7% to 11%; P ⫽ .02) of T24 cells evading the
G2 checkpoint, whereas the UM-UC-3 and TCC-SUP lines
had even greater attenuation of the response, with 15% (95%
CI ⫽ 11% to 21%; P⬍.001) of UM-UC-3 cells and 24%
(95% CI ⫽ 22% to 26%; P⬍.001) of TCC-SUP cells evading the
G2 checkpoint. Thus, although the efficiency of G2 checkpoint
response to DNA damage was statistically significantly reduced in
four of five bladder cancer lines relative to that in normal HUCs,
three of the bladder cancer lines responded to radiation effectively,
with greater than 90% inhibition of mitosis.
Decatenation Checkpoint Function in Normal HUCs and
Bladder Cancer Lines
The DNA topoisomerase II gate– blocking inhibitor ICRF193 was used to assess decatenation checkpoint function (43).
Fig. 2. Polyploidy checkpoint function in normal human fibroblasts
expressing the human catalytic subunit of telomerase (NHF1hTERT), which serves as a normal control, and bladder transitional
cell carcinoma cell lines. Cells growing in log-phase were incubated for 48 hours with colcemid, a mitosis inhibitor. Bromodeoxyuridine (BrdU) was added to the culture medium for the last
2 hours of incubation, and cells were then harvested and stained for
flow cytometry with a fluorescein isothiocyanate–labeled anti-BrdU
antibody (to identify S-phase nuclei) and with propidium iodide (to
label DNA). The T24 cell line reinitiated DNA synthesis in
polyploid nuclei (⬎4N, identified by the polygon) during incubation with colcemid, whereas the RT4 and NHF1-hTERT cell lines
did not. Representative data are shown.
1864 ARTICLES
Journal of the National Cancer Institute, Vol. 95, No. 24, December 17, 2003
Fig. 3. G2 checkpoint function in human uroepithelial cells (HUCs),
bladder transitional cell carcinoma lines, and normal human fibroblasts expressing the human catalytic subunit of telomerase (NHF1hTERT), which serves as a normal control. A) Duplicate cultures of
NHF1-hTERT, HUCs, and bladder cancer cell lines were shamtreated (sham) or ␥-irradiated with 1.5 Gy (gamma). Cells were
fixed in 70% ethanol 2 hours after irradiation and stained with
propidium iodide (to label DNA) and with an antibody against
phosphohistone H3 (PHH3; to determine the mitotic index by flow
cytometry), as described (16,38). Ellipses enclose the mitotic cells
with 4N DNA content and staining with PHH3 antibody. The
mitotic index (i.e., the fraction of cells in mitosis of 30 000 cells
counted) in the irradiated sample is shown near the ellipse and
expressed for the irradiated cells as a percentage of the shamtreated control and as a measure of the percentage of cells evading
the G2 checkpoint (36,37). Representative data are shown.
B) Mitotic indices were also determined in NHF1-hTERT, HUCs,
and the bladder cancer cell lines by fluorescence microscopy of
DAPI-stained nuclei (36,37). Mitotic indices were determined 2
hours after ␥-irradiation or sham treatment and are expressed as a
percentage relative to the sham-treated control. Graph shows the
mitotic indices determined by DAPI-stained nuclei (solid bars) and
by flow cytometry for PHH3 expression (hatched bars). Error
bars enclose the upper 95% confidence interval above the mean of
multiple experiments (n ⫽ 3). PHH3 results represent the average
of two separate experiments. Four of the bladder cancer cell lines
showed statistically significant attenuation of G2 checkpoint function relative to HUCs (*, P⬍.001; #, P ⫽ .002; and §, P ⫽ .004,
unadjusted for multiple comparisons). After adjustment for multiple comparisons using Tukey’s method, TCC-SUP, UM-UC-3, and
T24 were statistically significantly different from HUCs at the .05
level.
Daughter DNA molecules are intertwined (catenated) following
DNA replication, and topoisomerase II is needed to remove
DNA catenations before chromatid segregation at anaphase of
mitosis. Human cells appear to monitor decatenation during G2
and delay entry to mitosis when incubated with ICRF-193.
Similar to the DNA damage G2 checkpoint response, an effective decatenation checkpoint response produces a severe reduction in the mitotic index 2 hours after the addition of ICRF-193.
Cells with defective decatenation checkpoint function would be
expected to proceed from G2 into M with intertwined daughter
chromatids.
NHF1-hTERT fibroblasts and normal HUCs expressed a
highly reproducible and strong G2 delay in response to ICRF193, with mitosis being inhibited by approximately 95% (Fig. 4).
By contrast, all bladder TCC lines showed severe and statistically significant (P⬍.001) attenuation of the decatenation checkpoint response (Fig. 4). In fact, the numbers of cells in mitosis
after ICRF-193 treatment in the UM-UC-3 and T24 cell lines
exceeded the values seen in vehicle-treated control cells by
20%–30%. This was likely to be a result of ICRF-193 slowing
mitotic progression in cells that were unable to delay in G2 (44).
The bladder cancer lines displayed reduced G2 delay when
incubated with ICRF-193. Consequently, many cells progressed
into mitosis, with anaphase bridges often observed as an indicator of blocked decatenation (data not shown).
DISCUSSION
In this study, cell cycle checkpoint function was quantified in
several independent cultures of normal HUCs and five bladder
TCC lines to test whether defects in checkpoint function might
be associated with the genetic instability typically observed in
bladder cancer. Radiation-induced DNA damage and ICRF-193induced inhibition of DNA decatenation triggered effective
checkpoint responses in normal HUCs. However, although the
DNA damage G2 checkpoint function was effective in three
bladder cancer lines, with more than 90% of G2 cells arrested by
irradiation, the decatenation checkpoint function was uniformly
defective in bladder cancer lines, with less than 62% of G2 cells
arrested by the topoisomerase II inhibitor ICRF-193. Similarly,
Journal of the National Cancer Institute, Vol. 95, No. 24, December 17, 2003
ARTICLES 1865
Fig. 4. Decatenation checkpoint function in human uroepithelial
cells (HUCs), bladder transitional cell carcinoma lines, and normal
human fibroblasts expressing the human catalytic subunit of telomerase (NHF1-hTERT), which serves as a positive control. A) Mitotic indices were determined in cultures that were incubated for
2 hours with the DNA topoisomerase II inhibitor ICRF-193 or its
solvent dimethylsulfoxide, fixed and stained with propidium iodide
(to label DNA) and phosphohistone H3 (PHH3; to label mitotic
cells), described in Fig. 3. Circles enclose the mitotic cells with the
percentage of mitotic cells indicated. The mitotic index in cells
treated with ICRF-193 was expressed as a percentage of the
solvent-treated control as an index of the percentage of cells evading the decatenation checkpoint. Representative data are shown.
B) Mitotic indices were also determined in NHF1-hTERT, HUCs,
and the bladder cancer cell lines by fluorescence microscopy of
DAPI-stained nuclei (36,37). Mitotic indices (i.e., the number of
cells in mitosis of at least 2000 cells) were determined 2 hours after
treatment with ICRF-193 or solvent and are expressed as a percentage relative to the solvent-treated controls. Error bars enclose the
upper 95% confidence interval above the mean of multiple experiments (n ⫽ 3, P⬍.001). PHH3 results represent the average of two
separate experiments for J82, T24, and UM-UC-3 lines, three
experiments for NHF1-hTERT, and one experiment for RT4. All of
the bladder cancer cell lines showed statistically significant attenuation of decatenation checkpoint function relative to HUCs (*,
P⬍.001 unadjusted for multiple comparisons). After adjustment for
multiple comparisons using Tukey’s method, all bladder TCC lines
were statistically significantly different from HUCs at the .05 level.
all cancer lines displayed at least one defect in the functions of
the p53-dependent DNA damage and polyploidy checkpoints.
These functional defects in the bladder TCC lines (Table 1)
suggest that cell cycle checkpoints may represent barriers to
bladder carcinogenesis.
Accurate segregation of a replicated genome into two daughter cells requires that sister chromatids move to opposite poles of
the mitotic spindle during anaphase. It is thought that, after
replication, a small amount of sister chromatid DNA remains
catenated as a result of steric exclusion of topoisomerase I by the
replication machinery (45). This catenation may hold the sister
chromatids together until anaphase, when topoisomerase II–
mediated decatenation allows separation of the sister chromatids. ICRF-193 is a topoisomerase II catalytic inhibitor that does
not damage DNA. ICRF-193 binding to topoisomerase II holds
the enzyme in the closed-clamp conformation (43). When ICRF193 is bound to topoisomerase II, ATPase activity is diminished,
and the conformation change required to reopen the clamp is
prevented. In this study, we found that cell cycle progression
from G2 to mitosis was suppressed when HUCs were treated
1866 ARTICLES
with ICRF-193 but not when bladder TCC lines were treated
with ICRF-193. Thus, these bladder TCC lines had severe attenuation or complete ablation of the decatenation checkpoint
response. Failure of the decatenation checkpoint may result in
cells entering into mitosis with incompletely decatenated chromosomes. These entangled chromosomes cannot be segregated
properly, resulting in aneuploidy through nondisjunctional errors
or polyploidy after mitotic collapse (29,33). Insufficient decatenation before mitotic entry also can generate chromatid breaks
and exchanges, producing nonreciprocal translocations and severe chromosomal destabilization (16,29). Thus, defects in decatenation checkpoint function may produce abnormalities of
chromosome number and structure.
DNA damage checkpoints provide cells with additional time
to repair damaged DNA before initiating DNA synthesis and
mitosis. Repair of DNA lesions before DNA synthesis and
mitosis reduces the introduction of mutations and chromosomal
aberrations as permanent heritable changes within the genome.
We found that, after irradiation, G1 arrest occurred in HUCs,
with the early S-phase compartment emptying by an average of
Journal of the National Cancer Institute, Vol. 95, No. 24, December 17, 2003
68% relative to that in sham-treated controls 6 –9 hours postradiation. An absence or attenuation of the G1 checkpoint response has been shown in normal human prostate, breast, skin,
and bronchial epithelial cells (46 – 48), suggesting that epithelial
cells might generally display defective G1 checkpoint function.
However, an effective G1 checkpoint response to DNA damage
was shown in human thyroid epithelial cells (39) and rat hepatocytes in vivo and in vitro (41), suggesting that not all epithelial
cells are generally defective. Further work will be required to
determine whether the G1 checkpoint function is dependent on
epithelial cell type or is affected by conditions of cell culture.
Results of a recent study with rat mammary epithelial cells (49)
suggested that p53 function was developmentally regulated with
expression of p53 being induced by estrogen. Thus, cell type–
dependent variation in G1 checkpoint function may reflect differences in expression of critical effector genes such as p53.
DNA damage and polyploidy checkpoint functions depend on
p53 and RB proteins (28,50,51). Previous analyses of gene
expression and mutations in the bladder cancer lines revealed
high expression of mutant p53 in the UM-UC-3, TCC-SUP, and
J82 lines, low expression of mutant p53 in the T24 line, and low
expression of wild-type p53 in RT4 (Table 1) (52,53). In view of
these results (52,53), most of the functional defects we observed
in the DNA damage and polyploidy checkpoints were not surprising. The reduced DNA damage G1 checkpoint function in
the RT4 line, which expresses wild-type p53, may be explained
by its very low expression of RB and the homozygous deletions
of ARF and p16 (52,53). The effective polyploidy checkpoint
function in the TCC-SUP line was unexpected in view of the
mutation in p53, the absence of expression of RB in the nucleus,
and the severe attenuation of DNA damage G1 checkpoint function. A larger study with more cancer lines and primary tumors
is needed to establish an association between functional defects
in the DNA damage and polyploidy checkpoints in bladder
cancer.
The combination of defects in p53-dependent checkpoints
and the p53-independent decatenation checkpoint may amplify
genomic destabilization (33). Premature entry into mitosis by
cells with insufficiently decatenated chromatids should produce
stress on the mitotic spindle because chromatid separation is
impeded. If sufficient numbers of chromatids are affected, mitosis may be sufficiently retarded to induce mitotic collapse and,
in cells with intact p53 and RB signaling pathways, the
polyploidy checkpoint would block initiation of DNA synthesis.
Cells with defective polyploidy checkpoint function may be
more likely to undergo polyploidization after catenation-induced
mitotic collapse. Insufficient decatenation before mitosis could
also cause nondisjunction errors, leading to aneuploidy, or induce chromatid breaks (16), leading to translocations after repair. A recent analysis (54) described 10 translocations among
the 74 chromosomes in the T24 line, which is evidence of great
instability of chromosome number and structure. Our results
indicate that TCC of the bladder may possess a combination of
defects in cell cycle checkpoints that could increase genetic
instability and contribute to malignant progression.
A recent report (55) demonstrated that the decatenation
checkpoint requires WRN, a DNA helicase that is defective in
Werner syndrome, a familial cancer syndrome associated with
premature aging. Cells from Werner syndrome patients did not
arrest entry to mitosis when incubated with the bisdioxopiperazine ICRF-187, and expression of wild-type WRN protein in
these cells restored ICRF-187–induced G2 arrest. WRN was
required for ATR-dependent phosphorylation of BRCA1 in response to ICRF-187 (55). Decatenation checkpoint function thus
appears to require two genes—BRCA1 and WRN—that are
mutated in familial cancer syndromes and an essential gene—
ATR. Analysis of expression of mRNA and protein for these
genes may prove to be helpful for understanding the mechanisms of defective decatenation checkpoint function in bladder
cancer.
In this study, we showed that cell cycle checkpoint functions
may frequently be altered in bladder cancer lines. Defective
checkpoint function in the cancer lines was determined by direct
comparison with normal bladder uroepithelial cells. Beyond the
mutations in p53 and ARF/p16 and the reduced expression of
RB, we know nothing of the levels of expression of the many
other key regulators of cell cycle checkpoints in bladder TCC
and HUCs. Further studies with more cancer lines and HUCs
should try to connect functional defects with changes in the
activity or expression of key regulators such as BRCA1 and
WRN.
REFERENCES
(1) Soloway MS. Managing superficial bladder cancer: an overview. Urology
1992;40:5–10.
(2) Knowles MA, Elder PA, Williamson M, Cairns JP, Shaw ME, Law MG.
Allelotype of human bladder cancer. Cancer Res 1994;54:531– 8.
(3) Knowles MA, Currie GA. Genetic alterations in bladder cancer. Lancet
1993;342:1184.
(4) Rosin MP, Cairns P, Epstein JI, Schoenberg MP, Sidransky D. Partial
allelotype of carcinoma in situ of the human bladder. Cancer Res 1995;
55:5213– 6.
(5) Cordon-Cardo C, Sheinfeld J, Dalbagni G. Genetic studies and molecular
markers of bladder cancer. Semin Surg Oncol 1997;13:319 –27.
(6) Logothetis CJ, Xu HJ, Ro JY, Hu SX, Sahin A, Ordonez N, et al. Altered
expression of retinoblastoma protein and known prognostic variables in
locally advanced bladder cancer. J Natl Cancer Inst 1992;84:1256 – 61.
(7) Horowitz JM, Park SH, Bogenmann E, Cheng JC, Yandell DW, Kaye FJ,
et al. Frequent inactivation of the retinoblastoma anti-oncogene is restricted
to a subset of human tumor cells. Proc Natl Acad Sci U S A 1990;87:
2775–9.
(8) Ong F, Moonen LM, Gallee MP, ten Bosch C, Zerp SF, Hart AA, et al.
Prognostic factors in transitional cell cancer of the bladder: an emerging
role for Bcl-2 and p53. Radiother Oncol 2001;61:169 –75.
(9) Nouri AM, Cannell H, Dagini B, Dabare AA, Habib N, Fowler CG. Profile
of p53 expression in bladder and oral tumours. Effects of in vitro manipulations of p53 on the behaviour of established human tumour cell lines.
Eur J Cancer 2000;36:1853– 62.
(10) Keen AJ, Knowles MA. Definition of two regions of deletion on chromosome 9 in carcinoma of the bladder. Oncogene 1994;9:2083– 8.
(11) Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR
kinases. Genes Dev 2001;15:2177–96.
(12) Bartek J, Lukas J. Mammalian G1- and S-phase checkpoints in response to
DNA damage. Curr Opin Cell Biol 2001;13:738 – 47.
(13) Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, et al.
p53-dependent inhibition of cyclin-dependent kinase activities in human
fibroblasts during radiation-induced G1 arrest. Cell 1994;76:1013–23.
(14) Sherr CJ. Cancer cell cycles. Science 1996;274:1672–7.
(15) O’Connell MJ, Walworth NC, Carr AM. The G2-phase DNA-damage
checkpoint. Trends Cell Biol 2000;10:296 –303.
(16) Deming PB, Flores KG, Downes CS, Paules RS, Kaufmann WK. ATR
enforces the topoisomerase II-dependent G2 checkpoint through inhibition
of Plk1 kinase. J Biol Chem 2002;277:36832– 8.
(17) Liu F, Rothblum-Oviatt C, Ryan CE, Piwnica-Worms H. Overproduction
of human Myt1 kinase induces a G2 cell cycle delay by interfering with the
intracellular trafficking of Cdc2-cyclin B1 complexes. Mol Cell Biol 1999;
19:5113–23.
Journal of the National Cancer Institute, Vol. 95, No. 24, December 17, 2003
ARTICLES 1867
(18) Gatei M, Sloper K, Sorensen C, Syljuasen R, Falck J, Hobson K, et al.
ATM and NBS1 dependent phosphorylation of CHK1 on S317 in response
to IR. J Biol Chem 2003;278:14806 –11.
(19) Yarden RI, Pardo-Reoyo S, Sgagias M, Cowan KH, Brody LC. BRCA1
regulates the G2/M checkpoint by activating Chk1 kinase upon DNA
damage. Nat Genet 2002;30:285–9.
(20) Kumagai A, Dunphy WG. Binding of 14-3-3 proteins and nuclear export
control the intracellular localization of the mitotic inducer Cdc25. Genes
Dev 1999;13:1067–72.
(21) Yang J, Winkler K, Yoshida M, Kornbluth S. Maintenance of G2 arrest in
the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear
import. EMBO J 1999;18:2174 – 83.
(22) Toyoshima F, Moriguchi T, Wada A, Fukuda M, Nishida E. Nuclear export
of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint. EMBO J 1998;17:2728 –35.
(23) Hagting A, Karlsson C, Clute P, Jackman M, Pines J. MPF localization is
controlled by nuclear export. EMBO J 1998;17:4127–38.
(24) Toyoshima-Morimoto F, Taniguchi E, Shinya N, Iwamatsu A, Nishida E.
Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus
during prophase. Nature 2001;410:215–20.
(25) Paules RS, Levedakou EN, Wilson SJ, Innes CL, Rhodes N, Tlsty TD, et
al. Defective G2 checkpoint function in cells from individuals with familial
cancer syndromes. Cancer Res 1995;55:1763–73.
(26) Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, et al.
Requirement for p53 and p21 to sustain G2 arrest after DNA damage.
Science 1998;282:1497–501.
(27) Chan TA, Hwang PM, Hermeking H, Kinzler KW, Vogelstein B. Cooperative effects of genes controlling the G(2)/M checkpoint. Genes Dev
2000;14:1584 – 8.
(28) Andreassen PR, Lohez OD, Lacroix FB, Margolis RL. Tetraploid state
induces p53-dependent arrest of nontransformed mammalian cells in G1.
Mol Biol Cell 2001;12:1315–28.
(29) Downes CS, Clarke DJ, Mullinger AM, Gimenez A, Creighton AM,
Johnson RT. A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells. Nature 1994;372:467–70.
(30) Deming PB, Cistulli CA, Zhao H, Graves PR, Piwnica-Worms H, Paules
RS, et al. The human decatenation checkpoint. Proc Natl Acad Sci U S A
2001;98:12044 –9.
(31) Ishida R, Miki T, Narita T, Yui R, Sato M, Utsumi KR, et al. Inhibition of
intracellular topoisomerase II by antitumor bis(2,6-dioxopiperazine) derivatives: mode of cell growth inhibition distinct from that of cleavable
complex-forming type inhibitors. Cancer Res 1991;51:4909 –16.
(32) Iwai M, Hara A, Andoh T, Ishida R. ICRF-193, a catalytic inhibitor of
DNA topoisomerase II, delays the cell cycle progression from metaphase,
but not from anaphase to the G1 phase in mammalian cells. FEBS Lett
1997;406:267–70.
(33) Kaufmann WK, Campbell CB, Simpson DA, Deming PB, Filatov L,
Galloway DA, et al. Degradation of ATM-independent decatenation checkpoint function in human cells is secondary to inactivation of p53 and
correlated with chromosomal destabilization. Cell Cycle 2002;1:210 –9.
(34) Atala A. Creation of bladder tissue in vitro and in vivo. A system for organ
replacement. Adv Exp Med Biol 1999;462:31– 42.
(35) White AE, Livanos EM, Tlsty TD. Differential disruption of genomic
integrity and cell cycle regulation in normal human fibroblasts by the HPV
oncoproteins. Genes Dev 1994;8:666 –77.
(36) Kaufmann WK, Levedakou EN, Grady HL, Paules RS, Stein GH. Attenuation of G2 checkpoint function precedes human cell immortalization.
Cancer Res 1995;55:7–11.
(37) Kaufmann WK, Schwartz JL, Hurt JC, Byrd LL, Galloway DA, Levedakou
E, et al. Inactivation of G2 checkpoint function and chromosomal destabilization are linked in human fibroblasts expressing human papillomavirus
type 16 E6. Cell Growth Differ 1997;8:1105–14.
(38) Juan G, Traganos F, Darzynkiewicz Z. Methods to identify mitotic cells by
flow cytometry. Methods Cell Biol 2001;63:343–54.
(39) Namba H, Hara T, Tukazaki T, Migita K, Ishikawa N, Ito K, et al.
Radiation-induced G1 arrest is selectively mediated by the p53-WAF1/
Cip1 pathway in human thyroid cells. Cancer Res 1995;55:2075– 80.
1868 ARTICLES
(40) Cistulli CA, Kaufmann WK. p53-dependent signaling sustains DNA replication and enhances clonogenic survival in 254 nm ultraviolet-irradiated
human fibroblasts. Cancer Res 1998;58:1993–2002.
(41) Kaufmann WK, Behe CI, Golubovskaya VM, Byrd LL, Albright CD,
Borchet KM, et al. Aberrant cell cycle checkpoint function in transformed
hepatocytes and WB-F344 hepatic epithelial stem-like cells. Carcinogenesis 2001;22:1257– 69.
(42) Filatov L, Golubovskaya V, Hurt JC, Byrd LL, Phillips JM, Kaufmann WK.
Chromosomal instability is correlated with telomere erosion and inactivation of
G2 checkpoint function in human fibroblasts expressing human papillomavirus
type 16 E6 oncoprotein. Oncogene 1998;16:1825–38.
(43) Roca J, Wang JC. DNA transport by a type II DNA topoisomerase:
evidence in favor of a two-gate mechanism. Cell 1994;77:609 –16.
(44) Clarke DJ, Johnson RT, Downes CS. Topoisomerase II inhibition prevents
anaphase chromatid segregation in mammalian cells independently of the
generation of DNA strand breaks. J Cell Sci 1993;105:563–9.
(45) Holm C. Coming undone: how to untangle a chromosome. Cell 1994;77:
955–7.
(46) Gadbois DM, Lehnert BE. Cell cycle response to DNA damage differs in
bronchial epithelial cells and lung fibroblasts. Cancer Res 1997;57:3174 –9.
(47) Nigro JM, Aldape KD, Hess SM, Tlsty TD. Cellular adhesion regulates p53
protein levels in primary human keratinocytes. Cancer Res 1997;57:3635–9.
(48) Girinsky T, Koumenis C, Graeber TG, Peehl DM, Giaccia AJ. Attenuated
response of p53 and p21 in primary cultures of human prostatic epithelial
cells exposed to DNA-damaging agents. Cancer Res 1995;55:3726 –31.
(49) Sivaraman L, Conneely OM, Medina D, O’Malley BW. p53 is a potential
mediator of pregnancy and hormone-induced resistance to mammary carcinogenesis. Proc Natl Acad Sci U S A 2001;98:12379 – 84.
(50) Lanni JS, Jacks T. Characterization of the p53-dependent postmitotic
checkpoint following spindle disruption. Mol Cell Biol 1998;18:1055– 64.
(51) Di Leonardo, Khan SH, Linke SP, Greco V, Seidita G, Wahl GM. DNA
rereplication in the presence of mitotic spindle inhibitors in human and
mouse fibroblasts lacking either p53 or pRb function. Cancer Res 1997;
57:1013–9.
(52) Markl ID, Jones PA. Presence and location of TP53 mutation determines
pattern of CDKN2A/ARF pathway inactivation in bladder cancer. Cancer
Res 1998;58:5348 –53.
(53) Sanchez-Carbayo M, Socci ND, Charytonowicz E, Lu M, Prystowsky M,
Childs G, et al. Molecular profiling of bladder cancer lines using cDNA
microarrays: defining histogenesis and biological phenotypes. Cancer Res
2002;62:6973– 80.
(54) Harding MH, Arden KC, Gildea JW, Gildea JJ, Perlman EJ, Viars C, et al.
Functional genomic comparison of lineage-related human bladder cancer
cell lines with differing tumorigenic and metastatic potentials by spectral
karyotyping, comparative genomic hybridization, and a novel method of
positional expression profiling. Cancer Res 2002;62:6981–9.
(55) Franchitto A, Oshima J, Pichierri P. The G2-phase decatenation checkpoint
is defective in Werner syndrome cells. Cancer Res 2003;63:3289 –95.
(56) Heffernan TP, Simpson DA, Frank AR, Heinloth AN, Paules RS, CordeiroStone M, et al. An ATR- and Chk1-dependent S checkpoint inhibits
replicon initiation following UVC-induced DNA damage. Mol Cell Biol
2002;22:8552– 61.
NOTES
Supported in part by Public Health Service grants CA81343 and P30CA16086 (to W. K. Kaufmann) from the National Cancer Institute and P30ES10126 (to W. K. Kaufmann) from the National Institute of Environmental
Health Sciences, National Institutes of Health, Department of Health and Human
Services, and a scholarship (to S. C. Doherty) from the University of Ulster,
Northern Ireland, U.K..
We thank Drs. Fei Zou and Dominic Moore, Department of Biostatistics,
University of North Carolina School of Public Health for statistical advice.
Manuscript received May 16, 2003; revised October 7, 2003; accepted
October 23, 2003.
Journal of the National Cancer Institute, Vol. 95, No. 24, December 17, 2003