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Molecular Biology and Genetics |
Departments of Human Oncology [S. S., K. P. J., M. S. B., D. F. J., C. A. R.], Surgery [D. F. J., T. B. G.], and Ophthalmology [R.W. N., C. P.], and the University of Wisconsin Medical School and Comprehensive Cancer Center [S. S., K. P. J., M. S. B., D. F. J., C. A. R., R. W. N., C. P.], Madison, Wisconsin 53792; Unité 434 INSERM Instituté de Génétique Moléculaire, 75010 Paris, France [V. D. V.]; Faculti des Sciences, 86100 Poitiers, France [C-J. L.]; and Cancer Research Unit, Children’s Medical Research Institute, Wentworthville, 2145 New South Wales Australia [T. R. Y.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alterations in the pRb pathway are frequent in human cancers. In this
respect, inactivation of p16/CDKN2A is the second most
common alteration found across all human cancer types, including
bladder cancer (reviewed in Ref. 8
). Alterations involving
p16/CDKN2A have been identified in 
50% of human bladder
cancers (9–13)
. Several genetic/epigenetic mechanisms,
including mutation (14–16)
, de novo
methylation (17–19)
, and hemizygous and homozygous
deletions (10)
target the INK4a
p16/CDKN2A locus for inactivation. p16 inhibits cyclin
D1-Cdk4/6-dependent phosphorylation of pRb, resulting in the
sequestration of the E2F transcription factors and subsequent cell
cycle arrest (15, 20, 21)
. Our group was the first to
demonstrate that p16 elevation occurs at senescence in human epithelial
cells (uroepithelial and prostate epithelial cells), and thus we
proposed that these high p16 levels contribute to
G1 senescence cell cycle arrest (22, 23)
. p16 is elevated at senescence in other cell types as well
(21, 24, 25)
. Alterations in pRb are also frequent in
human cancers and are found in 30% of human bladder cancers
(26–28)
.
Inactivation of different components in the pRb pathway has different biological impacts. For example, either a p16 or pRb inactivation suffices for bypassing senescence (1) . However, only pRb inactivation also abrogates p53-dependent cell cycle arrest after DNA damage. Not surprisingly, pRb alterations are associated in vivo with later-stage and higher-grade bladder cancers, as well as with a greater probability of tumor progression and lower overall survival (27, 28) . Amplification of CCND1, which codes for cyclin D1, occurs frequently in many cancer types, including bladder cancer (29–31) , but its biological impact on bypassing senescence or tumor progression is unclear. Alterations in CDK4 and CDK6 are infrequently observed in human cancer. For example, a mutation in residue 24 of Cdk4 that alters the ability of Cdk4 to bind to or be inhibited by p16 has been identified in melanoma. However, this mutation does not alter its catalytic activity (32) . Thus, such mutations, although rare, might suffice for bypassing senescence and impact significantly on tumor progression.
Mutation of the tumor suppressor gene, TP53 is the most frequent alteration among all human cancer types (33, 34) . TP53 is thought to play an important role in maintaining genome stability by mechanisms including transactivation of genes involved in p53 stability (MDM2), DNA repair (GADD45), cell cycle arrest (p21WAF1/Cip1), and apoptosis (BAX). Mutant p53, which is usually increased or elevated compared with wild type, fails to transactivate these downstream genes or initiate these protective cascades. Inactivating alterations have also been identified in several downstream genes transactivated by p53. For example, a low percentage of bladder tumors contain inactivating mutations in p21WAF1/Cip1, altering the normal cellular downstream cell cycle response after DNA damage (35, 36) . MDM2 is found amplified (37) or overexpressed in a variety of tumors (38) . Because mdm2 binds to the NH2 terminus of p53, which is required for transactivation, its overexpression inhibits p53-mediated transactivation (39) . Thus, elevated mdm2 levels also result in abrogation of p53-mediated cell cycle arrest and apoptosis (40–42) . Another study shows that when MDM2 is overexpressed, wild-type p53 is elevated, representing an alternative mechanism for p53 inactivation (43) .
Most recently, a new member of the p53 pathway has been identified.
Studies with mouse embryo fibroblasts on the INK4a locus
that codes for p16/CDKN2A have led to the identification of
a second novel INK4a transcript that has a 3' sequence
identical to p16 but with a unique 5' end (44–47)
. This
transcript, called p19ARF in mice (the human
homologue is p14ARF), is derived from a distinct
first exon (exon 1ß), which is spliced to exon 2 that is shared with
p16 but in an ARF (48)
. Both
p19ARF and p14ARF have the
ability to arrest cell proliferation at both G1
and G2-M in a p53-dependent manner involving the
up-regulation of downstream target
p21WAF1/Cip1. This response is
abolished in cells lacking p53 (48, 49)
. However, two
ARF-negative cell lines (i.e., A375 in which exon
1ß is deleted and MCF7 in which exons 1
, 2, and 3 are deleted)
undergo cell cycle arrest when treated with actinomycin D, showing that
p14ARF expression is not involved in the response
to DNA damage (49)
. These data have also been confirmed in
mice, where ARF-null cells showed elevated p53 and
p21WAF1 levels after ionizing or UV irradiation,
thus exhibiting an intact p53 checkpoint pathway. Because mouse embryo
fibroblast lines (MEFs) that retained p19ARF
always sustained mutations in TP53 and vice
versa, these studies suggest that the functional losses of either
p53 or p19ARF are mutually exclusive events for
immortalization (50)
. Recent evidence also showed that
E2F1 could induce p14ARF expression by activating
the ARF promoter (51, 52)
.
p14ARF stabilizes p53 by binding and promoting
the degradation of another oncoprotein, mdm2 (53, 54)
.
p19ARF can be activated by other oncoproteins
including myc, ras, and E1A (55, 56)
. Thus, deregulated
E2F-1 activity, attributable to inactivation of pRb and activation of
other oncogenes, initiates abnormal proliferation, resulting in the
induction of a p53 response through activation of
p14ARF (5, 57–59)
. This would lead
to cell cycle arrest or apoptosis unless a second mutation occurred in
either ARF or TP53.
As summarized above, inactivation of both the pRb and p53 pathways is common in human cancers, including bladder cancer. We reported recently that an alteration in the pRb checkpoint pathway appears to be required for progression of superficial bladder tumors to invasive bladder cancer (1) . In the present study, we tested the hypothesis that an alteration in the p53 pathway is also required for transformation to invasive human bladder cancer. We then tested the hypothesis that different combinations of pathway alterations might have different impacts on tumor phenotype. We report here the presence of a p53 pathway alteration (p53, p14ARF, or p21WAF1) in 11 of 12 TCCs examined, and we identify four different combinations of pRb and p53 pathway alterations in these 12 invasive human TCCs. Only two of these combinations of alterations have been identified previously (2) ; thus two combinations are reported herein for the first time. We also report here for the first time that p14ARF can be concomitantly expressed with wild-type p53 in invasive TCCs. Thus, we show that a p14ARF or a p53 alteration is apparently not a required event in the development of invasive bladder cancer. Indeed, one invasive TCC failed to show an alteration of p53, p21WAF1, or p14ARF. Finally, we show that because of their unique genetic organization, inactivation of both p16 and p14ARF can be accomplished by a single alteration, but this was infrequent in our samples.
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MATERIALS AND METHODS |
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Protein Analysis.
The status of proteins in the pRb and p53 pathways was examined before
passage (P) 5 and again after cell line establishment (after P15) using
Western blot analysis as described previously (1)
. The
proteins examined included: pRb, cyclin D1, CDK4, CDK6, p16, p53,
p21WAF1, and mdm2. Briefly, 2–5 x 106 cells growing in late log phage were
lysed in ECB buffer [50 mM Tris-HCl (pH 8.0), 120
mM NaCl, 100 mM NaF, 0.2 mM
Na2VO4, 0.5% NP-40 with 10
mg/ml each of leupeptin, phenylmethylsulfonyl fluoride, and aprotinin
(Sigma)]. The proteins were quantified using the Bradford assay,
loaded using 50 µg/lane and run on a 12% SDS-polyacrylamide gel.
Proteins were transferred to a polyvinylidene difluoride membrane
(Immobilon-P; Millipore) and incubated with the appropriate primary
antibody. Monoclonal antibodies to p53 (Ab-2), p16 (Ab-1),
p21WAF1 (Ab-1), and -tubulin (Ab-1) were
obtained from Oncogene Research Products. Monoclonal antibody to pRb
(14001A) was obtained from PharMingen, and monoclonal antibodies to
mdm2 (SMP14) and cyclin D1 (HD-11) and polyclonal antibodies to Cdk6
(C-21) and Cdk4 (H-22) were obtained from Santa Cruz Biotechnology. A
well-characterized rabbit polyclonal antibody to
p14ARF was used (61)
. Bound antibody
was detected using enhanced chemiluminescence (Pierce). Equal loading
was verified using an antibody to -tubulin.
For p14 ARF detection, cells were lysed in RIPA buffer [1x PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, with 10 µl/ml each of phenylmethylsulfonyl fluoride (10 mg/ml) and Na2VO4 (100 mM) and 30 µl/ml of aprotinin (10 mg/ml)], and 75 µg protein/lane was run on a 15% SDS-polyacrylamide gel.
For pRb-E2F1 immunoprecipitation studies, 107
cells were lysed in RIPA buffer, as described above. The cell lysate
was incubated with 2 µg of primary antibody at 4°C for 1 h,
followed by an overnight incubation with 20 µl of Protein G
Plus-Agarose (Oncogene Research Products) with mixing at 4°C.
Precipitated proteins were collected by centrifugation, washed with
RIPA buffer, boiled for 2 min, separated on 12% SDS-polyacrylamide
gels, and immunoblotted as above. Monoclonal antibody to E2F-1 (KH95)
was obtained from Santa Cruz Biotechnology.
IHC.
IHC was used as a second assay to assess loss of wild-type pRb. Cells
were grown on chambered glass slides (Nunc) and fixed with
glutaraldehyde and formalin (62)
. The pRb monoclonal
antibody described above was used at 1:1000 dilution to detect
wild-type nuclear pRb using the Vectastain ABC kit from Vector
Laboratories and following the manufacturer instructions. The DAB
substrate kit (Vector Labs) for peroxidase was used for final
detection. Groups without primary pRb antibody and with an irrelevant
anti-SV40-T antigen antibody (Santa Cruz Biotechnology) were used as
controls. IHC was done at least twice for all samples and was scored
using coded samples.
Southern Analysis.
Southern blot analysis was used to detect p16/CDKN2A and p14
ARF/ARF deletions or altered
methylation in the TCCs. DNA was extracted from early-passage cells
using TNE buffer [0.5 M Tris-Cl,
0.1M NaCl, and 20 mM EDTA
with 100–200 µg/ml of proteinase K (Sigma)], phenol-chloroform
extraction, and dissolved in TE buffer [10 mM
Tris-Cl (pH 8.0) and 1 mM EDTA, pH 8.0) and
quantified. Methylation analysis was done as described
(1)
. Briefly, 10 µg of genomic DNA from each cell line
were digested with EcoRI and methylation-sensitive
SacII restriction enzymes (Promega Corp., Madison WI),
electrophoresed on a 1% agarose gel, and transferred to Hybond N+
membrane (Amersham Life Sciences). The blot was hybridized with a
p16/CDKN2A probe that spans the CpG islands of the promoter
and exon 1 (19)
in Rapid-hyb buffer (Amersham Life
Sciences). The probe was labeled with
[
-32P]dCTP using the Rediprime kit (Amersham
Life Sciences) according to the manufacturer’s instructions. The human
prostrate carcinoma cell line TSU-PR1 was used as a positive
control for methylation.
Northern Analysis.
Northern blot analysis was used to detect p16 and
p14ARF mRNA in TCC 97-15, TCC 97-18 and TCC
97-29. RNA was isolated using the RNeasy mini kit (Qiagen) according to
the manufacturer’s instructions. Ten µg of total RNA from each cell
line were run on a denaturing 1% formaldehyde-agarose gel and
transferred to a Hybond N+ nylon membrane. Hybridization was done
overnight at 65°C. A whole-length p16/CDKN2A DNA was used
to detect p16 mRNA. The p14ARF probe was made by
reverse transcription of total RNA from normal HUCs using the RNA PCR
core kit (Perkin-Elmer). The primers for PCR have been described
previously (63)
. The blots were stripped and hybridized
with a ß-actin cDNA probe to verify equal loading. The HeLa cell line
was used as a positive control; MCF7 and A375 cell lines were used as
negative controls because neither of these lines contains
p14ARF.
Irradiation and Cell Cycle Analysis.
Logarithmically growing cell cultures were irradiated with 18 Gy of
gamma radiation at a rate of 6 Gy/min using a
137Cs source. Twenty-four h after irradiation,
the cell cycle arrest was analyzed in duplicate using standard flow
cytometric techniques (64)
. Briefly, cells were incubated
for 2 h with 20 µM BrdUrd (Sigma) and fixed in 70%
ethanol. These cells were permeabilized and incubated with a primary
antibody against BrdUrd (Caltag), followed by another incubation with a
FITC-conjugated secondary antibody against mouse IgG (Sigma). After
labeling with 50 µg/ml propidium iodide, these cells were analyzed on
a FACScan (Becton Dickinson). Cell cycle distribution was determined
using CellQuest software package (Becton Dickinson). Irradiated cells
were also analyzed using Western blot analysis as described above.
DNA Sequencing.
Exons 1 and 2 of p16/CDKN2A and exons 1ß and 2 of
p14 ARF were PCR amplified, and the PCR products
were cloned into a plasmid using the TOPO TA cloning kit (Invitrogen)
according to the manufacturer’s instructions and sequenced at the
University of Wisconsin Biotechnology Center using an automated DNA
sequencer. The primers used to amplify exons 1, 1ß, and exon 2
have been described previously (19, 46, 20)
. PCR reactions
were performed in 50-µl volumes using 250 ng of genomic DNA as
template, 200 mM deoxynucleotide triphosphates,
1.5 mM MgCl2, 1
µM of each forward and reverse primers and 5
units of AmpliTaq DNA polymerase. Formamide (5%; Sigma) was added to
all PCR reactions for exon 1. Reagents for PCR were obtained from
Perkin-Elmer. The PCR conditions for exon 1 and 1ß were: an
initial denaturation step of 3 min at 95°C, 35 cycles of 45 s
each at 94°C, 62°C, and 72°C, followed by a final elongation step
of 5 min at 72°C. The PCR conditions for exon 2 included initial
denaturation of 5 min at 95°C; followed by 30 cycles of 60 s at
94°C; 30 s each at 68°C, 65°C, 62°C, 60°C, and 58°C;
60 s at 72°C; and a final elongation step of 5 min at 72°C.
Initial identification of TCCs with mutations in exons 5–9 of TP53 was done by cleavage fragment length polymorphism analyses using the protocol of Third Wave Technologies (Madison, WI.). TP53 exons 2–11 were then sequenced at early passage in culture (P < 5) in all 12 TCCs used in this study by Oncor, now Genelogic (Gaithersburg, MD), according to their published protocol.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Western analysis was performed at early passage after establishment
of tumor biopsies in vitro. Normal HUCs were used as
controls. All experiments were repeated at least twice. Results showed
that p16 was undetectable in 7 of 12 TCCs with wild-type levels of pRb
(Fig. 1A
). In contrast, pRb was undetectable in three TCCs that
showed elevated levels of p16 (Fig. 1A
). These latter p16
levels were comparable with the p16 levels seen at senescence in normal
HUCs (Fig. 1A
). One tumor, TCC 97-24, had elevated levels of
p16 along with a relatively strong pRb signal. However, the pRb signal
was consistent with a hypophosphorylated state (Fig. 1A
).
The functional status of pRb was determined by immunoprecipitation of
pRb and associated E2F-1, followed by Western analysis of the
immunoprecipitates with antibodies to pRb and E2F-1. Results confirmed
that in TCC 97-24, pRb existed primarily in a hypophosphorylated state
but also showed that pRb did not bind E2F-1 (Fig. 1B
). Thus,
pRb in TCC 97-24 was apparently inactivated. Another TCC, 97-7, showed
very low presenescence levels of p16 and wild-type pRb levels, a
combination not previously identified in TCCs. In this TCC,
immunoprecipitation studies revealed that pRb exists primarily in the
hyperphosphorylated state, and the levels of E2F-1 were relatively low
(Fig. 1B
). Thus, p16 is present in this TCC in low
quantities but appears functionally inactivated with regard to
inhibiting pRb phosphorylation and cell proliferation.
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Next, Cdk4, Cdk6, and cyclin D1 levels were examined by Western analysis (data not shown). Several alterations were identified. These included a diminished signal for Cdk4 in TCC 97-1 and for Cdk6 in TCC 97-29. In all other TCCs, both Cdk4 and Cdk6 signals were comparable with the normal HUC wild-type control. Thus, no TCC in this series lost both Cdk4 and Cdk6, which might have served to inactivate the pRb pathway. The signal for cyclin D1 was diminished compared with normal in TCC 96-2. Cyclin D1 was not found amplified in any of the TCCs in the sample.
p53 Pathway Alterations Identified in Invasive TCCs.
Next, TP53 status was studied in all 12 invasive TCCs. Exons
2–11 of TP53 were sequenced to investigate the underlying
mechanisms for altered p53 levels. The data are summarized in Table 2
. Briefly, no mutations in exons 2–11 were identified in four invasive
TCCs. Mutations in hot spots (exons 5–8) were found in the other eight
TCCs. The mutation in TCC 96-2 was a splice site mutation, resulting in
protein loss (Fig. 2A
and Table 2
). All other TCCs with mutations show
significantly elevated p53 levels (Fig. 2A
). Two TCC
biopsies taken from the same patient at different times including
97-18-I, a biopsy of primary invasive TCC, and TCC 97-21-M, a biopsy of
a nodal metastasis, had the same mutation in exon 6, suggesting that
metastasis occurred after the p53 mutation event. As reported
elsewhere, clinical biopsies of human bladder cancers show major and
minor clusters of mutational hot spots in exons 5 and 6 and in exons 7
and 8, respectively (64)
. Codon 8 mutations are in the
helical motif of p53 that is involved in DNA binding, whereas codon 5
mutations are involved in structural integrity (64)
. As
discussed below, all of the TCCs in this study with TP53
mutations showed functional loss consistent with p53 inactivation.
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), consistent with mutational inactivation of p53 function
(also see below). This result is consistent with p53 functional
inactivation by these mutations (Table 2)
. All of the TCCs with
wild-type p53 showed an apparently wild-type level of
p21WAF1, except for TCC 97-1, in which no
p21WAF1 was ever detected. Thus,
p21WAF1 was considered altered in this tumor, and
it was classified as -p16/-p21WAF1.
To further investigate the mechanism for increased levels of both
wild-type and mutant p53, the levels of mdm2 were next examined. These
were elevated in all TCCs with mutant p53, except TCC 96-2 in which a
splice mutation resulted in loss of p53 (Table 2)
. However, mdm2 levels
were also elevated in two TCCs (TCC 94-10 and TCC 97-1) with slightly
elevated wild-type p53 (Fig. 2A
). Thus, there was not a
correlation between p53 levels and mdm2 elevation in TCCs with
wild-type TP53.
Finally, p14ARF expression was examined by
Western analysis. Results reproducibly showed that
p14ARF was present in all TCCs, except TCC 97-29
and TCC 97-15 (Fig. 2B
). Notably, TCC 97-15 and TCC 97-29
were two of three TCCs in which a p53 pathway alteration was yet to be
identified. Mechanism(s) underlying p14ARF loss
in these TCCs are described below in the section on INK4a
inactivation. In summary, a p53 pathway alteration was identified in 11
of 12 invasive TCCs, of which 8 had mutant TP53, 2 had
p14ARF inactivation, and 1 lost
p21WAF1 (Table 2
; Fig. 2
).
Mechanisms of Inactivation of the 9p21 INK4a Locus
Genes.
Molecular analyses were performed to identify mechanisms of
p16/CDKN2A and p14ARF/ARF
inactivation in the TCCs. First p16/CDKN2A inactivation was
examined. No p16 signal was detected in seven TCCs (Fig. 3A
). Southern blot analysis (using a probe spanning exon 1)
was used to determine p16/CDKN2A promoter inactivation by
methylation (65)
. Absence of any signal in two TCCs
indicated the presence of homozygous deletion of exon 1 (Table 2)
.
TCC 94-10 showed one methylated allele and one normal allele (Fig. 3A
). Sequence analysis also revealed a mutation in TCC 94-10
(see below). Because protein was lacking in 94-10, it seems reasonable
to hypothesize that one allele is methylated and one is mutated.
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and 2 of p16/CDKN2A was used to
detect mutations that could account for the loss of p16 from these
TCCs. Results showed that TCC 97-21-M had a frameshift mutation in exon
1, codon 36. The mechanism of inactivation of the second allele was
not identified. TCC 94-10 had a frameshift mutation in exon 2 that
caused a premature termination signal. TCC 97-15 had a homozygous
deletion of exon 2, as confirmed by PCR using primers spanning exon 2
of p16/CDKN2A (data not shown). In TCC 97-18-I and TCC
97-29, no mutations were detected in exon 1 and exon 2. Northern
analysis was performed to determine whether p16 mRNA was present in
these lines. p16 mRNA was detected in TCC 97-18-I (which lacked p16
protein; Fig. 3C
), suggesting there might be some
posttranscriptional regulation in this line that would account for the
loss of the protein. The absence of p16 mRNA in TCC 97-29 was
consistent with p16 loss; however, a mechanism underlying the loss of
mRNA in this line could not be identified. In summary, mechanisms for
p16 inactivation were identified in six of seven TCCs that lost p16
(Table 2)
.
Loss of p14ARF was reproducibly demonstrated in
two TCCs, TCC 97-15 and TCC 97-29, using Western analysis (Fig. 3C
), but no mutations in exon 1ß could be identified in
either TCC by sequencing (Table 1)
. However, loss of
p14ARF expression in TCC 97-15 could be explained
by a homozygous deletion of exon 2 that is shared between
p16/CDKN2A and ARF (described above). Northern
analysis was performed to study ARF mRNA in the other TCC
lacking p14ARF, i.e., TCC 97-29. HeLa
and A375 cells were used as standard positive and negative controls,
respectively (61)
. No signal for ARF mRNA was detected in
TCC 97-29 (Fig. 3C
). This latter finding is consistent with
the lack of p14ARF by Western analysis in this
TCC. In contrast, ARF mRNA was identified in representative
TCCs in this study that showed protein by Western analysis,
i.e., TCC 92-1 and TCC 97-18-I.
In summary, molecular analyses identified mechanisms by which p16 and
p14ARF were inactivated in most of the TCCs
analyzed in this study. The exception was TCC 97-29 in which a
mechanism for the loss of mRNA (and subsequently protein) was not
identified (Table 2)
.
Different Biological Phenotypes Associated with Different
Combinations.
Several biological phenotypes in response to radiation were studied in
TCCs with different combinations of p53 and pRb pathway alterations.
Induction of p53 and its downstream targets, mdm2 and
p21WAF1, were examined after generation of 18 Gy
gamma radiation-induced DNA damage in the four TCCs with wild-type
TP53 and in three representative TCCs with mutant
TP53. Unirradiated TCCs and normal HUCs were used as
controls. Twenty-four h after irradiation, proteins were examined using
Western blot analysis (Fig. 4
). Briefly, p53 and mdm2 were elevated in TCCs with wild-type
TP53 after radiation. However, induction of
p21WAF1 was observed in only two TCCs,
i.e., TCC 97-29 (Fig. 4)
and TCC 97-15 (data not shown). One
TCC with wild-type p53 did not contain detectable
p21WAF1. Finally, one TCC with wild-type p53, TCC
94-10, contained p21WAF1 but did not show
induction after radiation. This was the only tumor in which no p53
alteration had yet been identified. As expected, none of the TCCs with
mutant p53 showed induction of and elevated levels of p53, mdm2, or
p21WAF1 in response to gamma radiation (Fig. 4)
.
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). As expected, TCCs with mutant TP53 failed to undergo cell
cycle arrest. The two TCCs (TCC 94-10 and TCC 97-1) with wild-type p53
lacking a p21WAF1 induction response after
irradiation (as described above), also failed to undergo cell cycle
arrest. However, two other TCCs, TCC 97-29 and TCC 97-15, with the
combination of -p16/-p14ARF that showed
downstream induction of p21WAF1, underwent cell
cycle arrest as indicated by a reduction in the percentage of S-phase
cells after radiation (Fig. 5)
. Thus, loss of
p14ARF in TCC 97-15 and in TCC 97-29 did not
affect their ability to show p53 downstream responses or to undergo
cell cycle arrest after radiation exposure.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This is the first study to our knowledge showing that both wild-type
p53 and wild-type p14ARF can be expressed
simultaneously in malignant tumors. Earlier models proposed that a
negative feedback loop exists between p53 and
p14ARF (49)
. Consistent with this
model, it was reported that tumor cell lines that expressed wild-type
p53 did not express p14ARF (49)
. In
contrast, the data presented herein show that
p14ARF and wild-type p53 can be present together
in invasive human cancers. The difference may be accounted for by the
use of early-passage cultures of tumor biopsies in the current study
rather than established cell lines. It has been shown that
p14ARF stabilizes both p53 and mdm2 by binding
and inhibiting the mdm2-induced turnover of p53 (49)
. Our
studies are somewhat consistent with this model. We showed that 10 of
12 TCCs that expressed p14ARF also had elevated
levels of mdm2 and p53 compared with normal. However, two TCCs that did
not express p14ARF showed comparatively lower
elevations of p53 and mdm2 (Figs. 2A
and 4). Thus, the
correlation is not perfect in this study.
Our data differ from results obtained in a recent analysis of bladder cancer cell lines showing that bladder cancers with wild-type TP53 or TP53 alterations in exons 1–4 (which are required for transactivation) always show alterations in p16/CDKN2A and ARF (2) . In fact, in the present study, TCC 97-1 and TCC 94-10, both with wild-type TP53, did not show any alterations in ARF at either the molecular or protein levels. p21WAF1 was absent in one of these tumors, but no p53 pathway alteration was detected in the other TCCs.
p21WAF1 is clearly a critical component of the downstream p53 pathway. Nonetheless, mutations in p21WAF1 are infrequently reported in cancers, including bladder tumors. However, in the small percentage of tumors that do harbor such mutations, no coexisting TP53 mutations have been identified (35) , suggesting that when p21WAF1 mutations occur, they are powerful enough to inactivate the p53 pathway. In our panel, 1 of 12 invasive TCCs, TCC 97-1, failed to show any signal for p21WAF1 by Western blot analysis and failed to undergo cell cycle arrest after radiation exposure. Thus, we assume that p21WAF1 is altered in this one TCC. Notably, there was one invasive TCC in which no p53 pathway alteration was detected. In summary, our study shows that most, but not all, invasive TCCs contain an inactivating alteration in p53, p21WAF1, or p14ARF.
We reported previously that invasive TCCs invariably have a pRb pathway alteration by inactivation of either p16 or pRb. Consistent with this previous observation, inactivation of the pRb pathway was observed in all TCCs in the current study. Loss of p16 was the most common pRb pathway alteration identified. Inactivation of pRb was identified in 4 of 12 samples. Interestingly, one TCC with wild-type pRb in the present study showed significantly decreased p16, rather than absent p16, as did the other TCC with pRb loss. We did not identify a mechanism for this apparent p16 down-regulation, but we did demonstrate that these low presenescent levels of p16 were insufficient to block pRb phosphorylation or cell proliferation in this TCC. Thus, most studies, including our own, show that a pRb or p16 alteration in the pRb pathway is almost always present in invasive human cancers. Nevertheless, the possibility of cancers with neither alteration still exists. Mutations in other critical components of the pRb pathway, such as CCND1, CDK4, or CDK6, could provide alternate mechanisms for bypassing the p16-mediated G1 senescence checkpoint, as discussed in the "Introduction." However, the relative importance of alterations in these other components of the pRb pathway and whether they can substitute in a small percentage of TCCs for a p16 or pRb alteration have not been determined.
As discussed above, much data support a model in which inactivation of
the 9p21 p16/CDKN2A tumor suppressor gene plays a critical
role in tumorigenesis in vivo and immortalization in
vitro of human cells. However, the more recent identification of a
second putative tumor suppressor gene at the INK4a locus,
ARF, led to the hypothesis that p14ARF
may be the more critical 9p21 tumor suppressor gene and/or that both
genes may be frequently inactivated concomitantly. Indeed, recent
studies demonstrate that p16 and p14ARFl losses
can occur concomitantly by a single alteration at the 9p21
INK4a locus. We observed this phenomenon in our TCC samples.
For example, in TCC 97-15 (and possibly TCC 97-29), a single mutation
at the INK4a locus targets p16 and
p14ARF, altering both pRb and p53 pathways.
However, such concomitant inactivation is clearly not obligatory, as
has been proposed. Specifically in our study, loss of p16 but not
p14ARF was observed in six of eight TCCs.
Furthermore, our study shows that homozygous deletions can inactivate
one INK4a gene but not the other. For example, TCC 97-1 and
TCC 96-1 lost p16 because of homozygous deletion of exon 1 but
expressed p14ARF. An even more powerful example
in this regard is TCC 94-10. In this TCC, p16 was inactivated by two
different mechanisms (promoter methylation and an exon 2 frameshift).
Because there is no p16 in this tumor, it is reasonable to propose that
each of these alterations is on a separate allele. Notably, neither the
exon 2 frameshift mutation nor the p16/CDKN2A promoter
methylation would affect p14ARF
(65)
. p16/CDKN2A- and ARF-specific
mRNA were absent in TCC 97-29. However, no mutations were identified in
exons 1, 1ß, or 2 of either p16/CDKN2A or
ARF. Thus, a mechanism for the loss of p16 and
p14ARF mRNAs in this TCC was not identified.
However, such loss could be attributable to several other mechanisms,
such as a mutation in the poly(A) site. Thus, these results elucidate
both the importance and complexity of the INK4a locus in
human bladder cancer pathogenesis.
Although most TCCs appear to have some alteration in both the pRb and
p53 pathways (Ref. 2
and our data), the impact of the
different possible combinations on tumor phenotype in vivo
(Table 1)
and biological phenotypes in vitro (as discussed
below) could differ and has not been reported. Many studies have
hypothesized that p53 status is the most useful prognostic indicator to
assess potential biological phenotypes in vivo, including
tumor recurrence, progression and metastases, and patient survival.
Significant data show that assessment of pRb status can alone serve as
an excellent prognostic indicator (66–68)
. Finally, more
recent studies suggest that alterations in pRb and p53 exert a
cooperative, synergistic effect on survival rates and bladder cancer
progression, and both markers should be used in screening assessments
(28)
. Interestingly, our data show that all TCCs with
altered pRb have altered p53 (Table 1)
. The combination of mutant pRb
and wild-type p53 was not observed by us, nor to our knowledge has it
been reported by others for human bladder cancer. Thus, our data
support studies showing that TCCs with altered pRb have a poor
prognosis but attribute this to the combination of altered p53 and pRb.
Supporting this hypothesis in another human epithelial cell type are
data showing that patients with early-stage, non-small cell lung
carcinoma with altered pRb and mutant p53 have relatively poor clinical
prognoses (69)
. Furthermore, mice mutant for pRb and p53
have reduced viability and exhibit increased tumor burden and
metastasis (70)
. In contrast to the TCCs with p53
alterations, TCCs with wild-type p53 and an alteration of another p53
pathway component (p14ARF or
p21WAF1) showed more favorable prognosis, even in
our small sample, consistent with the above clinical observations.
These data support the importance of a large clinical study to test the
statistical significance of this hypothesis.
We also examined some underlying mechanisms that might help to explain
the different biological phenotypes of the TCCs with different pRb and
p53 pathway alterations. Specifically, we studied the response of the
12 TCCs in our study to the DNA-damaging effects of gamma radiation.
Our control normal HUCs showed downstream induction of p53,
p21WAF1, and G1 cell cycle
arrest in response to radiation damage. As expected, none of these
p53-dependent responses were seen in TCCs with mutant p53 (Table 2)
.
Loss of the cell cycle arrest component was also seen in TCC 97-1 in
which p21WAF1 was never detected. Cell cycle
arrest repeatedly did not occur in TCC 94-10 in which p53 and mdm2, but
not p21WAF1, were elevated after radiation
exposure. The reason for this result is being investigated. On the
basis of many studies, loss of the cell cycle arrest response to DNA
damage is associated with failure to repair DNA damage and the
potential for the accumulation of genetic alterations needed for
progression. Consistent with that model, even in this small set of 12
TCCs, 2 that retained the p53 downstream responses stand out as the
least aggressive. The apoptotic responses and cell kill of this group
of tumors after exposure to gamma radiation would be relevant and of
interest to planning chemotherapy and radiation therapy.
A significant new finding in this study is that the TCCs with wild-type p53 and pRb and with the genotype of -p16/-p14ARF were phenotypically similar to normal HUCs in their downstream induction of p53, mdm2, p21WAF1, and G1 cell cycle arrest in response to radiation damage in vitro. Thus, there was an alteration in the p53 pathway in these TCCs, but the pathway was not inactivated by the criteria used in our study. However, wild-type TP53 alone was not always sufficient to predict downstream p53 functions of cell cycle arrest in response to DNA damage. One TCC, TCC 94-10 with wild-type p53, failed to show a p21WAF1 elevation after radiation. Studies on breast cancer show that overexpression of mdm2 increases the de novo resistance of cells to cytotoxic agents like doxorubicin. Therefore, mdm2 level is considered to be a novel marker for predicting lack of response to doxorubicin treatment in breast cancer patients (42) . A second tumor with wild-type p53, TCC 97-1, in which p21WAF1 was absent showed p53 and mdm2 induction and also failed to undergo cell cycle arrest in response to radiation. Thus, in these latter two tumors, some of the protective functions of p53 are lost.
In summary, our data contribute to the understanding of the genetic requirements for bladder cancer pathogenesis. We have identified two pRb and p53 pathway combinations not yet reported for human bladder cancers, i.e., -p16/-p21WAF1 and -p16/-p53. We showed that some TCCs have both wild-type p53 and wild-type p14ARF. Thus, we show that all TCCs do not have either a p53 or p14ARF alteration, as had been hypothesized previously for some tumor types. We identified a TCC with wild-type p53 but with p21WAF1 loss in which downstream responses to radiation were lost. We show that in one TCC, a common p53 pathway alteration (i.e., p53, p21WAF1, or p14ARF) was not present. Finally, we show for the first time in TCCs that invasive cancers with p53 pathway inactivation by p14ARF alteration retain downstream responses to gamma radiation. Thus, although p14ARF loss apparently fulfils the requirements for a p53 pathway alteration, the components of the p53 damage response pathway are intact. These new findings have potential impact on the use of pRb and p53 pathway alterations for diagnosis, prognosis, and treatment of human bladder cancers.
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FOOTNOTES |
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1 This work was supported by Grants RO1 CA29525
and RO1 CA67158 (to C. A. R.) and a pilot grant (to S. S.) from the
University of Wisconsin Comprehensive Cancer Center. 
2 To whom requests for reprints should be
addressed, at Wildrose Foster Children’s Home, 733 Lakewood Boulevard,
Madison, WI 53704. Phone: (608) 245-0793; Fax: (608) 265-8133;
E-mail: crezniko{at}facstaff.wisc.edu
3 The abbreviations used are: Cdk,
cyclin-dependent kinase; ARF, alternate reading frame; TCC,
transitional cell carcinoma; IHC, immunohistochemistry; HUC, human
uroepithelial cell; BrdUrd, bromodeoxyuridine.
Received 11/18/99. Accepted 5/10/00.
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