P53 point mutations in initial superficial bladder cancer occur only in tumors from current or recent cigarette smokers
Hélène LaRue,
Pierre Allard,
Maryse Simoneau,
Claire Normand,
Christian Pfister,
Lynne Moore,
Franciois Meyer,
Bernard Têtu and
Yves Fradet1
Centre de recherche en cancérologie de l'Université Laval, Pavillon L'Hôtel-Dieu de Québec, Centre Hospitalier Universitaire de Québec, 10 MacMahon, Québec G1R 2J6, Canada
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Abstract
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Sequencing of p53 exons 58 was carried out on 51 initial superficial bladder tumors selected on the basis of high grade and/or p53 overexpression (immunohistochemistry without antigen retrieval). Fourteen point mutations in 13 tumors and one 21 bp deletion in another tumor were identified. In addition, a germ-line mutation corresponding to a previously described polymorphism was detected in exon 6, in two tumors. Mostly G
A transitions (10) were found. Only three occurred at CpG sites, suggesting a major role for exogenous carcinogens in bladder tumorigenesis. Immunostaining for p53 and MDM2, using antigen retrieval, was carried out on the same tumors. A correlation was found between the percentage of p53-positive cells and the presence of p53 mutations (P = 0.005). No correlation was found between overexpression of p53 and MDM2 in this selected cohort of mostly high grade tumors. The presence of p53 mutations was also analyzed as a function of the smoking habits of the patients. A significant association was found between the presence of p53 point mutations and the number of years of smoking (P = 0.043). All patients with tumors carrying missense or nonsense p53 mutations had smoked for
30 years and if former smokers, had stopped for
5 years. However, no correlation was found between the presence of p53 point mutations and the number of cigarettes smoked. The deletion mutation was the only one present in a tumor from a non-smoker. The data suggest that duration of exposure to carcinogens is the most critical factor in p53 mutagenesis in bladder cancer.
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Introduction
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Mutations of the p53 tumor suppressor gene are present in approximately 50% of all human cancers and are the most frequent genetic alteration known in human cancers. The p53 protein can also be inactivated by mechanisms other than mutation, for example by complexing with viral or cellular proteins such as MDM2. It is clear that the malfunction of this protein plays a central role in the development of cancer. p53 acts as a transcription factor and is implicated in the regulation of the cell cycle and consequently in growth control (1). The current view of the p53 role in the cell suggests that it acts as the guardian of the genome, sensing damage or potential damage to the DNA and invoking a protective response either by blocking the cell cycle or by inducing apoptosis in the affected cell. Expression of high levels of wild-type p53 arrests the cell cycle in G0/G1 phase in response to DNA damage, allowing more time for DNA repair mechanisms. Loss of this gene function allows the propagation of cells with genetic damage and is a key step in the development of neoplasia (2).
p53 mutations occur in most tumor types. Up to 87% occur in exons 58 and 75% at G:C pairs (3). The frequency and type of mutations, however, tend to be unique to each cancer (35). The spectrum of mutations in tumors can provide clues to their etiology; mutations may be caused by both endogenous (spontaneous) biological processes and exogenous (environmental) carcinogens (6,7). Endogenous DNA alterations include the spontaneous deamination of mC at mCpG dinucleotides, resulting in G:C
A:T transitions (7). Carcinogens, on the other hand will often produce transversions; carcinogen-induced mutations occur preferentially on the non-transcribed strand (3).
The half-life of mutated p53 is increased and, consequently, the protein reaches a level detectable by immunohistochemistry methods which thus provide the simplest way of detecting p53 mutations. However, this approach is not entirely reliable as some mutations will abolish p53 expression while the protein can be stabilized by mechanisms other than mutations. In addition, antigen retrieval approaches have so increased the sensitivity of detection that in some circumstances, normal unmutated p53 can be detected (8).
In bladder cancer, p53 overexpression has been associated with a poor prognosis (912). In general, a good correlation has been observed between overexpression of the protein and the presence of mutations (1315). However, the concordance is not perfect suggesting that other mechanisms are also involved in the stabilization of this protein in bladder cancer. The MDM2 protein is known to bind to p53, blocking its transcription activation activity and targeting it for degradation (16). mdm2 transcription is known to be activated by p53. Thus, in normal conditions, an autoregulatory feedback mechanism exists between the two proteins by which p53 limits its own activity. We would therefore expect that MDM2 over-expression precludes alterations of p53 such as mutations or overexpression. Consistent with this model, a study on p53 and mdm2 in soft tissue sarcomas found no tumors containing alterations of both genes (17). In non-small cell lung carcinomas and in melanomas, MDM2 overexpression has been associated with p53 overexpression in the absence of p53 mutations (18,19). In bladder cancer, Lianes et al. (20) and Schmitz-Dräger et al. (21) found an association between overexpression of both proteins while Barbareschi et al. (22) detected immunoreactivities for each protein mostly in different subsets of tumors.
The pattern of p53 mutations has also been studied in bladder cancer. While many researchers have found a preponderance of G
A transitions (3), others have found mostly transversions (6,23,24). Epidemiological studies associate a 23-fold increased risk of developing bladder cancer for smokers than for non-smokers with 2560% of bladder cancers being related to tobacco-smoking (25,26). However, a correlation between p53 mutations and cigarette smoking has not yet been conclusively demonstrated. Kusser et al. (24) found no correlation between the presence of p53 mutations and smoking in bladder cancer, whereas Husgafvel-Pursiainen and Kannio (27) found a non-significant association between the two and Spruck et al. (23) suggested an association of cigarette smoking with the presence of double mutations. An association has, however, been reported between p53 overexpression and the number of cigarettes smoked per day (28).
We reported previously a low frequency of p53 overexpression, detected with a polyclonal antibody, in 15% of tumors in a cohort of 382 initial low-stage superficial bladder cancers (29). The p53-positive tumors were mostly of high grade and associated with shorter time to recurrence. A detailed history of smoking habits is available for these patients and it was observed that the smokers and past smokers presented a higher proportion of recurrences although smoking status was not correlated with time to first recurrence (30). We reasoned that these early tumors would provide a good opportunity to establish a possible association between smoking habits and p53 mutagenesis in bladder cancer. In the present study, we selected from this cohort all the grade III tumors for which we had frozen tissue to analyze p53 mutations by sequencing and correlate the findings with smoking habits. We also included in this study all the grade I and II tumors which had stained positively with the polyclonal anti p53 antibody. Moreover, p53 mutations were also correlated with p53 overexpression by immunostaining using antigen retrieval technique. MDM2 immunostaining was carried out to determine possible mechanisms of p53 overexpression.
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Materials and methods
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Tumors
Fifty-one bladder tumor patients were selected from a previously described cohort of patients with initial superficial tumors (29 T1, 21 TA, one undefined TA or T1) (30). For each patient, snap frozen and formalin-fixed tumoral tissue was available. As a correlation of p53 overexpression with high grade was observed in most studies, we selected for sequencing all the grade III tumors for which we had frozen tissue (36 tumors). We also included in the study all the grade I (two tumors) and II (13 tumors) tumors which stained positively with a polyclonal anti-p53 in a previous study (29).
Smoking data
The cigarette smoking history was obtained at the time of initial diagnosis, using a standardized questionnaire. Patients were classified as current smokers, past smokers or non-smokers. Information gathered included age at which smoking was initiated, time since stopping, as well as number of cigarettes smoked per day during the last week and during the last year of smoking.
DNA extraction
Genomic DNA was prepared from tumor specimens by proteinase K digestion of frozen 20 µm tissue sections or cell suspension as described previously (31). Blood leukocyte DNA was prepared from 200 µl whole blood also by proteinase K digestion (32). DNA concentrations were determined by the fluorometric diaminobenzoic acid assay (DABA).
DNA sequencing
Genomic DNA was amplified by nested PCR using the p53-specific primers shown in Table I
. The oligonucleotide primers were synthesized on an Applied Biosystems 391 DNA synthesizer excluding the biotinylated primers (General Synthesis and Diagnostics, Ontario, Canada). The first round of amplification was carried out on 100 ng of genomic DNA in a 100 µl reaction containing 50 mM KCl, 10 mM TrisHCl (pH 8.3), 2.0 mM MgCl2 (1.5 mM for exon 8), 100 µg/ml gelatin, 200 µM dNTPs, 50 pmol of each primer and 2.5 U of Taq DNA polymerase (Gibco BRL, Burlington, Ontario, Canada). For exon 7 amplification, 500 pmol of the sense primer was used. The reaction mixtures were subjected to 40 cycles of amplification in an automated thermocycler (Perkin-Elmer Cetus DNA thermal cycler), where each cycle consisted of 94°C for 1 min, 52°C for 1 min and 69°C for 2 min. The last cycle was followed by a final incubation at 69°C for 10 min. Negative controls containing no template DNA were included. For the second round of amplification, 1 µl of the first PCR reaction was used and amplification carried out using 10 pmol of biotinylated and non-biotinylated primers. Reaction conditions were as above except for the MgCl2 concentration which was set to 1.25 mM for all exons. Amplification reactions were carried out at least in duplicates and subsequently pooled to avoid sequencing mistakes due to PCR errors. The amplified DNA was then sequenced using fluorescent primers, listed in Table I
, on a Pharmacia A.L.F.TM automatic sequencer at the Montréal Cancer Institute. All scans were visually inspected and ambiguous cases were manually resequenced by the dideoxy method (T7 sequencing kit; Pharmacia Biotech, Baie d'Urfé, Québec, Canada) after purification of the amplified DNA using the QIAquick PCR purification kit (Qiagen, Mississauga, Ontario, Canada).
Immunohistochemistry
Both p53 and MDM2 staining were performed on formalin-fixed, paraffin-embedded sections using an antigen retrieval approach. Unmasking of both antigens was carried out in prewarmed 10 mM citrate buffer pH 6.0 by microwave treatment for 17 min at full power in a 900 W microwave oven for p53 and for 5 min in a pressure-cooker in the microwave oven for MDM2. For MDM2, endogenous peroxidase was inhibited by incubation in 3% H2O2 for 5 min. Monoclonal antibodies against p53 (1801, AB-2, 0.2 µg/ml; Oncogene Research Products, Calbiochem, Cambridge, MA) and MDM2 (IF2, AB-1, 2 µg/ml; Oncogene Research Products) were incubated at room temperature, overnight. Incubation with the second antibody (Signet multi-species ultra streptavidin detection system HRP; Dedham, MA) was for 20 min at room temperature. Diaminobenzidine (Zymed Laboratories, South San Francisco, CA) was used as the chromogen. Sections were counterstained with Mayer's hematoxylin. A negative control without the first antibody and appropriate positive controls (VMCUB3 bladder cancer cells for p53 and a positive colon tumor sections for MDM2) were included in each staining batch. Immunohistochemical evaluation was done by two independent investigators (H.L. and B.T. for p53, C.P. and B.T. for MDM2) who estimated the percentage of cells showing nuclear staining. Only intense nuclear staining was considered as positive and cytoplasmic staining was not considered as positive. The immunohistochemical analysis was performed in a blind fashion, without knowledge of the clinical data.
Statistical analysis
The relation between p53 overexpression and the presence of mutations and between p53 and MDM2 overexpression was analyzed by the
2 test. Correlation between the presence of p53 mutations and cigarette smoking habits was analyzed by the Wilcoxon rank sum test as the distribution of values was not normal. All reported P-values are two-sided.
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Results
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DNA sequencing
Sequencing of p53 exons 58 was carried out on 51 superficial bladder tumors. Sixteen single base changes and one 21 bp deletion were identified and are listed in Table II
. The mutations found were mostly transitions, with a marked strand bias: 10 G
A, three A
G and one C
T. Three mutations occurred at CpG sites: codons 175 (patients 2 and 3) and 248 (patient 9). Only two transversions were identified: G
C (patient 12) and A
T (patient 16). Three silent mutations were found. Two occurred at codon 213 in exon 6 (patients 6 and 7) and correspond to a previously reported rare polymorphism (33). Sequencing of blood leukocyte DNA confirmed that these were germ line mutations. The other silent mutation occurred at codon 287 in exon 8 (patient 15) and was associated with another mutation at codon 285. It thus appears to represent a case of multiple mutations in the presumed hot spot for p53 mutations in bladder tumors in the region of codons 280285 (34). Note that even if our total number of cases is low, we observed six point mutations out of 14 in this region (excluding the germ line mutations). As reported previously (34), most mutations occurred at G sites (11/16). A 21 bp deletion was detected in one tumor (patient 5) between codons 132138. This in-frame deletion would produce a protein missing seven amino acids. The presence of a direct repeat (CAAGA) at the 5' end of the deletion and immediately downstream of it suggests a mechanism of replication error for this mutation.
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Table II. Characteristics of tumors containing a p53 mutation. p53 and MDM2 staining were carried out on formaldehyde fixed sections, using antigen retrieval
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p53 and MDM2 immunostaining
Immunostaining using the anti-p53 monoclonal antibody 1801 and antigen retrieval technique was carried out on the 51 selected tumors. A clear correlation was found between the percentage of p53-positive cells and the presence of p53 mutations by
2 analysis (P = 0.003) (Table III
). No mutants were found in tumors with no positive cells. Three tumors (17%) with 0.55% p53-positive cells carried a p53 point mutation. One of these was a stop mutation (patient 16). Eleven tumors (55%) with >5% p53-positive cells carried a p53 mutation while eight tumors (57%) with >20% p53-positive cells carried a p53 mutation. This observation thus suggests a threshold of >5% positive cells, under these conditions, for defining a p53-positive tumor. Despite the correlation observed, two p53 mutants (other than the stop mutant) were found in the
5% category while nine tumors in which no p53 mutations were found fell in the >5% category, six of these expressing p53 in
30% of cells. A particularly puzzling situation occurred in patients 13, 14 and 15 who all carried the same mutation (G
A) in codon 285 (glu
lys) in their bladder tumor. Tumors 13 and 14 overexpressed p53 as expected while in tumor 15, only 12% of cells were p53 positive. This last tumor was the previously mentioned double mutant. However, the second mutation being silent, it cannot account for the absence of expression. This suggests that other damage exists elsewhere on the p53 gene or pathway.
In that perspective, we carried out MDM2 staining on the same tumors (Tables II and IV
). Twenty-six of 51 tumors were MDM2 positive (
20% stained nuclei) (20). Using the previously defined threshold of >5% positive cells for p53, we attempted to correlate p53 and MDM2 staining using the
2 test (Table IV
). No correlation was found. Ten tumors were found to express both antigens; of these, five (50%) carried a p53 mutation. Ten p53-positive tumors were MDM2 negative; of these, six (60%) carried a p53 mutation. We detected no p53 mutation in nine of these p53-positive tumors; of these nine tumors five overexpressed MDM2. Approximately 50% of the p53-negative tumors overexpressed MDM2 (16/31).
p53 mutations as a function of cigarette smoking
Complete information on smoking habits was available for 46 patients while partial information was available for the remaining five. We could thus analyze the presence of p53 mutations in relation to the smoking habits of the patients. With the exception of the deletion mutation which was found in a tumor from a non-smoker, all other p53 mutations were found in tumors from current or past smokers. When patients were classified according to their cigarette smoking status, all patients with a p53 mutation in their bladder tumor (with the exception of the deletion mutation) were found to have smoked for
30 years or, if past smokers, had stopped smoking for
5 years (Tables V and VI
). On the other hand, all patients with wild-type p53-tumors were non-smokers or had quit smoking for >5 years. There is a statistically significant correlation between the presence of p53 point mutations and the number of years of smoking (Table VI
). Patients without p53 mutations in their tumors had smoked for a median time of 34.5 years (066 years) while patients with p53 point mutations had smoked for a median time of 49.5 years (3067 years). Past smokers with a wild-type p53 tumor had stopped smoking for a median time of 15 years (0.236 years) compared with only 3 years (0.25 years) for those with a p53 point mutation (Table V
). Also all patients with p53 point mutations had smoked more than 200 000 cigarettes before their first tumor event (Figure 1
). However, no significant association was found between the presence of p53 mutations and the number of cigarettes smoked, as the five heaviest smokers (more than 600 000 cigarettes) carried no p53 mutations in their tumor. Three of these heavy smokers had stopped smoking for >5 years (for 6, 8 and 15 years, respectively) while the other two were current smokers, having smoked for 35 and 61 years, respectively.
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Table V. p53 point mutations in tumors according to smoking history of the patient as a function of smoking status
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Table VI. p53 point mutations in tumors according to smoking history of the patient as a function of number of years of smoking or having stopped smoking
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Fig. 1. p53 mutations as a function of the total number of cigarettes smoked until the first tumor event.
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No significant association could be made between p53 overexpression and smoking habits though only 7/26 (27%) of tumors from non-smokers and long-time past smokers (>5 years) overexpressed p53 compared with 12/24 (50%) in tumors from the smokers and short time non-smokers (
5 years). The bladder tumor carrying the deletion mutation is included among the tumors from non-smokers overexpressing p53.
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Discussion
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In this study, we have analyzed the presence of p53 mutations in a group of mostly high grade superficial bladder tumors, both by immunohistochemistry and by DNA sequencing. A significant association between the presence of p53 mutations and p53 overexpression was observed when using antigen retrieval and monoclonal antibody 1801. In our previous study (29) using a rabbit polyclonal primary antibody, and no antigen retrieval, although more mutants were found in the p53-positive tumors (8/27, 30%) than in the p53-negative ones (3/17, 17%) the difference was not statistically significant. In addition, under those conditions, patient 16's tumor presenting a stop codon at position 305 was p53-positive. However, the truncated protein derived from such a mutant p53 gene could not possibly be nuclear as it lacks the nuclear localization domain encoded by codons 316325 (35). In the conditions used in the present study, this tumor contained <5% p53-positive cells. The correlation between p53 mutations and protein accumulation was thus better with the use of the monoclonal antibody 1801 than the polyclonal antibody that we used in the past. To our knowledge, such a comparative study has not been reported previously although the 1801 monoclonal antibody has been reported to better predict survival than the polyclonal CM1 antibody (36). Even with the monoclonal anti-p53, however, we observed p53 overexpression in the absence of detectable mutations. This overexpression could result from a defective MDM2 as it is known that by binding to the p53 protein, MDM2 targets it for degradation and that the auto-regulation feedback mechanism existing between the two proteins help maintain their level. We found no correlation between the expression of the two proteins in this group of high grade tumors and thus cannot conclude that MDM2 plays a role in p53 overexpression in tumors without p53 mutations. However, an immunohistochemical study of p53 and MDM2 expression on a larger group of tumors from the same cohort found a correlation between expression of these two proteins but only in low grade tumors (37). In that same study, it was noted that p21 was less frequently expressed in tumors carrying a mutated p53 gene. As only exons 58 were sequenced, it is possible that mutations lie elsewhere on the gene as it has been reported that in bladder cancer, up to 28% of mutations may lie outside of these exons (3). Thus, while immunohistochemistry using an antigen retrieval technique and a monoclonal antibody remains a simple and efficient way of detecting p53 mutations, it is clearly not capable of detecting all such mutations.
The pattern of point mutations (excluding the germline mutations) found in this study is remarkably homogeneous, with 10/14 (71%) being G
A transitions. Of these, only three occurred at CpG sites. A predominance of G
A transitions has been reported in other studies though usually at a lower frequency (3). It probably reflects the homogeneity of our population of low stage initial tumors. The pattern of mutations and the strand bias observed are compatible with the action of carcinogens such as nitrosamines and thus with a tobacco-related disease (3). A predominance of G
A transitions has also been described in oesophagus and head and neck cancers, also related to tobacco smoking. In lung cancer, on the other hand, mostly G
T transversions are reported, suggesting the action of distinct carcinogens. These carcinogens could differ in solubility, nitrosamines being soluble while other inhaled products might not be. Such a hypothesis has been suggested for explaining different patterns of p53 mutations between cancers of the oral cavity compared with laryngeal and pharyngeal cancers (3).
We hypothesized that in early tumors such as those analyzed here an association between smoking habits and the presence of p53 mutations would be easier to demonstrate as there would have been less time for genetic damages to accumulate. Analysing the presence of p53 mutations in relation to the smoking habits of the patients, we found missense and nonsense mutations only in tumors from long-time smokers. A statistically significant correlation could be made between the presence of p53 point mutations and the number of years of smoking. In addition,we found no p53 mutations in bladder tumors from past smokers who quit smoking for >5 years. These observations suggest that time of exposure to carcinogens is a critical factor in p53 mutagenesis in bladder cancer and that p53 mutations are a late event in bladder carcinogenesis. Note that a study of smoking-related carcinogenDNA adducts in biopsies from human urinary bladders detected no difference in the levels of these adducts in the urinary bladders of non-smokers and past smokers with at least 5 years abstinence (38). In head and neck cancers, no significant difference in the presence of p53 mutations among smokers and former smokers was found suggesting that, contrary to what is seen in bladder cancer, p53 mutations in these tumors are an early event (7).
On the other hand, we could not demonstrate an association between the presence of p53 point mutations and the total number of cigarettes smoked because, surprisingly, no p53 mutations were detected in tumors from the heaviest smokers: more than 600 000 total cigarettes smoked or 5075 cigarettes per day (Figure 1
). However, when we examined the period over which these patients smoked, we found it varied considerably, between 22 and 61 years. Moreover, three of these patients had quit smoking for >5 years. Thus, the time of exposure again appears to be the critical element in p53 mutagenesis in bladder cancer, even more so than the number of cigarettes smoked. At least one study has reported a link between cigarette smoking and the presence of double mutations in p53 (23). We found only one such double mutation in our population of tumors which occurred in codons 285 and 287, in the bladder tumor mutation hotspot, where Spruck et al. (23) found three of their five double mutants. We also found a 21 bp deletion in one tumor that is most probably explained by a replication error, due to the presence of direct repeats. It was the only mutation found in a non-smoking patient.
It has recently been suggested that cigarette smoking increases the risk of recurrence of bladder cancer through its effect on p53 (39), a hypothesis supported by our data showing statistical correlation between p53 mutation and the number of years of cigarette smoking in a group of high-grade superficial bladder tumors. Moreover, in the cohort of patients from which these tumors were selected, current smokers presented a higher proportion of recurrence than non-smokers (57 versus 47%) while p53 overexpression (by polyclonal antibody) was significantly associated with earlier recurrence (30). Taken together these data suggest that superficial bladder tumor patients who smoke cigarettes increase their risk of mutating p53 and of experiencing a recurrence or progression as was recently suggested by a retrospective study on disease outcome according to smoking status in bladder cancer patients (40). Cessation of cigarette smoking after the initial diagnosis of superficial bladder tumors may perhaps have an impact on the outcome of the disease. As the correlation of p53 overexpression with smoking habit is not as good as with the presence of DNA mutations, we can speculate that the mechanisms of inducing p53, other than through DNA mutation, are not related to cigarette smoking. In that same cohort of patients, we also observed a significant correlation between the presence of chromosome 9 anomalies in bladder tumors and recurrence. However, in that study, patients with a smoking history had a similar frequency of chromosome 9 anomalies as non-smokers (41).
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Acknowledgments
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The authors are grateful to Nancy Roberge, Colette Dufour and Geneviève Beaudry for technical assistance, and to Alan Anderson for careful reading of the manuscript. This work was supported by the National Cancer Institute of USA, grant #CA47526 and by a joint program from the `Fonds de la Recherche en Santé du Québec/Hydro-Québec'. B.T. is recipient of a research clinical scholarship of the `Fonds de la Recherche en Santé du Québec'. C.P. was supported by fellowships from l'Association Franciaise d'Urologie, le Groupement des Entreprises Franciaises dans la Lutte contre le Cancer, la Ligue Contre le Cancer de Haute-Normandie, and la Société Franciaise du Cancer.
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Notes
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1 To whom correspondence should be addressed Email: yves.fradet{at}crhdq.ulaval.ca 
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Received May 17, 1999;
revised August 23, 1999;
accepted September 27, 1999.