Japanese Journal of Clinical Oncology Advance Access originally published online on January 17, 2006
Japanese Journal of Clinical Oncology 2006 36(2):116-120; doi:10.1093/jjco/hyi225
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© 2006 Foundation for Promotion of Cancer Research
Cancer Genetics Report |
Association of the PIG3 Promoter Polymorphism with Invasive Bladder Cancer in a Japanese Population
1 Department of Urology, Kyoto University Graduate School of Medicine, Kyoto and 2 Department of Urology, Akita University of Medicine, Akita, Japan
For reprints and all correspondence: Osamu Ogawa, Department of Urology, Kyoto University, Graduate School of Medicine, 54 Shogoin Kawaharacho, Sakyo-ku 606-8507, Kyoto, Japan. E-mail: ogawao{at}kuhp.kyoto-u.ac.jp
Received September 13, 2005; accepted November 30, 2005
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Abstract |
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 TOP Abstract INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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PIG3 (p53-induced gene 3) is one of the targets of TP53 and is involved in apoptosis. The promoter of PIG3 contains a variable number of tandem repeats (VNTRs) of pentanucleotides (TGYCC)n (Y = C or T) and the number of VNTRs was reported to be correlated with the activation by TP53. In this study, the clinical significance of the PIG3 promoter VNTRs was analyzed in the bladder cancer patients using the genome DNAs from 338 controls and 273 bladder cancer patients. There was no significant difference in the allele frequency of the PIG3 promoter VNTRs between them. However, the presence of 14 or less repeats allele was associated with higher cancer grade (P = 0.038) and higher stage in relative risk (adjusted odds ratio = 2.31, 95% confidence interval = 1.05–5.90). These data suggested that the PIG3 promoter VNTRs was associated with generation of invasive bladder cancer.
Key Words: PIG3 • bladder cancer • VNTRs
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INTRODUCTION |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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Bladder cancer is the 5th most common malignancy in males in Western society, and the 10th most common cause of cancer death (1). In Japan, it is the 12th most common cancer in males and approximately 1.5 people per 1 000 000 die of the disease annually (2,3). Histologically, 90% of cases present transitional cell carcinoma (TCC), and >70% of the bladder TCC present as the superficial and papillary subtype (4,5). They are usually treated with endoscopic transurethral resection (TUR), although >50% of them suffer recurrences in the bladder 5 years after TUR and 5–20% of them progress to life-threatening muscle invasive TCC (4,5). Invasive TCCs are highly aggressive and about half of them will recur with distant metastasis even if radical treatment is performed.
Mutations in the p53 gene (p53), one of the best characterized tumor suppressor genes, are the most common genetic events in human malignancies, including the bladder TCC (6). Alterations in p53 are linked to a high stage, a high grade and a poor prognosis of the bladder TCC (7–11). TP53 acts as a transcription factor and is involved in the regulation of cellular proliferation and apoptosis under genotoxic conditions (12). Recently, PIG3 (p53-induced gene 3) was identified as a result of the serial analysis of gene expression using the colorectal cancer cell line DLD-1 overexpressing TP53, and was defined as one of the genes associated with apoptosis (13). Interestingly, the promoter region of PIG3 contains a variable number of tandem repeats (VNTRs) of pentanucleotide sequences (TGYCC)n (Y = T or C), which were directly bound to by TP53, and the PIG3 promoter VNTRs were reported to correlate with the activation by TP53 in vitro (14). The previous data that PIG3 was related with apoptosis in the cancer cells provided the hypothesis that possession of the short allele of the PIG3 promoter was associated with susceptibility to cancers because of its reduced activation by TP53. In this study, we explored the association between the PIG3 promoter VNTRs, and the susceptibility and disease status of the bladder TCC.
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SUBJECTS AND METHODS |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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SUBJECTS
A total of 273 patients with the bladder TCC who were treated at Kyoto University Hospital in Kyoto Prefecture between June 1990 and December 2002 were enrolled in this study. All materials from the patients which have been preserved were used, although not all TCC patients treated during study periods were included because materials or informed consents were not available. Clinical and histopathological information was obtained from patient charts and pathological reports. The cancer stage and grade were assigned according to the tumor-node-metastasis (TNM) staging system (15) and the World Health Organization (WHO) criteria (16,17).
A total of 338 native Japanese people in Akita Prefecture, who visited community hospitals for a routine health checkup, were recruited for the study as controls. All control subjects were checked by routine urinalysis with microscopic examination of the urine sediment, urinary cytology, prostate specific antigen and ultrasonography to rule out the presence of urinary tract cancers. Except for clear evidence of urinary tract cancers, no exclusion criteria were provided for the recruitment of controls.
The group of TCC patients comprised of 207 males and 66 females and the group of controls comprised of 269 males and 69 females. The mean age ± SD was 61.2 ± 12.7 and 66.3 ± 11.6 years, respectively. The gender distributions and the mean ages were not statistically different between both groups. This study was approved by the Institutional Review Board of the Kyoto University Graduate School and the Akita University School of Medicine. Written informed consent to participate in the study was obtained from each patient before surgery, according to the ethical guidelines.
GENOTYPING OF THE PIG3 PROMOTER VNTRS
DNAs were extracted from blood samples collected from subjects using a QIAamp Blood Kit (QIAGEN, Hilden, Germany) or by the standard method with proteinase K digestion followed by phenol/chloroform extraction. The fragment encompassing the PIG3 promoter region was amplified using the specific forward primer 5'-TGCGGTGCCAGCCTGAGGCT-3' and fluorescent dye 6-FAM labeled reverse primer 5'-TTCCGGTCCTCCCGGCTTGT-3'. PCRs were carried out in a 25 µl volume containing 20 ng of genomic DNA, a commercial 1x PCR buffer, 0.2 mM of each dNTP (dATP, dCTP, dGTP and dTTP), 1.5 mM MgCl2, 50 pmol of each primer and 1.0 U of Ampli-Taq DNA polymerase (Perkin-Elmer, Branchburg, NJ). After a 10 min initial denaturation step at 95°C, 35 cycles of PCR consisting of 95°C for 30 s, 68°C for 30 s and 72°C for 60 s were carried out, followed by a 7 min final extension step at 72°C in a thermal cycler (TaKaRa PCR Thermal Cycler MP: TaKaRa BIOMEDICALS, Kusatsu, Japan). After confirmation of successful PCR amplification with 2.0% agarose gel electrophoresis, PCR products were run on an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems-Roche, Branchburg, NJ), and allele sizes were assigned using the GeneScan software (PE Applied Biosystems-Roche). The number of (TGYCC)n repeats was calculated from the size of the PCR products in relation to a series of standards and confirmed by the direct sequencing of PCR products in representative cases or 14% PAGE and silver nitrate staining.
TP53 IMMUNOHISTOCHEMISTRY
The status of the p53 protein in cancer tissue was assessed with immunohistochemistry using an anti-p53 antibody (18). In brief, after the deparaffinization and blocking of endogenous peroxidase with hydroxyl peroxide, antigen retrieval was performed using a microwave oven. The anti-p53 mouse monoclonal antibody Do-7 (Novocastra, Newcastle-upon-Tyne, UK) was incubated overnight at a dilution of 1:100 with specimens at 4°C. Staining was achieved using the Dako LSAB kit (Dako, Carpinteria, CA). Tumors with >10% immunoreactivity in nuclei were judged as positive stainings and defined as TP53 mutant-type cancers, and otherwise defined as TP53 wild-type cancers. Two investigators (J.W. and H.K.) independently assessed the results of immunostaining. When discordant diagnosis were judged by investigators, results were reevaluated and discussed until the agreement was reached.
STATISTICAL ANALYSIS
All data were entered into an access database and analyzed with Stat-View (version 5.0) software. In analyzing the relationship between the genotype and disease status of TCC, the genotype frequencies of each category of cancer grade and stage were compared against those of the controls. Data were analyzed with 2 x 2 contingency tables according to the genotype using the chi-square test. A multivariate logistic regression model was used to assess the relative risk [age and gender adjusted odds ratio (aOR) and 95% confidence interval (95% CI)] for bladder TCC risk and higher disease status. The frequencies of the PIG3 promoter VNTRs genotype between the TP53 status were compared using the chi-square test. All statistical results were considered significant if the P-value was <0.05.
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RESULTS |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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THE PIG3 PROMOTER VNTRS GENOTYPES AND THE RISK OF THE BLADDER TCC
Genotyping using PCR methods demonstrated that there were seven VNTRs in the PIG3 promoter; which were 10, 12, 14, 15, 16, 17 and 18 repeats of the pentanucleotide sequences (TGYCC) (Fig. 1). As for the allele frequency, there was no significant difference between the control group (10 = 6.7%, 12 = 0.2%, 14 = 0.4%, 15 = 88.6%, 16 = 2.8%, 17 =1.3% and 18 = 0%) and the TCC group (10 = 7.2%, 12 = 0%, 14 = 0.2%, 15 = 84.5%, 16 = 5.1%, 17 = 2.8% and 18 = 0.2%, P = 0.09). The genotype distribution of both groups was in the Hardy–Weinberg equilibrium (P = 0.94 in the control group and P = 0.72 in the TCC group). As the allele of 15 repeats was the most frequent and considered to be the standard one, the PIG3 promoter VNTRs genotypes were categorized into two groups; a short repeat subtype, which was defined as subjects who harbored at least one allele of 14 or less repeats of the VNTR (10/10, 10/15–17, 12/15 and 14/15) and a long repeat subtype, which included the other subjects. The distributions of the PIG3 promoter subtype were not significantly different between both genders. There was also no significant difference in the frequencies of the short repeat subtypes between the controls and the patients (12.4 and 14.3%, respectively, P = 0.500; Table 1).
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THE RELATIONSHIP OF THE PIG3 PROMOTER VNTRS AND CANCER GRADE/STAGE
To evaluate whether the PIG3 promoter VNTRs were associated with an advanced disease status of the bladder TCCs, the frequencies of the short repeat subtype were compared as for their cancer grades and stages. In terms of the cancer grade, the frequency of the short repeat subtype was significantly higher in the high grade (Grade 3) TCC patients than in the controls (20.4 and 12.4%, P = 0.038), whereas there were no significant differences in the frequencies of the short repeat subtype between the low grade (Grade 1–2) TCC patients and the controls (10.9 and 12.4%, P = 0.602; Table 1). As for the cancer stage, logistic regression analysis demonstrated that the subjects with the short repeat subtype had a significantly higher risk for high stage (
pT2) cancers compared with the controls (aOR = 2.31, 95% CI = 1.05–5.90, P = 0.038; Table 1). No significantly higher risk of low stage (
pT1) TCC against the controls was demonstrated (aOR = 0.90, 95% CI = 0.51–1.56, P = 0.699; Table 1).
THE STATUS OF TP53 AND THE PIG3 PROMOTER VNTRS SUBTYPES
We investigated the association of the TP53 status of cancer tissues and PIG3 promoter subtype. Among the 51 patients who were treated with radical cystectomy for invasive TCCs, 45 surgical specimens of the same number of patients were available for immunohistochemistry of TP53. Twenty-three cancers were judged as negative staining and defined as TP53 wild-type, and the other 22 cancers were defined as TP53 mutant-type. Among the 23 patients with TP53 wild-type cancers, 5 (21.7%) and 18 (78.3%) were classified into the short and the long repeat subtypes of the PIG3 promoter, respectively. On the other hand, all 22 patients with TP53 mutant-type cancers were of the long repeat subtype. The frequency of the short repeat subtype was statistically higher in the TP53 wild-type invasive TCCs than that in the TP53 mutant-type ones (P = 0.020; Table 2).
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DISCUSSION |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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In the present study, the PIG3 promoter VNTRs were analyzed in Japanese patients with bladder TCCs and healthy controls. In the healthy controls, the 15 repeats allele was dominant (88.6%), and the allele frequency for the 14 or less repeats was 7.3%. There were newly identified alleles of 12, 14 and 18 times repeats, which have not been reported in the German and Greek populations (14,19). The discordant result may come from racial differences.
The allele frequency of the 14 or less repeats in our series was not significantly different from those in the previous report from Germany (5.1%, P = 0.264 by chi-square test) (14). As for the significance of the PIG3 promoter VNTRs in the susceptibility to cancers, Gorgoulis et al. (19) reported that the frequencies of the VNTRs in breast and lung cancer patients were not significantly different from those of healthy controls, which was consistent with our results of those with bladder TCCs. However, when the short repeat subtype was defined as the patients with at least one allele of 14 or less repeats, the prevalence of the short repeat subtype was significantly higher in the high grade TCC patients than in the controls and was associated with a higher risk of advanced stage. The frequency of short repeat subtype of the PIG3 promoter VNTRs was not significantly different between the controls and high stage TCCs with chi-square test, but it was a significant risk factor of high stage TCCs with logistic regression analysis, which suggests that the advanced stage was also associated with the PIG3 promoter VNTRs. On the other hand, when the short repeat subtype was defined as those with at least one allele of 15 or less repeats, no significant difference was found between the normal and high-grade/stage TCCs (data not shown). Although there has been no report that analyzed the relation of the PIG3 promoter VNTRs to its expression level and the cellular phenotype, these data indicated that the existence of at least one allele of 14 or less repeats was associated with the susceptibility of high grade/high stage TCCs. In combination with the evidence that shorter repeats of VNTRs was related to lower expression levels of PIG3, the existence of at least one allele of 14 or less repeats might have resulted in the haploinsufficiency of PIG3 expression.
In the pathogenesis of bladder TCC, two different pathways have been proposed; the superficial TCC pathway and the invasive TCC pathway (9,20). Alterations of TP53 may play key roles in the development of invasive TCC, as the mutations in p53 are identified in over half of them, in contrast to superficial papillary TCC. Furthermore, the mutations in p53 may affect the sensitivity to chemotherapy and the prognosis (20). It was demonstrated in an in vitro study that the PIG3 promoter VNTRs is directly bound by TP53, and that the activity of PIG3 was influenced according to the number of repeats within it (14). Interestingly, our immunohistochemical analysis for TP53 indicated that the frequency of the PIG3 short repeat subtype was statistically higher in the patients with TP53 wild-type TCCs than those with TP53 mutant-type TCCs. TP53 seems to bind to the promoter of PIG3, and PIG3 was involved in TP53 mediated apoptosis. Mutations of TP53 or the presence of the short repeat of the promoter might down regulate the activation of PIG3, resulting in inhibition of apoptosis and induction of malignant phenotype. Although the sample number is too small (N = 45), these data suggested that the suppression of the TP53–PIG3 pathways might be critical for the tumorigenesis of high grade/high stage bladder TCC.
Originally, PIG3 was identified as one of the genes upregulated in the course of the TP53 induced, reactive oxygen species (ROS) mediated apoptosis (13,21,22) and has a homology with NAD (P) H quinone oxidoreductase-1 (NQO1), although its exact biological function has not been well elucidated. NQO1 suppresses DNA damage due to ROS by preventing the one-electron reduction of quinones by cytochrome P450 (23) and generates antioxidant forms of ubiquinone (24) and 
-tocopherol (25). Interestingly, polymorphisms of NQO1 have also been reported to be associated with the tumorigenesis of bladder cancer (26). Thus, PIG3 might play roles in regulating ROS and be associated with the invasive bladder TCC, like as NQO1. It would be interesting to analyze the function of PIG3 in the cancer cells.
Our preliminary results as for the association of the PIG3 promoter VNTRs with the progression of bladder cancer was marginally significant, but the result was limited due to relatively small number of T2 cases. Therefore, larger-scale study, that puts emphasis on the patients with T2 bladder cancer, would be needed to verify our results.
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Acknowledgments |
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This work was supported by Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports and Technology of Japan and by Suzuki Foundation for Urological Medicine.
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References |
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