Cyclin D1 gene polymorphism is associated with an increased risk of urinary bladder cancer

Lizhong Wang1,*, Tomonori Habuchi1,*, Takeshi Takahashi3, Kenji Mitsumori1, Toshiyuki Kamoto3, Yoshiyuki Kakehi3, Hideaki Kakinuma1, Kazunari Sato1, Akira Nakamura2, Osamu Ogawa3 and Tetsuro Kato1,4

1 Department of Urology and
2 Medical Information Science, Akita University School of Medicine, Akita 010-8543 and
3 Department of Urology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan

Abstract

Cyclin D1 is believed to play an important role in the genesis and/or progression of transitional cell cancer (TCC) of the urinary bladder. Cyclin D1 gene (CCND1) mRNA is alternatively spliced to produce two transcripts, and the splicing pattern may be modulated by a G to A single nucleotide polymorphism within the splice donor site of exon 4. This study was conducted to explore the association between the polymorphism and the susceptibility to and disease status of TCC of the bladder in 222 cases and 317 native Japanese controls. The relationship between the CCND1 polymorphism and the mRNA splicing pattern in TCC cells was evaluated by semi-quantitative reverse-transcription PCR. The CCND1 A allele was more frequently observed in the TCC group than the control group (P = 0.032) with a significant difference in the genotype frequency between the two groups (P = 0.029). The AA genotype was associated with a significantly higher risk of TCC compared with the AG+GG genotypes (adjusted odds ratio (aOR) = 1.76, 95% confidence interval (CI) = 1.09–2.84, P = 0.022). This association was observed more significantly in nonsmoking cases (aOR = 2.53; 95% CI = 1.28–4.51, P = 0.008). Looking at tumor grade, the presence of the A allele was associated with higher grade (= grade 3) tumors with a gene dosage effect (aOR = 1.77, CI = 1.16–2.69, P = 0.008). In tumor stage, although not significant, the AA + AG genotypes tended to be more frequently observed in cases with T1-4 tumors than those with Ta tumors (aOR = 1.94, 95% CI = 0.98–3.82, P = 0.057). The genotype seemed to influence the two alternatively spliced forms of the CCND1 mRNA because the ratio of the CCND1 transcript-b/transcript-a was significantly higher in cases with the AA genotype compared with those with the AG + GG genotypes. These data suggest that the CCND1 variant A allele may be associated with an increased risk of TCC of the bladder, especially in men without a history of smoking, and it may also have an effect on its disease status.

Abbreviations: CCND1, the cyclin D1 gene; TCC, transitional cell carcinoma; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OR, odds ratio; aOR, adjusted odds ratio; CI, confidence interval

Introduction

Transitional cell carcinoma (TCC) of the urinary bladder is the most common cancer of the urinary tract. Recent molecular genetic studies have shown that accumulation of activated oncogenes and inactivated suppressor genes leads to the genesis and progression of TCC (1,2). A frequent target in transitional cell carcinogenesis is the deregulation of G1-S phase progression in the cell cycle, whose transition through the G1 into S phase is regulated by cyclins, cyclin-dependent kinases and their inhibitors (1,2). Allelic deletions of the chromosomal region at 9p21 and 13q are frequent in TCCs, and the targets of such alterations are presumably p16 and Rb (3–8), which play an important role in the regulation of the G1/S phase transition (9). Cyclin D1 is an important positive regulator of the G1/S phase, which has been shown to be involved in various types of human cancer (10). The cyclin D1 gene (CCND1) is located at chromosome 11q13, and amplification of the chromosomal region is frequently detected in TCCs of the bladder (11,12). Furthermore, CCND1 overexpression occurs frequently in TCCs of the bladder (13–16) and may be associated with growth of low-grade papillary tumors (13,14). These findings suggest that CCND1 is a significant proto-oncogene in genesis and progression of TCC.

Recently, it has been reported that CCND1 mRNA is alternatively spliced to produce two transcripts (transcript-a and transcript-b), which are present simultaneously in a variety of normal tissues and cancer cells (17–19). The alternative splicing pattern seems to be modulated by a G to A single nucleotide polymorphism at codon 241, which corresponds to a conserved splice donor site of exon 4 (17,18). Studies using several kinds of cancer cells have demonstrated that the variant allele encoding A may be a major source of variant transcript-b (17,18). The CCND1 genotype was significantly correlated with clinical outcome in patients with non-small cell cancer of the lung, squamous cell carcinoma of the head and neck, squamous cell carcinoma of the oral/pharyngeal cavity, hereditary nonpolyposis colorectal cancer, and epithelial ovarian cancer, although the proposed risk allele was not consistent among these cancer types (17,20–23). These data suggest that the difference in the levels of the alternate CCND1 transcripts caused by the A/G polymorphism may influence the biological behavior in a variety of cancers.

This study was conducted to explore the association between the CCND1 polymorphism and the susceptibility to TCC or its disease status. We further evaluated the association stratified by patients' smoking history since epidemiological studies have shown that cigarette smoking is associated with a significantly higher risk of TCC (24,25). Furthermore, to clarify the biological significance of the polymorphism, we examined the relationship between the polymorphism and the alternative splicing pattern of CCND1 mRNA in TCC cells in vivo and in vitro.

Materials and methods

Subjects
A total of 222 patients with TCC of the bladder were enrolled in this study (Table IGo). The 222 comprised 90 patients who were treated at Akita University Medical Center in Akita Prefecture between April 1997 and April 2000, and 132 patients who were treated at Kyoto University Hospital in Kyoto Prefecture between June 1991 and December 1999. During the recruitment period, all patients were asked to provide blood samples for genetic analyses and consecutively entered with informed consent. All of them were diagnosed histologically with specimens obtained from biopsy or surgical resection. Clinical and histopathological information and a cigarette smoking history were obtained from patient charts, imaging studies, and pathological reports (Table IGo). In several patients, smoking history was further reviewed by additional interviews. The information was reviewed and the data were entered into the database. Tumor stage was assigned according to the tumor-node-metastasis (TNM) staging system (26). Pathological grading of the bladder cancer was determined according to the General Rule(s) for Clinical and Pathological Studies on Bladder Cancer by the Japanese Urological Association and the Japanese Society of Pathology, which is based on the World Health Organization (WHO) criteria (27,28). In two patients, no definite clinical stage was determined due to inadequate information. In four patients, the final pathological grade was not determined because of previous radiation therapy or primary carcinoma in situ. According to the history of cigarette smoking, patients with TCC of the bladder were divided into three groups: nonsmokers who had no history of a regular smoking habit, ex-smokers who had quit smoking over 10 years ago, and current smokers. All current smokers in this study were found to have smoked for >20 years. In nine patients, a smoking history could not be obtained. A total of 317 healthy native Japanese men and women 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 urine sediment to rule out the presence of TCC of the bladder. Except for no evidence of TCC of the bladder, no exclusion criteria were provided for the recruitment of controls. No information on smoking habits was obtained in the control subjects. Written informed consent was obtained from all the control subjects. The present study was approved by the Institutional Review Board of the Akita University School of Medicine, Akita, Japan.


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Table I. Characteristics of subjects and CCND1 genotypes
 
Genotyping of CCND1 polymorphism
DNA was extracted from blood samples collected from TCC patients, control subjects, and TCC tissues and cell lines using a QIAamp Blood Kit (QIAGEN, Germany) or by the standard method with proteinase K digestion followed by phenol/chloroform extraction. The 167 bp fragment encompassing the G to A polymorphic site in the CCND1 exon 4 terminal region was amplified using specific primers 5'-GTGAAGTTCATTTCCAATCCGC-3' in exon 4, and 5'-GGGACATCACCCTCACTTAC-3' in intron 4. PCR reactions were carried out in a 25 µl volume containing ~20 ng of genomic DNA, 1x PCR buffer supplied by a manufacturer, 0.2 mM of each dNTP (dATP, dCTP, dGTP, and dTTP), 1.5 mM MgCl2, 50 pmol of each primer, and 1.0 unit of Ampli-Taq Gold DNA polymerase (Perkin-Elmer, Branchburg, NJ). After a 10 min initial denaturation step at 95°C, 35 cycles of PCR reaction consisting of 95°C for 30 s, 55°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 (GeneAmp PCR System 9700: Perkin-Elmer, Branchburg, NJ). After confirmation of successful PCR amplification by 1.5% agarose gel electrophoresis, each PCR product was digested overnight with 5 units ScrFI enzyme at 37°C (New England Biolabs Inc., Beverly, MA) and was electrophoresed on 3.0% agarose gel. The 167 bp PCR fragment was divided into 146 bp and 22 bp fragments when the ScrFI site was present. The genotype was designated as G or A when the ScrFI restriction site was present or absent, respectively (17).

Semi-quantitation of alternatively spliced forms of CCND1 mRNA by multiplex reverse transcription-PCR (RT-PCR)
Ten TCC cell lines and 18 surgically resected TCC tissues of the bladder were analyzed for the relationship between the patterns of the two alternatively spliced CCND1 mRNA and the CCND1 genotypes. The ten TCC cell lines were T24, J82, 5637, RT112, TCCSUP, HT-1197, HT-1376, UMUC3, 253J and KY-BT1. Cell lines T24, J82, 5637 and HT-1197 were obtained from ATCC (American Type Culture Collection, Rockville, MD). KY-BT1 was established from invasive high-grade TCC of the bladder in the Department of Urology, Kyoto University Graduate School of Medicine. RT112, TCCSUP, HT-1376, UMUC3 and 253J were kindly provided by Dr M.A.Knowles at St. James's University Hospital, ICRF Cancer Medicine Unit, Leeds, UK. All cell lines were reported to be originated from TCC. These 10 cell lines were maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (JRH Biosciences A CSL Company, Lenexa, KS) and grown at 37°C in a humidified 5% CO2 atmosphere. The 18 TCC tissues were obtained at operation and were stored at –80°C.

Total RNA was isolated from the 10 TCC cell lines and 18 TCC tissues using the RNeasy Mini Kit (QIAGEN, Germany). First-strand cDNA was synthesized from 3 µg of total RNA using random primers according to the manufacturer's protocol (First-strand cDNA synthesis kit: Amersham Pharmacia Biotech, Piscataway, NJ). The efficacy of cDNA synthesis was monitored by the PCR amplification of a 769 bp fragment of the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase), using primers 5'-CGAGCCACATCGCTCAGACA (sense) and 5'-TAGACGGCAGGTCAGGTCCA (antisense). Semi-quantitation of alternatively spliced forms of CCND1 mRNA was evaluated by multiplex RT-PCR comparison with that of GAPDH RT-PCR amplification. Primers for amplification of transcript-a were as follows: 5'-CTCTGTGCCACAGATGTGAAGT (sense) located in exon 4 and 5'-GGGACATCACCCTCACTTAC (antisense) in exon 5. Primers for amplification of transcript-b were as follows: 5'-CTCTGTGCCACAGATGTGAAGT (sense) in exon 4 and 5'-CAAGGAGAATGAAGCTTTCCCTT (antisense) in intron 4. Multiplex PCR reactions for transcript-a were carried out in 25 µl volume containing cDNA derived from 50 ng of total RNA, 1x PCR Buffer, 0.2 mM of each dNTP, 1.5 mM MgCl2, 12 pmol of each transcript-a primer with 1.5 pmol of each GAPDH primer, and 1.0 unit of Ampli-Taq Gold DNA polymerase, and multiplex PCR reactions for transcript-b were carried out in a PCR mixture containing 24 pmol of each transcript-b primer with 1.5 pmol of each GAPDH primer. Note the concentration of GAPDH primers was reduced remarkably because preliminary results showed that the GAPDH PCR fragment was much more favorably amplified than transcript-a and transcript-b CCND1 fragments. The PCR condition was 10 min at 95°C followed by 30 cycles in transcript-a and 32 cycles in transcript-b for 30 s at 95°C, 30 s at 55°C, and 45 s at 72°C followed by a final extension at 72°C for 4 min. PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV illumination.

Evaluation of RT-PCR semi-quantification was performed in five separate tubes that underwent 23, 26, 29, 32, and 35 PCR cycles in transcript-a and 26, 29, 32, 35, and 38 PCR cycles in transcript-b (29,30). Furthermore, five sets of cDNA templates with various concentrations (x4, x2, x1, x1/2 and x1/4) were prepared for amplification. The GAPDH fragment was simultaneously amplified in each reaction at the same time. Each product was subjected to agarose gel electrophoresis. The bands of the products were scanned and quantified by densitometry (Lane and Spot Analyzer Software Version 5.0: ATTO Inc., Japan) and compared. The relative value of transcript-b/transcript-a was calculated by dividing the transcript-b/GAPDH value divided by the transcript-a/GAPDH value. All RT-PCR experiments were performed three times and the mean relative value of the three experiments in each sample was used for the statistical analysis.

Statistical analysis
All data were entered into an access database and analyzed by Excel 98 and SPSS (version 10.0J, SPSS) software. Hardy-Weinberg equilibrium analyses were performed to compare observed and expected genotype frequencies using a chi-square test (d.f. = 1). Case–control data were analyzed by two-sided 2 x 3 or 3 x 3 contingency tables according to the genotype using the Pearson {chi}2-test. The OR and 95% CI of CCND1 genotypes were calculated from a multivariate logistic regression model. In this study, we hypothesized that the presence of the A allele might be associated with higher risk of onset or progression of TCC of the bladder. However, because it was not clear whether the A allele had a dominant, recessive or gene-dosage effect, statistical modeling was performed on the relative risk of the AA genotype or the AG genotype against the GG genotype independently using a multivariate logistic regression model adjusted for age and sex. Furthermore, the gene dosage effect of the A allele was assessed by modeling a linear effect on the log odds scale for each A allele in the multivariate logistic regression. In addition, the relative risk of the AA genotype against the AG + GG genotypes, or the AA + AG genotypes against the GG genotype, was estimated using a multivariate logistic regression model. Age in years was entered as a continuous variable in this study. In addition, multivariate logistic regression analysis was also performed for the nonsmoking group and the smoking group, independently. In analyzing the relationship between the genotype and disease status of TCC, tumor grade and stage categories were transformed to binary data (grade 1 + 2 versus grade 3, Ta + 1 versus T2–4, Ta versus T1–4). Multivariate logistic regression analysis was performed adjusted for age, sex and smoking status to assess the relative risk of disease progression in the AA genotype or the AG genotype against the GG genotype, independently. The gene dosage effect of the A allele was then assessed as described above. Furthermore, the relative risk of the AA genotype against the AG + GG genotypes, or the AA + AG genotypes against the GG genotype, was estimated using a multivariate logistic regression model. In these logistic regression models, nonsmokers and current smokers were valued as `0' and `1', respectively. Since we do not know the relative effect of ex-smoking, we exclude the ex-smokers from the TCC group in these models. In addition, multivariate logistic regression analysis was separately performed adjusted for age and sex to assess the relative risk of having a high-grade (grade 3) tumor or high-stage (T1 or more) tumor for a nonsmoking or smoking group, independently.

Differences in the ratio of CCND1 transcript-b/transcript-a between the AA, AG, and GG genotypes were evaluated by the Kruskal–Wallis H-test, and the differences in the ratio between the AA and the AG + GG genotypes or between the AA + AG and the GG genotypes were evaluated by the Mann–Whitney U-test. The relationship between the number of PCR cycles or the amount of cDNA template and the density of amplified GAPDH, transcript-a or transcript-b PCR fragments was evaluated using Pearson's regression and correlation analysis. All statistical tests and P-values were two-tailed, and a result was considered significant if a P-value was <0.05.

Results

Subject characteristics
Brief clinical and pathological characteristics of the subjects in this study are presented in Table IGo. The TCC group comprised 172 males and 50 females, and the control group comprised 219 males and 98 females. The mean age ± SD of the TCC group and the control group was 65.69 ± 11.9 and 52.51 ± 11.7, respectively. Cigarette smoking history was available for 213 TCC patients. The 213 TCC patients comprised 77 nonsmokers, 11 ex-smokers who had quit smoking over 10 years ago, and 125 current smokers who had been smoking for >20 years at the diagnosis of TCC. The rate of current smokers was significantly higher in the male TCC subjects than the female TCC subjects (113/164, 68.9% versus 12/49, 24.5%; P = 0.002). In the control subjects, no information on smoking habits was obtained.

Genotypes of CCND1 polymorphism and risk of TCC of the bladder
The frequencies of the CCND1 genotype in the TCC subjects and the control subjects are shown in Table IGo, with brief clinical and pathological characteristics. There was a significant difference in the calculated allelic frequencies between the TCC group (A = 0.50, G = 0.50) and the control group (A = 0.43, G = 0.57) (P = 0.032). The allelic frequency in the control group was similar to those described previously (18,20,31,32). The genotype distribution of both groups was in Hardy–Weinberg equilibrium (P = 0.502 in the TCC group and P = 0.129 in the control group). Furthermore, there was no significant deviation from Hardy–Weinberg equilibrium in any of the subgroups presented in Table IGo. As for genotype frequency, there was a significant difference between the TCC group and the control group (P = 0.029; Table IGo). The difference in the genotype frequency was more significant between the nonsmoking TCC patients and the controls (P = 0.003), whereas no significant difference was found between TCC patients who currently smoke and the controls (P = 0.167), and TCC patients who currently smoke and nonsmoking TCC patients (P = 0.084) (Table IGo). In TCC patients, there was no significant difference in the genotype frequency (P = 0.982), the mean age (P = 0.883), the gender distribution (P = 0.678), and the frequency of current smokers (P = 0.374) between the case subjects recruited at the Akita University Medical Center and those recruited at Kyoto University Hospital (Table IGo and data not shown).

To evaluate the risk of TCC according to the CCND1 genotype, logistic regression analysis was conducted with adjustment for age at diagnosis and gender (Table IIGo). Compared with the GG genotype, no significant higher risk was found in cases with the AA genotype or cases with the AG genotype (Table IIGo). No significant gene dosage effect of the A allele was found (Table IIGo). When the AG and GG genotypes were combined, cases with the AA genotype had a 1.76-fold increased risk of TCC of the bladder compared with cases with the AG + GG genotypes (adjusted OR (aOR) = 1.76, 95% CI = 1.09–2.84, P = 0.022; Table IIGo). When the analysis was confined to the nonsmoking case subjects, the presence of the A allele was associated with an increased risk with the gene dosage effect (aOR = 1.77, 95% CI = 1.13–2.77, P = 0.013; Table IIGo), and the risk in the AA genotype was increased to 2.53-fold compared with that in the AG + GG genotypes (aOR = 2.53, 95% CI = 1.28–5.01, P = 0.008; Table IIGo). When confined to the smoking case subjects, no gene dosage effect with the A allele was found and the risk in the AA genotype was decreased and not significant (Table IIGo). When the smoking cases were compared with nonsmoking cases, there was no significant difference between the two groups (aOR = 1.42, 95% CI = 0.70–2.87, P = 0.327). When the AA + AG genotypes were compared with the GG genotype, no significant results were found (Table IIGo).


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Table II. Risk of TCC associated with CCND1 genotype according to different models
 
CCND1 polymorphism and tumor grade and stage
In terms of tumor grade, there was no significant difference in the CCND1 genotype frequency (P = 0.079; Table IGo). However, cases with the AA genotype had a significantly higher risk of high-grade (= grade 3) tumors compared with cases with the GG genotype (aOR = 3.09, 95% CI = 1.30–7.35, P = 0.010; Table IIIGo), and cases with the AG genotype had an intermediate risk (aOR = 2.14, 95% CI = 0.98–4.79, P = 0.064; Table IIIGo). The presence of the A allele was associated with high-grade tumors with a gene dosage effect (aOR = 1.77, 95% CI = 1.16–2.69, P = 0.008; Table IIIGo).


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Table III. Risk of high-grade (grade 3) TCC against low-grade TCC associated with CCND1 genotype
 
As for tumor stage, there was no significant difference in the CCND1 genotype frequency (P = 0.207; Table IGo). No difference was detected between cases with Ta tumors and those with T1–4 (P = 0.121), or between cases with Ta-1 and those with T2–4 (P = 0.111). No significant difference in the relative risk between cases with the three genotypes was found (Table IVGo). No gene dosage effect was found with the presence of the A allele (aOR = 1.42, 95% CI = 0.94–2.15, P = 0.096; Table IVGo). When cases with the AA + AG genotypes were combined, the AA + AG genotypes were more common in cases with T1–4 tumors than those with Ta tumors (aOR = 1.94, 95% CI = 0.98–3.82, P = 0.057; Table IVGo). The increased risk was significant in the smoking group (aOR = 2.47, 95% CI = 1.09–5.60, P = 0.031; Table IVGo), but not in the nonsmoking group (aOR = 1.07, 95% CI = 0.31–3.70, P = 0.911; Table IVGo). No significant difference was found when cases with Ta + T1 tumors were compared those with T2–4 tumors (data not shown).


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Table IV. Risk of high-stage (T1-4) TCC against low-stage (Ta) TCC associated with CCND1 genotype
 
Two transcripts of CCND1 with mRNA expression and genotypes
A preliminary RT-PCR experiment showed that both transcript-a and transcript-b were expressed in all the 10 TCC cell lines and 18 surgically resected TCC tissues. When the transcript-a and transcript-b fragments were amplified simultaneously with various reverse primer concentration ratios, no visible fragments were obtained for transcript-b. This finding was consistent with previous studies in which the mRNA expression level for transcript-b was much lower than that of transcript-a (17–19). Therefore, we evaluated the relative value of transcript-b/transcript-a by comparing those of the GAPDHmRNA levels in double multiplex PCR experiments. Since each primer concentration was modified to give evaluable amplification products, the calculated ratio of the two transcripts did not reflect the true ratio of mRNA transcription levels. A regression analysis of the intensity of amplified RT-PCR products for T24 cell line and the number of PCR cycles or the amount of cDNA templates gave a linear fit with a significant correlation coefficient, as shown by a straight line (Figure 1A–DGo). Thus, we examined the relation between the value of transcript-b/transcript-a mRNA and each genotype. The mean two-transcript ratio (transcript-b/transcript-a) of three independent experiments in 10 TCC cell lines and 18 TCC tissues was 2.23 ± 2.39 in the AA genotype, 0.40 ± 0.22 in the AG genotype and 0.55 ± 0.59 in the GG genotype (Figure 2A–CGo). There was no significant ratio difference among the three genotypes (P = 0.080; the Kruskal–Wallis H-test). However, in cells with the AA genotype, the value of the transcription ratio was significantly higher than that in cells with the AG + GG genotypes (P = 0.025; the Mann–Whitney U-test) (Figure 3Go). No significant difference in the value was found between the AA + AG genotype and the GG genotype (P = 0.313; the Mann–Whitney U-test).



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Fig. 1. Semi-quantitative analysis of transcript-a and transcript-b of CCND1 mRNA by multiplex RT-PCR in representative TCC cell line T24. (A) The intensity of simultaneously amplified fragments of transcript-a and GAPDH (Y-axis) and PCR cycles (X-axis). (B) The intensity of simultaneously amplified fragments of transcript-b and GAPDH (Y-axis) and PCR cycles (X-axis). (C) The intensity of simultaneously amplified fragments of transcript-a and GAPDH (Y-axis) and the various amounts of template cDNA cycles (X-axis) under a fixed PCR cycle condition. (D) The intensity of simultaneously amplified fragments of transcript-b and GAPDH (Y-axis) and the various amounts of template cDNA cycles (X-axis) under a fixed PCR cycle condition. The intensity of RT-PCR amplified fragments was measured with ethidium bromide staining using the Lane and Spot Analyzer (ATTO Inc.). 1x cDNA template amount corresponded to the cDNA reverse-transcribed from 50 ng total RNA of T24 cells.

 


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Fig. 2. CCDN1 genotype and semi-quantitative analysis of transcript-a and transcript-b of CCDN1 mRNA in 10 TCC cell lines. (A) CCND1 genotyping by PCR-restriction length fragment polymorphism analysis using ScrFI. (B) Multiplex RT-PCR amplification of transcript-a and GAPDH. (C) Multiplex RT-PCR amplification of transcript-b and GAPDH. The relative value of transcript-b/transcript-a was measured by dividing the transcript-b/GAPDH value by the transcript-a/GAPDH value. Number in parentheses is the size of each fragment (bp). M, molecular marker; N, negative control.

 


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Fig. 3. The mean value of transcript-a/transcript-b according to the CCND1 genotype in 10 TCC cell lines and 18 TCC tissues. The mean relative values calculated from three independent RT-PCR experiments for each sample were plotted according to the CCND1 genotype.

 
Discussion

Case–control analysis of our data showed that the CCND1 A allele was more frequently observed in the TCC group than the control group with a significant difference in the genotype frequency between the two groups (P = 0.029). The results suggested that the AA genotype in the CCND1 exon 4 was associated with ~1.8-fold increased risk of TCC of the bladder compared with the AG + GG genotypes. There was no significant gene dosage effect of the A allele. In addition, when the AA + AG genotypes were compared with the GG genotype, no significant association was found. These findings indicate that the CCND1 A allele has a recessive effect on the genesis of TCC. However, it should be noted that our study has several important limitations. Our control subjects were recruited in Akita Prefecture, whereas the case subjects were from university hospitals in Akita and Kyoto prefectures. Furthermore, no information on potential confounding factors, such as socioeconomic status, history of exposure to possible carcinogens, and family history of malignant disease, was obtained in both control and case subjects. Since both prefectures are located on the main island of Japan and all subjects were native Japanese, it is unlikely that there might be a significant difference in the CCND1 genotype between general populations in both prefectures. In fact, there was no difference in the genotype distribution, the mean age, the gender distribution, and the frequency of smokers between the case subjects from the two recruitment hospitals. Therefore, it is unlikely that the significant results were caused by the distinct recruitment areas.

The present data also indicated that the presence of the A allele might be associated with high-grade (grade 3) tumors with a gene dosage effect or it might have a dominant effect on the genesis of high-grade tumors. In tumor stage, the AA + AG genotypes were associated with higher-stage (T1–4) tumors compared with the GG genotype in the smoking cases, thus indicating a dominant effect of the A allele on the advanced disease status. However, due to the multiple comparisons performed in this study, it should be noted that these marginally significant results were caused by chance.

Tobacco consumption has been shown to be related to the onset of TCC of the bladder, and smokers may have a two- to five-times higher risk than nonsmokers (24,25). The higher risk can be attributed to tobacco-related chemical carcinogens (33). In this study, the increased risk of bladder cancer associated with the A allele was observed significantly when the nonsmoking cases were compared with the control subjects, whereas no such significant association was found in the smoking cases. On the other hand, the association of the A allele with the high-grade or high-stage tumors was more significantly found in the smoking cases. In a smoking population, various genetic factors involved in detoxifying and metabolizing chemical carcinogens may be determinants of susceptibility to TCC of the bladder, whereas the CCND1 genotype may be a determinant in disease progression. On the other hand, the CCND1 genotype may have a more significant impact on onset of TCC in a nonsmoking population. However, the result should be interpreted with caution because there was no significant difference as to the presence of the AA genotype between the smoking cases and the nonsmoking cases (P = 0.327, Table IGo), and no data on smoking habits were obtained in the control. Furthermore, the effect of the A allele on tumor grade may have been present in the nonsmoking population since the number of nonsmoking cases was smaller. Therefore, it remains to be clarified whether the effect of the A allele on susceptibility for bladder cancer is stronger in the nonsmoking population and whether the effect on the advanced disease status was more significant in the smoking population.

Our RT-PCR analysis results showed that all TCC cell lines and TCC tissues investigated expressed two forms of CCND1 mRNA transcripts, although the relative amount of transcript-b seemed much smaller than that of transcript-a. The present data were consistent with previous reports that the CCND1 genotype at the 3' end of exon 4 influences the ratio of the two alternatively spliced CCND1 transcripts (17,18). Transcript-b may be more preferentially transcribed from the A allele than the G allele and the transcript-b/transcript-a value is presumably higher in cancer cells with the AA genotype than in those with the AG + GG genotypes (Figure 3Go) (17,18). Although we found no obvious gene dosage effect of the allele on the transcript-b/transcript-a value, there might be a difference in the ratio, which could not detected in our evaluation method using semi-quantitative RT-PCR analysis, between cells with the AG genotype and GG genotype. It has been conjectured that altered protein encoded by transcript-b may have a longer half-life since it lacks the PEST-rich region (17,19,34). If there were a threshold in the amount of cyclin D1 protein for the regulation of cell cycle and such a threshold might vary under different cellular conditions, the difference in the amount of cyclin D1 protein caused by the absence or the presence of one or two A alleles may have a different effect on the regulation of the cell cycle. Under such circumstances, the A allele could exert a recessive effect on the cellular transformation in early carcinogenesis, while having a dominant effect in the progression to a more malignant phenotype on transformed cells.

Although our study is the first to examine the significance of the CCND1 polymorphism in TCC of the bladder, documents on other types of cancers may support our observation (17,21,23). In non-small cell lung cancers, the AG + AA genotypes were associated with a significantly shorter survival and a greater risk of local relapse compared with the AA genotype, indicating a dominant effect of the A allele on tumor progression (17). In hereditary nonpolyposis colorectal cancer, patients with at least one A allele developed colorectal cancer an average of 11 years earlier than those without the A allele, indicating a dominant effect of the A allele on tumor onset (21). In epithelial ovarian cancer, women with the CCND1 AA genotype were associated with early disease progression and reduced survival after postoperative chemotherapy (23). On the other hand, one group presented contradictory data on head and neck squamous cell carcinoma, i.e. that the GG genotype was associated with a poorly differentiated type and significantly reduced survival (20,22). In nonhereditary colorectal cancer, the CCND1 polymorphism had no significant relationship with disease progression (31). These discrepant results might come from differences in cellular conditions and origins in which cyclin D1 is involved in the regulation of the cell cycle because the function of cyclin D1 in the cell cycle is restricted by many tumor suppressor genes and oncogene products (9,10).

In conclusion, our data suggested that the variant A allele in the CCND1 polymorphism is associated with a higher risk of TCC, and that it may also have some effect on disease status of TCC. The increased risk may be caused by the difference in the ratio of two alternatively spliced forms of CCND1 mRNA influenced by the polymorphism. These findings warrant more controlled clinical studies in a larger study population to investigate the significance of this polymorphism as a marker for susceptibility and disease progression of TCC of the bladder.

Notes

4 To whom correspondence should be addressed at: Department of Urology, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan Email: tkato{at}med.akita-u.ac.jp Back

* The authors contributed equally to this work. Back

Acknowledgments

We thank Dr M.A.Knowles at ICRF Cancer Medicine Unit, Leeds, for kindly providing TCC cell lines and Itsuko Fujiwara and Tomoko Matsushita at Kyoto University for their technical help. We also thank Dr Sei-ichi Kitajima and Dr Kazuhiko Takano at Yuri-J.A. General Hospital for their kind help in collecting materials. This work is supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (B12470327, B10470331, B10470330, B10470336).

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Received May 11, 2001; revised October 18, 2001; accepted October 24, 2001.