A case-control study of cyclin D1 CCND1 870A->G polymorphism and bladder cancer

Victoria K. Cortessis1, Kimberly Siegmund, Shanyan Xue, Ronald K. Ross and Mimi C. Yu

Department of Preventive Medicine, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, MC-9175, Los Angeles, CA 90033, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of cyclin D1 is believed to lead to progression through the G1–S cell cycle checkpoint, and both experimental and pathological evidence suggest that over-expression of this protein may increase the risk of several cancers, including transition cell carcinoma of the bladder. Two major transcripts have been described for CCND1, the gene encoding cyclin D1. CCND1 870A->G, a common single nucleotide polymorphism in the splice donor region of exon 4, may modulate expression of these transcripts, with the A variant resulting in an increased pool of the isozyme encoded by transcript form b. A statistically significant 1.8-fold increased risk for bladder cancer among individuals possessing the A/A genotype was recently reported in a hospital-based case-control study conducted among native Japanese. We conducted a population-based case-control study of incidence of bladder cancer among non-Hispanic whites in Los Angeles County to examine the relationship between CCND1 870A->G genotypes and bladder cancer risk. No association between the A/A genotype and risk was observed (odds ratio = 0.90, 95% confidence interval 0.60–1.33). The null association was not appreciably modified by bladder cancer risk factors, including lifetime smoking history, or by histopathologic classification.

Abbreviations: 4-ABP, 4-aminobiphenyl; CDK, cyclin-dependent kinases; NSAIDS, non-steroidal anti-inflammatory drugs


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bladder cancer is the most common malignancy of the urinary tract, and the sixth most common cancer in the US. The most established risk factors for bladder cancer are occupational exposure to certain arylamines and exposure to cigarette smoke. Although the precise mechanism for the latter association is not fully clarified, cigarette smoke contains both 2-naphthylamine and 4-aminobiphenyl (4-ABP), which are known carcinogenic arylamines in occupational settings. Arylamines are also present in permanent hair dyes, and regular use of these products has recently been found to be associated with bladder cancer risk in a dose- and duration-dependent manner (1,2), especially among individuals with deficient arylamine-detoxification enzymes (13). Arylamines require metabolic activation to become fully carcinogenic. Active forms can adduct hemoglobin in the blood stream or be excreted through the kidney and urinary bladder, where they can adduct DNA of the bladder epithelium. Resulting damage to unspecified target genes, if not adequately repaired, is postulated to contribute to the malignant transformation of urinary epithelial cells (4). We recently reported elevated levels of 4-ABP hemoglobin adducts in lifelong non-smoking cases of bladder cancer relative to lifelong non-smoking control subjects, irrespective of hair dye use status, thus implicating a role for carcinogenic arylamines in non-smoking related bladder cancer that is independent of arylamine exposure from permanent hair dyes (5).

Cyclin D1 is a key regulator of cell cycle progression and a demonstrated oncogene. Over-expression of cyclin D1 is implicated in the etiology of several cancers, including urinary bladder carcinoma. Cyclin D1 regulates cell cycle progression by activating cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), which in turn phosphorylate the retinoblastoma protein (pRb). This reaction inactivates pRb and is postulated to lead to progression through a G1–S checkpoint, committing the cell to DNA replication. In some in vitro systems, controlled over-expression of cyclin D1 promotes progression through G1, with abundance of cyclin D1 being rate limiting. In chemically induced rat models of bladder carcinogenesis, over-expression of cyclin D1 occurs in a high proportion of pre-neoplastic lesions and tumors. The gene encoding cyclin D1, CCND1, is located on human chromosome 11q13, in a region amplified ~2- to 15-fold in ~20% of human bladder carcinoma. Moreover, cyclin D1 protein is over-expressed in a high proportion of human bladder tumors (610).

Alternate splicing of CCND1 produces two major transcripts, designated forms a and b. Form b is not spliced at the downstream exon 4 boundary and encodes a cyclin D1 protein whose predicted sequence has an altered C-terminus, with 14 amino acids encoded by a read through into intron 4 replacing the entire exon 5 encoded sequence. The exon 5 encoded sequence includes a PEST sequence postulated to target protein for rapid degradation (11). Absence of the PEST sequence in the form b encoded isoform may lead to over-expression of cyclin D1. Several authors have suggested that splicing to produce the two transcripts is modulated by CCND1 870A->G, a common single nucleotide polymorphism in the splice donor region of CCND1 exon 4 (1214). They concur that the A allele may be the major source of transcript b, although both transcripts are detected at some level regardless of genotype.

Wang et al. (14) investigated the possibility that the CCND1 870A allele confers elevated risk of bladder cancer. In a hospital-based case-control study, they found an association between the A/A genotype and bladder cancer [odds ratio (OR), 1.76; 95% confidence interval (CI), 1.09–2.84], with more pronounced risk among non-smoking cases and for bladder cancer of higher grade and stage. We report here a population-based study of the CCND1 870A->G polymorphism in relation to bladder cancer in Los Angeles County, California.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects and in-person interviews
Recruitment of subjects in the Los Angeles Bladder Cancer Study has been described in detail previously (15). Briefly, incident cases of bladder cancer were identified through the Los Angeles County Cancer Surveillance Program, the population-based Surveillance, Epidemiology and End Results (SEER) cancer registry of Los Angeles County. Eligibility criteria include histologically confirmed cancer diagnosed between January 1, 1987 and April 30, 1996, among non-Asians between the ages of 25 and 64 years. Enrolled cases (n = 1679) represented roughly 75% of total eligible patients. For each enrolled case patient, an invariant procedure was followed to recruit a control subject from the neighborhood of residence of the index case at the time of cancer diagnosis, who was matched to the index case by age (within 5 years), sex and racial/ethnic group (non-Hispanic white, Hispanic white, African-American). A matched control subject could not be found for 5% (n = 86) of interviewed cases. Therefore, 1593 case-control pairs were interviewed for the study. Twenty-one control subjects were not matched to the index case by racial/ethnic group. All of the study subjects signed informed consent forms (separate forms for interview, and donation of blood and/or urine) approved by the University of Southern California Human Subjects Committee.

In-person, structured interviews were conducted in subjects' homes. The questionnaire requested information up to 2 years prior to the diagnosis of cancer for cases and 2 years prior to diagnosis of cancer of the index case for matched controls (i.e. reference year). The questionnaire included information on demographic characteristics, height, weight, lifetime use of tobacco and alcohol, usual adult dietary habits, lifetime occupational history, prior medical conditions and prior use of medications.

Collection of blood samples; determination of GSTM1, GSTT1, GSTP1 and NAT1 genotypes; and measurement of ABP hemoglobin adducts
Beginning in January 1992, all case patients and their matched control subjects were asked for a blood donation at the end of the in-person interviews. We obtained a blood sample from 771 of 1044 (74%) case patients, and from 775 of 979 (79%) control subjects. Interviewed cases who donated a blood specimen were comparable with total eligible patients in terms of histology, racial/ethnic group, sex and age at diagnosis. Specifically, 95% of total eligible patients were diagnosed with transitional cell type versus 95% in enrolled cases with blood. Corresponding figures for non-Hispanic white race, male sex and mean age (standard deviation) in years were 88 versus 91%, 78 versus 78% and 56.2 (7.2) versus 56.4 (7.6), respectively. Similarly, controls who donated a blood sample were comparable with all controls in terms of age, gender and race.

Various blood components (serum, plasma, buffy coat, red blood cells) were isolated and stored at -80°C until analysis. Of those subjects who donated blood, 3- and 4-ABP hemoglobin adduct values were determined in 761 (99%) case patients and 770 (100%) control subjects as described by Skipper (16). Because tobacco use is a major source of ABP exposure in the US (17), all subjects who donated blood were asked additional detailed questions about their use of tobacco products during the 60 days before blood was drawn. DNA was isolated from peripheral blood by standard methods. Genotypic analysis was conducted as described by Bell et al. (18) to identify null and non-null variants of GSTM1. A modification of the method described by Arand et al. (19) was used to discriminate between null and non-null variants of GSTT1. Genotypic analysis of GSTP1 was performed using the method of Harries et al. (20). Genotypic analysis identifying the NAT1 alleles NAT1*3, NAT1*10 and NAT1*11 was conducted using the method described by Bell et al. (21). Among those who donated a blood sample, GSTM1, GSTT1, GSTP1 and NAT1 genotypes were obtained for 756 (98%), 752 (98%), 751 (97%) and 740 (96%) case patients and 766 (99%), 759 (98%), 763 (98%) and 753 (97%) control subjects, respectively.

Collection of urine samples, and determination of CYP1A2 and NAT2 phenotypic indices
Caffeine was used as a probe substrate to measure CYP1A2 and NAT2 phenotypic indices as urinary metabolite ratios. Beginning in January 1992, all case patients and their matched controls subjects were asked to donate an overnight urine specimen following caffeine consumption in the afternoon. Each subject was given ~70 mg caffeine in instant coffee between 3 and 6 pm and asked to collect an overnight urine sample (including the first morning void). Urine specimens were picked up the same day, acidified using 400 mg ascorbic acid/20 ml urine, then stored at -20°C. Urinary caffeine metabolite levels and phenotypic indices were determined using the method of Kalow and Tang (17,22). We obtained urine samples from 724 (69%) case patients and from 689 (70%) control subjects. Of those who donated a urine sample, NAT2 and CYP1A2 phenotypes were determined in 716 (99%) cases and in 683 (99%) control subjects.

Determination of CCND1 genotypes
We used a PCR–RFLP assay to distinguish between A and G variants in the CCND1 870A->G polymorphism. 167 bp fragments containing the polymorphic nucleotide were amplified from 12 ng genomic DNA using 4 pmol each of the forward primer 5'-GTGAAGTTCATTTCCAATCCGC-3' and reverse primer 5'-GGGACATCACCCTCACTTAC-3' (12) in a reaction mix with 1.5 mM MgCl2. The thermocycling profile was 10 min of initial denaturation at 95°C; 35 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 55°C and 30 s of elongation at 72°C; and 10 min of final extension at 72°C. PCR products were submitted to MspI (C-CGG) digestion (New England BioLabs, Beverly, MA) followed by gel electrophoresis in 3.5% high-resolution agarose (Ambion, Austin, TX). PCR products with G at the polymorphic site were digested to two fragments of 145 and 22 bp, while those with A were not. The 22 bp fragment was not retained. Thus, samples yielding a single 145 bp fragment were scored G/G, those with a single 167 bp fragment were scored A/A, and those with both were scored A/G. Results were scored independently by two readers, and any sample for which a discrepancy was found was re-analyzed. Each PCR assay included a negative control in which additional H2O was substituted for template. Each set of samples subjected to restriction digestion included A/G and G/G controls, both sequences having been confirmed by direct sequencing.

Statistical methods
Because of possible heterogeneity between genetic factors and bladder cancer risk, we limited the present study to non-Hispanic whites, the primary racial/ethnic group in the Los Angeles Bladder Cancer Study. We measured CCND1 genotype on 515 cases and 612 control subjects.

We assessed Hardy–Weinberg equilibrium using a likelihood ratio statistic of one degree of freedom. We compared the frequencies of genotypes encoded by this polymorphism (A/A, A/G, G/G) between cases and controls using both conditional logistic regression and unconditional logistic regression adjusting for the matching variables (23); finding similar results using both approaches, we report detailed results of the unconditional analyses only. We also compared genotypes between each of two groups of cases categorized by histology [non-invasive (papillary, carcinoma in situ) versus invasive] and controls. These analyses were adjusted for two sets of variables found previously to be associated with risk. In the first set of analyses, we controlled for the effects of the matching variables: sex and age at bladder cancer diagnosis for cases or age at matched case's diagnosis for controls. In the second set, we controlled for sex, age and seven additional factors found to be associated with bladder cancer in earlier analyses of the Los Angeles Bladder Cancer Study: level of education (24), use of non-steroidal anti-inflammatory drugs (NSAIDs) (24), use of permanent hair dyes (1,2), lifetime smoking history (15), dietary intake of carotenoids (unpublished data), NAT2 phenotype (unpublished data) and GSTM1 genotype (unpublished data). Results of both analyses were quite similar, and we provide detailed results of analyses adjusted for the second set of variables.

We tested whether the effect of the CCND1 870A->G genotype was modified by use of permanent hair dyes, use of NSAIDs, gender, 4-ABP hemoglobin adduct level or lifetime smoking history, adjusting for all other variables described above. Genotype was coded using the number of A alleles (0,1,2), and modification of the genotype effect across the different levels of exposure was tested using interaction terms in the logistic regression model. Since tobacco smoke is a source of 4-ABP, we used separate criteria for individuals who were non-smokers or smokers at the time of blood collection to categorize 4-ABP hemoglobin adduct levels as ‘low’ or ‘high’. Among non-smokers at blood draw, individuals with 4-ABP levels less than the median among non-smokers (3.08 pg 4-ABP/g hemoglobin) were scored ‘low’ and others were scored ‘high’. Among smokers at blood draw, individuals for whom the residual of 4-ABP level on the natural log-scale regressed on number of cigarettes smoked per day was less than the median among smokers (1.00 pg 4-ABP/g hemoglobin) were scored ‘low’, and others were scored ‘high’.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genomic DNA obtained from 515 non-Hispanic white cases and 612 non-Hispanic white controls was subjected to genotypic analysis of the CCND1 870A->G polymorphism located within the splice donor site of exon 4. We estimate the frequency of the A allele to be the same (47%) among both cases and controls, and alleles at this polymorphism appear to be in Hardy–Weinberg equilibrium (among cases PHWE = 0.89, among controls PHWE = 0.67). As shown in Table I, we found no association between genotype at this polymorphism and occurrence of bladder cancer, either overall or by histologic subgroups.


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Table I. Distribution of CCND1 870A->G genotypes among bladder cancer cases and controls in Los Angeles County, 1992–1999

 
We looked for modification of the association between the CCND1 870A->G polymorphism and disease by gender, and by bladder cancer risk factors identified previously in the Los Angeles Bladder Cancer Study—smoking history (never versus ever smoking; never versus former versus current smoking in reference year), habitual use of permanent hair dyes (a non-occupational source of arylamine exposure), level of 4-ABP hemoglobin adducts (a biomarker for 4-aminobiphenyl exposure)—and by one putative protective factor, use of non-steroidal anti-inflammatory drugs. As shown in Table II, none of these factors appear to modify the effect of CCND1 870A->G genotypes on risk of bladder cancer.


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Table II. Association of CCND1 870A->G genotypes and bladder cancer risk among 515 cases and 612 controlsa, stratified by selected bladder cancer risk factors

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We did not find an association between genotypes defined by the CCND1 870A->G polymorphism and occurrence of bladder cancer. Our results contrast with those of Wang et al. (14), who reported an association between the A/A genotype and bladder cancer. There were several notable differences between these studies. In the present study, exposure and biomarker data were systematically collected from all subjects, while in the somewhat smaller study of Wang et al. (222 cases and 317 controls), smoking was assessed only for cases, and additional risk factors were not measured for either cases or controls. Subjects in the present study were population-based incident bladder cancer cases and controls. Wang et al., on the other hand, conducted a hospital-based study in which the hospital controls were not sampled from hospitals where the cases were diagnosed and treated. In light of uncertainties about comparability of cases and controls in the study of Wang et al., we noted genotypic frequencies observed among cases and controls in both studies. Frequencies of the A/A genotype were similar for three of these groups, cases and controls in the present study and cases studied by Wang et al. (22.3, 21.6 and 26.1%, respectively), while they were somewhat lower among controls studied by Wang et al. (16.7%). It will be useful to compare these observed frequencies with additional estimates among population-based samples of disease-free individuals of known racial/ethnic composition, as such estimates appear in the literature.

The difference between the findings of Wang et al. (14) and those presented here may also reflect different distributions of other important cofactors in the varying populations of the two studies (Japanese versus non-Hispanic whites). This could occur under at least three possible scenarios. First, it is possible that both somatic and inherited alterations in CCND1 lead to disease, with a higher proportion of US whites versus Japanese experiencing somatic changes. Since over-expression can result from gene amplification, and amplification of the chromosome 11q13 region containing CCND1 is reported to be relatively frequent in bladder cancer, it would be useful to compare the frequency of this event in bladder cancer cases in US whites versus Japanese. A second possibility is that etiologic effects of the CCND1 870A allele occur in the context of variants in one or more additional molecules, with prevalence of these unmeasured variants differing between the two populations. Future studies should address joint effects of this CCND1 polymorphism and other molecules, beginning with candidates known to influence cyclin D1 function, such as its catalytic partners CDK4 and CDK6, and regulators of CCND1 expression such as FAK (25). A third possibility is that functional differences in cyclin D1 arise from sequence variants other than the polymorphism measured in this study. The observed results would be expected if a functional variant were in linkage disequilbrium with CCND1 870A among native Japanese, but not among non-Hispanic whites residing in Los Angeles. More complete description of variants in the CCND1 region and their distributions between racial/ethnic groups will be useful in evaluating this possibility.

Interpretation of association studies of CCND1 870A->G is currently hampered by important gaps in our understanding of the functional consequences of this polymorphism. It already seems clear that (i) the 870A->G polymorphism is located within the splice donor site of CCND1 exon 4, (ii) there are two major CCND1 transcripts that encode distinct protein isoforms and (iii) the form b transcript lacks the exon 5 sequence containing the PEST region and includes sequence read from intron 4. What remain inadequately understood are (i) the functional consequences of the distinct protein isoforms and (ii) whether the CCND1 870A->G genotypic polymorphism leads to differential expression of the alternate transcripts or isoforms. The limited genotype-specific expression data now available (1214) relate genotype to relative abundance of the two mRNA transcripts, rather than the protein isoforms they encode. These observations are based on semi-quantitative measures of mRNA, and they do not reveal a distinct relationship between presence of the CCND1 870A allele and form b transcript; instead, all these studies found both transcripts to be present in all samples, irrespective of genotype. It is possible that the isoform encoded by transcript b has altered abundance or function that could lead to disease, but that the 870A->G polymorphism does not directly measure occurrence of this isoform, or does so too poorly to be a reliable marker.

The present study has an expected power of 98% to detect a relative risk of 1.76 for bladder cancer in A/A homozygotes, as observed by Wang et al. (14). Therefore, it is unlikely that lack of statistical power was the reason the results we present here contrast with those of Wang et al. (14). It is also useful to view the results of Wang et al. (14) in the context of a Bayesian perspective, which indicates that in the presence of a low prior probability of a hypothesized association, the statistical significance of the observed association needs to be extremely high (e.g. P << 0.01) in order for the posterior probability of a false-positive result to be low. It is reasonable to assume a low prior probability of an association between the CCND1 870A->G polymorphism and bladder cancer, given the limited information on the functionality of this polymorphism and the specific role of cyclin D1, in relation to other—presumably many—candidate genes, in influencing the development of bladder cancer. Therefore, the level of statistical significance for the association reported by Wang et al. (P = 0.022) is compatible with a high probability of finding a false-positive result.


    Notes
 
1 To whom correspondence should be addressed Email: cortessi{at}usc.edu Back


    Acknowledgments
 
We thank Susan Roberts, Kazuko Arakawa and Hansong Wang, Keck School of Medicine at the University of Southern California, for their assistance in data collection, management and analysis. This study was supported by grants P01 CA17054, R35 CA53890 and R01 CA655726 from the United States National Cancer Institute and grant P30 E507048 from the United States National Institute of Environmental Health Sciences.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 7, 2003; revised July 18, 2003; accepted July 23, 2003.