The effect of cyclin D1 (CCND1) G870A-polymorphism on breast cancer risk is modified by oxidative stress among Chinese women in Singapore
Michela Ceschi,
Can-Lan Sun 1,
David Van Den Berg 2,
Woon-Puay Koh 3,
Mimi C. Yu 1 and
Nicole Probst-Hensch *
Cancer Registry and Molecular Epidemiology, University Hospital, Vogelsangstrasse 10, 8091 Zurich, Switzerland, 1 University of Minnesota Cancer Center, Minneapolis, MN 55455, USA, 2 USC/Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA 90033-0800, USA and 3 Department of Community, Occupational and Family Medicine, Faculty of Medicine, National University of Singapore, Singapore 117597
* To whom correspondence should be addressed. Tel: +41 0 1 255 56 34; Fax: +41 0 1 255 56 36; Email: Nicole.Probst{at}usz.ch
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Abstract
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Cyclin D1 (CCND1), an intracellular cell-cycle regulatory protein with checkpoint function, can promote cell proliferation or induce growth arrest and apoptosis depending on the cellular context. We hypothesized that the direction of the association between the (CCND1) G870A-polymorphism and breast cancer risk may be modified by dietary and genetic factors influencing the oxidantantioxidant balance, such as a dietary pattern with a high intake of n-6 fatty acids and a low intake of n-3 fatty acids, or a genetic profile that is deficient in glutathione S-transferases. We tested our hypothesis in a casecontrol study nested into the Singapore Chinese Health Study, a prospective investigation of diet and cancer in 63 000 Chinese men and women. Genomic DNA collected from 258 incident cases of breast cancer and 670 female cohort controls was examined for CCND1, GSTM1, GSTT1 and GSTP1 genes using fluorogenic 5'-nuclease assay. Unconditional logistic regression models were used to assess the effects with adjustment for potential confounders. All statistical tests were two-sided. The heterozygous CCND1 GA genotype significantly reduced the breast cancer risk in all subjects (OR = 0.67, 95% CI 0.450.99) when compared with the GG genotype. The association was restricted to women with a high (above median value) intake level of n-6 fatty acids (OR = 0.51, 95% CI 0.300.87), a low (below median value) intake level of the antagonistic marine n-3 fatty acids (OR = 0.54, 95% CI 0.320.93) or a total lack of the antioxidative GSTM1 (OR = 0.44, 95% CI 0.250.80) or GSTT1 genes (OR = 0.46, 95% CI 0.240.87). The effects were consistently stronger in cases with advanced disease. The AA genotype did not affect breast cancer risk. The results of this study are compatible with the hypothesis that the oxidantantioxidant balance in cells is an important determinant of the direction of the cyclin D1 effect, leading either to cell proliferation or cell death.
Abbreviations: BMI, body mass index; CCND1, Cyclin D1 gene; CI, confidence interval; GST, glutathione S-transferase; OR, odds ratio; ROS, reactive oxygen species; Th1, T-helper 1
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Introduction
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Cyclin D1, a protein encoded by the CCND1 gene located on chromosome 11q13, is a key cell-cycle regulatory protein modulating the restriction point early in the G1-phase [reviews in (14)]. Cyclin D1 gene (CCND1) is amplified or overexpressed in a variety of tumours (2,5,6). In up to 20% of breast cancers, CCND1 is amplified and >50% of mammary tumours overexpress it (7,8). CCND1 exhibits a common A/G polymorphism at nt 870, which modulates alternate splicing of CCND1. Both alleles lead to the expression of two different transcripts, but at different proportions. Several studies found the A-allele to be the major source of transcript form b, which encodes a cyclin D1 protein with an altered C-terminus. It lacks a PEST sequence postulated to target protein for rapid degradation (911). Carriers of one or two A-alleles may thus possess a longer protein half-life. Several epidemiological studies (1217) found an increased risk for different cancer types among carriers of the A-allele in accordance with the cell proliferating role of cyclin D1. But the absence of an association with cancer risk or inverse associations between cancer risk or survival and the A-allele also were reported (1823). Additional, modifiable risk factors were seldom taken into consideration in these prior studies.
The direction of the biological impact of cyclin D1 overexpression depends on the state of the cell in accordance with its checkpoint function. While cyclin D1 is best known for its proliferating effect (4,24,25), experimental evidence suggests that under conditions such as oxidative stress (24,2629) or senescence (3033), cyclin D1 can inhibit S-phase entry and DNA replication and promote growth arrest as well as apoptosis. The context-dependent dual role of cyclin D1 on cell proliferation and growth arrest, may explain the inconsistent associations observed between CCND1 genotype and cancer risk (12,14,20,34), and emphasizes the importance of assessing the interaction between CCND1 genotype and factors that modify the cellular micromilieu.
A potential modifier of the CCND1 genotype/breast cancer association is oxidative stress. First, reactive oxygen species (ROS) can activate signalling cascades that ultimately lead to cell-cycle arrest and apoptosis by altering the expression of DNA damage inducible genes including cyclin D1 and its inhibitors (3537). Glutathione S-transferase (GST) enzymes which play a major role in the elimination of oxidative by-products were found to interact with CCND1 in previous cancer susceptibility studies (38,39). The GST, GSTp, has been identified as an important regulator of JNK signalling, a member of the stress kinase family of genes involved in cell-cycle control (36,40). Second, oxidative stress from various sources has been proposed as a risk factor for breast cancer (4143). A shift in the oxidantantioxidant balance possibly underlies the inverse association between marine n-3 fatty acid intake and breast cancer risk reported by us (44), that was confined to GSTM1, GSTT1 and GSTP1 deficient women and was more pronounced in patients with an advanced stage of disease (45). Kikugawa et al. (46), in accordance with experimental data (4749), reported a protective effect of fish oil against oxidative stress induced DNA damage in rat liver in vivo, that was attributed to the lipid peroxidation products resulting from marine n-3 fatty acids. These lipid peroxidation metabolites are excreted less efficiently by women possessing low activity GST genotypes. We also found a dose-dependent increase in breast cancer risk with increasing intake of n-6 fatty acids among women with low intake of marine n-3 fatty acids (44). The tumour-enhancing effect of n-6 fatty acids has been related to oxidative DNA damage (47). In accordance with the impact of oxidative stress on the cell-cycle checkpoint, marine n-3 fatty acids inhibited (50) and n-6 promoted (30) CCND1-expression in cancer cells.
In this report, we investigated whether the association of the CCND1 polymorphism with breast cancer risk was modified by the intake levels of n-3 and n-6 fatty acids, GST genotypes and stage of the disease as markers of oxidative burden.
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Materials and methods
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Study population
The subjects were participants of the Singapore Chinese Health Study, a population-based, prospective investigation of diet and cancer risk. The study design has been described previously (51). Briefly, from April 1993 through December 1998, we recruited 63 257 Chinese women and men from two major dialect groups in Singapore (Hokkien and Cantonese). Subjects were between the ages of 45 and 74 years and residing in government housing estates at the time of enrollment. During the period of study enrollment, 86% of the Singapore population resided in such facilities. Only women (n = 35 298) were considered in this report. At recruitment, a face-to-face interview was conducted by a trained interviewer, using a structured questionnaire to elicit information on demographics, lifetime use of tobacco, physical activity, menstrual and reproductive history, medical history and family history of cancer. The questionnaire included a dietary component assessing usual intake pattern (including frequency and portion size) during the previous 12 months on 165 food and beverage items, which were subsequently validated against a series of 24-h recalls (51). Average daily intake of n-6 and marine n-3 fatty acids, expressed as %kcal to adjust for total energy intake in the analyses, was computed for each study subject via linkage to the Singapore Food Composition Table (51). The food frequency questionnaire listed 14 seafood items commonly consumed by Chinese in Singapore, including fresh fish, fresh shellfish, dried/salted fish and canned fish. Major sources of n-6 fatty acids were meat (10%), grain products (20%) and cooking oils (40%) (44).
Between April 1994 and July 1999, we collected blood and single-void urine specimens from a random 3% sample of study enrollees. A 20 ml blood sample was obtained from each consenting subject and stored in a liquid nitrogen tank at 180°C until August 2001, when they were moved to a 80°C freezer for long-term storage. If the subject refused to donate blood, he/she was asked to donate buccal cells, which were collected through the use of a modified mouthwash protocol based on published methods (52).
Out of 1059 female cohort participants contacted for biospecimen donation, blood (n = 514) and buccal cells (n = 164) were collected from 678 subjects, representing a participation of 64%. The control group of the present study consisted of the 670 women (203 premenopausal, 467 post-menopausal) who remained free of breast cancer as of April 30, 2002 (8 women in this subgroup of cohort subjects were first diagnosed with breast cancer between enrollment and April 30, 2002). Demographic characteristics (age, dialect group and education) of the control women who donated blood or buccal cells were comparable with all women in the cohort.
We identified incident breast cancer cases through the population-based cancer registry in Singapore (53). As of April 30, 2002, a total of 399 cases of incident breast cancer had developed among female cohort subjects. Histological and staging information on all breast cancer diagnoses were confirmed by manual review of the pathology reports and clinical charts. Blood (n = 198) or buccal (n = 60) specimens were available for 258 (65%) breast cancer cases. Of these 258 cases of breast cancer, 33 cases had in situ cancers, 68 had stage I, 110 had stage II, 23 had stage III and 18 had stage IV tumours, while staging information was not available for 6 cases. The 151 cases that had stage II or higher tumours (regional and metastatic disease) are classified hereafter as having advanced disease. Breast cancer cases who did not give a blood or buccal cell sample were less educated than those who provided such a sample (44 versus 30% had no formal education). More Cantonese donated specimens (69%) compared with Hokkien (60%). The two groups were otherwise similar with respect to age at cancer diagnosis (mean age of 61 versus 59 years). In all analyses, subjects with non-informative genotypes were excluded (3 cases and 4 controls).
Informed consent forms were completed by all participants at baseline interviews and at the time of collection of biological specimens. The Institutional Review Boards at the University of Southern California and the National University of Singapore had approved this study.
Genotyping methods
DNA was purified from buffy coats of peripheral blood and buccal cell samples using a QIAamp 96 DNA Blood Kit (Qiagen, Valencia, CA). Genotyping for CCND1, GSTM1, GSTT1 and GSTP1 was performed using the fluorogenic 5'-nuclease assay (TaqMan Assay) (54).
The TaqMan assays were performed using a TaqMan PCR Core Reagent kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.
The CCND1 polymorphism (A/G) was previously described (14). The oligonucleotide primers for amplification of the polymorphic region of CCND1 were GC091for (5'-CCCCAACAACTTCCTGTCCTACTA-3') and GC091rev (5'-AGGCTGCCTGGGACATCA-3'). In addition, the fluorogenic oligonucleotide probes (TaqMan MGB Probes; ABI) used to detect each of the alleles were GC091F (5'-CCTCCTTACCGGGTCA-3') labelled with 6-FAM to detect the G allele and GC091V (5'-CCTCCTTACTGGGTCA-3') labelled with VIC to detect the A allele. PCR amplification using
10 ng of genomic DNA was performed in a thermal cycler (MWG Biotech, High Point, NC) with an initial step of 95°C for 10 min followed by 50 cycles of 95°C for 25 s and 62°C for 1 min. The fluorescence profile of each well was measured in an ABI 7900HT Sequence Detection System and the results analysed with Sequence Detection Software (ABI). Experimental samples were compared with 12 controls to identify the 3 genotypes at each locus (A/A, A/G, G/G). Any samples outside the parameters defined by the controls were identified as non-informative and were retested.
The oligonucleotide primers for amplification of the polymorphic region of GSTP1 were GC070for (5'-CCTGGTGGACATGGTGAATG-3') and GC070rev (5'-TGCTCACACCATAGTTGGTGTAGATGA-3'). In addition, the fluorogenic MGB oligonucleotide probes used to detect each of the alleles were GC070F (5'-TGCAAATACGTCTCCCT-3') labelled with 6-FAM and GC070V (5'-TGCAAATACATCTCCCT-3') labelled with VIC (Applied Biosystems). PCR amplification consisted in the same procedure described above for CCND1.
Genotyping of the GSTT1 and GSTM1 loci using the TaqMan assay consisted of separate assays for GSTT1, GSTM1 and the albumin control genes. The oligonucleotide primers for amplification of the GSTT1, GSTM1 and albumin genes were GC003 for (5'-GTGCAAACACCTCCTGGAGAT-3') and GC003rev (5'-AGTCCTTGGCCTTCAGAATGA-3'), GC004 for (5'-CTTGGAGGAACTCCCTGAAAAG-3') and GC004rev (5'-TGGAACCTCCATAACACGTGA-3'), GC005 for (5'-CGATTTTCTTTTTAGGGCAGTAGC-3') and GC005rev (5'-TGGAAACTTCTGCAAACTCAGC-3'), respectively. Fluorescent oligonucleotide probes, for detection of PCR reaction products, were synthesized to contain the dye 6-FAM (BioSearch Technologies, Novato, CA). The probes for the GSTT1, GSTM1 and albumin genes were GC003FAM (5'-ATGCTGCCCATCCCTGCCC-3'), GC004FAM (5'-AAGCGGCCATGGTTTGCAGG-3') and GC005FAM (5'-CGCCTGAGCCAGAGATTTCCCA-3'), respectively. PCR amplification using
10 ng of genomic DNA was performed in an ABI 7900HT Sequence Detection System (Applied Biosystem) with an initial step of 95°C for 10 min followed by 50 cycles of 95°C for 25 s and 62°C for 1 min. The fluorescence profile of each well was measured in real time during the PCR amplification and the results analysed with Sequence Detection Software (ABI). Any sample with a fluorescence signal that crossed a threshold of 0.2
Rn before cycle 40 was considered positive for the loci analysed. Samples negative for both GSTT1 and GSTM1 must be positive for albumin to be called; otherwise, the sample was designated non-informative and retested. All analyses were carried out blind to case or control status.
Statistical analysis
Although we sampled our controls randomly from the whole cohort, our study is more casecontrol than casecohort in design since time of follow-up was relatively short and comparable between cases and the subcohort, with only eight subjects in the latter group developing breast cancer during the observation period. We used unconditional logistic regression methods to examine the effect of the G870A-polymorphism of CCND1 alone and together with fatty acid intake levels, and GST genotypes on breast cancer risk. Indicator variables for the three genotypes of CCND1 (GG, AG, AA) were created using the GG genotype as the reference category. The quartile distribution of marine n-3 and n-6 fatty acids (in units of %kcal) among female cohort members formed the basis for categorization of subjects. The median values of n-3 and n-6 fatty acid intake were used as cutoff points to define individuals with high (third and fourth quartiles) and low (first and second quartiles) intake levels (0.19 and 4.34 %kcal, respectively). GSTM1 and GSTT1 null genotypes were subjects homozygous for the respective gene deletions. GSTP1 AA genotype was considered the high-activity genotype compared with the AB/BB genotypes. The strength of the genecancer associations was measured by odds ratio (OR) and its 95% confidence interval (CI). Age at recruitment (years), year of recruitment (19931998), dialect group (Hokkien, Cantonese), level of education (no formal education, primary school only, secondary school or higher), number of livebirths (0, 12, 34 or 5+), and age when period became regular (
12, 1314, 1516, 17+ or never regular) were included in all models as covariates. Addition of a term for body mass index (BMI) (<20, 20 to <24, 24 to <28 or
28 kg/m2) did not appreciably alter the results and thus, was not retained in the final models. Polytomous logistic regression models (55) were used to compare cases, stratified by the stage of disease, with all controls.
Statistical analysis was carried out using the SAS version 9.0 (SAS Institute, Cary, NC). All P-values are two-sided and P < 0.05 were considered statistically significant.
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Results
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The characteristics of the study population are summarized in Table I. Included in the study were 670 women without and 258 women with breast cancer. Consistent with the present understanding of breast cancer risk, factors positively associated with risk were the level of education and age at first birth; characteristics inversely associated with risk were the number of livebirths, age when period became regular and age at menopause (56). Mean age at recruitment was similar for case and control groups [55.6 years (SD 7.4) and 55.8 (SD 8.0), respectively].
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Table I. Distribution of selected variables in female breast cancer patients and controls in Singapore Chinese women
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The distribution of CCND1 genotype by casecontrol status and disease stage is summarized in Table II. The frequency of the A-allele in control women was 0.58. The genotype distribution among controls was in HardyWeinberg equilibrium (P = 0.47). Women carrying the heterozygous GA genotype showed an overall reduced risk of breast cancer (OR = 0.67, 95% CI = 0.450.99), while women carrying the AA genotype exhibited an OR of 0.93 (95% CI = 0.631.38). Restricting the analysis to patients with advanced stage disease accentuated the protective effect of the GA genotype (OR = 0.52, 95% CI = 0.320.84). Menopausal status did not modify the association between CCND1 genotype and breast cancer (data not shown).
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Table II. Distribution of cyclin D1 (CCND1) genotype in breast cancer patients and controls overall and according to stage of disease in Singapore Chinese women
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Table III shows the results of the CCND1 genotype effects on breast cancer risk stratified by the intake level of marine n-3 and n-6 fatty acids. The association between the GA genotype and breast cancer protection was confined to women with low intake of n-3 fats or high intake of n-6 fats. The strongest association between GA genotype and risk reduction was found in women with both low n-3 and high n-6 fats intake (OR = 0.33, 95% CI = 0.150.73). The protective effects of the GA genotype were consistently stronger in patients with advanced disease. The power for assessment of the GA genotype effect among advanced stage patients with both low n-3 and high n-6 fat intake was insufficient.
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Table III. Cyclin D1 (CCND1) genotype in relation to breast cancer risk stratified by marine n-3 and n-6 fatty acids intake level overall and according to stage of disease in Singapore Chinese women
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Table IV presents the effects of CCND1 genotype on breast cancer risk according to subjects' specific GST genotypes. The protective effect of the CCND1-GA genotype on breast cancer risk when compared with GG-genotype was restricted to women possessing either the GSTM1-null or GSTT1-null genotypes (OR 0.44, 95% CI 0.250.80 and 0.46, 0.240.87, respectively). Again, these protective effects were stronger and statistically significant only in subjects with an advanced stage of the disease. There was no evidence of a modification effect of the CCND1 genotypebreast cancer association by GSTP1 genotype.
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Table IV. Cyclin D1 (CCND1) genotype in relation to breast cancer risk stratified by GST genotypes overall and according to stage of disease in Singapore Chinese women
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Restricting the analyses to subjects with both low activity GST genotypes and low n-3 or high n-6 intake levels, resulted in sparse data and unstable OR estimates. Nonetheless, the GA genotype effects on breast cancer risk were uniformly stronger (data not shown). The OR for GA versus GG genotypes for subjects with low n-3 fat intake and GSTM1-null, GSTT1-null or GSTP1-AB/AA genotypes were 0.19 (95% CI 0.080.43), 0.29 (95% CI 0.110.73) and 0.56 (95% CI 0.231.36), respectively. The corresponding OR for subjects with high n-6 fats intake and GSTM1-null, GSTT1-null, or GSTP1-AB/AA genotypes were 0.25 (95% CI 0.110.56), 0.43 (95% CI 0.181.00) and 0.47 (95% CI 0.191.17), respectively.
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Discussion
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In the present study, we found a protective effect of the heterozygous CCND1 GA genotype on breast cancer risk which was restricted to situations of elevated oxidative stress characterized by high intake level of n-6 fatty acids, low intake level of the antagonistic marine n-3 fatty acids and absence in the host of the antioxidative GSTM1 and GSTT1 enzymes. The observed genecancer effects are more pronounced among cases with advanced disease, which is compatible with the hypothesized interaction between cyclin D1 and oxidative stress, since cancer cells constitutively produce redox products (36).
ROS are a significant endogenous and exogenous source of DNA damage and thus trigger cell-cycle arrest at different cell-cycle checkpoints including G1 (57). Activation of p53 and cyclin dependent kinase inhibitors plays an important role in the response of cells to oxidants (58). In vitro experiments of breast tumour and other tumour cells exposed to oxidative stressors demonstrate that cyclin D1 activation and overexpression is also able to activate molecular pathways resulting in cell-cycle arrest and apoptosis (24,28,29,59). According to these results, cyclin D1 modulates growth arrest and cell death in a p53-dependent way following exposure to ionizing radiation and oxidative DNA damage. Turner et al. (27) reported an increased radiosensitivity in breast cancer cells among individuals overexpressing cyclin D1 and provided in vivo evidence of cyclin D1 as a caretaker gene offering downstream protection against oxidative damage. Our findings extend this evidence to situations of more moderate oxidative burden than the one caused by ionizing radiation. If confirmed, they suggest that modulation of the biological function of cyclin D1 by dietary factors may lead to differential impact of the CCND1 gene on breast cancer susceptibility and progression. The demonstration of an interactive effect between the CCND1 gene and intake of marine n-3 and n-6 fatty acids, also strengthens our previously proposed hypothesis that the observed effects of marine n-3 and n-6 fatty acids on breast carcinogenesis are due to their respective impact on cellular oxidative burden (44,45).
Ours is the first study to investigate the association between the G870A CCND1 polymorphism and breast cancer risk stratified by markers of oxidative stress. Only two other studies, a hospital-based casecontrol study in an Australian population (18) and a population-based casecontrol study in Austria (19), have analysed the impact of the same cyclin D1 polymorphism on breast cancer risk. Neither study found an association with breast cancer risk overall or following stratification by tumour characteristics, such as size, histological grade, lymph node involvement, estrogens receptor status or overall survival of patients. No covariates or effect modifiers were considered in either study. Previous studies have linked the CCND1 A allele to increased susceptibility or clinical outcome in non-small cell lung cancer (11,60) and prostate cancer (15). However, discordant results between the CCND1 A/G polymorphism and cancer risk have been observed for urinary bladder cancer (14,20) and colorectal cancer (16,17, 61). An increased susceptibility to squamous cell carcinoma of the head and neck was reported for individuals with the AA genotype (13), but two other investigations linked the AA genotype to a longer disease-free interval than the GG genotype (23,39). These published results are compatible with the notion of a differential role of the CCND1 polymorphism on cancer risk as opposed to cancer progression, and are consistent with the current study observation of stronger CCND1 effects in an advanced stage of the disease. A few studies have examined GA genotype separately from the AA genotype, which mainly found no significant GA genotype association with cancer risk (1216). But the CCND1 GA genotype has been linked to differential overall and disease-free survival in patients with ovarian and colorectal cancers (62,63). Molecular heterosis, in which subjects heterozygous for a specific genetic polymorphism show stronger effects than subjects homozygous for either alleles, has been described (64). This differs from the classical notion of a genedose effect on cancer risk, and is intriguing in light of the checkpoint function of cyclin D1 and dual biological activity with distinct molecular pathways, leading to either cell-cycle promotion or programmed cell death (26,30,31,65,66). A one-directional, genedose dependent model may be applicable only when gene products possess a singularly defined biological activity. The two transcripts expressed by the two alleles of the CCND1 A870G polymorphism are both able to inhibit cell proliferation (10). Our results suggest that the heterozygous CCND1 genotype provides the optimal proportion of transcript form a to transcript form b for the induction of proliferation arrest under conditions of oxidative stress. It is of interest that heterosis also has been observed for another gene with important regulatory activity. The cytokine IL-12 plays a critical role in promoting the development of T-helper 1 (Th1) cells and suppressing the release of Th2 cytokines. Heterozygotes in IL12 are associated with decreased expression relative to the two homozygous genotypes and with severe asthma in children (67). The molecular mechanism underlying heterosis are poorly understood. Comings and MacMurray (64) proposed several levels at which molecular heterosis may be operatingat the level of gene regulation or at the level of interaction between protein subunits. As a result, heterozygotes may produce just the right amount of protein for optimal biological activity, or they may possess a broader range of gene expression products allowing for functional plasticity. The latter explanation may be of special relevance with regard to the checkpoint function of cyclin D1.
This study has several strengths. Dietary assessment was conducted using a validated, semi-quantitative food frequency questionnaire. All exposure assessments occurred prior to cancer diagnosis, and therefore, could be presumed to be free of recall bias. Finally, the study population is genetically homogeneous since they are full-blooded descendents of natives from two contiguous prefectures in southern China. There is a theoretical concern for selection bias arisen from the higher availability of blood specimens among Cantonese versus Hokkien cases with breast cancer and the higher levels of education among cases with blood specimens versus those without blood specimens. We examined if CCND1 genotype is related to dialect group or education among our cohort participants, and no associations were found. Given that dialect group and education are not confounders of the hypothesized CCND1breast cancer association in our study population, the unequal distributions of these two factors between cases with and without blood specimens should not impact on the validity of our study findings.
A major limitation of our study is its low statistical power for the assessment of geneenvironment and genegene interactions, due to the rather modest number of incident cases of breast cancer occurring within the relatively short period of follow-up (an average of 6.0 ± 2.5 years per subject) of this cohort of Singapore Chinese women. However, as this long-term, prospective investigation continues to accrue incident cases of breast cancer, we will have the opportunity to revisit this hypothesis with sufficient statistical power. Our reason for publishing these findings now is because they are internally consistent and are biologically plausible. We hope the publication of this set of preliminary results will spur others to conduct confirmatory epidemiologic studies and mechanistic laboratory studies.
Our study results indicate that most genotype effects on disease risk cannot be generalized across diverse populations, since environmental factors may play important modifying roles. Our findings are consistent with the notion that the oxidantantioxidant balance in cells is one important determinant of the direction of the cyclin D1 checkpoint function, leading to either cell proliferation or cell death.
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Acknowledgments
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We thank Siew-Hong Low for supervising the field work of the Singapore Chinese Health Study, and Ms Kazuko Arakawa for the development and management of the cohort study database. The Singapore Chinese Health Study has been supported by Grants R01 CA55069, R35 CA53890 and R01 CA80205 from the National Cancer Institute, Bethesda, MD. Drs Ceschi and Probst-Hensch are grateful to the Swiss National Science Foundation for the financial support.
Conflict of Interest Statement: None declared.
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Received December 3, 2004;
revised March 18, 2005;
accepted April 10, 2005.