Genetic Susceptibility Research Core
Research Accomplishments - 2006
In this sixth year of activity, the Genetic Susceptibility Research Core has continued promoting interdisciplinary research in mechanisms of environmental disease. The GSRC Members have outstanding records of research accomplishment and productivity. Here we describe the variety of collaborations that have formed among Center faculty in pursuit of common research goals fostered by the Center.
Head and Neck Cancer
Dr. Olshan recently completed a large case-control study of head and neck cancer in North Carolina. The study includes 1,380 cases and 1,400 controls and will investigate polymorphisms of genes involved in carcinogen metabolism and DNA repair. Dr. Olshan is collaborating in a recently funded CEHS pilot project with Dr. Perou. Dr. Perou identified novel genes in an expression profiling of head and neck tumors. The pilot project resequenced the TALD01 gene and is now evaluating the association of sequence variants and disease risk in a subset of 350 cases and 350 controls from Dr. Olshan’s head and neck cancer study. In addition, Drs. Perou and Olshan are examining the association of p53 gene variants in the same case and control sample. In collaboration with Dr. Millikan, Dr. Olshan recently completed a preliminary study of DNA repair polymorphisms and the risk of birth defects. These findings are being extended to evaluate more candidate repair genes and SNPs and birth defects in a large national case-control study of birth defects.
Melanoma
Dr. Cordeiro-Stone is a member of a multidisciplinary group of basic and physician scientists interested in the underlying mechanisms of melanoma development. This group has submitted a program project grant application to the NIEHS which is currently pending. Dr. Cordeiro-Stone is an active collaborator with Drs. Kaufmann and Kaufman. These collaborations have resulted in the discovery of new origins of DNA replication in human cells (Cell Cycle 2006;5:869-872; Exp Mol Pathology 2006, in press), and new information on DNA repair and checkpoint function as markers of breast cancer risk (Carcinogenesis 2006, in press) and metal-induced toxicity in human cells (Toxicology and Applied Pharmacology, 2006, in press).
Dr. Kaufmann’s laboratory has collaborated with Drs. Cordeiro-Stone and Sancar to demonstrate a new element of intra-S checkpoint response to sunlight-induced DNA damage. The intra-S checkpoint response to UVC was shown not to include ATM-dependent degradation of Cdc25A, nor inhibition of cyclin E/Cdk2, unlike the response provoked by ionizing radiation. Instead UVC appeared to signal through ATR and Chk1 to Dbf4/Cdc7 complexes to inhibit replicon initiation. Additional studies showed that the Tim-Tipin replication fork protection complex was a mediator of the UVC-induced intra-S checkpoint and confirmed a new element of intra-S signaling. Not only does the intra-S checkpoint signal to inhibit initiation of DNA synthesis at origins of replication (replicon initiation) it also appears to signal to slow the rate of DNA chain elongation in active replicons. This slowing of chain elongation also provides more time for excision repair to remove DNA adducts before DNA replication, thereby increasing the ability of excision repair to protect against carcinogen-induced mutations and chromosomal aberrations. Dr. Kaufmann also organized an inter-disciplinary collaboration among CEHS members and other UNC-CH investigators to develop and submit to the NIEHS a DISCOVER center application to study the mechanisms whereby sunlight induces the environmental cancer, melanoma.
Breast Cancer
Dr. Gammon is principal investigator of the Long Island Breast Cancer Study Project (LIBCSP), a population-based case-control study that was specifically undertaken to identify environmental risk factors for the disease. Recent efforts include a continued focus on understanding the impact of exposure to polycyclic aromatic hydrocarbons (PAH) and breast cancer development, particularly to identify women who are genetically susceptible to the effects of PAH, including sources such as cigarette smoke (which, as we reported this past year, is the primary source of PAH-DNA adduct formation among those who are non-occupationally exposed (Shantakumar 2005)). For example, in 2006 we reported in Carcinogenesis (Ahn 2006), on glutathione S-transferases (GSTs), which are phase II enzymes that are involved in the detoxification of a wide range of carcinogens. The novel GSTA1*A and GSTA1*B genetic polymorphism results in differential expression, with lower transcriptional activation of GSTA1*B (variant) than that of GSTA1*A (common) allele. Considering that cruciferous vegetables induce GSTs, which metabolize tobacco smoke carcinogens, we hypothesized that the variant GSTA1*B genotype may predispose women to breast cancer, particularly among low cruciferous vegetable consumers and among smokers. Thus, we evaluated potential relationships between GSTA1 polymorphisms and breast cancer risk, in relation to vegetable consumption and smoking status in the LIBCSP. Genotyping (1036 cases and 1089 controls) was performed, and putative breast cancer risk factors and usual dietary intakes were assessed. Having GSTA1*A/*B or *B/*B genotypes was not associated with increased breast cancer risk, compared to having the common *A/*A genotype. However, among women in the lowest two tertiles of cruciferous vegetable consumption, *B/*B genotypes were associated with increased risk (OR (95% CI)=1.73 (1.10-2.72) for 0-1 servings/week), compared to women with *A/*A genotypes. Among women with *B/*B genotypes, a significant inverse trend between cruciferous vegetable consumption and breast cancer risk was observed (P for trend=0.05), and higher consumption (4+ servings/week) ameliorated the increased risk associated with the genotype. Current smokers with *B/*B genotypes had a 1.89-fold increase in risk (OR (95% CI)=1.89 (1.09-3.25)), compared with never smokers with *A/*A genotypes. These data indicate that GSTA1 genotypes related to reduced GSTA1 expression are associated with increased breast cancer primarily among women with lower consumption of cruciferous vegetables and among current smokers. In another example, Dr. Gammon explored the role of immunoglobulin mu-binding protein 2 (IGHMBP2), which is involved in DNA repair, replication and recombination. Our findings, which were published in 2006 in Breast Cancer Research and Treatment (Shen 2006), included an increased risk among women who had detectable PAH-DNA adducts and carried at least one variant-G allele (OR = 1.4, 95% CI = 1.0-1.8, p for trend = 0.01) compared to women carrying the wild-type AA genotype and with non-detectable adducts. Smokers carrying the IGHMBP2 variant-G allele had no significant increased breast cancer risk compared with non-smoking women with the AA genotype. Heavy smokers (>31 pack years) had a statistically significant association with breast cancer risk (OR=2.0, 95% CI=1.2-3.3) relative to nonsmokers with the AA genotype though the magnitude of association was not different than heavy smokers (> 31 pack years) with the AA genotype (OR=1.6, 95% CI=0.9-2.6). Overall our study observes only modestly higher effect estimates for PAH-DNA adduct exposure and cigarette smoking among those with the high-risk genotype, but these differences are not statistically significant. Additional studies focused on the biological function of the variant-G allele and interactions with other genetic polymorphisms are necessary to confirm our findings.
Current research topics in Dr. Robert Millikan’s laboratory include investigation of genetic and environmental risk factors for breast cancer, colon cancer, and malignant melanoma; epidemiologic studies of Fogo Selvagem (an environmentally-caused autoimmune disease in Brazil), cardiovascular disease, birth defects and injury. Work with Dr. Swenberg discovered that rare functional alleles are found within common haplotypes and contribute to cancer risk. Drs. Millikan and Perou collaborated in a paper in the Journal of the American Medical Association showing that African American women exhibit a high prevalence of a specific molecular subtype of breast cancer that confers a poor prognosis. Major funding includes the renewal of the Specialized Program of Research Excellence (SPORE) in Breast Cancer, (PI of Project 1: Carolina Breast Cancer Study, 2006-2011) in collaboration with Dr. Perou. Dr. Millikan also collaborated with Drs. Kaufmann and Cordeiro-Stone to demonstrate that sporadic breast cancer was not associated with defects in repair of chromatid breaks or checkpoint response to DNA double strand breaks.
Liver Cancer and Damage
Dr. Rusyn's laboratory continued their studies on the mechanisms of liver carcinogenesis. We are using environmental exposures to model liver disease, including cancer. For example, choline-deficient model of hepatocellular carcinoma provides a unique opportunity to unveil the molecular events associated with various stages of liver injury and carcinogenesis and to distinguish between causal and consecutive changes. We have used gene expression profiling and global histone modification status to identify temporal patterns in molecular signatures that occur during progression of hepatocellular carcinomas of various etiologies (J Nutr, in press; Hepatology, 2005; and Cancer Sci, 2005). In addition, we have recently observed that patients with greater intra-hepatic oxidative stress have a higher incidence of HCC recurrence, and demonstrated that oxidative stress biomarkers could potentially be used as a useful clinical diagnostic tool to predict the duration of disease-free survival in patients with HCV-associated HCC (Ann Surg Oncol, in press). Dr. Rusyn is serving as an expert consultant in support of the preparation of the 12th Report on Carcinogens by the National Toxicology Program (NTP) and a member of the Working Group to develop International Agency for Research on Cancer (IARC) Monographs Volume 96 on "Alcoholic beverage consumption, acetaldehyde and urethane."
Over the last year, the Watkins lab has identified the active components present in grapefruit juice that account for drug interactions produced by this juice (1,2). In addition, they used these substances to “knock out” the intestinal enzyme CYP3A4 in a human study and thereby establish the relative roles of this enzyme in the oral availability of saquinavir, a drug commonly used to treat HIV infection). Finally, Dr Watkins reported that recurrent therapeutic dosing of acetaminophen produces low grade hepatotoxicity in approximately one third of healthy volunteers (4). This establishes a novel human model to study mechanisms underlying susceptibility to hepatotoxicity.
DNA Replication and Damage
David G. Kaufman studies the organization of DNA replication during the S phase and seeks targets for DNA damage by carcinogens. Together with Drs. Kaufmann and Cordeiro-Stone he showed the effects of DNA damage on the progression of DNA replication to characterize the S phase checkpoint. Visualization of altered replication dynamics directly showed inhibition of both new origin activation and DNA strand elongation (Cell Cycle, in press). With Dr. Swenberg he showed visually that formation of apurinic/apyrimidinic sites in genomic DNA is non-random (FASEB Journal, in press) which may have relevance to how environmental oxidants and estrogens can contribute to the development of tissue toxicity and cancer. In collaboration with Dr. Cordeiro-Stone he found two origins of DNA replication that are activated late in the S phase (Cell Cycle 5: 869-872, 2006; Experimental and Molecular Pathology, in press) for comparison with previously identified origins activated early in the S phase. These late replicating genes are Fmr1 and Fmr2 which are associated with the Fragile X Syndrome. Knowledge of the relationship between the locations of the origin of replication and the triple repeat in the Fmr1 gene, which expands in the disease, may contribute to our understanding of the mechanism of this disease.
Dr. Stephen Chaney has collaborated with Dr. Swenberg and the Biomarker Facility Core to develop a highly sensitive LC-MS/MS method for measuring the number of DNA Pt-d(GpG) cross-links and determining the biological effects of various platinum-DNA adducts that are formed by the chemotherapeutic drug cisplatin and carboplatin. The structural basis for differential protein recognition of cisplatin (CP)-DNA and oxaliplatin (OX)-DNA adducts has not been determined and could be important for the design of more effective platinum anticancer agents. Dr. Chaney’s group recently reported high resolution solution NMR structures of OX-GG, CP-GG adducts and undamaged DNA dodecamers in the AGGC (G = G coordinated to platinum) sequence context. A comparison of the structures of these platinated-DNA adducts revealed that the conformation of CP and OX in the AGGC sequence is significantly different than that observed previously in other sequence contexts, which may relate to the increased mutagenicity of CP adducts in the AGGC sequence context. A comparison between the solution NMR structures of OX-TGGT and the OX-AGGC adducts solved in the Chaney laboratory also revealed significant differences. The 1H NMR study of the temperature dependence of the exchangeable protons of the OX-DNA adducts demonstrated an unprecedented dynamic motion for the 5'-flanking residue to the platinated adduct site in the TGGT sequence context. Compared to the OX-AGGC adduct, the OX-TGGT adduct exhibited less buckle, greater propeller twist, and less opening for the 5'- and 3'-G•C base pairs. In addition, compared to the OX-AGGC adduct, the OX-TGGT adduct exhibited a greater roll, slide, and twist for the 5'-flanking base pair step and greater tilt and less twist for the 3'-flanking base-pair steps. In addition to the helical parameters, a greater chi angle value for the C4 and T20 residues of OX-TGGT adduct compared to those of the OX-AGGC adduct indicated a higher anti-character for the C4 and T20 residues of OX-TGGT adduct. The differences observed in the NMR structural features of OX-TGGT and OX-AGGC adducts highlight the importance of sequence context of DNA in explaining the discrimination shown for the translesion synthesis past the DNA-platinum adduct sites.
Dr. Swenberg’s laboratory has been utilizing DT40 chicken B-lymphocytes and their isogenic cells with varying genetic alterations in DNA damage response genes to better understand DNA damage response to the naphthalene metabolites and formaldehyde. Isogenic DT40 cells were exposed to 1,2-naphthalenediol (NCAT), 1,2-naphthoquinone (1,2-NQ), 1,4-naphthalenediol (NHQ) or 1,4-naphthoquinone (1,4-NQ). The impact on the cell survival of the naphthalene metabolites in the wild-type (wt) DT40 cells was ranked in decreasing order as NHQ=1,4-NQ>>NCAT=1,2-NQ. Compared to the wt cells, survival assays in mutant cell lines showed that NHQ induced greater cell death in cells deficient in mismatch repair, homologous recombination, long-patch base excision repair pathways, and error-prone translesion DNA synthesis polymerases, but not in the short-patch base excision repair pathway. These DNA damage response data suggest that bulky adducts or oxidative DNA lesions, but not abasic sites, appear to play important roles on DNA damage response to NHQ. Likewise, this system has been used to show that deficiencies in homologous recombination and mismatch repair pathways make cells hypersensitive to formaldehyde. DNA and globin adducts are being used to evaluate the role of nucleotide excision repair and epoxide hydrolase on mutagenesis in wild type, XPA -/- and EH -/- mice exposed to butadiene (BD). Likewise, in collaboration with the Biomarker Facility Core, we have greatly improved the pyr-Val assay for BD diepoxide globin adducts and are finalizing the data for two large molecular epidemiology studies on BD workers. Upon completion of the analyses, the exposure codes will be broken and the data will be examined for exposure response and interactions with a comprehensive set of mutational endpoints, urinary metabolites and genetic polymorphisms. With the increase in sensitivity of the assay, we can now measure pyr-Val in smokers and possibly in individuals exposed to environmental tobacco smoke. A battery of oxidative DNA lesions is being evaluated to understand which adducts are sensitive to nucleotide excision repair using wild type and XPA -/- mice exposed to carbon tetrachloride.
