Environmental cancer pilot projects

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Genotoxicity of epoxides from photochemical oxidation of biogenic volatile organic compounds contributing to SOA
Principal Investigator:  Zhenfa Zhang, Ph.D., Research Assistant Professor, Department of Environmental Science and Engineering
Award: $25,000

Abstract
Atmospheric photochemical oxidation of volatile organic compounds (VOC) forms gas phase products that are often more toxic than the parent compound. We recently demonstrated that oxidation of isoprene yields epoxydiol (IEPOX) isomers under low-NOx conditions and methacrylic acid epoxide (MAE) under high NOx conditions. Preliminary work shows that oxidation of 2-methyl-3-butene-2-ol (MBO) by ozone leads to two isomeric epoxides. These epoxides have not been previously identified in air; epoxides in general are genotoxic via alkylation of DNA. The hypothesis of this study is that health effects linked to SOA and VOC exposure may be attributable to genotoxicity induced by gas phase epoxides formed from photo-oxidation of BVOCs. To test the hypothesis, we propose the following specific aims: 1). Examine the capacity of IEPOX isomers, MAE and MBO epoxides to cause DNA damage in the DT40 DNA damage response assay and the Ames assay. In preliminary DT40 analysis REV1-/- and PCNAK164R/K164R cells are much more sensitive to IEPOX-2 and MAE than to MNU. 2) Characterize DNA adducts formed from deoxynucleosides or DNA exposed to authentic epoxides.

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Formaldehyde-mediated bone barrow toxicity in FancD2 knock-out mice induced by methanol treatment
Principal Investigator:  Scott Bultman, Ph.D., Assistant Professor of Genetics
Award: $25,000

Abstract
Formaldehyde has been classified by the IARC as a known human carcinogen that causes nasopharyngeal cancer and leukemia. However, the limited evidence for inhaled formaldehyde causing hematolymphopoietic cancers and the biological implausibility of the hypothesis that inhaled formaldehyde causes leukemia has raised many questions. While inhaled formaldehyde does not reach the bone marrow cells, methanol treatment (p.o.) significantly elevates the amount of formaldehyde DNA adducts in bone marrow cells in rats through metabolic activation in bone marrow. In this proposal, we will address whether Fanconi anemia group D2 (Fancd2) mutant mice are more susceptible to bone marrow cell toxicity and genotoxicity caused by methanol-derived formaldehyde than wild-type mice. The significance of this project is not only to demonstrate potential bone marrow toxicity caused by methanol-derived formaldehyde, but also to understand the formaldehyde-derived DNA adduct levels sufficient to initiate bone marrow toxicity in Fancd2-/- mice, which appear to be the most sensitive animal model of formaldehyde-mediated toxicity. These results will also be used as positive controls for future grant proposals to understand the potential for inhaled formaldehyde causing bone marrow toxicity.

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Circadian oscillation of XPA expression in human hair follicles and PBMCs
Principal Investigator:  Shobhan Gaddameedhi, Ph.D.,   Post-doctoral Fellow, Department of Biochemistry & Biophysics, School of Medicine
Award: $25,000

Abstract
Skin cancer is the most common form of cancer in the United States. Recently, we found that the rate of Nucleotide Excision Repair (NER) due to XPA protein oscillates with a circadian rhythm in mouse skin with a minimum in the morning and a maximum in the evening. As a consequence, mice exposed to UV radiation (UVR) in early morning display an earlier onset and increased squamous cell carcinoma than mice exposed to UVR in the evening. The goal of this proposal is to compare the human XPA expression as a function of time of day by analyzing human hair follicles and PBMCs and to translate the basic science findings to potential health ramifications. If XPA oscillates in humans as a time of day, it is likely that the mutagenicity and carcinogenicity of sunlight and tanning beds may be strongly affected by the time of day of light exposure and it might be advisable for humans, to the extent possible, to restrict their occupational, therapeutic, recreational and cosmetic UVR exposure to a given time of day.

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Role of the circadian clock in UV-induced skin carcinogenesis
Principal Investigator: Shobhan Gaddameedhi, Ph.D., Post-doctoral Fellow, Department of Biochemistry and Biophysics, School of Medicine.
Award: $25,000

Abstract
Skin cancer is the most common form of cancer in the United States. Solar ultraviolet radiation (UVR) is a well-known human skin carcinogen. Exposure to the UVR causes DNA damage by generating photoproducts in DNA. In humans, these photoproducts are solely repaired by the process of nucleotide excision repair, and the loss of this repair system is strongly correlated with the development of skin cancer. We recently discovered that the circadian clock regulates nucleotide excision repair in mouse. Our preliminary results suggest that UV-induced DNA repair capacity varies in mouse skin as a function of time of day reaching its maximum in the evening (4 pm) and its minimum in the morning (4 am).The overall goal of this project is to understand how the circadian clock controls cellular responses to UV-induced DNA damage and to determine whether UV exposure at certain times of the day is more likely to cause skin cancer. These studies establish a rational for chrono-photo biology and suggests at what time of the day would be best for sunlight exposure and use of tanning beds.

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Melanoma: Small signal molecules may characterize the disease and guide rational design of therapeutics
Principal Investigator: Clark D. Jeffries, Research Professor, Department of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy.
Award: $25,000

Abstract
Several labs have reported (Lu 2005) that TaqMan microRNA (miRNA) assays can reliably discern cancer types and stages. Applied Biosystems technology has enabled exceedingly specific and sensitive, simultaneous RT-qPCR assays of 373 miRNA species in one 384-well plate. After confirming performance reported by others (Chen 2005, Orina 2009), we are now prepared to apply the technology. A notable publication by Jukic et al. (Jukic 2010) described TaqMan results for FFPE samples from melanoma patients, including certain miRNAs dramatically differentiated. Our preliminary tests with RNA isolated from seven cell lines obtained from the UNC Program in Melanoma subsequently indicated that cell lines indeed capture the miRNA biology of melanoma concordantly with FFPE samples. Numerous advantages clearly attend use of cell lines. We must now increase the number of data points substantially, hence our pilot grant application. Funding for confirmation of our preliminary results will promptly enable a scientific paper and applications to major funding sources.

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Genome-wide analysis of DNA replication fork stalling due to environmentally-induced DNA damage
Principal Investigator: Cyrus Vaziri, Ph.D.; Associate Professor of Pathology and Laboratory Medicine
Award: $25,000

Abstract
Benzo[a]pyrene (B[a]P) and Ultraviolet (UV) radiation are ubiquitous environmental agents that damage DNA and cause cancer. During S-phase DNA replication forks are particularly vulnerable to the detrimental consequences of DNA damage. Failure to accurately replicate and repair DNA leads to many hallmarks of cancer cells including mutagenesis and gross chromosomal rearrangements. This project will test the hypothesis that DNA replication defects arising from environmental DNA-damaging agents are over-represented at specific loci in the human genome. We have devised an innovative protocol for isolating chromatin at sites where replication fork stalling occurs specifically in response to B[a]P- or UV-induced DNA damage. We will perform next-generation sequencing and Microarray-based Comarative Genomic Hybridization (CGH) to perform genome-wide analysis of replication events in occurring in the absence and presence of genotoxins. The CEHS Biostatistics and Bioinformatics core facility will provide expertise and assistance for alignment of sequence reads on the human genome and identification of sites of significant over-representation. We anticipate we will be able to identify novel DNA sequences and epigenetic marks associated with replication fork stalling due to environmental carcinogens.

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Inactivation of Timeless in murine melanocytes enhances melanomagenesis
Principal Investigator: Norman Sharpless, M.D., Associate Professor of Medicine and Genetics.
Award: $25,000

Abstract
Effort in this pilot project will generate a novel murine “floxed” allele of an essential gene, Timeless. Timeless is a component of the replication fork protection complex that stabilizes DNA replication forks that are stalled at natural fork barriers and sites of UV-induced DNA damage. Reduced expression of Timeless should produce a condition of genomic instability leading to oncogenic events. UNC environmental melanoma investigators will use a conditionally activatable, melanocyte-specific CRE recombinase allele to selectively delete Timeless in melanocytes. Reduction in Timeless should enhance melanoma development and malignant progression. Additional studies will combine Timeless inactivation with other oncogenic events such as Ras activation to facilitate melanoma formation. To determine the contribution of Timeless to suppression of melanomagenesis, we propose the following specific aims: 1) to generate germ-line transmission of the floxed Timeless (TimLOXP) allele in C57Bl/6 mice; and 2) to breed TimLOXP mice with Tyr-Cre-ERT2 mice to generate mice with deletion of Timeless in melanocytes, and characterize the effects of somatic Timeless deletion in melanocytes. This project will determine whether an essential checkpoint mediator suppresses melanomagenesis in a murine model.

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Mapping methylated DNA sites associated with arsenical-induced skin disease
Principal Investigator: Rebecca Fry, Assistant Professor, Department of Environmental Sciences & Engineering, SPH
Award: $30,000

Abstract
Tens of millions of people worldwide are currently exposed to high levels of arsenic. Consumption of drinking water contaminated with inorganic arsenic (iAs) results in arsenicosis or chronic arsenic poisoning. Advanced stages of arsenicosis are associated with cancer of the skin. While the exact basis for iAs-induced skin disease is not known, a potential mechanism is that iAs causes epigenetic alterations that influence the expression levels of tumor-associated genes. We hypothesize that epigenomic alterations, specifically deregulation of DNA methylation, contribute in a significant way to the skin disease manifestations of arsenicosis. We will test this hypothesis by defining the epigenome-wide sites of DNA methylation that are associated with iAs-induced skin disease, using DNA isolated from residents of arsenicosis-endemic areas of Mexico. We will evaluate associations between DNA methylation, iAs exposure and metabolism, and skin disease. This pilot study will identify differential sites of DNA methylation that are associated with susceptibility to iAs-induced skin disease. This research is directly in line with the mission of the CEHS.

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Heterotypic interactions in breast responses to ionizing radiation
Principal Investigator: Melissa A. Troester, Ph.D., M.P.H., Assistant Professor of Epidemiology.
Award: $40,000

Abstract
Heterotypic interactions between initiated epithelium and surrounding stroma play a critical role in development and progression of cancer. There is strong evidence that the stroma does not just passively respond to initiated cells, but plays a more active role in carcinogenesis. This project will test the hypothesis that stromal-epithelial interactions determine the ionizing radiation (IR) response phenotype of the breast. In Aim 1, the gene expression changes induced by stromal-epithelial interactions will be assessed in coculture. This aim will develop a model culture system for characterizing cell-cell interactions. In Aim 2, we will use this culture system to study IR responses. Using cells in monoculture and coculture, we will identify gene expression changes that occur in coculture and are correlated with phenotypic endpoints such as growth arrest and apoptosis. Responses will also be compared with what is observed in cultures of tissue explants. This project will develop and validate a model coculture system for testing hypotheses about the role of heterotypic interaction in environmental carcinogenesis and will provide important biological insights about breast responses to ionizing radiation.

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Preclinical evaluation of genistein and soy extract in combination with conventional cytotoxic and hormonal treatment regimens for endometrial cancer
Principal Investigator: Victoria Bae-Jump, MD, PhD, Assistant Professor of Gynecologic Oncology
Award: $25,000

Abstract
Treatment of women with recurrent or advanced endometrial cancer has been met with limited success. This has prompted a search for an additional agent which could be used in combination with more traditional therapies to dramatically increase efficacy while not increasing toxicities. Genistein, the bioactive isoflavone of soybeans, acts as a potential radiosensitizer for prostate cancer and has been shown to enhance the cytotoxicity of chemotherapeutic drugs in a variety of tumor types. Thus, our overall goal is to evaluate cell proliferation, telomerase activity and apoptosis in a novel endometrial cancer co-culture model system after exposure to genistein or soy extract in combination with cytotoxic and hormonal chemotherapeutic agents commonly used in the treatment of this disease. The effects of genistein or soy extract on estrogen and progesterone receptor signaling will also be explored. We hope that this will provide valuable evidence that phytoestrogens may potentiate the effects of other cytotoxic and hormonal agents in endometrial cancer, and that combination therapy may be a more effective treatment option for women with recurrent or advanced stage disease.

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Gastrointestinal stem cells and risk factors for colorectal adenoma
Principal Investigator: Professor P. Kay Lund, Ph.D., Departments of Cell and Molecular Physiology, Nutrition and Pediatrics.
Award: $25,000

Abstract
Colorectal cancer is thought to be initiated by inappropriate survival or expansion of crypt stem cells. Low apoptosis of crypt cells strongly predicts precancerous adenomas in multiple patient populations supporting this model. Elevated insulin, obesity, and high fat diet increase adenoma risk, and correlate with low apoptosis. However, definitive evidence for a role of stem cells in adenoma, or an effect of environmental factors on stem cells is lacking because no valid stem cell markers were available. Recent findings by others, and here at UNC, define GPR49/Lgr5 and SOX9 as markers of multipotent intestinal stem cells, and SOX4 as a potential biomarker of a subset of stem cells. Our studies in mice demonstrate that insulin signaling promotes survival of SOX9-positive stem cells and colon tumorigenesis. This pilot aims to translate these exciting findings to humans and directly test the hypothesis that adenoma, elevated plasma insulin, obesity, or high fat diet promote expansion of GPR49/Lgr5, SOX9 or SOX4 positive stem cells in normal colon, leading to selective expansion of the same stem-cell sub-type in adenomas.

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Mutations in B-Raf increase UV genotoxicity in melanocytes through attenuation of DNA-damage induced cell-cycle checkpoints
Principal Investigator: Dennis A. Simpson, PhD; Research Instructor, Dept. of Pathology and Laboratory Medicine, School of Medicine

Abstract
Defects in DNA damage responses may underlie genetic instability and malignant progression in melanoma. Indeed melanomas harboring the most common mutation [B-Raf(V600E)] all exhibit attenuation in their DNA damage induced G2 checkpoint response. The molecular basis and biological result of this correlation between the B-Raf mutation and checkpoint attenuation is not clear. This proposal aims to begin to test the hypothesis that mutations in B-Raf result in attenuation of the DNA damage G2 checkpoint response and that this attenuation results in an increased sensitivity to UV induced mutations in additional genes such as CDKN2A, a gene frequently mutated in melanoma. This hypothesis will be tested by introduction of the B-Raf(V600E) allele into normal human melanocytes followed by precise measurements of the affects on cell cycle, including the DNA damage induced G2 checkpoint response. The majority of time in this proposal will be spent deriving the cell lines with which to test the hypothesis. We anticipate that these cell lines will be useful reagents for further studies of the early stages of melanoma progression regardless of the validity of the hypothesis.

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PCaP-GIS: A geospatial analysis of environmental risk factors for prostate cancer severity in North Carolina
Principal Investigator: Jane C. Schroeder DVM, PhD, Assistant Professor of Epidemiology, SPH

Abstract
African Americans are at greater risk of dying of prostate cancer than whites, in part because of racial differences in tumor characteristics that may reflect differences in etiologic mechanisms. Prior studies suggest that pesticide exposures may increase the risk or severity of prostate cancer, and it has been hypothesized that African Americans may be more highly exposed or susceptible to pesticide-mediated carcinogenesis than whites. The PCaP Geographic Information Study (PCaP-GIS) study will be based on existing data from approximately 450 African American and 450 white North Carolina prostate cancer patients enrolled in the North Carolina Louisiana Prostate Cancer Project (PCaP), a population-based study of racial disparities in prostate cancer aggressiveness. Community-level markers of pesticide exposures and healthcare availability will be derived using spatial statistics, and will be analyzed in combination with PCaP interview data concerning race, farming, pesticide-associated occupations and healthcare access to estimate race-specific associations between pesticides and prostate cancer aggressiveness. The PCaP-GIS will provide a unique and cost-efficient opportunity to evaluate environmental factors that may contribute to racial differences in prostate cancer severity among North Carolina men.

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Effects of cadmium on DNA synthesis, cell cycle control and microsatellite mutation rate in human cells in culture
Principal Investigator: Jayne C. Boyer, PhD, assistant professor, Department of Pathology and Laboratory Medicine

Abstract
Cadmium is an environmental human carcinogen that increases oxidative DNA damage and inhibits DNA repair. This study will determine the effects of cadmium on DNA replication and mismatch repair (MMR) in diploid human fibroblasts. DNA fiber immunofluorescence microscopy will be used to determine whether cadmium-induced DNA damage triggers the intra-S checkpoint to inhibit the initiation of new replicons and/or slows the rate of DNA chain elongation in active replicons. Analysis of Chk1 phosphorylation will test whether cadmium inhibits ATR signaling in response to replicative stress. For statistical significance, oxidative DNA damage will be measured and correlated with effects on DNA replication in five different lines of cadmium-treated human fibroblasts. Cadmium has been shown to increase microsatellite mutation rates in yeast and inhibit MMR in human cell extracts. Using a plasmid-based reporter system microsatellite mutation rates will be quantified in five lines of human fibroblasts after chronic exposure to cadmium. These studies will test the hypothesis that cadmium contributes to human carcinogenesis by inhibiting MMR and thereby increasing mutation rates in microsatellites as well as in non-repetitive sequences.

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Correlating breast cancer and obesity: Detection of biomarkers of susceptibility using an obese mouse model
Principal Investigator: Christoph H. Borchers, Ph.D., Assistant Professor of Biochemistry and Biophysics

Abstract
Breast cancer is a leading cause of cancer-related deaths among women, second only to lung cancer. Many epidemiological studies have uncovered a correlation between obesity and breast cancer. Not all obese women, however, will get breast cancer. Furthermore, the mitigating effects of exercise have also been postulated.

Our hypothesis is that there is a genetic basis for this difference in susceptibility and that it should be reflected in a difference in the proteome. The specific aim of this proposal is to discover protein biomarkers for breast cancer susceptibility in obese mice, which can be used to uncover the corresponding human biomarker. We will use quantitative proteomic approaches on blood from obese mice which did not develop breast cancer, vs. blood from mice which did develop breast cancer.

The results obtained will be used for a joint R01 between Dr. Borchers and Dr. Threadgill with the goal of using these biomarkers to study the mitigating effects of exercise in susceptible mice. Ultimately, we will develop a protein chip for screening human subjects in order to target prevention strategies to susceptible individuals.

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Identification of polymorphisms in smoking induced genes
Principal Investigator: Charles M. Perou, Ph.D., Assistant Professor of Genetics and of Pathology & Laboratory Medicine

Abstract
The diversity of clinical behaviors of squamous cell carcinoma of the head and neck (SCCHN; cancer of the oral cavity, pharynx, and larynx) poses a challenge to the prevention and treatment of this disease. Our laboratory has used genome-wide expression profiles to classify SCCHN into four distinct and reproducible subtypes. These subtypes showed statistically significant differences in recurrence-free survival. One subtype showed high expression of antioxidant enzymes that are involved in xenobiotic metabolism including Glutathione S-Transferase M3, Thioredoxin Reductase 1, Glutathione Peroxidase 2, Aldo-Keto Reductase 1, and two genes involved in the pentose phosphate cycle (Transaldolase 1 and Phosphogluconate Dehydrogenase). We propose a unique translational study to: 1) “resequence” these four of these genes from 80 SCHHN tumors to identify Single Nucleotide Polymorphisms (SNP) and determine haplotype structure and 2) to evaluate the population allele frequencies for these SNP using samples from an ongoing UNC SCCHN case-control study. The proposed “resequencing” project will not only provide clues to the importance of genetic variation in these smoking-related genes but will also provide polymorphic markers for evaluation in many other population studies.

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Melanoma expression signatures and heterogeneity
Principal Investigator: Nancy E. Thomas, MD, PhD, Associate Professor, Department of Dermatology, SOM & Lineberger Comprehensive Cancer Center

Abstract
Melanoma is an environmentally-induced cancer which has increased in incidence at an alarming rate during the past few decades. A primary cause of melanoma is DNA damage due to ultraviolet radiation (UV) from sunlight. However, the wavelength, intensity, and pattern of UV exposure on the human body that lead to malignant melanoma have not been sufficiently characterized; and, based upon tumor site, histology, and different genetic alterations that occur, it is likely that melanoma is a heterogeneous disease with differing etiologies related to different exposures. Further studies of etiology would be greatly facilitated by a better understanding of melanoma heterogeneity, and we hypothesize that some of this heterogeneity can be elucidated through gene expression profiling. We propose to do gene expression profiling in the context of known mutations in melanoma. This pilot project would begin that process by identifying gene expression patterns associated with BRAF mutation in frozen and formalin-fixed paraffin-embedded melanoma tissues. This pilot study will provide the foundation for future studies of melanomagenesis in the context of environmental exposures.

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The role of metabolism in the modulation of toxic and cancer promoting effects of arsenic in human urinary bladder
Principal Investigator: Zuzana Drobná

Abstract
Cancer of the urinary bladder is one of the most common forms of cancer associated with chronic exposure to inorganic arsenic (iAs) from drinking water. In humans the metabolism of iAs yields methylarsenic (MAs) and dimethylarsenic (DMAs) metabolites that contain arsenic in +3 or +5 oxidation state. The key enzyme in this pathway, AsIII-methyltransferase (Cyt19), is expressed in liver, but not in normal human urothelial (UROtsa) cells. Recent studies have shown that methylated trivalent arsenicals, methylarsonous acid (MAsIII) and dimethylarsinous acid (DMAsIII), are more potent than iAs as cytotoxins, genotoxins, and enzyme inhibitors. We have shown that in human bladder cells, MAsIII and DMAsIII are considerably more potent than iAsIII in activating ERK-mediated AP-1 DNA binding, a mechanism that is known to regulate cell proliferation and transcription of genes involved in bladder carcinogenesis (e.g., TGF-a). Notably, preliminary experiments have shown that iAsIII and methylated trivalent arsenicals do induce TGF-a expression by UROtsa cells. Thus the rate of the production and the yields of MAsIII and DMAsIII may be a critical step in cancer promoting effects of iAs. Recombinant wild-type (w/t) and mutated forms of rat and human Cyt19 are now available in our laboratory. The rates and patterns of in vitro methylation reactions catalyzed by these proteins depend strongly on the extent and character of mutation. Using a retroviral vector encoding w/t and mutated forms of Cyt19, we plan to established a genetically modified UROtsa cell lines with modified methylation capacities. We plan to use this cell line to study the role of enzymatic methylation in the modulation of toxic and cancer promoting effects of iAs in human bladder cells.

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Human responses to UV-induced DNA damage
Principal Investigator: Marila Cordeiro-Stone, Ph.D., Professor of Pathology and Toxicology

Abstract
This pilot project seeks to acquire preliminary data in support of a competitive application for a program project grant on “Human responses to UV-induced DNA damage.” Four independent researchers and a junior investigator with expertise in biochemistry, proteomics, molecular biology and radiobiology will pursue interdisciplinary research projects focused on the responses of human cells to UV-induced DNA damage. Project investigators independently have established strong records of productivity and accomplishment in the fields of DNA repair and cell cycle checkpoints. The joint effort by these investigators will be directed toward developing in vitro systems that are amenable to biochemical manipulations and responsive to DNA damage. The primary goal will be to demonstrate that these model systems could support mechanistic studies on the primary components of DNA damage checkpoints, mainly damage sensors, mediators, signal transducers, and effectors of specific responses. The focus of this pilot project will be on the intra-S phase checkpoint and the signal transduction reactions that underlie the inhibition of replicon initiation. Synergistic approaches fostered by this pilot project will lead to a new program of study to investigate the multiple interactions among cell cycle checkpoint and DNA repair proteins in human keratinocytes and fibroblasts damaged by UV. Solar UV radiation is a ubiquitous environmental carcinogen accounting for over a million new cases of skin cancer each year. Information gained by study of human responses to UV will facilitate understanding of responses to other environmental toxins that damage DNA and induce cancer at internal sites.

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Functional ATM polymorphisms
Principal Investigator: William K. Kaufmann
Biostatistics Consultant: Fei Zou

Abstract
This project will determine whether certain missense mutations in ATM produce a trait of G2-irradiation chromosomal hypersensitivity. A case-control study is underway at UNC-CH in which peripheral blood lymphocytes are tested for mutations in ATM and chromosomal hypersensitivity following treatment with ionizing radiation in G2. Cases represent women with newly diagnosed breast cancer and controls represent hospital patients matched for age and race but without cancer. To date ATM mutations and chromosomal hypersensitivity have been detected in both cases and controls. The frequency of the traits is increased about three-fold in cases relative to controls suggesting that ATM mutations and chromosomal hypersensitivity predispose to development of breast cancer. Several patient samples have been identified which display both mutations in ATM and chromosomal hypersensitivity. To prove that the trait of chromosomal hypersensitivity seen in these samples was a consequence of the mutation in ATM, a functional assay must be developed. We propose to clone ATM cDNA into a retroviral vector for efficient, stable transduction and expression of ATM in ATM-null cell lines. Expression of ATM in AT cells should restore DNA damage checkpoint functions and reduce chromosomal sensitivity to irradiation. Having a system in which ATM function can be demonstrated by restoration of checkpoint function and chromosomal repair, we will test the effect of introducing specific mutations in the ATM cDNA by site-directed mutagenesis. Missense mutations that were seen in lymphocytes with chromosomal hypersensitivity will be transduced alone or in combination with wildtype ATM. We will determine whether the missense mutations reduce the ability of ATM to enforce cell cycle checkpoints and enhance repair of chromatid breaks, and whether the mutations can override the function of wildtype ATM (dominant negative). This effort will help to define functional polymorphisms in a DNA repair gene.