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Michele K. Evans, M.D., Investigator Chief, DNA Repair Unit and Deputy Scientific Director, NIA Laboratory of Cellular and Molecular Biology Email: me42v@nih.gov |
| Biography: Dr. Michele K. Evans, a board certified internist and medical oncologist, received her medical degree from the University of Medicine and Dentistry of New Jersey-The Robert Wood Johnson Medical School in Piscataway. She received her postgraduate training in internal medicine at Emory University School of Medicine and fellowship training in medical oncology within the Medicine Branch of the Clinical Oncology Program at the National Cancer Institute (NCI). Interest in human cancer prone disorders and DNA repair led her to study the role of DNA repair in cancer susceptibility as a Senior Clinical Investigator in the Laboratory of Molecular Pharmacology, NCI. At the National Institute on Aging (NIA), her major research interest centers on the clinical implications of eukaryotic DNA repair in cancer pathogenesis and aging. She also conducts epidemiologic work in the area of health disparities. In addition, Dr. Evans serves as Deputy Scientific Director, NIA. |
| Research: DNA repair mechanisms are believed to play a vital role in the maintenance of genome integrity. Loss of fidelity in the replicative mechanism, accumulation of genetic lesions, and faulty DNA repair mechanisms facilitate tumorigenesis. Similarly, aging or cellular senescence is characterized by random accumulation of damage or mutation in DNA, RNA, or proteins and perhaps a diminished ability to repair DNA. The increased incidence of cancer as a function of age underscores the mechanistic relatedness of these two cellular processes. The diminished ability to repair DNA appears to be the crucial and convergent factor highlighting the important clinical manifestations associated with defects in DNA repair mechanisms. The overall thrust of our work has been to understand the role of DNA repair in cellular senescence and tumorigenesis in order to uncover ways to use measured DNA repair capacity as a clinical tool in the diagnosis and treatment of cancer and age-related disease and disability. |
| Breast Cancer and DNA Repair: Breast cancer accounts for 15-18% of all deaths among women every year, with about 180,000 new cases being diagnosed every year. Even though the causes of breast cancer remain unknown, several lines of evidence suggest that accumulation of DNA damage coupled with defects in DNA repair play an important role in breast cancer. It has been speculated that DNA base damage may lead to mutations that subsequently can be carcinogenic. Of primary importance are the base lesions caused by reactive oxygen species (ROS). Cellular DNA is exposed to ROS either endogenously by cellular metabolism or through exogenous exposure to environmental mutagens. ROS induce a wide range of DNA lesions. Thymine glycol (Tg) and 8-hydroxyguanine (8-oxoG) are some of the most deleterious oxidative base lesions. Thymine glycol is a toxic lesion that blocks DNA replication and transcription, causing cell death. 8-oxoG is a premutagenic lesion. In order to avoid the harmful effects of 8-oxoG, organisms have developed mechanisms for repairing this damage. Studies using High Performance Liquid Chromatography and Gas Chromatography-Mass Spectrometry have revealed increased levels of 8-oxoG in invasive ductal breast carcinomas relative to normal breast tissue implicating oxidative damages in the etiology of breast cancer. It has been shown that 8-oxoG is repaired via the base excision repair (BER) pathway. To date, there are no reports on the removal of 8-oxoG or other oxidative DNA base lesions in breast cancer cells. Therefore, it remains to be established whether BER of oxidative lesions is altered during breast carcinogenesis. We therefore, hypothesized that the transformation from normal to malignant breast tissue may result from defects in oxidative DNA damage repair, consequently leading to mutations in important genes. Such a defect may occur in the nuclear and/or the mitochondrial genome. Mitochondrial DNA (mtDNA) encodes 13 proteins that are involved in oxidative phosphorylation. Oxidatively induced mutations in the mtDNA can lead to dysfunctional mitochondria, and have been implicated in degenerative diseases, cancer and aging. Therefore, effective oxidative damage repair processes are essential in order for the cell to maintain the integrity of the mitochondrial genome. We examined the ability of nuclear and mitochondrial extracts from a non-neoplastic mammary epithelial cell line and breast cancer MCF-7 and MDA-MB-468 cell lines to incise 8-oxoG and Tg lesions from duplex oligonucleotides. We have reported three important findings in this study: first, mitochondrial extracts from both MCF-7 and MDA-MB-468 breast cancer cell lines are deficient in the removal of 8-oxoG. Both breast cancer cell lines exhibited more than two-fold decrease in their ability to incise 8-oxoG relative to the wild type. This defect was specific for 8-oxoG since the incision of Tg by the same mitochondrial extracts was comparable to that of wild type cells. Second, nuclear extracts from both breast cancer cell lines removed 8-oxoG more rapidly and efficiently than mitochondrial extracts. Third, nuclear extracts were shown to remove Tg more rapidly than 8-oxoG. We have shown for the first time that mitochondria from human breast cancer cell lines are defective in the repair of 8-oxoG. This defective repair of 8-oxoG may imply that breast cancer cells have a high incidence of mtDNA mutations. The genetic status of mtDNA from these breast cancer cells remains to be determined through sequence analyses. Therefore, we conclude that repair of 8-oxoG in the mitochondrial genome may be crucial in the development of breast cancer. Our studies may provide a basis for novel molecular interventions of breast cancer. We further propose that other forms of cancer may be defective in oxidative DNA damage repair. We have also hypothesized that mitochondrial DNA of these cells may have excessive oxidative damage caused by defective oxidative repair. To address this hypothesis, mitochondrial and genomic DNA from these and other breast cancer cell lines will be analyzed by LC/GC mass spectrophotometry to determine the basal level oxidative damage. We will also assess induction of oxidative DNA damage by treating cells with specific oxidative damaging agents (e.g., Menadione, gamma irradiation, or hydrogen peroxide), for analysis of rates of lesion formation via LC/GC mass spectrophotometry. |
| In our most recent work, we have begun to evaluate the role of the BRCA 1 gene in oxidative damage repair. We are using two cell lines (CRL2336 and CRL2337) that are either homozygous or heterozygous for BRCA-1 mutation. The wt control for this project is the AG10009 lymphoblast cell line. Preliminary data suggests that nuclear repair of oxidative lesions, 8-oxoG, thymine glycol and 5-hydroxycytosine is reduced in cells homozygous for the BRCA-1 mutation relative to wild-type cells. Mitochondrial repair of oxidative lesions in this mutant cell line is comparable to that of wild-type cells. Once we have confirmed the repair phenotype of the BRCA1 mutant cell lines, further investigation will be directed to examining whether the specific repair enzymes involved in oxidative lesion repair (e.g., human endonuclease III (hNTH1) for thymine glycol) complexes with BRCA1 and other members of the BASC complex (Brca1-associated genome surveillance complex) as defined Wang et al. (BRCA1, ATM, NBS1, BLM, MRE-11, RAD50, MSH2, MLH1, MSH6). It is possible that the BRCA1 gene may play an important role in oxidative DNA repair in mammary tissue possibly partially explaining one of its roles in breast tumorigenesis. |
| The clinical relevance of nucleotide excision and base excision repair defects in tumor cells may lie in potential use of this DNA repair profiling as a tool in assessing metastatic potential of a specific tumor or in deciding upon appropriate cytotoxic chemotherapy. |
| Prostate Cancer and DNA Repair: Prostate cancer is the most prevalent cancer among American men and is classified as the second leading cause of their cancer mortality. In the United States, there will be 220,900 new cancer cases in 2003 making prostate cancer one of the cancers with the fastest rising incidence in this country as well as in Western Europe. While certain dietary, genetic, lifestyle and environmental factors are implicated in prostate cancer risk, the molecular mechanisms underlying the etiology of the disease are largely unknown. |
| Mutagenic oxidative DNA base damage increases with age in prostatic tissue. Many factors may influence this increase including: increased production of reactive oxygen species, increased susceptibility to oxidative stress, alterations in detoxifying enzyme levels or defects in DNA repair. Several research groups have begun to identify genes associated with heritable forms of prostate cancer and genes, in which somatic mutations or other somatic alterations may set the stage for the development and/or progression of the disease. To this end, it has been shown by several groups that hypermethylation of the p-class glutathione S-transferase gene (GSTP1) promoter region inhibits transcription of the gene and is associated with prostate cancer development. The function of GSTP1 has been proposed as a gene that defends genomic DNA in prostate cells from environmental or endogenous DNA-damaging agents. Environmental carcinogens such as heterocyclic amines and polycyclic aromatic hydrocarbons that result from cooking meat at high temperatures may play a role as it has been shown that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine can induce prostate cancer in rats. |
| Reactive oxygen species (ROS), most notably the hydroxyl radical, generated endogenously by cellular metabolism are known to cause oxidative DNA damage that has been implicated in prostate carcinogenesis. Research on the development of prostate cancer suggests that symptomatic and asymptomatic chronic and acute inflammation occurs in the prostate over the life span and acts in synergy with environmental, genetic, and dietary factors to cause injury to prostatic epithelium. In response to this injury, cellular proliferation has been shown to occur. This proliferation is accompanied by oxidative stress that is related to the ongoing inflammatory process that in turn may result in high rates of oxidative damage to DNA. Other findings that implicate a role for oxidative DNA damage in prostate carcinogenesis include work by Bostwick et al. showing that SOD1, SOD2 and catalase levels are lower in prostate intraepithelial neoplasia and prostate cancer relative to benign prostate epithelium. There is also a significant increase in the proportion of mutagenic oxidatively induced DNA base lesions, 8-hydroxyadenine (8-oxoA) and 8-hydroxyguanine (8-oxoG) present in malignant prostatic tissue as well as an increase in the levels of these lesions in benign prostatic tissue with aging. Further evidence supporting the hypothesis that defective repair of oxidative DNA damage may be pivotal in prostate carcinogenesis has been provided by work on genetic polymorphisms in the base excision repair (BER) gene OGG1. Taken together these data suggest that reactive oxygen species and oxidative DNA damage may be critical in the development of prostate cancer. |
| Using LC/MS and GC/MS, we show increased levels of oxidative DNA base damage over the baseline in PC-3 and DU-145 prostate cancer cells following exposure to ionizing radiation and a repair period. Nuclear extracts of PC-3 and DU-145 prostate cancer cell lines have defective incision of the DNA base lesions, 8-hydroxyguanine (8-oxoG), 5-hydroxycytosine (5OHC) and thymine glycol (TG) when compared to the non-malignant prostate cell line. Concomitantly, the levels of NEIL1 and NEIL2, enzymes that incise these lesions, are reduced in both cancer cell lines. Mitochondrial extracts from PC-3 and DU-145 also have defective incision of 8-oxoG compared to the control. PC-3 mitochondrial extracts are severely defective in the incision of TG and 5OHC. Consistent with the incision data, NTH1 and OGG1�2a protein levels are decreased in mitochondria of PC-3 cells. The antioxidant enzymes, glutathione peroxidase (GPx), catalase, and superoxide dismutases (SOD1, SOD2) have altered expression patterns in the cancer cell lines. Genetic analysis of the OGG1 gene reveals that both PC-3 and DU-145 cell lines harbor polymorphisms associated with a higher susceptibility to certain cancers. These data suggest that the malignant phenotype in PC-3 and DU-145 cell lines is associated with defects in base excision repair (BER), alterations in expression of BER and antioxidant enzymes, and OGG1 genetic polymorphisms. |
| Interaction of Human 8-oxoguanine-DNA Glycosylase with Proliferating Cell Nuclear Antigen: Strand Discrimination as a Mutation Avoidance Strategy: Human 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme for repairing 7-8, dihydro-8-oxoguanine (8-oxoG), a pre-mutagenic guanine base lesion produced by reactive oxygen species (ROS). The mutagenicity of 8-oxoguanine lies in its propensity to mispair with adenine during DNA replication. The importance of 8-oxoguanine and its repair by OGG1 are underscored by the frequent absence of the OGG1 allele in human lung tumors and the increased incidence of lung tumors in mice lacking a functional OGG1. 8-oxoguanine can occur in DNA by the oxidation of guanine in a G:C pair and by the incorporation of 8-oxoG into the newly synthesized nascent strand opposite cytosine or adenine during DNA replication or repair synthesis. Mispairings of 8-oxoG, when repaired by OGG1, could fix mutations if 8-oxoG in the parental strand is removed from a mispair with adenine. Accordingly, OGG1 should act only to remove 8-oxoG formed in DNA in situ and newly incorporated 8-oxoG in the nascent strand. If 8-oxoG in the parental strand becomes mispaired during DNA replication and is subsequently removed by OGG1, a G to T transversion mutation could result. Using co-immunoprecipitation, we identified an interaction between OGG1 and proliferating cell nuclear antigen (PCNA). PCNA is a multi-functional protein involved in DNA replication, repair synthesis and cell cycle regulation. The interaction of OGG1 with PCNA is of particular interest because known PCNA-binding proteins, such as DNA polymerases and components of the mismatch repair system, perform their functions on newly synthesized DNA are directed to the nascent strand via a directional interaction with PCNA. Using an in vitro binding assay and mutant OGG1 proteins, we have identified a functional consensus PCNA binding motif in the C-terminus of OGG1. Additionally, using immunofluorescence, we have shown that OGG1 and PCNA co-localize at sites of DNA synthesis in vivo. The association of OGG1 and PCNA suggests a bimodal mechanism of OGG1-mediated repair of 8-oxoguanine. In non-dividing cells, OGG1 and perhaps other DNA repair proteins may indiscriminately remove 8-oxoguanine as it occurs in DNA. During replication however, the OGG1-PCNA interaction may serve to direct OGG1 to the nascent strand in order to prevent fixation of mutations in the parental strand. The functional consequences of the interaction of OGG1 with PCNA, which are likely to be highly significant in vivo, are currently being investigated. |
| The Role of Uracil DNA Glycosylase in Base Excision Repair: Uracil is a normal base in RNA but a miscoding lesion in DNA. Incorporation of uracil in the genome can occur through deamination of cytosine and also through occasional use of dUTP instead of TTP during DNA replication resulting in premutagenic U:G or U:A base pairs. Unrepaired uracil in DNA mispairs with adenine resulting in mutagenic phenotype that is potentially carcinogenic. |
| To avoid the mutagenesis associated with unrepaired or insufficient repair of uracil, most organisms harbor uracil DNA glycosylase (UDG) in their cells. This enzyme is encoded by the ung gene in the nuclear genome but a splice variant is translocated to the mitochondria of higher organisms. Ung-/- mice have a modest increase in spontaneous mutation frequency (Nielsen et al., Mol. Cell, 2000, 5: 1059-1065). Quite recently, Nilsen and co-workers showed that ung deficient mice have increased incidence of B-cell lymphomas at old age (Nilsen et al., Oncogene; 2003, 22: 5381-5386). These data provide the first evidence of cancer development due to a deficiency in a DNA glycosylase in mice model in addition to a possible role of ung in the immune system. Furthermore, the data by Nilsen and co-workers also provide a link between carcinogenesis, DNA repair and the aging process. |
| The UDG is a highly ubiquitous DNA repair enzyme that initiates the repair of uracil through the versatile DNA repair pathway known as base excision repair (BER). The major steps in BER include: scission of the bond between the inappropriate base and the sugar (glycosylic activity) by a DNA glycosylase leaving an apurinic/apyrimidinic (AP) site, phosphodiester bond cleavage by AP lyase activity of the same enzyme or by an AP endonuclease (AP lyase activity), addition of the correct nucleotide by DNA polymerases (polymerization) and ligation by DNA ligases. The polymerization step has been shown to diverge into two sub pathways: the short- and the long patch. In the short-patch BER sub pathway, only one nucleotide is incorporated after the glycosylic step, whereas, in the long-patch sub pathway, more than one nucleotide is incorporated. To date, the in vivo significance and regulation of the two BER sub pathways in the repair of uracil remains unclear. We have hypothesized that uracil repair in the mitochondria is accomplished via the short-patch mechanism and that the sub pathways of BER are largely determined by the nature of DNA glycosylases involved. Our preliminary results obtained using mitochondrial extracts of human lymphoblastoid origin suggest that uracil repair in the human system is accomplished exclusively via the short-patch BER sub pathway. |
| In order to understand the mechanism of uracil BER in the mammalian mitochondria, we have engaged in a collaborative study with Dr. Samuel H. Wilson�s Laboratory of Structural Biology at the National Institute of Environmental Health Sciences, NIH. In this study, we are using wild type and UDG knockout mouse fibroblasts, which were established by Dr. Wilson�s group and oligonucleotides containing a single uracil at specific location. Using this model system, we are assessing glycosylic activities of mitochondrial and nuclear isoforms of UDG. In addition, we are examining the mechanism of nucleotide incorporation (repair synthesis) in uracil BER. We are also studying the size (nucleotides) of the repair patch generated in the course of uracil repair. Furthermore, we are using this system to determine the nature of protein-protein interactions involved during the repair of uracil in mouse system. Since UDG is a pure DNA glycosylase without an associated AP lyase activity, it must perform the glycosylic bond scission and then hand over the resulting AP site to an AP endonuclease for further processing in order to complete the repair process. This notion would be consistent with the �passing the button� model proposed some years ago by Dr. Wilson. To ascertain if this is the case, we intend to perform repair synthesis reactions using cell-free extracts from mouse UDG-knockout and wild type cells in the presence of purified AP endonuclease. Proficient repair synthesis is expected in the presence of both UDG and AP endonuclease if the proposed model is true. However, the lack of UDG in the knockout cells may not support proficient repair synthesis even if AP endonuclease is present. This project may also allow us to determine if the mouse system harbors back-up DNA repair pathways for uracil. |
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