This is a revised application to study mutations in human cells which may result from deoxynucleotide precursor [dNTP] pool imbalance during DNA replication in human cells, and to ferret out their mechanisms. (Recent evidence has demonstrated that mutator phenotypes play an important roll in the development of colon and other cancers.) Recent evidence suggests that mutagenic damage from dNTP pool imbalances caused by environmental agents which alter metabolism of nucleotides may have important roles in the development of cancers. (Nucleoide pool imbalances can be caused in human cells by nutritional deprivation, by a number of agents used in cancer chemotherapy and by exposure to a variety of carcinogenic environmental genotoxins.) The goal of this proposal is to dissect mechanisms which may result in deoxynucleotide precursor [dNTP] pool imbalance-induced errors during DNA replication in human cells. These studies will employ transposable EBV vectors carrying the lacI gene to determine the mutational specificity of anti-folate induced mutations in normal and repair deficient human colon cancer lines . The mechanisms leading to formation of these hot spots will then be dissected by employing vectors in which DNA sequence surrounding hot spots for mutation have been altered by site specific approaches to change the DNA sequence context of the mutation site. The rationale for these studies is that sites where mutants are recovered at exceptionally high frequency have long been the most distinctive feature of the spectra of mutations caused by a wide variety of agents which damage DNA. Such sites have the potential to be the most accessible fingerprint of mutagens. In spite of our knowledge of this relationship, however, direct evidence for the role of DNA sequence context in causation of mutations has largely been inferred from studies in vitro and supported in living cells and organisms by relatively few investigations. In this proposal, mechanisms leading to formation of a hot spot mutation caused by dNTP pool imbalances will be explored. These investigations will examine the response of the susceptible DNA sequence in cells with both normal and defective mismatch repair in order to determine the impact of alterations in post-replicative repair capacity on inducible hot spots that require an interplay of multiple mechanisms for expression. Understanding how DNA sequence contributes to dNTP pool imbalance-induced hot spots will facilitate assessment of cancer risk from environmental exposure to toxic agents and aid in a rational choice of therapy for individuals with inherited or acquired defects in processes assuring faithful duplication of their DNA.
| Meyers, Mark; Hwang, Arlene; Wagner, Mark W et al. (2003) A role for DNA mismatch repair in sensing and responding to fluoropyrimidine damage. Oncogene 22:7376-88 |
| Ma, A H; Xia, L; Littman, S J et al. (2000) Somatic mutation of hPMS2 as a possible cause of sporadic human colon cancer with microsatellite instability. Oncogene 19:2249-56 |
| Chen, W D; Eshleman, J R; Aminoshariae, M R et al. (2000) Cytotoxicity and mutagenicity of frameshift-inducing agent ICR191 in mismatch repair-deficient colon cancer cells. J Natl Cancer Inst 92:480-5 |
| Veigl, M L; Kasturi, L; Olechnowicz, J et al. (1998) Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci U S A 95:8698-702 |
