The overall goal of the proposed research is to determine whether DNA damage from genotoxic chemical carcinogens is distributed non-randomly in the genome, i.e., is """"""""targeted"""""""" to specific genes in vivo. The specific objective of this research is to determine the molecular basis for the strong preferential effects of carcinogens on the expression of inducible genes, principally using the DNA crosslinking agent, mitomycin C (MMC), as a model genotoxic agent, and the hormone-inducible phosphoenolpyruvate carboxykinase (PEPCK) gene as a model inducible gene. Based on our previous work, we have hypothesized that specific regions of inducible promoters represent the target for carcinogen effects on gene expression, and that the chromatin structure of these regions is an important determinant for carcinogen sensitivity.
Our specific aims will be to examine the following questions: 1. Are specific regulatory regions within the PEPCK promoter responsible for the preferential effects of MMC on PEPCK gene expression? We will determine the molecular target(s) for carcinogen effects on basal and hormone-inducible PEPCK gene expression using: a) stably transfected H4IIE cell lines containing integrated PEPCK promoter/CAT reporter gene constructs to determine which regions of the PEPCK promoter confer carcinogen sensitivity; and b) LM-PCR-based in vivo footprinting to examine carcinogen-induced alterations in regulatory factor-DNA interactions within these regions. We will also examine whether specific PEPCK promoter regions can confer carcinogen sensitivity to the constitutive beta-actin gene promoter. 2. Do specific chromatin structural features within the PEPCK promoter play an important role in determining the responsiveness of the PEPCK gene to glucocorticoid hormones and/or MMC? We will examine the structure of the PEPCK promoter i detail, using DNase I and micrococcal nuclease (MNase) mapping techniques. We will compare the structural features of these promoters with levels of basal and glucocorticoid-inducible expression, and with MMC sensitivity, in: a) stably transfected H4IIE cell lines containing randomly integrated intact PEPCK promoter/CAT reporter constructs; b) stably transfected H4IIE cell lines containing various genetically altered PEPCK promoter/CAT reporter constructs; and in c) chick embryo liver in vivo between 10 and 16 days of development, during which both hormone responsiveness and carcinogen sensitivity are concomitantly altered. Determining the mechanisms for such preferential effects would have important implications for understanding the molecular basis for the initiation of the cancer process by chemical carcinogens. These studies may also provide insight into new ways of modulating the actions of chemotherapeutic crosslinking agents such as MMC or cisplatin, with the aim of more selectively killing cancer cells while reducing the toxicity of these drugs to the patient.
