ALTERATION OF NAD METABOLISM BY CHEMICAL CARCINOGENS

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Cancer results when cells survive DNA damage and progressively lose genomic integrity. Compelling evidence now demonstrates that poly(ADP- ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are part of a complex network of proteins that function to oppose the loss of genomic integrity by detecting DNA damage and mediating cellular responses, which can range from DNA repair leading to recovery of normal cell function to the elimination of damaged cells by apoptosis or necrosis. The hypothesis to be tested in this proposal is that the coordinated activities of PARP and PARG are essential for the maintenance of genomic integrity. Specific aim 1 is to determine specific functions of PARG. This will be accomplished by the controlled depletion of the cellular content of PARG, using inducible expression of a stably transfected antisense clone of PARG cDNA in 3T3L1 cells. The objective is to approach a PARG null phenotype. Specific aim 2 is to determine if an optimal ratio of PARP to PARG is required for modulation of cellular responses to DNA damage. The cellular content of PARG will be progressively elevated by the inducible overexpression of a sense clone of PARG in 3T3L1 cells with a normal content of PARP and in cells derived from animals containing a disrupted PARP gene. In both specific aims 1 and 2, biochemical and biological functions affected by the genetic modulation of PARG will be assessed. In the absence of genotoxic stress, cell viability and inherent genomic stability will be determined by colony formation, rates of growth, and SCE frequency. Following genotoxic stress, the effects of modulation on the frequency of SCE, chromosomal aberrations, DNA base excision repair, p53 function, and cytotoxic responses involving apoptosis and necrosis will be determined. Analysis of alterations of the kinetics of NAD and ADP-ribose polymer metabolism will be used to evaluate the function of PARG in these cellular responses. Specific aim 3 is to determine specific functions of PARG structures revealed by cDNA cloning, including a putative regulatory domain and a nuclear location signal. Antibodies specific to the regulatory domain will be produced to follow its cellular fate and genetic modulation of this domain will be used to search for its function. Understanding how PARG functions is essential to the long term objectives of this proposal, which are to enhance protective cellular responses that maintain genomic integrity and to design therapies that target this pathway to facilitate the destruction of cancer cells.