Cancer cells and normal cells frequently differ in the way they respond to DNA damage. The goal of this project is to define differences in the response to DNA damage between cells with and without functional p53, the gene most commonly mutated in human cancer. DNA recombination is now recognized as an important mechanism in the repair of DNA double- strand breaks, such as those caused by ionizing radiation. Dr. Powell has reported that tumor cells lacking functional p53 have a rate of spontaneous DNA recombination up to 10,000 times greater than normal cells. The biological consiequences of high recombination rates are assumed to be genetic instability and chromosome abnormalities. The impact of high recombination rates on the repair of double-strand DNA damage is the focus of this renewal application.
The first aim will test the hypothesis that DNA recombination induced by double-strand cleavage is a determinant of the lethal effects of ionizing radiation. DNA damage-induced recombination will be measured using a panel of integrating DNA vectors that contain sequences which can be cleaved following integration into the genome. Two cell systems will be studied. First, radio-sensitive mutants which show recombination abnormalities (e.g. ataxia-telangiectasia (A-T) cells and scid cells) will be used to test whether genetic correction which restores radiation-resistance also corrects the recombination defects. Second, in cells either null or mutant for p53 (which have minor changes in radio-sensitivity in the absence of apoptosis) the frequency and type of DNA recombination will be compared with normal cells. A lack of G1/S arrest in response to DNA damage is seen in p53(-) cells and A-T cells.
The second aim i s to determine whether the high rate of DNA recombination seen in these cells is related to the lack of G1/S arrest. The cyclin-dependent kinase inhibitor p21 (WAF1, cip1), transactivated by p53 in response to DNA damage, will be both """"""""knocked out"""""""" and """"""""added back"""""""" in cells lacking functional p53. In addition, cell synchrony will be used to exclude cells from specific cell-cycle transitions, and DNA recombination will be measured at early time-points using an assay based on the polymerase chain reaction. The goal of this work is to identify DNA repair processes altered in cells without functional p53, to modify repair in these cells, and ultimately, achieve therapeutic gain by targeted action against the many cancer cells which have p53 mutations.
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