DNA interstrand cross-linking agents produce devastating point mutations, deletions, chromosomal aberrations, and cell death. However, these toxic effects are beneficial when directed at rapidly dividing tumor cells, leading to powerful antitumor activity. The mechanism of carcinogenicity versus chemotherapeutic potential within the cell remains unexplained. The proposed research will examine the effect of cellular proteins on DNA cross-linking by carcinogenic diepoxyalkanes and will additionally investigate the cross-linking reactions of natural diepoxides found in certain mushrooms. The antitumor drug cisplatin forms DNA intrastrand and interstrand cross-links, both of which induce a bent DNA structure. The resulting lesions are targeting be HMG1 and related proteins that recognize altered DNA structures. Changes in DNA global structure may therefore have cytotoxic significance via HMG proteins. Geometries of diepoxide- cross-linked DNA's will be examined through native polyacrylamide gel electrophoresis (PAGE) to verify experimentally the bending suggested by previous computer modeling studies. Binding to HMG1 by diepoxide- cross-linked DNA will then be monitored by a gel-shift assay. These studies will allow the exploration of the relationship between the geometry of a DNA cross-linked lesion and recognition by HMG proteins. This information could provide a new avenue for drug design: agents designed to trigger HMG binding to specific DNA sequences and thus induce cancer cell death. Because DNA does not exist free in the cell but rather is complexed with histone proteins to form chromatin, cross-linked products may differ in vivo from those found in vitro with free DNA. The histone core constrains DNA structure to generate differences in hydroxyl radical reactivity within the nucleosome. Therefore, it is likely that these DNA-protein interactions will modulate diepoxide cross-linking reactions relative to free DNA. PAGE will be used to define the sites of attack and relative efficiencies for diepoxyalkanes within defined-sequence nucleosomes. This work will provide further information about the genomic sites of cross-linking for chemically analogous agents with differing biological activities. Denaturing PAGE will also be used to pinpoint putative sites of interstrand cross-linking by a cytotoxic diepoxide found in Hydnum rapandum, perhaps providing data for the development of new antitumor drugs.
