In cancer, particularly in melanomas, the resistance to radiation is a major handicap in the successful treatment of these diseases. The mechanisms and genes involved in resistance to radiation are not well identified. The applicant has previously discovered that a transcription factor ATF2 that bind to the UV response element plays a major role in apoptosis induced by the radiation in normal cells. The resistance to radiation develops when the cells do not undergo apoptosis upon exposure to UV or ionizing radiation. In preliminary studies, the applicant has found that another enzyme which is involved in phospholipid metabolism namely P13K may also play a role in transducing the signal from membrane to the nucleus. The applicant now wishes to explore the role of P13K in radiation induced resistance in greater detail. There are three specific aims in the presented research proposal. First, the applicant will study the phospholipids and activity of related enzymes like phosphatidylinositol kinase and phospholipases involved in the UV response using cells whose radiation resistance has been altered through expression of dominant negative ATF2 or P13K constructs. Second, he will determine the relationship between P13K and ATF2 in conferring radiation resistance. He will also determine whether P13K uses the same pathway as ATF2. To this end, he wil transfect melanoma cells which show reduced resistance to a radiation dominant negative mutant of regulatory subunit p85 or catalytic subunit P110 of P13K. The earlier stage melanoma cells that exhibit elevated resistance because of P110 expression will be transfected with wild type or mutant ATF2 constructs. All cell lines will be evaluated for radiation resistance, phospholipid turnover, P13K and ATF2 transcriptional activities. Third, he will study the mechanisms by which P13K can force radiation resistance. The applicant will us melanoma cells transfected with P85 (dominant negative) and P110 (over expresser) subunit of P13K as well as with selective ATF2 fragments, to identify the downstream changes which mediate altered resistance of melanoma cells to radiation. The end point for the radiation resistance will be measurement of apoptosis and DNA fragmentation.
