The overall goal of this application is to determine if DNA damage produced by clinically used alkylating anti-tumor agents can alter the expression of activated oncogenes which are critical for the maintenance of the tumorigenic phenotype. Using Maxam-Gilbert DNA sequencing methodology initial studies will determine if nitrogen mustard (HN-2), L-phenylalanine mustard (L-PAM), and two cyclophosphamide derivatives (4-HC and C2) have DNA sequence preferences for attack in cloned coding regions from several activated oncogenes. The effects of this DNA damage on transcription of the damaged oncogene sequence will be studied in an in vitro RNA transcription system. The sequence selectivity of the agents will then be studied in treated tumor cells by determining if alkylation patterns in oncogenes and other critical cellular genes differ with the different anti-tumor agents. These experiments will also allow the study of the repair of DNA damage in activated oncogenes. The drugs will then be studied for DNA damaging properties, cytotoxicity as measured by colony formation assays, and effects on oncogene expression (as measured by Dot and Northern blot analysis) in human tumor cell lines having these oncogenes activated. Cell lines having single copy over-expressed oncogenes will be compared to cell lines having amplified copies of the same oncogene to determine if the anti- tumor agents are more, or less, effective at oncogene inactivation and cell killing depending on the oncogene copy number. From these studies it is hoped that anti-tumor agents might be selected for use against specific oncogenes due to the ability of the particular anti-tumor agent to efficiently inactivate critical oncogenes in the tumor cell.
