Data from this and other laboratories indicate that the haloethylnitrosoureas and related antitumor agents exert their therapeutic effects by modifying DNA. This hypothesis has been supported by evidence that Mer+ cell lines are resistant to the cytotoxic effects of the haloethylnitrosoureas and that a resistant cell line has a different profile of DNA modification than a sensitive cell line. While the presence of 06-alkylguanine-DNA alkyltransferase in Mer+ cells explains the absence of cross-links which originate with alkylation of the 06-position of guanine, the absence of other modified bases in the profiles of resistant cells suggests that other DNA modifications may also be cytotoxic. Furthermore, since these bases are not removed by 06-alkylguanine-DNA alkyltransferase, there is an implication that other significant repair mechanisms are present. The studies proposed here will use the observed differences in DNA modification between sensitive and resistant cells to attack the resistance problem. First, DNA modifications which are absent in cell lines resistant to the haloethylnitrosoureas will be structurally identified since their absence suggests that they may be cytotoxic in sensitive cells. Then, our observation that bacterial 3-methyladenine DNA glycosylase II can release these depleted peaks will be pursued; a sensitive assay for enzymatic release of these modified bases will be developed and used to assay this activity in resistant human tumor cell lines. The repair assay will also be used to develop potential inhibitors which could be used to restore sensitivity to the resistant cell lines. Initially, bases released enzymatically will be synthesized and tested for their activity as inhibitors. In view of the potential clinical advantages of newer compounds related to the haloethylnitrosoureas, these studies will also be extended to include clomesone and cyclodisone. DNA modifications will be identified and a search for cytotoxic lesions will be undertaken with a particular emphasis on identifying any cross-linked structures which may be present. The activity of known DNA repair enzymes on DNA modified by these new agents will be determined. As resistant cell lines become available, correlations will be sought between the development of resistance on the one hand, and profiles of DNA modification and the presence of repair activities on the other hand.
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