The objective of this proposal is to determine the mechanisms and significance of early response gene activation by antitumor drugs which inhibit DNA topoisomerases. Although these agents damage DNA by stabilization of a """"""""cleavable-complex"""""""" topoisomerase-DNA reaction intermediate, additional events are believed necessary for cell death. Activation of specific genes may be among such events, as there are known bacterial, yeast, and mammalian stress responses which involve induction of gene transcription. Preliminary work has demonstrated that etoposide, a topoisomerase II inhibitor, and camptothecin, a topoisomerase I inhibitor, induce expression of c-jun and other early response genes. These findings will be extended by using other inhibitors of these enzymes and by determining the effects of these agents on expression at both the RNA and protein levels (Specific Aim 1). The mechanism responsible for induction of c-jun expression will be determined by nuclear run-on, mRNA stability, reporter-gene, and electrophoretic mobility-shift assays (Specific Aim 2). Preliminary data also indicate that a protein kinase, possibly protein kinase C, is involved in the induction of c-jun by camptothecin. Consequently, experiments are planned to confirm and extend this finding by directly measuring phosphorylation of specific peptides and by determining effects of inhibitors of this activity on drug-induced c-jun expression (Specific Aim 3). The relationship between induction of c-jun expression and other events, such as programmed cell death, will be determined by the use of a trans-dominant negative Jun mutant capable of suppressing transactivation by wild-type Jun (Specific Aim 4). Finally, the induction of c-jun expression by topoisomerase inhibitors will be studied-in cells resistant to the lethal effects of these agents (Specific Aim 5). Taken together, these studies should improve the current understanding of the cellular response to anticancer agents which inhibit topoisomerases.
| Rubin, E; Wood, V; Bharti, A et al. (1995) A phase I and pharmacokinetic study of a new camptothecin derivative, 9-aminocamptothecin. Clin Cancer Res 1:269-76 |
| Rubin, E; Pantazis, P; Bharti, A et al. (1994) Identification of a mutant human topoisomerase I with intact catalytic activity and resistance to 9-nitro-camptothecin. J Biol Chem 269:2433-9 |
