The biochemical and molecular perturbations which mediate the cytostatic and cytotoxic effects of antineoplastic drugs which damage DNA are not understood. The studies proposed in this application are predicated upon the hypothesis that down regulation of the expression of the c-myc oncogene and alterations in the levels and activity of the myc oncoprotein may be critical components of one pathway of growth arrest in response to DNA damage& in MCF-7 breast tumor cells. In order to test this hypothesis, we propose to demonstrate that transfection of MCF-7 cells with a c-myc construct driven by a constitutive promoter reduces or abrogate sensitivity to the topoisomerase II inhibitors, VM-26 and m-AMSA; in contrast, a similar transfection of K562 human leukemic cells (where c-myc appears to be uninvolved in growth regulation) with constitutively expressed c-myc should fail to alter cell sensitivity to these drugs. The nature of c-myc down-regulation will be defined by discriminating between effects of VM-26 and m-AMSA at the level of transcription (transcript initiation and elongation) and transcript stability; the involvement of the c-myc promoter region in the cellular response to VM-26 and m-AMSA will be established by monitoring drug effects on CAT activity using a myc promoter-CAT construct transfected into MCF-7 cells. The association of the myc oncoprotein with growth arrest 'will be defined by determining the influence of VM-26 and m-AMSA on oncoprotein levels, the phosphorylation state of the oncoprotein, and binding of the oncoprotein to its consensus sequence. Determination of the capacity of various DNA damaging drugs (and ionizing radiation) to produce collateral modulation of c-myc expression and growth arrest will serve to establish whether c-myc is uniformly involved in the cellular response to DNA damage in MCF-7 cells. Finally, the paradigm relating down-regulation of c-myc expression to growth arrest in response to DNA damage will be evaluated in other experimental models of breast cancer.
