The major goal of this project is to understand how the nature of the carrier ligand affects resistance platinum drugs. In particular, we are interested in carrier ligand effects involving diaminocyclohexane (dach)-Pt compounds, since one of these compounds, ormaplatin (OP), is currently in clinical trial. Our data show no significant carrier ligand effects on bulk DNA repair of Pt adducts in the L1210 cell lines, but do not eliminate the possibility of carrier ligand effects on gene specific repair. Thus, we propose to collaborate with Dr. Vilhelm Bohr to determine whether there are any carrier ligand effects on gene specific repair in the L1210 cell lines. We have characterized carrier ligand effects in some detail with the human cell lines, but have not completely resolved the issue of which of these carrier ligand effects are most predictive dach-Pt sensitivity in cisplatin (CP)-resistant tumors. Thus, we plan to: (a) compare carrier ligand effects uptake and efflux, tolerance of PT-DNA adducts, repair and bypass replication for A2780/DDP, which shows no carrier ligand effects on resistance, and A2780/CP8, another CP-resistant derivative of A2780 which retains sensitivity to dach-Pt compounds; (b) confirm carrier ligand effects on repair by an independent assay; and (c) determine carrier ligand effects on bypass replication. Recent studies have suggested that decreased accumulation of platinum may be an important mechanism of resistance in many 11 lines. Unfortunately, the mechanism of platinum accumulation is not known in detail, in part because of the lack of suitable radiolabeled platinum compounds and in part because of the difficulty in separating efflux from the metabolism of platinum complexes. Our data show that the pattern of uptake OP and CP is essentially the same in the human cell lines we are studying. Thus, we plan to: (a) use the 3H- OP we have prepared previously to characterize uptake and efflux in detail; (b) use the HPLC techniques we have devised to determine the effects of intracellular metabolism on uptake and efflux; and prepare a non-metabolizable platinum complex for uptake and efflux studies. Finally, our data suggest at both repair and bypass replication may be inducible. Thus, we plan to: (a) determine the extent to which both repair and bypass replication are inducible, whether the induction of those processes is required for the resistant phenotype, and whether carrier ligand effects are expressed at the level of induction; (b) use the transient chloramphenical acetyltransferase expression assay to characterize the induction of gene expression in our cell lines; and (c) determine if the signal for the induction of gene expression is Pt damage on DNA. By identifying the sites and mechanisms of carrier ligand effects on resistance, it is hoped that is research will: (1) lead to improved strategies (eg combination chemotherapy) for overcoming resistance with dach-Pt compounds; (2) help identify the tumor types most likely to respond to dach-Pt drugs; and (3) lead to the design of even more effective platinum compounds.
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