cis-Diamminedichloroplatinum (II) (CP) is one of the most important antineoplastic agents used to treat solid tumors. The bases of its selective toxicity to tumor tissue and development of resistance by malignant tissue to CP are incompletely understood. The overall objective of this grant proposal is to determine the biochemical mechanisms by which signal transduction pathways control the antitumor activity of CP and to develop strategies to improve the therapeutic efficacy of CP. Specifically, I intend to use human cervical cancer (HeLa) cells that are sensitive and resistant to CP to investigate how protein kinase C (PKC), a key signal transducing element, regulates the antiproliferative activity of CP.
The Specific Aims and the methodologies to be used are as follows: 1. Determine the effects of PKC inhibitors on cellular sensitization to CP. The ability of PKC inhibitors to decrease CP sensitivity or to antagonize PKC activator-mediated CP sensitization will be investigate by using novel synthetic inhibitors of PKC or monoclonal antibody to PKC. 2. Elucidate the role of PKC isoforms in the drug sensitization phenomenon. The contribution of a particular isoform of PKC in the drug sensitization phenomenon will be investigated by comparing: (i) the change in expression of PKC isoforms induced by agents that sensitize cells to CP, (ii) cell-type specific expression of isoforms either constitutively or following treatment with PKC activators, and (iii) CP sensitivity of cells that overexpress a specific PKC isoform due to introduction of expression vector containing PKC cDNA corresponding to the alpha, beta or gamma isoform. 3. Elucidate the mechanism(s) of PKC-mediated sensitization to CP. The functional significance of PKC-mediated sensitization to CP will be investigated by studying the effects of specific PKC activators and inhibitors on: (i) cellular drug accumulation (uptake and efflux), (ii) subcellular distribution, and (iii) DNA damage and repair. 4. Determine if phosphorylation of a specific protein is involved in PKC- mediated cellular sensitization to CP. Protein phosphorylation will be examined in intact cells under conditions that would either induce or reverse CP sensitization to establish a link between phosphorylation of a particular protein and CP sensitization.
