Cisplatin is thought to impart its chemotherapeutic efficacy via the formation of coordinate-covalent DNA adducts and the subsequent induction of programmed cell death, or apoptosis. Repair of the DNA damage by the nucleotide excision repair (NER) pathways is detrimental to the cytotoxic activity of this drug. Cisplatin is also used clinically as a sensitizer to ionizing radiation (IR), however, the exact mechanism of how cisplatin exerts this activity is unclear. The applicant's preliminary data support a mechanism involving inhibition of the DNA-dependent protein kinase (DNA-PK). The goals of the research described in this proposal are to characterize the cellular proteins that bind cisplatin-damaged DNA and determining how these interactions contribute to the in vivo activities of cisplatin. The applicant hypothesizes that both the sensitization and cytotoxic activity of cisplatin is potentiated by the interplay of protein factors that bind the cisplatin-damaged DNA. To address the hypothesis and achieve these goals, four specific aims will be completed. The applicant's hypothesis predicts shielding proteins will have a preferential kinetic interaction with cisplatin-damaged DNA compared to NER proteins. Therefore, in Aim 1, the applicant proposes to investigate the kinetics of cisplatin-DNA damage recognition and binding by NER and HMG1 shielding proteins. The applicant's hypothesis also predicts that in cells treated with cisplatin, shielding proteins will be associated with cisplatin-damaged-DNA and block the access of NER proteins to the damaged sites. The experiments described in Aim 2 will assess the in vivo interaction of NER and shielding proteins with cisplatin-damaged DNA via indirect immunofluorescence. To test the hypothesis that the sensitization activity of cisplatin is a result of DNA-PK inhibition, a series of in vitro and in vivo experiments are proposed in Aims 3 and 4. The effect of cisplatin-DNA damage on DNA-PK activity and double-strand DNA break repair will be assessed in vitro and DSB repair and sensitivity to IR will be assessed in vivo. Achieving the goals described in this proposal will provide important information on the interaction of mammalian proteins with cisplatin-damaged DNA and how these interactions alter the cytotoxic and sensitization activity of the drug cisplatin in vivo. A better understanding of these interactions will allow the development of more effective cancer treatment protocols that may include inhibiting DNA repair pathways to achieve greater cytotoxicity or increase the sensitivity of cancer cells to IR.
Showing the most recent 10 out of 35 publications
