| Summary: | Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1999. === Vita. === Includes bibliographical references. === The anticancer drug cisplatin is a small inorganic molecule that forms several types of covalent adducts on DNA. There is evidence that at least some of the consequences of this genetic damage are mediated by proteins that bind to the cisplatin-DNA cross-links or influence cellular pathways in response to the genotoxic stress. In either case, such factors can regulate the processing of the cisplatin lesions and thereby affect cellular sensitivity to the drug. Identification of these proteins and exploration of their cellular functions has implicated multiple systems including several classes of DNA repair, transcription, cell cycle and cell death responses. Chapter one reviews the current information of how components of these pathways respond to cisplatin-DNA adducts and contribute to the cytotoxic mechanism of action. Chapters two and three explore the capacity of one cellular defense mechanism, the nucleotide excision repair pathway, to remove cisplatin- DNA damage, and factors that can modulate this activity were examined. The cisplatin intrastrand cross-links are all excised by human nucleotide excision repair, however removal of the minor interstrand cross-link was not detected. Experiments performed with a reconstituted system of purified nucleotide excision components demonstrated that structural differences among the types of cisplatin-DNA lesions affect the relative excision repair rates. Attempts to correlate the repair capacity of human tissue with cisplatin sensitivity were unsuccessful, however the use of several model systems suggest that there are cellular factors that can modulate the removal of the cisplatin-DNA adducts. Members of the HMG-domain family of proteins specifically blocked the in vitro repair of the 1,2-intrastrand cross-links, an effect that was observed with a single HMG-domain polypeptide, and in the reconstituted repair assay. These results suggest that the ability of HMGdomain proteins to recognize cisplatin-modified DNA could affect cellular removal of the lesions, and that HMG-domain proteins with a selective expression pattern could contribute to cisplatin sensitivity. In particular, the testis-specific proteins tested were the most effective at inhibiting repair, which may be relevant to the favorable clinical response of testicular tumors to cisplatin-based chemotherapy. No repair inhibition by a mismatch repair protein was detected, although binding to cisplatin-DNA adducts was observed. Another factor that could explain the efficacy of cisplatin in treating testicular cancer is that, unlike other human neoplasms, a low frequency of mutations in the p53 tumor suppressor gene are observed in this tumor type. Previous studies have investigated the relationship between p53 status and cisplatin sensitivity, but have provided conflicting results. The p53-mediated responses in murine testicular teratocarcinoma cells exposed to cisplatin were examined in chapter four. Cisplatin exposure of cells with a wild-type p53 gene resulted in accumulation of p53 protein through posttranscriptional mechanisms; induction of p53-target genes was also observed. Drug treatment resulted in rapid apoptosis in p53-wild-type cells but not in a p53-/- teratocarcinoma line . In the latter cells, cisplatin exposure caused prolonged cell cycle arrest in the G1/early S phase, accompanied by induction of the p21 gene. Clonogenic assays demonstrated that the p53 mutation did not confer resistance to cisplatin. These experiments suggest that cisplatin inhibits cellular proliferation of testicular teratocarcinoma cells by two possible mechanisms, p53-dependent apoptosis and p53-independent cell cycle arrest. In the final chapter, the role of HMG-domain proteins in the mechanism of cisplatin cytotoxicity was further investigated. A human cervical carcinoma cell line was constructed which ectopically expressed a testis-specific protein, tsHMG, under the control of an inducible promoter. Examination of tsHMG, expression and cisplatin-induced apoptosis on a cellular level revealed that the nuclear protein can modulate the cytotoxic consequences of cisplatin. However, the effects of tsHMG depend on the drug treatment conditions and the extent of cell death. The results may be relevant 4 to the clinical requirement of achieving a balance between the antineoplastic and toxic properties of cisplatin. A model proposed to explain the observations will provide a basis for future studies of cellular factors that can affect cisplatin sensitivity, and for the design of more comprehensive antineoplastic therapies. === by Deborah B. Zamble. === Ph.D.
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