Cisplatin is the most effective chemotherapy drug for the treatment of ovarian cancer. Its usefulness in the clinic is restricted by dose-limiting nephrotoxicity and the development of drug-resistance within the tumor. During the previous funding period our studies demonstrated that the nephrotoxicity of cisplatin is due to the metabolism of cisplatin-glutathione conjugate through a GGT- mediated pathway in the kidney. We further demonstrated that this GGT-mediated toxicity is specific to the kidney. When GGT was transfected into tumor cells it reduced the toxicity of cisplatin rather than increasing it as was seen in the kidney. These findings indicate that there are two distinct mechanisms of cisplatin toxicity. The delineation of these two mechanisms would provide the opportunity to inhibit the side effects of cisplatin without compromising its antitumor activity. The current proposal focuses on defining the mechanisms by which cisplatin exerts its toxic effects and identifying the mechanism by which tumor cells develop resistance to cisplatin. The first specific aim is to further characterize the unstable cisplatin-glutathione conjugate that is a substrate for GGT and follow its metabolism within the kidney. A series of cisplatin-glutathione conjugates will be tested as substrates for GGT-mediated activation to nephrotoxins by LLC-PK1 cells and human renal proximal tubule cells. Cisplatin-glutathione conjugates will be isolated by HPLC and their structure determined by Mass Spectrometry. The pathway by which they are metabolized will be determined with the use of selective inhibitors of enzymes within the pathways. Inhibitors that block the activation of cisplatin in vitro will be tested for their ability to block the nephrotoxicity of cisplatin in vivo. The second specific aim is to identify the mechanism by which tumors become resistant to cisplatin in vivo. Human epithelial ovarian tumors will be propagated in nude mice. Half the mice will be treated with cisplatin. Cisplatin resistant tumors will be isolated after several courses of treatment. mRNA will be isolated from these tumors and differentially expressed genes will be isolated by representational differential analysis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA057530-08
Application #
2859778
Study Section
Special Emphasis Panel (ZRG1-PTHB (01))
Program Officer
Wolpert, Mary K
Project Start
1992-08-01
Project End
1999-08-31
Budget Start
1999-06-01
Budget End
1999-08-31
Support Year
8
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Virginia
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
West, Matthew B; Partyka, Katie; Feasley, Christa L et al. (2014) Detection of distinct glycosylation patterns on human ?-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology. BMC Biotechnol 14:101
West, Matthew B; Wickham, Stephanie; Parks, Eileen E et al. (2013) Human GGT2 does not autocleave into a functional enzyme: A cautionary tale for interpretation of microarray data on redox signaling. Antioxid Redox Signal 19:1877-88
Wickham, Stephanie; Regan, Nicholas; West, Matthew B et al. (2012) Divergent effects of compounds on the hydrolysis and transpeptidation reactions of ?-glutamyl transpeptidase. J Enzyme Inhib Med Chem 27:476-89
Hanigan, M H; Dela Cruz, B L; Shord, S S et al. (2011) Optimizing chemotherapy: concomitant medication lists. Clin Pharmacol Ther 89:114-9
West, Matthew B; Wickham, Stephanie; Quinalty, Leslie M et al. (2011) Autocatalytic cleavage of human gamma-glutamyl transpeptidase is highly dependent on N-glycosylation at asparagine 95. J Biol Chem 286:28876-88
Wickham, Stephanie; West, Matthew B; Cook, Paul F et al. (2011) Gamma-glutamyl compounds: substrate specificity of gamma-glutamyl transpeptidase enzymes. Anal Biochem 414:208-14
West, Matthew B; Hanigan, Marie H (2010) ?-Glutamyl transpeptidase is a heavily N-glycosylated heterodimer in HepG2 cells. Arch Biochem Biophys 504:177-81
West, Matthew B; Segu, Zaneer M; Feasley, Christa L et al. (2010) Analysis of site-specific glycosylation of renal and hepatic ?-glutamyl transpeptidase from normal human tissue. J Biol Chem 285:29511-24
King, Jarrod B; West, Matthew B; Cook, Paul F et al. (2009) A novel, species-specific class of uncompetitive inhibitors of gamma-glutamyl transpeptidase. J Biol Chem 284:9059-65
Hanigan, Marie H; Townsend, Danyelle M; Cooper, Arthur J L (2009) Metabolism of cisplatin to a nephrotoxin. Toxicology 257:174-5; author reply 176-7

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