The candidate, during the prior fellowship training at the UWCCC (years 1+2-clinical training, 3+4-preclinical) has had the opportunity to gain experience in all stage of a research project. Clinical experience was gained as the """"""""phase I fellow"""""""" which involved writing protocols, monitoring the studies, and publishing the results. Due to the candidate's prior work and interest in preclinical drug modulation, research experience was sought in the laboratory of Prof. R.T. Mulcahy, an active member of the Phase I program who has an extensive history of working in the field of experimental therapeutics and drug modulators. Preliminary work examining the glutathione (GSH) redox system has provided valuable experience in tissue culture and molecular biology techniques as well as work with enzyme assays, all necessary to successfully complete the CIA research proposal. Elevations in GSH, a ubiquitous tripeptide, or in GSH S-transferases, a related enzyme, have been found in an increasing number of drug-resistant cell lines. Unfortunately, there has been minimal work exploring the etiology of the increased GSH levels. Is it related to increased biosynthetic enzymatic activity and if so, does this originate at the gene level of post-translationally? Also, could increased expression of a biosynthetic enzyme be a better marker of drug resistance? In order to provide insight into this, the activity of the enzymes of the GSH redox system will be examined in multiple drug-resistant tumor cell lines with elevations in GSH or related enzymes. The human cDNAs for gamma- glutamylcysteine synthase (which was cloned in the Mulcahy lab) and gamma-glutamyl transpeptidase and the necessary biochemical techniques have been successfully used in the Mulcahy lab to initiate studies to hopefully answer these questions. Another important aspect of the proposal is the candidate's role as the study chair of recent and any future clinical trials with Buthionine Sulfoximine (BSO) a modulator of GSH. This provides a unique opportunity to take the observations from the laboratory directly to the clinic or vice versa. The UWCCC Phase I program (a NCI contract awardee for > 10 years) provides an excellent environment to accomplish the clinical aspects of this proposal. The preclinical research would continue to be done in Prof. Mulcahy's laboratory with continued use of UWCCC shared facilities and resources, all of which provide the necessary equipment, space, and personnel to complete the proposal.
