Development of drug resistance is a major problem in the management of human plasma cell neoplasms. The mechanisms underlying resistance are still undefined, a serious obstacle in planning strategies for chemotherapy. For a better understanding of drug resistance, we have developed mouse and human plasma cell tumor model that are resistant to cyclophosphamide (CY) and closely related structural analogs such as 4- hydroperoxycyclophosphamide (4HC). CY is frequently used for treating a wide variety of tumors. This drug is inactive in the native form and is metabolized by live enzymes in to cytotoxic alkylating molecules. Using 4HC, an active analog of CY, we observed that drug transport is identical in CY resistant and sensitive mouse LPC-1 plasmacytoma but drug metabolism is different in these two tumor lines. A search for biochemical differences revealed increased glutathione S-transferase activity (GST) in CY resistant mouse and human tumor cells. GST are key enzymes in the detoxification of alkylating agents and other toxic chemicals. The overall objective of this proposal is to determine whether CY resistance is due to increased activity of GST enzyme and hence altered drug metabolism in CY resistant tumor cells (CY-R). To achieve this objective, we will pursue the following specific aims. 1. To characterize the relationship between GST and CY resistance, we will measure and correlate GST activity with development of resistance. 2. To determine whether GST in CY-R tumor cells inactivate toxic metabolites by conjugation with glutathione, we will analyze metabolic products in tumor cell homogenates after incubating with radiolabeled 4HC and glutathione by high pressure liquid chromatography and mass spectrometry. 3. To determine the molecular basis of CY resistance we will characterize the biochemical characteristics of tumor GST and determine whither increased GST activity in CY-R Tumor cells is due to gene mutation, gene amplification, increased transcription, translation or altered turnover using antibodies and available cDNA probes for GST. 4. To rule out the possibility that reduced levels of DNA crosslinking in CY resistant cells is due to increased repair rather than decreased availability of drug to produce DNA lesions, we will examine the extent of DNA crosslinking at multiple time points following drug treatment. These studies may pave the way for a more rational method of drug selection and novel approaches to modify tumor cell responsiveness to alkylating agents.
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