Chemotherapy is successful in alleviating and, in some cases, curing some forms of human cancer. The major reason for treatment failure is the overgrowth of a subpopulation of cancer cells resistant to these antineoplastic agents. In order to develop more effective methods for treating human cancers, the molecular mechanisms responsible for this drug resistance must be delineated. The alkylating agents, such as cyclophosphamide and the nitrogen mustards are some of the most commonly used antineoplastic agents. Cancer cells which are resistant to treatment with these agents often show a decreased level of drug uptake, increased levels of glutathione (GSH) adn glutathione-S-transferase (GST) activity, and an increase in DNA repair activity. Glutathione is thought to play a role in the intracellular inactivation of the drugs and also has been implicated in cancer cell resistance to radiotherapy and hyperthermic therapy. This proposal outlines a series of studies designed to directly assess the contribution of GSH and GSH-mediated processes to the overall drug resistance mechanism. A combination of nuclear magnetic resonance spectroscopy, radiotracer, HPLC, and mass spectrometric techniques will be used to probe the mechanisms involved in the inactivation of alkylating agents in two lines of MCF-7 human mammary adenocarcinoma cells. One line is sensitive to treatment with cyclophosphamide and phosphoramide mustard, and one line displays a five-fold resistance to these agents. The steady-stater levels of GSH and the rate of GSH turnover will be measured in these cell lines. The structure and the rate of formation of the inactive metabolites of these alkylating agents will be determined in cell lysates and whole cells.. Initially, studies will be performed in buffer solution. This will be followed by studies in cell lysates, whole cells in monolayer, and finally, intact perfused cells. This unified approach, progressing from buffer solution through cell lysates and finally to whole cells, will provide a better understanding of the drug resistance mechanism. These experiments will provide direct evidence for the mechanisms primarily responsible for drug resistance in cultured human cancer cells. With this knowledge, new treatment protocols may be designed which will increase the effectiveness of chemotherapy.
| Gamcsik, M P; Ludeman, S M; Shulman-Roskes, E M et al. (1993) Protonation of phosphoramide mustard and other phosphoramides. J Med Chem 36:3636-45 |
