MECHANISMS OF NON-P-GLYCOPROTEIN MULTIDRUG RESISTANCE

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Resistance to chemotherapy continues to be a major impediment to the successful treatment of many types of cancer. Failure of chemotherapy may be in part due to the emergence of drug resistance. Attention has been directed towards the multiple drug resistance phenotype (MDR) in which cells selected for resistance to a given antineoplastic agent simultaneously develop resistance to other drugs which are structurally and functionally unrelated. This phenotype appears to be associated with the expression of a 170 kD membrane glycoprotein designated as the P-glycoprotein (PGP) which is the product of the mdr-1 gene. Recent studies suggest that tumor cells may also exhibit an MDR phenotype without expression of the mdr-1 gene. The focus of this proposal is to identify and characterize those mechanisms of multidrug resistance alternative to mdr-1. The principle model system to be studied is the human breast carcinoma cell line MCF-7 and a subline selected for resistance to mitoxantrone, MCF-7/MITOX. Selection for resistance to this agent in vitro results in cells which are cross resistant to anthracyclines, vinca alkaloids, and epipodophyllotoxins. There is no overexpression of PGP nor of the mdr-1 gene in these cells although there is a marked decrease in drug accumulation which is energy dependent. No evidence for an alteration in the intracellular target of this drug, DNA topoisomerase II, has been found to date. Considering the similar patterns of drug resistance and transport in non-PGP MDR as compared to PGP-mediated MDR, we postulate that there may be an alternative drug transport system operating in the non-PGP cell lines. To identify and characterize this system, we have constructed cDNA expression libraries from the drug-resistant MCF-7/MITOX and the drug-sensitive MCF-7/S cell lines and are screening them for differentially expressed genes through the use of subtractive hybridization techniques. In addition, we have produced monoclonal antibodies directed against proteins uniquely expressed in the drug-resistant cell line and will use them to screen the cDNA libraries as well. Differentially expressed cDNA clones identified by our screening procedures will be characterized by northern blot analysis and DNA sequencing. Their relationship to known genes and proteins will be ascertained through screening currently existing data bases such as GenBank and the National Biomedical Research Foundation protein data base and their relationship to previously cloned genes ascertained. To demonstrate a causative role in the drug resistant phenotype, full length cDNA clones will be isolated and transfected into drug-sensitive host cells. By studying this form of drug resistance means may be developed which will lead to increased diagnostic capabilities and a potential target for alternative therapy with chemosensitizers.