ISOLATION OF A SELECTED DRUG RESISTANT GENE

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The emergence of tumors refractory to further chemotherapy is a major obstacle for the successful treatment of cancer. Overexpression of MDR1 has been associated with clinical multidrug resistance. However, clinical trials have indicated that other as yet unidentified mechanisms occur in clinical multidrug resistance. This laboratory has selected mitoxantrone resistant human cell lines which is associated with an energy dependent drug efflux but is not related to the overexpression of known drug transporters, MDR1 and MRP. These cell lines provide a model of a novel multidrug resistant phenotype. We have recently determined by chromosome transfer that the resistance associated with mitoxantrone selection is due to a dominant genetic event. This provides the rationale for the cloning of the gene responsible for mitoxantrone selected resistance and is the goal of this proposal. A major undertaking of this proposal is the development of probes to screen a cDNA library. We have invoked strategies that utilize photoaffinity analogs of mitoxantrone as well as ATP photoaffinity analogs to identify putative proteins involved in resistance. Purification of proteins which are tagged in resistant cell lines by the photo affinity analogs will ultimately allow us to develop antibodies for immunoscreening a cDNA expression library. Purified proteins will also be sequenced which will allow us to design degenerate oligos for screening of a cDNA library. Finally, we propose that if we are unable to identify probes as outlined in this proposal we will proceed to physically map the gene. This will be accomplished by utilizing YAC libraries generated from chromosome transfer hybrids to clone the mitoxantrone selected resistant gene.