MULTIDRUG RESISTANCE IN MAMMALIAN CELLS

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The development of resistance to chemotherapeutic agents is observed in the clinic, and has been studied extensively in tissue culture cells. One unusual phenotype is that of multidrug resistance, in which cells that are challenged with any one of a variety of cytotoxic drugs develop resistance not only to the selective agent but also cross-resistance to other, seemingly, unrelated, compounds. This multidrug-resistant (mdr) phenotype is associated in many cases with the overproduction of a small family of membrane glycoproteins called p-glycoproteins (pgp's), which are thought to act as ATP-activated efflux pumps. Of the three family members in hamster, it is the homolog of the human mdr1 protein (known to be overexpressed in human tumors), pgp1, that is most frequently involved with the establishment and maintenance of multidrug-resistance in tissue culture cell lines, and is the subject of this application. Little is known concerning the efflux mechanism attributed to pgp1 or the manner in which cross-resistance patterns are established. Moreover it remains unclear whether pgp1 functions alone or in combination with other factors to sustain efflux. In addition to these questions about function, regulation of pgp1 gene expression appears to be complex and to occur at both the transcriptional and translational levels. Using a combination of recombinant DNA technology, gene transfection assays, nuclear run-on experiments and in situ mutagenesis techniques. We propose to 1) carry out a molecular genetic study of transmembrane domain 6 of the pgp1 transporter, a region of the protein known to be involved with mediation of cross-resistance; 2) establish the molecular basis and functional significance of alternate splicing forms of pgp1 mRNAs that have been found to exist in mdr cells and to potentially encode novel pgp1's; 3) clarify whether additional factors are required to sustain pgp1 mediated efflux and address the question of whether pgp1 overexpression in hamster fibroblasts decreases the oncogenic potential of these cells, and 4) demonstrate that amplified pgp1 genes may be regulated at the level of transcription, independent of gene copy number.