Retroviral-mediated transfer and expression of the multidrug resistance protein 1 gene (MRP1) protect human hematopoietic cells from antineoplastic drugs

Multidrug resistance protein (MRP1) is a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily that confers multidrug resista nce. The transfer and expression of the MRP1 gene in human hematopoietic st em cells may be a useful alternative to multidrug resistance (MDR1) gene tr ansfer for protection from the myelosuppressive effects of chemotherapy in cancer patients. We constructed a gibbon ape leukemia virus packaging cell line (PG13) using the human MRP1 cDNA in a Moloney murine leukemia virus (M oMuLV) backbone containing a modified LTR. This PG13-based cell line, desig nated MRP1-PG13, produces retroviral vectors bearing the MRP1 gene at a tit er of 1.7 X 10(5) viral particles/ml. Transduction of the human leukemic ce ll line K562 showed that viral MRP1-PG13 supernatants routinely transfer th e MRP1 gene to similar to 35% of target K562 cells, of which at least one t hird are capable of prolife:rating in the presence of otherwise toxic conce ntrations of etoposide. Southern blot analyses indicated that most clones h ad only one proviral integration. Northern blot analysis of expanded K562 c lones showed the presence of a major full-length similar to 8-kb MRP1 trans cript as well as a minor similar to 6-kb transcript in all clones. Flow cyt ometric analysis of the producer cells and clones of transduced K562 cells demonstrated significantly increased MRP1 expression in these cells (simila r to 30-fold increase). Human bone marrow mononuclear cells and CD34(+) cel ls were also transduced with MRP1-PG13 supernatants on fibronectin-coated c ulture flasks in the presence of SCF, IL-3, and IL-6. PCR analysis of indiv idual hematopoietic colonies in methylcellulose cultures demonstrated provi ral DNA in similar to 10% of unselected human hematopoietic progenitor cell s cultured from nonsorted mononuclear cell samples and in up to similar to 75% of progenitors when CD34-enriched cell populations were targeted. To as sess functional MRP1 gene expression, normal human hematopoietic progenitor s and K562 cells were cultured in methylcellulose assays containing vincris tine or etoposide. All transduced samples gave rise to similar to 10% drug- resistant colonies, which were shown to be provirus-positive by PCR. Our st udies document the development of an amphotropic MRP1 rertroviral vector pr oducer cell line and pave the way for large animal and preclinical studies of chemoprotection by MRP1 gene transfer.