Retroviral-Mediated Transfer and Expression of the Multidrug Resistance Protein 1 Gene (MRP1) Protect Human Hematopoietic Cells from Antineoplastic Drugs

Abstract

Multidrug resistance protein (MRP1) is a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily that confers multidrug resistance. The transfer and expression of the MRP1 gene in human hematopoietic stem cells may be a useful alternative to multidrug resistance (MDR1) gene transfer 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 (MoMuLV) backbone containing a modified LTR. This PG13-based cell line, designated MRP1-PG13, produces retroviral vectors bearing the MRP1 gene at a titer of 1.7 X 105 viral particles/ml. Transduction of the human leukemic cell line K562 showed that viral MRP1-PG13 supernatants routinely transfer the MRP1 gene to ca.35% of target K562 cells, of which at least one third are capable of proliferating in the presence of otherwise toxic concentrations of etoposide. Southern blot analyses indicated that most clones had only one proviral integration. Northern blot analysis of expanded K562 clones showed the presence of a major full-length ca.8-kb MRP1 transcript as well as a minor ca.6-kb transcript in all clones. Flow cytometric analysis of the producer cells and clones of transduced K562 cells demonstrated significantly increased MRP1 expression in these cells (ca.30-fold increase). Human bone marrow mononuclear cells and CD34⁺ cells were also transduced with MRP1-PG13 supernatants on fibronectin-coated culture flasks in the presence of SCF, IL-3, and IL-6. PCR analysis of individual hematopoietic colonies in methylcellulose cultures demonstrated proviral DNA in ca.10% of unselected human hematopoietic progenitor cells cultured from nonsorted mononuclear cell samples and in up to ca.75% of progenitors when CD34-enriched cell populations were targeted. To assess functional MRP1 gene expression, normal human hematopoietic progenitors and K562 cells were cultured in methylcellulose assays containing vincristine or etoposide. All transduced samples gave rise to ca.10% drug-resistant colonies, which were shown to be provirus-positive by PCR. Our studies document the development of an amphotropic MRP1 retroviral vector producer cell line and pave the way for large animal and preclinical studies of chemoprotection by MRP1 gene transfer.