title: Retroviral transfer of human MDR1 gene to hematopoietic cells: effects of drug selection and of transcript splicing on expression of encoded P-glycoprotein.
abstract: Protection of hematopoietic cells of patients undergoing anticancer chemotherapy by MDR1 gene transfer is currently being studied in clinical trials. From animal studies, it has been suggested that aberrant splicing due to cryptic donor and acceptor sites in the MDR1 cDNA could be a major reason for failure to obtain high-level expression of P-glycoprotein in bone marrow. We investigated effects of drug selection on protein expression levels and on splicing of MDR1 transcripts in murine bone marrow cells (BMCs) in vitro. To this end, retroviruses were generated through an identical plasmid, pHaMDR1/A, introduced into different packaging cells. GP + E86- but not PA317-derived producer cells were found to express truncated in addition to full-length message. In BMCs transduced with GP + E86-derived viruses, both messages were increased after treatment with colchicine or daunomycin. Similar results were obtained with NIH 3T3 fibroblasts. However, transduced and drug-selected BMCs displayed the spliced transcript even if the respective PA317-derived producer cells contained no truncated RNA as detected in transduced NIH 3T3 fibroblasts. Short-term drug selection in BMCs transduced with either ecotropic or amphotropic retroviruses resulted in a striking increase in P-glycoprotein expression. Thus, aberrant splicing failed to abrogate P-glycoprotein expression in BMCs. We also studied a vector in which MDR1 was coexpressed with glucocerebrosidase, using an internal ribosomal entry site. Although chemoprotection was less efficient than with pHaMDR1/A, augmentation of protein expression was observed at low selecting drug concentrations. Our study shows that drug selection can partially compensate for inefficient transduction of hematopoietic cells, and may help to develop strategies by which unstable expression of transduced genes can be overcome.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
authors.forename: T
authors.initials: T
authors.lastname: Licht
authors.forename: J M
authors.initials: JM
authors.lastname: Aran
authors.forename: S K
authors.initials: SK
authors.lastname: Goldenberg
authors.forename: W D
authors.initials: WD
authors.lastname: Vieira
authors.forename: M M
authors.initials: MM
authors.lastname: Gottesman
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D017398:Alternative Splicing; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism*; D003078:Colchicine / Q000494:pharmacology; D003630:Daunorubicin / Q000494:pharmacology; D018014:Gene Transfer Techniques; D019450:Genes, MDR; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003078:Colchicine; D003630:Daunorubicin