Novel bicistronic retroviral vector expressing gamma-glutamylcysteine synthetase and the multidrug resistance protein 1 (MRP1) protects cells from MRP1-effluxed drugs and alkylating agents.
We have constructed two retroviral vectors, one expressing multidrug resistance protein 1 (MRP1) alone (SF91MRP) and the other expressing MRP1 and gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione biosynthesis (SF91GCS-MRP). We have utilized the hybrid FMEV (Friend mink cell focus-forming/murine embryonic stem cell virus) backbone, previously shown to be efficient in early hematopoietic cells, even when coexpressing two distinct genes. In SF91GCS-MRP, the cDNAs were combined via an internal ribosomal entry site (IRES) sequence from poliovirus, resulting in a bicistronic mRNA produced via the long terminal repeat (LTR). Producer Fly-eco clones were established by trans-infection with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped retroviral supernatants. Drug-resistant producer clones were subsequently selected with antimony potassium tartrate, a nonmutagenic MRP1 substrate. By RNA slot-blot and transduction of 3T3 fibroblasts, titers of both SF91MRP and SF91GCS-MRP were found to be greater than 10(6) viral particles/ml. The correct viral integration in the genome was established by Southern blotting. By flow cytometry, both MRP1 and bicistronic clones showed an increase in expression of the MRP1 protein. The bicistronic producer clones, as well as 3T3 cells transduced with SF91GCS-MRP, presented an increase in intracellular glutathione levels, compared with the parental counterparts. Producer cells, 3T3 fibroblasts transduced with either SF91MRP or SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were resistant to MRP1-effluxed drugs. However, only bicistronic producers, 3T3 fibroblasts transduced with SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were also resistant to alkylating agents. We conclude that the retrovirus SF91GCS-MRP has features that make it a suitable vector to induce bone marrow resistance to multiple classes of chemotherapeutic agents. The strategy of coexpressing gamma-GCS and MRP1 may help to design an effective in vivo selection for various clinical protocols of gene therapy.
Rappa
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G
Department of Tumor Biology, Norwegian Radium Hospital, Montebello, Oslo 0310, Norway. germanar@labmed.uio.no
Lorico
A
A
Hildinger
M
M
Fodstad
Ø
Ø
Baum
C
C
D016475:3T3 Cells; D000477:Alkylating Agents / Q000494:pharmacology*; D000818:Animals; D000965:Antimony / Q000494:pharmacology; D015139:Blotting, Southern; D002478:Cells, Cultured; D002699:Chlorambucil / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D005622:Friend murine leukemia virus / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral; D005721:Glutamate-Cysteine Ligase / Q000096:biosynthesis* / Q000235:genetics*; D005978:Glutathione / Q000096:biosynthesis / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D008297:Male; D008558:Melphalan / Q000494:pharmacology; D008562:Membrane Glycoproteins; D051379:Mice; D008807:Mice, Inbred BALB C; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis* / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D020079:Terminal Repeat Sequences; D014161:Transduction, Genetic; D014759:Viral Envelope Proteins / Q000378:metabolism
D000477:Alkylating Agents; D000965:Antimony; D002699:Chlorambucil; D002945:Cisplatin; D018076:DNA, Complementary; D004317:Doxorubicin; D005047:Etoposide; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D008558:Melphalan; D008562:Membrane Glycoproteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012313:RNA; D012333:RNA, Messenger; D014759:Viral Envelope Proteins; C028391:G protein, vesicular stomatitis virus; C421109:multidrug resistance-associated protein 1