Coexpression of rat glutathione S-transferase A3 and human cytidine deaminase by a bicistronic retroviral vector confers in vitro resistance to nitrogen mustards and cytosine arabinoside in murine fibroblasts

Abstract

The transfer of drug resistance genes into hematopoietic cells is an experimental approach to protect patients from drug-induced myelosuppression. Because anti-cancer drugs are often administered in combination to increase their clinical efficacy, vectors that express two drug resistance genes are being developed to broaden the spectrum of chemoprotection. We have constructed a bicistronic vector, MFG/GST-IRES-CD (MFG/GIC) coexpressing rat glutathione S-transferase (GST) A3 isoform (rGST Yc 1 ) and human cytidine deaminase (CD). Murine NIH 3T3 fibroblast cells transduced with this vector were evaluated for their resistance to nitrogen mustards and cytosine nucleoside analogs. GIC-transduced polyclonal cell populations (GIC cells) demonstrated marked increases in selenium-independent glutathione peroxidase (peroxidase) and CD activities, as well as increased resistance to melphalan (2.3-fold), chlorambucil (3.4-fold), and cytosine arabinoside (Ara-C) (8.1-fold). After selection with Ara-C, the peroxidase and CD activities of GIC cells were augmented 2.6-and 2.9-fold, respectively, in comparison with unselected cells, and the resistance to melphalan, chlorambucil, and Ara-C was further increased to 3.7-, 5.9-, and 53-fold, respectively. Melphalan selection of GIC cells likewise augmented their peroxidase (2.3-fold) and CD (1.9-fold) activities. GIC cells proliferated in the simultaneous presence of melphalan and Ara-C at drug concentrations that completely inhibited the growth of untransduced cells. The growth rate of unselected GIC cells exposed to the drug combination averaged 18% that of drug-free cultures. The growth rate of GIC cells exposed to the drug combination increased to 30% of controls after Ara-C selection and to 50% after melphalan selection. Our results suggest that retroviral transfer of MFG/GIC may be useful for chemoprotection against the toxicities of nitrogen mustards and cytosine nucleoside analogs. Cancer Gene Therapy (2000) 7, 757-765

Gene therapy with drug resistance genes

“…In other studies, several bicistronic vectors were developed to coexpress GST transgene together with a second drug resistance marker with the aim to broaden the scope of chemoprotection. 83,84 Additional studies with GST are warranted to determine the usefulness of GST for in vivo selection and myeloprotection.…”

Increased resistance to nitrogen mustards and antifolates following in vitro selection of murine fibroblasts and primary hematopoietic cells transduced with a bicistronic retroviral vector expressing the rat glutathione S-transferase A3 and a mutant dihydrofolate reductase

“…The construction of pBluescript/3 0 MFG-GST, which contains a fragment of the MFG vector sequence, was previously described. 45 The 3 0 MFG-GSTA3-IRES-DHFR L22Y fragment was released from the pBluescript plasmid by digestion with AgeI and BamHI, and ligated between the same sites into the MFG vector (obtained from R Mulligan, Whitehead Institute, Cambridge, MA). The construction of MFG/GST and MFG/lac-Z was previously described.…”

“…45 A measure of 10 mg of genomic DNA from different cell lines was digested with NheI, run on a 1% agarose gel and finally transferred to a nylon membrane. Graded quantities of NheI-digested pMFG/ GST and pMFG/GID plasmids, supplemented with 10 mg of NIH 3T3 genomic DNA, were used to provide a copy number/cell standard.…”

“…The virus titer of virus-producing cells was determined in a clonogenic assay by selecting transduced clones with TMTX for the GP+E-86/GID cell line (titer ¼ 1.2 Â 10 5 CFU/ml), or by staining with Xgal for the GP+E-86/b-gal cell line (titer ¼ 8 Â 10 4 CFU/ ml). NIH/GID, NIH/GST, and NIH/DHFR were produced by exposing the NIH 3T3 cells to filtered medium from, respectively, GP+envAM12/GID, GP+envAM12/ GST, 45 and GP+envAM12/DHFR, daily for 3 consecutive days, in presence of 4 mg/ml polybrene.…”

“…45 Retrovirus-mediated transduction of hematopoietic cells and drug selection C3H/HeJ syngenic mice (Jackson Lab, Bar Harbour, ME) were housed in a conventional animal facility and were used between 10 and 18 weeks of age. BMCs were harvested from the hind leg femurs and tibias of C3H/HeJ donors treated intraperitoneally with 150 mg/kg 5-fluorouracil 48 hours prior to marrow collection.…”