Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors

Abstract

We previously reported the protection of hematopoietic cells from methotrexate (MTX) toxicity using an N2-based double copy vector containing serine 31 (S31)-mutated dihydrofolate reductase (DHFR) (DC/SV6S31). To examine whether the use of SFG-based dicistronic vectors will lead to improvement in gene transfer over the DC/SV6 vector, we compared the protection provided by MTX to NIH3T3 cells and hematopoietic progenitor cells infected with these retroviral constructs containing the S31 variant DHFR cDNA. In NIH3T3 cells, the 50% effective dose values of MTX conferred by the SFG vector were 8-fold higher than those obtained with the DC/SV6 vector. DHFR mRNA levels were 22-fold and 38-fold higher than that seen for the DC/SV6 vector according to Northern blot and real-time polymerase chain reaction analysis, respectively. However, DHFR protein expression and DHFR enzyme activity were only 1.5-fold and 2-fold higher in the SFG vector, respectively, indicating that the mRNA from the SFG vector is translated less efficiently than the mRNA generated from the DC/SV6 vector. Furthermore, the degree of MTX protection conferred by each vector in both mouse and human hematopoietic cells was the same. These results indicate that the in vitro transduction efficiency and transgene expression of human DHFR in hematopoietic progenitor cells is equally conferred by both vectors. Cancer Gene Therapy (2000) 7, 910 -919

Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells

“…Here, increases in the order of 1.3-6-fold for MGMT, 29 but also up to 40-fold for mutant DHFR 23,30 have been described. One obvious way to increase resistance levels would be better transgene expression.…”

Resistance to antifolates

“…This approach facilitates enrichment of populations of progenitor cells transfected with mutant enzyme on exposure to antifolate in vitro. The strategy has been utilized to achieve resistance to MTX with mutated DHFR in murine progenitor cells (Banerjee et al, 1994;Flasshove et al, 1995a;Takebe et al, 2000) and human CD34 þ peripheral human cells in vitro and in vivo (Flasshove et al, 1995b;Sorrentino et al, 1999). Bicistronic retroviral vectors have been used to expand the spectrum of resistance in progenitors, as with the transduction of both mutated DHFR and Pgp into murine bone marrow progenitors (Galipeau et al, 1997), or with DHFR and cytidine deaminase conferring resistance to both MTX and cytosine arabinoside (Sauerbrey et al, 1999), or with cotransfection of mutated DHFR and wild-type aldehyde dehydrogenase to achieve resistance to both MTX and cyclophosphamide (Takebe et al, 2001).…”

p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

“…Cells were grown for various times in the presence or absence of 1 g/ml doxycycline, collected, and lysed; mRNA was isolated using the Invitrogen mRNA isolation kit, and reverse transcriptase reaction performed using the Invitrogen cDNA cycle kit. The relative message quantity was analyzed using probe and primers specific for DHFR cDNA as described previously (21). In brief, PCR was performed by using a TaqMan 1,000 RXN Gold with buffer A kit and the ABIPrism 7700 thermocycler (Applied Biosystems).…”