Comparison of the Protection of Cells from Antifolates by Transduced Human Dihydrofolate Reductase Mutants

Abstract

Retroviral transduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into cells can increase their resistance to the cytotoxic effects of these drugs. We evaluated the ability of wild-type hDHFR and 20 mutant enzymes (13 with single-amino acid substitutions, 7 with two substitutions) to prevent growth inhibition in antifolate-treated CCRF-CEM cells. The wild-type enzyme and all of the variants significantly protected transduced cells from trimetrexate (TMTX)-induced growth inhibition. However, only half of the variants conferred more protection than does the wild-type enzyme. For the variants tested, the observed protective effect was higher for TMTX than for methotrexate (< or =7.5-fold increased resistance), piritrexim (< or =16-fold), and edatrexate (negligible). Transduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (approximately 200-fold). Protection from loss of cell viability was similar to protection from growth inhibition. The protection associated with a particular mutant hDHFR did not result from the level of expression: Efficient protection resulted from low affinity of the variant for antifolates, reasonable catalytic activity, and good thermal stability. Clones isolated from a polyclonal population of transduced cells varied by as much as 30-fold in their resistance to TMTX, the resistance differences depending on hDHFR expression levels.

Multiple Conformers in Active Site of Human Dihydrofolate Reductase F31R/Q35E Double Mutant Suggest Structural Basis for Methotrexate Resistance

“…The high % error on K m DHF results from the low values of K m DHF and the correspondingly low spectrophotometric signal, which was enhanced by the use of 10-cm path length cuvettes. Kinetic data for the F31R variant had previously been reported (10), and compares well with our data, although we determined a K m DHF value that is 6-fold lower than previously reported. Because we expected a low value for K m DHF and a correspondingly low spectrophotometric signal, we used 10-cm cuvettes in K m DHF determination, rather than 1-cm cuvettes (10).…”

“…Kinetic data for the F31R variant had previously been reported (10), and compares well with our data, although we determined a K m DHF value that is 6-fold lower than previously reported. Because we expected a low value for K m DHF and a correspondingly low spectrophotometric signal, we used 10-cm cuvettes in K m DHF determination, rather than 1-cm cuvettes (10). This enabled a more precise measurement in the target range.…”

“…In addition, we have recently shown that this variant is an efficient selectable marker for various mammalian cell types, including murine hematopoietic stem cells (18). 7 Because mutations giving rise to MTX resistance are not observed in mammals, and because MTX is approved for human treatment, engineered resistant DHFRs offer great potential as human selective markers ex vivo or in vivo (10,27,28). To better understand the effect of either amino acid substitution on each ligand, a kinetic double mutant cycle was constructed with the simple variants F31R and Q35E.…”

Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors

“…The first monkey (RC701) received a transplant with G-CSF/SCFmobilized PB CD34 ϩ stem cells 29 transduced with an MSCV vector 30 containing a yellow fluorescent protein (YFP) marker in the upstream position and an IRES-driven DHFR resistance gene containing the L22Y and F31R substitutions 31 in the downstream position (Table 1). This vector was produced in stable packaging cells expressing the VSV-G envelope protein.…”

Gene therapy with drug resistance genes

“…Double mutations, L22Y and F31S or L22Y and F31R, of DHFR provide much higher resistance to MTX than each single mutant. 36,37 Expression of mutant DHFR can protect hematopoiesis of transplanted mice from the cytotoxic effects of methotrexate and trimetrexate. 20,21 The advantage of DHFR over other drug-reistance genes, such as MGMT (discussed below), is that the drugs used for in vivo selection of DHFR (MTX and TMTX) are not genotoxic whereas the drugs used for selection of MGMT, (BCNU and TMZ) are genotoxic.…”