title: Retrovirally mediated gene transfer of Arg22 and Tyr22 forms of dihydrofolate reductase into the hematopoietic cell line K562: a comparison of methotrexate resistance.
abstract: Mutations in the enzyme dihydrofolate reductase (DHFR) can confer resistance to the inhibitory effects of folate analogs such as methotrexate (Mtx) and trimetrexate (Ttx). Retroviral vectors expressing the DHFR-Arg22 mutants and the newly described DHFR-Tyr22 mutant were used to transduce the hematopoietic cell line K562. In vitro selection of vector-containing cells was documented via polymerase chain reaction and Southern analysis. When proliferation of selected vector-containing cells was evaluated over a range of Mtx concentrations (0.01 to 10 micromol/L), both Arg22 and Tyr22 provided protection from Mtx, but Tyr22 proved superior to Arg22 in conferring Mtx resistance at low concentrations. Ttx proved to be 10- to 100-fold more potent than Mtx in inhibiting proliferation of nontransduced K562, but the relative effectiveness of individual mutants in conferring drug resistance was similar to that of Mtx. Decreasing the amount of folate in the culture medium to more physiological concentrations increased the potency of administered Mtx and Ttx. Drug resistance in retrovirally transduced K562 cells is consistent with the enzymatic characteristics of the individual mutants. Our findings suggest that the new Tyr22 form of DHFR may prove better in conferring drug resistance than the previously reported Arg22 mutant.
authors.affiliation: Section of Hematology, Indiana University School of Medicine, Indianapolis, USA.
authors.forename: S E
authors.initials: SE
authors.lastname: Braun
authors.forename: R S
authors.initials: RS
authors.lastname: McIvor
authors.forename: A S
authors.initials: AS
authors.lastname: Davidson
authors.forename: M
authors.initials: M
authors.lastname: Hanna
authors.forename: C M
authors.initials: CM
authors.lastname: Traycoff
authors.forename: D A
authors.initials: DA
authors.lastname: Berebetsky
authors.forename: R
authors.initials: R
authors.lastname: Gonin
authors.forename: H E
authors.initials: HE
authors.lastname: Broxmeyer
authors.forename: K
authors.initials: K
authors.lastname: Cornetta
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001120:Arginine / Q000737:chemistry; D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D002455:Cell Division; D002460:Cell Line; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D018014:Gene Transfer Techniques; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D012190:Retroviridae / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000964:Antimetabolites, Antineoplastic; D014443:Tyrosine; D001120:Arginine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate