Protection of CCRF-CEM human lymphoid cells from antifolates by retroviral gene transfer of variants of murine dihydrofolate reductase.
Expression of certain variants of dihydrofolate reductase (DHFR) in mammalian cells protects them from methotrexate. Retroviral transfer of the gene for such a variant DHFR into hematopoietic cells might permit selection of modified cells in vivo by antifolate administration or alleviate antifolate-induced myelosuppression in patients receiving antifolate therapy. We examined protection of cells of the human lymphoblastoid line, CCRF-CEM, transduced with variants of mouse DHFR. In transduced cells selected with G418 but not with antifolate, the variant that had arginine substituted for leucine 22 did not protect against either methotrexate or trimetrexate; however, four other variants did offer protection, with the best having leucine 22 changed to tyrosine. Polyclonal cultures transduced with the different variants express DHFR at about the same level, but clones within each polyclonal population differ in DHFR expression levels and in resistance. These differences in expression were shown to reflect different integration sites for proviral DNA. Exposure to trimetrexate selects highly resistant clones, with high expression due to both high copy number and integration sites that are favorable for expression. Differences in the resistance of cultures expressing different variants at the same level are due to differences in the catalytic activity of the expressed DHFR, its affinity for antifolates, and its stability.
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
S M
SM
Mareya
B P
BP
Sorrentino
R L
RL
Blakley
D000818:Animals; D003001:Cloning, Molecular; D019008:Drug Resistance, Neoplasm; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D018628:Gene Dosage; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D016597:Trimetrexate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D016597:Trimetrexate; D008727:Methotrexate