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 ma mmalian 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 a lleviate anti folate-induced myelosuppression in patients receiving an tifolate therapy. We examined protection of cells of the human lymphob lastoid line, CCRF-CEM, transduced with variants of mouse DHFR. In tra nsduced cells selected with G418 but not with antifolate, the variant that had arginine substituted for leucine 22 did not protect against e ither methotrexate or trimetrexate; however, four other variants did o ffer protection, with the best having leucine 22 changed to tyrosine. Polyclonal cultures transduced with the different variants express DHF R at about the same level, but clones within each polyclonal populatio n differ in DHFR expression levels and in resistance. These difference s 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 si tes that are favorable for expression. Differences in the resistance o f cultures expressing different variants at the same level are due to differences in the catalytic activity of the expressed DHFR, its affin ity for antifolates, and its stability.