Maintenance of dihydrofolate reductase enzyme after disappearance of DHFR mRNA during muscle cell differentiation.

Abstract

Terminally differentiating mouse muscle cells were used to examine the relationship between myogenic withdrawal from the cell cycle and the levels of dihydrofolate reductase (DHFR) mRNA and DHFR activity. Differentiation was induced by removal of fibroblast growth factor activity from the medium. DHFR mRNA was measured by a RNase protection assay. DHFR activity was measured by a spectrophotometric assay and by a [3H]methotrexate binding assay. Proliferative myoblasts contained four DHFR mRNA molecules and 1.8 X 10(5) DHFR enzyme molecules. By 12.5 h after induction, when [3H]thymidine labeling indices showed all cells had withdrawn from the cell cycle, DHFR mRNA levels had declined to 0.7 copies per cell. In contrast, myogenic withdrawal did not result in reduced DHFR activity. Qualitatively similar results, i.e. down-regulation of mRNA and constitutive expression of activity, were observed in a methotrexate-selected muscle cell line with greater than 50-fold amplification of the DHFR gene. Enzyme synthesis rate and stability measurements indicated that persistence of DHFR activity in postreplicative cells was due to a long enzyme lifetime rather than to continued synthesis from residual normal DHFR mRNA or an alternative mRNA species not detected by the RNase protection assay. Unlike DHFR, thymidine kinase (TK) activity disappeared rapidly as muscle cells differentiated. Both DHFR mRNA and TK mRNA are expressed in a replication-dependent manner; however, the enzymes encoded by these messages are subject to different fates in postreplicative cells.