Requirement of de novo protein synthesis for aminopterin-induced apoptosisin a mouse myeloma cell line

Cells synthesize nucleotides through de novo and salvage pathways that requ ire the activities of dihydrofolate reductase (DHFR) and hypoxanthine-guani ne phosphoribosyltransfease (HGPRT), respectively. Aminopterin, an inhibito r of dihydrofolate reductase, has been demonstrated to allow HGPRT(-) cells to be negatively selected. However, the pathway by which aminopterin leads to cell death remains to be clarified. In this study, we characterized fea tures of cellular responses induced by aminopterin treatment in P3-X63-Ag8. 653, a mouse HGPRT- myeloma cell line. Upon treatment with aminopterin, the cells readily underwent an apoptotic process, as assessed by DNA fragmenta tion assay and electron microscopic analysis. Aminopterin-induced apoptosis was drastically reduced by addition of actinomycin D and cycloheximide, in dicating that active RNA and protein synthesis is required for the apoptoti c effect of aminopterin. Interestingly; the induction of c-myc gene express ion preceded the activity of DNA fragmentation in aminopterin-treated cells . Taken together, these results suggest that cells deficient in the salvage pathway of purine biosynthesis are susceptible to aminopterin-induced apop tosis that requires de novo synthesis of proapoptotic factors, including My c oncoprotein. (C) 2001 Elsevier Science B.V. All rights reserved.