Posttranscriptionally mediated decreases in folylpolyglutamate synthetase gene expression in some folate analogue-resistant variants of the L1210 cell. Evidence for an altered cognate mRNA in the variants affecting the rate of de novo synthesis of the enzyme.
L1210 cell variants resistant to edatrexate (EDX) were isolated by selection in vivo during therapy with this folate analogue. Among the variants selected, seven (L1210/EDX-4 to -7 and L1210/EDX-12 to -14) were found to exhibit 2-23-fold lower levels of folylpolyglutamate synthetase (FPGS) activity compared with parental L1210 cells. Lower levels of FPGS activity in cell-free extract from these variants using EDX as substrate were characterized by the same relative decrease in value for Vmax with no change in apparent Km. The results of an analysis of FPGS activity in mixtures of variant and parental cell extract suggested that no endogenous inhibitors in the variant cells or stimulatory factors in parental cells accounted for the differences observed. Also, FPGS from variant and parental cells showed no difference in thermostability. Decreases in a 60-61-kDa protein as shown by immunoblotting with anti-FPGS peptide antibody were found to occur commensurately with the decrease in FPGS activity in cell extract from the variants compared with parental cells. However, no evidence was obtained for a difference in turnover of FPGS protein during measurement of the decay of FPGS activity in cycloheximide-treated variant and parental cells. In addition, Northern blotting of poly(A)+ RNA did not reveal any difference in the size or level of FPGS mRNA among these various cell types. Studies of in vitro translation of hybridization-selected FPGS mRNA from L1210 cells showed that both mitochondrial and cytosolic forms of FPGS were generated during the reaction. Moreover, FPGS mRNA from the variant cells was significantly less effective in mediating formation of the FPGS peptide product in a manner correlating with FPGS activity and protein found in the cytosol of the various cell types. These results suggest that FPGS gene expression in these variants is posttranscriptionally altered at the level of the cognate mRNA itself and that this alteration constitutively down-regulates the steady-state level of FPGS in these variants.
Roy
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Program in Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Egan
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Sirlin
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Sirotnak
F M
FM
D000630:Aminopterin / Q000008:administration & dosage / Q000031:analogs & derivatives*; D000818:Animals; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics*; D005493:Folic Acid Antagonists / Q000008:administration & dosage*; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000235:genetics*; D014158:Transcription, Genetic
D005493:Folic Acid Antagonists; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; C040210:edatrexate; D000630:Aminopterin