Different antifolate-resistant L1210 cell variants with either increased or decreased folylpolyglutamate synthetase gene expression at the level of mRNA transcription.
L1210 cell variants selected in the presence of the lipophilic dihydrofolate reductase inhibitor, metoprine, expressed increased levels of one-carbon, reduced folate transport inward (Sirotnak, F. M., Moccio, D. M., and Yang, C.-H. (1984) J. Biol. Chem. 259, 13139-13144). Growth of one of these variants (L1210/R69), with metoprine in the presence of decreasing concentrations of 1,L5-CHO-folateH4 (natural diastereoisomer of 5-formyl-tetrahydrofolate), resulted in the selection of other variants (L1210/R82, R83, and R84) with further reduction in one-carbon, reduced folate transport and in two cases (L1210/R83 and R84) with 3-8-fold increased folylpolyglutamate synthetase (FPGS) activity and folate compound polyglutamate formation in situ. Metoprine resistance was further increased, and the requirement for exogenous folate during growth was decreased as well in these variants. The increase in FPGS activity observed in L1210/R83 and R84 was characterized by 3- and 8-fold increases in value for Vmax with no change in Km and the same increase in a 60-61-kDa protein as shown by immunoblotting. Northern blotting revealed the same increases in these two variants in the level of a 2.3-kilobase FPGS mRNA when compared with control, while Southern blotting of genomic DNA did not reveal any increase in FPGS gene-copy number or restriction polymorphisms. Also, no difference in stability of FPGS mRNA was found between parental and variant cells. In contrast, nuclear run-on assays revealed differences among these cell types in the rate of FPGS mRNA transcription that correlated with increased FPGS activity, protein, and mRNA level in the variants. Similar studies with a transport-defective, methotrexate-resistant L1210 cell variant (L1210/R25) documented a 2-3-fold decrease in FPGS activity, protein, and mRNA levels that was accounted for by a decrease in FPGS mRNA transcription. These results provide the first examples of constitutively altered transcriptional regulation of FPGS activity associated with acquired resistance to antifolates.
Roy
K
K
Program in Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Mitsugi
K
K
Sirlin
S
S
Shane
B
B
Sirotnak
F M
FM
D000595:Amino Acid Sequence; D000818:Animals; D004351:Drug Resistance; D005493:Folic Acid Antagonists / Q000494:pharmacology; D005784:Gene Amplification; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D007939:Leukemia L1210 / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D011739:Pyrimethamine / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D005493:Folic Acid Antagonists; D012333:RNA, Messenger; C006308:metoprine; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D011739:Pyrimethamine