Abstract
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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U33557
Similar studies with a transport-defective, methotrexate-resistant L1210 cell variant (L1210/R25) documented a 2–3-fold decrease in folylpolyglutamate synthetase (FPGS) activity, protein, and mRNA levels that was accounted for by a decrease in FPGS mRNA transcription. These results provide the first examples of constituitively altered transcriptional regulation of FPGS activity associated with acquired resistance to antifolates
Their metabolism to polyglutamates and that of folate analogues are mediated [1,2,3,4,5] by the enzyme, folylpolyglutamate synthetase (FPGS),1 and metabolic turnover of these anabolites appears to be modulated by folylpolyglutamate hydrolase after their mediated entry into lysosomes
Summary
Cells and Culture Conditions—Methods for the isolation of variant L1210 cells with elevated one-carbon reduced folate transport and FPGS activity were similar to those reported earlier [25,26,27,28,29] from this laboratory. Derivation of a Murine FPGS cDNA Probe—A murine FPGS cDNA (ZAP-L1210/R83–1) was obtained by hybridization screening [32] of an L1210 cell cDNA library in gt (Stratagene, La Jolla, CA) using a human cDNA, pTZ 18U [24], as a probe. This gt cDNA construct incorporates a 2.296-kilobase insert ligated at the XhoI polylinker site including 3Ј- and 5Ј-untranslated region sequences of 461 and 74 base pairs, respectively, and an open reading frame of 1761 base pairs, which codes for a putative mitochondrial leader peptide as well as the enzyme protein. Folyl-polyglutamate hydrolase activity was measured as described previously [12]
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