Different antifolate-resistant L1210 cell variants with either increased or decreased folylpolyglutamate synthetase gene expression at the level of mRNA transcription Journal Article
| Authors: | Roy, K.; Mitsugi, K.; Sirlin, S.; Shane, B.; Sirotnak, F. M. |
| Article Title: | Different antifolate-resistant L1210 cell variants with either increased or decreased folylpolyglutamate synthetase gene expression at the level of mRNA transcription |
| 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-folate H4 (natural diastereoisomer of 5- formyltetrahydrofolate), 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 V(max) with no change in K(m) 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 thai 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. |
| Keywords: | nonhuman; methotrexate; animal cell; mouse; animal; mice; gene amplification; gene expression; transcription, genetic; drug resistance; tumor cells, cultured; gene number; transcription regulation; amino acid sequence; molecular sequence data; rna, messenger; folinic acid; folic acid; folic acid antagonist; folic acid antagonists; rna transcription; northern blotting; southern blotting; michaelis constant; enzyme stability; variation (genetics); peptide synthases; leukemia l1210; human; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; pyrimethamine; leukemia l 1210; folylpolyglutamate synthase; metodiclorofen |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 270 |
| Issue: | 45 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 1995-11-10 |
| Start Page: | 26918 |
| End Page: | 26922 |
| Language: | English |
| DOI: | 10.1074/jbc.270.45.26918 |
| PUBMED: | 7592937 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 28 August 2018 -- Source: Scopus |
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