The Changes in the Levels of Dihydrofolate Reductase mRNA and its Gene Dosage in 5-Fluorouracil-Resistant L1210 Cells

Yukari Konishi, Ryunosuke Kanamaru, Chikashi Ishioka, Akira Ishikawa, Hiroyuki Shibata, Akira Wakui, Toshio Dei

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

KONISHI, Y., KANAMARU, R., ISHIOKA, C, ISHIKAWA, A., SHIBATA, H., WAKUI, A. and DEI, T. The Changes in the Levels of Dihydrofolate Reductase mRNA and Its Gene Dosage in 5-Fluorouracil-Resistant L1210 Cells. Tohoku J. Exp. Med., 1990, 161 (1), 33-42 — 5-Fluorouracil (5-FU)-resistant L1210 cell line (L1210/5-FU-l) was established in this laboratory, and maintained by serial passage in the peritoneal cavities of BDF: mice. This and another 5-FU-resistant cell line (L1210/5-FU-2) showed approximately 50-fold increase in resistance to 5-FU, i.e., IC50 of 5-FU determined for wild type L1210 cells was 3 × 10-7 M, whereas those for 5-FU-resistant lines, L1210/5-FU-1 and L1210/5-FU-2 were 1.65×10-5 M and 1.35×l0-5 M, respectively. The incorporation of 3H-5-FU into L1210/5-FU-1 cells was about 57% of that observed in wild type L1210 cells. Northern blot analysis of DHFR mRNA obtained from 5-FU-sensitive and -resistant cell lines revealed four distinct bands of 1.6 kb, 1.2 kb, 1.0 kb and 0.75 kb in length. Although all these bands showed higher density in autoradiography in 5-FU-resistant lines than in wild type, no extra band was observed. Southern blot analysis of DHFR DNA, digested with the restriction enzymes, EcoRI, BamHI or HindIII, revealed no rearrangement. However, all the fragments were expanded, showing that DHFR gene increased in 5-FU-resistant cells. The karyotype analysis carried out for L1210/5-FU-1 showed abnormal banding region in a part of chromosome X, and this chromosomal aberration was considered to be the reflection of the amplification of DHFR gene. Many investigators have reported that thymidylate synthetase (TS), a target enzyme for 5-FU, increased in 5-FU-resistant cells and that the increase of TS was responsible for the drug resistance to 5-FU. The increase both in DHFR mRNA and DHFR DNA suggested the increase in DHFR and also in N5, N10 methylenetetrahydrofolate (methylene THF), a coenzyme of TS. The increase of methylene THF, together with the increase of TS, might result in the resistance of the cells to 5-FU. — drug resistance ; 5-fluorouracil; L1210 cell; dihydrofolate reductase.

Original languageEnglish
Pages (from-to)33-42
Number of pages10
JournalTohoku Journal of Experimental Medicine
Volume161
Issue number1
DOIs
Publication statusPublished - 1990

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Dive into the research topics of 'The Changes in the Levels of Dihydrofolate Reductase mRNA and its Gene Dosage in 5-Fluorouracil-Resistant L1210 Cells'. Together they form a unique fingerprint.

Cite this