Maintenance of Dihydrofolate Reductase Enzyme After Disappearance of DHFR MRNA During Muscle Cell Differentiation

Overview

Journal In Vitro Cell Dev Biol

Specialties Anatomy & Histology
Cell Biology

Date 1989 Aug 1

PMID 2768131

Citations 2

Authors

E E Schmidt

G F Merrill

Affiliations

Soon will be listed here.

Abstract

Terminally differentiating mouse muscle cells were used to examine the relationship between myogenic withdrawal from the cell cycle and the levels of dihydrofolate reductase (DHFR) mRNA and DHFR activity. Differentiation was induced by removal of fibroblast growth factor activity from the medium. DHFR mRNA was measured by a RNase protection assay. DHFR activity was measured by a spectrophotometric assay and by a [3H]methotrexate binding assay. Proliferative myoblasts contained four DHFR mRNA molecules and 1.8 X 10(5) DHFR enzyme molecules. By 12.5 h after induction, when [3H]thymidine labeling indices showed all cells had withdrawn from the cell cycle, DHFR mRNA levels had declined to 0.7 copies per cell. In contrast, myogenic withdrawal did not result in reduced DHFR activity. Qualitatively similar results, i.e. down-regulation of mRNA and constitutive expression of activity, were observed in a methotrexate-selected muscle cell line with greater than 50-fold amplification of the DHFR gene. Enzyme synthesis rate and stability measurements indicated that persistence of DHFR activity in postreplicative cells was due to a long enzyme lifetime rather than to continued synthesis from residual normal DHFR mRNA or an alternative mRNA species not detected by the RNase protection assay. Unlike DHFR, thymidine kinase (TK) activity disappeared rapidly as muscle cells differentiated. Both DHFR mRNA and TK mRNA are expressed in a replication-dependent manner; however, the enzymes encoded by these messages are subject to different fates in postreplicative cells.

Citing Articles

BAC TG-EMBED: one-step method for high-level, copy-number-dependent, position-independent transgene expression.

Bian Q, Belmont A Nucleic Acids Res. 2010; 38(11):e127.

PMID: 20385594 PMC: 2887973. DOI: 10.1093/nar/gkq178.

Changes in dihydrofolate reductase (DHFR) mRNA levels can account fully for changes in DHFR synthesis rates during terminal differentiation in a highly amplified myogenic cell line.

Schmidt E, Merrill G Mol Cell Biol. 1991; 11(7):3726-34.

PMID: 2046674 PMC: 361140. DOI: 10.1128/mcb.11.7.3726-3734.1991.

References

Slater C . Control of myogenesis in vitro by chick embryo extract. Dev Biol. 1976; 50(2):264-84. DOI: 10.1016/0012-1606(76)90151-2. View

Goldman I, LICHTENSTEIN N, OLIVERIO V . Carrier-mediated transport of the folic acid analogue, methotrexate, in the L1210 leukemia cell. J Biol Chem. 1968; 243(19):5007-17. View

Borun T, Scharff M, Robbins E . Rapidly labeled, polyribosome-associated RNA having the properties of histone messenger. Proc Natl Acad Sci U S A. 1967; 58(5):1977-83. PMC: 223893. DOI: 10.1073/pnas.58.5.1977. View

Linkhart T, Clegg C, Hauschika S . Myogenic differentiation in permanent clonal mouse myoblast cell lines: regulation by macromolecular growth factors in the culture medium. Dev Biol. 1981; 86(1):19-30. DOI: 10.1016/0012-1606(81)90311-0. View

Baram J, Chabner B, Drake J, Fitzhugh A, Sholar P, Allegra C . Identification and biochemical properties of 10-formyldihydrofolate, a novel folate found in methotrexate-treated cells. J Biol Chem. 1988; 263(15):7105-11. View

Leys E, Crouse G, Kellems R . Dihydrofolate reductase gene expression in cultured mouse cells is regulated by transcript stabilization in the nucleus. J Cell Biol. 1984; 99(1 Pt 1):180-7. PMC: 2275613. DOI: 10.1083/jcb.99.1.180. View

Thompson C, Challoner P, Neiman P, Groudine M . Levels of c-myc oncogene mRNA are invariant throughout the cell cycle. Nature. 1985; 314(6009):363-6. DOI: 10.1038/314363a0. View

Leys E, Kellems R . Control of dihydrofolate reductase messenger ribonucleic acid production. Mol Cell Biol. 1981; 1(11):961-71. PMC: 369718. DOI: 10.1128/mcb.1.11.961-971.1981. View

Hofbauer R, Mullner E, Seiser C, Wintersberger E . Cell cycle regulated synthesis of stable mouse thymidine kinase mRNA is mediated by a sequence within the cDNA. Nucleic Acids Res. 1987; 15(2):741-52. PMC: 340464. DOI: 10.1093/nar/15.2.741. View

10.

Jaynes J, Chamberlain J, Buskin J, Johnson J, Hauschka S . Transcriptional regulation of the muscle creatine kinase gene and regulated expression in transfected mouse myoblasts. Mol Cell Biol. 1986; 6(8):2855-64. PMC: 367853. DOI: 10.1128/mcb.6.8.2855-2864.1986. View

11.

KONIGSBERG I . Diffusion-mediated control of myoblast fusion. Dev Biol. 1971; 26(1):133-52. DOI: 10.1016/0012-1606(71)90113-8. View

12.

Osborn M, Huennekens F . Enzymatic reduction of dihydrofolic acid. J Biol Chem. 1958; 233(4):969-74. View

13.

Nadal-Ginard B . Commitment, fusion and biochemical differentiation of a myogenic cell line in the absence of DNA synthesis. Cell. 1978; 15(3):855-64. DOI: 10.1016/0092-8674(78)90270-2. View

14.

Stokstad E, Koch J . Folic acid metabolism. Physiol Rev. 1967; 47(1):83-116. DOI: 10.1152/physrev.1967.47.1.83. View

15.

Farnham P, Schimke R . Transcriptional regulation of mouse dihydrofolate reductase in the cell cycle. J Biol Chem. 1985; 260(12):7675-80. View

16.

Mariani B, Slate D, Schimke R . S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1981; 78(8):4985-9. PMC: 320316. DOI: 10.1073/pnas.78.8.4985. View

17.

Liu H, Gibson C, Hirschhorn R, Rittling S, Baserga R, Mercer W . Expression of thymidine kinase and dihydrofolate reductase genes in mammalian ts mutants of the cell cycle. J Biol Chem. 1985; 260(6):3269-74. View

18.

Kaufman R, Sharp P . Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes. Mol Cell Biol. 1983; 3(9):1598-608. PMC: 370013. DOI: 10.1128/mcb.3.9.1598-1608.1983. View

19.

Esch F, Baird A, Ling N, Ueno N, Hill F, Denoroy L . Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci U S A. 1985; 82(19):6507-11. PMC: 390746. DOI: 10.1073/pnas.82.19.6507. View

20.

Doering J, Fischman D . The in vitro cell fusion of embryonic chick muscle without DNA synthesis. Dev Biol. 1974; 36(2):225-35. DOI: 10.1016/0012-1606(74)90046-3. View