Abstract
It is widely accepted that the cellular oncogene c-myc plays an important role in the control of cell proliferation and that its expression diminishes in differentiated cells. We examined whether there is a correlation between c-myc expression and cell proliferation or differentiation by using a subclone of a rat skeletal muscle cell line L6E9. Myoblasts irreversibly withdraw from the cell cycle, fuse to form multinucleated myotubes, and express muscle-specific genes (terminal differentiation). Muscle-specific genes can also be expressed in the absence of fusion (biochemical differentiation). Such mononucleated but biochemically differentiated cells can be stimulated to reenter the cell cycle. c-myc was induced by insulin, insulin-like growth factor, or serum factors in G0-arrested cells, whereas induction by protein synthesis inhibitors or superinduction by protein synthesis inhibitors in combination with serum factors occurred in all physiological states tested. We found that c-myc expression was reduced in biochemically and terminally differentiated cells as well as in quiescent undifferentiated cells but that it remained inducible by growth factors in all three physiological states. Results of nuclear runoff transcription assays suggested that the induction of c-myc mRNA by growth factors and its deinduction in these physiological states were regulated mainly at the transcriptional level. In contrast, induction and superinduction of c-myc mRNA by protein synthesis inhibitors alone and in combination with growth factors, respectively, were regulated posttranscriptionally mainly by stabilization of c-myc mRNA. Moreover, c-myc and muscle-specific genes could be simultaneously transcribed in both biochemically and terminally differentiated cells. These results indicate that irreversible repression of c-myc is not required for terminal myogenic differentiation and that its expression is insufficient by itself to suppress the differentiated phenotype.
Full text
PDF![1412](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/df40/367665/0c7e46323d51/molcellb00089-0060.png)
![1413](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/df40/367665/9d05e8f3645c/molcellb00089-0061.png)
![1414](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/df40/367665/57ceeffd1bb7/molcellb00089-0062.png)
![1415](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/df40/367665/968354c8410c/molcellb00089-0063.png)
![1416](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/df40/367665/d46df12e19e1/molcellb00089-0064.png)
![1417](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/df40/367665/fb774a95e593/molcellb00089-0065.png)
![1418](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/df40/367665/33a1294d3830/molcellb00089-0066.png)
![1419](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/df40/367665/b1f93506d493/molcellb00089-0067.png)
![1420](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/df40/367665/55213b228938/molcellb00089-0068.png)
![1421](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/df40/367665/ce77d2086ed5/molcellb00089-0069.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Armelin H. A., Armelin M. C., Kelly K., Stewart T., Leder P., Cochran B. H., Stiles C. D. Functional role for c-myc in mitogenic response to platelet-derived growth factor. Nature. 1984 Aug 23;310(5979):655–660. doi: 10.1038/310655a0. [DOI] [PubMed] [Google Scholar]
- Bader D., Masaki T., Fischman D. A. Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J Cell Biol. 1982 Dec;95(3):763–770. doi: 10.1083/jcb.95.3.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Benoff S., Nadal-Ginard B. Transient induction of poly(A)-short myosin heavy chain messenger RNA during terminal differentiation of L6E9 myoblasts. J Mol Biol. 1980 Jun 25;140(2):283–298. doi: 10.1016/0022-2836(80)90106-0. [DOI] [PubMed] [Google Scholar]
- Blanchard J. M., Piechaczyk M., Dani C., Chambard J. C., Franchi A., Pouyssegur J., Jeanteur P. c-myc gene is transcribed at high rate in G0-arrested fibroblasts and is post-transcriptionally regulated in response to growth factors. Nature. 1985 Oct 3;317(6036):443–445. doi: 10.1038/317443a0. [DOI] [PubMed] [Google Scholar]
- Bravo R., Burckhardt J., Curran T., Müller R. Stimulation and inhibition of growth by EGF in different A431 cell clones is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. EMBO J. 1985 May;4(5):1193–1197. doi: 10.1002/j.1460-2075.1985.tb03759.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Campisi J., Gray H. E., Pardee A. B., Dean M., Sonenshein G. E. Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation. Cell. 1984 Feb;36(2):241–247. doi: 10.1016/0092-8674(84)90217-4. [DOI] [PubMed] [Google Scholar]
- Caravatti M., Minty A., Robert B., Montarras D., Weydert A., Cohen A., Daubas P., Buckingham M. Regulation of muscle gene expression. The accumulation of messenger RNAs coding for muscle-specific proteins during myogenesis in a mouse cell line. J Mol Biol. 1982 Sep;160(1):59–76. doi: 10.1016/0022-2836(82)90131-0. [DOI] [PubMed] [Google Scholar]
- Coughlin S. R., Lee W. M., Williams P. W., Giels G. M., Williams L. T. c-myc gene expression is stimulated by agents that activate protein kinase C and does not account for the mitogenic effect of PDGF. Cell. 1985 Nov;43(1):243–251. doi: 10.1016/0092-8674(85)90029-7. [DOI] [PubMed] [Google Scholar]
- Dalla-Favera R., Gelmann E. P., Martinotti S., Franchini G., Papas T. S., Gallo R. C., Wong-Staal F. Cloning and characterization of different human sequences related to the onc gene (v-myc) of avian myelocytomatosis virus (MC29). Proc Natl Acad Sci U S A. 1982 Nov;79(21):6497–6501. doi: 10.1073/pnas.79.21.6497. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dani C., Blanchard J. M., Piechaczyk M., El Sabouty S., Marty L., Jeanteur P. Extreme instability of myc mRNA in normal and transformed human cells. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7046–7050. doi: 10.1073/pnas.81.22.7046. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Devlin B. H., Konigsberg I. R. Reentry into the cell cycle of differentiated skeletal myocytes. Dev Biol. 1983 Jan;95(1):175–192. doi: 10.1016/0012-1606(83)90016-7. [DOI] [PubMed] [Google Scholar]
- Dony C., Kessel M., Gruss P. Post-transcriptional control of myc and p53 expression during differentiation of the embryonal carcinoma cell line F9. Nature. 1985 Oct 17;317(6038):636–639. doi: 10.1038/317636a0. [DOI] [PubMed] [Google Scholar]
- Emerson C. P., Jr, Beckner S. K. Activation of myosin synthesis in fusing and mononucleated myoblasts. J Mol Biol. 1975 Apr 25;93(4):431–447. doi: 10.1016/0022-2836(75)90238-7. [DOI] [PubMed] [Google Scholar]
- Ewton D. Z., Florini J. R. Effects of the somatomedins and insulin on myoblast differentiation in vitro. Dev Biol. 1981 Aug;86(1):31–39. doi: 10.1016/0012-1606(81)90312-2. [DOI] [PubMed] [Google Scholar]
- Falcone G., Tatò F., Alemà S. Distinctive effects of the viral oncogenes myc, erb, fps, and src on the differentiation program of quail myogenic cells. Proc Natl Acad Sci U S A. 1985 Jan;82(2):426–430. doi: 10.1073/pnas.82.2.426. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
- Fischbach G. D. Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Dev Biol. 1972 Jun;28(2):407–429. doi: 10.1016/0012-1606(72)90023-1. [DOI] [PubMed] [Google Scholar]
- Florini J. R., Nicholson M. L., Dulak N. C. Effects of peptide anabolic hormones on growth of myoblasts in culture. Endocrinology. 1977 Jul;101(1):32–41. doi: 10.1210/endo-101-1-32. [DOI] [PubMed] [Google Scholar]
- Garfinkel L. I., Periasamy M., Nadal-Ginard B. Cloning and characterization of cDNA sequences corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin. J Biol Chem. 1982 Sep 25;257(18):11078–11086. [PubMed] [Google Scholar]
- Gonda T. J., Metcalf D. Expression of myb, myc and fos proto-oncogenes during the differentiation of a murine myeloid leukaemia. Nature. 1984 Jul 19;310(5974):249–251. doi: 10.1038/310249a0. [DOI] [PubMed] [Google Scholar]
- Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
- Groudine M., Peretz M., Weintraub H. Transcriptional regulation of hemoglobin switching in chicken embryos. Mol Cell Biol. 1981 Mar;1(3):281–288. doi: 10.1128/mcb.1.3.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gusella J. F., Weil S. C., Tsiftsoglou A. S., Volloch V., Neumann J. R., Keys C., Housman D. E. Hemin does not cause commitment of murine erythroleukemia (MEL) cells to terminal differentiation. Blood. 1980 Sep;56(3):481–487. [PubMed] [Google Scholar]
- Hastings K. E., Emerson C. P., Jr cDNA clone analysis of six co-regulated mRNAs encoding skeletal muscle contractile proteins. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1553–1557. doi: 10.1073/pnas.79.5.1553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horn J. P., Wood T. G., Murphy E. C., Jr, Blair D. G., Arlinghaus R. B. A selective temperature-sensitive defect in viral RNA expression in cells infected with a ts transformation mutant of murine sarcoma virus. Cell. 1981 Jul;25(1):37–46. doi: 10.1016/0092-8674(81)90229-4. [DOI] [PubMed] [Google Scholar]
- Kaczmarek L., Hyland J. K., Watt R., Rosenberg M., Baserga R. Microinjected c-myc as a competence factor. Science. 1985 Jun 14;228(4705):1313–1315. doi: 10.1126/science.4001943. [DOI] [PubMed] [Google Scholar]
- Kelly K., Cochran B. H., Stiles C. D., Leder P. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell. 1983 Dec;35(3 Pt 2):603–610. doi: 10.1016/0092-8674(83)90092-2. [DOI] [PubMed] [Google Scholar]
- Knight E., Jr, Anton E. D., Fahey D., Friedland B. K., Jonak G. J. Interferon regulates c-myc gene expression in Daudi cells at the post-transcriptional level. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1151–1154. doi: 10.1073/pnas.82.4.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lachman H. M., Skoultchi A. I. Expression of c-myc changes during differentiation of mouse erythroleukaemia cells. Nature. 1984 Aug 16;310(5978):592–594. doi: 10.1038/310592a0. [DOI] [PubMed] [Google Scholar]
- Leder P., Battey J., Lenoir G., Moulding C., Murphy W., Potter H., Stewart T., Taub R. Translocations among antibody genes in human cancer. Science. 1983 Nov 18;222(4625):765–771. doi: 10.1126/science.6356357. [DOI] [PubMed] [Google Scholar]
- Leof E. B., Wharton W., van Wyk J. J., Pledger W. J. Epidermal growth factor (EGF) and somatomedin C regulate G1 progression in competent BALB/c-3T3 cells. Exp Cell Res. 1982 Sep;141(1):107–115. doi: 10.1016/0014-4827(82)90073-8. [DOI] [PubMed] [Google Scholar]
- Lim R. W., Hauschka S. D. A rapid decrease in epidermal growth factor-binding capacity accompanies the terminal differentiation of mouse myoblasts in vitro. J Cell Biol. 1984 Feb;98(2):739–747. doi: 10.1083/jcb.98.2.739. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Linkhart T. A., Clegg C. H., Hauschika S. D. Myogenic differentiation in permanent clonal mouse myoblast cell lines: regulation by macromolecular growth factors in the culture medium. Dev Biol. 1981 Aug;86(1):19–30. doi: 10.1016/0012-1606(81)90311-0. [DOI] [PubMed] [Google Scholar]
- Lomedico P., Rosenthal N., Efstratidadis A., Gilbert W., Kolodner R., Tizard R. The structure and evolution of the two nonallelic rat preproinsulin genes. Cell. 1979 Oct;18(2):545–558. doi: 10.1016/0092-8674(79)90071-0. [DOI] [PubMed] [Google Scholar]
- Makino R., Hayashi K., Sugimura T. C-myc transcript is induced in rat liver at a very early stage of regeneration or by cycloheximide treatment. Nature. 1984 Aug 23;310(5979):697–698. doi: 10.1038/310697a0. [DOI] [PubMed] [Google Scholar]
- McCormack J. E., Pepe V. H., Kent R. B., Dean M., Marshak-Rothstein A., Sonenshein G. E. Specific regulation of c-myc oncogene expression in a murine B-cell lymphoma. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5546–5550. doi: 10.1073/pnas.81.17.5546. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Medford R. M., Nguyen H. T., Nadal-Ginard B. Transcriptional and cell cycle-mediated regulation of myosin heavy chain gene expression during muscle cell differentiation. J Biol Chem. 1983 Sep 25;258(18):11063–11073. [PubMed] [Google Scholar]
- Medford R. M., Wydro R. M., Nguyen H. T., Nadal-Ginard B. Cytoplasmic processing of myosin heavy chain messenger RNA: evidence provided by using a recombinant DNA plasmid. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5749–5753. doi: 10.1073/pnas.77.10.5749. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Merlie J. P., Buckingham M. E., Whalen R. G. Molecular aspects of myogenesis. Curr Top Dev Biol. 1977;11:61–114. doi: 10.1016/s0070-2153(08)60743-7. [DOI] [PubMed] [Google Scholar]
- Müller R., Bravo R., Burckhardt J., Curran T. Induction of c-fos gene and protein by growth factors precedes activation of c-myc. Nature. 1984 Dec 20;312(5996):716–720. doi: 10.1038/312716a0. [DOI] [PubMed] [Google Scholar]
- Nadal-Ginard B. Commitment, fusion and biochemical differentiation of a myogenic cell line in the absence of DNA synthesis. Cell. 1978 Nov;15(3):855–864. doi: 10.1016/0092-8674(78)90270-2. [DOI] [PubMed] [Google Scholar]
- Nguyen H. T., Medford R. M., Nadal-Ginard B. Reversibility of muscle differentiation in the absence of commitment: analysis of a myogenic cell line temperature-sensitive for commitment. Cell. 1983 Aug;34(1):281–293. doi: 10.1016/0092-8674(83)90159-9. [DOI] [PubMed] [Google Scholar]
- Okazaki K., Holtzer H. Myogenesis: fusion, myosin synthesis, and the mitotic cycle. Proc Natl Acad Sci U S A. 1966 Nov;56(5):1484–1490. doi: 10.1073/pnas.56.5.1484. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Periasamy M., Wydro R. M., Strehler-Page M. A., Strehler E. E., Nadal-Ginard B. Characterization of cDNA and genomic sequences corresponding to an embryonic myosin heavy chain. J Biol Chem. 1985 Dec 15;260(29):15856–15862. [PubMed] [Google Scholar]
- Piechaczyk M., Yang J. Q., Blanchard J. M., Jeanteur P., Marcu K. B. Posttranscriptional mechanisms are responsible for accumulation of truncated c-myc RNAs in murine plasma cell tumors. Cell. 1985 Sep;42(2):589–597. doi: 10.1016/0092-8674(85)90116-3. [DOI] [PubMed] [Google Scholar]
- Pinset C., Whalen R. G. Manipulation of medium conditions and differentiation in the rat myogenic cell line L6. Dev Biol. 1984 Apr;102(2):269–277. doi: 10.1016/0012-1606(84)90192-1. [DOI] [PubMed] [Google Scholar]
- Pledger W. J., Stiles C. D., Antoniades H. N., Scher C. D. Induction of DNA synthesis in BALB/c 3T3 cells by serum components: reevaluation of the commitment process. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4481–4485. doi: 10.1073/pnas.74.10.4481. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reitsma P. H., Rothberg P. G., Astrin S. M., Trial J., Bar-Shavit Z., Hall A., Teitelbaum S. L., Kahn A. J. Regulation of myc gene expression in HL-60 leukaemia cells by a vitamin D metabolite. Nature. 1983 Dec 1;306(5942):492–494. doi: 10.1038/306492a0. [DOI] [PubMed] [Google Scholar]
- Sariban E., Mitchell T., Kufe D. Expression of the c-fms proto-oncogene during human monocytic differentiation. Nature. 1985 Jul 4;316(6023):64–66. doi: 10.1038/316064a0. [DOI] [PubMed] [Google Scholar]
- Schwartz R. J., Haron J. A., Rothblum K. N., Dugaiczyk A. Regulation of muscle differentiation: cloning of sequences from alpha-actin messenger ribonucleic acid. Biochemistry. 1980 Dec 9;19(25):5883–5890. doi: 10.1021/bi00566a034. [DOI] [PubMed] [Google Scholar]
- Shani M., Zevin-Sonkin D., Saxel O., Carmon Y., Katcoff D., Nudel U., Yaffe D. The correlation between the synthesis of skeletal muscle actin, myosin heavy chain, and myosin light chain and the accumulation of corresponding mRNA sequences during myogenesis. Dev Biol. 1981 Sep;86(2):483–492. doi: 10.1016/0012-1606(81)90206-2. [DOI] [PubMed] [Google Scholar]
- Stein G. H., Yanishevsky R. Autoradiography. Methods Enzymol. 1979;58:279–292. doi: 10.1016/s0076-6879(79)58143-9. [DOI] [PubMed] [Google Scholar]
- Stiles C. D., Capone G. T., Scher C. D., Antoniades H. N., Van Wyk J. J., Pledger W. J. Dual control of cell growth by somatomedins and platelet-derived growth factor. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1279–1283. doi: 10.1073/pnas.76.3.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Westin E. H., Gallo R. C., Arya S. K., Eva A., Souza L. M., Baluda M. A., Aaronson S. A., Wong-Staal F. Differential expression of the amv gene in human hematopoietic cells. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2194–2198. doi: 10.1073/pnas.79.7.2194. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Westin E. H., Wong-Staal F., Gelmann E. P., Dalla-Favera R., Papas T. S., Lautenberger J. A., Eva A., Reddy E. P., Tronick S. R., Aaronson S. A. Expression of cellular homologues of retroviral onc genes in human hematopoietic cells. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2490–2494. doi: 10.1073/pnas.79.8.2490. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yaffe D. Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc Natl Acad Sci U S A. 1968 Oct;61(2):477–483. doi: 10.1073/pnas.61.2.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Bustros A., Baylin S. B., Berger C. L., Roos B. A., Leong S. S., Nelkin B. D. Phorbol esters increase calcitonin gene transcription and decrease c-myc mRNA levels in cultured human medullary thyroid carcinoma. J Biol Chem. 1985 Jan 10;260(1):98–104. [PubMed] [Google Scholar]