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. 1994 Sep;14(9):5661–5670. doi: 10.1128/mcb.14.9.5661

Nuclear c-Myc plays an important role in the cytotoxicity of tumor necrosis factor alpha in tumor cells.

R U Jänicke 1, F H Lee 1, A G Porter 1
PMCID: PMC359091  PMID: 8065303

Abstract

The phosphoprotein c-Myc has the potential to kill cells by apoptosis. To investigate whether c-Myc is involved in tumor necrosis factor alpha (TNF-alpha)-mediated cell killing, we have examined two HeLa cell lines (D98 and H21) which show dramatic differences in their susceptibilities to TNF-alpha cytotoxicity. Northern (RNA) blot analyses showed that there were no significant differences between these cell lines in basal or TNF-alpha-induced mRNA expression for a variety of proteins, including manganous superoxide dismutase, A20 zinc finger protein, plasminogen activator inhibitor type 2, and hsp70, all of which are known to influence the susceptibility of certain cells to TNF-alpha killing. On the other hand, there was a dramatic increase in c-Myc mRNA expression in TNF-alpha-sensitive D98 cells, but not in TNF-alpha-resistant H21 cells, which was only observed when the cells were treated with cycloheximide. Western blot (immunoblot) analyses revealed that even in the absence of TNF-alpha or cycloheximide, c-Myc was detectable only in nuclear extracts of TNF-alpha-sensitive D98 cells, implying a role for preexisting c-Myc in TNF-alpha killing. In support of this interpretation, a c-myc antisense oligonucleotide specifically inhibited the TNF-alpha killing of D98 cells, provided that the oligonucleotide was added 6 h prior to TNF-alpha treatment. Either dexamethasone treatment or transient expression of c-myc antisense cDNA fragments decreased nuclear c-Myc in D98 cells and rendered the cells more resistant to TNF-alpha cytotoxicity. Nuclear c-Myc was also detectable in a TNF-alpha-sensitive human HT-1080 fibrosarcoma cell line, but it was undetectable in a derivative of HT-1080 (SS-HT-1080) known to be resistant to TNF-alpha killing because of overexpression of plasminogen activator inhibitor type 2. HT-1080 cells transfected with antisense c-myc cDNA had significantly less nuclear c-Myc and were resistant to TNF-alpha cytotoxicity. Together, these data indicate that a nuclear transcription factor, c-Myc, plays an important role in sensitizing two different tumor cell types to TNF-alpha cytotoxicity.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abrams H. D., Rohrschneider L. R., Eisenman R. N. Nuclear location of the putative transforming protein of avian myelocytomatosis virus. Cell. 1982 Jun;29(2):427–439. doi: 10.1016/0092-8674(82)90159-3. [DOI] [PubMed] [Google Scholar]
  2. Agarwal S., Drysdale B. E., Shin H. S. Tumor necrosis factor-mediated cytotoxicity involves ADP-ribosylation. J Immunol. 1988 Jun 15;140(12):4187–4192. [PubMed] [Google Scholar]
  3. Andus T., Heinrich P. C., Bauer J., Tran-Thi T. A., Decker K., Männel D., Northoff H. Discrimination of hepatocyte-stimulating activity from human recombinant tumor necrosis factor alpha. Eur J Immunol. 1987 Aug;17(8):1193–1197. doi: 10.1002/eji.1830170817. [DOI] [PubMed] [Google Scholar]
  4. Askew D. S., Ashmun R. A., Simmons B. C., Cleveland J. L. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene. 1991 Oct;6(10):1915–1922. [PubMed] [Google Scholar]
  5. Ayer D. E., Kretzner L., Eisenman R. N. Mad: a heterodimeric partner for Max that antagonizes Myc transcriptional activity. Cell. 1993 Jan 29;72(2):211–222. doi: 10.1016/0092-8674(93)90661-9. [DOI] [PubMed] [Google Scholar]
  6. Bissonnette R. P., Echeverri F., Mahboubi A., Green D. R. Apoptotic cell death induced by c-myc is inhibited by bcl-2. Nature. 1992 Oct 8;359(6395):552–554. doi: 10.1038/359552a0. [DOI] [PubMed] [Google Scholar]
  7. Blackwood E. M., Eisenman R. N. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science. 1991 Mar 8;251(4998):1211–1217. doi: 10.1126/science.2006410. [DOI] [PubMed] [Google Scholar]
  8. Cheley S., Anderson R. A reproducible microanalytical method for the detection of specific RNA sequences by dot-blot hybridization. Anal Biochem. 1984 Feb;137(1):15–19. doi: 10.1016/0003-2697(84)90339-7. [DOI] [PubMed] [Google Scholar]
  9. Chen M. J., Holskin B., Strickler J., Gorniak J., Clark M. A., Johnson P. J., Mitcho M., Shalloway D. Induction by E1A oncogene expression of cellular susceptibility to lysis by TNF. Nature. 1987 Dec 10;330(6148):581–583. doi: 10.1038/330581a0. [DOI] [PubMed] [Google Scholar]
  10. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  11. Defilippi P., Poupart P., Tavernier J., Fiers W., Content J. Induction and regulation of mRNA encoding 26-kDa protein in human cell lines treated with recombinant human tumor necrosis factor. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4557–4561. doi: 10.1073/pnas.84.13.4557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Duerksen-Hughes P., Wold W. S., Gooding L. R. Adenovirus E1A renders infected cells sensitive to cytolysis by tumor necrosis factor. J Immunol. 1989 Dec 15;143(12):4193–4200. [PubMed] [Google Scholar]
  13. Evan G. I., Littlewood T. D. The role of c-myc in cell growth. Curr Opin Genet Dev. 1993 Feb;3(1):44–49. doi: 10.1016/s0959-437x(05)80339-9. [DOI] [PubMed] [Google Scholar]
  14. Evan G. I., Wyllie A. H., Gilbert C. S., Littlewood T. D., Land H., Brooks M., Waters C. M., Penn L. Z., Hancock D. C. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 1992 Apr 3;69(1):119–128. doi: 10.1016/0092-8674(92)90123-t. [DOI] [PubMed] [Google Scholar]
  15. Fanidi A., Harrington E. A., Evan G. I. Cooperative interaction between c-myc and bcl-2 proto-oncogenes. Nature. 1992 Oct 8;359(6395):554–556. doi: 10.1038/359554a0. [DOI] [PubMed] [Google Scholar]
  16. Fiers W. Tumor necrosis factor. Characterization at the molecular, cellular and in vivo level. FEBS Lett. 1991 Jul 22;285(2):199–212. doi: 10.1016/0014-5793(91)80803-b. [DOI] [PubMed] [Google Scholar]
  17. Goh C. R., Loh C. S., Porter A. G. Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin). Protein Eng. 1991 Oct;4(7):785–791. doi: 10.1093/protein/4.7.785. [DOI] [PubMed] [Google Scholar]
  18. Hann S. R., King M. W., Bentley D. L., Anderson C. W., Eisenman R. N. A non-AUG translational initiation in c-myc exon 1 generates an N-terminally distinct protein whose synthesis is disrupted in Burkitt's lymphomas. Cell. 1988 Jan 29;52(2):185–195. doi: 10.1016/0092-8674(88)90507-7. [DOI] [PubMed] [Google Scholar]
  19. Hayakawa M., Ishida N., Takeuchi K., Shibamoto S., Hori T., Oku N., Ito F., Tsujimoto M. Arachidonic acid-selective cytosolic phospholipase A2 is crucial in the cytotoxic action of tumor necrosis factor. J Biol Chem. 1993 May 25;268(15):11290–11295. [PubMed] [Google Scholar]
  20. Hirata F., Schiffmann E., Venkatasubramanian K., Salomon D., Axelrod J. A phospholipase A2 inhibitory protein in rabbit neutrophils induced by glucocorticoids. Proc Natl Acad Sci U S A. 1980 May;77(5):2533–2536. doi: 10.1073/pnas.77.5.2533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Itoh N., Yonehara S., Ishii A., Yonehara M., Mizushima S., Sameshima M., Hase A., Seto Y., Nagata S. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell. 1991 Jul 26;66(2):233–243. doi: 10.1016/0092-8674(91)90614-5. [DOI] [PubMed] [Google Scholar]
  22. Jarvis W. D., Kolesnick R. N., Fornari F. A., Traylor R. S., Gewirtz D. A., Grant S. Induction of apoptotic DNA damage and cell death by activation of the sphingomyelin pathway. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):73–77. doi: 10.1073/pnas.91.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jättelä M., Wissing D., Bauer P. A., Li G. C. Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity. EMBO J. 1992 Oct;11(10):3507–3512. doi: 10.1002/j.1460-2075.1992.tb05433.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Khandjian E. W. UV crosslinking of RNA to nylon membrane enhances hybridization signals. Mol Biol Rep. 1986;11(2):107–115. doi: 10.1007/BF00364822. [DOI] [PubMed] [Google Scholar]
  25. Kohase M., Henriksen-Destefano D., Sehgal P. B., Vilcek J. Dexamethasone inhibits feedback regulation of the mitogenic activity of tumor necrosis factor, interleukin-1, and epidermal growth factor in human fibroblasts. J Cell Physiol. 1987 Aug;132(2):271–278. doi: 10.1002/jcp.1041320211. [DOI] [PubMed] [Google Scholar]
  26. Kulkarni A. B., Reinke R., Feigelson P. Acute phase mediators and glucocorticoids elevate alpha 1-acid glycoprotein gene transcription. J Biol Chem. 1985 Dec 15;260(29):15386–15389. [PubMed] [Google Scholar]
  27. Kull F. C., Jr, Jacobs S., Cuatrecasas P. Cellular receptor for 125I-labeled tumor necrosis factor: specific binding, affinity labeling, and relationship to sensitivity. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5756–5760. doi: 10.1073/pnas.82.17.5756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kumar S., Baglioni C. Protection from tumor necrosis factor-mediated cytolysis by overexpression of plasminogen activator inhibitor type-2. J Biol Chem. 1991 Nov 5;266(31):20960–20964. [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Larrick J. W., Wright S. C. Cytotoxic mechanism of tumor necrosis factor-alpha. FASEB J. 1990 Nov;4(14):3215–3223. doi: 10.1096/fasebj.4.14.2172061. [DOI] [PubMed] [Google Scholar]
  31. Laster S. M., Wood J. G., Gooding L. R. Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. J Immunol. 1988 Oct 15;141(8):2629–2634. [PubMed] [Google Scholar]
  32. Lillie J. W., Green M., Green M. R. An adenovirus E1a protein region required for transformation and transcriptional repression. Cell. 1986 Sep 26;46(7):1043–1051. doi: 10.1016/0092-8674(86)90704-x. [DOI] [PubMed] [Google Scholar]
  33. Lüscher B., Eisenman R. N. New light on Myc and Myb. Part I. Myc. Genes Dev. 1990 Dec;4(12A):2025–2035. doi: 10.1101/gad.4.12a.2025. [DOI] [PubMed] [Google Scholar]
  34. Meikrantz W., Gisselbrecht S., Tam S. W., Schlegel R. Activation of cyclin A-dependent protein kinases during apoptosis. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3754–3758. doi: 10.1073/pnas.91.9.3754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Niitsu Y., Watanabe N., Neda H., Yamauchi N., Maeda M., Sone H., Kuriyama H. Induction of synthesis of tumor necrosis factor in human and murine cell lines by exogenous recombinant human tumor necrosis factor. Cancer Res. 1988 Oct 1;48(19):5407–5410. [PubMed] [Google Scholar]
  36. Opipari A. W., Jr, Hu H. M., Yabkowitz R., Dixit V. M. The A20 zinc finger protein protects cells from tumor necrosis factor cytotoxicity. J Biol Chem. 1992 Jun 25;267(18):12424–12427. [PubMed] [Google Scholar]
  37. Reid T. R., Torti F. M., Ringold G. M. Evidence for two mechanisms by which tumor necrosis factor kills cells. J Biol Chem. 1989 Mar 15;264(8):4583–4589. [PubMed] [Google Scholar]
  38. Schreiber E., Matthias P., Müller M. M., Schaffner W. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic Acids Res. 1989 Aug 11;17(15):6419–6419. doi: 10.1093/nar/17.15.6419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schulze-Osthoff K., Beyaert R., Vandevoorde V., Haegeman G., Fiers W. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J. 1993 Aug;12(8):3095–3104. doi: 10.1002/j.1460-2075.1993.tb05978.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shi Y., Glynn J. M., Guilbert L. J., Cotter T. G., Bissonnette R. P., Green D. R. Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas. Science. 1992 Jul 10;257(5067):212–214. doi: 10.1126/science.1378649. [DOI] [PubMed] [Google Scholar]
  41. Shrivastava A., Saleque S., Kalpana G. V., Artandi S., Goff S. P., Calame K. Inhibition of transcriptional regulator Yin-Yang-1 by association with c-Myc. Science. 1993 Dec 17;262(5141):1889–1892. doi: 10.1126/science.8266081. [DOI] [PubMed] [Google Scholar]
  42. Sprang S. R. The divergent receptors for TNF. Trends Biochem Sci. 1990 Oct;15(10):366–368. doi: 10.1016/0968-0004(90)90228-4. [DOI] [PubMed] [Google Scholar]
  43. Suen T. C., Rodriguez R. U., Hung M. C., Klostergaard J. V-abl confers resistance and growth advantage to TNF-alpha in NIH3T3 cells. Lymphokine Res. 1990 Spring;9(1):15–26. [PubMed] [Google Scholar]
  44. Sugarman B. J., Aggarwal B. B., Hass P. E., Figari I. S., Palladino M. A., Jr, Shepard H. M. Recombinant human tumor necrosis factor-alpha: effects on proliferation of normal and transformed cells in vitro. Science. 1985 Nov 22;230(4728):943–945. doi: 10.1126/science.3933111. [DOI] [PubMed] [Google Scholar]
  45. Tartaglia L. A., Ayres T. M., Wong G. H., Goeddel D. V. A novel domain within the 55 kd TNF receptor signals cell death. Cell. 1993 Sep 10;74(5):845–853. doi: 10.1016/0092-8674(93)90464-2. [DOI] [PubMed] [Google Scholar]
  46. Tsujimoto M., Okamura N., Adachi H. Dexamethasone inhibits the cytotoxic activity of tumor necrosis factor. Biochem Biophys Res Commun. 1988 May 31;153(1):109–115. doi: 10.1016/s0006-291x(88)81196-3. [DOI] [PubMed] [Google Scholar]
  47. Tsujimoto M., Yip Y. K., Vilcek J. Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7626–7630. doi: 10.1073/pnas.82.22.7626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wallach D. Cytotoxins (tumour necrosis factor, lymphotoxin and others): molecular and functional characteristics and interactions with interferons. Interferon. 1986;7:89–124. [PubMed] [Google Scholar]
  49. Watanabe-Fukunaga R., Brannan C. I., Copeland N. G., Jenkins N. A., Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature. 1992 Mar 26;356(6367):314–317. doi: 10.1038/356314a0. [DOI] [PubMed] [Google Scholar]
  50. White E. Death-defying acts: a meeting review on apoptosis. Genes Dev. 1993 Dec;7(12A):2277–2284. doi: 10.1101/gad.7.12a.2277. [DOI] [PubMed] [Google Scholar]
  51. White E., Sabbatini P., Debbas M., Wold W. S., Kusher D. I., Gooding L. R. The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha. Mol Cell Biol. 1992 Jun;12(6):2570–2580. doi: 10.1128/mcb.12.6.2570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wong G. H., Elwell J. H., Oberley L. W., Goeddel D. V. Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell. 1989 Sep 8;58(5):923–931. doi: 10.1016/0092-8674(89)90944-6. [DOI] [PubMed] [Google Scholar]
  53. Yarden A., Kimchi A. Tumor necrosis factor reduces c-myc expression and cooperates with interferon-gamma in HeLa cells. Science. 1986 Dec 12;234(4782):1419–1421. doi: 10.1126/science.3097823. [DOI] [PubMed] [Google Scholar]
  54. Yonish-Rouach E., Resnitzky D., Lotem J., Sachs L., Kimchi A., Oren M. Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature. 1991 Jul 25;352(6333):345–347. doi: 10.1038/352345a0. [DOI] [PubMed] [Google Scholar]
  55. Yuh Y. S., Thompson E. B. Glucocorticoid effect on oncogene/growth gene expression in human T lymphoblastic leukemic cell line CCRF-CEM. Specific c-myc mRNA suppression by dexamethasone. J Biol Chem. 1989 Jun 25;264(18):10904–10910. [PubMed] [Google Scholar]

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