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. 1995 Jul;15(7):3470–3478. doi: 10.1128/mcb.15.7.3470

The nerve growth factor-responsive PC12 cell line does not express the Myc dimerization partner Max.

R Hopewell 1, E B Ziff 1
PMCID: PMC230583  PMID: 7791753

Abstract

Heterodimerization of Max with the nuclear oncoprotein Myc and the differentiation-associated proteins Mad and Mxi1 enables these factors to bind E-box sites in DNA and control genes implicated in cell proliferation and differentiation. We show that in the PC12 pheochromocytoma tumor cell line, functional Max protein is not expressed because of the synthesis of a mutant max transcript. This transcript encodes a protein incapable of homo- or heterodimerization. Furthermore, the mutant Max protein, unlike wild-type Max, is incapable of repressing transcription from an E-box element. Synthesis of mutant max transcripts appears to be due to a homozygous chromosomal alteration within the max gene. Reintroduction of max into PC12 cells results in repression of E-box-dependent transcription and a reduction in growth rate, which may explain the loss of Max expression either during the growth of the pheochromocytoma or in subsequent passage of the PC12 cell line in vitro. Finally, the ability of these cells to divide, differentiate, and apoptose in the absence of Max demonstrates for the first time that these processes can occur via Max- and possibly Myc-independent mechanisms.

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Selected References

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  1. Amati B., Brooks M. W., Levy N., Littlewood T. D., Evan G. I., Land H. Oncogenic activity of the c-Myc protein requires dimerization with Max. Cell. 1993 Jan 29;72(2):233–245. doi: 10.1016/0092-8674(93)90663-b. [DOI] [PubMed] [Google Scholar]
  2. Amati B., Littlewood T. D., Evan G. I., Land H. The c-Myc protein induces cell cycle progression and apoptosis through dimerization with Max. EMBO J. 1993 Dec 15;12(13):5083–5087. doi: 10.1002/j.1460-2075.1993.tb06202.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amin C., Wagner A. J., Hay N. Sequence-specific transcriptional activation by Myc and repression by Max. Mol Cell Biol. 1993 Jan;13(1):383–390. doi: 10.1128/mcb.13.1.383. [DOI] [PMC free article] [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., Eisenman R. N. A switch from Myc:Max to Mad:Max heterocomplexes accompanies monocyte/macrophage differentiation. Genes Dev. 1993 Nov;7(11):2110–2119. doi: 10.1101/gad.7.11.2110. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Batistatou A., Greene L. A. Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity. J Cell Biol. 1991 Oct;115(2):461–471. doi: 10.1083/jcb.115.2.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Batistatou A., Merry D. E., Korsmeyer S. J., Greene L. A. Bcl-2 affects survival but not neuronal differentiation of PC12 cells. J Neurosci. 1993 Oct;13(10):4422–4428. doi: 10.1523/JNEUROSCI.13-10-04422.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Berberich S., Hyde-DeRuyscher N., Espenshade P., Cole M. max encodes a sequence-specific DNA-binding protein and is not regulated by serum growth factors. Oncogene. 1992 Apr;7(4):775–779. [PubMed] [Google Scholar]
  10. Blackwell T. K., Huang J., Ma A., Kretzner L., Alt F. W., Eisenman R. N., Weintraub H. Binding of myc proteins to canonical and noncanonical DNA sequences. Mol Cell Biol. 1993 Sep;13(9):5216–5224. doi: 10.1128/mcb.13.9.5216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Blackwell T. K., Kretzner L., Blackwood E. M., Eisenman R. N., Weintraub H. Sequence-specific DNA binding by the c-Myc protein. Science. 1990 Nov 23;250(4984):1149–1151. doi: 10.1126/science.2251503. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Blackwood E. M., Lüscher B., Eisenman R. N. Myc and Max associate in vivo. Genes Dev. 1992 Jan;6(1):71–80. doi: 10.1101/gad.6.1.71. [DOI] [PubMed] [Google Scholar]
  14. Boulukos K. E., Ziff E. B. Adenovirus 5 E1A proteins disrupt the neuronal phenotype and growth factor responsiveness of PC12 cells by a conserved region 1-dependent mechanism. Oncogene. 1993 Feb;8(2):237–248. [PubMed] [Google Scholar]
  15. Burstein D. E., Greene L. A. Nerve growth factor has both mitogenic and antimitogenic activity. Dev Biol. 1982 Dec;94(2):477–482. doi: 10.1016/0012-1606(82)90364-5. [DOI] [PubMed] [Google Scholar]
  16. Charron J., Malynn B. A., Fisher P., Stewart V., Jeannotte L., Goff S. P., Robertson E. J., Alt F. W. Embryonic lethality in mice homozygous for a targeted disruption of the N-myc gene. Genes Dev. 1992 Dec;6(12A):2248–2257. doi: 10.1101/gad.6.12a.2248. [DOI] [PubMed] [Google Scholar]
  17. Cogliati T., Dunn B. K., Bar-Ner M., Cultraro C. M., Segal S. Transfected wild-type and mutant max regulate cell growth and differentiation of murine erythroleukemia cells. Oncogene. 1993 May;8(5):1263–1268. [PubMed] [Google Scholar]
  18. Coppola J. A., Cole M. D. Constitutive c-myc oncogene expression blocks mouse erythroleukaemia cell differentiation but not commitment. Nature. 1986 Apr 24;320(6064):760–763. doi: 10.1038/320760a0. [DOI] [PubMed] [Google Scholar]
  19. Dang C. V., McGuire M., Buckmire M., Lee W. M. Involvement of the 'leucine zipper' region in the oligomerization and transforming activity of human c-myc protein. Nature. 1989 Feb 16;337(6208):664–666. doi: 10.1038/337664a0. [DOI] [PubMed] [Google Scholar]
  20. Davis A. C., Wims M., Spotts G. D., Hann S. R., Bradley A. A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice. Genes Dev. 1993 Apr;7(4):671–682. doi: 10.1101/gad.7.4.671. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Ferré-D'Amaré A. R., Prendergast G. C., Ziff E. B., Burley S. K. Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain. Nature. 1993 May 6;363(6424):38–45. doi: 10.1038/363038a0. [DOI] [PubMed] [Google Scholar]
  23. Freytag S. O., Dang C. V., Lee W. M. Definition of the activities and properties of c-myc required to inhibit cell differentiation. Cell Growth Differ. 1990 Jul;1(7):339–343. [PubMed] [Google Scholar]
  24. Gilladoga A. D., Edelhoff S., Blackwood E. M., Eisenman R. N., Disteche C. M. Mapping of MAX to human chromosome 14 and mouse chromosome 12 by in situ hybridization. Oncogene. 1992 Jun;7(6):1249–1251. [PubMed] [Google Scholar]
  25. Gizang-Ginsberg E., Ziff E. B. Nerve growth factor regulates tyrosine hydroxylase gene transcription through a nucleoprotein complex that contains c-Fos. Genes Dev. 1990 Apr;4(4):477–491. doi: 10.1101/gad.4.4.477. [DOI] [PubMed] [Google Scholar]
  26. Greenberg M. E., Greene L. A., Ziff E. B. Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells. J Biol Chem. 1985 Nov 15;260(26):14101–14110. [PubMed] [Google Scholar]
  27. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Gu W., Bhatia K., Magrath I. T., Dang C. V., Dalla-Favera R. Binding and suppression of the Myc transcriptional activation domain by p107. Science. 1994 Apr 8;264(5156):251–254. doi: 10.1126/science.8146655. [DOI] [PubMed] [Google Scholar]
  29. Hagemeier C., Bannister A. J., Cook A., Kouzarides T. The activation domain of transcription factor PU.1 binds the retinoblastoma (RB) protein and the transcription factor TFIID in vitro: RB shows sequence similarity to TFIID and TFIIB. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1580–1584. doi: 10.1073/pnas.90.4.1580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Harel-Bellan A., Ferris D. K., Vinocour M., Holt J. T., Farrar W. L. Specific inhibition of c-myc protein biosynthesis using an antisense synthetic deoxy-oligonucleotide in human T lymphocytes. J Immunol. 1988 Apr 1;140(7):2431–2435. [PubMed] [Google Scholar]
  31. Hateboer G., Timmers H. T., Rustgi A. K., Billaud M., van 't Veer L. J., Bernards R. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8489–8493. doi: 10.1073/pnas.90.18.8489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Heikkila R., Schwab G., Wickstrom E., Loke S. L., Pluznik D. H., Watt R., Neckers L. M. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1. 1987 Jul 30-Aug 5Nature. 328(6129):445–449. doi: 10.1038/328445a0. [DOI] [PubMed] [Google Scholar]
  33. Holt J. T., Redner R. L., Nienhuis A. W. An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Mol Cell Biol. 1988 Feb;8(2):963–973. doi: 10.1128/mcb.8.2.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kato G. J., Lee W. M., Chen L. L., Dang C. V. Max: functional domains and interaction with c-Myc. Genes Dev. 1992 Jan;6(1):81–92. doi: 10.1101/gad.6.1.81. [DOI] [PubMed] [Google Scholar]
  35. Kerkhoff E., Bister K., Klempnauer K. H. Sequence-specific DNA binding by Myc proteins. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4323–4327. doi: 10.1073/pnas.88.10.4323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kretzner L., Blackwood E. M., Eisenman R. N. Myc and Max proteins possess distinct transcriptional activities. Nature. 1992 Oct 1;359(6394):426–429. doi: 10.1038/359426a0. [DOI] [PubMed] [Google Scholar]
  37. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  38. Langdon W. Y., Harris A. W., Cory S., Adams J. M. The c-myc oncogene perturbs B lymphocyte development in E-mu-myc transgenic mice. Cell. 1986 Oct 10;47(1):11–18. doi: 10.1016/0092-8674(86)90361-2. [DOI] [PubMed] [Google Scholar]
  39. Larsson L. G., Pettersson M., Oberg F., Nilsson K., Lüscher B. Expression of mad, mxi1, max and c-myc during induced differentiation of hematopoietic cells: opposite regulation of mad and c-myc. Oncogene. 1994 Apr;9(4):1247–1252. [PubMed] [Google Scholar]
  40. Li L. H., Nerlov C., Prendergast G., MacGregor D., Ziff E. B. c-Myc represses transcription in vivo by a novel mechanism dependent on the initiator element and Myc box II. EMBO J. 1994 Sep 1;13(17):4070–4079. doi: 10.1002/j.1460-2075.1994.tb06724.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Maheswaran S., Lee H., Sonenshein G. E. Intracellular association of the protein product of the c-myc oncogene with the TATA-binding protein. Mol Cell Biol. 1994 Feb;14(2):1147–1152. doi: 10.1128/mcb.14.2.1147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Maruyama K., Schiavi S. C., Huse W., Johnson G. L., Ruley H. E. myc and E1A oncogenes alter the responses of PC12 cells to nerve growth factor and block differentiation. Oncogene. 1987;1(4):361–367. [PubMed] [Google Scholar]
  43. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Mukherjee B., Morgenbesser S. D., DePinho R. A. Myc family oncoproteins function through a common pathway to transform normal cells in culture: cross-interference by Max and trans-acting dominant mutants. Genes Dev. 1992 Aug;6(8):1480–1492. doi: 10.1101/gad.6.8.1480. [DOI] [PubMed] [Google Scholar]
  45. Murre C., McCaw P. S., Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 1989 Mar 10;56(5):777–783. doi: 10.1016/0092-8674(89)90682-x. [DOI] [PubMed] [Google Scholar]
  46. Philipp A., Schneider A., Väsrik I., Finke K., Xiong Y., Beach D., Alitalo K., Eilers M. Repression of cyclin D1: a novel function of MYC. Mol Cell Biol. 1994 Jun;14(6):4032–4043. doi: 10.1128/mcb.14.6.4032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pittman R. N., Wang S., DiBenedetto A. J., Mills J. C. A system for characterizing cellular and molecular events in programmed neuronal cell death. J Neurosci. 1993 Sep;13(9):3669–3680. doi: 10.1523/JNEUROSCI.13-09-03669.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Prendergast G. C., Lawe D., Ziff E. B. Association of Myn, the murine homolog of max, with c-Myc stimulates methylation-sensitive DNA binding and ras cotransformation. Cell. 1991 May 3;65(3):395–407. doi: 10.1016/0092-8674(91)90457-a. [DOI] [PubMed] [Google Scholar]
  49. Prendergast G. C., Ziff E. B. Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region. Science. 1991 Jan 11;251(4990):186–189. doi: 10.1126/science.1987636. [DOI] [PubMed] [Google Scholar]
  50. Reddy C. D., Dasgupta P., Saikumar P., Dudek H., Rauscher F. J., 3rd, Reddy E. P. Mutational analysis of Max: role of basic, helix-loop-helix/leucine zipper domains in DNA binding, dimerization and regulation of Myc-mediated transcriptional activation. Oncogene. 1992 Oct;7(10):2085–2092. [PubMed] [Google Scholar]
  51. Ribon V., Leff T., Saltiel A. R. c-Myc does not require max for transcriptional activity in PC-12 cells. Mol Cell Neurosci. 1994 Jun;5(3):277–282. doi: 10.1006/mcne.1994.1032. [DOI] [PubMed] [Google Scholar]
  52. Ron D., Dressler H. pGSTag--a versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins. Biotechniques. 1992 Dec;13(6):866–869. [PubMed] [Google Scholar]
  53. Roy A. L., Carruthers C., Gutjahr T., Roeder R. G. Direct role for Myc in transcription initiation mediated by interactions with TFII-I. Nature. 1993 Sep 23;365(6444):359–361. doi: 10.1038/365359a0. [DOI] [PubMed] [Google Scholar]
  54. Rukenstein A., Rydel R. E., Greene L. A. Multiple agents rescue PC12 cells from serum-free cell death by translation- and transcription-independent mechanisms. J Neurosci. 1991 Aug;11(8):2552–2563. doi: 10.1523/JNEUROSCI.11-08-02552.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Rustgi A. K., Dyson N., Bernards R. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product. Nature. 1991 Aug 8;352(6335):541–544. doi: 10.1038/352541a0. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. Shibuya H., Yoneyama M., Ninomiya-Tsuji J., Matsumoto K., Taniguchi T. IL-2 and EGF receptors stimulate the hematopoietic cell cycle via different signaling pathways: demonstration of a novel role for c-myc. Cell. 1992 Jul 10;70(1):57–67. doi: 10.1016/0092-8674(92)90533-i. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. Sollenberger K. G., Kao T. L., Taparowsky E. J. Structural analysis of the chicken max gene. Oncogene. 1994 Feb;9(2):661–664. [PubMed] [Google Scholar]
  60. Trent J. M., Kaneko Y., Mitelman F. Report of the committee on structural chromosome changes in neoplasia. Cytogenet Cell Genet. 1989;51(1-4):533–562. doi: 10.1159/000132807. [DOI] [PubMed] [Google Scholar]
  61. Wagner A. J., Le Beau M. M., Diaz M. O., Hay N. Expression, regulation, and chromosomal localization of the Max gene. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3111–3115. doi: 10.1073/pnas.89.7.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wenzel A., Cziepluch C., Hamann U., Schürmann J., Schwab M. The N-Myc oncoprotein is associated in vivo with the phosphoprotein Max(p20/22) in human neuroblastoma cells. EMBO J. 1991 Dec;10(12):3703–3712. doi: 10.1002/j.1460-2075.1991.tb04938.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zervos A. S., Gyuris J., Brent R. Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites. Cell. 1993 Jan 29;72(2):223–232. doi: 10.1016/0092-8674(93)90662-a. [DOI] [PubMed] [Google Scholar]

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