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. 1995 Nov;15(11):6386–6397. doi: 10.1128/mcb.15.11.6386

Doxorubicin-induced Id2A gene transcription is targeted at an activating transcription factor/cyclic AMP response element motif through novel mechanisms involving protein kinases distinct from protein kinase C and protein kinase A.

M Kurabayashi 1, S Dutta 1, R Jeyaseelan 1, L Kedes 1
PMCID: PMC230890  PMID: 7565791

Abstract

We have recently shown that doxorubicin (Dox), an antineoplastic drug and an inhibitor of terminal differentiation of myogenic and adipogenic cells, induces expression of Id, a gene encoding a helix-loop-helix transcriptional inhibitor. In this study we have investigated the molecular mechanisms underlying Dox-induced Id2A expression. We have also attempted to determine whether the genetic responses to Dox are related to the UV response, a well-characterized set of reactions to UV and DNA-damaging compounds that is partly mediated by AP-1. Transient transfection of a series of deletions and point mutation derivatives of the human Id2A promoter sequence shows that two closely spaced and inverted short elements similar to an activating transcription factor (ATF) binding site or a cyclic AMP response element (CRE) are necessary and sufficient for a full response to Dox. We refer to this element as the IdATF site. Sequences containing an IdATF site conferred Dox inducibility on a minimal heterologous promoter. An electrophoretic mobility shift assay showed nuclear proteins specifically interacting with the IdATF sequence. While oligonucleotides containing either legitimate ATF/CRE or AP-1 binding sequences competed for binding, antibody supershift experiments suggested that neither CREB/ATF-1 nor AP-1 are major factors binding to IdATF. Several independent criteria suggest that Dox inducibility was independent of Ca2+/phospholipid-dependent protein kinase (protein kinase C), cyclic AMP-dependent protein kinase (protein kinase A), and tyrosine kinase. Moreover, we found that Dox also induces transcription from promoters of immediate-early genes through an AP-1-independent pathway. Taken together, our results suggest that Dox elicits a novel genetic response distinct from the classical UV response.

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Selected References

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  1. Angel P., Baumann I., Stein B., Delius H., Rahmsdorf H. J., Herrlich P. 12-O-tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5'-flanking region. Mol Cell Biol. 1987 Jun;7(6):2256–2266. doi: 10.1128/mcb.7.6.2256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angel P., Hattori K., Smeal T., Karin M. The jun proto-oncogene is positively autoregulated by its product, Jun/AP-1. Cell. 1988 Dec 2;55(5):875–885. doi: 10.1016/0092-8674(88)90143-2. [DOI] [PubMed] [Google Scholar]
  3. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
  4. Angel P., Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991 Dec 10;1072(2-3):129–157. doi: 10.1016/0304-419x(91)90011-9. [DOI] [PubMed] [Google Scholar]
  5. Baeuerle P. A. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim Biophys Acta. 1991 Apr 16;1072(1):63–80. doi: 10.1016/0304-419x(91)90007-8. [DOI] [PubMed] [Google Scholar]
  6. Baichwal V. R., Tjian R. Control of c-Jun activity by interaction of a cell-specific inhibitor with regulatory domain delta: differences between v- and c-Jun. Cell. 1990 Nov 16;63(4):815–825. doi: 10.1016/0092-8674(90)90147-7. [DOI] [PubMed] [Google Scholar]
  7. Benezra R., Davis R. L., Lockshon D., Turner D. L., Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell. 1990 Apr 6;61(1):49–59. doi: 10.1016/0092-8674(90)90214-y. [DOI] [PubMed] [Google Scholar]
  8. Brindle P. K., Montminy M. R. The CREB family of transcription activators. Curr Opin Genet Dev. 1992 Apr;2(2):199–204. doi: 10.1016/s0959-437x(05)80274-6. [DOI] [PubMed] [Google Scholar]
  9. Brindle P., Linke S., Montminy M. Protein-kinase-A-dependent activator in transcription factor CREB reveals new role for CREM repressors. Nature. 1993 Aug 26;364(6440):821–824. doi: 10.1038/364821a0. [DOI] [PubMed] [Google Scholar]
  10. Bruder J. T., Heidecker G., Rapp U. R. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev. 1992 Apr;6(4):545–556. doi: 10.1101/gad.6.4.545. [DOI] [PubMed] [Google Scholar]
  11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  12. Christy B., Nathans D. Functional serum response elements upstream of the growth factor-inducible gene zif268. Mol Cell Biol. 1989 Nov;9(11):4889–4895. doi: 10.1128/mcb.9.11.4889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Devary Y., Gottlieb R. A., Lau L. F., Karin M. Rapid and preferential activation of the c-jun gene during the mammalian UV response. Mol Cell Biol. 1991 May;11(5):2804–2811. doi: 10.1128/mcb.11.5.2804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Devary Y., Gottlieb R. A., Smeal T., Karin M. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell. 1992 Dec 24;71(7):1081–1091. doi: 10.1016/s0092-8674(05)80058-3. [DOI] [PubMed] [Google Scholar]
  15. Devary Y., Rosette C., DiDonato J. A., Karin M. NF-kappa B activation by ultraviolet light not dependent on a nuclear signal. Science. 1993 Sep 10;261(5127):1442–1445. doi: 10.1126/science.8367725. [DOI] [PubMed] [Google Scholar]
  16. Fisch T. M., Prywes R., Roeder R. G. c-fos sequence necessary for basal expression and induction by epidermal growth factor, 12-O-tetradecanoyl phorbol-13-acetate and the calcium ionophore. Mol Cell Biol. 1987 Oct;7(10):3490–3502. doi: 10.1128/mcb.7.10.3490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gonzalez G. A., Montminy M. R. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell. 1989 Nov 17;59(4):675–680. doi: 10.1016/0092-8674(89)90013-5. [DOI] [PubMed] [Google Scholar]
  18. Gustafson T. A., Kedes L. Identification of multiple proteins that interact with functional regions of the human cardiac alpha-actin promoter. Mol Cell Biol. 1989 Aug;9(8):3269–3283. doi: 10.1128/mcb.9.8.3269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hagmeyer B. M., König H., Herr I., Offringa R., Zantema A., van der Eb A., Herrlich P., Angel P. Adenovirus E1A negatively and positively modulates transcription of AP-1 dependent genes by dimer-specific regulation of the DNA binding and transactivation activities of Jun. EMBO J. 1993 Sep;12(9):3559–3572. doi: 10.1002/j.1460-2075.1993.tb06030.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hai T. W., Liu F., Coukos W. J., Green M. R. Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev. 1989 Dec;3(12B):2083–2090. doi: 10.1101/gad.3.12b.2083. [DOI] [PubMed] [Google Scholar]
  21. Hai T., Curran T. Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3720–3724. doi: 10.1073/pnas.88.9.3720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Han T. H., Lamph W. W., Prywes R. Mapping of epidermal growth factor-, serum-, and phorbol ester-responsive sequence elements in the c-jun promoter. Mol Cell Biol. 1992 Oct;12(10):4472–4477. doi: 10.1128/mcb.12.10.4472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hannun Y. A., Foglesong R. J., Bell R. M. The adriamycin-iron(III) complex is a potent inhibitor of protein kinase C. J Biol Chem. 1989 Jun 15;264(17):9960–9966. [PubMed] [Google Scholar]
  24. Herrlich P., Ponta H., Rahmsdorf H. J. DNA damage-induced gene expression: signal transduction and relation to growth factor signaling. Rev Physiol Biochem Pharmacol. 1992;119:187–223. doi: 10.1007/3540551921_7. [DOI] [PubMed] [Google Scholar]
  25. Hibi M., Lin A., Smeal T., Minden A., Karin M. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 1993 Nov;7(11):2135–2148. doi: 10.1101/gad.7.11.2135. [DOI] [PubMed] [Google Scholar]
  26. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984 Oct 9;23(21):5036–5041. doi: 10.1021/bi00316a032. [DOI] [PubMed] [Google Scholar]
  27. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hunter T., Karin M. The regulation of transcription by phosphorylation. Cell. 1992 Aug 7;70(3):375–387. doi: 10.1016/0092-8674(92)90162-6. [DOI] [PubMed] [Google Scholar]
  29. Ito H., Miller S. C., Billingham M. E., Akimoto H., Torti S. V., Wade R., Gahlmann R., Lyons G., Kedes L., Torti F. M. Doxorubicin selectively inhibits muscle gene expression in cardiac muscle cells in vivo and in vitro. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4275–4279. doi: 10.1073/pnas.87.11.4275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kaibuchi K., Fukumoto Y., Oku N., Hori Y., Yamamoto T., Toyoshima K., Takai Y. Activation of the serum response element and 12-O-tetradecanoylphorbol-13-acetate response element by the activated c-raf-1 protein in a manner independent of protein kinase C. J Biol Chem. 1989 Dec 15;264(35):20855–20858. [PubMed] [Google Scholar]
  31. Kaibuchi K., Fukumoto Y., Oku N., Takai Y., Arai K., Muramatsu M. Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C. J Biol Chem. 1989 Aug 15;264(23):13489–13496. [PubMed] [Google Scholar]
  32. Kaszubska W., Hooft van Huijsduijnen R., Ghersa P., DeRaemy-Schenk A. M., Chen B. P., Hai T., DeLamarter J. F., Whelan J. Cyclic AMP-independent ATF family members interact with NF-kappa B and function in the activation of the E-selectin promoter in response to cytokines. Mol Cell Biol. 1993 Nov;13(11):7180–7190. doi: 10.1128/mcb.13.11.7180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kedar P. S., Widen S. G., Englander E. W., Fornace A. J., Jr, Wilson S. H. The ATF/CREB transcription factor-binding site in the polymerase beta promoter mediates the positive effect of N-methyl-N'-nitro-N-nitrosoguanidine on transcription. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3729–3733. doi: 10.1073/pnas.88.9.3729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kim S. J., Wagner S., Liu F., O'Reilly M. A., Robbins P. D., Green M. R. Retinoblastoma gene product activates expression of the human TGF-beta 2 gene through transcription factor ATF-2. Nature. 1992 Jul 23;358(6384):331–334. doi: 10.1038/358331a0. [DOI] [PubMed] [Google Scholar]
  35. Kurabayashi M., Dutta S., Kedes L. Serum-inducible factors binding to an activating transcription factor motif regulate transcription of the Id2A promoter during myogenic differentiation. J Biol Chem. 1994 Dec 9;269(49):31162–31170. [PubMed] [Google Scholar]
  36. Kurabayashi M., Jeyaseelan R., Kedes L. Antineoplastic agent doxorubicin inhibits myogenic differentiation of C2 myoblasts. J Biol Chem. 1993 Mar 15;268(8):5524–5529. [PubMed] [Google Scholar]
  37. Kurabayashi M., Jeyaseelan R., Kedes L. Doxorubicin represses the function of the myogenic helix-loop-helix transcription factor MyoD. Involvement of Id gene induction. J Biol Chem. 1994 Feb 25;269(8):6031–6039. [PubMed] [Google Scholar]
  38. Kurabayashi M., Jeyaseelan R., Kedes L. Two distinct cDNA sequences encoding the human helix-loop-helix protein Id2. Gene. 1993 Nov 15;133(2):305–306. doi: 10.1016/0378-1119(93)90658-p. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Lee K. A., Green M. R. A cellular transcription factor E4F1 interacts with an E1a-inducible enhancer and mediates constitutive enhancer function in vitro. EMBO J. 1987 May;6(5):1345–1353. doi: 10.1002/j.1460-2075.1987.tb02374.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  42. Liu L. F. DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem. 1989;58:351–375. doi: 10.1146/annurev.bi.58.070189.002031. [DOI] [PubMed] [Google Scholar]
  43. Luckow B., Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. doi: 10.1093/nar/15.13.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Miwa T., Boxer L. M., Kedes L. CArG boxes in the human cardiac alpha-actin gene are core binding sites for positive trans-acting regulatory factors. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6702–6706. doi: 10.1073/pnas.84.19.6702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Montminy M. R., Sevarino K. A., Wagner J. A., Mandel G., Goodman R. H. Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6682–6686. doi: 10.1073/pnas.83.18.6682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Morgan R. W., Christman M. F., Jacobson F. S., Storz G., Ames B. N. Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8059–8063. doi: 10.1073/pnas.83.21.8059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nakano H., Kobayashi E., Takahashi I., Tamaoki T., Kuzuu Y., Iba H. Staurosporine inhibits tyrosine-specific protein kinase activity of Rous sarcoma virus transforming protein p60. J Antibiot (Tokyo) 1987 May;40(5):706–708. doi: 10.7164/antibiotics.40.706. [DOI] [PubMed] [Google Scholar]
  48. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
  49. Nomura S., Oishi M. Indirect induction of erythroid differentiation in mouse Friend cells: evidence for two intracellular reactions involved in the differentiation. Proc Natl Acad Sci U S A. 1983 Jan;80(1):210–214. doi: 10.1073/pnas.80.1.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Offringa R., Gebel S., van Dam H., Timmers M., Smits A., Zwart R., Stein B., Bos J. L., van der Eb A., Herrlich P. A novel function of the transforming domain of E1a: repression of AP-1 activity. Cell. 1990 Aug 10;62(3):527–538. doi: 10.1016/0092-8674(90)90017-9. [DOI] [PubMed] [Google Scholar]
  51. Olson R. D., Mushlin P. S. Doxorubicin cardiotoxicity: analysis of prevailing hypotheses. FASEB J. 1990 Oct;4(13):3076–3086. [PubMed] [Google Scholar]
  52. Posada J., Vichi P., Tritton T. R. Protein kinase C in adriamycin action and resistance in mouse sarcoma 180 cells. Cancer Res. 1989 Dec 1;49(23):6634–6639. [PubMed] [Google Scholar]
  53. Radler-Pohl A., Sachsenmaier C., Gebel S., Auer H. P., Bruder J. T., Rapp U., Angel P., Rahmsdorf H. J., Herrlich P. UV-induced activation of AP-1 involves obligatory extranuclear steps including Raf-1 kinase. EMBO J. 1993 Mar;12(3):1005–1012. doi: 10.1002/j.1460-2075.1993.tb05741.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sassone-Corsi P., Ransone L. J., Verma I. M. Cross-talk in signal transduction: TPA-inducible factor jun/AP-1 activates cAMP-responsive enhancer elements. Oncogene. 1990 Mar;5(3):427–431. [PubMed] [Google Scholar]
  55. Singal P. K., Deally C. M., Weinberg L. E. Subcellular effects of adriamycin in the heart: a concise review. J Mol Cell Cardiol. 1987 Aug;19(8):817–828. doi: 10.1016/s0022-2828(87)80392-9. [DOI] [PubMed] [Google Scholar]
  56. Smith M. R., Greene W. C. Identification of HTLV-I tax trans-activator mutants exhibiting novel transcriptional phenotypes. Genes Dev. 1990 Nov;4(11):1875–1885. doi: 10.1101/gad.4.11.1875. [DOI] [PubMed] [Google Scholar]
  57. Stein B., Rahmsdorf H. J., Steffen A., Litfin M., Herrlich P. UV-induced DNA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein. Mol Cell Biol. 1989 Nov;9(11):5169–5181. doi: 10.1128/mcb.9.11.5169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Sun X. H., Copeland N. G., Jenkins N. A., Baltimore D. Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins. Mol Cell Biol. 1991 Nov;11(11):5603–5611. doi: 10.1128/mcb.11.11.5603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
  60. Tamaoki T. Use and specificity of staurosporine, UCN-01, and calphostin C as protein kinase inhibitors. Methods Enzymol. 1991;201:340–347. doi: 10.1016/0076-6879(91)01030-6. [DOI] [PubMed] [Google Scholar]
  61. Taylor A., Erba H. P., Muscat G. E., Kedes L. Nucleotide sequence and expression of the human skeletal alpha-actin gene: evolution of functional regulatory domains. Genomics. 1988 Nov;3(4):323–336. doi: 10.1016/0888-7543(88)90123-1. [DOI] [PubMed] [Google Scholar]
  62. Triton T. R., Yee G. The anticancer agent adriamycin can be actively cytotoxic without entering cells. Science. 1982 Jul 16;217(4556):248–250. doi: 10.1126/science.7089561. [DOI] [PubMed] [Google Scholar]
  63. Trush M. A., Kensler T. W. An overview of the relationship between oxidative stress and chemical carcinogenesis. Free Radic Biol Med. 1991;10(3-4):201–209. doi: 10.1016/0891-5849(91)90077-g. [DOI] [PubMed] [Google Scholar]
  64. Walton K. M., Rehfuss R. P., Chrivia J. C., Lochner J. E., Goodman R. H. A dominant repressor of cyclic adenosine 3',5'-monophosphate (cAMP)-regulated enhancer-binding protein activity inhibits the cAMP-mediated induction of the somatostatin promoter in vivo. Mol Endocrinol. 1992 Apr;6(4):647–655. doi: 10.1210/mend.6.4.1350057. [DOI] [PubMed] [Google Scholar]
  65. 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]
  66. Williams T., Admon A., Lüscher B., Tjian R. Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements. Genes Dev. 1988 Dec;2(12A):1557–1569. doi: 10.1101/gad.2.12a.1557. [DOI] [PubMed] [Google Scholar]
  67. Yamamoto K. K., Gonzalez G. A., Biggs W. H., 3rd, Montminy M. R. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature. 1988 Aug 11;334(6182):494–498. doi: 10.1038/334494a0. [DOI] [PubMed] [Google Scholar]
  68. van Dam H., Offringa R., Meijer I., Stein B., Smits A. M., Herrlich P., Bos J. L., van der Eb A. J. Differential effects of the adenovirus E1A oncogene on members of the AP-1 transcription factor family. Mol Cell Biol. 1990 Nov;10(11):5857–5864. doi: 10.1128/mcb.10.11.5857. [DOI] [PMC free article] [PubMed] [Google Scholar]

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