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Molecular and Cellular Biochemistry

, Volume 195, Issue 1–2, pp 25–36 | Cite as

Growth arrest and induction of apoptosis in breast cancer cells by antisense depletion of protein kinase A-RIα subunit: p53-independent mechanism of action

  • Rakesh K. Srivastava
  • Aparna R. Srivastava
  • Prem Seth
  • Sudhir Agrawal
  • Yoon S. Cho-Chungi
Article

Abstract

The enhanced expression of the RIα subunit of cyclic AMP-dependent protein kinase type 1 (PKA-I) has been correlated with cancer cell growth. We have investigated the effects of sequence-specific inhibition of RIα gene expression on the growth of MCF-7 human breast cancer cells. We report that RIα antisense treatment results in a reduction in RIα expression at both mRNA and protein levels and inhibition of cell growth. The growth inhibition was accompanied by changes in cell morphology, cleavage of poly(ADP-ribose) polymerase (PARP) and appearance of apoptotic nuclei. In addition, bcl-2 protein level was reduced and p53 expression increased in growth arrested cells. Interestingly, RIα antisense inhibited cell viability and induced apoptosis in the absence of p53, suggesting that these actions of RIα antisense are exerted independent of p53. In contrast, two- and four-base mismatched control oligonucleotides had no effect on either cell growth or morphology. These results demonstrate that the RIα antisense, which efficiently depletes the growth stimulatory molecule RIα, induces cell differentiation and apoptosis, providing a new approach to combat breast cancer cell growth.

antisense oligonucleotide apoptosis cAMP-dependent protein kinase cancer cells growth inhibition 

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References

  1. 1.
    Krebs EG, Beavo JA: Phosphorylation-dephosphorylation of enzymes. Ann Rev Biochem 88: 923–939, 1979Google Scholar
  2. 2.
    Beebe SJ, Corbin JD: Cyclic nucleotide-dependent protein kinases. In: PD Boyer, EG Krebs (eds). The Enzymes: Control by Phosphorylation, Part A, Vol. 17. Academic Press, New York, 1986, pp 43–111Google Scholar
  3. 3.
    McKnight GS, Clegg CH, Uhler MD, Chrivia JC, Cadd GG, Correll LA, Otten AD: Analysis of the cAMP dependent protein kinase system using molecular genetic approaches. Rec Prog Horm Res 44: 307–335, 1988PubMedGoogle Scholar
  4. 4.
    Levy FO, Øyen O, Sandberg M, Tasken K, Eskild W, Hansson V, Jahnsen T: Molecular cloning, complementary deoxyribonucleic structure and predicted full-length amino aid sequence of the hormone-inducible regulatory subunit of 3¢,5¢-cyclic adenosine monophosphate-dependent protein kinase from human testis. Mol Endocrinol 2: 1364–1373, 1988PubMedGoogle Scholar
  5. 5.
    Lehmann SM, Waiter U: Regulation of the cellular and subcellular concentrations and distribution of cyclic nucleotide-dependent protein kinase. Adv Cyclic Nucleotide Protein Phosphorylation Res 18: 63–117, 1984PubMedGoogle Scholar
  6. 6.
    Cho-Chung YS: Role of cyclic AMP receptor proteins in growth, differentiation, and suppression of malignancy: New approach to therapy [perspective in cancer research]. Cancer Res 50: 7093–7100, 1990PubMedGoogle Scholar
  7. 7.
    Schwartz DA, Rubin CS: Identification and differential expression of two forms of regulatory subunits (RII) of cAMP-dependent protein kinase II in Friend erythroleukemic cells. J Biol Chem 260: 6296–6303, 1985PubMedGoogle Scholar
  8. 8.
    Tortora G, Yokozaki H, Pepe S, Clair T, Cho-Chung YS: Differentiation of HL-60 leukemia by type 1 regulatory subunit antisense oligodeoxynucleotide of cAMP-dependent protein kinase. Proc Natl Acad Sci USA 88: 2011–2015, 1991PubMedGoogle Scholar
  9. 9.
    Yokozaki H, Budillon A, Tortora G, Meissner S, Beaucage SL, Miki K, Cho-Chung YS: An antisense oligonucleotide that depletes RIα subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells. Cancer Res 53: 868–872, 1993PubMedGoogle Scholar
  10. 10.
    Nesterova M, Cho-Chung YS: A single-injection protein kinase A-directed antisense treatment to inhibit tumor growth. Nature Medicine 1: 528–533, 1995PubMedGoogle Scholar
  11. 11.
    Bursch W, Oberhammer F, Schulte-Hermann R: Cell death by apoptosis and its protective role against disease. Trends Pharmacol Sci 13: 245–251, 1992PubMedGoogle Scholar
  12. 12.
    Liebermann DA, Hoffman B, Steinman RA: Molecular controls of growth arrest and apoptosis: p53-dependent and independent pathways. Oncogene 11: 199–210, 1995PubMedGoogle Scholar
  13. 13.
    Haldar S, Negrini M, Monne M, Sabbioni S, Croce CM: Downregulation of bcl-2 by p53 in breast cancer cells. Cancer Res 54: 2095–2097, 1994PubMedGoogle Scholar
  14. 14.
    Hockenbery D: Defining apoptosis. Am J Pathol 146: 16–19, 1995PubMedGoogle Scholar
  15. 15.
    Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC: Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature (London) 371: 346–347, 1994Google Scholar
  16. 16.
    Martin SJ, Green DR: Protease activation during apoptosis. Death by a thousand cuts? Cell 82: 349–352, 1995CrossRefPubMedGoogle Scholar
  17. 17.
    Zhang R, Lu Z, Zhao H, Zhang X, Diasio RB, Habus I, Jiang Z, Iyer RP, Yu D, Agrawal S: In vivo stability, disposition and metabolism of a ‘hybrid’ oligonucleotide phosphorothioate in rats. Biochem Pharmacol 50: 545–546, 1995PubMedGoogle Scholar
  18. 18.
    Fan S, Smith ML, Rivet DJ, Duba D, Zhan Q, Kohn KW, Fornace AJ Jr, O'Conner PM: Disruption of p53 function sensitizes breast cancer cells to cisplatin and pentoxifylline. Cancer Res 55: 1649–1654, 1995PubMedGoogle Scholar
  19. 19.
    Scheffner M, Huibregtse JM, Howley PM: Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53. Proc Natl Acad Sci USA 91: 8797–8801, 1994PubMedGoogle Scholar
  20. 20.
    Oberhammer FA, Pavelka M, Shanna S, Tiefenbacher R, Purchio AF, Bursch W, Schulte-Hermann R: Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor β1. Proc Natl Acad Sci USA 89: 5408–5412, 1992PubMedGoogle Scholar
  21. 21.
    Milligan JF, Matteucci MD, Martin JC: Current concepts in antisense drug design. Med Chem 36: 1923–1937, 1993PubMedGoogle Scholar
  22. 22.
    Stein CA, Cheng YC: Antisense oligonucleotides as therapeutic agents-is the bullet really magical? Science 261: 1004–1012, 1993PubMedGoogle Scholar
  23. 23.
    Shibahara S, Mukai S, Morisawa H, Nakashima H, Kobayashi S, Yamamoto N: Inhibition of human immunodeficiency virus (HIV-1) replication by synthetic oligo-RNA derivatives. Nuc Acids Res 17: 239–252, 1989Google Scholar
  24. 24.
    Metelev V, Lisziewiez J, Agrawal S: Study of antisense oligonucleotide phosphorothioates containing segments of oligodeoxynucleotides and 2′-O-methyloligoribonucleotides. Bioorg Med Chem Lett 4: 2929–2934, 1994Google Scholar
  25. 25.
    Monia BP, Lesnik EA, Gonzalez C, Lima WF, McGee D, Guinosso CJ, Kawasaki AM, Cook PD, Freier SM: Evaluation of 2′-modified oligonucleotides containing 2′-deoxygaps as antisense inhibitors of gene expression. J Biol Chem 268: 14514–14522, 1993PubMedGoogle Scholar
  26. 26.
    Fisher DE: Apoptosis in cancer therapy: Crossing the threshold. Cell 78: 539–542, 1994PubMedGoogle Scholar
  27. 27.
    Otten AD, Parenteau LA, Doskeland S, McKnight GS: Hormonal activation of gene transcription in ras transformed NIH3T3 cells overexpressing RIIα and RIIβ subunits of the cAMP-dependent protein kinase. J Biol Chem 266: 23074–23082, 1991PubMedGoogle Scholar
  28. 28.
    Wyllie AH: Cell death: The significance of apoptosis. Int Rev Cytol 5: 131–136, 1980Google Scholar
  29. 29.
    el-Deiry WS, Tokino T, Velculeseu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B: WAF1, a potential mediator of the p53 tumor suppression. Cell 75: 817–825, 1993PubMedGoogle Scholar
  30. 30.
    Kastan MB, Zhan Q, el-Deiry WS, Carrier F, Jacks T, Walsh WV, Plunkett BS, Vogelstein B, Fomace AJ Jr.: A mammalian cell cyclic check point pathway utilizing p53 and GADD45 is defective in ataxia telangiectasia. Cell 71: 587–597, 1992PubMedGoogle Scholar
  31. 31.
    Perry ME, Piette J, Zawadzki JA, Harvey D, Levine A: The mdm-2 gene is induced in response to UV light in a p53-dependent manner. Proc Natl Acad Sci USA 90: 11623–11627, 1993PubMedGoogle Scholar
  32. 32.
    Vaux DL, Cory S, Adams JM: Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335: 440–442, 1988Google Scholar
  33. 33.
    Haldar S, Jena N, DuBois GC, Takayama S, Reed JC, Fu SS, Croce CM: Purification and characterization of bcl-2 protein. Arch Biochem Biophys 315: 483–488, 1994PubMedGoogle Scholar
  34. 34.
    Miyashita T, Reed JC: Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 11: 151–157, 1993Google Scholar
  35. 35.
    Haidar S, Jena N, Croce CM: Inactivation of bcl-2 by phosphorylation. Proc Natl Acad Sci USA 92: 4507–4511, 1995PubMedGoogle Scholar
  36. 36.
    Tortora G, Pepe S, Bianco C, Baldassarre G, Budillon A, Clair T, Cho-Chung YS, Bianco AR, Ciardiello F: The RIα subunit of protein kinase A controls serum dependency and entry into cell cycle of human mammary epithelial cells. Oncogene 9: 3233–3240, 1994PubMedGoogle Scholar
  37. 37.
    Gadbois DM, Crissman HA, Tobey RA, Bradbury EM: Multiple kinase arrest points in the G1 phase of nontransformed mammalian cells are absent in transformed cells. Proc Natl Acad Sci USA 89: 8626–8630, 1992PubMedGoogle Scholar
  38. 38.
    Pepe S, Sandomenico C, Damiano V, Ruggiero A, Nesterova M, Bianco AR, Tortora G, Cho-Chung YS: Role of protein kinase A type I (PKAI) in the regulation of HL-60 human promyelocytic leukemia cell cycle kinetics. Proc Am Assoc Cancer Res 37: (abstr) 33, 1996Google Scholar
  39. 39.
    Cvijic ME, Yang W-L, Chin K-V: Cisplatin resistance in cAMPdependent protein kinase mutants. Pharmacol Ther, 1997 (in press)Google Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Rakesh K. Srivastava
    • 1
    • 2
  • Aparna R. Srivastava
    • 1
    • 2
  • Prem Seth
    • 1
    • 2
  • Sudhir Agrawal
    • 1
    • 2
  • Yoon S. Cho-Chungi
    • 1
    • 2
  1. 1.Cellular Biochemistry Section, Laboratory of Tumor Immunology and Biology [R. K.S., Y.S. C-CNational Cancer Institute, National Institutes of HealthBethesdaU.S.A
  2. 2.Hybridon Inc.CambridgeUSA [S.A

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