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Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators

Abstract

PKC-δ is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-δ slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-δ dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-δ using a Zn2+ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-δ caused rapid and significant downregulation of cyclin D1 and cyclin E proteins (50% decreases, P<0.05), while mRNA levels remained unchanged. The PKC agonist, phorbol 12-myristate 13-acetate (TPA, 100 nM, 4 h), induced two-fold higher protein and mRNA levels of p21Waf1, a cyclin-dependent kinase (cdk) inhibitor in PKC-δ transfectants compared with empty vector (EV) transfected cells, whereas the PKC-δ specific inhibitor rottlerin (3 μ M) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21Waf1 expression. Concomitantly, compared to EV control cells, PKC-δ upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-δ increased binding of cdk inhibitor p27Kip1 to cdk4. These alterations in cyclin-cdks and their inhibitors are predicted to decrease G1 cyclin kinase activity. As an independent confirmation of the direct role PKC-δ plays in cell growth and cell cycle regulation, we knocked down PKC-δ using specific siRNA oligonucleotides. PKC-δ specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKC-δ protein by more than 80% in Caco-2 cells. Moreover, PKC-δ knockdown enhanced cell proliferation (1.4-2-fold, P<0.05) and concomitantly increased cyclin D1 and cyclin E expression (1.7-fold, P<0.05). This was a specific effect, as nontargeted PKC-ζ was not changed by PKC-δ siRNA oligonucleotides. Consistent with accelerated apoptosis in PKC-δ transfectants, compared to EV cells, PKC-δ upregulation increased proapoptotic regulator Bax two-fold at mRNA and protein levels, while antiapoptotic Bcl-2 protein was decreased by 50% at a post-transcriptional level. PKC-δ specific siRNA oligonucleotides inhibited Bax protein expression by more than 50%, indicating that PKC-δ regulates apoptosis through Bax. Taken together, these results elucidate two critical mechanisms regulated by PKC-δ that inhibit cell cycle progression and enhance apoptosis in colon cancer cells. We postulate these antiproliferative pathways mediate an important tumor suppressor function for PKC-δ in colonic carcinogenesis.

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Abbreviations

PKC:

protein kinase C

PKC-δ:

PKC-delta

EV:

empty vector

SDS–PAGE:

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

FACS:

fluorescence-activated cell sorter

BisA:

Bistratene A

TPA:

12-O-tetradecanoylphorbol 13-acetate

IP:

immunoprecipitation

cdk:

cyclin dependent kinase

SS:

scrambled sequence

DAG:

diacylglycerol

PC:

phosphatidylcholine

PI:

phophatidylinositol

PLC:

phospholipase C

RTK:

receptor tyrosine kinase

GPCR:

G protein coupled receptors

References

  • Abraham C, Scaglione-Sewell B, Skarosi SF, Qin W, Bissonnette M, Brasitus TA . (1998). Gastroenterology 114: 503–509.

  • Acevedo-Duncan M, Patel R, Whelan S, Bicaku E . (2002). Cell Prolif 35: 23–36.

  • Akiyama T, Ohuchi T, Sumida S, Matsumoto K, Toyoshima K . (1992). Proc Natl Acad Sci USA 89: 7900–7904.

  • Andre F, Rigot V, Remacle-Bonnet M, Luis J, Pommier G, Marvaldi J . (1999). Gastroenterology 116: 64–77.

  • Arber N, Doki Y, Han EK, Sgambato A, Zhou P, Kim NH et al. (1997). Cancer Res 57: 1569–1574.

  • Arber N, Hibshoosh H, Moss SF, Sutter T, Zhang Y, Begg M et al. (1996). Gastroenterology 110: 669–674.

  • Arita Y, Buffolino P, Coppock DL . (1998). Exp Cell Res 242: 381–390.

  • Ashton AW, Watanabe G, Albanese C, Harrington EO, Ware JA, Pestell RG . (1999). J Biol Chem 274: 20805–20811.

  • Assert R, Kotter R, Bisping G, Scheppach W, Stahlnecker E, Muller KM et al. (1999). Int J Cancer 80: 47–53.

  • Atten MJ, Godoy-Romero E, Attar BM, Milson T, Zopel M, Holian O . (2005). Invest New Drugs 23: 111–119.

  • Avazeri N, Courtot AM, Lefevre B . (2004). J Cell Sci 117: 4969–4978.

  • Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM et al. (1989). Science 244: 217–221.

  • Banan A, Fields JZ, Farhadi A, Talmage DA, Zhang L, Keshavarzian A . (2002). J Pharmacol Exp Ther 303: 17–28.

  • Barrett CM, Lewis FL, Roaten JB, Sweatman TW, Israel M, Cleveland JL et al. (2002). Mol Cancer Ther 1: 469–481.

  • Bartek J, Lukas J . (2001). FEBS Lett 490: 117–122.

  • Bartkova J, Lukas J, Strauss M, Bartek J . (1994). Int J Cancer 58: 568–573.

  • Besson A, Yong VW . (2000). Mol Cell Biol 20: 4580–4590.

  • Black JD . (2000). Front Biosci 5: D406–D423.

  • Black JD . (2001). Gastroenterology 120: 1868–1872.

  • Boise LH, Gottschalk AR, Quintans J, Thompson CB . (1995). Curr Top Microbiol Immunol 200: 107–121.

  • Bosari S, Moneghini L, Graziani D, Lee AK, Murray JJ, Coggi G et al. (1995). Hum Pathol 26: 534–540.

  • Brasitus TA, Bissonnette M . (1998). Gastroenterology 115: 225–227.

  • Brodie C, Blumberg PM . (2003). Apoptosis 8: 19–27.

  • Bronner MP, Culin C, Reed JC, Furth EE . (1995). Am J Pathol 146: 20–26.

  • Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y . (1982). J Biol Chem 257: 7847–7851.

  • Cayrol C, Knibiehler M, Ducommun B . (1998). Oncogene 16: 311–320.

  • Cerda SR, Bissonnette M, Scaglione-Sewell B, Lyons MR, Khare S, Mustafi R et al. (2001). Gastroenterology 120: 1700–1712.

  • Chen A, Davis BH, Bissonnette M, Scaglione-Sewell B, Brasitus TA . (1999). J Biol Chem 274: 35505–35513.

  • Clark JA, Black AR, Leontieva OV, Frey MR, Pysz MA, Kunneva L et al. (2004). J Biol Chem 279: 9233–9247.

  • Craven PA, DeRubertis FR . (1992). Cancer Res 52: 2216–2221.

  • Craven PA, DeRubertis FR . (1994). Dig Dis Sci 39: 481–489.

  • Darzynkiewicz Z . (1994). Cell Biology: A Laboratory Handout. Academic Press: New York, pp 261–271.

    Google Scholar 

  • Davidson LA, Jiang YH, Derr JN, Aukema HM, Lupton JR, Chapkin RS . (1994). Arch Biochem Biophys 312: 547–553.

  • Davies SP, Reddy H, Caivano M, Cohen P . (2000). Biochem J 351: 95–105.

  • Denning MF, Wang Y, Nickoloff BJ, Wrone-Smith T . (1998). J Biol Chem 273: 29995–30002.

  • Di Mari JF, Mifflin RC, Powell DW . (2005). Gastroenterology 128: 2131–2146.

  • Ding QM, Ko TC, Evers BM . (1998). Am J Physiol 275: C1193–C1200.

  • Djelloul S, Forgue-Lafitte ME, Hermelin B, Mareel M, Bruyneel E, Baldi A et al. (1997). FEBS Lett 406: 234–242.

  • Donnellan R, Chetty R . (1999). FASEB J 13: 773–780.

  • Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW et al. (1994). Cell 76: 1013–1023.

  • Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T . (2001). Nature 411: 494–498.

  • el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM et al. (1993). Cell 75: 817–825.

  • Evers BM, Ko TC, Li J, Thompson EA . (1996). Am J Physiol 271: G722–G727.

  • Fearon ER, Vogelstein B . (1990). Cell 61: 759–767.

  • Fima E, Shtutman M, Libros P, Missel A, Shahaf G, Kahana G et al. (2001). Oncogene 20: 6794–6804.

  • Fishman DD, Segal S, Livneh E . (1998). Int J Oncol 12: 181–186.

  • Frey MR, Clark JA, Leontieva O, Uronis JM, Black AR, Black JD . (2000). J Cell Biol 151: 763–778.

  • Frey MR, Saxon ML, Zhao X, Rollins A, Evans SS, Black JD . (1997). J Biol Chem 272: 9424–9435.

  • Fukumoto S, Nishizawa Y, Hosoi M, Koyama H, Yamakawa K, Ohno S et al. (1997). J Biol Chem 272: 13816–13822.

  • Gartel AL, Goufman E, Najmabadi F, Tyner AL . (2000a). Oncogene 19: 5182–5188.

  • Gartel AL, Najmabadi F, Goufman E, Tyner AL . (2000b). Oncogene 19: 961–964.

  • Gartel AL, Serfas MS, Gartel M, Goufman E, Wu GS, el-Deiry WS et al. (1996). Exp Cell Res 227: 171–181.

  • Gavrielides MV, Frijhoff AF, Conti CJ, Kazanietz MG . (2004). Curr Drug Targets 5: 431–443.

  • Geiges D, Marks F, Gschwendt M . (1995). Exp Cell Res 219: 299–303.

  • Goerke A, Sakai N, Gutjahr E, Schlapkohl WA, Mushinski JF, Haller H et al. (2002). J Biol Chem 277: 32054–32062.

  • Goldstein DR, Cacace AM, Weinstein IB . (1995). Carcinogenesis 16: 1121–1126.

  • Graham MA, Rawe I, Dartt DA, Joyce NC . (2000). Invest Ophthalmol Vis Sci 41: 4124–4132.

  • Greenlee RT, Murray T, Bolden S, Wingo PA . (2000). CA Cancer J Clin 50: 7–33.

  • Gschwendt M . (1999). Eur J Biochem 259: 555–564.

  • Gschwendt M, Muller HJ, Kielbassa K, Zang R, Kittstein W, Rincke G et al. (1994). Biochem Biophys Res Commun 199: 93–98.

  • Hosokawa Y, Arnold A . (1998). Genes Chromosomes Cancer 22: 66–71.

  • Irie N, Sakai N, Ueyama T, Kajimoto T, Shirai Y, Saito N . (2002). Biochem Biophys Res Commun 298: 738–743.

  • Jackson DN, Foster DA . (2004). FASEB J 18: 627–636.

  • Jang SJ, Park YW, Park MH, Lee JD, Lee YY, Jung TJ et al. (1999). Eur J Surg Oncol 25: 157–163.

  • Jones SM, Kazlauskas A . (2001). FEBS Lett 490: 110–116.

  • Kahl-Rainer P, Karner-Hanusch J, Weiss W, Marian B . (1994). Carcinogenesis 15: 779–782.

  • Kahl-Rainer P, Sedivy R, Marian B . (1996). Gastroenterology 110: 1753–1759.

  • Khare S, Bissonenette M, Scaglione-Sewell B, Wali RK, Sitrin MD, Brasitus TA . (1999). Am J Physiol 276: G993–G1004.

  • Kikkawa U, Matsuzaki H, Yamamoto T . (2002). J Biochem (Tokyo) 132: 831–839.

  • King RW, Deshaies RJ, Peters JM, Kirschner MW . (1996). Science 274: 1652–1659.

  • Kitamura K, Mizuno K, Etoh A, Akita Y, Miyamoto A, Nakayama K et al. (2003). Genes Cells 8: 311–324.

  • Klein IK, Ritland SR, Burgart LJ, Ziesmer SC, Roche PC, Gendler SJ et al. (2000). Cancer Res 60: 2077–2080.

  • Krajewski S, Krajewska M, Shabaik A, Miyashita T, Wang HG, Reed JC . (1994). Am J Pathol 145: 1323–1336.

  • La Porta CA, Porro D, Comolli R . (2002). Melanoma Res 12: 297–307.

  • Laemmli UK . (1970). Nature 227: 680–685.

  • Lee YJ, Soh JW, Dean NM, Cho CK, Kim TH, Lee SJ et al. (2002). Cell Growth Differ 13: 237–246.

  • Leontieva OV, Black JD . (2004). J Biol Chem 279: 5788–5801.

  • Lewis AE, Susarla R, Wong BC, Langman MJ, Eggo MC . (2005). Cell Signal 17: 253–262.

  • Li W, Jiang YX, Zhang J, Soon L, Flechner L, Kapoor V et al. (1998). Mol Cell Biol 18: 5888–5898.

  • Li W, Michieli P, Alimandi M, Lorenzi MV, Wu Y, Wang LH et al. (1996). Oncogene 13: 731–737.

  • Li W, Zhang J, Bottaro DP, Pierce JH . (1997). J Biol Chem 272: 24550–24555.

  • Lin SY, Liang YC, Ho YS, Tsai SH, Pan S, Lee WS . (2002). Mol Carcinog 35: 21–28.

  • Lipkin M . (1971). Cancer 28: 38–40.

  • Liu JJ, Chao JR, Jiang MC, Ng SY, Yen JJ, Yang-Yen HF . (1995). Mol Cell Biol 15: 3654–3663.

  • Liu WS, Heckman CA . (1998). Cell Signal 10: 529–542.

  • Livneh E, Fishman DD . (1997). Eur J Biochem 248: 1–9.

  • Lu Z, Hornia A, Jiang YW, Zang Q, Ohno S, Foster DA . (1997). Mol Cell Biol 17: 3418–3428.

  • MacLachlan TK, Sang N, Giordano A . (1995). Crit Rev Eukaryot Gene Exp 5: 127–156.

  • Mandal M, Olson DJ, Sharma T, Vadlamudi RK, Kumar R . (2001). Gastroenterology 120: 71–78.

  • McCracken MA, Miraglia LJ, McKay RA, Strobl JS . (2003). Mol Cancer Ther 2: 273–281.

  • McGarrity TJ, Peiffer LP . (1994). Dig Dis Sci 39: 458–463.

  • McMillan L, Butcher SK, Pongracz J, Lord JM . (2003). Br J Cancer 88: 748–753.

  • Meyer E, Vollmer JY, Bovey R, Stamenkovic I . (2005). Cancer Res 65: 4261–4272.

  • Milde-Langosch K, Bamberger AM, Methner C, Rieck G, Loning T . (2000). Int J Cancer 87: 468–472.

  • Moss SF, Liu TC, Petrotos A, Hsu TM, Gold LI, Holt PR . (1996). Gastroenterology 111: 1425–1432.

  • Nabha SM, Glaros S, Hong M, Lykkesfeldt AE, Schiff R, Osborne K et al. (2005). Oncogene 24: 3166–3176.

  • Nakagawa M, Oliva JL, Kothapalli D, Fourier A, Assoian RK, Kazanietz MG . (2005). J Biol Chem 280: 33926–33934.

  • Newton AC . (1997). Curr Opin Cell Biol 9: 161–167.

  • Nishizuka Y . (1995). FASEB J 9: 484–496.

  • Ogura E, Senzaki H, Yamamoto D, Yoshida R, Takada H, Hioki K et al. (1999). Oncol Rep 6: 365–369.

  • Page K, Li J, Corbit KC, Rumilla KM, Soh JW, Weinstein IB et al. (2002). Am J Respir Cell Mol Biol 27: 204–213.

  • Palmiter RD . (1994). Proc Natl Acad Sci USA 91: 1219–1223.

  • Parekh D, Ziegler W, Yonezawa K, Hara K, Parker PJ . (1999). J Biol Chem 274: 34758–34764.

  • Perletti G, Marras E, Dondi D, Osti D, Congiu T, Ferrarese R et al. (2005). Int J Cancer 113: 42–53.

  • Perletti G, Marras E, Osti D, Felici L, Zaro S, de Eguileor M . (2004). J Cell Mol Med 8: 563–569.

  • Perletti GP, Concari P, Brusaferri S, Marras E, Piccinini F, Tashjian Jr AH . (1998). Oncogene 16: 3345–3348.

  • Perletti GP, Marras E, Concari P, Piccinini F, Tashjian Jr AH . (1999). Oncogene 18: 1251–1256.

  • Pinto M, Robine-Leon S, Appay M-D, Kedinger M, Triadou N, Dussaulx E et al. (1983). Biol Cell 47: 323–330.

  • Pongracz J, Webb P, Wang K, Deacon E, Lunn OJ, Lord JM . (1999). J Biol Chem 274: 37329–37334.

  • Polyak K, Hamilton SR, Vogelstein B, Kinzler KW . (1996). Am J Pathol 149: 381–387.

  • Potten CS, Wilson JW, Booth C . (1997). Stem Cells 15: 82–93.

  • Qiao L, Koutsos M, Tsai LL, Kozoni V, Guzman J, Shiff SJ et al. (1996). Cancer Lett 107: 83–89.

  • Ruemmele FM, Dionne S, Qureshi I, Sarma DS, Levy E, Seidman EG . (1999). Cell Death Differ 6: 729–735.

  • Rybin VO, Sabri A, Short J, Braz JC, Molkentin JD, Steinberg SF . (2003). J Biol Chem 278: 14555–14564.

  • Ryves WJ, Evans AT, Olivier AR, Parker PJ, Evans FJ . (1991). FEBS Lett 288: 5–9.

  • Santiago-Walker AE, Fikaris AJ, Kao GD, Brown EJ, Kazanietz MG, Meinkoth JL . (2005). J Biol Chem 280: 32107–32114.

  • Sauma S, Yan Z, Ohno S, Friedman E . (1996). Cell Growth Differ 7: 587–594.

  • Savickiene J, Gineitis A, Stigbrand T . (1999). Cell Death Differ 6: 698–709.

  • Saxon ML, Zhao X, Black JD . (1994). J Cell Biol 126: 747–763.

  • Scaglione-Sewell B, Abraham C, Bissonnette M, Skarosi SF, Davidson NO, Wali RK et al. (1998). Cancer Res 58: 1074–1081.

  • Scaglione-Sewell BA, Bissonnette M, Skarosi S, Abraham C, Brasitus TA . (2000). Endocrinology 141: 3931–3939.

  • Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW . (2000). Anal Biochem 285: 194–204.

  • Seki T, Matsubayashi H, Amano T, Shirai Y, Saito N, Sakai N . (2005). Genes Cells 10: 225–239.

  • Shanmugam M, Krett NL, Maizels ET, Murad FM, Rosen ST, Hunzicker-Dunn M . (2001). Cancer Lett 172: 43–53.

  • Shanmugathasan M, Jothy S . (2000). Pathol Int 50: 273–279.

  • Sherr CJ . (1996). Science 274: 1672–1677.

  • Sherr CJ, Roberts JM . (1999). Genes Dev 13: 1501–1512.

  • Sitailo LA, Tibudan SS, Denning MF . (2004). J Invest Dermatol 123: 434–443.

  • Soh JW, Weinstein IB . (2003). J Biol Chem 278: 34709–34716.

  • Srivastava J, Procyk KJ, Iturrioz X, Parker PJ . (2002). Biochem J 368: 349–355.

  • Steinberg SF . (2004). Biochem J 384: 449–459.

  • Thompson CB . (1995). Science 267: 1456–1462.

  • Tillman DM, Izeradjene K, Szucs KS, Douglas L, Houghton JA . (2003). Cancer Res 63: 5118–5125.

  • Toyoda M, Gotoh N, Handa H, Shibuya M . (1998). Biochem Biophys Res Commun 250: 430–435.

  • Umar S, Sellin JH, Morris AP . (2000). Am J Physiol Gastrointest Liver Physiol 278: G765–G774.

  • Verstovsek G, Byrd A, Frey MR, Petrelli NJ, Black JD . (1998). Gastroenterology 115: 75–85.

  • Villalba M . (1998). Neuroreport 9: 2381–2385.

  • Wakino S, Kintscher U, Liu Z, Kim S, Yin F, Ohba M et al. (2001). J Biol Chem 276: 47650–47657.

  • Wali RK, Frawley Jr BP, Hartmann S, Roy HK, Khare S, Scaglione-Sewell BA et al. (1995). Cancer Res 55: 5257–5264.

  • Wang Q, Wang X, Evers BM . (2003). J Biol Chem 278: 51091–51099.

  • Wang Q, Wang X, Zhou Y, Evers BM . (2006). Int J Cancer 118: 326–334.

  • Wang X, Wang Q, Hu W, Evers BM . (2004). Oncogene 23: 1885–1895.

  • Watters D, Garrone B, Coomer J, Johnson WE, Brown G, Parsons P . (1998). Biochem Pharmacol 55: 1691–1699.

  • Weinstein IB . (1991). Princess Takamatsu Symp 22: 277–283.

  • Weller SG, Klein IK, Penington RC, Karnes Jr WE . (1999). Gastroenterology 117: 848–857.

  • Wetsel WC, Khan WA, Merchenthaler I, Rivera H, Halpern AE, Phung HM et al. (1992). J Cell Biol 117: 121–133.

  • Yin L, Bennani-Baiti N, Powell CT . (2005). J Biol Chem 280: 5533–5541.

  • Yokoyama G, Fujii T, Tayama K, Yamana H, Kuwano M, Shirouzu K . (2005). Biochem Biophys Res Commun 327: 720–726.

  • Zang Q, Lu Z, Curto M, Barile N, Shalloway D, Foster DA . (1997). J Biol Chem 272: 13275–13280.

  • Zeng YX, el-Deiry WS . (1996). Oncogene 12: 1557–1564.

  • Zezula J, Sexl V, Hutter C, Karel A, Schutz W, Freissmuth M . (1997). J Biol Chem 272: 29967–29974.

  • Zhu GH, Wong BC, Slosberg ED, Eggo MC, Ching CK, Yuen ST et al. (2000). Gastroenterology 118: 507–514.

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Acknowledgements

This research was supported in part by National Institutes of Health Grant 2R01CA36745 (MB), the American Cancer Society Illinois Division Grant 04-14 (SRC), and the Digestive Disease Research Core Center Grant P30DK42086 at the University of Chicago.

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Cerda, S., Mustafi, R., Little, H. et al. Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators. Oncogene 25, 3123–3138 (2006). https://doi.org/10.1038/sj.onc.1209360

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