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Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells

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Abstract

Protein phosphorylation plays an indispensable role in cellular regulation of mitosis, metabolism, differentiation, and death. We previously reported that the protein phosphatase inhibitor okadaic acid (OKA) induces apoptosis in renal epithelial cells in culture. In the present study, we examined the role of phosphotidylinositol 3 (PI3) kinase signaling in okadaic acid-induced apoptosis by pre-treating normal rat kidney renal epithelial cells expressing human bcl-2 with the PI3 kinase inhibitors, LY294002 and wortmannin, followed by apoptosis-inducing concentrations of okadaic acid. Given the reported cell survival activity of PI3 kinase signaling mostly attributed to Akt kinase activation, we hypothesized that inhibition of PI3 kinase would enhance okadaic-induced apoptosis. Surprisingly, our data show that pretreatment with LY294002, but not wortmannin, attenuated okadaic acid-induced apoptosis. In contrast, to LY294002, wortmannin enhanced apoptosis. Interestingly, we also found that LY294002 treatment increased bcl-2 protein levels in normal rat kidney epithelial cells expressing bcl-2 (NRK-bcl-2). In untreated cells, bcl-2 appeared to be mainly perinuclear, coincident with the nuclear membrane, or in the cytosol. In OKA treated cells that were pre-treated with Ly294002, bcl-2 was highly co-localized with mitochondria, but in cells treated with okadaic acid alone, bcl-2 was associated with fragmented chromatin. In this model, it appears that LY294002 may exert anti-apoptotic effects by a previously unreported treatment related increase in bcl-2. Although it is widely accepted that bcl-2 protein can inhibit apoptosis, we propose that the subcellular location of bcl-2 is an important determinant in whether bcl-2 effectively inhibits apoptosis.

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References

  1. Franke TF, Kaplan DR, Cantley LC PI3K: Downstream AKTion blocks apoptosis, Cell 1997; 88: 435–437.

    Google Scholar 

  2. Rondinone CM, Zarnowski MJ, Londos C, Smith UP The inhibitory effect of staurosporine on insulin action is prevented by okadaic acid. Evidence for an important role of serine/threonine phosphorylation in eliciting insulin-like effects. Biochim Biophys Acta 1996; 1314: 49–56.

    Google Scholar 

  3. Rondinone CM, Smith U Okadaic acid exerts a full insulin-like effect on glucose transport and glucose transporter 4 translocation in human adipocytes. Evidence for a phosphatidylinositol 3-kinase-independent pathway. J Biol Chem 1996; 271: 18148–18153.

    Google Scholar 

  4. Syed NA, Khandelwal RL Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells [In Process Citation]. Mol Cell Biochem 2000; 211: 123–136.

    Google Scholar 

  5. Schubert KM, Scheid MP, Duronio V Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473. J Biol Chem 2000; 275: 13330–13335.

    Google Scholar 

  6. Chen D, Fucini RV, Olson AL, Hemmings BA, Pessin JE Osmotic shock inhibits insulin signaling by maintaining Akt/protein kinase B in an inactive dephosphorylated state. Mol Cell Biol 1999; 19: 4684–4894.

    Google Scholar 

  7. Wymann MP, Bulgarelli-Leva G, Zvelebil MJ, et al Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cell Biol 1996; 16: 1722–1733.

    Google Scholar 

  8. Yatomi Y, Hazeki O, Kume S, Ui M Suppression by wortmannin of platelet responses to stimuli due to inhibition of pleckstrin phosphorylation. Biochem J 1992; 285: 745–751.

    Google Scholar 

  9. Barker SA, Caldwell KK, Hall A, et al Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking. Mol Biol Cell 1995; 6: 1145–1158.

    Google Scholar 

  10. Vlahos CJ, Matter WF, Hui KY, Brown RF A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994; 269:5241–5248.

    Google Scholar 

  11. de Larco JE, Todaro GJ Epithelioid and fibroblastic rat kidney cell clones: Epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation, J Cell Physiol 1978; 94: 335–342.

    Google Scholar 

  12. Davis MA, Smith MW, Chang SH, Trump BF Characterization of a renal epithelial cell model of apoptosis using okadaic acid and the NRK-52E cell line. Toxicol Pathol 1994; 22: 595–605.

    Google Scholar 

  13. Davis MA, Chang SH, Trump BF Differential sensitivity of normal and H-ras oncogene-transformed rat kidney epithelial cells to okadaic acid-induced apoptosis.Toxicol Appl Pharmacol 1996; 141: 93–101.

    Google Scholar 

  14. Chan Y, Walmsley RP Learning and understanding the Kruskal-Wallis one-way analysis-of-variance-by-ranks test for differences among three or more independent groups. Phys Ther 1997; 77: 1755–1762. Erratum Phys Ther 1998; 78(3): 322.

    Google Scholar 

  15. Theodorsson-Norheim E. Kruskal-Wallis test: BASIC computer program to perform nonparametric one-way analysis of variance and multiple comparisons on ranks of several independent samples. Comput Methods Programs Biomed 1986; 23: 57–62.

    Google Scholar 

  16. Blagosklonny MV, Schulte T, Nguyen P, Trepel J, Neckers LM Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. Cancer Res 1996; 56: 1851–1854.

    Google Scholar 

  17. Liu Y, Bhalla K, Hill C, Priest DG Evidence for involvement of tyrosine phosphorylation in taxol-induced apoptosis in a human ovarian tumor cell line. Biochem Pharmacol 1994; 48: 1265–1272.

    Google Scholar 

  18. Ishikawa Y, Sugiyama H, Stylianou E, Kitamura M Bioflavonoid quercetin inhibits interleukin-1-induced transcriptional expression of monocyte chemoattractant protein-1 in glomerular cells via suppression of nuclear factor-kappaB. J Am Soc Nephrol 1999; 10: 2290–2296.

    Google Scholar 

  19. Ishikawa Y, Kitamura M Bioflavonoid quercetin inhibits mitosis and apoptosis of glomerular cells in vitro and in vivo. Biochem Biophys Res Commun 2000; 279: 629–634.

    Google Scholar 

  20. Ishikawa Y, Kitamura M Anti-apoptotic effect of quercetin: Intervention in the JNK-and ERK-mediated apoptotic pathways. Kidney Int. 2000; 58: 1078–1087.

    Google Scholar 

  21. Yokoo T, Kitamura M Unexpected protection of glomerular mesangial cells from oxidant-triggered apoptosis by bioflavonoid quercetin. Am J Physiol 1997; 273: F206-F212.

    Google Scholar 

  22. Srivastava AK, Chiasson JL Effect of quercetin on serine/ threonine and tyrosine protein kinases. Prog Clin Biol Res 1986; 213: 315–318.

    Google Scholar 

  23. Srivastava AK, Inhibition of phosphorylase kinase, and tyrosine protein kinase activities by quercetin. Biochem Biophys Res Commun 1985; 131: 1–5.

    Google Scholar 

  24. Walker HE, Pacold EM, Perisic O, et al Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell 2000; 6: 909–919.

    Google Scholar 

  25. Pugazhenthi S, Miller E, Sable C, et al Insulin-like growth factor-I induces bcl-2 promoter through the transcription factor cAMP-response element-binding protein. J Biol Chem 1999; 274: 27529–27535.

    Google Scholar 

  26. Schandl CA, Li S, Re GG, Fan W, Willingham MC Mitotic chromosomal bcl-2. II. Localization to interphase nuclei. J Histochem Cytochem 1999; 47: 151–158.

    Google Scholar 

  27. Schandl CA, Li S, Re GG, Fan W, Willingham MC Mitotic chromosomal bcl-2. I. Stable expression throughout the cell cycle and association with isolated chromosomes. J Histochem Cytochem 1999; 47: 139–149.

    Google Scholar 

  28. Willingham MC, Bhalla K Transient mitotic phase localization of bcl-2 oncoprotein in human carcinoma cells and its possible role in prevention of apoptosis. J Histochem Cytochem 1994; 42: 441–450.

    Google Scholar 

  29. Collado M, Medema RH, Garcia-Cao I, et al Inhibition of the phosphoinositide 3-kinase pathway induces a senescence-like arrest mediated by p27Kip1. J Biol Chem 2000; 275: 21960–21968.

    Google Scholar 

  30. Tresini M, Mawal-Dewan M, Cristofalo VJ, Sell C A phosphatidylinositol 3-kinase inhibitor induces a senescent-like growth arrest in human diploid fibroblasts [see comments]. Cancer Res 1998; 58: 1–4.

    Google Scholar 

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Authors and Affiliations

  1. Department of Pathology, University of Maryland, School of Medicine, Baltimore, MD, 21201, USA

    D. E. Carbott, L. Duan & M. A. Davis

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  1. D. E. Carbott
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  2. L. Duan
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  3. M. A. Davis
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Carbott, D.E., Duan, L. & Davis, M.A. Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells. Apoptosis 7, 69–76 (2002). https://doi.org/10.1023/A:1013517013069

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