Abstract
Caspases play important roles in the initiation and progression of apoptosis. In experimental models of ATP depletion, we have demonstrated the activation of caspase-9, -8, and -3, which is followed by the development of apoptotic morphology. To determine the specific contribution of caspase-9 to ATP depletion-induced apoptosis, we transfected renal epithelial cells with its endogenous dominant-negative inhibitor caspase-9S. Two cell clones with stable transfection were obtained. These clones expressed caspase-9S, and the cytosol isolated from these cells was resistant to cytochrome c-induced caspase activation in vitro. The clones were then examined for ATP depletion-induced apoptosis. Compared with the wild-type cells, the caspase-9S clones were markedly resistant to apoptosis in this model. Caspase activation was also inhibited. Surprisingly, these clones also showed significantly less cytochrome c release during ATP-depletion. Moreover, Bax translocation to mitochondria was inhibited, suggesting that these clones were resistant to apoptosis not only at the cytosolic caspase activation level but also at the upstream mitochondrial level. To gain insights into the mitochondrial resistance, we analyzed the expression of Bcl-2 family proteins. While the expression of Bax, Bak, and Bcl-2 was comparable to the wild-type cells, the selected clones showed specific up-regulation of Bcl-XL, an anti-apoptotic protein. We conclude that the selected clones were resistant to apoptosis at two levels. In the cytosol, they expressed dominant negative caspase-9, and at the mitochondria they up-regulated Bcl-XL.
Similar content being viewed by others
References
Danial NN, Korsmeyer SJ: Cell death: critical control points. Cell 116: 205–219, 2004
Green DR: Apoptotic pathways: the roads to ruin. Cell 94: 695–698, 1998
Ashkenazi A, Dixit VM: Death receptors: signaling and modulation. Science 281: 1305–1308, 1998
Budihardjo I, Oliver H, Lutter M, Luo X, Wang X: Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15: 269–290, 1999
Kaushal GP, Basnakian AG, Shah SV: Apoptotic pathways in ischemic acute renal failure. Kidney Int 66: 500–506, 2004
Padanilam BJ: Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Am J Physiol 284: F608–F627, 2003
Kelly KJ, Plotkin Z, Dagher PC: Guanosine supplementation reduces apoptosis and protects renal function in the setting of ischemic injury. J Clin Invest 108: 1291–1298, 2001
Schumer M, Colombel MC, Sawczuk IS, Gobe G, Connor J, O'Toole KM, Olsson CA, Wise GJ, Buttyan R: Morphologic, biochemical, and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol 140: 831–838, 1992
Kaushal GP, Singh AB, Shah SV: Identification of gene family of caspases in rat kidney and altered expression in ischemia-reperfusion injury. Am J Physiol 274: F587–F595, 1998
Basnakian AG, Ueda N, Kaushal GP, Mikhailova MV, Shah SV: DNase I-like endonuclease in rat kidney cortex that is activated during ischemia/reperfusion injury. J Am Soc Nephrol 13: 1000–1007, 2002
Daemen MA, van't Veer C, Denecker G, Heemskerk VH, Wolfs TG, Clauss M, Vandenabeele P, Buurman WA: Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J Clin Invest 104: 541–549, 1999
Kelly KJ, Sutton TA, Weathered N, Ray N, Caldwell EJ, Plotkin Z, Dagher PC: Minocycline inhibits apoptosis and inflammation in a rat model of ischemic renal injury. Am J Physiol 287: F760–F766, 2004
Wang J, Wei Q, Wang CY, Hill WD, Hess DC, Dong Z: Minocycline up-regulates Bcl-2 and protects against cell death in mitochondria. J Biol Chem 279: 19948–19954, 2004
Saikumar P, Dong Z, Patel Y, Hall K, Hopfer U, Weinberg JM, Venkatachalam MA: Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene 17: 3401–3415, 1998
Dong Z, Saikumar P, Patel Y, Weinberg JM, Venkatachalam MA: Serine protease inhibitors suppress cytochrome c-mediatedcaspase-9 activation and apoptosis during hypoxia-reoxygenation. Biochem J 347 Pt 3: 669–677, 2000
Dong Z, Wang J: Hypoxia selection of death-resistant cells. A role for Bcl-X(L). J Biol Chem 279: 9215–9221, 2004
Seol DW, Billiar TR: A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis. J Biol Chem 274: 2072–2076, 1999
Srinivasula SM, Ahmad M, Guo Y, Zhan Y, Lazebnik Y, Fernandes-Alnemri T, Alnemri ES: Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis. Cancer Res 59: 999–1002, 1999
Woost PG, Orosz DE, Jin W, Frisa PS, Jacobberger JW, Douglas JG, Hopfer U: Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats. Kidney Int 50: 125–134, 1996
Dong Z, Wang J, Zhong Q: Postmitochondrial regulation of apoptosis by bicarbonate. Exp Cell Res 288: 301–312, 2003
Wei Q, Alam MM, Wang MH, Yu F, Dong Z: Bid activation in kidney cells following ATP depletion in vitro and ischemia in vivo. Am J Physiol 286: F803–F809, 2004
Jiang M, Yi X, Hsu S, Wang CY, Dong Z: Role of p53 in cisplatin-induced tubular cell apoptosis: Dependence on p53 transcriptional activity. Am J Physiol 287: F1140–F1147, 2004
Charalampopoulos I, Tsatsanis C, Dermitzaki E, Alexaki VI, Castanas E, Margioris AN, Gravanis A: Dehydroepiandrosterone and allopregnanolone protect sympathoadrenal medulla cells against apoptosis via antiapoptotic Bcl-2 proteins. Proc Natl Acad Sci USA 101: 8209–8214, 2004
Wei Q, Wang J, Wang MH, Yu F, Dong Z: Inhibition of Apoptosis by Zn2+ in Renal Tubular Cells Following ATP-depletion. Am J Physiol 287: F492–F500, 2004
Dong Z, Wang JZ, Yu F, Venkatachalam MA: Apoptosis-resistance of hypoxic cells: Multiple factors involved and a role for IAP-2. Am J Pathol 163: 663–671, 2003
Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, Green DR: Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process. J Cell Biol 153: 319–328, 2001
Nomura M, Shimizu S, Sugiyama T, Narita M, Ito T, Matsuda H, Tsujimoto Y: 14-3-3 Interacts directly with and negatively regulates pro-apoptotic Bax. J Biol Chem 278: 2058–2065, 2003
Nishiyama J, Yi X, Venkatachalam MA, Dong Z: cDNA cloning and promoter analysis of rat caspase-9. Biochem J 360: 49–56, 2001
Dong Z, Patel Y, Saikumar P, Weinberg JM, Venkatachalam MA: Development of porous defects in plasma membranes of adenosine triphosphate-depleted Madin-Darby canine kidney cells and its inhibition by glycine. Lab Invest 78: 657–668, 1998
Adams JM, Cory S: The Bcl-2 protein family: Arbiters of cell survival. Science 281: 1322–1326, 1998
Mikhailov V, Mikhailova M, Pulkrabek DJ, Dong Z, Venkatachalam MA, Saikumar P: Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane. J Biol Chem 276: 18361–18374, 2001
Clem RJ, Cheng EH, Karp CL, Kirsch DG, Ueno K, Takahashi A, Kastan MB, Griffin DE, Earnshaw WC, Veliuona MA, Hardwick JM: Modulation of cell death by Bcl-XL through caspase interaction. Proc Natl Acad Sci USA 95: 554–559, 1998
Fujita N, Nagahashi A, Nagashima K, Rokudai S, Tsuruo T: Acceleration of apoptotic cell death after the cleavage of Bcl-XL protein by caspase-3-like proteases. Oncogene 17: 1295–1304, 1998
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Yi, X., Wang, J., Seol, DW. et al. Characterization of cell clones stably transfected with short form caspase-9: Apoptotic resistance and Bcl-XL expression. Mol Cell Biochem 282, 1–12 (2006). https://doi.org/10.1007/s11010-006-1089-0
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11010-006-1089-0