The effect of an HIV-1 viral protease inhibitor on staurosporine-induced apoptosis in immortalized mesangial cells
Keywords:
Immortalized mesangial cell, Progressive glomerular sclerosis, ApoptosisAbstract
CONTEXT: Progressive glomerular sclerosis is a condition characterized by the accumulation of glomerular extracellular matrix and a decrease in the number of glomerular cells. The mechanisms involved in the progressive loss of glomerular cells are not well understood but may involve the process of apoptosis. The principal mediators for the apoptotic pathway are a class of protease enzymes called caspases. It is not known how other therapeutic protease inhibitors affect the caspase cascade and therefore whether they would be effective in preventing excessive apoptosis in the late stages of progressive glomerular sclerosis. OBJECTIVE: To evaluate whether an inhibitor of the HIV1 viral protease Ac-Leu-Val-phenylalanine (PI) could inhibit apoptosis in immortalized mesangial cells. DESIGN: Experimental. SETTING: Nephrology Division, Universidade Federal de São Paulo/Escola Paulista de Medicina. PARTICIPANTS: Immortalized mesangial cells. PROCEDURES: Cell culture. MAIN MEASUREMENTS: Viability and rate of apoptosis. RESULTS: Immortalized mesangial cells were treated with staurosporine (at concentrations of 10-100 nM for 8-28 hours) to induce apoptosis. Staurosporine at 10 nM for 8 hours had no effect on viability, but did cause a significant increase in the rate of apoptosis (p = 0.0411, n = 6). Increasing the incubation time elicited a greater increase in the rate of apoptosis (p = 0.0001, n = 6), although there was also a significant decrease in viability (p=0.0002). Increasing the concentration of staurosporine to 100 nM resulted in a marked increase in apoptosis (p <0.0001) but resulted in unacceptable viability (<40%, p <0.0001, n = 6). CONCLUSIONS: Incubation of immortalized mesangial cells with PI (900 nM) alone for 2-24 hours had no effect on cell viability or the rate of apoptosis when compared with vehicle (methanol) controls. Coincubation of the cells with staurosporine (10 nM) and PI for 24 hours had no significant effect on the rate of apoptosis. Therefore, in immortalized mesangial cells, staurosporine-induced apoptosis was not significantly affected by the HIV-1 viral protease inhibitor Ac-Leu-Val-phenylalanine.
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References
Sugiyama H, Kashihara N, Makino H,YamasakiY, Ota Z. Apoptosis in glomerular sclerosis. Kidney International 1996;49:103-11.
Wang W, Tzanidis A, Divjak M, Thomson NM, Stein-Oakley AN. Altered signaling and regulatory mechanisms of apoptosis in focal and segmental glomerulosclerosis Journal of the American Society of Nephrology 2001;12(7):1422-33.
Daha MR. Mechanisms of mesangial injury in glomerular diseases. Journal of Nephrology 2000;3:89-95.
Haas CS, Schocklmann HO, Lang S, Krelewski M, Sterzel, RB. Regulatory mechanism in glomerular mesangial cell proliferation. Journal of Nephrology 1999;12(6):405-15.
Ortiz A. Nephrology forum: apoptotic regulatory proteins in renal injury. Kidney International 2000;58(1):467-85.
Savill J. Apoptosis in post-streptococcal glomerulonephritis. Kidney International 2001;60(3):1203-14.
Thompson EB. Special topic: apoptosis. Annual Reviews in Physiology 1998;60:525-32.
Thornberry NA, Lazenbrik Y. Caspases: enemies within. Science 1998;281:1312-16.
Amstad PA, Yu G, Johnson GL, Lee BW, Dhawan S, Phelps DJ. Detection of caspases activation in situ fluorochrome- labeled caspase inhibitors. Biotechniques 2001;31(3):608-10.
Pong K, Doctrow SR, Huffman K, Adinolf CA, Baudry M. Attenuation of staurosporine-induced apoptosis, oxidative stress, and mitochondrial dysfunction by synthetic superoxide dismutase and catalase mimetics, in cultured cortical neurons. Experimental Neurology 2001;171(1):84-97.
Gorman A, McCarthy J, Finucane D, Reville W, Cotter TG. Morphological assessment of apoptosis. In: Cotter TG, Martin SJ, editors. Techniques in apoptosis: a user’s guide. London: Portland Press; 1996:1-20.
Caponigro F, French RC, Kaye SB. Protein kinase C: a worthwhile target for anticancer drugs? Anticancer Drugs 1997;8:26-33.
Tee AR, Proud CG. Staurosporine inhibits phosphorylation of translational regulators linked to m TOR. Cell Death Differentiation 2001;8(8):841-9.
Fujita E, Egashira J, Urase K, Kuida K, Momoi T. Caspase-9 processing by caspase-3 via a feedback amplification loop in vivo. Cell Death Differentiation 2001;8(4):335-44.
