IOU/flu 1 of Nc‘uro«heinisIr~ Lippincott—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry Characterization of CPP3 2-Like Protease Activity Following Apoptotic Challenge in SH-SY5Y Neuroblastoma Cells *tRand Posmantur, tKim McGinnis, tRavi Nadimpalli, *Rjchard B. Gilbertsen, and lHKevin K. W. Wang Departments of *Immunopathologv and ~Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research, Warner-Lanibert Company, Ann Arbor, Michigan, U.S.A Abstract: We characterized the activation of interleukin- 1/3-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following chal- lenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehy- drogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h fol lowing staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PAAP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate ace- tyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after stauro- sporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The local- ization of CPP32 in untreated SH-SY5Y cells was exclu- sively restricted to the cytoplasm. Following stauro- sporine challenge there was a condensing of CPP32 im- munofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PAAP immunofluorescence was evenly distributed in the nu- cleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. lt is important that a pan ICE inhibitor [carbo- benzoxy-Asp-CH 2OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following stauro- sporine challenge. Key Words: Interleukin-1/3-convert- ing enzyme-like proteases—H uman SH-SY5Y neu- roblastoma cells—Apoptosis—CPP32—Poly(ADP- ribose) polymerase. J. Neurochem. 68, 2328—2337 (1997). Apoptosis (programmed cell death) is a physiologi- cal process involved in the elimination of excess neu- rons during nervous system development and matura- tion (Homma et al., 1994; Zamenhof and Guthrie, 1995; Yaginuma et al., 1996). Apoptotic cell death has been associated with several hallmark morphological changes, including condensation of the nucleus, frag- mentation of chromatin, membrane blebbing, and the formation of apoptotic bodies (Kerr and Harmon, 1991; Gavrieli et al., 1992; Brown et al., 1993; Martin et al., 1995). Recently, the involvement of unregulated apoptosis has been implicated in several neurological disorders, including cerebral ischemia (Linnik et al., 1993; MacManus et al., 1993; Hill et al., 1995; Koh et al., 1995), Huntington‘s disease (Portera-Cailliau et al., 1995; Zeitlin et al., 1995; Goldberg et al., 1996), Alzheimer‘s disease (Nixon et al., 1994; Satou et al., 1995; Gschwind et al., 1996; Vito et al.. 1996; Yamat- suji et al., 1996), amyotrophic lateral sclerosis (Troost et al., 1995; Marx et al., 1996), and Parkinson‘s dis- ease (Ziv et al., 1994; Walkinshaw and Waters, 1995). A new class of proteases has been implicated in apoptotic cell death belonging to the interleukin-l/3- converting enzyme (ICE)/CED-3 or caspase family of cysteine proteases. These consist of at least 10 different homologues, including ICE, CPP32, lch-l (Nedd2), lch-2, lCE~111, Mch-2, Mch-3, Mch-4, Mch-5, and Mch-6 (for reviews, see Alnemri et al., 1996; Fraser and Evan, 1996; Vaux and Strasser, 1996). The overall common feature of this family of proteases is the con- servation of the active site QACXG (where X is R, Q, or G) pentapeptide to induce apoptosis and the absolute requirement for proteolytic cleavage of proen- zymes and substrates at a conserved aspartate residue Received January 8, 1997; revised manuscript received February 12, 1997; accepted February 13, 1997. Address correspondence and reprint requests to Dr. R. Posmantur at Department of Neuroscience Therapeutics. Parke-Davis Pharma- ceutical Research, Warner-Lambert Company. 2800 Plymouth Road, Ann Arbor, Ml 48105. U.S.A. Abbreviations used: Ac-DEVD-MCA, acetyl-DEVD-7-amido-4- methylcoumarin; Ac-YVAD-MCA. acctyl-YVAD-7-amido-4-meth- ylcoumarin; ICE, interleukin- I ß-converting enzyme: LDH, lactate dehydrogenase; PAGE, polyacrylamide gel electrophoresis; PARP. poly( ADP-ribose) polymerase; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate: Z-D-DCB, carbobenzoxy-Asp- CH2OC (0) -2.6-dichlorobenzene. 2328