Title: Caspase-3 Is Required for α-Fodrin Cleavage but Dispensable for Cleavage of Other Death Substrates in Apoptosis
Abstract: Although the commonly activated death protease caspase-3 appears not to be essential for apoptosis during development except in the brain, it was not shown whether substrates known to be cleaved by caspase-3 are still proteolyzed in its absence. We have addressed this question with MCF-7 breast carcinoma cells that we recently showed lack caspase-3 owing to the functional deletion of theCASP-3 gene. Tumor necrosis factor- or staurosporine-induced apoptosis of caspase-3-deficient MCF-7 cells resulted in cleavage of the death substrates PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45, but not α-fodrin. In contrast, all these substrates including α-fodrin were cleaved in apoptotic HeLa cells expressing caspase-3. Introduction ofCASP-3 cDNA, but not CASP-10 cDNA, into MCF-7 cells restored α-fodrin cleavage. In addition, tumor necrosis factor- or staurosporine-induced apoptosis of MCF-7 cells stably expressing pro-caspase-3 also resulted in α-fodrin cleavage. Although the specific caspase inhibitory peptides Z-VAD-fmk and Z-DEVD-fmk prevented apoptosis of MCF-7 cells, we were unable to detect activation of caspases 2 and 7, which are known to be inhibited by Z-DEVD-fmk. Together our results suggest that caspase-3 is essential for cleavage of α-fodrin, but dispensable for the cleavage of PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45 and imply that one or more caspases other than caspases 2, 3, and 7 is activated and plays a crucial role in the cleavage of these substrates in MCF-7 cells. Although the commonly activated death protease caspase-3 appears not to be essential for apoptosis during development except in the brain, it was not shown whether substrates known to be cleaved by caspase-3 are still proteolyzed in its absence. We have addressed this question with MCF-7 breast carcinoma cells that we recently showed lack caspase-3 owing to the functional deletion of theCASP-3 gene. Tumor necrosis factor- or staurosporine-induced apoptosis of caspase-3-deficient MCF-7 cells resulted in cleavage of the death substrates PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45, but not α-fodrin. In contrast, all these substrates including α-fodrin were cleaved in apoptotic HeLa cells expressing caspase-3. Introduction ofCASP-3 cDNA, but not CASP-10 cDNA, into MCF-7 cells restored α-fodrin cleavage. In addition, tumor necrosis factor- or staurosporine-induced apoptosis of MCF-7 cells stably expressing pro-caspase-3 also resulted in α-fodrin cleavage. Although the specific caspase inhibitory peptides Z-VAD-fmk and Z-DEVD-fmk prevented apoptosis of MCF-7 cells, we were unable to detect activation of caspases 2 and 7, which are known to be inhibited by Z-DEVD-fmk. Together our results suggest that caspase-3 is essential for cleavage of α-fodrin, but dispensable for the cleavage of PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45 and imply that one or more caspases other than caspases 2, 3, and 7 is activated and plays a crucial role in the cleavage of these substrates in MCF-7 cells. Programmed cell death (apoptosis) is an essential mechanism for controlling cell numbers in metazoan organisms (1Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2428) Google Scholar). Among the many known effectors and regulators of apoptosis, a distinct class of aspartyl proteases (ICE 1The abbreviations used are: ICE, interleukin 1β-converting enzyme; TNF, tumor necrosis factor; DED, death effector domains; PARP, poly(ADP-ribose) polymerase; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; PKC, protein kinase C; Chx, cycloheximide; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 1,4-piperazinediethanesulfonic acid; pNA, p-nitroanilide; Rb, retinoblastoma protein. -like proteases or caspases) stands out as being crucial for apoptosis in almost all cell types examined (2Nicholson D.W. Nature Biotechnol. 1996; 14: 297-301Crossref PubMed Scopus (239) Google Scholar, 3Kumar S. Lavin M.F. Cell Death Differ. 1996; 3: 255-267PubMed Google Scholar, 4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2201) Google Scholar). Caspases are present as inactive zymogens containing an N-terminal prodomain and large and small catalytic subunits. They are activated either by autocatalytic processing and/or cleavage by other caspases at internal Asp residues following a variety of death stimuli including tumor necrosis factor (TNF), Fas ligand, staurosporine, or etoposide. Ten distinct caspases have been identified that can be grouped into three subfamilies based on their substrate specificities (4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2201) Google Scholar, 5Talanian R.V. Quinlan C. Trautz S. Hackett M.C. Mankovich J.A. Banach D. Ghayur T. Brady K.D. Wong W.W. J. Biol. Chem. 1997; 272: 9677-9682Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar, 6Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1872) Google Scholar). Group I caspases (caspases 1, 4, and 5) prefer the tetrapeptide sequence WEHD and are believed to play a role mainly in inflammation, whereas members of group II (caspases 2, 3, and 7) and group III (caspases 6, 8, 9, and 10) with the optimal peptide recognition motifs DExD and (I/L/V)ExD, respectively, are mainly involved in apoptosis (4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2201) Google Scholar, 5Talanian R.V. Quinlan C. Trautz S. Hackett M.C. Mankovich J.A. Banach D. Ghayur T. Brady K.D. Wong W.W. J. Biol. Chem. 1997; 272: 9677-9682Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar, 6Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1872) Google Scholar). The fact that caspases 8 and 10 each contain two N-terminal located death effector domains (DED) that enable them to associate with death receptors, places these two caspases most upstream in the apoptotic activation pathway (7Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (698) Google Scholar, 8Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2122) Google Scholar, 9Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2757) Google Scholar). In contrast, caspase-3 is believed to play the role of the executioner most downstream in the apoptotic pathways as it is commonly activated in cells by various death stimuli (10Faleiro L. Kobayashi R. Fearnhead H. Lazebnik Y. EMBO J. 1997; 16: 2271-2281Crossref PubMed Scopus (342) Google Scholar, 11MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar, 12Martins L.M. Kottke T. Mesner P.W. Basi G.S. Sinha S. Frigon Jr., N. Tatar E. Tung J.S. Bryant K. Takahashi A. Svingen P.A. Madden B.J. McCormick D.J. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 1997; 272: 7421-7430Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 13Takahashi A. Hirata H. Yonehara S. Imai Y. Lee K.-K. Moyer R.W. Turner P.C. Mesner P.W. Okazaki T. Sawai H. Kishi S. Yamamoto K. Okuma M. Sasada M. Oncogene. 1997; 14: 2741-2752Crossref PubMed Scopus (106) Google Scholar). Many different substrates are apparently cleaved by caspase-3, notably DNA fragmentation factor (DFF-45) (14Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1655) Google Scholar), the actin regulatory protein gelsolin (15Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1050) Google Scholar), the DNA repair enzymes poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs), structural proteins such as α-fodrin (16Martin S.J. O'Brien G.A. Nishioka W.K. McGahon A.J. Mahboubi A. Saido T.C. Green D.R. J. Biol. Chem. 1995; 270: 6425-6428Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar,17Cryns V.L. Bergeron L. Zhu H. Li H. Yuan J. J. Biol. Chem. 1996; 271: 31277-31282Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), the signaling enzymes protein kinase Cδ (PKCδ), cytosolic phospholipase A2 (cPLA2), sterol-regulatory element-binding proteins, and p21-activated kinase 2 (PAK2) (4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2201) Google Scholar,18Rudel T. Bokoch G. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (606) Google Scholar, 19Porter A.G. Ng P. Jänicke R.U. BioEssays. 1997; 19: 501-507Crossref PubMed Scopus (166) Google Scholar, 20Villa P. Kaufmann S.H. Earnshaw W.C. Trends Biochem Sci. 1997; 22: 388-393Abstract Full Text PDF PubMed Scopus (551) Google Scholar). Although it seems likely that the selective cutting of these and other key proteins is detrimental to the cell, it is unclear which of the substrates must be cleaved to commit the cell to die and to ensure apoptosis. Elucidating the substrate specificities of the individual proteases in vivo is another largely unsolved problem and is complicated by the fact that most caspases cleave more than one caspase precursor and/or substrate in vitro (4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2201) Google Scholar, 19Porter A.G. Ng P. Jänicke R.U. BioEssays. 1997; 19: 501-507Crossref PubMed Scopus (166) Google Scholar,20Villa P. Kaufmann S.H. Earnshaw W.C. Trends Biochem Sci. 1997; 22: 388-393Abstract Full Text PDF PubMed Scopus (551) Google Scholar). Mice with a homozygous deletion in the CASP-3 gene show hyperplasia and abnormalities only in the brain, demonstrating that caspase-3 is essential for normal brain development (21Kuida K. Zheng T.S. Na S. Kuan C-Y. Yang D. Karasuyama H. Rakic P. Flavell R.A. Nature. 1996; 384: 368-372Crossref PubMed Scopus (1721) Google Scholar). Furthermore, using the MCF-7 breast carcinoma cell line that lacks caspase-3 due to the functional deletion of the CASP-3 gene (22Jänicke R.U. Sprengart M.L Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar), we and others have recently demonstrated that caspase-3 is required for DNA fragmentation and blebbing (22Jänicke R.U. Sprengart M.L Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar) and for apoptosis induced by cytochromec (23Li F. Srinivasan A. Wang Y. Armstrong R.C. Tomaselli K.J. Fritz L.C. J. Biol. Chem. 1997; 272: 30299-30305Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), but is not essential for TNF-, staurosporine- (22Jänicke R.U. Sprengart M.L Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar), or Fas-induced apoptosis (23Li F. Srinivasan A. Wang Y. Armstrong R.C. Tomaselli K.J. Fritz L.C. J. Biol. Chem. 1997; 272: 30299-30305Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). However, with the exception of PARP (21Kuida K. Zheng T.S. Na S. Kuan C-Y. Yang D. Karasuyama H. Rakic P. Flavell R.A. Nature. 1996; 384: 368-372Crossref PubMed Scopus (1721) Google Scholar), it was not shown whether substrates known to be cleaved by caspase-3 are still proteolyzed in its absence, which other caspases are activated in apoptotic caspase-3-deficient cells, and whether various cleavage events are necessary for the cell to die. These questions are addressed in more detail in this paper. All cell lines were maintained in RPMI 1640 supplemented with 10% fetal calf serum, 10 mm glutamine, and 50 μg (each) of streptomycin and penicillin/ml (24Jänicke R.U. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1994; 14: 5661-5670Crossref PubMed Scopus (94) Google Scholar). The human breast carcinoma cell line MCF-7 was obtained from the ATCC. The caspase inhibitory peptides benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) and Z-Asp-Glu-Val-Asp-fmk (Z-DEVD-fmk) were obtained fromCLONTECH, and the protease inhibitors aprotinin, bacitracin, antipain, leupeptin, and phenylmethylsulfonyl fluoride as well as staurosporine were purchased from Sigma. TNF was recombinant human TNF-α with a specific activity of 4 × 107units/mg of protein. Purified, active recombinant human caspases 3, 6, 7, and 8 were purchased from Pharmingen. The following antibodies were used: monoclonal antibodies to gelsolin were from Sigma. The monoclonal antibodies to CPP32 (caspase-3), Ich-1L (caspase-2), and gelsolin were purchased from Transduction Laboratories Inc. The polyclonal antibodies raised against ICE (caspase-1), TX (caspase-4), Mch4 (caspase-10), Mch5 (caspase-8), DNA-PKcs, and PAK2, as well as the monoclonal antibodies to Rb (Rb-IF8) were bought from Santa Cruz Biotechnology. Polyclonal caspase-7 and caspase-9 antibodies were generated by immunizing rabbits with the synthetic peptides KPDRSSFVPSLFSKKKKN and MDEADRRLLRR corresponding to the N terminus of the putative p20 subunit of caspase-7 and to the N terminus of the prodomain of caspase-9, respectively (25Duan H. Chinnaiyan A.M. Hudson P.L. Wing J.P. He W.-W. Dixit V.M. J. Biol. Chem. 1996; 271: 1621-1625Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 26Duan H. Orth K. Chinnaiyan A.M. Poirier G.G. Froelich C.J. He W.-W. Dixit V.M. J. Biol. Chem. 1996; 271: 16720-16724Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). The monoclonal anti-non-erythroid spectrin (α-fodrin; mAb1622) antibodies were from Chemicon International Inc., and the monoclonal anti-PARP antibodies were obtained from G. G. Poirier. The polyclonal antibodies raised against DFF-45 were from X. Wang. Nuclear and cell extracts were prepared as described (24Jänicke R.U. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1994; 14: 5661-5670Crossref PubMed Scopus (94) Google Scholar, 27Jänicke R.U. Lin X.Y. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1996; 16: 5245-5253Crossref PubMed Scopus (56) Google Scholar) in the presence of 1 mm phenylmethylsulfonyl fluoride and 10 μg/ml each of aprotinin, bacitracin, antipain, and leupeptin. To confirm equal loadings, protein concentrations were determined with the Bio-Rad protein assay. For detection of Rb, PARP, DNA-PKcs, and α-fodrin cleavage, nuclear extracts and cell extracts were separated in 0.1% SDS, 6.5% polyacrylamide gels and subjected to Western blotting as described (27Jänicke R.U. Lin X.Y. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1996; 16: 5245-5253Crossref PubMed Scopus (56) Google Scholar). The cell extracts for the detection of PAK2, gelsolin, DFF-45, and the various caspases were fractionated in 0.1% SDS, 12.5% polyacrylamide gels. The proteins were visualized by the Amersham Pharmacia Biotech ECL kit. The plasmid pcDNA3 containing the full-length Yama (CASP-3) cDNA was provided by V. Dixit. The cDNA encoding the full-length Mch4 (CASP-10) cDNA (7Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (698) Google Scholar) was obtained by polymerase chain reaction and cloned into the expression vector pcDNA3 (Invitrogen). For the microscopic assessment of apoptotic cell death, 1 × 105 MCF-7 cells per well of a 24-well plate were transiently transfected using the SuperFect Reagent (Qiagen) with 0.3 μg of pCMV-β-galactosidase in the presence of 0.9 μg of pcDNA3 containing full-length cDNA of CASP-3 orCASP-10. At 40 h post-transfection, cells were fixed and stained for β-galactosidase expression as described (28Kumar S. Kinoshita M. Noda M. Copeland N.G. Jenkins N.A. Genes Dev. 1994; 8: 1613-1626Crossref PubMed Scopus (591) Google Scholar). Transfection efficiency was approximately 40–60%. For Western blot analysis of death substrates, 5 × 105 MCF-7 cells in a 60-mm dish were transiently transfected with 5 μg of plasmids (without the pCMV-β-galactosidase construct). At 40 h post-transfection, nuclear extracts and cell extracts were prepared and analyzed as described above. MCF-7 cells were stably transfected with the CASP-3 cDNA as described (22Jänicke R.U. Sprengart M.L Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar). To induce apoptosis, cells were either treated with a combination of TNF (30 ng/ml) and cycloheximide (Chx; 10 μg/ml) (Sigma) or staurosporine (1 μm). Cell death was either assessed by microscopic examination, trypan blue uptake or was measured with the standard TNF cytotoxicity assay as described previously (24Jänicke R.U. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1994; 14: 5661-5670Crossref PubMed Scopus (94) Google Scholar). The DEVD activity assay which is based on the spectrophotometric detection of the chromophore paranitroanilide (pNA) after cleavage from the labeled substrate DEVD-pNA by caspase-3 (or a caspase-3-like) protease was performed with the ApoAlertTMCPP32 Colorimetric Assay Kit (CLONTECH) according to the protocol of the manufacturer. For the protease cleavage assays, Rb was synthesized in the presence of [35S]methionine as described (29Jänicke R.U. Walker P.A. Lin X.Y. Porter A.G. EMBO J. 1996; 15: 6969-6978Crossref PubMed Scopus (223) Google Scholar). Cleavage reactions were performed for 2 h at 37 °C using 3 μl of in vitro translated Rb and 200 ng of active caspases in 30 μl containing 20 mm PIPES (pH 7.2), 100 mm NaCl, 10 mm dithiothreitol, 1 mm EDTA, 0.1% CHAPS, and 10% sucrose, and analyzed in 0.1% SDS-6.5% polyacrylamide gels followed by autoradiography. We have recently demonstrated that the human MCF-7 breast carcinoma cell line is devoid of caspase-3 owing to the functional deletion of the CASP-3 gene (22Jänicke R.U. Sprengart M.L Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar). Despite the lack of caspase-3, MCF-7 cells are still sensitive to a variety of apoptosis inducers such as transforming growth factor-β1 (30Oberhammer F. Wilson J.W. Dive C. Morris I.D. Hickman J.A. Wakeling A.E. Walker P.R. Sikorska M. EMBO J. 1993; 12: 3679-3684Crossref PubMed Scopus (1169) Google Scholar), Fas (23Li F. Srinivasan A. Wang Y. Armstrong R.C. Tomaselli K.J. Fritz L.C. J. Biol. Chem. 1997; 272: 30299-30305Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), and TNF or staurosporine (22Jänicke R.U. Sprengart M.L Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar) indicating that other caspases may be activated. To examine whether MCF-7 cells specifically lack only pro-caspase-3 and to determine which pro-caspases are activated in TNF/Chx- or staurosporine-induced apoptosis, control and apoptotic cell lysates of MCF-7 cells were probed with antibodies to various caspases. As a control, lysates of similarly treated HeLa D98 and H21 cells which are highly or only marginally sensitive to TNF, respectively (24Jänicke R.U. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1994; 14: 5661-5670Crossref PubMed Scopus (94) Google Scholar), were included. Besides caspase-3, MCF-7 cells also lack detectable levels of pro-caspase-1 which, however, is present in both HeLa cell lines (Fig. 1). All other pro-caspases tested (pro-caspases 2, 5, 7, 8, 9, and 10) were present in all three cell lines examined (Fig. 1). There was some variation in the protein levels of pro-caspases 2 and 7 in HeLa and MCF-7 cells, but pro-caspases 5, 9, and 10 were equally expressed in all three cell types (Fig. 1). In addition to the observed activation of caspase-3 in HeLa D98 and HeLa H21 cells following a death stimulus (Fig. 1), only caspase-8, but none of the other caspases, was activated by both death stimuli to various degrees as demonstrated by the appearance of a singlet band or doublet of approximately 24–26 kDa (Fig. 1, lanes 2,3, 5, 6, and 8). These proteins which were present only in apoptotic lysates most likely represent intermediate cleavage products of caspase-8, as demonstrated previously in vitro and in vivo (31Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1045) Google Scholar). Interestingly, in all three cell lines examined, caspase-8 was efficiently and similarly activated by TNF/Chx (Fig. 1,lanes 2, 5, and 8), despite the fact that only 18% of the HeLa H21 cells were killed by this death stimulus (Fig. 1). In contrast, all three cell lines were killed to a similar extent following a 16-h staurosporine treatment (Fig. 1), which, however, resulted only in a marginal and almost undetectable activation of caspase-8 in HeLa H21 and in MCF-7 cells, respectively (Fig. 1, lanes 6 and 9). These data are in agreement with reports demonstrating an important role for caspase-8 predominantly in TNF- or Fas-mediated apoptosis (8Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2122) Google Scholar, 9Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2757) Google Scholar, 31Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1045) Google Scholar), but also suggest that caspase-8 (like caspase-3) may not always be essential for apoptosis, even though both caspases are commonly activated by a variety of death stimuli (10Faleiro L. Kobayashi R. Fearnhead H. Lazebnik Y. EMBO J. 1997; 16: 2271-2281Crossref PubMed Scopus (342) Google Scholar, 13Takahashi A. Hirata H. Yonehara S. Imai Y. Lee K.-K. Moyer R.W. Turner P.C. Mesner P.W. Okazaki T. Sawai H. Kishi S. Yamamoto K. Okuma M. Sasada M. Oncogene. 1997; 14: 2741-2752Crossref PubMed Scopus (106) Google Scholar, 31Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1045) Google Scholar, 32Chinnaiyan A.M. Orth K. O'Rourke K. Duan H. Poirier G.G. Dixit V.M. J. Biol. Chem. 1996; 271: 4573-4576Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar). Having established that MCF-7 cells lack pro-caspases 1 and 3, but express other apoptosis-related pro-caspases, we examined control and apoptotic cell lysates of HeLa D98, HeLa H21, and MCF-7 cells for the status of various death substrates known to be cleaved by caspase-3 or a caspase-3-like protease (4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2201) Google Scholar, 19Porter A.G. Ng P. Jänicke R.U. BioEssays. 1997; 19: 501-507Crossref PubMed Scopus (166) Google Scholar). The same experimental conditions were applied as described in Fig. 1. The cleavages of PARP, the retinoblastoma protein (Rb), α-fodrin, and DNA-PKcs could be readily assessed by the appearance of the corresponding fragments in apoptotic lysates, whereas proteolysis of PAK2, gelsolin, and DFF-45 was judged by the decrease in the intensity of the full-length proteins. Two different antibodies to PAK2 and gelsolin both gave the same results, but no fragments were detected. Nevertheless, with the exception of α-fodrin, all substrates tested including PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45 were still cleaved in caspase-1- and caspase-3-deficient MCF-7 cells (Fig. 2, lanes 8 and9). Except for gelsolin which was only cleaved in staurosporine-treated, but not in TNF/Chx-treated, MCF-7 cells (a pattern also observed in D98 cells; Fig. 2, lanes 2 and 3), all substrates were cleaved to a similar extent in MCF-7 cells treated with TNF/Chx or staurosporine (Fig. 2, lanes 8 and 9). This is in contrast with the cleavage patterns obtained in the two HeLa cell lines, in which most of the substrates were cleaved more efficiently in TNF/Chx-treated cells (Fig. 2, lanes 1–6). Together with our results demonstrating that of all the caspases tested only caspase-8 was activated in MCF-7 cells by TNF/Chx but not by staurosporine (Fig. 1), these data provide evidence for the existence of other as yet unknown caspases that cleave PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45. Interestingly, α-fodrin, known to be cleaved by caspase-3 but not by caspases 1 and 2 (16Martin S.J. O'Brien G.A. Nishioka W.K. McGahon A.J. Mahboubi A. Saido T.C. Green D.R. J. Biol. Chem. 1995; 270: 6425-6428Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 17Cryns V.L. Bergeron L. Zhu H. Li H. Yuan J. J. Biol. Chem. 1996; 271: 31277-31282Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), was the only substrate tested that was resistant to caspase cleavage in apoptotic lysates of TNF/Chx- or staurosporine-treated MCF-7 cells, based on the absence of the typical 120-kDa fragment (Fig. 2, lanes 8 and9). Although both death stimuli induced the formation of a 150-kDa α-fodrin fragment in all three cell lines including MCF-7 cells (Fig. 2, lanes 2, 3,5, 6, 8, and 9), this fragment was recently shown to be the product of the thiol protease calpain, an enzyme unrelated to caspases (33Nath R. Raser K.J. Stafford D. Hajimohammadreza I. Posner A. Allen H. Talanian R.V. Yuen P. Gilbertsen R.B. Wang K.W. Biochem. J. 1996; 319: 683-690Crossref PubMed Scopus (398) Google Scholar). Taken together, these results imply that of all the substrates tested, only cleavage of α-fodrin appears to require the activation of caspase-3. To assess whether certain cleavage events are necessary for apoptosis, MCF-7 cells were transiently transfected with a pSV-β-galactosidase plasmid in the presence of a 3-fold excess of vector alone or an expression construct encoding full-length pro-caspase-3. As a further control, an expression construct encoding pro-caspase-10 was also introduced into MCF-7 cells. Almost all CASP-10-transfected MCF-7 cells were dead (95%) in contrast to only 50–60% of MCF-7 cells transfected with theCASP-3 cDNA (Fig. 3 A). This difference can be most likely explained by the facts that caspase-10 is a more upstream protease that is capable of activating other caspases (7Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (698) Google Scholar) and that caspase-3 may not be able to autocatalyze as efficiently, which is supported by the presence of some inactive caspase-3 precursor remaining in CASP-3-transfected MCF-7 cells (Fig. 3 B, upper panel, lane 4). Regardless of the explanation, these results indicate that caspase-3 can activate a death pathway in MCF-7 cells. More importantly, Western blot analysis showed that the overexpression of pro-caspase-3 (but not pro-caspase-10) led to the cleavage of α-fodrin giving rise to the typical 120-kDa fragment (Fig. 3 B, compare lanes 4 and 5). Surprisingly, Rb was not cleaved in the presence of caspase-3 (Fig. 3 B, lane 4), although the cleavage site in Rb (DEAD↓G) appears to be close to a typical caspase-3 recognition motif (29Jänicke R.U. Walker P.A. Lin X.Y. Porter A.G. EMBO J. 1996; 15: 6969-6978Crossref PubMed Scopus (223) Google Scholar, 34Tan X. Martin S.J. Green D.R. Wang J.Y.J. J. Biol. Chem. 1997; 272: 9613-9616Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Likewise, little or no Rb cleavage was observed in MCF-7 cells transfected with CASP-10 (Fig. 3 B, lane 5). PARP, on the other hand, was cleaved into the signature 85-kDa apoptotic fragment in MCF-7 cells transfected with either the CASP-3 or CASP-10 cDNAs (Fig. 3 B, lanes 4 and5). To further investigate whether caspase-3 is required for α-fodrin cleavage, MCF-7 cells stably expressing pro-caspase-3 (22Jänicke R.U. Sprengart M.L Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar) were treated with TNF/Chx or staurosporine, and α-fodrin cleavage was monitored by Western blotting. Both death stimuli resulted in the activation of caspase-3 in three individual MCF-7 clones stably expressing pro-caspase-3 (Fig. 4, upper panels). Consistent with the caspase-3 activation profile obtained in apoptotic HeLa cells (Fig. 1), TNF/Chx treatment of the three pro-caspase-3-expressing MCF-7 clones resulted in a more efficient activation of this protease than the treatment with staurosporine (Fig. 4, upper panels). More importantly, processing of pro-caspase-3 in these cells was accompanied by the cleavage of α-fodrin into the typical 120-kDa fragment (Fig. 4, lower panels). Similar to the results obtained with parental caspase-3-deficient MCF-7 cells (Figs. 2 and3 B), TNF/Chx or staurosporine treatment of vector-transfected MCF-7 cells did not result in the appearance