Different Subcellular Distribution of Caspase-3 and Caspase-7 following Fas-induced Apoptosis in Mouse Liver

Caspases plays a key role in the execution phase of apoptosis. “Initiator” caspases, such as caspase-8, activate “effector” caspases, such as caspase-3 and -7, which subsequently cleave cellular substrates thereby precipitating the dramatic morphological changes of apoptosis. Following treatment of mice with an agonistic anti-Fas antibody to induce massive hepatocyte apoptosis, we now demonstrate a distinct subcellular localization of the effector caspases-3 and -7. Active caspase-3 is confined primarily to the cytosol, whereas active caspase-7 is associated almost exclusively with the mitochondrial and microsomal fractions. These data suggest that caspases-3 and -7 exert their primary functions in different cellular compartments and offer a possible explanation of the presence of caspase homologs with overlapping substrate specificities. Translocation and activation of caspase-7 to the endoplasmic reticulum correlates with the proteolytic cleavage of the endoplasmic reticular-specific substrate, sterol regulatory element-binding protein 1. Liver damage, induction of apoptosis, activation and translocation of caspase-7, and proteolysis of sterol regulatory element-binding protein 1 are all blocked by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.fmk). Our data demonstrate for the first time the differential subcellular compartmentalization of specific effector caspases following the induction of apoptosis in vivo. Caspases plays a key role in the execution phase of apoptosis. “Initiator” caspases, such as caspase-8, activate “effector” caspases, such as caspase-3 and -7, which subsequently cleave cellular substrates thereby precipitating the dramatic morphological changes of apoptosis. Following treatment of mice with an agonistic anti-Fas antibody to induce massive hepatocyte apoptosis, we now demonstrate a distinct subcellular localization of the effector caspases-3 and -7. Active caspase-3 is confined primarily to the cytosol, whereas active caspase-7 is associated almost exclusively with the mitochondrial and microsomal fractions. These data suggest that caspases-3 and -7 exert their primary functions in different cellular compartments and offer a possible explanation of the presence of caspase homologs with overlapping substrate specificities. Translocation and activation of caspase-7 to the endoplasmic reticulum correlates with the proteolytic cleavage of the endoplasmic reticular-specific substrate, sterol regulatory element-binding protein 1. Liver damage, induction of apoptosis, activation and translocation of caspase-7, and proteolysis of sterol regulatory element-binding protein 1 are all blocked by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.fmk). Our data demonstrate for the first time the differential subcellular compartmentalization of specific effector caspases following the induction of apoptosis in vivo. Apoptosis is a crucial mechanism by which multicellular organisms control cell numbers and ensure the removal of damaged or potentially harmful cells (1Arends M.J. Wyllie A.H. Int. Rev. Exp. Pathol. 1991; 32: 223-254Crossref PubMed Scopus (1394) Google Scholar). Administration of an agonistic anti-Fas antibody results in ligation of the Fas (CD95, APO-1) receptor, extensive hepatocyte apoptosis, and liver damage (2Ogasawara J. Watanabe-Fukunaga R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1815) Google Scholar). The intracellular death domain of the Fas receptor binds to FADD/MORT1, which in turn recruits and activates caspase-8 (MACH/FLICE/Mch5) through its N-terminal death effector domain (3Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 4Muzio 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 (2743) Google Scholar, 5Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar). Recombinant caspase-8 cleaves and activates all other known caspases and has been proposed to be at the apex of a hypothetical caspase cascade (3Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 4Muzio 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 (2743) Google Scholar, 5Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar). Caspases are a family of aspartate-specific cysteine proteases, which pre-exist in the cytoplasm as single chain inactive zymogens (6Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 7Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar). They are proteolytically processed to active heterodimeric enzymes during the execution phase of apoptosis. Caspases may be divided into “initiator” caspases with long prodomains (caspases-8, -9, and -10), which activate “effector” caspases with short prodomains (caspases-3, -6, and -7), which in turn cleave intracellular substrates, resulting in the dramatic morphological and biochemical changes of apoptosis (6Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 7Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 8Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). Following Fas-induced apoptosis of cells in vitro, activation of a number of caspases, including caspases-3, -4, -6, -7, -8, and a caspase-1-like activity have all been reported (4Muzio 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 (2743) Google Scholar,9Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar, 10Enari M. Hug H. Nagata S. Nature. 1995; 375: 78-81Crossref PubMed Scopus (798) Google Scholar, 11Schlegel J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 12Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (969) Google Scholar, 13Kamada S. Washida M. Hasegawa J.-I. Kusano H. Funahashi Y. Tsujimoto Y. Oncogene. 1997; 15: 285-290Crossref PubMed Scopus (93) Google Scholar). To date, a family of at least 10 caspases have been identified, but it is not known precisely which of these caspase(s) are activated in vivo and which are responsible for the cleavage of particular substrates. Many of the caspases have overlapping substrate specificities, suggesting that there may be redundancy (6Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 7Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 14Thornberry 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 (1852) Google Scholar). Many cellular proteins are cleaved during the execution phase of apoptosis at a DXXD motif by the effector caspases-3 and -7 (reviewed in Refs. 6Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar and 7Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar). Relatively little is known about the subcellular distribution of the caspases. Caspase-1 is found predominantly in the cytosol (15Ayala J.M. Yamin T.-T. Egger L.A. Chin J. Kostura M.J. Miller D.K. J. Immunol. 1994; 153: 2592-2599PubMed Google Scholar) although some has been localized to the external cell surface membrane (16Singer I.I. Scott S. Chin J. Bayne E.K. Limjuco G. Weidner J. Miller D.K. Chapman K. Kostura M.J. J. Exp. Med. 1995; 182: 1447-1459Crossref PubMed Scopus (140) Google Scholar). Other caspases have been considered to be cytosolic, a conclusion based largely on data with caspase-1 and on the isolation and purification of caspase-3 (17Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar). In this study, we demonstrate for the first time the differential subcellular distribution of specific caspases during the induction of apoptosisin vivo. Following Fas-induced apoptosis in vivo, active caspase-3 is found primarily in the cytosol, whereas active caspase-7 is associated almost exclusively with the mitochondrial and microsomal fractions. Both the activation of caspase-7 in the endoplasmic reticulum and the cleavage of the endoplasmic reticular-specific substrate, sterol regulatory element-binding protein 1 (SREBP-1), 1The abbreviations used are: SREBP, sterol regulatory element-binding protein; Z-VAD.fmk, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone; Z-DEVD.afc, benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; DEVDase, proteolytic activity to cleave Z-DEVD.afc; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate; PAGE, polyacrylamide gel electrophoresis. are blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.fmk). These results support the hypothesis that during the execution phase of apoptosis, different caspase homologs cleave specific substrates in different cellular compartments. In this study 6–8-week-old (20 g) Balb/c males were used. All mice were bred in the Biomedical Sciences Department of the University of Leicester. Mice were injected either with 10 μg of purified hamster monoclonal antibody to mouse Fas (JO2) (PharMingen, Los Angeles, CA) (2Ogasawara J. Watanabe-Fukunaga R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1815) Google Scholar) in 160 μl of 0.9% (w/v) saline, 12.5% (v/v) Me2SO, or 160 μl of 0.9% (w/v) saline, 12.5% (v/v) Me2SO (controls). Where indicated, mice were injected with JO2 antibody (10 μg) in 80 μl of 0.9% (w/v) saline followed 5 min later by Z-VAD.fmk (500 μg) (Enzyme Systems Ltd., Dublin, CA) in 80 μl of 0.9% (w/v) saline, 25% (v/v) Me2SO. Animals were sacrificed at the indicated times by cervical dislocation. Livers were removed and fixed in 10% formaldehyde in buffered saline. Representative sections of the left lateral, median, and posterior lobes were stained with hematoxylin and eosin and examined for apoptosis. Following removal of the livers, excess hair and blood were removed by washing several times in buffer A (0.3 m mannitol, 5 mm MOPS, 1 mmEGTA, 4 mm KH2PO4). The livers were then chopped up and homogenized using a dounce homogenizer in 5 ml of buffer A supplemented with protease inhibitors (20 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, 2 mmphenylmethylsulfonyl fluoride). The crude homogenates were centrifuged at 650 × g for 10 min at 4 °C and the resultant supernatant centrifuged at 10,000 × g for 15 min at 4 °C to sediment the mitochondria. The mitochondria were washed in supplemented buffer A and pelleted. The microsomal and cytosolic fractions were obtained following centrifugation of the 10,000 ×g supernatant at × g for min at 4 of the cytosolic, and microsomal fractions by using to G. 1997; PubMed Scopus Google Scholar) by M. and is on the mitochondrial membrane T. H. T. H. K. R. R. S. 1996; 272: PubMed Scopus Google is a cytosolic J.D. Rev. Biochem. Biol. 1995; PubMed Scopus Google and is in the endoplasmic reticulum R. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). that and of and and of were in the cytosolic, and microsomal found almost exclusively in the endoplasmic reticulum 4 and data not and were on liver fractions as M. K. Alnemri E.S. G.M. J. Biol. 1997; PubMed Scopus Google Scholar). The were using a antibody to the of caspase-3 by J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, G. Nicholson D.W. Oncogene. 1996; Google a antibody to the of caspase-7 M. K. Alnemri E.S. G.M. J. Biol. 1997; PubMed Scopus Google and a antibody to of The proteolytic activity of the liver fractions using a with (Enzyme Systems Dublin, CA) as substrate as M. K. Alnemri E.S. G.M. J. Biol. 1997; PubMed Scopus Google Scholar). of the the of the proteolytic of caspases-3 and -7 to as The agonistic Fas receptor antibody JO2 extensive liver damage and in Balb/c with of 4 1 (2Ogasawara J. Watanabe-Fukunaga R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1815) Google Scholar). The caspase inhibitor, Z-VAD.fmk blocked Fas-induced liver damage and and hepatocyte apoptosis to 1 to the agonistic antibody not The by Z-VAD.fmk a role for the activation of caspases in Fas-induced apoptosis in vivo, with I. K. C. Nagata S. J. Exp. Med. 1996; PubMed Scopus Google Scholar, N. T. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). To date, on Fas-induced apoptosis have been in vivo, in S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar, 10Enari M. Hug H. Nagata S. Nature. 1995; 375: 78-81Crossref PubMed Scopus (798) Google Scholar, 11Schlegel J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 12Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (969) Google Scholar, 13Kamada S. Washida M. Hasegawa J.-I. Kusano H. Funahashi Y. Tsujimoto Y. Oncogene. 1997; 15: 285-290Crossref PubMed Scopus (93) Google Scholar). In to the of caspases in apoptosisin vivo, their activation by in crude liver homogenates control and activity is primarily a of caspase-3 and caspase-7 although there may be a other caspases 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 (1852) Google Scholar, D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar, M. O'Rourke K. S. Dixit V.M. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, T. A. R. J. Yu G. Litwack G. Alnemri E.S. 1995; Google Scholar). with the agonistic Fas antibody in a in liver 4 in with that in These results the activation of the effector caspase-3 caspase-7 in Fas-induced apoptosis in vivo in with in which have the activation of these caspases following treatment of cells with Fas or J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, H. Chinnaiyan A.M. J.P. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google A.M. K. O'Rourke K. H. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). In to the role of caspases in Fas-induced apoptosis in vivo, we examined and caspase in subcellular liver fractions control and Fas treatment and in in cytosolic, and mitochondrial In all Z-VAD.fmk the in Z-VAD.fmk blocked apoptosis either by the activity of caspases-3 and -7 or by an such as is in control cells as an inactive J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, M. K. Alnemri E.S. G.M. J. Biol. 1997; PubMed Scopus Google Scholar, A.M. K. O'Rourke K. H. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). induction of apoptosis, it is processed at the and a which is processed at and to and (17Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar, T. J. S.M. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). In control in the cytosolic with in the mitochondrial or microsomal fractions 1 and Following treatment with the Fas of with the of its active in the cytosolic In of and were The in all the cytosolic and microsomal fractions the of this is not it has been proposed to be to of caspase-3 following cleavage at a which is not processed to active caspase-3 G. Nicholson D.W. Oncogene. 1996; Google Scholar). with Z-VAD.fmk in almost of the of of the and the of caspase-3 by the of a of an which may be either to of Z-VAD.fmk to the or to of the of the M. K. Alnemri E.S. G.M. J. Biol. 1997; PubMed Scopus Google Scholar, J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The primarily in the cytosolic of livers mice with little in other These data demonstrate that following Fas induction of apoptosis, active caspase-3 is primarily in the as an inactive in control cells M. K. Alnemri E.S. G.M. J. Biol. 1997; PubMed Scopus Google Scholar, H. Chinnaiyan A.M. J.P. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. Alnemri E.S. G.M. M. Biochem. J. 1997; PubMed Scopus Google Scholar). induction of apoptosis it is activated by at the and followed by cleavage at to the active M. K. Alnemri E.S. G.M. J. Biol. 1997; PubMed Scopus Google Scholar, T. A. R. J. Yu G. Litwack G. Alnemri E.S. 1995; Google Scholar, H. Chinnaiyan A.M. J.P. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). In livers control caspase-7 as an in the cytosolic and microsomal fractions with 4 and caspase-7 has been in the cytoplasm of cells H. Chinnaiyan A.M. J.P. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google this is the first time it has been to have a microsomal or in the mitochondrial control mouse livers Following treatment with the Fas of in the cytosolic and microsomal fractions 5 and of caspase-7 in the cytosolic little the active of caspase-7 in the mitochondrial and microsomal fractions 2 and that the in the mitochondrial to microsomal the endoplasmic protein exclusively in the microsomal 4 and data not In the using the caspase-3 in the microsomal control or livers and not in the mitochondrial 1 and The of the of caspase-7 in the microsomal following Fas-induced apoptosis the of in control liver and These results that caspase-7 the cytosol to the following its activation by an The data demonstrate that following Fas induction of apoptosis in mouse caspase-7 is processed to its active which is found primarily in the mitochondrial and microsomal fractions with little in the Z-VAD.fmk the Fas-induced cleavage of as as the of the in all subcellular fractions and Z-VAD.fmk blocked the of an in the microsomal with the caspase-3 data suggest that Z-VAD.fmk the of caspase-3 and it is not known precisely which caspase activates caspase-3 and caspase-7 during Fas-induced apoptosis, caspase-8 has been considered the (3Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 4Muzio 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 (2743) Google Scholar, 5Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar). have that binds to in a and H. A. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google D. I. S.M. Ahmad M. Alnemri E.S. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). results in the activation of which in turn cleaves and activates caspase-3 D. I. S.M. Ahmad M. Alnemri E.S. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanism of activation of is not it may be to activation by caspase-8 S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar) or to a mechanism and D. I. S.M. Ahmad M. Alnemri E.S. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google but it not to be to a activation by caspase-3 H. A. S. S. H. T. K. M. J. Exp. Med. PubMed Scopus Google Scholar). activation of caspase-8 is a following Fas-induced apoptosis, it is not this is to the activation of caspases following mitochondrial damage with the of and the activation of Z-VAD.fmk may exert its by the caspase cascade by caspase-8 and support for the hypothesis that different effector caspases are responsible for the activity in different subcellular compartments by the data with in cells apoptosis is to be primarily to activation of caspase-3 and caspase-7 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 (1852) Google Scholar, D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar, M. O'Rourke K. S. Dixit V.M. Cell. 1995; Full Text PDF PubMed Scopus Google T. A. R. J. Yu G. Litwack G. Alnemri E.S. 1995; Google Scholar). the the polyacrylamide gel the cytosolic (20 that either the mitochondrial or microsomal fractions and 15 2 and the antibody to caspase-7 the in the mitochondrial and microsomal fractions 2 and that a caspase other caspase-7 primarily responsible for in the cytosolic this based on the data that the active of caspase-3 primarily in the cytosol results suggest that the in the microsomal and mitochondrial fractions is to caspase-7, in the cytosolic it is to results suggest that following its caspase-7 is to the microsomal and mitochondrial it is responsible for the cleavage of specific substrates in these distinct subcellular compartments. A study using an in the of active caspases in the and cytosol cell S. P.A. A. 1997; PubMed Google Scholar). results with the study the about different active caspases may be to different subcellular Many have overlapping substrate of caspases-3 and -7. using substrates substrate to caspases-3 and -7 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 (1852) Google and enzymes cleave (17Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google M. O'Rourke K. S. Dixit V.M. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, T. A. R. J. Yu G. Litwack G. Alnemri E.S. 1995; Google Scholar). has been that there is a redundancy for caspases, which may be to the of this in damaged or cells (6Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 7Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar). results with caspase-3 which apoptosis in that some caspases may in a K. S. D. H. Nature. 1996; PubMed Scopus Google Scholar). data suggest at least in some caspases with overlapping substrate may exert their functions in different cellular the cleavage of specific substrates. To this we examined the of of the endoplasmic proteins known to be cleaved in apoptosis R. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, J. J. J. 1996; 15: PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). to the family of and are in the of sterol R. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). the induction of apoptosis and are cleaved by the hamster homologs of caspases-3 and -7 J. J. J. 1996; 15: PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). In livers control exclusively associated with the microsomal with in the cytosolic or mitochondrial fractions 1 and data not of the in liver obtained mice 4 with the agonistic Fas antibody The antibody to the but not the cleaved The Fas-induced cleavage of largely by Z-VAD.fmk active caspase-7 and the subcellular it is possible that in vivo caspase-7 is responsible for the Fas-induced cleavage of based on data and the of the cytosol and endoplasmic we the that may be at least in by have that following Fas-induced apoptosis in vivo, active caspase-3 primarily in the cytosol, whereas active caspase-7 associated with the mitochondrial and microsomal fractions. Our data the first of the differential subcellular distribution of specific caspases in vivo of apoptosis. K. and for their