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Ionizing Radiation-induced Apoptosis in Ataxia-Telangiectasia Fibroblasts

2001; Elsevier BV; Volume: 276; Issue: 31 Linguagem: Inglês

10.1074/jbc.m010525200

ISSN

1083-351X

Autores

Yin Zhang, Alexandre Dimtchev, Anatoly Dritschilo, Mira Jung,

Tópico(s)

Carcinogens and Genotoxicity Assessment

Resumo

Ionizing radiation (IR) has been shown to induce apoptosis to a greater extent in a fibroblast cell line AT5BIVA derived from an individual with ataxia-telangiectasia (AT) than in control fibroblasts. However, the signaling pathway that underlies IR-induced apoptosis in AT cells has remained unknown. The mechanism of apoptosis in response to γ-irradiation has now been examined in three AT fibroblast lines (AT3BIVA, AT4BIVA, and AT5BIVA) derived from different individuals with AT. The apoptotic indexes of these cell lines at 72 h after irradiation were 12, 31, and 35%, respectively, compared with a value of 2.3% for control fibroblasts. Immunoblot analysis and fluorometric assays revealed that the extents of IR-induced activation of caspase-3 and caspase-9 were markedly greater in AT4BIVA and AT5BIVA cells than in AT3BIVA and control cells. Furthermore, the basal abundance of the apoptotic inhibitor, a cellular inhibitor of apoptosis proteins (c-IAP-1), was markedly reduced in AT4BIVA and AT5BIVA cells compared with that in AT3BIVA and control cells. The overexpression of either caspase-9 mutant forms or recombinant c-IAP-1 in AT5BIVA cells inhibited the IR-induced activation of caspases-3 and 9 and reduced the apoptotic index of the irradiated cells. These results indicate that the extent of IR-induced apoptosis in different AT cell lines is inversely related to the abundance of c-IAP-1 and directly related to the extent of activation of caspases-3 and 9. Ionizing radiation (IR) has been shown to induce apoptosis to a greater extent in a fibroblast cell line AT5BIVA derived from an individual with ataxia-telangiectasia (AT) than in control fibroblasts. However, the signaling pathway that underlies IR-induced apoptosis in AT cells has remained unknown. The mechanism of apoptosis in response to γ-irradiation has now been examined in three AT fibroblast lines (AT3BIVA, AT4BIVA, and AT5BIVA) derived from different individuals with AT. The apoptotic indexes of these cell lines at 72 h after irradiation were 12, 31, and 35%, respectively, compared with a value of 2.3% for control fibroblasts. Immunoblot analysis and fluorometric assays revealed that the extents of IR-induced activation of caspase-3 and caspase-9 were markedly greater in AT4BIVA and AT5BIVA cells than in AT3BIVA and control cells. Furthermore, the basal abundance of the apoptotic inhibitor, a cellular inhibitor of apoptosis proteins (c-IAP-1), was markedly reduced in AT4BIVA and AT5BIVA cells compared with that in AT3BIVA and control cells. The overexpression of either caspase-9 mutant forms or recombinant c-IAP-1 in AT5BIVA cells inhibited the IR-induced activation of caspases-3 and 9 and reduced the apoptotic index of the irradiated cells. These results indicate that the extent of IR-induced apoptosis in different AT cell lines is inversely related to the abundance of c-IAP-1 and directly related to the extent of activation of caspases-3 and 9. cellular inhibitor of apoptosis ataxia-telangiectasia ionizing radiation poly(ADP-ribose) polymerase 7-methoxycoumarin-4-yl)acetyl 2,4-dinitrophenyl 4-methyl-coumaryl-7-amide N-acetyl-S-farnesyl-l-cysteine gray Apoptosis is a genetically regulated and highly conserved mechanism of cell death, impairments of which contribute to human diseases such as cancer and autoimmune disorders (1Herman S. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2430) Google Scholar, 2Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6190) Google Scholar, 3Golstein P. Science. 1997; 275: 1081-1082Crossref PubMed Scopus (336) Google Scholar, 4Hetts S.W. J. Am. Med. Assoc. 1998; 279: 300-307Crossref PubMed Scopus (473) Google Scholar). The principal effectors of apoptosis in mammalian cells are members of a family of cysteine-containing aspartate-specific proteases known as caspases (5Nicholson D.W. Nat. Biotechnol. 1996; 14: 297-301Crossref PubMed Scopus (239) Google Scholar). Several caspases have been isolated from human cells and characterized as cell type-specific and stimulus-dependent (6Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6157) Google Scholar). Each caspase is synthesized as an inactive precursor (zymogen) that is converted by proteolytic cleavage to an active heterodimer when cells are exposed to a variety of insults (7Srinivasula S. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (482) Google Scholar, 8Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar). Once activated, caspases cleave a host of cellular substrates, resulting in the morphological hallmarks of apoptosis such as DNA fragmentation and condensation of cellular organelles (9Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2180) Google Scholar). The characterization of the regulation of various caspases in mammalian cells has led to the definition of two distinct apoptotic signaling pathways (10Deveraux Q.L. Roy N. Stennicke H.R. van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1242) Google Scholar). In one of these pathways, procaspase-8 is recruited by the cytosolic domains of members of the tumor necrosis factor receptor family (11Wallach D. Boldin M. Varfolomeev E. Beyaert R. Vandenabeele P. Fiers W. FEBS Lett. 1997; 410: 96-106Crossref PubMed Scopus (211) Google Scholar). Such recruitment results in the proteolytic cleavage of procaspase-8, and the activated enzyme then induces the activation of downstream effector caspases, such as caspases-3, 6, and 7 (7Srinivasula S. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (482) Google Scholar, 8Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar). In the second pathway, cytochromec is released from mitochondria into the cytosol (12Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4461) Google Scholar, 13Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4276) Google Scholar, 14Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4410) Google Scholar), resulting in its high affinity interaction with apoptotic protease-activating factor-1 and subsequent activation of caspase-9 (15Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1137) Google Scholar, 16Hu Y. Benedict M.A. Wu D. Inohara N. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4386-4391Crossref PubMed Scopus (497) Google Scholar). The active caspase-9 in turn activates downstream effector caspases that mediate the execution phase of apoptosis (6Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6157) Google Scholar, 9Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2180) Google Scholar,14Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4410) Google Scholar, 15Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1137) Google Scholar). The activation of various caspases is regulated by cellular inhibitor of apoptosis proteins (c-IAP)1 (10Deveraux Q.L. Roy N. Stennicke H.R. van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1242) Google Scholar, 15Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1137) Google Scholar, 17Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. Elia A. de la Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe S.W. Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar), which are conserved among various species including humans (18Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1719) Google Scholar, 19Faleiro L. Kobayashi R. Fearnhead H. Lazebnik Y. EMBO J. 1997; 16: 2271-2281Crossref PubMed Scopus (341) Google Scholar). These proteins inhibit apoptosis induced by a variety of stimuli by interacting with and inhibiting the activity of caspases (15Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1137) Google Scholar, 16Hu Y. Benedict M.A. Wu D. Inohara N. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4386-4391Crossref PubMed Scopus (497) Google Scholar, 17Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. Elia A. de la Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe S.W. Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar, 18Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1719) Google Scholar). For example, they block the cytochrome c-induced activation of caspase-9 (10Deveraux Q.L. Roy N. Stennicke H.R. van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1242) Google Scholar). Thus, the basal concentration of these inhibitors appears to be an important determinant of cell survival (19Faleiro L. Kobayashi R. Fearnhead H. Lazebnik Y. EMBO J. 1997; 16: 2271-2281Crossref PubMed Scopus (341) Google Scholar, 20Budihardjo I. Oliver H. Lutter M. Luo X. Wang X. Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2268) Google Scholar). Cells from individuals with the genetic disease ataxia telangiectasia (AT) exhibit defects in cell cycle checkpoints, DNA repair, and signaling pathways associated with regulation of apoptosis in response to ionizing radiation (IR) (21Meyn M.S. Cancer Res. 1995; 55: 5991-6001PubMed Google Scholar, 22Shafman T.D. Saleem A. Kyriakis J. Weichselbaum R. Kharbanda S. Kufe D. Cancer Res. 1995; 55: 3242-3245PubMed Google Scholar, 23Jung M. Zhang Y. Dimtchev A. Dritschilo A. Radiat. Res. 1998; 149: 596-601Crossref PubMed Scopus (53) Google Scholar). Both the tumor suppressor protein p53 and the protein ATM encoded by the gene mutated in individuals with AT contribute to IR-induced cell death (24Meyn M.S. Strasfeld L. Allen C. Int. J. Radiat. Biol. 1994; 66: S141-S149Crossref PubMed Scopus (104) Google Scholar, 25Barlow C. Brown K.D. Deng C.-X. Tagle D.A. Wynshaw-Boris A. Nat. Genet. 1997; 17: 453-456Crossref PubMed Scopus (190) Google Scholar, 26Herzog K.H. Chong M.J. Kapsetaki M. Morgan J.I. McKinnon P.J. Science. 1998; 280: 1089-1091Crossref PubMed Scopus (372) Google Scholar, 27Westphal C.H. Rowan S. Schmaltz C. Elson A. Fisher D.E. Leder P. Nat. Genet. 1997; 16: 397-401Crossref PubMed Scopus (212) Google Scholar). We have previously shown that the apoptotic index of AT5BIVA cells, a cell line derived from an individual with AT, is increased by γ-irradiation to a greater extent than that of control cells (23Jung M. Zhang Y. Dimtchev A. Dritschilo A. Radiat. Res. 1998; 149: 596-601Crossref PubMed Scopus (53) Google Scholar). With the use of additional AT cell lines each derived from a different individual with AT, we have now shown that IR-induced apoptosis in these cells is mediated by a series of events that include the activation of caspases-3 and 9. Furthermore, our data suggest that a deficiency of c-IAP-1 may contribute to IR-induced programmed cell death in AT fibroblasts. AT5BIVA fibroblasts were obtained from the Human Genetic Mutant Cell Repository (Bethesda, MD). AT3BIVA, AT4BIVA, and MRC5CV1 (normal fibroblasts) were kindly provided by C. Arlett (University of Sussex, Brighton, UK). The AT and MRC5CV1 cells are immortalized as a result of the expression of SV40 large T-antigen. AT5BIVA/C9DN and /C9Mut were transfected with plasmid DNA containing a dominant negative form (28Srinivasula S.M. Ahmad M. Guo Y. Zhan Y. Lazebnik Y. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1999; 59: 999-1002PubMed Google Scholar) and the active site mutant (C287A) of caspase-9 (29Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (967) Google Scholar) by using Lipofectin reagent as suggested by the manufacturer (Life Technologies, Inc.). Both plasmid vectors containing either a dominant negative form or an active site mutant on C287A of caspase-9 cDNA were generously provided by Dr. Emad Alnemri at Thomas Jefferson University (Philadelphia, PA). AT5BIVA-c-IAP-1/CL3 cells were isolated as a G418-resistant clone (400 µg/ml) after stable transfection of AT5BIVA cells with a plasmid (pCMV-c-IAP-1N) containing the cDNA for a hemagglutinin epitope-tagged NH2-terminal fragment (residues 1–342) of human c-IAP-1 (15Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1137) Google Scholar, 30Ambrosini G. Adida C. Altieri D. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3012) Google Scholar) under the control of the cytomegalovirus promoter. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 100 µg/ml of streptomycin, 100 µg/ml of penicillin, and either 10 mm sodium pyruvate and 10% fetal bovine serum for MRC5CV1 cells or 10 mm nonessential amino acids, 2 mml-glutamine, 1 µg/ml of hydrocortisone, and 20% fetal bovine serum for AT cells. They were maintained in a 5% CO2 incubator at 37 °C. All cell lines were determined to be free of mycoplasma infection by testing at three-month intervals. Adherent and floating cells were collected at various times after exposure to 5 Gy of γ-radiation. The apoptotic index was determined morphologically by staining cells with acridine orange and ethidium bromide, counting the number of apoptotic cells (those with condensed chromosomes or fragmented nuclei) with a fluorescence microscope and expressing their number as a percentage of the total number of cells (23Jung M. Zhang Y. Dimtchev A. Dritschilo A. Radiat. Res. 1998; 149: 596-601Crossref PubMed Scopus (53) Google Scholar). Cells (5 × 106) were collected at various times after irradiation and subjected to four cycles of freezing and thawing in 300 µl of a lysis buffer containing 50 mm Hepes-NaOH, pH 7.4, 50 mm KCl, 5 mm EGTA, 2 mmMgCl2, 20 µm cytochalasin B, 1 mmdithiothreitol, 2 mm phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin, 1 µg/ml of pepstatin A, and 10 µg/ml of aprotinin. The resulting cell lysates were centrifuged at 16,000 × g for 15 min at 4 °C, and the resulting supernatants were stored in −80 °C until use. Mitochondrial and cytosolic fractions were prepared as described (31Han Z. Li G. Bremner T.A. Lange T.S. Zhang G. Jemmerson R. Wyche J.H. Hendrickson E.A. Cell Death Differ. 1998; 5: 469-479Crossref PubMed Scopus (27) Google Scholar). In brief, cells (5 × 107) were incubated for 10 min on ice in 0.2 ml of a lysis buffer containing 10 mmHepes-NaOH (pH 7.4), 5 mm MgCl2, 5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, as well as leupeptin, aprotinin, and antipain each at 50 µg/ml. They were then subjected to six passages through a syringe fitted with a 26-gauge needle. The resulting lysate was mixed with 1.25 ml of the same lysis buffer containing 0.3 m sucrose. The mixture was centrifuged at 500 × g for 10 min at 4 °C to remove the nuclei, and the resulting supernatant was centrifuged at 10,000 × g for 10 min at 4 °C. The pellet and supernatant from this second centrifugation were stored at −80 °C as the mitochondrial and cytosolic fractions, respectively. Cell lysates (20 µg of protein) or mitochondrial and cytosolic fractions (50 µg of protein) were resolved by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot analysis with antibodies to poly(ADP-ribose) polymerase (PARP) (Enzyme Synthesis, Dublin, CA), to caspases-3 and 9, cytochrome c, Bcl-2 or Bax (Santa Cruz Biotechnology, Santa Cruz, CA), or to c-IAP-1 (PharMingen, San Diego, CA). Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents (Amersham Pharmacia Biotech). Equal loading and transfer of proteins among lanes were verified by immunoblot analysis with antibodies to actin (Amersham Pharmacia Biotech) or by staining the membrane with Ponceau S. The activities of caspases-3, 8, and 9 were assayed fluorometrically with specific fluorogenic substrates: MOCAc-Asp-Glu-Val-Asp-Ala-Pro-Lys(DNP)-NH2(Peptide Institute, Osaka, Japan), Ac-Ile-Glu-Thr-Asp-MCA (Peptide Institute), and Ac-Leu-Glu-His-Asp-AFC (Calbiochem), respectively. Cell extracts (60 µg of protein) were incubated for 1 h at 37 °C with 600 nm substrate in a reaction mixture (500 µl) containing 10 mm Hepes-NaOH, pH 7.4, 40 mm β-glycerophosphate, 50 mm NaCl, 2 mm MgCl2, 5 mm EGTA, 1 mm dithiothreitol, 2 mm ATP, 10 mmcreatine phosphate, and 50 µg/ml of creatine kinase. The fluorescence of the cleaved substrates was measured with a Hitachi F-4500 spectrophotometer at excitation and emission wavelengths, respectively, of 328 and 393 nm for MOCAc-DEVDAPK(DNP)-NH2, 380 and 460 nm for Ac-IETD-MCA, and 400 and 505 nm for Ac-LEHD-AFC. Caspase activities did not differ substantially among the AT and control cell lines under basal conditions. We have previously shown that γ-radiation induces a markedly greater increase in the apoptotic index of AT5BIVA cells than it does in that of control MRC5CV1 cells (23Jung M. Zhang Y. Dimtchev A. Dritschilo A. Radiat. Res. 1998; 149: 596-601Crossref PubMed Scopus (53) Google Scholar). We, therefore, investigated the effects of IR on the apoptotic index of two additional AT fibroblast lines, AT3BIVA and AT4BIVA. Apoptotic cells were apparent within 24 h after irradiation of AT3BIVA, AT4BIVA, and AT5BIVA fibroblasts, although the apoptotic index varied. At 72 h after irradiation, the apoptotic indexes of AT4BIVA (31%) and AT5BIVA (35%) cells were similar to each other and markedly greater than that of AT3BIVA cells (12%) (Fig. 1 a), suggesting differences in the cell death programs among these AT cell lines. The apoptotic index of MRC5CV1 cells 72 h after irradiation was only 2.3%. Various proteins that contribute to the maintenance of cellular integrity are cleaved by caspases during apoptosis. For example, the nuclear enzyme PARP, which plays an important role in the repair of DNA damage and in the maintenance of genomic integrity (32Kaufmann S.H. Desnoyers S. Ottaviano Y. Davidson N.E. Poirier G.G. Cancer Res. 1993; 53: 3976-3985PubMed Google Scholar, 33Lazebnik Y.A. Kaufman S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2347) Google Scholar), is specifically cleaved between its NH2-terminal DNA-binding domain and its multifunctional COOH-terminal domain by caspase-3 or caspase-3-like proteases early during the execution phase of apoptosis (9Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2180) Google Scholar). Therefore, we monitored the amounts of PARP after exposure of AT and control cells to IR. Immunoblot analysis revealed the presence of intact PARP (116 kDa) in all untreated cells (Fig. 1 b). A cleavage product of PARP (89 kDa) appeared within 24–48 h after irradiation in AT5BIVA and AT4BIVA cells but not in AT3BIVA or control cells, correlating with the effects of IR on the apoptotic indexes of these cells. These observations suggest the importance of caspase-3 as a downstream protease responsible for the cleavage of PARP during IR-induced apoptosis in AT4BIVA and AT5BIVA cells. In addition, the abundance of intact PARP gradually increased after the exposure of AT3BIVA and AT4BIVA cell lines to IR. We also measured the activities of caspases-1, 3, 6, and 8 in cell extracts prepared from AT and control cells at various times after irradiation. Fluorometric determination of the cleavage of specific fluorogenic peptide substrates revealed little activity of caspases-1, 6, and 8 (data not shown), but a marked increase in caspase-3 activity in AT5BIVA and AT4BIVA cells that was apparent at 24 h and maximal at 48 h after exposure to IR (Fig. 1 c). In contrast, only a small increase in caspase-3 activity was apparent in AT3BIVA or MRC5CV1 cells by 72 h after irradiation. Immunoblot analysis also revealed that the time course of the conversion of procaspase-3 to the active protease in AT4BIVA and AT5BIVA cells was consistent with the results obtained with the fluorometric activity assay (Fig.1 d). Also consistent with the activity data, the cleavage of procaspase-3 was not detected in AT3BIVA and control cells up to 72 h after irradiation. We next examined the effects of γ-irradiation on the activity of caspase-9 in AT and control cells. Fluorometric assay revealed that IR induced a marked time-dependent increase in caspase-9 activity that was apparent as early as 24 h after irradiation in both AT4BIVA and AT5BIVA cells (Fig. 2 a). In contrast, the activity of caspase-9 was increased only slightly by IR in AT3BIVA and MRC5CV1 cells. These results were confirmed by immunoblot analysis of the conversion of procaspase-9 to the active enzyme. Thus, whereas IR induced the proteolytic processing of procaspase-9 to active caspase-9 in AT4BIVA and AT5BIVA cells, no such effect was apparent in AT3BIVA or control cells (Fig. 2 b). IR did not induce the cleavage of procaspase-8 in any of the four cell lines examined (data not shown). Taken together, these data implicated caspases-3 and 9 as effectors of IR-induced apoptosis in AT4BIVA and AT5BIVA cells. To determine a causal relationship between caspase-9 and 3 activities, AT5BIVA cells were transfected with plasmid DNA containing either a dominant negative form, which is an alternatively spliced isoform and lacks the central large subunit caspase domain (28Srinivasula S.M. Ahmad M. Guo Y. Zhan Y. Lazebnik Y. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1999; 59: 999-1002PubMed Google Scholar), or the active site mutant (C287A) form of caspase-9 cDNA (29Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (967) Google Scholar). At 24 h posttransfection, cells were exposed to IR and harvested at indicated intervals. As shown in Fig. 3, in the expression of either mutant form of caspase-9, the apoptotic index was significantly reduced (∼50%) within 48 h after irradiation (Fig. 3 a). Both caspase-9 and 3 activities were also markedly inhibited as well (Fig. 3, b and c); however, the vector alone did not modify the IR-induced apoptotic index and caspases-9 and 3 activities. These data show that the inhibition of caspase-9 activity attenuates a cellular apoptotic response and the activation of caspase-3, supporting caspase-9 as an upstream effector of caspase-3. In turn, caspase-9 inhibition reduced caspase-3 activity. This observation is consistent with recent reports suggesting that caspase-9 can initiate a caspase-cascade involving caspase-3 (28Srinivasula S.M. Ahmad M. Guo Y. Zhan Y. Lazebnik Y. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1999; 59: 999-1002PubMed Google Scholar, 29Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (967) Google Scholar,34Thornberry 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 (1849) Google Scholar, 35Green D. Kroemer G. Trends Cell Biol. 1998; 8: 267-271Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar, 36Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). The role of the mitochondrial pathway of apoptosis in the IR-induced death of AT cells was examined by immunoblot analysis of the abundance of Bcl-2, an antiapoptotic protein, and of Bax, an effector of this pathway, in a mitochondrial fraction. These proteins regulate apoptosis by controlling the release of cytochromec from mitochondria (13Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4276) Google Scholar, 14Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4410) Google Scholar). The basal amounts of Bax and of Bcl-2 did not differ markedly among the three AT cell lines, although the abundance of these proteins in the AT cells was greater than that in MRC5CV1 cells (Fig.4). The abundance of Bax in the mitochondrial fraction of AT cells but not in that of control cells was increased 48 h after irradiation (Fig. 4 a). In contrast, the amount of Bcl-2 in the AT cell lines but not that in control cells was decreased 48 h after irradiation (Fig.4 b). We next examined whether the IR-induced activation of caspase-9 in AT4BIVA and AT5BIVA cells is mediated by the release of mitochondrial cytochrome c. A cytosolic fraction was prepared from irradiated cells under conditions that maintain the integrity of mitochondria (31Han Z. Li G. Bremner T.A. Lange T.S. Zhang G. Jemmerson R. Wyche J.H. Hendrickson E.A. Cell Death Differ. 1998; 5: 469-479Crossref PubMed Scopus (27) Google Scholar). Immunoblot analysis of this fraction revealed that the basal amounts of cytochrome c in AT cells were similar to each other and greater than that in control cells (data not shown). The amount of cytochrome c released from mitochondria into the cytosol was markedly increased within 24–48 h after irradiation of AT4BIVA or AT5BIVA cells (Fig. 5). In contrast, cytochrome oxidase (subunit II), which was assayed as a mitochondrial marker protein, was undetectable in the cytosolic fractions prepared from these cell lines before or after irradiation (data not shown) confirming that the presence of cytochromec in the cytosol was not simply attributable to contamination by mitochondria. Together, these data suggest that IR-induced apoptosis in AT4BIVA and AT5BIVA cells is mediated, at least in part, by the mitochondrial-signaling pathway. The activation of caspase-9 in the mitochondrial-signaling pathway of apoptosis is inhibited by c-IAP proteins (10Deveraux Q.L. Roy N. Stennicke H.R. van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1242) Google Scholar, 15Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1137) Google Scholar, 17Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. Elia A. de la Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe S.W. Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar). To investigate the role of c-IAP-1 and c-IAP-2 in IR-induced apoptosis in AT cells, we determined the effects of γ-irradiation on the abundance of these proteins. The basal amounts of c-IAP-1 in AT4BIVA and AT5BIVA cells were substantially smaller than those in AT3BIVA or control cells (Fig.6 a). Densitometric analysis revealed that the abundance of c-IAP-1 in AT4BIVA, AT3BIVA, and control cells was 2, 6, and 8 times, respectively, that in AT5BIVA cells. The amount of c-IAP-1 was not affected by IR in any of the four cell lines examined (data not shown). Furthermore, c-IAP-2 was expressed at only a low level in the AT and control cell lines (data not shown). To further investigate the role of c-IAP-1 in IR-induced apoptosis in AT5BIVA cells, which exhibited the highest apoptotic index in response to IR as well as the lowest abundance of c-IAP-1, we transfected these cells with a plasmid encoding an hemagglutinin-tagged fragment of human c-IAP (residues 1–342) under the control of the cytomegalovirus promoter. After G418 selection and subcloning, one G418-resistant clone (AT5BIVA-c-IAP-1/CL3) that overexpressed hemagglutinin-tagged c-IAP-1 (Fig. 6 b) was subjected to further analysis. The extent of IR-induced apoptosis (as reflected by the apoptotic index) in the transfected cells was reduced by ∼50% compared with that observed in the parental AT5BIVA cells (data not shown). The extent of IR-induced cleavage of PARP was also reduced in AT5BIVA-c-IAP-1/CL3 cells (Fig.7 a). Furthermore, fluorometric assays revealed that the IR-induced increases in the activities of caspase-3 (Fig. 7 b) and caspase-9 (Fig. 7 c) were substantially reduced in the transfected cells compared with those apparent in the parental cells. These data suggest that c-IAP-1 inhibits the activation of caspases-3 and 9, and that the abundance of this protein is a critical factor in determining the extent of IR-induced apoptosis in AT cells. Apoptotic cell death is one response to the genotoxic effects of ionizing radiation. We previously showed that radiosensitive AT5BIVA cells undergo apoptosis in response to γ-irradiation (23Jung M. Zhang Y. Dimtchev A. Dritschilo A. Radiat. Res. 1998; 149: 596-601Crossref PubMed Scopus (53) Google Scholar). We have now shown that the apoptotic index differs among three AT cell lines after exposure to IR. Furthermore, IR-induced apoptotic signaling in AT4BIVA and AT5BIVA cells that showed the highest apoptotic indexes appears to be mediated by the release of mitochondrial cytochromec and the activation of caspases-9 and 3. The IR-induced release of mitochondrial cytochrome c into the cytosol in these two AT cell lines coincided with a decrease in the abundance of Bcl-2. The basal intracellular concentration of c-IAP-1 in AT and control cell lines also appeared to be inversely related to the apoptotic index after exposure to IR, suggesting a role for c-IAP-1 in limiting the extent of caspase-9 activation and apoptosis in these cells. This conclusion was supported by the observation that overexpression of mutant caspase-9 and c-IAP-1 in AT5BIVA cells reduced the extent of apoptosis as well as that of caspase-9 activation in response to γ-irradiation. The activation of caspases is reflected in the proteolysis of intracellular proteins associated with cellular integrity. PARP is a major substrate of caspase-3 during apoptosis. We have now shown that the extent of IR-induced cleavage of PARP is consistent with the apoptotic index of AT and control cells, indicating the importance of caspase-3 as a downstream protease during IR-induced apoptosis in AT4BIVA and AT5BIVA cells. The mechanism and role of the IR-induced increase in the abundance of PARP that are also apparent in AT3BIVA and AT4BIVA cell lines examined remain unknown. The activation of caspase-9 is mediated by a mitochondrial signaling that involves the release of cytochrome c and the activation of caspase-3 (12Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4461) Google Scholar, 13Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4276) Google Scholar, 14Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4410) Google Scholar). Using dominant negative and active site mutant forms of caspase-9, we demonstrate that caspase-9 is involved in IR-induced apoptotic signaling and activation of caspase-3. Furthermore, the amount of cytochrome c in the cytosol of AT4BIVA and AT5BIVA cells was markedly increased by exposure to IR. The basal abundance of cytochrome c in the cytosol of AT cells was greater than that apparent in control cells, which possibly contributes to cell death, results from the impairment in mechanisms that protect against oxidative stress in AT cells (37Watters D. Kedar P. Spring K. Bjorkman J. Chen P. Gatei M. Birrell G. Garrone B. Srinivasa P. Crane D.I. Lavin M.F. J. Biol. Chem. 1999; 274: 34277-34282Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 38Takao N. Li Y. Yamamoto K.-i. FEBS Lett. 2000; 472: 133-136Crossref PubMed Scopus (86) Google Scholar). Taken together, our data suggest that IR-induced apoptosis in AT fibroblasts is mediated by this mitochondrial-signaling pathway involving cytochrome c release and the activation of caspases-9 and 3. The ratio between the amounts of the proapoptotic Bax and the antiapoptotic Bcl-2 is an important determinant of cell survivalversus cell death (39Reed J.C. Jurgensmeier J.M. Natsuyama S. Biochim. Biophys. Acta. 1998; 1366: 127-137Crossref PubMed Scopus (351) Google Scholar). Both proteins regulate the release of cytochrome c from mitochondria with Bax promoting this process and Bcl-2 preventing it (40Tsujimoto Y. Genes Cells. 1998; 3: 697-707Crossref PubMed Scopus (541) Google Scholar, 41Kane D.J. Sarafian T.A. Anton R. Hahn H. Gralla E.B. Valentine J.S. Ord T. Bredesen D.E. Science. 1993; 262: 1274-1277Crossref PubMed Scopus (1614) Google Scholar, 42Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (940) Google Scholar, 43Rosse T. Olivier R. Monney L. Rager M. Conus S. Fellay I. Jansen B. Borner C. Nature. 1998; 391: 496-499Crossref PubMed Scopus (796) Google Scholar, 44Hockenbery D.M. Oltvai Z.N. Yin X.M. Milliman C.L. Korsmeyer S.J. Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3294) Google Scholar, 45Alnemri E.S. J. Cell. Biochem. 1997; 64: 33-42Crossref PubMed Scopus (290) Google Scholar, 46Slee E.A. Harte M.T. Kluck R.M. Wolf B.B. Casiano C.A. Newmeyer D.D. Wang H.G. Reed J.C. Nicholson D.W. Alnemri E.S. Green D.R. Martin S.J. J. Cell Biol. 1999; 144: 281-292Crossref PubMed Scopus (1681) Google Scholar, 47Amarante-Mendes G.P. McGahon A.J. Nishioka W.K. Afar D.E. Witte O.N. Green D.R. Oncogene. 1998; 16: 1383-1390Crossref PubMed Scopus (199) Google Scholar). IR induced an increase in the amount of Bax and a decrease in the abundance of Bcl-2 in the AT cell lines examined, suggesting that this change in the Bax/Bcl-2 ratio contributes to the release of mitochondrial cytochrome c and apoptosis in these AT cells. Furthermore, the basal abundance of c-IAP-1 in AT4BIVA and AT5BIVA cells was less than that apparent in AT3BIVA and control cells, consistent with the differences in the apoptotic indexes among these cells and the fact that the abundance of c-IAP is a critical factor in the apoptotic process (20Budihardjo I. Oliver H. Lutter M. Luo X. Wang X. Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2268) Google Scholar). The overexpression of c-IAP-1 in AT5BIVA cells resulted in a marked inhibition of IR-induced apoptosis, PARP cleavage, and an activation of caspase-9, suggesting that the low concentration of c-IAP-1 in AT5BIVA cells contribute to their high sensitivity to IR. These data are consistent with observations by other laboratories demonstrating that IAPs directly or indirectly inhibit the activation of caspases-3, 7, and 9, and that the endogenous threshold level of the IAPs is correlated with cellular sensitivities to apoptosis inducing stimuli in various cell types (10Deveraux Q.L. Roy N. Stennicke H.R. van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1242) Google Scholar, 18Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1719) Google Scholar, 20Budihardjo I. Oliver H. Lutter M. Luo X. Wang X. Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2268) Google Scholar, 48Takahashi R. Deveraux Q.L. Tamm I. Welsh K. Assa-Munt N. Salvesen G.S. Reed J.C. J. Biol. Chem. 1998; 273: 7787-7790Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). In summary, the apoptotic index was shown to differ markedly among three AT fibroblast lines after exposure to γ-radiation being greatest in the AT4BIVA and AT5BIVA lines. Apoptosis in these cells appeared to be mediated by the mitochondrial-signaling pathway involving the release of cytochrome c into the cytosol and the activation of caspases-9 and 3. A reduced abundance of c-IAP in these two AT cell lines appeared to be a critical factor in their increased susceptibility to IR-induced apoptosis. Different mutations in the atm gene thus may contribute to the variation in the response of the three AT fibroblast lines examined to IR. We thank Drs. Emad S. Alnemri and Srinivasa Srinivasula at Thomas Jefferson University for providing vectors containing mutant forms of caspase-9, J. Tuturea for technical assistance, and E. North for help in the preparation of the manuscript.

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