Initiation of DNA Fragmentation during Apoptosis Induces Phosphorylation of H2AX Histone at Serine 139
2000; Elsevier BV; Volume: 275; Issue: 13 Linguagem: Inglês
10.1074/jbc.275.13.9390
ISSN1083-351X
AutoresEmmy P. Rogakou, Wilberto Nieves‐Neira, Chye Boon, Yves Pommier, William M. Bonner,
Tópico(s)DNA Repair Mechanisms
ResumoHistone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A histones by the presence of an evolutionarily conserved C-terminal motif, -KKATQASQEY. The serine residue in this motif becomes rapidly phosphorylated in cells and animals when DNA double-stranded breaks are introduced into their chromatin by various physical and chemical means. In the present communication we show that this phosphorylated form of H2AX, referred to as γ-H2AX, appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments. γ-H2AX forms before the appearance of internucleosomal DNA fragments and the externalization of phosphatidylserine to the outer membrane leaflet. γ-H2AX formation is inhibited byN-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the inhibitor of caspase-activated DNase, and it is induced when DNase I and restriction enzymes are introduced into cells, suggesting that any apoptotic endonuclease is sufficient to induce γ-H2AX formation. These results indicate that γ-H2AX formation is an early chromatin modification following initiation of DNA fragmentation during apoptosis. Histone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A histones by the presence of an evolutionarily conserved C-terminal motif, -KKATQASQEY. The serine residue in this motif becomes rapidly phosphorylated in cells and animals when DNA double-stranded breaks are introduced into their chromatin by various physical and chemical means. In the present communication we show that this phosphorylated form of H2AX, referred to as γ-H2AX, appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments. γ-H2AX forms before the appearance of internucleosomal DNA fragments and the externalization of phosphatidylserine to the outer membrane leaflet. γ-H2AX formation is inhibited byN-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the inhibitor of caspase-activated DNase, and it is induced when DNase I and restriction enzymes are introduced into cells, suggesting that any apoptotic endonuclease is sufficient to induce γ-H2AX formation. These results indicate that γ-H2AX formation is an early chromatin modification following initiation of DNA fragmentation during apoptosis. caspase-activated DNase inhibitor of CAD N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone DNA fragmentation factor acetic acid/urea/Triton X-100 acetic acid/urea/cetyltrimethylammonium bromide phosphate-buffered saline megabase pairs terminal DNA deoxynucleotidylexotransferase end labeling In multicellular organisms, programmed cell death is an essential mechanism in morphogenesis, development, differentiation, and homeostasis, which occurs primarily through an evolutionarily conserved process termed apoptosis. Typical apoptosis is distinguished from necrosis as the former involves the activation of specific pathways that result in characteristic morphological features including DNA fragmentation, chromatin condensation, cytoplasmic and nuclear pycnosis, and the formation of apoptotic bodies (1.Kerr J.F.R. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12927) Google Scholar, 2.Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4163) Google Scholar, 3.Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2257) Google Scholar, 4.Vaux D.L. Korsmeyer S.J. Cell. 1999; 96: 245-254Abstract Full Text Full Text PDF PubMed Scopus (1369) Google Scholar). Although many stimuli can trigger various pathways to apoptosis, all these pathways converge to a common process involving the activation of a cascade of highly homologous endopeptidases, named caspases, that specifically cleave substrates at aspartic acid residues (5.Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1943) Google Scholar). Subsequently, the activated execution caspases (e.g. caspase-3, -6, and -7) cleave other polypeptide substrates including lamin (6.Takahashi A. Alnemri E.S. Lazebnik Y.A. Fernandes-Alnemri T. Litwack G. Moir R.D. Goldman R.D. Poirier G.G. Kaufmann S.H. Earnshaw W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8395-8400Crossref PubMed Scopus (472) Google Scholar), actin (7.Mashima T. Naito M. Noguchi K. Miller D.K. Nicholson D.W. Tsuruo T. Oncogene. 1997; 14: 1007-1012Crossref PubMed Scopus (218) Google Scholar), poly(ADP-ribose) polymerase (8.Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2351) Google Scholar), D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family of GTPases (9.Na S. Chuang T.-H. Cunningham A. Turi T.G. Hanke J.H. Bokoch G.M. Danley D.E. J. Biol. Chem. 1996; 271: 11209-11213Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), nuclear scaffold attachment factor A (10.Gohring F. Schwab P.N. Leist M. Fackelmayer F.O. EMBO J. 1997; 16: 7361-7371Crossref PubMed Scopus (124) Google Scholar), p21-activated kinase 2 (11.Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (605) Google Scholar), and DNA-dependent protein kinase (12.Han Z. Nusrat M. Carter T. Reeves W.H. Wyche J.H. Hendrickson E.A. J. Biol. Chem. 1996; 271: 25035-25040Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13.Song Q. Lees-Miller S.P. Kumar S. Zhang Z. Chan D.W. Smith G.C. Jackson S.P. Alnemri E.S. Litwack G. Khanna K.K. Lavin M.F. EMBO J. 1996; 15: 3238-3246Crossref PubMed Scopus (330) Google Scholar). Although the significance of these cleavages in apoptosis is not fully understood, it seems that execution caspases exert their roles either by blocking pathways that might interfere with the apoptotic program or by activating pathways that advance the program (14.Wyllie A. Nature. 1998; 391: 20-21Crossref PubMed Scopus (53) Google Scholar). Whereas caspase activation follows a decision-making step, where the Bcl-2 family members seem to play a crucial role (15.Reed J.C. Nature. 1997; 387: 773-776Crossref PubMed Scopus (1391) Google Scholar), activated caspases can also be controlled by endogenous inhibitors (16.Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1724) Google Scholar). These factors attest to the complex organization and tight control of the apoptotic program. A caspase-activated DNase (CAD)1 and its inhibitor (ICAD) have been identified in mouse lymphoma cells, whereas in human cells DNA fragmentation factor (DFF) has been identified as a heterodimer of 40- (DFF40) and 45-kDa subunits (17.Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar, 18.Enari M. Sakahira H. Yokoyamma H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2812) Google Scholar, 19.Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1427) Google Scholar, 20.Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar). DFF40 has been shown to induce chromatin condensation and formation of apoptotic bodies in isolated nuclei (20.Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar). Although chromatin changes are a hallmark of apoptosis, little is known about the specific events and mechanisms involved in this process. H2AX is a member of the H2A histone family, which includes three distinct subfamilies as follows: H2A1-H2A2, H2AZ, and H2AX (21.West M.H.P. Bonner W.M. Biochemistry. 1980; 19: 3238-3245Crossref PubMed Scopus (219) Google Scholar). Each H2A member occupies an analogous position in different nucleosomes, an attribute leading to considerable nucleosomal diversity. Our laboratory has recently demonstrated that histone H2AX becomes phosphorylated on serine residue 139 in the unique C-terminal motif SQEY within seconds after the induction of DNA double-strand breaks in mammalian cells and tissues (22.Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4203) Google Scholar). The rejoining of DNA double-strand breaks is essential if a cell is to remain capable of proliferation. However, during apoptosis a cell is rendered not only incapable of proliferation but is fragmented into apoptotic bodies to be reabsorbed by the organism. We examined whether H2AX is phosphorylated on serine residue 139 (a form referred to as γ-H2AX) as a result of apoptotic DNA fragmentation. We demonstrate that γ-H2AX formation is a cellular response to endonuclease-mediated DNA fragmentation downstream from caspase activation during apoptosis. The HL60 cell line was a gift from Dr. T. Breitman (NCI). The Jurkat and MCF7 cell lines were obtained from ATCC (Manassas, VA). The NCI/ADR-RES cell line, formerly known as MCF7/adr, was obtained from the DTP Human Tumor Cell Line Screen (Developmental Therapeutics Program, NCI). All cell lines were cultured in RPMI 1640 medium containing 10% fetal calf serum at 37 °C in a humidified atmosphere. For Jurkat cell cultures, apoptosis was induced by addition of 0.5 μg/ml of a mouse monoclonal activating antibody to human FAS (IgM, CH-11, Upstate Biotechnology, Lake Placid, NY), 0.5 μm staurosporine (Sigma), or 50 μm etoposide (Calbiochem). For HL60 cells, apoptosis was induced with 0.5 μm staurosporine or 10 mmEGTA. For NCI/ADR-RES and MCF7 cells, apoptosis and cell death was induced with 1 μm 7-hydroxystaurosporine (UCN-01) (Kyowa Hakko Co., Tokyo, Japan). For measurements of MCF7 and NCI/ADR-RES cell viability, 300 cells were seeded in triplicate in T25 flasks and allowed to attach overnight. The cells were then incubated with 1 μm UCN-01 for 24 h. After extensive washing, growth was continued for 10–14 days. Colonies were fixed in methanol, stained with 0.05% methylene blue, and counted. Suspension cultures were pelleted by centrifugation at 0 °C at 1,000 ×g for 20 min. Attached cultures were scraped and pelleted as described above. The pellets were washed twice in PBS and histones were extracted in 3 volumes of 0.5 n HCl for 30 min on ice. Histones were subjected to high resolution gel electrophoresis (23.Bonner W.M. West M.H.P. Stedman J.D. Eur. J. Biochem. 1980; 109: 17-23Crossref PubMed Scopus (194) Google Scholar), and quantitative analysis was performed as described in Rogakouet al. (22.Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4203) Google Scholar). Briefly, the acid extracts were prepared and loaded onto 8 m urea, 10% first dimension acetic acid/urea/Triton X-100 (AUT) gels. After electrophoresis, proteins were stained with Coomassie Blue R-250. The regions of interest of the first dimension gels were excised and subjected to electrophoresis in second dimension acetic acid/urea/cetyltrimethylammonium bromide (AUC) gels. Images of the H2A region of the Coomassie Blue R-250-stained two-dimensional gels were imaged as TIF files in an EagleEye II gel documentation system with Eaglesight software (version 3.2, Stratagene Cloning Systems, La Jolla, CA). The γ-H2AX and unmodified H2AX spots were quantitated with ImageQuant software (version 2, Molecular Dynamics Corp., San Diego, CA). The amount of γ-H2AX is expressed as its percentage of total H2AX. DNA fragmentation was assayed by three methods. For nucleosomal ladders, the phenol/chloroform/isoamyl alcohol procedure was used to extract DNA from aliquots of cell lysates (5 × 106 cells per sample) that had been digested with proteinase K. The DNA was ethanol-precipitated, dissolved in Tris-EDTA buffer, incubated with RNase A (50 μg/ml) for 30 min at 37 °C, and then subjected to analysis by electrophoresis in 1.5% agarose gels. For quantitative studies, DNA filter elution analysis was performed as described in Bertrand et al. (24.Bertrand R. Kohn K.W. Solary E. Pommier Y. Drug Dev. Res. 1995; 34: 138-144Crossref Scopus (35) Google Scholar). Briefly, exponentially growing cells were labeled with [methyl-14C]thymidine (58.6 μCi/mmol, NEN Life Science Products) at 0.02 μCi/ml for 24–30 h. After an overnight chase, the cells were harvested, loaded onto Metricel filters (0.8 μm pore size, Gelman Sciences, Ann Arbor, MI), washed with 0.02m EDTA, and lysed with LS-10 buffer (0.2% sodium Sarkosyl, 2 m NaCl, 0.04 m EDTA, pH 10.0). The wash and lysis fractions were allowed to drip through the filters into scintillation vials. The filters were placed in scintillation vials, and the DNA was solubilized in 1 n HCl at 65 °C. After measuring the radioactivity in each sample, DNA fragmentation was determined as the percentage of dpm in the lysis and wash fractions (fragmented DNA) divided by the total dpm (total DNA). The background fragmentation was subtracted in order to compare the results of different experiments. In order to determine the timing of the initial large size fragmentation of DNA during apoptosis, aliquots of Jurkat cell cultures were taken at various times after addition of anti-Fas and subjected to pulse-field gel analysis according to standard protocols. Briefly, cells were embedded in agarose plugs and digested with proteinase K. The plugs were placed in the wells of a long 0.8% agarose gel in 0.5× TBE. Electrophoresis was ramped for 144 h at 60 V from 600 to 3600 s in a CHEF II apparatus (Bio-Rad). Gels were stained with ethidium bromide and imaged with the EagleEye II (Stratagene Cloning Systems, La Jolla, CA). DNA breaks in single cells were detected using the TUNEL assay (APO-BRDUTM kit, PharMingen, San Diego, CA). Subsequently, samples were processed through a fluorescent-activated cell analyzer, using the Cell Quest software (version 1.2, FACScan, Becton-Dickinson, Franklin Lakes, NJ). 105–106 Jurkat cells were centrifuged at 1000 × g for 10 min at 20 °C and washed with 0.5 ml of cold PBS. Phosphatidylserine externalization to the outer cell membrane leaflet was assayed with annexin-V (TACSTM Annexin-V FITC Kit, Trevigen). Cell suspensions were subsequently processed using the FACScan as described above. pEF-mICAD-Ldm, which carries FLAG-tagged caspase-resistant mouse ICAD-L in a mammalian expression vector pEF-BOS (18.Enari M. Sakahira H. Yokoyamma H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2812) Google Scholar, 19.Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1427) Google Scholar), was a generous gift from Dr. S. Nagata. pEF-mICAD-Ldm was transiently transfected into Jurkat cells with FuGENE 6 reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions. Flag epitope was detected under confocal microscope (Nikon eclipse TE300, PCM 2000) with anti-Flag antibody (clone M2, Eastman Kodak Co.). Electroporation was performed as described (25.Morgan W.F. Day J.P. Methods Mol. Biol. 1995; 48: 63-71PubMed Google Scholar). Briefly, Jurkat cells were washed with PBS and resuspended in electroporation buffer at a concentration of 6.5 × 106 cells/ml. Aliquots containing 0.8 ml of the cell suspension were transferred to electroporation cuvettes (0.4-cm electrode gap). Bovine serum albumin (BSA), RNase-free DNase I (final concentrations of 125 or 375 units/ml, Roche Molecular Biochemicals), AluI, and Sau3AI (final concentrations of 30 units/ml, New England Biolabs, Beverly, MA) were added to the cell samples that were subjected to electroporation with a Gene Pulser (Bio-Rad) set at 0.3 kV and 125 microfarads. After electroporation, cells were incubated in complete RPMI 1640 for 20 min at 37 °C. Each sample was then split into 2 aliquots for parallel γ-H2AX analysis by gel electrophoresis and DNA break analysis by the TUNEL assay as described above. Our laboratory recently reported that H2AX becomes phosphorylated on serine 139 when double-strand breaks are introduced into the DNA of cells exposed to a variety of environmental and exogenous agents (22.Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4203) Google Scholar). These DNA-damaging agents include ionizing radiation, radiomimetic compounds, and 365 nm light when the cellular DNA contains bromodeoxyuridine (22.Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4203) Google Scholar). Since chromatin fragmentation involving DNA double-stranded break formation is a hallmark of apoptosis, we examined whether H2AX phosphorylated on serine 139, termed γ-H2AX, was formed in several cell lines undergoing apoptosis in response to a variety of inducers including some that do not interact with DNA (Fig. 1). Jurkat cells, derived from a human acute T cell leukemia, undergoes apoptosis upon incubation with a variety of chemical and biological agents (Fig. 1,A–D). The protein kinase inhibitor staurosporine, a general and highly effective apoptotic inducer (26.Shao R.-G. Shimizu T. Pommier Y. Exp. Cell Res. 1997; 234: 388-397Crossref PubMed Scopus (86) Google Scholar, 27.Bertrand R. Solary E. Kohn K.W. O'Connor P.O. Pommier Y. Exp. Cell Res. 1994; 211: 314-321Crossref PubMed Scopus (471) Google Scholar), also induced γ-H2AX formation (Fig. 1 B). Thus, staurosporine apparently does not inhibit the kinase responsible for γ-H2AX formation during apoptosis. Etoposide (VP-16), a specific inhibitor of topoisomerase II, was also found to induce γ-H2AX during apoptosis in Jurkat cells (Fig.1 C). Jurkat plasma membranes contain Fas receptors that become aggregated when the cells are exposed to the appropriate antibody (28.Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Abstract Full Text PDF PubMed Scopus (2678) Google Scholar). The antibody acts similarly to an endogenously formed apoptotic inducer, Fas ligand. Thus this system mimics biologically programmed initiation of apoptosis (31.Watanabe-Fukunaga R. Brannan C.I. Copeland N.G. Jenkins N.A. Nagata S. Nature. 1992; 356: 314-317Crossref PubMed Scopus (2737) Google Scholar, 32.Ogasawara J. Watanabe-Fukunaga R. Adachi M. Matsuzawa A. Kasugal T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1815) Google Scholar, 33.Schneider P. Bodmer J.L. Holler N. Mattmann C. Scuderi P. Terskikh A. Peitsch M.C. Tschopp J. J. Biol. Chem. 1997; 272: 18827-18833Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). γ-H2AX was formed when Jurkat cell cultures were incubated with anti-Fas (Fig. 1 D), and internucleosomal DNA fragmentation was formed in Jurkat cells in response to the three apoptotic inducers (Fig. 1, B–D, insets). HL60 is a human leukemia cell line that also easily undergoes apoptosis upon incubation with a variety of chemical agents (29.Solary E. Bertrand R. Pommier Y. Leuk. & Lymphoma. 1994; 15: 21-32Crossref PubMed Scopus (86) Google Scholar). As with Jurkat cells, staurosporine induced the formation of both γ-H2AX and internucleosomal DNA fragmentation in HL60 cells (Fig. 1 F, main panel and inset). EGTA, a chelating agent of divalent cations, particularly Ca+2, also induced both DNA fragmentation and γ-H2AX formation (Fig. 1 G, main panel and inset). Whereas many types of endonuclease activities require divalent cations in vitro, DNA fragmentation does take place in HL60 cells in EGTA-containing medium. In contrast to the Jurkat and HL60 cell lines that grow in suspension, NCI/ADR-RES is an attached human breast carcinoma line that undergoes apoptosis when incubated with 7-hydroxystaurosporine (UCN-01), a clinical derivative of staurosporine (30.Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-402Crossref PubMed Scopus (119) Google Scholar). The DNA of NCI/ADR-RES cells does become fragmented during apoptosis but not extensively enough to form internucleosomal DNA fragmentation (30.Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-402Crossref PubMed Scopus (119) Google Scholar). γ-H2AX was found in NCI/ADR-RES cells undergoing apoptosis (Fig. 1, H andI). Thus, the results obtained with three cell lines and five inducers of apoptosis indicate that the appearance of γ-H2AX is a general characteristic of apoptosis. The demonstration that γ-H2AX formation was detectable in several cell lines during apoptosis (Fig. 1) prompted a more detailed examination to determine the timing of γ-H2AX formation. Since the formation of internucleosomal DNA fragmentation is a late event in apoptotic progression, resulting from digestion of large DNA fragments formed earlier in apoptosis (34.Bicknell G.R. Snowden R.T. Cohen G.M. J. Cell Sci. 1994; 107: 2483-2489Crossref PubMed Google Scholar, 35.Walker R.P. Sikorska M. Biochem. Cell Biol. 1994; 72: 615-623Crossref PubMed Scopus (88) Google Scholar), the filter elution procedure (24.Bertrand R. Kohn K.W. Solary E. Pommier Y. Drug Dev. Res. 1995; 34: 138-144Crossref Scopus (35) Google Scholar) was utilized to detect large double-stranded DNA fragments in a quantitative manner. DNA analysis by filter elution was coupled with histone gel analysis to examine Jurkat cell cultures incubated for various times in the presence of anti-Fas (Fig. 2 and Fig. 3,A and B). γ-H2AX was not detectable in the control culture or in the treated culture at 0.5 h (Fig. 2,A and B) and was at the limit of detection at 1 h (Fig. 2 C). However, γ-H2AX was clearly detectable at 1.5 h (Fig. 2 D) and increased further until 3 h (Fig. 2, E–G). The amount of γ-H2AX then declined somewhat but remained at significant levels even at 8 h (Fig.2 I). When γ-H2AX formation was quantified (Fig.3 A) and compared with DNA fragmentation measured by neutral filter elution in parallel samples (Fig. 3 B), the former appeared abruptly at 1.5 h while γ-H2AX increased gradually and became substantially above background after 1.5 h.Figure 3Kinetics of γ-H2AX formation compared with DNA fragmentation during apoptosis.γ-H2AX formation (A and C) and DNA fragmentation (B and D) were quantitated using filter elution analysis for the latter (“Experimental Procedures”).A and B, Jurkat cell cultures incubated with 0.5 μg/ml anti-Fas for the times indicated. C andD, NCI/ADR-RES (solid lines) and MCF7 (dotted lines) cell cultures incubated with 1 μm UCN-01 for the times indicated. Each data point represents the average of three determinations; error barsindicate the standard deviation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These results suggested γ-H2AX to be a sensitive and early indicator of the double-stranded DNA fragmentation that occurs during apoptosis. Similar studies were performed with NCI/ADR-RES cells, a human breast carcinoma cell line that undergoes apoptosis more slowly than Jurkat or HL60 cells when incubated with UCN-01 (29.Solary E. Bertrand R. Pommier Y. Leuk. & Lymphoma. 1994; 15: 21-32Crossref PubMed Scopus (86) Google Scholar). In these cells, γ-H2AX was found in substantial amounts by 8 h (Fig. 3 C), while DNA fragmentation was not detected until after 8 h (Fig.3 D). These data show that, irrespective of the different stimuli and different apoptosis initiation events, γ-H2AX appears early during apoptosis concurrently with small levels of initial double-stranded breakage of the genomic DNA. In contrast to NCI/ADR-RES, MCF7, another breast carcinoma from the NCI Anticancer Drug Screen, undergoes cell death without exhibiting the typical apoptotic phenotype (30.Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-402Crossref PubMed Scopus (119) Google Scholar). 2W. Nieves-Neira and Y. Pommier, unpublished observations. This difference is possibly because MCF7 cells lack caspase-3 (37.Janicke 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 (1727) Google Scholar), an enzyme known to activate downstream apoptotic events including DNA fragmentation. In the presence of 1 μm UCN-01, both the MCF7 and the NCI/ADR-RES cell lines had greatly decreased clonogenic survival values, 2% for MCF7 and 8% for NCI/ADR-RES. These results indicated that cell death had occurred in both lines to similar extents. However, the MCF7 cells, when incubated with UCN-01, exhibited neither DNA fragmentation (Fig. 3 D) nor γ-H2AX formation (Fig.3 C). This observation indicates that γ-H2AX formation is observed in dying cells only if and when their DNA is fragmented. As shown above (Fig. 2 D), γ-H2AX is present in substantial amounts in Jurkat cultures 1.5 h after addition of anti-Fas. Changes in the plasma membranes of cells are also known to occur early in apoptosis. One of these changes, externalization of phosphatidylserine to the outer plasma membrane, can be assayed by annexin-V binding (38.Naito M. Nagashima K. Mashima T. Tsuruo T. Blood. 1997; 89: 2060-2066Crossref PubMed Google Scholar). When Jurkat cells undergoing apoptosis were examined by flow cytometry for the externalization of phosphatidylserine, the major increase in the number of cells capable of binding annexin-V was apparent after 3 h incubation with anti-Fas, although smaller increases were apparent at 2 and 2.5 h (Fig. 4). However, since γ-H2AX was already present in substantial amounts at 1.5 h (Figs.2 D and 3 A), these results demonstrate that γ-H2AX is formed in the chromatin before phosphatidylserine externalization is detectable in the outer plasma membrane of apoptotic cells. DNA fragmentation during apoptosis is the result of an endonuclease that becomes activated by caspase action (17.Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar, 18.Enari M. Sakahira H. Yokoyamma H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2812) Google Scholar). DNA fragmentation can be inhibited by incubating cultures withN-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD), a peptide that inhibits caspase activity (39.Dolle R.E. Hoyer D. Prasad C.V.C. Schmidt S.J. Helaszek C.T. Miller R.E. Ator M.A. J. Med. Chem. 1994; 37: 563-564Crossref PubMed Scopus (177) Google Scholar, 40.Armstrong R.C. Aja T. Xiang J. Gaur S. Krebs J.F. Hoang K. Bai X. Korsmeyer S.J. Karanewsky D.S. Fritz L.C. Tomaselli K.J. J. Biol. Chem. 1996; 271: 16850-16855Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). When Jurkat cell cultures were incubated for 3 or 8 h with anti-Fas in the continuous presence or absence of ZVAD, the ZVAD-containing cultures exhibited background levels of DNA fragmentation and no detectable γ-H2AX formation (Fig. 5, Aand B). Similarly, NCI/ADR-RES cultures, when incubated with 1 μm UCN-01 plus 50 μm ZVAD for 12 or 24 h, exhibited only background levels for both DNA fragmentation and γ-H2AX formation (Fig. 5, C and D). 10 μm ZVAD, which only partially inhibited DNA fragmentation in this system, also only partially inhibited γ-H2AX formation (data not shown). These results suggest that γ-H2AX formation during apoptosis is a downstream consequence of caspase activation. The initial DNA fragments formed during apoptosis are in the size range 0.3–2.0 Mbp (34.Bicknell G.R. Snowden R.T. Cohen G.M. J. Cell Sci. 1994; 107: 2483-2489Crossref PubMed Google Scholar, 35.Walker R.P. Sikorska M. Biochem. Cell Biol. 1994; 72: 615-623Crossref PubMed Scopus (88) Google Scholar). In order to determine when γ-H2AX appears relative to the 0.3–2.0-Mbp DNA fragmentation, aliquots from Jurkat cell cultures undergoing apoptosis with anti-Fas were subjected to pulse-field electrophoretic analysis (Fig.6). The 0.3–2.0-Mbp DNA fragments appear in substantial amounts 1.5 h after the addition of anti-Fas; with longer times, more DNA material is seen at progressively shorter lengths. Whereas the apoptotic samples on the pulse-field gel contained increasing amounts of DNA fragments as large as 2.0 Mbp, there was no detectable amount of DNA in the 3–6- Mbp region, even in the samples taken at 0.5 and 1 h. This observation substantiates that the 0.3–2.0-Mbp fragments may be the initial fragments cleaved from intact chromatin during apoptosis (34.Bicknell G.R. Snowden R.T. Cohen G.M. J. Cell Sci. 1994; 107: 2483-2489Crossref PubMed Google Scholar, 35.Walker R.P. Sikorska M. Biochem. Cell Biol. 1994; 72: 615-623Crossref PubMed Scopus (88) Google Scholar). The presence of ZVAD in the cultures prevented the formation of DNA fragments of any size (Fig. 6). Thus, these findings indicate that γ-H2AX is formed concurrently with the initiation of DNA double-stranded breaks resulting from the apoptotic endonuclease activation. Caspases are known to trigger multiple pathways that result in the global apoptotic phenotype. One major pathway involves the activation of CAD/DFF40, which leads to DNA fragmentation and apparently to chromatin condensation (17.Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar). mICAD was transiently transfected into Jurkat cells (Fig. 7). Nineteen hours after transfection, transfectants and mock transfectants were treated with 1 μm staurosporine for 3 h. γ-H2AX formation was inhibited when mICAD was overexpressed (Fig. 7, A andB). The extent of inhibition was similar to the fraction of the cells found to express the FLAG epitope of the recombinant ICAD. Yet, the possibility that other apoptotic
Referência(s)