Subunit Structures and Stoichiometries of Human DNA Fragmentation Factor Proteins before and after Induction of Apoptosis
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m303807200
ISSN1083-351X
AutoresPiotr Widłak, Joanna Łanuszewska, Robert B. Cary, William T. Garrard,
Tópico(s)DNA Repair Mechanisms
ResumoDNA fragmentation factor (DFF) is one of the major endonucleases responsible for internucleosomal DNA cleavage during apoptosis. Understanding the regulatory checkpoints involved in safeguarding non-apoptotic cells against accidental activation of this nuclease is as important as elucidating its activation mechanisms during apoptosis. Here we address these issues by determining DFF native subunit structures and stoichiometries in human cells before and after induction of apoptosis using the technique of native pore-exclusion limit electrophoresis in combination with Western analyses. For comparison, we employed similar techniques with recombinant proteins in conjunction with atomic force microscopy. Before induction of apoptosis, the expression of DFF subunits varied widely among the cell types studied, and the chaperone/inhibitor subunits DFF45 and DFF35 unexpectedly existed primarily as monomers in vast excess of the latent nuclease subunit, DFF40, which was stoichiometrically associated with DFF45 to form heterodimers. DFF35 was exclusively cytoplasmic as a monomer. Nuclease activation upon caspase-3 cleavage of DFF45/DFF35 was accompanied by DFF40 homo-oligomer formation, with a tetramer being the smallest unit. Interestingly, intact DFF45 can inhibit nuclease activity by associating with these homo-oligomers without mediating their disassembly. We conclude that DFF nuclease is regulated by multiple pre- and post-activation fail-safe steps. DNA fragmentation factor (DFF) is one of the major endonucleases responsible for internucleosomal DNA cleavage during apoptosis. Understanding the regulatory checkpoints involved in safeguarding non-apoptotic cells against accidental activation of this nuclease is as important as elucidating its activation mechanisms during apoptosis. Here we address these issues by determining DFF native subunit structures and stoichiometries in human cells before and after induction of apoptosis using the technique of native pore-exclusion limit electrophoresis in combination with Western analyses. For comparison, we employed similar techniques with recombinant proteins in conjunction with atomic force microscopy. Before induction of apoptosis, the expression of DFF subunits varied widely among the cell types studied, and the chaperone/inhibitor subunits DFF45 and DFF35 unexpectedly existed primarily as monomers in vast excess of the latent nuclease subunit, DFF40, which was stoichiometrically associated with DFF45 to form heterodimers. DFF35 was exclusively cytoplasmic as a monomer. Nuclease activation upon caspase-3 cleavage of DFF45/DFF35 was accompanied by DFF40 homo-oligomer formation, with a tetramer being the smallest unit. Interestingly, intact DFF45 can inhibit nuclease activity by associating with these homo-oligomers without mediating their disassembly. We conclude that DFF nuclease is regulated by multiple pre- and post-activation fail-safe steps. Apoptosis, or programmed cell death, is a fundamental process essential for both development and maintenance of tissue homeostasis (for review, see Refs. 1Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar and 2Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4559) Google Scholar). Two major apoptotic pathways exist, the death receptor pathway and the mitochondrial pathway (3Green D.R. Cell. 2000; 102: 1-4Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar, 4Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar). Multiple apoptotic stimuli, which include activation of Fas receptors, serum starvation, ionizing radiation, or various drugs that target DNA, trigger the activation of proteases called caspases, which in turn initiate and execute the apoptotic program (5Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2451) Google Scholar). One of the hallmarks of the terminal stages of apoptosis is DNA breakdown, which has functional significance (6Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4153) Google Scholar, 7Wyllie A.H. Morris R.G. Smith A.L. Dunlop D. J. Pathol. 1984; 142: 66-77Crossref Scopus (1442) Google Scholar, 8Nagata S. Exp. Cell Res. 2000; 256: 12-18Crossref PubMed Scopus (741) Google Scholar, 9Zhang J. Xu M. Trends Cell Biol. 2002; 12: 84-89Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Although cell death can occur without significant DNA degradation, cell-autonomous DNA breakdown of tumor or virally infected cells functionally prepares the resulting apoptotic corpses for engulfment by phagocytes and as such eliminates the transforming potential of any hazardous oncogenes. Interestingly, patients or an animal model with defective apoptotic DNA processing have a predisposition to auto-immune disease, which is known to be associated with the appearance of anti-DNA and anti-nucleosomal antibodies (10Yasutomo K. Nat. Genet. 2001; 28: 313-314Crossref PubMed Scopus (474) Google Scholar, 11Nupirei M. Karsunky H. Zevnik B. Stephan H. Mannherz H.G. Möröy T. Nat. Genet. 2000; 25: 177-181Crossref PubMed Scopus (662) Google Scholar). Finally, the execution of apoptotic DNA fragmentation and the cell death program appears to be reversible at a low frequency, and under such conditions chromosomal translocations mediated by non-homologous end joining of proto-oncogenes are thought to be one mechanism of cellular transformation (12Betti C.J. Villalobos M.J. Diaz M.O. Vaughan A.T.M. Cancer Res. 2001; 61: 4550-4555PubMed Google Scholar). In particular, it is well documented that treatment of leukemias with proapoptotic drugs, which target DNA, can lead to secondary tumors that exhibit new chromosomal translocations (13Roulston D. Espinosa III, R. Nucifora G. Larson R.A. Le Beau M.M. Rowley J.D. Blood. 1998; 92: 2879-2885Crossref PubMed Google Scholar). Taken together, it seems clear that understanding the regulation of proteins that are involved in DNA processing events during apoptosis is of fundamental importance, as well as how these processes are safeguarded from accidental activation in non-apoptotic cells. In the current study we address these issues for a major apoptotic nuclease complex, termed DFF. 1The abbreviations used are: DFF, DNA fragmentation factor; AFM, atomic force microscopy; CAD, caspase-activated deoxyribonuclease (also termed DFF40); DFF45, 45-kDa subunit of DFF; DFF40, 40-kDa subunit of DFF; DFF35, 35-kD subunit of DFF; ICAD-L and ICAD-S, inhibitors of CAD, long and short forms (also termed DFF45 and DFF35, respectively); CPAN, caspase-activated nuclease (also termed DFF40 and CAD). Apoptotic cell genomic DNA cleavage is known to occur in at least two stages, initial cleavage at ≥50-kilobase intervals, a size consistent with chromatin loop domains, followed by a second stage of internucleosomal DNA cleavage (also called DNA laddering) (14Oberhammer F. Wilson J.W. Dive C. Morris I.D. Hickman J.A. Waleling A.E. Walker P.R. Sikorska M. EMBO J. 1993; 12: 3679-3684Crossref PubMed Scopus (1161) Google Scholar). Apoptosis-inducing factor (15Susin S.A. Lorenzo H.K. Zamzami N. 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Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar, 26Liu X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 40Wolf B.B. Schuler M. Echeverri F. Green D.R. J. Biol. Chem. 1999; 274: 30651-30656Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). DFF40 nuclease activity on naked DNA substrates can be further activated by specific chromosomal proteins, such as histone H1 or high mobility group 1/2 (22Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar, 26Liu X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 27Widlak P. Li P. Wang X. Garrard W.T. J. Biol. Chem. 2000; 275: 8226-8232Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 41Toh S.Y. Wang X. Li P. Biochem. Biophys. Res. Commun. 1998; 250: 598-601Crossref PubMed Scopus (40) Google Scholar) and topoisomerase II (27Widlak P. Li P. Wang X. Garrard W.T. J. Biol. Chem. 2000; 275: 8226-8232Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 42Durrieu F. Samejima K. Fortune J.M. Kandels-Lewis S. Osheroff N. Earnshaw W.C. Curr. Biol. 2000; 10: 923-926Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Other gene products also appear to participate in the DFF pathway. Two proteins encoded by genes called CIDEs (cell death-inducing DFF45-like effectors) that exhibit homology to the N-terminal domain of DFF45 can activate apoptosis in a DFF45-inhibitable fashion, but their precise functions and mechanisms of action remain to be elucidated (43Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (283) Google Scholar). It should be noted that the physiological significance of DFF in triggering DNA laddering during apoptosis has been unequivocally proven. Homozygous deletions of the single copy genes encoding DFF45 or DFF40 have been created in the mouse germ line and in cultured chicken cells (44Zhang J. Liu X. Scherer D.C. Kaer L.V. Wang X. Xu M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12480-12485Crossref PubMed Scopus (159) Google Scholar, 45Samejima K. Tone S. Earnshaw W.C. J. Biol. Chem. 2001; 276: 43427-43432Abstract Full Text Full Text PDF Scopus (93) Google Scholar, 46Kawane K. Fukuyama H. Yoshida H. Nagase H. Ohsawa Y. Uchiyama Y. Okada K. Iida T. Nagata S. Nat. Immunol. 2003; 4: 138-144Crossref PubMed Scopus (194) Google Scholar). In addition, dominant negative forms of DFF45 have been expressed in a cell line and transgenic mice (18Sakahira H. Enari M. Ossawa Y. Uchiyama Y. Nagata S. Curr. 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In conclusion, it is well established experimentally that DFF plays a major and regulated role in cell-autonomous apoptotic DNA fragmentation. Developing an understanding of the steps involved in safe-guarding normal cells against accidental activation of this nuclease is as important as elucidating its activation mechanisms during apoptosis. To gain insight on the regulation of these processes, in the present study we have determined the native subunit structures and stoichiometries of these proteins before and after induction of apoptosis using the technique of native pore-exclusion limit electrophoresis in combination with Western analyses (50Clos J. Westwood J.T. Becker P.B. Wilson S. Lambert K. Wu C. Cell. 1990; 63: 1085-1097Abstract Full Text PDF PubMed Scopus (267) Google Scholar) and have utilized similar techniques with recombinant proteins along with atomic force microscopy. We find that DFF activation is regulated by multiple fail-safe steps and that its inhibitory subunits unexpectedly are expressed at vast stoichiometric excess relative to its latent nuclease subunit in several tumor cells studied. Expression, Purification, Activation, and Inhibition of Recombinant Forms of DFF—His6-tagged human recombinant DFF40/DFF45 heterodimer, human recombinant DFF45 monomer, and hamster recombinant caspase-3 were expressed in Escherichia coli and purified using nickel affinity columns as described previously (22Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar). DFF40/DFF45 heterodimer was incubated with caspase-3 at room temperature in reaction buffer consisting of 10 mm KCl, 100 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, and 20 mm Tris-HCl, pH 7.5. After 30 min of incubation caspase-3 was inhibited with 10 μm acetyl-Asp-Glu-Val-Asp-aldehyde. In some experiments fresh DFF45 (10-fold molar excess as compared with the starting DFF40/DFF45 heterodimer concentration) was then added, and the reaction mixture was incubated on ice for an additional 15 min. Nuclease activity was assayed as described previously (27Widlak P. Li P. Wang X. Garrard W.T. J. Biol. Chem. 2000; 275: 8226-8232Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Cell Culture and Treatments—Several established human cell lines were used for experiments: HL60 and K562 myeloid leukemia cells (DSMZ ACC 107 and EC ACC 89121407, respectively), Jurkat T cell leukemia cells (DSMC ACC 282), HeLa S3 cervical carcinoma cells (ATTC CC1 2.2), MCF7 breast adenocarcinoma cells (IZSBS BS TCL 37), A549 lung carcinoma cells (DSMZ ACC 107), and Me45 melanoma cells. As non-cancer cells, we used primary fibroblasts (passage 15), resting total lymphocytes (80% T cells), and proliferating T lymphocytes. Lymphocytes and leukemia cells were cultured in RPMI1640 medium, whereas other cells were cultured in Dulbecco's modified Eagle's medium, all supplemented with 10% fetal calf serum, at 37 °C under 5% CO2. Lymphocytes were freshly isolated from circulating blood by centrifugation on Ficoll Lymphoprep™ (ICN). To obtain proliferating T cells, isolated lymphocytes were incubated for 3 days in RPMI1640 medium supplemented with 10% fetal calf serum and 10 mg/liter lectin (Sigma). To induce apoptosis, HL60 cells were incubated for 5 h with 34 μm etoposide (Sigma). DNA fragmentation in such cells was assayed as described previously (51Widlak P. Palyvoda O. Kumala S. Garrard W.T. J. Biol. Chem. 2002; 277: 21683-21690Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Isolation of Cytoplasmic and Nuclear Extracts—Cell growing in log phase were collected and washed twice with ice-cold phosphate-buffered saline. Washed cells were incubated for 15 min on ice in lysis buffer consisting of 0.5% Nonidet P-40, 0.25 m sucrose, 10 mm KCl, 4 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, and 20 mm Tris-HCl, pH 7.5, supplemented with a mixture of protease inhibitors Complete™ (Roche Applied Science). After incubation, nuclei were collected by centrifugation for 5 min at 600 × g. Cell lysates were then centrifuged for 15 min at 10,000 × g, and the resulting supernatants are referred to as cytoplasmic extracts. In some experiments nuclei were obtained by a non-detergent lysis procedure in which HeLa cells washed with phosphate-buffered saline were suspended in the lysis buffer depleted of Nonidet P-40 and sucrose, incubated with 10 μm cytochalasin B for 15 min on ice, then lysed by passing 5 times through 25-gauge needles. Nuclei were washed with the lysis buffer depleted of Nonidet P-40 by low speed centrifugation and incubated for 15 min on ice in the nuclear extract buffer consisting of 0.5% Triton X-100, 0.25 m sucrose, 10 mm KCl, 200 mm NaCl, 4 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, and 20 mm Tris-HCl, pH 7.5, supplemented with a mixture of protease inhibitors (Complete™ (Roche Applied Science)). Nuclei were then centrifuged for 15 min at 10,000 × g, and the resulting supernatants were referred to as nuclear extracts. Western Blot Analyses—Total cell protein (80 μg/lane) and the corresponding fractions of cytoplasmic extracts, nuclei and nuclear extracts, or protein from cytoplasmic extracts alone (50 μg/lane) were separated on SDS, 14% polyacrylamide gels and electrophoretically transferred onto nitrocellulose membranes (Amersham Biosciences or Schleicher & Schuell). Membrane-immobilized proteins were probed with the following commercial antibodies: rabbit anti-human DFF40 polyclonal antibodies (Pharmingen or Axxora), rabbit anti-human DFF45 N terminus polyclonal antibodies (Pharmingen), rabbit anti-human DFF45 C terminus polyclonal antibodies (Axxora), and mouse anti-human β-actin monoclonal antibody (Oncogene). The antigen-antibody complexes were visualized using enhanced chemiluminescence (ECL) Western-blotting detection reagents (Amersham Biosciences). Atomic Force Microscopy—Protein samples were diluted to a final concentration of ∼1 μg/ml in 50 mm HEPES, pH 7.5. Immediately before imaging, 2-μl aliquots of diluted protein samples were introduced to the surface of freshly cleaved mica and rinsed with 200 μl of H2O followed by dehydration using a graded series of ethanol solutions consisting of 20, 40, 80, and 100% ethanol. Images were collected under ambient conditions in tapping mode using a Nanoscope IIIa (Digital Instruments Inc., Santa Barbara, CA) equipped with a vertical engage J-scanner operated at a scan rate of 1.97 Hz. Volume data were calculated as previously described (52Smith G.C.M. Cary R.B. Lakin N.D. Hann B.C. Teo S. Chen D.J. Jackson S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11134-11139Crossref PubMed Scopus (146) Google Scholar). Native Pore-exclusion Limit Electrophoresis—Native pore-exclusion limit electrophoresis was performed as described elsewhere (50Clos J. Westwood J.T. Becker P.B. Wilson S. Lambert K. Wu C. Cell. 1990; 63: 1085-1097Abstract Full Text PDF PubMed Scopus (267) Google Scholar), with minor modifications. Briefly, soluble proteins from cytoplasmic extracts (300 μg of protein/lane) were separated on linear 4–24% gradient polyacrylamide gels in 0.1 m Tris borate, pH 9.5, 1 mm β-mercaptoethanol. Gels were calibrated by co-electrophoresis of ovalbumin (45 kDa), bovine serum albumin (66 kDa), lactate dehydrogenase (140 kDa), with α-actin (monomer of actin G and a series of its oligomers) and also with Kaleidoscope pre-stained standards (Bio-Rad 161-0324). Electrophoresis was continued for 16 h at 300 V, ∼10 mA, at 4 °C. Gels were soaked with 0.1% SDS, 25 mm Tris-Cl, 250 mm glycine, pH 8.5, and then proteins were electrophoretically transferred onto nitrocellulose membranes. Membrane-immobilized proteins were probed with antibodies as described above. In one experiment (in-gel DFF40 oligomerization) proteins from cytoplasmic extracts were electrophoresed briefly (∼1 cm) into a native 5% polyacrylamide gel, and the appropriate gel fragment was excised. The gel fragment was then incubated for 30 min at room temperature with 10 μg of caspase-3, washed, and polymerized at the top of a 5–24% gradient gel. When cytoplasmic extracts were analyzed by two-dimensional electrophoresis, protein were first separated on native gradient gel, the gel fragment was soaked in 2% SDS, 0.1 m β-mercaptoethanol, and 50 mm Tris-HCl, pH 6.8, and placed on the top of standard SDS-14% polyacrylamide gel. DFF Levels Vary Widely among Both Normal and Transformed Human Cells—Previous studies have noted varied expression of DFF45/35 and DFF40 among different human and mouse cell lines and tissues by Northern analyses of mRNA levels but not by Western analyses of protein levels (36Lechardeur D. Drzymala L. Sharma M. Zylka D. Kinach R. Pacia J. Hicks C. Usmani N. Rommens J.M. Lukacs G.L. J. Cell Biol. 2000; 150: 321-334Crossref PubMed Scopus (78) Google Scholar, 48Zhang J. Wang X. Bove K.E. Xu M. J. Biol. Chem. 1999; 274: 37450-37454Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 53Mukae N. Enari M. Sakahira H. Fukuda Y. Inazawa J. Toh H. Nagata S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9123-9128Crossref PubMed Scopus (172) Google Scholar). Because we were interested in determining the native subunit structures and relative stoichiometries of these proteins, it was first necessary to survey several cell sources to identify those that possessed adequate levels for subsequent analyses. For this purpose we performed Western analyses on suitably prepared cell extracts using antibodies specific for DFF40, DFF45 (with antibodies against the C terminus of DFF45), and both DFF45 and DFF35 (with antibodies against the N terminus of DFF45). As shown in Fig. 1, DFF species levels varied more than 10-fold between the cell sources studied, with levels undetectable in resting lymphocytes and A549 lung carcinoma cells, barely detectable in fibroblasts and stimulated lymphocytes, and quite robust in MCF-7 breast adenocarcinoma, HeLa cervical carcinoma, and Jurkat, HL-60 and K562 leukemia cells. Thus, DFF levels in some tumor cells, but not others, far exceed the levels exhibited by normal cells. Whether there is any relationship between these differences in DFF levels and sensitivities to killing by various apoptotic stimuli remain to be determined. Nevertheless, based on these results, we selected Jurkat, HeLa, and HL-60 cells for further studies. By relating results on na
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