|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/1998//637 $5.00
The Journal of Cell Biology, Volume 140, Number 3,
, 1998 637-645
Article |
E4orf4, a Novel Adenovirus Death Factor That Induces p53-independent Apoptosis by a Pathway That Is Not Inhibited by zVAD-fmk
In the absence of E1B, the 289-amino acid product of human adenovirus type 5 13S E1A induces p53-independent apoptosis by a mechanism that requires viral E4 gene products (Marcellus, R.C., J.C. Teodoro, T. Wu, D.E. Brough, G. Ketner, G.C. Shore, and P.E. Branton. 1996. J. Virol. 70:6207–6215) and involves a mechanism that includes activation of caspases (Boulakia, C.A., G. Chen, F.W. Ng, J.G. Teodoro, P.E. Branton, D.W. Nicholson, G.G. Poirier, and G.C. Shore. 1996. Oncogene. 12:529–535). Here, we show that one of the E4 products, E4orf4, is highly toxic upon expression in rodent cells regardless of the p53 status, and that this cytotoxicity is significantly overcome by coexpression with either Bcl-2 or Bcl-XL. Conditional expression of E4orf4 induces a cell death process that is characterized by apoptotic hallmark features, such as externalization of phosphatidylserine, loss of mitochondrial membrane potential, cytoplasmic vacuolation, condensation of chromatin, and internucleosomal DNA degradation. However, the wide-spectrum inhibitor of caspases, tetrapeptide zVAD-fmk, does not affect any of these apoptogenic manifestations, and does not alter the kinetics of E4orf4-induced cell death. Moreover, E4orf4 expression does not result in activation of the downstream effector caspase common to most apoptosis-inducing events, caspase-3 (CPP32). We conclude, therefore, that in the absence of E1A, E4orf4 is sufficient by itself to trigger a p53-independent apoptosis pathway that may operate independently of the known zVAD-inhibitable caspases, and that may involve an as yet uncharacterized mechanism.
Abbreviations used in this paper: CPP32, caspase-3; DAPI, 4',6-diamidino-Z-phenylindole; HA, hemagglutinin; ICE, interleukin-1β–convertase enzyme; PARP, poly(ADP-ribosyl) polymerase; PP, protein phosphatase; rtTA, reverse tetracycline; TMRE, tetramethylrhodamine ethyl ester perchlorate.
TISSUE homeostasis is maintained by a tight balance between cellular proliferation and cell death processes such as apoptosis. Apoptosis is a cell suicide mechanism culminating in an autolytic cellular degradation that can be triggered by a wide range of stimuli, such as viral infection (White and Gooding, 1994; Teodoro and Branton, 1997), growth factor withdrawal (Evan et al., 1992; Rao et al., 1992; Wagner et al., 1994; Lin and Benchimol, 1995; Sakamuro et al., 1995), TNF-
and Fas ligand (for review see Nagata, 1997), and DNA damage after either irradiation (Clarke et al., 1993; Lowe et al., 1993b; Strasser et al., 1994; Han et al., 1995), or treatment with chemotherapeutic drugs (Lowe et al., 1993a). Apoptotic cell death may occur in either a p53-dependent or -independent manner (for review see Liebermann et al., 1995). The role of p53 as a general tumor suppressor gene is well established, and its ability to function as a sequence-specific transcription factor appears to be directly linked to its role in G1-arrest of the cell cycle in response to detrimental conditions (Bates and Vousden, 1996). The mechanism by which p53 regulates induction of apoptosis, however, remains unclear, but may rely on the ability of p53 to control expression of proapoptotic inducers such as Bax (Reed, 1997). The molecular determinants of p53-independent apoptotic responses remain poorly understood.
Although the initial triggering phase of apoptosis may take several routes, in most cases the execution phase of this process converges on a common pathway characterized by activation of the caspase family of interleukin-1β–convertase enzyme (ICE)1-like cysteine proteases (Oltvai and Korsmeyer, 1994; Steller, 1995; Chinnaiyan and Dixit, 1996; Fraser and Evan, 1996). Additionally, however, recent evidence suggests that other pathways, which do not rely on activation of the known caspases, can also lead to cell death and manifestation of the morphological features of apoptosis (Xiang et al., 1996; Boise and Thompson, 1997). In both cases, irreversible commitment to cell death may involve a loss of the mitochondrial membrane potential (
m) through opening of the permeability transition pore complex (Kroemer et al., 1995; Petit et al., 1995; Zamzami et al., 1995a,b; Castedo et al., 1996; Zamzami et al., 1996; Kroemer, 1997). Regardless of the biochemical transformations leading to apoptosis, however, the final phase — the degradation phase — is usually characterized by typical morphological changes, including plasma membrane blebbing, cytoplasmic boiling and vacuolation, chromatin condensation, internucleosomal cleavage of DNA, cell shrinkage, and fragmentation of the cell into membrane-bound apoptotic bodies that are ultimately engulfed by other cells (Wyllie, 1980). Not surprisingly, both the initiation and execution of apoptosis are tightly regulated. One element of this control is provided by a family of proteins related to Bcl-2, which act as general suppressors of apoptosis in many contexts, including those leading to p53-dependent or -independent death (for review see Farrow and Brown, 1996; Yang and Korsmeyer, 1996).
Adenoviruses have provided important model systems to study the regulation of DNA synthesis, cell proliferation, transformation, and apoptosis (Dyson and Harlow, 1992; Moran, 1993; White and Gooding, 1994; Teodoro and Branton, 1997). The products of early region 1A (E1A 13S and E1A 12S) are the triggers of these cellular effects, determined by the context in which E1A genes operate. In the presence of E1B products, for example, the cytotoxic consequences of E1A expression are blocked, allowing its growth-promoting properties to be realized (White et al., 1991; Rao et al., 1992). E1A produces two major mRNAs of 13S and 12S that encode proteins of 289 and 243 residues, respectively, and that are identical except for a central conserved region, termed CR3, in the 289-residue product that transactivates expression of other early viral units (E2, E3, E4; Kimelman et al., 1985). p53 is required for E1A-induced apoptosis in cells infected by an E1B- defective Ad5 mutant expressing the 12S E1A mRNA (Debbas and White, 1993; Lowe and Ruley, 1993; Lowe et al., 1994; Teodoro et al., 1995; Querido et al., 1997). However, the alternatively spliced E1A 13S transcript can also induce apoptosis independently of p53 and appears to rely on CR3 and additional early viral genes to do so (Teodoro et al., 1995). Identification of E4 as the requisite early region providing these cooperating transcripts has come from mapping experiments conducted in p53-null SAOS-2 cells infected with various mutant viruses (Marcellus et al., 1996). More recent studies suggest the contribution of the E4orf4 protein and possibly, E4orf62. The ability of E4 proteins to contribute to viral cytotoxicity may relate to viral particle transmission during infection, in which accumulation of progeny virus inside apoptotic bodies permits the spreading of the virus to neighboring cells while evading host immune surveillance (Teodoro and Branton, 1997).
In the present study, we report that one of the E4 adenoviral proteins, E4orf4, is sufficient by itself to induce p53-independent cell death. Conditional expression of this 14-kD product in rodent cells triggers an apoptotic pathway that may involve a novel signaling event. In contrast to induction of apoptosis that involves E1A and E1A-dependent activation of caspase-3 (Boulakia et al., 1996; Sabbatini et al., 1997), we detect no involvement of the zVAD–fmk-inhibitable caspases in the mechanism of apoptosis caused by the solitary delivery of Ad2 E4orf4.
 |
Materials and Methods |
|---|
 TopAbstract Materials and Methods ResultsDiscussionReferences |
|---|
MEM supplemented with 10% fetal bovine serum and streptomycin sulfate/penicillin (100 U/ml). Cells were grown in a humidified 5% CO2 atmosphere at 37°C. To establish the CHO-rtTA-orf4 cell lines, the reverse tetracycline (rtTA) transactivator plasmid (pUHD17-1) was first cotransfected in CHO cells together with the pCDNA3 vector encoding the neomycin resistance gene. Stable transfectants were isolated after a 15-d selection period in the presence of G-418 (800 µg/ml), and screened on the basis of their responsivness to doxycycline, as previously described (Gossen et al., 1995). Clone CHO-13 was selected for its low basal and highly doxycycline-induced transactivation activity. A hemagglutinin (HA) epitope tag (Chen et al., 1996) was placed at the 5' end of Ad2 E4orf4 coding sequence by standard PCR using the sense and antisense oligos, 5' GGGGTACC ATG GCG TAC CCA TAC GAT GTT CCA GAT TAC GCT ATG GTT CTT CCA GCT CTT CC 3' and 5' CTA CTG TAC GGA GTG CGCC 3', respectively. The resulting DNA fragment was sequenced and then cloned into the SacII/XbaI sites of PUHD10-3, containing the hCMV minimal promoter with heptamerized tetoperators (Gossen and Bujard, 1992). Studies confirmed that the HA tag did not interfere with the ability of orf4 to induce cytotoxicity. HA-orf4/PUHD10-3 plasmid was cotransfected together with pJK-puro, encoding the puromycin resistance gene into CHO-13 inducible clonal cell line. Stable transfectants were isolated after a 15-d selection period in the presence of puromycin (2 µg/ml) and then screened for induction of orf4 protein after addition of doxycycline at 1 µg/ml (Sigma Chemical Co., St. Louis, MO). Two clones, named CHO-orf4-7 and CHO-orf4-4, were selected on the basis of their high doxycycline-induced HA-orf4 protein expression. The percentage of cells expressing HA-orf4 was evaluated after induction in the presence of doxycycline for varying periods, by immunolocalization studies (50% for clone CHO-orf4-7, and 38% for clone CHO-orf4-4, respectively).
Transient Transfection and Luciferase Assay
CHO LR73 and HyA4 cells were seeded at a density of 150,000 cells per well in 24-well plates and then cotransfected by the calcium phosphate method with 0.2 µg of the reporter plasmid pRSV-luc, together with 2.0 µg of expression vector containing either E4orf4 or E4orf2 coding sequences (Öhman et al., 1993) cloned into pCDNA3 as described2, and 3.8 µg of sheared salmon sperm DNA, or 3.8 µg of expression vector containing Bcl-2 (Nguyen et al., 1994) or Bcl-XL sequences (Ng et al., 1997). 48–72 h after transfection, cells were lysed, and then aliquots were assayed for luciferase activity as previously described (Lavoie et al., 1996a). Data are representative for at least three independent experiments performed in duplicate and are expressed as relative luciferase units, which were calculated relative to the luciferase activity in cells cotransfected with the corresponding empty vectors set to 1 U. For screening of the CHO-rtTA clonal cell lines, cells were transfected with 0.2 µg of pTRE-luc/pUHD10-3 (Gossen and Bujard, 1992) and 4.8 µg of sheared salmon sperm DNA. 24 h after transfection, doxycycline was added to the culture medium at 1 µg/ml for 36 h and then the luciferase activity was measured.
Cell Viability and DNA Degradation Assay
CHO-orf4 cell lines were seeded in 12-well plates and incubated in the presence of 1 µg/ml doxycycline for various periods of time. Nonadherent and adherent cells were collected, and then aliquots were mixed with an equal volume of 0.4% trypan blue (GIBCO BRL, Gaithersburg, MD). The percentage of cells that picked up the dye was determined and considered to be dead cells. Data are representative of at least three independent culture dishes per time point and these are corrected for the percentage of cells expressing orf4 as revealed by immunofluorescence on parallel cultures induced with doxycycline. Measurement of internucleosomal DNA cleavage was achieved in CHO-orf4-7 cells incubated in the presence of 1 µg/ml doxycycline or 1.0 µg/ml anti-human Fas antibody (clone CH-11; Upstate Biotechnology Inc., Lake Placid, NY) for various periods of time. Where required, zVAD-fmk was added simultaneously with the apoptotic triggering signal at a final concentration of 50 µM (Enzyme System Products, Dublin, CA). Adherent and nonadherent cells from a 60-mm Petri dish were collected, washed in PBS, and resuspended gently in 0.5 ml of lysis buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.2% Triton X-100). After a 30-min incubation at room temperature, the samples were centrifuged at 12,000 rpm for 30 min. 150 µl of 5 M NaCl and 500 µl of 100% ethanol were added to the supernatant, and DNA was then precipitated overnight at –20°C. Samples were centrifuged at 12,000 rpm for 30 min, and then pellets were resuspended in Tris/EDTA (TE). Extracted nucleic acids were treated with RNaseA at 37°C for 1 h and then analyzed on 1% agarose gels stained with ethidium bromide.
Immunoblotting and Immunofluorescence Analysis
Cells were lysed in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2.3% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.05% bromphenol blue, 1 mM phenylmethylsulfonyl fluoride), and then equal amounts of protein from whole cell lysates were separated by SDS-PAGE in 12–14% acrylamide gels. Proteins were detected immunologically after electrotransfer onto nitrocellulose membranes as described previously (Lavoie et al., 1995). Blots were developed with either mouse anti-HA HA.11 (BAbCO, Richmond, CA), rabbit polyclonal CPP32 antibody raised against the recombinant p17 subunit of CPP32 (Boulakia et al., 1996), provided by D.W. Nicholson (Merck-Frosst Center for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada), or rabbit polyclonal poly(ADP-ribosyl) polymerase (PARP) antibody 422, raised against the automodification domain of bovine PARP as described (Duriez et al., 1997), provided by G. Poirier (Centre Hospitalier du l'Université Laval Research Center, Sainte-Foy, Quebec, Canada). Horseradish peroxidase-linked goat anti–rabbit or anti–mouse immunoglobulin G (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), revealed by the enhanced chemiluminescence detection system (Amersham Corp., Arlington Heights, IL), was used to detect the antigen–antibody complexes. Protein concentrations were measured using the Bio-Rad (Hercules, CA) assay. For immunolocalization of HA-orf4, cells were plated on glass coverslips and then incubated in the presence of doxycycline for various periods of time. Cells were washed in PBS containing 1 mM MgCl2 and then fixed in 3.7% formaldehyde/PBS for 20 min. Permeabilization of cells was performed in 0.2% Triton X-100/ PBS for 5 min, and then mouse anti-HA HA.11, followed by Texas red– conjugated anti–mouse immunoglobulin G (Molecular Probes, Inc., Eugene, OR) were used to detect HA-orf4. The nuclear morphology of cells was analyzed by staining of DNA with 4'-6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes, Inc.). Annexin V binding assays were performed by incubating the cells at 37°C in the presence of fluorescein-conjugated human annexin V at a final concentration of 0.1 µg/ml for 15 min. Cells were then fixed in binding buffer (10 mM Hepes, NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 3.7% formaldehyde, 0.1 µg/ml annexin V) for 10 min in the dark and then visualized. Mitochondrial membrane potential was monitored by incubating cells grown on coverslips in culture medium containing 137 mM KCl to abolish the plasma membrane potential, and 1 µg/ml tetramethylrhodamine ethyl ester perchlorate (TMRE; Molecular Probes, Inc.) at 37°C for 10 min. Coverslips were rinsed in warm dye-free culture medium containing 137 mM KCl and then mounted in a living cell chamber made of 0.7-mm-thick rubber containing the same medium, as previously described (Chen, 1989). In parallel experiments, cells were preincubated in the presence of 10 µM CCCP at 37°C for 20 min to abolish the mitochondrial membrane potential before in vivo staining, as a control for the specificity of the dye (data not shown).
Electron Microscopy
CHO-orf4-7 cells were incubated in the presence of 1 µg/ml doxycycline and 50 µM zVAD–fmk for 36 h. Nonadherent and adherent cells were collected, washed in sucrose buffer, and then fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2–7.4, for 90 min at room temperature. Postfixation was performed with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at 4°C, followed by en bloc staining with 2% uranyl acetate for 30 min. Samples were dehydrated and embedded in Epon (Polysciences, Inc., Warington, PA). Thin sections (75-nm) were analyzed by transmission electron microscopy using an electron microscope (model 410; Philips Electron Optics, Eindhoven, The Netherlands).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Conditional Expression of E4orf4 in Rodent Cells Causes a Cell Death Process That Is Characterized by Typical Apoptotic Hallmark Features
To better characterize E4orf4 cytotoxicity and to determine whether its expression is sufficient to induce cell death, we established a rtTA-inducible system in CHO LR73 cells (CHO-orf4). An HA-tagged version of Ad2 E4orf4 was used in this system to facilitate the detection of the viral product. Clonal inducible CHO-orf4 cell lines were isolated as described (refer to Materials and Methods). HA-orf4 protein was induced to a significant level in CHO-orf4-7 cells within 7 h of exposure to doxycycline and increased further to reach a maximum level of expression per milligram total cell protein at 48 h after treatment (Fig. 2 A). Induction of HA-orf4 resulted in early cell lifting and a progressive increase in cell death, with the onset of membrane permeability to trypan blue occurring at 
16 h after induction, and reaching 40–50% of the cell population expressing HA-orf4 within 48 h of doxycycline exposure (Fig. 2 B).
|
|
Finally, isolation of low molecular weight DNA from CHO-orf4-7 cells incubated with doxycycline for various periods of time revealed the presence of oligonucleosomal DNA ladders, which became detectable after 24 h but reached a significant intensity 64 h after induction of HA-orf4 (Fig. 3 C). As expected, such apoptotic DNA degradation was observed at relatively late times after HA-orf4 expression, as compared to changes such as DNA condensation and externalization of phosphatidylserine.
E4orf4-induced Apoptosis Is Not Inhibited by zVAD-fmk
To initially characterize the signaling pathway implicated in E4orf4-mediated apoptosis, activation of caspase-3 (CPP32/ apopain/Yama), an ICE-like cysteine protease that has been shown to perform a central role in many apoptosis systems (Fernandes-Alnemri et al., 1994; Nicholson et al., 1995; Tewari et al., 1995; Casciola et al., 1996; Schlegel et al., 1996), was monitored. CHO-orf4-7 cells were incubated in the presence of either doxycycline, to induce HA-orf4– mediated apoptosis, or hygromycin B, to induce apoptosis (Chen et al., 1995) in the absence of the viral protein. Immunoblotting of total cell extracts was performed using an antibody raised against the recombinant p17 subunit of caspase-3 (Boulakia et al., 1996), or the 422–anti-PARP antibody, which recognizes the full-length PARP and the 85-kD cleaved product observed after apoptotic stimuli (Duriez et al., 1997). Processing of procaspase-3 upon an apoptotic signal yields two subunits of 17 and 12 kD, which assemble to form the active tetrameric holoenzyme (Nicholson et al., 1995). Treatment of CHO-orf4-7 cells with hygromycin B for 48 h triggered processing of procaspase-3, as revealed by the appearance of the 17-kD subunit, as well as cleavage of PARP, a substrate for active caspase-3 (Nicholson et al., 1995; Tewari et al., 1995) (Fig. 4). In contrast, no 17-kD band was detectable in CHO-orf4-7 after a 48-h incubation in the presence of doxycycline (Fig. 4), when HA-orf4 expression was maximal (Fig. 2 A). Consistent with this, no PARP cleavage was observed. A detailed kinetic analysis performed at various times after induction of HA-orf4 failed to reveal at any time processing of procaspase-3 or cleavage of PARP in cells undergoing HA-orf4–mediated cell death (data not shown).
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The proapoptotic member of the Bcl-2 family, Bax (Oltvai and Korsmeyer, 1994), exhibits several of the properties that might be expected of such a p53 downstream event that can be substituted by E4orf4. First, evidence has been obtained that BAX is a target of p53 transcriptional activation (Miyashita et al., 1994; Selvakumaran et al., 1994; Miyashita and Reed, 1995). Second, although Bax expression induces activation of procaspase-3, it is also capable of inducing all of the apoptotic manifestations that are also observed for E4orf4 when expressed in the presence of zVAD-fmk (Xiang et al., 1996). Finally, Bcl-XL, a heterodimerizing partner of Bax, is also capable of functioning at a step downstream of the caspases, protecting cells against the loss of mitochondrial membrane potential. This is consistent with our finding that Bcl-2/Bcl-XL protects cells against E4orf4 cytotoxicity. It remains to be determined, however, if E4orf4 is simply acting like a partially functional analogue of Bax. Sequence analysis of E4orf4, for example, does not reveal the presence of strong similarities to the structural hallmarks of the Bax family, notably the BH3 domain (Yang and Korsmeyer, 1996). The one known function of E4orf4 relates to its ability to bind the heterotrimeric form of protein phosphatase (PP)2A and to increase the activity of the holoenzyme (Kleinberger and Shenk, 1993), but whether or not this increase in PP2A is sufficient to explain the apoptosis inducing properties of E4orf4 is not yet known. It is additionally relevant, however, that regulators of the Bcl-2 family setpoint are subject to phosphorylation/dephosphorylation events. These include Bad (Zha et al., 1996), as well as Bcl-2 itself (Haldar et al., 1995; Strack et al., 1996; Chang et al., 1997). Another possibility, therefore, is that E4orf4 links PP2A to these potential targets at sites where the Bcl-2 family proteins function downstream of the caspases (Boise and Thompson, 1997). Finally, it may be that E4orf4 is multifunctional, despite its relatively small size (14-kD), and that its role in apoptosis is different than its effect on PP2A. Future characterization of E4orf4 interaction(s) with cellular proteins should provide insights into the signaling pathways involved in mediating E4orf4 p53–independent apoptosis.
|
Acknowledgments |
|---|
Submitted: 6 October 1997
Revised: 12 December 1997
This work was financed by operating grants from the National Cancer Institute of Canada (grant number 007307) and the Medical Research Council of Canada (grant number MT6192). J.N. Lavoie is supported by a Centennial Postdoctoral Fellowship from the Medical Research Council of Canada.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bates S & Vousden KH. p53 in signaling checkpoint arrest or apoptosis, Curr Opin Genet Dev, 1996, 6, 12–19.[Medline]
Boise LH & Thompson CB. BclxL can inhibit apoptosis in cells that have undergone Fas-induced protease activation, Proc Natl Acad Sci USA, 1997, 94, 3759–3764.
Boulakia CA, Chen G, Ng FW, Teodoro JG, Branton PE, Nicholson DW, Poirier GG & Shore GC. Bcl-2 and adenovirus E1B 19 kDA protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose) polymerase, Oncogene, 1996, 12, 529–535.[Medline]
Casciola RL, Nicholson DW, Chong T, Rowan KR, Thornberry NA, Miller DK & Rosen A. Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death, J Exp Med, 1996, 183, 1957–1964.
Castedo M, Hirsh T, Susin SA, Zamzami N, Marchetti P, Macho A & Kroemer G. Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis, J Immunol, 1996, 157, 512–521.[Abstract]
Chang BS, Minn AJ, Muchmore SW, Fesik SW & Thompson CB. Identification of a novel regulatory domain in Bcl-X(L) and Bcl-2, EMBO (Eur Mol Biol Organ) J, 1997, 16, 968–977.[Medline]
Chen G, Branton PE & Shore GC. Induction of p53-independent apoptosis by hygromycin B: suppression by Bcl-2 and adenovirus E1B 19-kDa protein, Exp Cell Res, 1995, 221, 55–59.[Medline]
Chen G, Branton PE, Yang E, Korsmeyer SJ & Shore GC. Adenovirus E1B 19-kDa death suppressor protein interacts with Bax but not with Bad, J Biol Chem, 1996, 271, 24221–24225.
Chen LB. Fluorescent labeling of mitochondria, Methods Cell Biol, 1989, 29, 103–123.[Medline]
Chinnaiyan AM & Dixit VM. The cell-death machine, Curr Biol, 1996, 6, 555–562.[Medline]
Clarke AR, Purdie CA, Harrison DJ, Morris RG, Bird CC, Hooper ML & Wyllie AH. Thymocyte apoptosis induced by p53- dependent and independent pathways, Nature, 1993, 362, 849–852.[Medline]
Cohen JJ. Programmed cell death in the immune system, Adv Immunol, 1991, 50, 55–85.[Medline]
Debbas M & White E. Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B, Genes Dev, 1993, 7, 546–554.
Duriez PJ, Desnoyers S, Hoflack JC, Shah GM, Morelle B, Bourassa S, Poirier G & Talbot B. Characterization of anti-peptide antibodies directed towards the automodification domain and apoptotic fragment of poly (ADP-ribose) polymerase, Biochim Biophys Acta, 1997, L1334, 65–72.[Medline]
Dyson N & Harlow E. Adenovirus E1A targets key regulators of cell proliferation, Cancer Surv, 1992, 12, 161–195.[Medline]
Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ & Hancock DC. Induction of apoptosis in fibroblasts by c-myc protein, Cell, 1992, 69, 119–128.[Medline]
Farrow SN & Brown R. New members of the Bcl-2 family and their protein partners, Curr Opin Genet Dev, 1996, 6, 45–49.[Medline]
Fernandes-Alnemri T, Litwack G & Alnemri ES. CPP32, a novel human apoptotic protein with homology to Caenorhabditis eleganscell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme, J Biol Chem, 1994, 269, 30761–30764.
Fraser A & Evan G. A license to kill, Cell, 1996, 85, 781–784.[Medline]
Gossen M & Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters, Proc Natl Acad Sci USA, 1992, 89, 5547–5551.
Gossen M, Freundlieb S, Bender G, Muller G, Hillen W & Bujard H. Transcriptional activation by tetracyclines in mammalian cells, Science, 1995, 268, 1766–1769.
Haldar S, Jena N & Croce CM. Inactivation of Bcl-2 by phosphorylation, Proc Natl Acad Sci USA, 1995, 92, 4507–4511.
Han, Z., D. Chatterjee, D.M., J. Early, P. Pantazis, J.H. Wyche, and E.A. Hendrickson. 1995. Evidence for a G2 checkpoint in p53-independent apoptosis induction by X-irradiation. Mol. Cell. Biol. 15:5849–5857.
Kimelman DJ, Miller JS, Porter D & Roberts BE. E1A regions of the human adenoviruses and of the highly oncogenic simian adenovirus 7 are closely related, J Virol, 1985, 53, 399–409.
Kleinberger T & Shenk T. Adenovirus E4orf4 protein binds to protein phosphatase 2A, and the complex down regulates E1A-enhanced junB transcription, J Virol, 1993, 67, 7556–7560.
Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis, Nature Med, 1997, 3, 614–620.[Medline]
Kroemer G, Petit PX, Zamzami N, Vayssière J-L & Mignotte B. The biochemistry of apoptosis, FASEB (Fed Am Soc Exp Biol) J, 1995, 9, 1277–1287.[Abstract]
Lavoie JN, Lambert H, Hickey E, Weber LA & Landry J. Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27, Mol Cell Biol, 1995, 15, 505–516.[Abstract]
Lavoie JN, L'Allemain G, Brunet A, Müller R & Pouysségur J. Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway, J Biol Chem, 1996a, 271, 20608–20616.
Lavoie JN, Rivard N, L'Allemain G & Pouysségur J. A temporal and biochemical link between growth factor-activated MAP kinases, cyclin D1 induction and cell cycle entry, Prog Cell Cycle Res, 1996b, 2, 49–58.[Medline]
Levine AJ. The tumor suppressor genes, Annu Rev Biochem, 1993, 62, 623–651.[Medline]
Liebermann DA, Hoffman B & Steinman RA. Molecular controls of growth arrest and apoptosis: p53-dependent and independent pathways, Oncogene, 1995, 11, 199–210.[Medline]
Lin Y & Benchimol S. Cytokines inhibit p53-dependent apoptosis but not p53-mediated G1 arrest, Mol Cell Biol, 1995, 15, 6045–6054.[Abstract]
Lowe SW & Ruley HE. Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis, Genes Dev, 1993, 7, 535–545.
Lowe SW, Jacks T, Housman DE & Ruley HE. Abrogation of oncogene-associated apoptosis allows transformation of p53-deficient cells, Proc Natl Acad Sci USA, 1994, 91, 2026–2030.
Lowe SW, Ruley HE, Jacks T & Housman DE. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents, Cell, 1993a, 74, 957–967.[Medline]
Lowe SW, Schmitt EM, Smith SW, Osborne BA & Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes, Nature, 1993b, 362, 847–849.[Medline]
Marcellus RC, Teodoro JC, Wu T, Brough DE, Ketner G, Shore GC & Branton PE. Adenovirus 5 early region 4 is responsible for E1A-induced p53-independent apoptosis, J Virol, 1996, 70, 6207–6215.[Abstract]
Martin SJ, Reutelingsperger CPM, McGahon AJ, Rader JA, van Schie RCAA, LaFace DM & Green DR. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl, J Exp Med, 1995, 182, 1545–1556.
Miyashita T & Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene, Cell, 1995, 80, 293–299.[Medline]
Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, Hoffman B & Reed JC. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo, Oncogene, 1994, 9, 1799–1805.[Medline]
Moran E. DNA tumor virus transforming proteins and the cell cycle, Curr Opin Genet Dev, 1993, 3, 63–70.[Medline]
Nagata S. Apoptosis by death factor, Cell, 1997, 88, 355–365.[Medline]
Ng FWH, Nguyen M, Kwan T, Branton PE, Nicholson DW, Cromlish JA & Shore GC. p28 BAP31, a Bcl-2/Bcl-XL– and Procaspase 8–associated protein in the endoplasmic reticulum, J Cell Biol, 1997, 139, 327–338.
Nguyen M, Branton PE, Walton PA, Oltvai ZN, Korsmeyer SJ & Shore GC. Role of membrane anchore domain of Bcl-2 in suppression of apoptosis caused by E1B-defective adenovirus, J Biol Chem, 1994, 269, 16521–16524.
Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA et al.. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis, Nature, 1995, 376, 37–43.[Medline]
Öhman K, Nordqvist K & Akusjärvi G. Two adenovirus proteins with redundant activities in virus growth facilitates tripartite leader mRNA accumulation, Virology, 1993, 194, 50–58.[Medline]
Oltvai ZN & Korsmeyer SJ. Checkpoints of dueling dimers foil death wishes, Cell, 1994, 79, 189–192.[Medline]
Petit PX, LeCoeur H, Zorn E, Dauguet C, Mighotte B & Gourgeon ML. Alterations of mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis, J Cell Biol, 1995, 130, 157–167.
Polverino AJ & Patterson SD. Selective activation of caspases during apoptotic induction in HL-60 cells. Effects of a tetrapeptide inhibitor, J Biol Chem, 1997, 272, 7013–7021.
Querido E, Teodoro JG & Branton PE. Accumulation of p53 induced by the adenovirus E1A protein requires regions involved in the stimulation of DNA synthesis, J Virol, 1997, 71, 3526–3533.[Abstract]
Rao L, Debbas M, Sabbatini P, Hockenbery D, Korsmeyer S & White E. The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins, Proc Natl Acad Sci USA, 1992, 89, 7742–7746.
Reed JC. Double identity for proteins of the Bcl-2 family, Nature, 1997, 387, 773–776.[Medline]
Sabbatini P, Han J, Chiou SK, Nicholson DW & White E. Interleukin 1 β converting enzyme-like proteases are essential for p53-mediated transcriptionally dependent apoptosis, Cell Growth Differ, 1997, 8, 643–653.[Abstract]
Sakamuro D, Eviner V, Elliott KJ, Showe L, White E & Prendergast GC. c-myc induces apoptosis in epithelial cells by both p53-dependent and p53-independent mechanisms, Oncogene, 1995, 11, 2411–2418.[Medline]
Schlegel J, Peters I, Orrenius S, Miller DK, Thornberry NA, Yamin TT & Nicholson DW. CPP32/apopain is a key interleukin 1 βeta converting enzyme-like protease involved in Fas-mediated apoptosis, J Biol Chem, 1996, 271, 1841–1844.
Selvakumaran M, Lin HK, Miyashita T, Wang HG, Krajewski S, Reed JC, Hoffman B & Liebermann D. Immediate early up-regulation of bax expression by p53 but not TGF beta 1: a paradigm for distinct apoptotic pathways, Oncogene, 1994, 9, 1791–1798.[Medline]
Slee EA, Zhu H, Chow SC, MacFarlane M, Nicholson DW & Cohen GM. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32, Biochem J, 1996, 315, 21–24.[Medline]
Stanbridge EJ & Nowell PC. Origins of human cancer revisited, Cell, 1990, 63, 867–874.[Medline]
Strack PR, Frey MW, Rizzo CJ, Cordova B, George HJ, Meade R, Ho SP, Corman J, Tritch R & Korant BD. Apoptosis mediated by HIV protease is preceded by cleavage of Bcl-2, Proc Natl Acad Sci USA, 1996, 93, 9571–9576.
Strasser A, Harris AW, Jacks T & Cory S. DNA damage can induce apoptosis in proliferating lymphiod cells via p53-independent mechanisms inhibitable by Bcl-2, Cell, 1994, 79, 329–339.[Medline]
Steller H. Mechanisms and genes of cellular suicide, Science, 1995, 267, 1445–1449.
Teodoro JG & Branton PE. Regulation of apoptosis by viral gene products, J Virol, 1997, 71, 1739–1746.[Medline]
Teodoro JG, Shore GC & Branton PE. Adenovirus E1A proteins induce apoptosis by both p53-dependent and p53-independent mechanisms, Oncogene, 1995, 11, 467–474.[Medline]
Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS & Dixit VM. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase, Cell, 1995, 81, 801–809.[Medline]
Thompson CB. Apoptosis in the pathogenesis and treatment of disease, Science, 1995, 267, 1456–1462.
Wagner AJ, Kokontis JM & Hay N. Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/Cip1, Genes Dev, 1994, 8, 2817–2830.
White, E., and L.R. Gooding. 1994. Regulation of apoptosis by human adenoviruses. In Apoptosis: The Molecular Basis for Cell Death II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 111–141.
White E, Cipriani R, Sabbatini P & Danton A. Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins, J Virol, 1991, 65, 2968–2978.
Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation, Nature, 1980, 284, 555–556.[Medline]
Xiang J, Chao DT & Korsmeyer SJ. Bax-induced cell death may not require interleukin 1β-converting enzyme-like proteases, Proc Natl Acad Sci USA, 1996, 93, 14559–14563.
Yang E & Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and cell death, Blood, 1996, 88, 386–401.
Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B & Kroemer G. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death, J Exp Med, 1995a, 182, 367–377.
Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssière J-L, Petit PX & Kroemer G. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo, J Exp Med, 1995b, 181, 1661–1672.
Zamzami N, Susin SA, Marchetti P, Hirsch T, Castedo M & Kroemer G. Mitochondrial control of nuclear apoptosis, J Exp Med, 1996, 183, 1533–1544.
Zha J, Harada H, Yang E, Jockel J & Korsmeyer SJ. Serine phosphorylation of death agonist Bad in response to survival factor results in binding to 14-3-3 not BclxL, Cell, 1996, 87, 619–628.[Medline]
Zhu H, Fearnhead HO & Cohen GM. An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells, FEBS (Fed Eur Biochem Soc) Lett, 1995, 374, 303–308.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
