Bax-mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity
1998; Springer Nature; Volume: 17; Issue: 13 Linguagem: Inglês
10.1093/emboj/17.13.3576
ISSN1460-2075
AutoresHarris Perlman, Masataka Sata, Aude Le Roux, Thomas W. Sedlak, Didier Branellec, Kenneth Walsh,
Tópico(s)RNA Interference and Gene Delivery
ResumoArticle1 July 1998free access Bax-mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity Harris Perlman Harris Perlman Division of Cardiovascular Research, St Elizabeth's Medical Center, Tufts University School of Medicine, 736 Cambridge Street, Boston, MA, 02135 USA Program in Cell, Molecular and Developmental Biology, Tufts University, Sackler School of Biomedical Studies, 136 Harrison Avenue, Boston, MA, 02111 USA Search for more papers by this author Masataka Sata Masataka Sata Division of Cardiovascular Research, St Elizabeth's Medical Center, Tufts University School of Medicine, 736 Cambridge Street, Boston, MA, 02135 USA Search for more papers by this author Aude Le Roux Aude Le Roux Rhone-Poulenc Rorer Gencell, Centre de Recherche de Vitry-Alfortville, 94403 Vitry-sur-Seine, France Search for more papers by this author Thomas W. Sedlak Thomas W. Sedlak Division of Molecular Oncology, Departments of Medicine and Pathology, Howard Hughes Medical Institute, Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Didier Branellec Didier Branellec Rhone-Poulenc Rorer Gencell, Centre de Recherche de Vitry-Alfortville, 94403 Vitry-sur-Seine, France Search for more papers by this author Kenneth Walsh Corresponding Author Kenneth Walsh Division of Cardiovascular Research, St Elizabeth's Medical Center, Tufts University School of Medicine, 736 Cambridge Street, Boston, MA, 02135 USA Program in Cell, Molecular and Developmental Biology, Tufts University, Sackler School of Biomedical Studies, 136 Harrison Avenue, Boston, MA, 02111 USA Search for more papers by this author Harris Perlman Harris Perlman Division of Cardiovascular Research, St Elizabeth's Medical Center, Tufts University School of Medicine, 736 Cambridge Street, Boston, MA, 02135 USA Program in Cell, Molecular and Developmental Biology, Tufts University, Sackler School of Biomedical Studies, 136 Harrison Avenue, Boston, MA, 02111 USA Search for more papers by this author Masataka Sata Masataka Sata Division of Cardiovascular Research, St Elizabeth's Medical Center, Tufts University School of Medicine, 736 Cambridge Street, Boston, MA, 02135 USA Search for more papers by this author Aude Le Roux Aude Le Roux Rhone-Poulenc Rorer Gencell, Centre de Recherche de Vitry-Alfortville, 94403 Vitry-sur-Seine, France Search for more papers by this author Thomas W. Sedlak Thomas W. Sedlak Division of Molecular Oncology, Departments of Medicine and Pathology, Howard Hughes Medical Institute, Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Didier Branellec Didier Branellec Rhone-Poulenc Rorer Gencell, Centre de Recherche de Vitry-Alfortville, 94403 Vitry-sur-Seine, France Search for more papers by this author Kenneth Walsh Corresponding Author Kenneth Walsh Division of Cardiovascular Research, St Elizabeth's Medical Center, Tufts University School of Medicine, 736 Cambridge Street, Boston, MA, 02135 USA Program in Cell, Molecular and Developmental Biology, Tufts University, Sackler School of Biomedical Studies, 136 Harrison Avenue, Boston, MA, 02111 USA Search for more papers by this author Author Information Harris Perlman1,2, Masataka Sata1, Aude Le Roux3, Thomas W. Sedlak4, Didier Branellec3 and Kenneth Walsh 1,2 1Division of Cardiovascular Research, St Elizabeth's Medical Center, Tufts University School of Medicine, 736 Cambridge Street, Boston, MA, 02135 USA 2Program in Cell, Molecular and Developmental Biology, Tufts University, Sackler School of Biomedical Studies, 136 Harrison Avenue, Boston, MA, 02111 USA 3Rhone-Poulenc Rorer Gencell, Centre de Recherche de Vitry-Alfortville, 94403 Vitry-sur-Seine, France 4Division of Molecular Oncology, Departments of Medicine and Pathology, Howard Hughes Medical Institute, Washington University School of Medicine, St Louis, MO, 63110 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:3576-3586https://doi.org/10.1093/emboj/17.13.3576 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Tissues with the highest rates of proliferation typically exhibit the highest frequencies of apoptosis, but the mechanisms that coordinate these processes are largely unknown. The homeodomain protein Gax is down-regulated when quiescent cells are stimulated to proliferate, and constitutive Gax expression inhibits cell proliferation in a p21WAF/CIP-dependent manner. To understand how mitogen-induced proliferation influences the apoptotic process, we investigated the effects of deregulated Gax expression on cell viability. Forced Gax expression induced apoptosis in mitogen-activated cultures, but quiescent cultures were resistant to cell death. Though mitogen activation was required for apoptosis, neither the cdk inhibitor p21WAF/CIP nor the tumor suppressor p53 was required for Gax-induced cell death. Arrest in G1 or S phases of the cell cycle with chemical inhibitors also did not affect apoptosis, further suggesting that Gax-mediated cell death is independent of cell cycle activity. Forced Gax expression led to Bcl-2 down-regulation and Bax up-regulation in mitogen-activated, but not quiescent cultures. Mouse embryonic fibroblasts homozygous null for the Bax gene were refractive to Gax-induced apoptosis, demonstrating the functional significance of this regulation. These data suggest that the homeostatic balance between cell growth and death can be controlled by mitogen-dependent pathways that circumvent the cell cycle to alter Bcl-2 family protein expression. Introduction Homeostasis is achieved through a balance of cell growth and cell death. Many recent studies have shown that the proper regulation of cell cycle activity is crucial for cell viability. Deregulated expression of immediate early genes (Evan et al., 1992), cell cycle regulators (Hoang et al., 1994; Shi et al., 1994) or cell cycle-dependent transcription factors (Qin et al., 1994; Kowalik et al., 1995; Liu and Kitsis, 1996) can initiate an abortive cell cycle followed by apoptosis. In addition, the withdrawal of growth factors in vitro (Batistatou and Greene, 1993; Wang and Walsh, 1996) or in vivo (Colombel et al., 1992) from differentiated cells also induces abortive cell cycle progression and apoptosis. These data suggest that cell cycle activity can markedly influence the susceptibility of cells to apoptosis and that aberrant cell cycle progression can induce apoptosis. However, the mechanisms by which cell proliferation and cell death are coordinately regulated are largely unknown. The Bcl-2 family of proteins are apoptotic regulators that function as molecular rheostats to control cellular survival (Yang and Korsmeyer, 1996). Expression of the apoptotic antagonists, Bcl-2 or Bcl-xL, protects cells against various apoptotic stimuli (Hockenbery et al., 1990; Hockenbery, 1995), while overexpression of apoptotic accelerators, such as Bax, Bad or Bcl-xS, induces apoptosis (Oltvai et al., 1993; Yang and Korsmeyer, 1996). However, the effects of cell proliferation on Bcl-2 family protein expression remains to be elucidated. The smooth muscle cells that comprise the vessel wall can undergo high rates of proliferation (Clowes et al., 1983; Kearney et al., 1997; Wei et al., 1997) and apoptosis (Bochaton-Piallat et al., 1995; Han et al., 1995; Isner et al., 1995; Perlman et al., 1997) in a number of vascular disorders. Mitogen-regulated transcription factors function as regulators of proliferation of vascular smooth muscle cells (VSMCs) in response to vessel injury (Walsh and Perlman, 1996). One such transcription factor, the homeoprotein Gax (growth arrest-specific homeobox), was isolated from adult rat aorta cDNA (Gorski et al., 1994). Gax is expressed in quiescent VSMCs and is down-regulated rapidly by conditions which promote proliferation both in vitro and in vivo (Gorski et al., 1993; Weir et al., 1995; Yamashita et al., 1997). The regulation of gax by mitogens resembles the gas (growth arrest-specific) and gadd (growth arrest DNA-damaging) family of genes (Hoffman and Liebermann, 1994). gas and gadd genes are down-regulated rapidly by mitogens and up-regulated by conditions that promote growth arrest. Presumably the down-regulation of gas and gadd genes is required for proper cell cycle progression in response to mitogen activation. Indeed, the constitutive expression of gas1, gas3, gadd45 or gadd153 inhibits S-phase entry (Del Sal et al., 1992; Zhan et al., 1994; Zoidl et al., 1995). Analyses of Gax action in vitro have revealed that its overexpression inhibits proliferation in the G1 phase of the cell cycle (Smith et al., 1997). This growth inhibition is associated with the p53-independent up-regulation of the cyclin-dependent kinase inhibitor p21WAF/CIP. Fibroblasts homozygous null for the p21WAF/CIP gene are not susceptible to Gax-mediated growth inhibition, demonstrating that p21WAF/CIP is essential for Gax-induced growth arrest (Smith et al., 1997). Furthermore, gax overexpression inhibits the formation of injury-induced vascular lesions that are comprised of proliferating VSMCs (Maillard et al., 1997; Smith et al., 1997). Based on previous investigations demonstrating interrelationships between cell proliferation and apoptosis, we examined the effects of gax expression on cell viability. gax-transduced cultures were viable under conditions of quiescence, but underwent apoptosis between 24 and 48 h following serum stimulation. In contrast to Gax-induced cell cycle arrest, Gax-induced apoptosis was independent of the cdk inhibitor p21WAF/CIP, demonstrating that its effects on cell cycle and apoptosis are mediated by separate pathways. Gax-induced cell death was associated with mitogen-dependent up-regulation of Bax, and fibroblasts homozygous null for bax were refractory to Gax-induced cell death. These data suggest that mitogen-regulated transcription factors can coordinate cell proliferation and death through their ability to modulate Bcl-2 family protein expression independent of cell cycle activity. Results Gax expression reduces viability of mitogen-activated VSMCs We previously demonstrated that Gax arrests VSMCs in the G1 phase of the cell cycle following the stimulation of quiescent cells with serum (Smith et al., 1997). However, the effect of Gax expression on cell viability was not addressed previously. Quiescent VSMC cultures were infected with a replication-deficient adenovirus expressing the hemagglutinin-tagged Gax cDNA (Ad-Gax) (Smith et al., 1997) and incubated with serum. At 48 h post-serum stimulation, the Ad-Gax-infected cells displayed a morphology that differed markedly from that of control cells. Light microscopic analyses revealed cytoplasmic shrinkage and detachment from the plate surface in the Ad-Gax-infected cultures (Figure 1A) that overexpress the epitope-tagged Gax protein (Figure 1B). Cultures treated with a replication-deficient adenovirus containing the β-galactosidase cDNA (Ad-β-gal) remained viable and attached to the plate surface under these conditions. A cell viability assay using trypan blue exclusion (Raffo et al., 1995) demonstrated a marked decrease in cell viability at 48 h post-serum stimulation in the Ad-Gax-infected VSMCs compared with mock- or Ad-β-gal-transduced cells (Figure 1C). In contrast, quiescent cells transduced with Ad-Gax remained viable over this time course. Figure 1.Adenovirus-mediated Gax expression reduces viability of serum-stimulated but not quiescent cells. Primary cultures of rat VSMCs were made quiescent for 72 h in low mitogen medium (0.5% FBS). Cells were mock-transduced or transduced at an m.o.i. of 750 p.f.u./cell with either Ad-β-gal or Ad-Gax for 12 h, after which the virus was removed and cultures were returned to low mitogen medium for an additional 12 h. High serum medium was then added for 48 h to stimulate proliferation. For analysis of quiescent cells, parallel cultures were retained in low mitogen media and harvested 72 h after infection. (A) Representative phase-contrast micrographs of quiescent control cells and Ad-Gax-transduced cells that were stimulated with mitogens for 48 h. (B) Immunoblot analysis of control quiescent (CQ) cells, that express endogenous Gax, and serum-stimulated (SS) cells transduced with Ad-Gax. VSMC whole-cell extract (50 μg) was subjected to SDS–PAGE, and immunoblot analysis was performed with antibodies directed against the Gax protein. The adenovirus-encoded Gax protein is tagged with the hemagglutinin epitope leading to an increase in molecular weight. (C) Quantitative analysis of VSMC viability under mitogen deprivation and mitogen activation (48 h). Cells treated as described above were harvested by trypsinization, fixed and counted with a hemacytometer using the trypan blue exclusion method. Each time point represents the mean ± standard error of three determinations from a representative experiment. Download figure Download PowerPoint Gax induces the apoptotic phenotype in mitogen-activated cells Apoptosis is characterized by membrane blebbing, cellular and cytoplasmic shrinkage, chromosomal condensation, and activation of endonuclease-dependent chromosomal fragmentation (Yang and Korsmeyer, 1996). To determine whether the reduction in cell number in Ad-Gax-transduced VSMC cultures can be attributed to apoptosis, transmission electron microscopic analysis was performed to identify morphological features that are characteristic of apoptosis. The Ad-Gax-infected VSMCs displayed cytoplasmic blebbing and chromatin condensation while retaining membrane-bound organelles (Figure 2A). These features were not detected in control cells. Figure 2.Ad-Gax-infected cells exhibit apoptotic features following mitogen activation. (A) Transmission electron microscopic analysis of uninfected quiescent VSMCs and VSMCs were transduced with either Ad-β-gal or Ad-Gax at an m.o.i. of 750 p.f.u./cell and stimulated with serum for 48 h. The quiescent (5200×) and Ad-β-gal-transduced (2950×) VSMCs displayed normal chromatin structure, membrane-bound organelles and visible nucleoli in the plane of focus. The Ad-Gax-transduced VSMCs (3900×) display cytoplasmic blebbing (black arrow) and chromatin condensation (white arrow). (B) Gax-transduced VSMCs are TUNEL positive. Uninfected quiescent VSMCs and mitogen-stimulated VSMCs that had been transduced with the indicated adenovirus construct were stained with TUNEL and Hoechst 33258. Download figure Download PowerPoint DNA fragmentation was analyzed in individual cells using the TdT-mediated dUTP-fluorescein nick-end labeling (TUNEL) analysis. Ad-Gax-treated VSMCs displayed a high frequency of TUNEL-positive labeling, while Ad-β-gal-treated cultures displayed little or no detectable TUNEL-positive cells (Figure 2B). Furthermore, DNA extracted from VSMCs at 48 h post-serum stimulation displayed a characteristic 180 bp increment DNA ladder in the Ad-Gax-treated cultures, but not in mock- or Ad-β-gal-infected cultures (not shown). Flow-cytometric analysis was also employed to detect cell populations containing hypodiploid DNA content (<2N) which is indicative of DNA fragmentation. At 24 h post-serum stimulation, the Ad-Gax-treated VSMCs predominately displayed G0/G1 growth arrest (Figure 3A), consistent with our previous report (Smith et al., 1997). However, by 48 h post-serum stimulation, the Ad-Gax-treated VSMCs displayed a marked increase in hypodiploid DNA, while parallel cultures of saline- or Ad-β-gal-treated cells showed normal DNA content. A population of cells containing hypodiploid DNA was not detected in serum-stimulated VSMCs transduced with an adenoviral vector expressing the general cdk inhibitor p21WAF/CIP, though G0/G1 cell cycle arrest was evident (Figure 3A). Figure 3.Gax-induced DNA fragmentation requires mitogen activation. (A) FACS analysis in serum-stimulated VSMC cultures. Quiescent VSMC cultures were infected by the indicated adenoviral construct at an m.o.i. of 750 p.f.u./cell for 12 h after which the virus was removed and cultures were returned to low mitogen medium for an additional 12 h. Cultures were transferred to high serum medium for the indicated period of time prior to analysis by flow cytometry. (B) FACS analysis of quiescent cultures. Quiescent VSMC cultures were infected with the indicated adenoviral construct at an m.o.i. of 750 p.f.u./cell for 12 h and then transferred to low serum for 60 h prior to analysis by flow cytometry to determine DNA content. Download figure Download PowerPoint Quiescent VSMCs express endogenous Gax protein (Gorski et al., 1993), yet they do not undergo apoptosis. Quiescent VSMCs were also refractory to adenovirus-mediated Gax overexpression (Figure 3B). In contrast, VSMCs infected with a replication-defective adenovirus expressing Fas ligand (Ad-FasL), a known apoptosis inducer (Sata et al., 1998), displayed hypodiploid DNA content under both quiescent and mitogen-activated conditions. G1- and S-phase inhibitors do not block Gax-induced cell death Chemical inhibitors of the cell cycle can prevent abortive cell cycle and apoptosis (Farinelli and Greene, 1996). Therefore, the effects of cell cycle inhibition at different phases was examined in VSMCs transduced with either Ad-β-gal or Ad-Gax. Addition of the G1-phase inhibitor rapamycin (Figure 4A), which blocks Rb phosphorylation (Marx et al., 1995), arrested Ad-β-gal-transduced VSMCs in G1 and had no effect on viability. However, parallel cultures of Ad-Gax-transduced VSMCs underwent similar frequencies of apoptosis in the presence or absence of either rapamycin (Figure 4B) or mimosine (not shown). In addition, the late G1/S inhibitor hydroxyurea or the S-phase inhibitor aphidicolin (Farinelli and Greene, 1996) also did not affect the frequency of cell death induced by the combination of mitogen activation and Gax expression. Figure 4.G1- and S-phase inhibitors do not affect Gax-induced cell death. Quiescent VSMC cultures were infected with the indicated adenoviral construct at an m.o.i. of 750 p.f.u./cell for 12 h. After virus removal, cultures were incubated in low mitogen medium for an additional 12 h and then transferred to high serum medium in the absence (saline) or presence of the cell cycle inhibitors, rapamycin, hydroxyurea or aphidicolin for 48 h prior to analysis by flow cytometry. (A) Percentage of cells with ≥2N DNA content in G1, S and G2/M phases following infection with Ad-β-gal or Ad-Gax and treatment with the indicated cell cycle inhibitor. (B) Percentage of total cells with <2N DNA content. Download figure Download PowerPoint Gax induces apoptosis independent of p21WAF/CIP and p53 It was of interest to determine whether forced Gax expression could induce apoptosis in mouse embryonic fibroblasts null homozygous for both p21WAF/CIP alleles (p21−/− MEFs) which previously have been shown to be refractory to Ad-Gax-induced growth arrest (Smith et al., 1997). Flow-cytometric analysis was not utilized since p21−/− MEFs are polyploid (Deng et al., 1995; H.Perlman, unpublished observations), thereby confounding the analysis of hypodiploid DNA content. Instead, viability was assessed by directly counting cells using trypan blue exclusion (Raffo et al., 1995). p21−/− MEF cultures transduced with Ad-Gax displayed a 79% decrease in cell viability compared with the quiescent control cultures (Figure 5). In contrast, Ad-β-gal-infected cultures increased in cell number during this time course, as did mock-infected cultures (not shown). TUNEL analyses of saline-, Ad-β-gal- or Ad-Gax-infected p21−/− MEF cultures revealed that a large portion of the Ad-Gax-transduced cells were TUNEL positive and displayed chromatin condensation, while the control cultures were TUNEL negative (not shown). Figure 5.Gax overexpression induces apoptosis independently of p21WAF/CIP. Quiescent p21−/− MEFs were infected either with Ad-β-gal or Ad-Gax at an m.o.i. of 750 p.f.u./cell for 12 h. Virus was removed and cultures were returned to low mitogen medium for an additional 12 h prior to incubation in high serum media for 48 h. Parallel quiescent cultures were incubated for 72 h in low mitogen media without viral transduction. Cultures were harvested by trypsinization, fixed and cell number was determined by the trypan blue exclusion method (Raffo et al., 1995). Each time point represents the mean ± standard error of three determinations from a representative experiment. Download figure Download PowerPoint To determine if Gax-induced apoptosis requires p53, spontaneously immortalized MEFs containing deletions of both p53 alleles (10.1 MEFs) (Harvey and Levine, 1991) were either treated with saline or infected with the adenoviral constructs and subjected to flow-cytometric analyses at 48 h post-serum stimulation. Ad-Gax-infected cells displayed hypodiploid DNA content while uninfected or Ad-β-gal-infected cells displayed normal DNA profiles (Figure 6A). In addition, Ad-Gax-infected cultures exhibited nuclei with condensed chromatin that stained positive for TUNEL (not shown). Figure 6.Gax overexpression induces apoptosis independently of p53. (A) Quiescent 10.1 MEFs that are null for p53 were infected with Ad-β-gal or Ad-Gax at an m.o.i. of 750 p.f.u./cell for 12 h. The virus was removed, cultures were returned to low mitogen medium for an additional 12 h and then transferred to growth medium for 48 h. DNA content was analyzed by FACS analysis. An uninfected quiescent culture of 10.1 MEFs is shown for comparison. (B) 10.1 MEFs co-transfected with β-galactosidase and wild-type Gax expression plasmids displayed reduced number of β-galactosidase-positive cells. Shown is a representative micrograph of 10.1 MEFs co-transfected with pCMV-β-galactosidase and plasmids that express wild-type Gax or a mutant Gax lacking the homeodomain. Quiescent 10.1 MEFs transfected with 1 μg of the β-galactosidase (pCMV-β-gal) and 4 μg of either pCGN-Gax (Gax) or pCGN-ΔHD-Gax (ΔHD-Gax) expression vectors using LipofectAmine (Gibco-BRL). Following transfection, cultures were transferred to high serum for 48 h and then fixed and stained with X-gal. Inclusion of 50 μM NAC increased the number of β-galactosidase-positive cells that were co-transfected with pCGN-Gax. (C) Representative micrograph demonstrating co-localization of pyknotic nuclei and Gax protein from the pCGN-Gax expression plasmid. Transfected 10.1 MEFs were fixed and stained with anti-Gax antibody, followed by treatment with a rhodamine (red)-conjugated secondary antibody. Cellular DNA was stained with Hoechst 33258. (D) Quantitative analysis of 10.1 MEFs transfected with pCMV-β-gal and either pCGN-Gax (Gax) or pCGN-ΔHD-Gax (ΔHD-Gax) expression plasmids as described above in the presence or absence of 50 μM NAC. Cultures were fixed and stained with X-gal and the number of β-galactosidase-positive cells was determined. Each time point represents the mean ± standard error of three determinations from a representative experiment. (E) Bcl-2 overexpression rescues 10.1 MEFs transfected with the Gax expression plasmid. 10.1 MEFs were transfected with 1 μg of pCMV-β-gal and either 2 μg of pCDNA-Bcl-2 (Bcl-2), 2 μg of empty vector (−), 2 μg of pCGN-Gax (Gax) or 2 μg of pCDNA-Bcl-2 and 2 μg of pCGN-Gax (Gax & Bcl-2). Some mixes contained additional pCGN empty vector such that all transfections were performed with 5 μg of plasmid DNA. Values represent the mean ± standard error and were compared for statistical significance by ANOVA and student t-test analysis (P <0.05). Download figure Download PowerPoint Since the adenovirus E4 region can influence apoptosis (Marcellus et al., 1996), it was necessary to determine whether the apoptosis induced by the Gax transgene was modulated by adenoviral genes. An established cell death assay (Miura et al., 1993; Boyd et al., 1995; Chittenden et al., 1995) was performed using expression plasmids encoding Gax or other test genes transfected in combination with a β-galactosidase expression plasmid. This assay allows assessment of changes in cell viability as determined by variations in the number of β-galactosidase-positive cells. Expression vectors were utilized that encoded either wild-type Gax or a mutant form of Gax lacking the homeodomain (ΔHD-Gax), which is ineffective at inducing cell cycle arrest and transactivating the p21WAF/CIP promoter (Smith et al., 1997). ΔHD-Gax- and empty vector-transfected 10.1 MEFs (Figure 6B, D and E), p21−/− MEFs (not shown) and A7r5 VSMCs (not shown) displayed numerous β-galactosidase-positive cells compared with the cultures transfected with wild-type Gax expression plasmid. These data suggest that the homeodomain is essential for Gax-induced cell death. Apoptosis induced by the Gax expression plasmid was also indicated by the presence of Gax-positive pyknotic nuclei (Figure 6C). Incubation of Gax-transfected cultures with 50 μM N-acetyl-cysteine (NAC), an antioxidant that inhibits apoptosis (Hockenbery et al., 1993; Fabbretti et al., 1995), significantly increased the number of β-galactosidase-positive cells (Figure 6B and D). Furthermore, co-transfection of equal amounts of pCDNA-Bcl-2 expression plasmid (Figure 6E) with pCGN-Gax expression plasmid also increased the number of β-galactosidase-positive MEFs, suggesting that Bcl-2 overexpression can rescue cells from Gax-induced cell death (P <0.05). Constitutive Gax expression modulates Bcl-2 and Bax proteins The expression levels of the Bcl-2 family of proteins were determined by Western blot analyses on extracts prepared from quiescent and serum-stimulated cultures treated with either saline (mock-infected), Ad-β-gal or Ad-Gax. Relative to control cultures, serum-stimulated cultures infected with Ad-Gax displayed elevated Bax levels and decreased Bcl-2 levels (Figure 7A). Bad levels also increased under these conditions (not shown), but no changes were observed in levels of the Bcl-2 family proteins Bcl-xL, Bak or Bag, while Bcl-xS was not detected in any samples (not shown). In contrast, Ad-Gax infection had little or no effect on Bax and Bcl-2 expression when VSMC cultures were retained in low mitogen media (Figure 7B). Figure 7.Mitogen-dependent modulation of Bcl-2 family proteins by the Gax homeoprotein. (A–D) Immunoblot analyses of Bcl-2 family proteins in VSMCs, 10.1 MEFs and p21−/− MEFs. Whole-cell extracts (50 μg) were prepared from the indicated quiescent and serum-stimulated cultures that were either mock-infected (saline) or infected with Ad β-gal or Ad-Gax. Immunoblot analysis was performed with antibodies directed against the indicated proteins. Tubulin or Bcl-xL immunoblots indicate equal loading of protein. Antibody specificity was demonstrated by loss of signal when a molar excess of immunogenic peptide was pre-incubated with antibody (not shown). (E) RNase protection assay of bcl-2 family transcripts. Quiescent cultures of VSMCs were infected with Ad-β-gal, Ad-Gax or mock-infected (saline) and serum-stimulated for 48 h. Total RNA was prepared and hybridized to a multi-probe template set and fractionated by acrylamide gel electrophoresis. (F) Immunohistochemical analysis of Bax. Quiescent VSMCs or VSMCs stimulated with serum after infection with Ad-β-gal or Ad-Gax were fixed and stained with an anti-Bax primary antibody. The signal was detected with rhodamine-conjugated anti-rabbit secondary antibody and DNA was stained with Hoechst 33258. Download figure Download PowerPoint Infection with Ad-Gax markedly up-regulated Bax and down-regulated Bcl-2 in 10.1 MEFs, that lack p53, and in p21−/− MEFs that are sensitive to Gax-induced cell death (Figure 7C and D). RNase protection analysis using a multi-probe template set revealed that Gax overexpression had little or no effect on bax or other bcl-2 gene family transcript levels in VSMCs (Figure 7E), indicating that Bax up-regulation occurs at a post-transcriptional level. Expression of Bax was evaluated further in individual cells by immunofluorescence (Figure 7F). Control VSMCs displayed diffuse staining of Bax protein, while Ad-Gax-infected cultures displayed an intense staining pattern. Bcl-2 expression was detected in the control cells, but not in the Ad-Gax-transduced cultures (not shown). Collectively, these data demonstrate that the up-regulation of Bax and down-regulation of Bcl-2 consistently occurs in cell types that are susceptible to Gax-induced cell death. Bax is essential for Gax-induced cell death MEFs with homozygous disruptions in both bax alleles (Bax−/− MEFs) (Knudson et al., 1995) were analyzed to determine whether this Bcl-2 family protein is essential for death signal transmission induced by forced Gax expression. Serum-stimulated Bax−/− MEFs transduced with Ad-Gax did not undergo apoptosis since normal DNA profiles, devoid of hypodiploid DNA, were observed by flow-cytometric analysis (Figure 8A). In contrast, isogenic wild-type MEFs transduced with Ad-Gax exhibited decreased cell viability (not shown) and 40% hypodiploid DNA content (Figure 8B). Bax−/− and wild-type MEFs transduced with Ad-β-gal displayed normal DNA profiles that were similar to saline-treated cultures. These data demonstrate that Gax-induced apoptosis is dependent on the function of Bax. Figure 8.Bax is essential for Gax-induced apoptosis. Wild-type MEF and isogenic Bax−/− MEF cultures were mock-infected (saline) or infected with Ad-β-gal and Ad-Gax at an m.o.i. of 750 p.f.u./cell for 12 h. The virus was then removed and cultu
Referência(s)