TGFbeta-induced Apoptosis Research Articles

Par6 is an essential mediator of apoptotic response to transforming growth factor beta in NMuMG immortalized mammary cells

BackgroundWe previously observed that the TGFbeta-Par6 pathway mediates loss of polarity and apoptosis in NMuMG cells. Here we investigate the contribution of Par6 versus TGFbeta receptor I activation to TGFbeta-induced apoptosis in association with changes in apico-basal polarity. We focus on the effect of Par6 activation on alpha6beta4 integrin expression and localization, and Nuclear Factor-kappaB (p65/RelA) activation, previously shown to mediate polarity-dependent cell survival.MethodsUsing immunoblotting and/or immunofluorescence we investigated the effect of TGFbeta1 on apoptosis, alpha6, beta4 and beta1 integrin expression/localization, and p65/RelA phosphorylation/localization in monolayer and three-dimensional (3D) cultures of NMuMG cells with an overactive or inactive Par6 pathway. Results were quantified by band densitometry or as percent of 3D structures displaying a phenotype. Differences among means were compared by two-way ANOVA.ResultsBlocking Par6 activation inhibits TGFbeta-induced apoptosis. Par6 overactivation enhances TGFbeta-induced apoptosis, notably after 6-day exposure to TGFbeta (p < 0.001), a time when parental NMuMG cells no longer respond to TGFbeta apoptotic stimuli. 48-hour TGFbeta treatment reduced beta4 integrin levels in NMuMG monolayers and significantly reduced the basal localization of alpha6 (p < 0.001) and beta4 (p < 0.001) integrin in NMuMG 3D structures, which was dependent on both Par6 and TGFbeta receptor I activation and paralleled apoptotic response. After 6-day exposure to TGFbeta, Par6-dependent changes to beta4 integrin were no longer apparent, but there was reduced phosphorylation of p65/RelA (p < 0.001) only in Par6 overexpressing cells. Differences in p65/RelA localization were not observed among the different cell lines after 48-hour TGFbeta exposure.ConclusionsPar6 and TGFbeta receptor I activation are both necessary for TGFbeta-induced apoptosis in NMuMG cells. Importantly, Par6 overexpression enhances the sensitivity of NMuMG to TGFbeta-induced apoptosis, notably upon prolonged exposure to this growth factor, when NMuMG parental cells are usually apoptosis-resistant. Thus, endogenous Par6 level might be important in determining whether TGFbeta will function as either a pro-apoptotic or pro-survival factor in breast cancer, and potentially aid in predicting patient’s prognosis and therapy response.

Runx1 Is a Co-activator with FOXO3 to Mediate Transforming Growth Factor β (TGFβ)-induced Bim Transcription in Hepatic Cells

Transforming growth factor beta (TGFbeta) regulates essential cellular functions such as cellular proliferation, differentiation, and apoptosis. The Bcl-2 family of proteins has been implicated as mediators of TGFbeta-induced apoptosis. We demonstrated previously that TGFbeta induces the expression of Bim (Bcl-2-interacting mediator of cell death), a member of the BH3-only family of pro-apoptotic Bcl-2 proteins, to induce cell death in B-lymphocytes. Here, we investigated the mechanism of TGFbeta-mediated Bim expression in two hepatocyte cell lines that undergo apoptosis with TGFbeta, AML-12 and Hep3B. We show that TGFbeta induces Bim protein and mRNA levels, and its expression is sufficient to induce cell death. Gene array results revealed that Runx1, a member of the Runx family of transcription factors, was induced by TGFbeta, and this induction was confirmed at the mRNA and protein levels. Interestingly, TGFbeta specifically induced the expression of Runx1 protein from an internal ribosome entry site (IRES)-dependent, cap-independent, mRNA transcript, and its overexpression was sufficient to induce hepatocyte apo pto sis. Deletion and mutation analyses of the murine Bim promoter identified a putative forkhead binding element, at position -174 to -168 from the transcription start site, as the mediator of Runx1 induction. Co-immunoprecipitation, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays demonstrated that Runx1 does not bind directly to the identified forkhead binding element but rather binds the transcriptional regulator FOXO3, which occupies this site. Finally, small interfering RNA knockdown of Runx1 or FOXO3 decreased TGFbeta-induced Bim expression. Our results support a mechanism in which TGFbeta stimulates Bim transcription by up-regulating Runx1 expression, which binds FOXO3, and the two cooperate in the transcriptional induction of Bim.

Transforming growth factor ß1 induces apoptosis by suppressing FLICE‐like inhibitory protein in DU145 prostate epithelial cells

Transforming growth factor beta (TGFbeta) is a paracrine mediator of prostate epithelial cell apoptosis. In rodents, castration induces production of TGFbeta by stromal cells, which leads to apoptosis of epithelial cells. To identify potential mediators of this cell death pathway, we developed a model using DU145 cells, a tumorigenic human prostate epithelial cell line. We discovered that at low density, in low mitogen media, DU145 cells apoptose when treated with TGFbeta1. Prior to the onset of death, TGFbeta1 treatment downregulated the expression of the caspase inhibitor FLICE-like inhibitory protein (FLIP), at both the mRNA and protein level, suggesting a causal role between FLIP downregulation and cell death. To confirm the importance of FLIP in TGFbeta1-induced apoptosis, we employed small interfering RNA (siRNA) to silence FLIP expression. Doing so led to apoptosis, which is consistent with the hypothesis that FLIP prevents death in these cells. Furthermore, inhibition of caspase-8 by siRNA knockdown partially rescued the apoptotic effects of TGFbeta1, suggesting a role for death receptor signaling components in TGFbeta-mediated death of prostate epithelial cells.

Pivotal role of Smad3 in a mouse model of T cell–mediated hepatitis

Transforming growth factor beta (TGFbeta) promotes hepatocellular apoptosis and suppresses hepatic lymphocyte responses in part through activation of Smad3. The purpose of the current study was to determine the importance of Smad3 signaling in an experimental model of autoimmune hepatitis induced by concanavalin A (ConA), a process involving T cell activation and hepatocellular apoptosis. C57Bl/6 wild-type (Wt) or Smad3-deficient (Smad3(-/-)) mice were injected intravenously with 15 mg/kg ConA or vehicle. Nine hours post ConA injection, Wt mice presented with severe hepatitis as assessed by increased liver transferases. This injury was associated with eosinophil accumulation and preceded at 3 hours post-injection by significant increases in hepatic T helper 1 (interferon gamma) and T helper 2 (interleukin-4) cytokine production. Absence of Smad3 significantly blunted hepatocellular injury 9 hours post ConA injection, which was associated with reduced early T helper 1 and T helper 2 cytokine production and eosinophil accumulation. Smad3(-/-) livers also showed significant reductions in hepatocellular apoptosis as assessed by terminal UTP nick-end labeling when compared to ConA-treated Wt mice in conjunction with reduced caspase 3 cleavage, which was likely mediated by a Smad3-dependent inhibition of the survival factor extracellular signal-regulated kinase 1/2. In vitro, Smad3(-/-) hepatocytes were resistant to TGFbeta-induced apoptosis, and this protection was dependent on extracellular signal-regulated kinase activation. Together, these results show, for the first time, the significance of Smad3 signaling in autoimmune hepatitis, underlining the control of Smad3-dependent TGFbeta signaling on proinflammatory cytokine production, eosinophil recruitment, and hepatocellular apoptosis. Interruption of this pathway could be beneficial clinically to limit acute fulminant liver pathologies.

Upregulation of two BH3-only proteins, Bmf and Bim, during TGFβ-induced apoptosis

Transforming growth factor-beta (TGFbeta)-activated signalling pathways can lead to apoptosis, growth arrest or promotion of malignant behaviour, dependent on cellular context. The molecular mechanisms involved in TGFbeta-induced apoptosis remain controversial; although changes in gene expression are thought to be pivotal to the process, several different candidate apoptotic initiators and mediators have been proposed. Smad4, a critical component of the TGFbeta-induced transcriptional machinery, is shown here to be essential for induction of apoptosis. Gene expression analysis identified the proapoptotic Bcl-2 family members, Bmf and Bim, as induced by TGFbeta, dependent on both Smad4 and p38 function and the generation of reactive oxygen species. TGFbeta-induced Bmf and Bim localize to cellular membranes implicated in apoptosis. Inhibition of the TGFbeta-induced expression of both these proteins together provides significant protection of cells from apoptosis. The TGFbeta-triggered cell death programme thus involves induction of multiple BH3-only proteins during the induction of apoptosis.

Daxx is required for stress-induced cell death and JNK activation

Daxx has been implicated in the modulation of apoptosis in response to various stimuli. In the nucleus, Daxx interacts and colocalizes with the promyelocytic leukemia protein (PML) into the PML-nuclear body. Moreover, overexpressed Daxx positively modulates FAS-ligand and TGFbeta-induced apoptosis. However, recent reports indicate that Daxx can also act as an antiapoptotic factor. As most studies on the role of Daxx in cell death have been conducted using tumour cell lines, we analysed the function of Daxx in physiological settings. We found that Daxx is induced upon exposure to ultraviolet (UV) irradiation and hydrogen peroxide treatment. We employed RNA interference to downregulate Daxx in primary fibroblasts. Remarkably, Daxx-depleted cells are resistant to cell death induced by both UV irradiation and oxidative stress. Furthermore, the downregulation of Daxx results in impaired MKK/c-Jun-N-terminal kinase (JNK) activation. This is the first evidence that Daxx promotes cell death and JNK activation in physiological conditions.

Heparin-binding epidermal growth factor and its receptors mediate decidualization and potentiate survival of human endometrial stromal cells.

Heparin-binding epidermal growth factor (HB-EGF) has pleiotropic biological functions in many tissues, including those of the female reproductive tract. It facilitates embryo development and mediates implantation and is thought to have a function in endometrial receptivity and maturation. The mature HB-EGF molecule manifests its activity as either a soluble factor (sol-HB-EGF) or a transmembrane precursor (tm-HB-EGF) and can bind two receptors, EGFR and ErbB4/HER4. In this study, we identify factors that modulate expression of HB-EGF, EGFR, and ErbB4 in endometrial stromal cells in vitro. We demonstrate that levels of sol- and tm-HB-EGF, EGFR, and ErbB4 are increased by cAMP, a potent inducer of decidualization of the endometrial stroma. We also show that production of sol- and tm-HB-EGF is differentially modulated by TNF alpha and TGF beta. Our data suggest that HB-EGF has a function in endometrial maturation in mediating decidualization and attenuating TNF alpha- and TGF beta-induced apoptosis of endometrial stromal cells.

Regulation of TGFbeta1-mediated growth inhibition and apoptosis by RUNX2 isoforms in endothelial cells.

Runx transcription factors regulate viral growth, hematopoiesis, bone formation, angiogenesis, and gastric epithelial development through specific DNA-binding motifs on target gene promoters. Vascular endothelial cells (ECs) express RUNX genes that are activated by angiogenic factors. The RUNX2 gene also activates the vascular endothelial growth factor promoter. Alternatively spliced forms of RUNX genes have been described, but their functions in angiogenesis have not been elucidated. In this study, expression of a novel alternatively spliced variant of RUNX2 (RUNX2Delta8), lacking the region encoded by exon 8, was detected in aortic tissue undergoing angiogenesis in vitro and in ECs. Expression of RUNX2 and RUNX2Delta8 increased in vascular sprouts concomitant with expression of cellular proteases and cytokines known to mediate angiogenesis. RUNX2 DNA-binding activity was expressed in proliferating but not quiescent ECs. Ectopic expression of RUNX2 in ECs increased cell sprouting, cell proliferation, DNA synthesis, and phosphorylation of phosphorylated retinoblastoma relative to control transfectants while RUNX2, but not RUNX2Delta8 transfectants, acquired resistance to growth inhibition by transforming growth factor (TGFbeta1). Furthermore, RUNX2Delta8-transfected cells were more sensitive to TGFbeta1-induced apoptosis than RUNX2 transfectants. Consistent with these data, the RUNX2 gene was a strong repressor of the promoter of the cyclin-dependent kinase inhibitor, p21(CIP1), while RUNX2Delta8 was a competitive inhibitor of RUNX2 and exhibited weak repression activity. These results support the hypothesis that ECs regulate growth and apoptosis, in part, by alternative splicing events in the RUNX2 transcription factor to affect the TGFbeta1 signaling pathway. The exon 8 domain of RUNX2 may contribute to the strong repression activity of RUNX2 for some target gene promoters.

TGFbeta1-induced suppression of glutathione antioxidant defenses in hepatocytes: caspase-dependent post-translational and caspase-independent transcriptional regulatory mechanisms.

TGFbeta1-induced hepatocyte apoptosis involves the production of reactive oxygen species. An effective cellular defense mechanism against oxidative stress is the tripeptide glutathione (GSH), and the rate-limiting step in GSH biosynthesis is catalyzed by the heterodimeric holoenzyme glutamate cysteine ligase (GCL). Here, we demonstrate that TGFbeta1-induced apoptosis in the TAMH murine hepatocyte cell line is accompanied by both the cleavage and loss of the catalytic subunit of GCL (GCLC) and the down-regulation of GCLC gene expression resulting in a reduction in GCL activity and depletion of intracellular GSH. TGFbeta1-induced apoptosis is also accompanied by a reduction in Bcl-XL, an effect that may facilitate TGFbeta1-induced apoptosis as Bcl-XL overexpression inhibits TGFbeta1-induced caspase activation and cell death. Interestingly, Bcl-XL overexpression prevents TGFbeta1-induced cleavage of GCLC protein but not down-regulation of GCLC mRNA. Furthermore, TGFbeta1-induced down-regulation of GCLC mRNA is prevented by inhibition of histone deacetylase activity, suggesting that this is an active repression of GCLC gene transcription. These findings suggest that the suppression of GSH antioxidant defenses associated with the caspase-dependent cleavage of GCLC protein, caspase-independent suppression of GCLC gene expression, and depletion of intracellular GSH may play a role in enhancing TGFbeta1-induced oxidative stress and potentiating apoptotic cell death.

Smad3 Potentiates Transforming Growth Factor β (TGFβ)-induced Apoptosis and Expression of the BH3-only Protein Bim in WEHI 231 B Lymphocytes

Transforming growth factor-beta (TGFbeta) is a potent growth inhibitor and inducer of apoptosis in B lymphocytes and is essential for immune regulation and maintenance of self-tolerance. Here we show that exogenous overexpression of Smad3 potentiates TGFbeta-induced apoptosis and expression of the pro-apoptotic protein Bim in WEHI 231 B lymphocytes. Overexpression of dominant-negative forms of Smad3 abrogate these TGFbeta-induced responses. We also demonstrate that TGFbeta induces Bim protein expression concomitant with its induction of apoptosis in the mouse progenitor B lymphocyte cell line, Ba/F3. Enhanced expression of Bim protein induced by TGFbeta is associated with an increased association of Bim with Bcl-2 and a concomitant loss of mitochondrial membrane potential. Furthermore, we find that the anti-apoptotic effect of the pro-survival cytokine CD40 results in the abrogation of TGFbeta-mediated Bim induction. Our data provide the first evidence of Bim expression levels that are increased by the addition of a pro-apoptotic cytokine, TGFbeta, and also suggest that the TGFbeta-specific transcription factor Smad3 plays a role in mediating Bim expression levels and apoptosis.

Mitochondrial and microsomal derived reactive oxygen species mediate apoptosis induced by transforming growth factor-beta1 in immortalized rat hepatocytes.

Transforming growth factor-beta1 (TGFbeta1) is a multifunctional cytokine that is over expressed during liver hepatocytes injury and regeneration. SV40-transformed CWSV-1 rat hepatocytes that are p53-defective undergo apoptosis in response to choline deficiency (CD) or TGFbeta1, which mediates CD-apoptosis. Reactive oxygen species (ROS) are essential mediators of apoptosis. We have shown that apoptosis induced by TGFbeta1 is accompanied by ROS generation and the ROS-trapping agent N-acetylcysteine (NAC) inhibits TGFbeta1-induced apoptosis. While persistent induction of ROS contributes to this form of apoptosis, the source of ROS generated downstream of TGFbeta1 is not clear. The mitochondria and the endoplasmic reticulum both harbor potent electron transfer chains that might be the source of ROS essential for completion of TGFbeta1-apoptosis. Here we show that CWSV-1 cells treated with cyclosporine A, which prevents opening of mitochondrial membrane pores required for ROS generation, inhibits TGFbeta1-induced apoptosis. A similar effect was obtained by treating these cells with rotenone, an inhibitor of complex 1 of the mitochondrial electron transfer chain. However, we demonstrate that TGFbeta1 induces cytochrome P450 1A1 and that metyrapone, a potent inhibitor of cytochrome P450 1A1, inhibits TGFbeta1-induced apoptosis. Therefore, our studies indicate that concurrent with promoting generation of ROS from mitochondria, TGFbeta1 also promotes generation of ROS from the cytochrome P450 electron transfer chain. Since inhibition of either of these two sources of ROS interferes with apoptosis, it is reasonable to conclude that the combined involvement of both pathways is essential for completion of TGFbeta1-induced apoptosis.

Transforming growth factor beta 1 induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells.

Transforming growth factor beta (TGF-beta) induces apoptosis in a variety of cells. We have previously shown that TGF-beta 1 rapidly induces apoptosis in the FaO rat hepatoma cell line. We have now studied the effect of TGF-beta 1 on the expression of different members of the Bcl-2 family in these cells. We observed no detectable changes in the steady-state levels of Bcl-2, Bcl-X(L), and Bax. However, TGF-beta 1 induced caspase-dependent cleavage of BAD at its N terminus to generate a 15-kDa truncated protein. Overexpression of the 15-kDa truncated BAD protein enhanced TGF-beta 1-induced apoptosis, whereas a mutant BAD resistant to caspase 3 cleavage blocked TGF-beta 1-induced apoptosis. Overexpression of Smad3 dramatically enhanced TGF-beta 1-induced cleavage of BAD and apoptosis, whereas antisense Smad3 blocked TGF-beta 1-induced apoptosis and BAD cleavage. These results suggest that TGF-beta 1 induces apoptosis through the cleavage of BAD in a Smad3-dependent mechanism.

Transforming growth factor-beta1-induced Smad signaling, cell-cycle arrest and apoptosis in hepatoma cells

Transforming growth factor-beta1 (TGFbeta) is involved in the regulation of liver cell proliferation and apoptosis, and escape of hepatoma cells from the growth restraining signals of TGFbeta has been suggested to contribute to tumor development. TGFbeta modulates gene transcription by receptor-mediated activation of Smad proteins which act as transcription factors. TGFbeta-mediated primary signaling responses as well as effects on the cell cycle and apoptosis were investigated in the human hepatoblastoma line HepG2, the rat hepatoma line FTO-2B and the mouse hepatoma line 55.1c. Activation of a Smad (Sma and Mad homolog) response-element-driven luciferase reporter by TGFbeta was very similar in all three cell lines, indicating functionality of the primary TGFbeta signaling pathway. Moreover, TGFbeta-inducible early gene was transiently activated by TGFbeta in all cell lines as shown by RT-PCR. HepG2 cells, however, were completely resistant to TGFbeta-induced growth arrest and apoptosis and 55.1c cells were only slightly susceptible to TGFbeta-induced apoptosis. By contrast, treatment of FTO-2B cells with TGFbeta led to a partial G0/G1 arrest and a strong induction of apoptosis. TGFbeta-induced apoptosis of FTO-2B cells was inhibited by dexamethasone, insulin, phenobarbital and dieldrin. Of these agents, only insulin led to a significant reduction of TGFbeta-stimulated Smad-reporter activity, suggesting that the other compounds interfere with TGFbeta-induced apoptosis downstream of Smad-mediated primary transcriptional responses at a level that may be constitutively altered in apoptosis-resistant hepatoma cell lines.

The increase in bladder carcinoma cell population induced by the free beta subunit of human chorionic gonadotrophin is a result of an anti-apoptosis effect and not cell proliferation.

Ectopic production of free beta human chorionic gonadotrophin (hCGβ) by bladder carcinoma is well described and occurs in approximately 35% of cases. hCGβ secreting tumours are more aggressive, radioresistant and have a greater propensity to metastasize. We proposed that the ectopic production of hCGβ was contributing in an autocrine fashion to the radioresistance and metastatic potential of such secreting tumours. Though we demonstrated that the addition of hCGβ to the culture media of bladder, cervical and endometrial carcinoma cell lines brought about an increase in cell populations this was not accompanied by a significant increase in the rate of replication. Since a cell population size is a balance of mitosis and mortality, we proposed that hCGβ was inhibiting apoptosis. Here we have demonstrated that following incubation with recombinant hCGβ, bladder carcinoma cells refrain from undergoing apoptosis. Quantitation of apoptotic bodies was carried out by immunoassay and corrected to cell number as determined by MTT assay. In each cell line, addition of hCGβ reduced the number of apoptotic bodies dose-dependently, indicating a diminished apoptotic rate. Furthermore, TGFβ1-induced apoptosis could be dose-dependently inhibited by co-incubation with hCGβ. We propose, therefore, that such a decline in apoptosis may account for the cell population increase previously reported. It may also explain the radioresistance and aggressive nature of hCGβ-secreting tumours and the poor prognosis associated therein. © 2000 Cancer Research Campaign

Peroxisome proliferators induce apoptosis and decrease DNA synthesis in hepatoma cell lines.

We examined the effects of various peroxisome proliferators (PPs) such as the hypolipidaemic agents clofibric acid (CLO), bezafibrate (BEZA), ciprofibrate (CIPRO) and nafenopin (NAFE) and the plasticizer di-(2-ethylhexyl)phthalate (DEHP) on peroxisomal enzyme activities, apoptosis and DNA synthesis in rat FaO and human HepG2 hepatoma cell lines. Both growing and confluent cultures were treated with PPs (250 microM) for 48 or 72 h. In accordance with our previous observations in PP-treated primary hepatocyte cultures of rat and human origin, the various PPs increased peroxisomal enzyme activities in rat FaO cells but not in human HepG2 cells. PPs strongly induced apoptosis in FaO cells. They did not affect TGFbeta-induced apoptosis, with the exception of DEHP and NAFE, respectively blocking and increasing induced apoptosis in confluent cultures. Moreover, PPs produced a minor, but significant, decrease in DNA synthesis in FaO cells. PPs also decreased DNA synthesis in growing HepG2 cells, and CLO, CIPRO and NAFE induced apoptosis in confluent HepG2 cultures. This is in opposition with the effects of PPs on primary hepatocyte cultures, i.e. inhibition of both spontaneous and TGFbeta-induced apoptosis and increases in DNA synthesis in rat hepatocytes, and unchanged mitosis-apoptosis balance in human hepatocytes.

Role of caspases and possible involvement of retinoblastoma protein during TGFbeta-mediated apoptosis of human B lymphocytes.

In this study, we investigated the involvement of caspases in TGFbeta-induced apoptosis in human B cells. Our results show that TGFbeta-mediated nuclear fragmentation, observed in the Epstein-Barr virus-negative Burkitt's Lymphoma cell line BL41, was abolished in the presence of the tripeptide caspase inhibitor zVAD-fmk or the specific caspase-3 inhibitor DEVD-fmk. Other apoptotic manifestations such as cell shrinkage, surface phosphatidylserine expression and chromatin condensation were strongly inhibited by zVAD-fmk but only partially by DEVD-fmk. This suggests that other caspases in addition to caspase-3 control these apoptotis-associated features. Specific activation of caspase-3 during TGFbeta-induced apoptosis was demonstrated by the DEVD-fmk-sensitive expression of the active p17 subunit of caspase-3 and by in vivo cleavage of PARP. In addition, TGFbeta treatment of BL41 promoted the expression of both dephosphorylated and truncated forms of the retinoblastoma protein. Inhibition of caspase-3 activity abolished both nuclear fragmentation and expression of the truncated retinoblastoma protein, without modifying the G1 cell cycle arrest induced by TGFbeta. Our data thus demonstrate that TGFbeta-induced apoptosis of lymphoma B lymphocytes is dependent on caspase activation and involves caspase-dependent cleavage of the retinoblastoma protein.

Suppression of hepatocyte apoptosis and induction of DNA synthesis by the rat and mouse hepatocarcinogen diethylhexylphlathate (DEHP) and the mouse hepatocarcinogen 1,4-dichlorobenzene (DCB).

Nongenotoxic rodent hepatocarcinogens do not damage DNA but cause liver tumours in the rat and mouse, associated with the induction of hepatic DNA synthesis. Previously, we have demonstrated that nongenotoxic hepatocarcinogens such as phenobarbitone and the peroxisome proliferator (PP), nafenopin, also suppress rat hepatocyte apoptosis. The nongenotoxic chemicals 1,4-dichlorobenzene (DCB) and the PP, diethylhexyl phthalate (DEHP), both induce high levels of DNA synthesis in rat liver in vivo, but only DEHP is hepatocarcinogenic in this species. Here, we investigate whether the difference in rat carcinogenicity of these two hepatic mitogens may be due to differences in their ability to suppress hepatocyte apoptosis. In rat hepatocytes in vitro, MEHP (the active metabolite of DEHP) induced DNA synthesis 2.5-fold (P = 0.001) and suppressed 10- and 4-fold, respectively both spontaneous (P = 0.0008) and transforming growth factor beta1 (TGFbeta1)-induced (P = 0.0001) apoptosis. DCB gave a small (1.7-fold) increase in DNA synthesis (P = 0.03) and a small (1.7- to 2-fold) suppression of both spontaneous (P = 0.022) and TGFbeta1-induced (P = 0.015) apoptosis. We next analysed the induction of DNA synthesis and the suppression of apoptosis in rat liver in vivo. Both DEHP and DCB were able to induce DNA synthesis although, as seen in vitro, the induction by DCB (4.2-fold; P = 0.023) was less marked than that with DEHP (13.4-fold; P = 0.007). Similarly, DEHP and DCB were both able to suppress rat hepatocyte apoptosis in vivo but the magnitude of the suppression was comparable; apoptosis was reduced to undetectable levels in four out of five animals with DCB and three out of five with DEHP. Since both chemicals suppressed apoptosis and induced DNA synthesis in rat liver but, overall, DCB was less potent, the disparate hepatocarcinogenic potential of these two chemicals could arise from differences in the magnitude of growth perturbation. To test this hypothesis, we repeated the studies in mouse, a species where both DCB and DEHP are hepatocarcinogenic. Both in vitro and in vivo, DCB and DEHP/MEHP were able to suppress apoptosis and induce hepatocyte DNA synthesis in the mouse with comparable potencies. The data support the hypothesis that the carcinogenicity of nongenotoxic hepatocarcinogens is associated strongly with the ability to perturb hepatocyte growth regulation. However, the ability to effect such changes is not unique to nongenotoxic carcinogens and is common to some noncarcinogenic chemicals, such as DCB, suggesting that the growth perturbation may need to exceed a threshold for carcinogenesis.

The effect of non-genotoxic carcinogens, phenobarbital and clofibrate, on the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis.

Phenobarbital and clofibrate, two non-genotoxic carcinogens, have been investigated regarding the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis in primary cultures of rat hepatocytes. Low toxicity concentrations, 200 and 100 microg/ml for phenobarbital and clofibrate respectively, were used to examine their effect on spontaneous or transforming growth factor beta1 (TGFbeta1)-induced apoptosis and on the expression of antioxidant defence enzymes (superoxide dismutases and catalase). The increased incidence of apoptotic nuclei was visualized in TGFbeta1-treated cultures with the fluorescent dye Hoechst 33258 and was quantified under all experimental conditions by measurement of the hypodiploid peak in DNA histograms obtained by flow cytometry. Both substances, when added separately to hepatocyte cultures and incubated for 24 and 48 h, significantly diminished spontaneous apoptosis and exhibited a slight suppression of TGFbeta1-induced apoptosis. Endogenous peroxide production by hepatocytes increased with TGFbeta1, phenobarbital or clofibrate and the increase was greater with phenobarbital and in the presence of TGFbeta1 with both drugs. Gene expression of catalase and Mn- and Cu,Zn superoxide dismutases (SOD) was evaluated by northern blot analysis of hepatocytes incubated in the presence of phenobarbital or clofibrate with or without TGFbeta1 and the following differences were detected: phenobarbital induced a significant decrease in both dismutases (to 56%, P < 0.05, and 55%, P < 0.05, for Mn- and Cu,Zn-SOD respectively) and a 2-fold increase (P < 0.01) in catalase; clofibrate induced a slight decrease in both SODs and a 4-fold increase (P < 0.05) in catalase; TGFbeta1 significantly decreased to 37% (P < 0.05) expression of catalase while not significantly affecting expression of both SODs. We conclude that inhibition of spontaneous apoptosis induced by either phenobarbital or clofibrate is accompanied by increases in the endogenous levels of peroxides and by significant induction of catalase gene expression. Furthermore, the lack of effect of both compounds on TGFbeta1-induced apoptosis could be a consequence of the inability of these two compounds to counteract the depressing effect of TGFbeta1 on expression of catalase.

Decreased levels of p26-Bcl-2, but not p30 phosphorylated Bcl-2, precede TGFbeta1-induced apoptosis in colorectal adenoma cells.

Bcl-2 expression is confined to the base of the colonic crypt, whereas transforming growth factor beta (TGFbeta) is expressed in the upper crypt, as are the apoptotic death promoters, Bak and Bax. In colonic adenoma cells, TGFbeta induces a growth arrest. In some adenoma cell lines, this is accompanied by apoptosis and in others it is not. In this study, we used two human colonic adenoma cell lines: RG/C2, in which TGFbeta induces a G1 arrest without apoptosis, and BH/C1, in which TGFbeta induces both a G1 arrest and apoptosis. TGFbeta does not induce apoptosis in RG/C2 cells even if hydrocortisone and insulin are removed from the culture medium. In BH/C1 cells, TGFbeta induces apoptosis in the presence of insulin and hydrocortisone. Apoptosis induced by TGFbeta is preceded by a reduction in p26-Bcl-2 protein levels. There was no change in the levels of the p30 phosphorylated form of Bcl-2 or in levels of the proapoptotic proteins Bax or Bak. RG/C2 cells did not show decreased Bcl-2 levels in response to TGFbeta-induced growth inhibition. Therefore, TGFbeta regulates Bcl-2 expression in colonic adenoma cells which undergo apoptosis in response to TGFbeta, but not in those which are growth inhibited, but resistant to TGFbeta-induced apoptosis. TGFbeta may play an important role in the colonic epithelium, not only in the inhibition of cell proliferation, but also in the regulation of apoptosis.

ICE-like protease (caspase) is involved in transforming growth factor beta1-mediated apoptosis in FaO rat hepatoma cell line.

Transforming growth factor-beta1 (TGF-beta1) arrests growth and/or stimulates apoptosis of a variety of cells. The biochemical pathways involved in the apoptotic processes, however, remain poorly defined. TGF-beta1 induces DNA fragmentation together with morphological changes, which are characteristic of apoptosis in the FaO rat hepatoma cell line. Histones were remarkably enriched in lysates of these cells during TGF beta1-induced apoptosis. We identified U1-70 kd as a death substrate which is cleaved following TGF-beta1 treatment. The tetrapeptide caspase inhibitor carbobenzoxy-valyl-alanly-aspartyl-(beta-O-methyl)-fluoromethyl ketone (ZVAD-FMK) prevented TGF beta1-induced apoptotic DNA fragmentation and cleavage of the U1-70 kd protein, showing that caspase(s) are involved in TGF beta1-mediated apoptosis. To identify specific caspases involved in apoptosis induced by TGF-beta1 in FaO cells, proteolytic activation of several of these caspases and their substrates were studied as a function of time following TGF beta1-treatment. TGF beta1-treatment induced the progressive proteolytic processing of caspase-2 (ICH-1L/Nedd-2), whereas caspase-1 itself did not show any cleavage from the precursor. Pretreatment with ZVAD-FMK abrogated the maturation of caspase-2 and blocked the apoptotic progress. These results suggest that caspase-2, but not caspase-1, may play a crucial role in TGF beta1-induced apoptosis in these cells.