TGFβ-induced Apoptosis Research Articles

Primary cirrhotic hepatocytes resist TGFβ-induced apoptosis through a ROS-dependent mechanism

Background/Aims The cirrhotic liver manifests dysregulated hepatocyte growth by poor regenerative capacity, formation of regenerative nodules, and malignant transformation to hepatocellular carcinoma. The purpose of this study was to determine if dysregulated hepatocyte growth occurs through deficient apoptosis. Methods Hepatocytes were isolated from normal and CCl 4-treated mice and treated with TGFβ, TNFα, and UV-C, known apoptotic agents. Results Cirrhotic hepatocytes were less sensitive to TGFβ- (45±5 vs. 15±3%; P<0.003), TNFα- (59±21 vs. 21±8%; P=0.02), and UV-C-induced (31±4 vs. 17±4%; P<0.03) apoptosis compared to normal hepatocytes. In normal hepatocytes, TGFβ-induced apoptosis occurred through a ROS-, MPT-, and caspase-dependent pathway. Cirrhotic hepatocytes lacked caspase activation, had decreased procaspase-8 expression, failed to undergo the MPT, and had increased basal ROS activity compared to normal hepatocytes. After treatment with trolox, an antioxidant that reduced basal ROS activity, cirrhotic hepatocytes underwent apoptosis in response to TGFβ treatment. Conclusions These findings suggest that increased ROS activity in cirrhotic hepatocytes plays a critical role in mediating cirrhotic hepatocyte resistance to apoptosis. Cirrhotic hepatocyte resistance to TGFβ-induced apoptosis is ROS-dependent and is a mechanism of dysregulated growth in the chronically inflamed liver.

Possible mechanisms underlying the mitogenic actionof heptachlor in rat hepatocytes

The worldwide use of the organochlorine pesticide heptachlor has led to widespread contamination in the environment. Like many other organochlorine pesticides, heptachlor is considered to pose a threat to human health. It has been shown that heptachlor is a tumor-promoting agent, but the mechanisms involved still remain unclear. The negative response of heptachlor in in vitro genotoxicity test suggests that this pesticide displays its carcinogenicity through epigenetic pathways. With the growing evidence that proliferation accounts for the tumor-promoting effects of many agents, the purpose of this work was to investigate the mechanisms involved in the mitogenic activity of heptachlor in quiescent rat hepatocytes and to understand the properties of this compound as a tumor promoter in the liver. Heptachlor triggered significant proliferation in quiescent rat hepatocytes. Two mechanisms were delineated to support the mitogenic effect in the hepatocyte: activation of key kinases in signaling pathways and inhibition of apoptosis. Exposure to heptachlor led to activation of protein kinase C mitogenactivated protein kinases. Moreover, these results indicate that like many tumor promoters, heptachlor strongly inhibited TGFβ-induced apoptosis and cytochrome c release into the cytosol. The levels of the anti-apoptotic protein Bcl-2 were also increased in the presence of heptachlor. In conclusion, these results indicate that heptachlor alters basic cell function by interfering with key cellular signaling pathways.

Ras and TGF[beta] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways.

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling.

Transforming Growth Factor β (TGFβ) Mediates Schwann Cell DeathIn VitroandIn Vivo: Examination of c-Jun Activation, Interactions with Survival Signals, and the Relationship of TGFβ-Mediated Death to Schwann Cell Differentiation

In some situations, cell death in the nervous system is controlled by an interplay between survival factors and negative survival signals that actively induce apoptosis. The present work indicates that the survival of Schwann cells is regulated by such a dual mechanism involving the negative survival signal transforming growth factor beta (TGFbeta), a family of growth factors that is present in the Schwann cells themselves. We analyze the interactions between this putative autocrine death signal and previously defined paracrine and autocrine survival signals and show that expression of a dominant negative c-Jun inhibits TGFbeta-induced apoptosis. This and other findings pinpoint activation of c-Jun as a key downstream event in TGFbeta-induced Schwann cell death. The ability of TGFbeta to kill Schwann cells, like normal Schwann cell death in vivo, is under a strong developmental regulation, and we show that the decreasing ability of TGFbeta to kill older cells is attributable to a decreasing ability of TGFbeta to phosphorylate c-Jun in more differentiated cells.

P38-mediated regulation of an Fas-associated death domain protein-independent pathway leading to caspase-8 activation during TGFbeta-induced apoptosis in human Burkitt lymphoma B cells BL41.

On binding to its receptor, transforming growth factor beta (TGFbeta) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGFbeta-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGFbeta-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGFbeta-treated cells. TGFbeta induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGFbeta induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGFbeta-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGFbeta-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGFbeta-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGFbeta induced an apoptotic pathway via FADD-independent activation of caspase-8.

Effect of Rodent Hepatocarcinogenic Peroxisome Proliferators on Fatty Acyl-CoA Oxidase, DNA Synthesis, and Apoptosis in Cultured Human and Rat Hepatocytes

The effects of the rodent hepatocarcinogens clofibric acid and ciprofibrate on the activity of the peroxisomal fatty acyl-CoA oxidase, DNA synthesis, and apoptosis were compared in cultured rat and human hepatocytes. Rat hepatocytes expressed a 10-fold greater level of the peroxisomal fatty acyl-CoA oxidase compared to human hepatocytes. At the highest concentration (1.0 mM), both drugs induced a two- to threefold increase in this enzyme activity in both rat and human hepatocytes. Ciprofibrate (0.1 and 0.2 mM) caused a twofold increase in DNA synthesis in rat hepatocytes, whereas clofibric acid had no effect on DNA synthesis in these cells. In contrast, increasing concentrations of both clofibric acid and ciprofibrate produced inhibition of DNA synthesis in human hepatocytes. By using the terminal transferase dUTP–biotin nick end labeling technique, it was observed that 0.1 and 0.2 mM clofibric acid and ciprofibrate suppressed transforming growth factor-β (TGFβ)-induced apoptosis by 50% in rat hepatocytes, but they had no effect on TGFβ-induced apoptosis in human hepatocytes. Although clofibric acid and ciprofibrate diminished TGFβ-induced apoptosis, they had no effect on the basal apoptotic levels in the rat hepatocyte cultures. However, both drugs significantly increased the percent of apoptotic cells in the human hepatocyte cultures. It is concluded that primary rat and human hepatocyte cultures respond differently to peroxisome proliferators. The differences in effects on DNA synthesis and apoptosis support the hypothesis that human liver cells are refractory to peroxisome proliferator-induced hepatocarcinogenesis.