Induction of Fas (CD95/APO-1) ligand is essential for p53-dependent apoptosis in an in vitro renal carcinoma model system

Purpose The Fas/CD95/APO-1 ligand (FasL) is a death cytokine that binds to cell surface Fas/CD95/APO-1 receptor, yet a possible role of FasL expression in p53-dependent apoptosis is not fully understood in many human malignancies, including renal carcinoma. Methods By Northern blot and Western blot analyses, we determined the effect of p53 on the FasL and Fas receptor expression. To do this, we employed an in vitro renal carcinoma model system that was previously established by stably co-transfecting a temperature-sensitive mutant allele of the p53 tumor suppressor (ts-p53) with either the c-Myc oncogene or adenovirus E1A oncogene in baby rat kidney (BRK) epithelial cells. The ts-p53 is activated only at a permissive temperature. The transactivation activity of p53 was assessed by luciferase reporter assays. The sub-G1 cell population in the cell cycle representing apoptotic cell death was measured by flow cytometric analysis. Results We found that the level of endogenous FasL, but not Fas receptor, was increased at a permissive temperature with delayed kinetics when compared with p21WAF1 expression, but was coincident with p53-induced apoptosis, whereas an apoptosis-defective mutant p53, which lacks the PxxP region (P: Proline, x: any amino acid), failed to induce FasL expression and hence apoptosis. Notably, p53-induced apoptosis was completely blocked by overexpressing a dominant negative inhibitor of the FADD/Mort-1, a pro-apoptotic adaptor that lies immediately downstream of the FasL/Fas receptor. Conclusions These results suggest that the FasL is a critical downstream effector of p53-dependent apoptosis in a cultured BRK renal carcinoma model system.     

Liver apoptosis

Apoptosis or programmed cell death occurs in the liver as in other organs. In the normal state it is not a frequent mode of hepatic cell destruction. Morphological and biochemical characteristics of liver cell apoptosis do not differ from what is observed in other cells. The Fas receptor pathway, a frequent hepatic apoptotic pathway among various others, involves intra-cellular signals amplified by mitochondria. Although hepatic apoptosis may occur by following several others pathways, Fas, which is abundantly expressed in the plasma membrane of hepatocytes, is very often involved in hepatocyte demise during B or C viral hepatitis irrespective of their clinical form, alcoholic hepatitis, cholestasis due to accumulation of hepatic biliary salts, or certain types of drug-induced hepatitis. Fas is also probably responsible for the death of biliary cells in primary biliary cirrhosis. In contrast one of the causes of resistance to apoptosis of hepatic cancerous cells could be related to an alteration of the Fas receptor. This is why much experimental work is presently performed to achieve inhibition of the Fas receptor either at the mRNA level or at the level of Fas-inductible proteolytic enzymes called caspases. One perspective is a specific treatment of apoptosis as an adjuvant treatment of liver diseases.     

Apoptose hépatique

Apoptosis or programmed cell death occurs in the liver as well as in other organs. In the normal state it is not a frequent event of hepatic cell destruction. Nevertheless morphological and biochemical characteristics of liver apoptosis do not differ from that it is observed in other cells. Between the various hepatic apoptotic pathways the Fas receptor pathway is frequently involved and its intra-cellular signal is amplified by mitochondria. Although hepatic apoptosis may occur through several others pathways, Fas which is abundantly expressed on the plasma membrane of hepatocytes and biliary cells, is very often involved in cell destruction during B or C viral hepatitis, whatever their clinical form, alcoholic hepatitis, primary biliary cirrhosis, cholestasis due to hepatic biliary salt accumulation, or drug hepatitis. In contrast, one of the causes for the resistance of hepatic cancerous cells to apoptosis could be due to an alteration of the Fas receptor. It is the reason why many experimental works are presently performed to inhibit the Fas receptor either at the level of its mRNA or at the level of caspases which are Fas-inductible proteolytic enzymes.     

Involvement of the Fas/Fas ligand system in p53-mediated granulosa cell apoptosis during follicular development and atresia

In the present study we have examined the presence of Fas, Fas ligand (FasL), and p53 in rat granulosa cells during follicular development and atresia, especially in relation to the granulosa cell cycle progression and the onset of granulosa cell apoptosis. Fas, FasL, and p53 proteins were immunolocalized, and their contents were determined by Western blotting. Granulosa cell apoptosis was assessed by DNA fragmentation analyses (DNA ladder) and in situ terminal deoxynucleotidyl transferase mediated deoxy-UTP-biotin nick end labeling (TUNEL) as well as by flow cytometry. Ovaries not exposed to gonadotropins (control) consisted predominantly of preantral and early (small) antral follicles, the latter of which were mostly atretic and demonstrated intense TUNEL staining in granulosa cells exhibiting positive immunoreactivities for FasL and Fas. Granulosa cells isolated from these follicles were apoptotic, as evident by clear ladder pattern of DNA fragmentation upon electrophoretic analysis and the high percentage (>10%) of the cell population in the A0 phase of the cell cycle. After gonadotropin treatment, these features completely disappeared during each of the 3 days of follicular growth to the medium to large antral stages. Cell cycle analysis showed significantly higher proportion of the cells in S and G2/M phases compared with controls, which was accompanied by marked decrease in immunoreactivities for Fas, FasL, and p53. By days 4 and 5, widespread atresia and extensive granulosa cell apoptosis were noted in large antral and preovulatory follicles and were coincidental to increased expression of p53 and Fas, but not of FasL, as well as an apparent arrest of granulosa cell G1/S progression, as evident by an increased cell population in G0/G1 and a decrease in the S and G2/M. Granulosa cells from equine CG-primed ovaries exhibited marked increases in p53 and Fas protein contents and apoptosis after adenoviral p53-sense complementary DNA infection in vitro and were more responsive to Fas activation by an agonistic Fas monoclonal antibody challenge. Taken together, these findings are consistent with the well accepted concept that gonadotropin plays a central role as a survival factor in the regulation of granulosa cell Fas/FasL and p53 expression during ovarian follicular development. In addition, the control of granulosa cell apoptosis may involve two consecutive cellular/molecular events: cell cycle arrest at G1/S and exit from G0 into A0 phase, via regulation of the p53 and Fas/FasL death pathways.     

Sustained phosphorylation of Bid is a marker for resistance to Fas-induced apoptosis during chronic liver diseases

BACKGROUND & AIMS: Increased rates of apoptosis have been reported to play a role in the pathophysiology of many disorders, including liver diseases. Conversely, genetic mutations that result in impairment of programmed cell death have been associated with cancer development. However, apoptosis resistance can also be the result of nongenetic stress adaptation, as seen in the cancer-prone metabolic liver disease hereditary tyrosinemia. To clarify whether stress-induced apoptosis resistance is a general feature of chronic liver diseases, an animal model of chronic cholestasis was examined. METHODS: Studies were performed with mice before and 2 weeks following bile duct ligation and with Fah-/- and Fah/p21-/- mice before and after NTBC withdrawal. RESULTS: Here we show that bile duct ligation induced profound resistance against Fas monoclonal antibody-mediated hepatocyte death. The apoptosis signaling pathway was blocked downstream of caspase-8 activation and proximal to mitochondrial cytochrome c release. In controls, activation of the Fas receptor resulted in rapid dephosphorylation of Bid and its subsequent cleavage, whereas Bid remained phosphorylated and uncleaved in chronic cholestasis and other models of hepatic apoptosis resistance. CONCLUSIONS: We propose a model in which the phosphorylation status of Bid determines the apoptotic threshold of hepatocytes in vivo. Furthermore, resistance to apoptosis in chronic cholestasis may contribute to the long-term risk of cancer in this setting.     

Fas (CD95)- and tumor necrosis factor-mediated apoptosis in liver endothelial cells: role of caspase-3 and the p38 MAPK.

Recently, we showed that TNF enhances the susceptibility of endothelial cells from murine liver sinusoids (LEC) to Fas-mediated apoptosis, suggesting that signals transduced by Fas and TNF receptors may synergistically increase intracellular death signals in these cells. In this work we evaluated whether caspase-3 and p38 are involved in LEC apoptosis induced by Fas and TNF. Here we show that LEC treated with Fas agonist (Jo2 mAb at 0.1 microg/ml) and TNF had a greater caspase-3 activity (twofold increase) than cells treated with each factor alone. There was a strong correlation between caspase-3 activity and cell killing induced by Jo2/TNF, indicating that this caspase plays a critical role in this process. Likewise, there was a significant increase in caspase-8 activity in LEC treated with Jo2 and TNF, compared with untreated cells or cells treated with each factor alone. Apoptosis of LEC induced by Jo2/TNF was partially reversed by SB203580, a p38 inhibitor, suggesting that p38 is involved in apoptosis of these cells. To our knowledge, this is the first report that apoptosis induced by Fas/TNF in LEC is associated with coactivation of both caspase-3 and p38. Potentially, both caspase-3 and p38 may be of great importance in endothelial cell pathology as molecular targets for preventing vascular damage due to endothelial cell apoptosis.     

Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth.

Loss of activity of the p53 tumor suppressor gene product has been postulated in the pathogenesis of human restenosis. Although the antioncogenes p53 and retinoblastoma (Rb) susceptibility gene have been reported to play a pivotal role in cell cycle progression in various cells, the role of p53 and Rb in the growth of human vascular smooth muscle cells (VSMC) has not yet been clarified. We used antisense strategy against p53 and Rb genes by the viral envelope-liposomal method. Transfection of antisense p53 oligodeoxynucleotides (ODN) alone resulted in an increase in DNA synthesis compared with control (P<0.01). Similarly, transfection of antisense Rb ODN alone resulted in a higher DNA synthesis rate than control (P<0.01). Moreover, increase in VSMC number was only induced by transfection of antisense p53 ODN alone or cotransfection of p53/Rb ODN (P<0.01), whereas a single transfection of antisense Rb ODN had little effect on cell number. Therefore, we hypothesized that this discrepancy is due to the induction of apoptosis mediated by p53. Interestingly, apoptotic cells were markedly increased in VSMC transfected with antisense Rb ODN alone, accompanied by the induction of p53 protein. The number of apoptotic cells was attenuated by cotransfection of antisense p53 ODN (P<0.01). We finally examined the molecular mechanisms of apoptosis induced by the absence of Rb. In VSMC transfected with antisense Rb ODN, bax, a promoter of apoptosis, was significantly increased in VSMC transfected with antisense Rb ODN (P<0.01), whereas bcl-2 and Fas did not play a pivotal role in the induction of apoptosis. Overall, these data first demonstrated that the antioncogenes p53 and Rb negatively regulated the cell cycle in VSMC, suggesting that the modulation of their activity may mediate VSMC growth such as that in restenosis and atherosclerosis. The presence of p53 plays a pivotal role in the regulation of apoptosis in human VSMC growth, probably through the bax pathway. These results provide evidence that p53 is a functional link between cell growth and apoptosis in VSMC.     

Mechanisms of cell death induced by suicide genes encoding purine nucleoside phosphorylase and thymidine kinase in human hepatocellular carcinoma cells in vitro

For gene therapy of hepatocellular carcinoma (HCC), the Escherichia coli purine nucleoside phosphorylase (PNP)/fludarabine suicide gene system may be more useful than the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system as a result of a stronger bystander effect. To analyze the molecular mechanisms involved in PNP/fludarabine-mediated cell death in human HCC cells in comparison with HSV-tk/GCV, we transduced human HCC cells of the cell lines, HepG2 and Hep3B, with PNP or HSV-tk using adenoviral vectors, followed by prodrug incubation. Both systems predominantly induced apoptosis in HepG2 and Hep3B cells. PNP/fludarabine induced strong p53 accumulation and a more rapid onset of apoptosis in p53-positive HepG2 cells as compared with p53-negative Hep3B cells, but efficiency of tumor cell killing was similar in both cell lines. In contrast, HSV-tk/GCV-induced apoptosis was reduced in p53-negative Hep3B cells as compared with p53-positive HepG2 cells. HSV-tk/GCV, but not PNP/fludarabine, caused up-regulation of Fas in p53-positive HepG2 cells and of Fas ligand (FasL) in both HCC cell lines. These results demonstrate cell line-specific differences in response to treatment with PNP/fludarabine and HSV-tk/GCV, respectively, and indicate that PNP/fludarabine may be superior to HSV-tk/GCV for the treatment of human HCC because of its independence from p53 and the Fas/FasL system.     

The role of Fas-mediated apoptosis in thyroid disease

Recent evidence has emphasized the importance of apoptosis in the maintenance of tissue homeostasis and the pathogenesis of malignant and immune diseases. Autoimmune thyroid diseases such as Hashimoto's thyroiditis and Graves' disease, as well as other autoimmune endocrine diseases, have been associated with dysregulation of apoptotic signaling pathways. In particular, dysfunction of the Fas apoptotic pathway or production of soluble factors including soluble Fas and soluble Fas ligand may be involved in the pathogenesis of these disorders. On the other hand, malignant thyroid cells may avoid Fas-mediated suicide possibly by expression of inhibitors of apoptosis and evade the immune system by inducing apoptosis on infiltrating lymphocytes. The delicate balance between cell proliferation and cell death through the Fas pathway may also play an important role in the control of thyroid cell mass and goitrogenesis. This review analyzes the current evidence on the role of Fas-mediated apoptosis in the pathogenesis of thyroid diseases including Hashimoto's thyroiditis, Graves' disease, thyroid cancer and goiter. However, the exact mechanisms involved in the regulation of apoptosis in thyroid disease remain unclear. Further investigation is needed.     

Induction of cell proliferation arrest and apoptosis in hepatoma cells through adenoviral-mediated transfer of p53 gene

BACKGROUND/AIM: Loss of p53 function is common in hepatocellular carcinoma and is associated with an extremely poor prognosis. The aim of the study was to evaluate the biologic effect of adenoviral-mediated gene transfer of wild-type p53 gene in four hepatoma cell lines with different p53 genetic makeup. METHODS: Recombinant adenovirus expressing wild-type p53 was used. Recombinant adenoviruses with either an empty expression cassette or expressing beta-galactosidase gene served as controls. RESULTS: High-level expression of wild-type p53 was achieved with adenoviral-mediated gene transfer. The expressed p53 protein showed nuclear localization and its expression was associated with an induction of p21 and bax expression. Expression of the p53 gene was associated with inhibition of tumor cell proliferation and induction of apoptosis. Expression of p53 was also associated with an upregulation of CD95 (Apo-1/Fas) gene expression, which may predispose the tumor cells to undergo apoptosis induced by the Fas Ligand/Fas cytolytic pathway. An additional anti-tumor effect, in terms of allowing the replication-defective adenovirus to replicate, was observed in hepatoma cells with homozygous deletion of p53 genes and to a lesser extent, hepatoma cells with mutated p53 genes. CONCLUSIONS: These data showed that adenoviral-mediated gene transfer is effective in delivering p53 gene to tumor cells, and the multiple pathways involved in their antitumor activities.     

Expression of Apo-1/Fas (CD95), Bcl-2, Bax and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status

The down-regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl-2, which inhibits apoptosis, of Bax, which counteracts Bcl-2, of Bcl-x_L and Bcl-x_S, which act in an anti- and pro-apoptotic fashion, respectively, and of Apo-1/Fas, which induces programmed cell death, when activated by the Apo-1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl-2, but displayed different amounts of Bax and Bcl-x proteins. The Apo-1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo-1/Fas correlated with that of Bax, but not with that of Bcl-2 or Bcl-x subtypes. Furthermore, the effectiveness of the Apo-1/Fas mab was associated with the relative expression levels of the Apo-1/Fas and with that of the Bax antigen, but not with that of the Bcl-2 and Bcl-x antigens. We further showed that wild-type p53 function is not required for Apo-1/Fas-induced apoptosis, nor is it necessary for the expression of Bax or Apo-1/Fas antigens in myeloma.   In conclusion, our results suggest a p53-independent co-regulation of Apo-1/Fas and Bax, as well as a role for Bax in Apo-1/Fas-induced apoptosis in myeloma.     

Apoptosis in experimental NASH is associated with p53 activation and TRAIL receptor expression

Background and Aims:  We examined extrinsic and intrinsic (endogenous) mitochondrial apoptosis pathways in experimental non-alcoholic steatohepatitis (NASH).
Methods:  To assess extrinsic pathways, we measured hepatic expression of death-inducing cytokine receptors (tumor necrosis factor-α-receptor (TNF-R)1, TNF-R2, Fas, and TNFα-related apoptosis-inducing ligand-receptor (TRAIL-R) mRNA, TUNEL, caspase 3 activation, liver injury and liver pathology in mice fed a methionine and choline deficient (MCD) diet. For endogenous stress pathways, we determined serum insulin-like growth factor-1 (IGF-1), hepatic p53, Bcl-XL, tBid and p21 expression.
Results:  Methionine and choline deficient feeding increased alanine aminotransferase (ALT) and apoptosis from day 10, without increases in TNF-R1, TNF-R2, and Fas. However, murine TRAIL receptors, particularly decoyTRAIL-R1/TNFRSFH23 and Killer/DR5 mRNA increased. MCD feeding enhanced hepatic p53 expression, corresponding to ∼50% fall in serum IGF-1, decreased Bcl-XL, enhanced Bid cleavage to tBid, and up-regulation of p21. Nutritional restitution experiments showed that correcting either methionine or choline deficiency suppressed liver inflammation (extrinsic pathway), but failed to correct apoptosis, IGF-1 or p53.
Conclusions:  Methionine and choline deficiency lower IGF-1 to de-repress p53 during induction of steatohepatitis. The p53 induced by nutritional stress is biologically active in mediating mitochondrial cell death pathways, but may also be responsible for TRAIL receptor expression, thereby linking intrinsic and exogenous apoptosis pathways in NASH.     

Apoptosis and the liver: Relevance for the hepato-biliary-pancreatic surgeon

Cell death by apoptosis is thought to be involved in various pathophysiological situations involving the liver. Indeed, an understanding of apoptosis is becoming increasingly helpful for understanding disease and for patients' care. In this article, we review current scientific and clinical concepts of apoptosis, including death factors such as Fas ligand and tumor necrosis factor, apoptotic signal transduction mechanisms, and the role of intracellular proteinases called caspases. We also discuss apoptosis in the liver, as related to ischemia/reperfusion injury, cholestasis, and cancer, circumstances which physicians often face in the field of the liver surgery.     

Involvement of Fas system-mediated apoptosis in pathogenesis of viral hepatitis

Enhanced Fas system-mediated hepatocyte apoptosis is observed in hepatitis C virus (HCV)- and hepatitis B virus (HBV)-associated chronic liver diseases. In these forms of viral hepatitis, liver-infiltrating lymphocytes that recognize the viral antigen on hepatocytes become activated and express cytolytic Fas ligand (FasL) molecules. In contrast, hepatocytes exhibit enhanced Fas expression and become susceptible to FasL-mediated death. Augmentation of the Fas system has also been observed in other liver diseases and biliary disorders. Moreover, the Fas system is involved in removing aged hepatocytes. Thus, Fas-mediated apoptosis plays an important role in several liver diseases and in maintaining normal liver homeostasis.     

Human parvovirus B19 non-structural protein (NS1) induces apoptosis through mitochondria cell death pathway in COS-7 cells

Human parvovirus B19 has been found in various tissues in addition to erythroid lineage cells, and non-structural protein (NS1) is reported to induce cytotoxicity and apoptosis in erythroid lineage cells, but the mechanism in non-permissive cells is still unclear. To address this issue, we have constructed the NS1 gene in a cytomegalovirus episomal vector, pEGFP-C1 and transfected it into monkey epithelial cells, COS-7. EGFP-NS1 expression in transfected cells was monitored and assessed by fluorescence microscopy, RT-PCR and Western blot. The flow cytometric analysis showed that the NS1-transfected cells were arrested at G1 phase by paclitaxel treatment and there was increased apoptosis. The expression of p53, an important molecule in apoptosis and cell cycle regulation, and its downstream cell cycle kinase inhibitors p16(INK4) and p21(WAF1/CIP1) were up-regulated in the NS1-transfected cells. Also, increased expression of the pro-apoptotic Bcl-2 members Bax, Bad and activation of caspase 3 and caspase 9, but not the activation of caspase 8 or Fas were detected in the NS1-transfected cells. p53-induced Bax expression and subsequent activation of caspase 9 is probably the apoptotic pathway in NS1-transfected cells since activation of the caspase 9 was suppressed by the p53 inhibitor and apoptosis was significantly inhibited by the caspase 9 inhibitor. Our results suggest that the cell death of the NS1-transfected cells is associated with mitochondria related apoptosis. These findings might provide alternative information for further study and characterization of B19 NS1 protein in B19 non-permissive cells.     

The roles of apoptosis in lung injury]

Apoptosis serves important roles in organ development, cell differentiation, and the maintenance of homeostasis. Lung injury studies have underlined the role of fibroblast and endothelial cell apoptosis during lung repair from acute lung injuries, and demonstrated apoptosis of alveolar epithelial cells in association with diffuse alveolar damage. Pulmonary fibrosis is characterized by the loss of lung epithelial cells and the proliferation of fibroblasts. It is possible that Fas, Fas ligand, p 53, p 21, and other apoptosis-regulating proteins may play important roles in the pathophysiology of lung injury and fibrosis.     

Peg3/Pw1 promotes p53-mediated apoptosis by inducing Bax translocation from cytosol to mitochondria

Mitochondria is believed to play a central role in p53-mediated apoptosis. However, the signal transduction pathways leading to mitochondria remain unclear. Here, we report that translocation of Bax protein from cytosol to mitochondria is required for p53-induced apoptosis. Cytosolic Bax is unable to induce apoptosis, and blocking Bax translocation inhibits cell death. Expression of Bcl-2 blocks cytochrome c release and apoptosis but has no effect on Bax translocation, suggesting that Bax translocation acts upstream of Bcl-2. We further demonstrate that Peg3/Pw1, a protein up-regulated in p53-mediated cell death process, induces Bax translocation independent of apoptosis. The results suggest that Bax translocation represents an important regulatory step in p53-mediated apoptosis, and Peg3/Pw1 functions as a modulator downstream of p53 to regulate Bax redistribution in the cells, thus favoring the cellular decision toward apoptosis over growth arrest following p53 induction.     

Survival and apoptosis: a dysregulated balance in liver cancer.

BACKGROUND/AIMS: Dysregulation of the balance between proliferation and cell death represents a protumorigenic principle in human hepatocarcinogenesis. This article aims to provide a review of the current findings about how physiological hepatocyte apoptosis is regulated and whether or not its dysregulation might contribute to the progression towards a hepatocellular carcinoma (HCC) process. RESULTS: Although some physiological proapoptotic molecules are downregulated or inactivated in HCC, such as Fas, p53, Bax or Bid, dysregulation of the balance between death and survival is mainly due to overactivation of antiapoptotic signals. Thus, some growth factors that mediate cell survival are upregulated in HCC, as well as the molecules involved in the machinery responsible for cleavage of their proforms to an active peptide. The expression of the pten gene is reduced or absent in almost half the HCCs and the Spred family of Ras/ERK inhibitors is also dysregulated in HCC, which consequently lead to the overactivation of relevant survival kinases: AKT and ERKs. Alterations in the expression and/or activity of molecules involved in counteracting apoptosis, such as NF-kappaB, Bcl-X(L), Mcl-1 or c-IAP1, have also been observed in HCC. CONCLUSIONS: Therefore, therapeutic strategies to inhibit selectively antiapoptotic signals in tumour cells have the potential to provide powerful tools to treat liver cancer.