Expression of Bcl—2 inhibited Fas—mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Apoptosis plays an important role in embryonic development,tissue remodeling,immune regulation and tumor regression.Two groups of molecules(Bcl-2 family and “Death factor” family) are involved in regulating apoptosis.In order to know about the effect of Bcl-2 on apoptosis induced by Fas,a typical member of “Death factor” family,the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells,a human hepatocellular carcinoma cell line which expresses endogenous Fas,but not FasL and Bcl-2.The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells.However,the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis.Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis.These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.     

Induction of apoptosis and change of bcl—2 expression in macrophage Ana—1 cells by all—trans retinoic acid

Macrophage cells play an important role in the initiation and regulation of the immune response.All-trans retinoic acid (ATRA) and its natural and synthetic analogs (retinoids)affect a large number of biological processes.Recently,retinoids have been shown promise in the therapy and prevention of various cancers.However,many interesting questions related to the activities of retinoids remain to be answered:(I) Molecular mechanisms by which retinoids exert their effects;（Ⅱ)why the clinical uses of retinoids give undesirable side effects of varying severity with a higher frequency of blood system symptoms;(Ⅲ)little is known for its impacts on macrophage cells etc.We set up this experiment,therefore,to examine the apoptosis of ATRA on macrophage Ana-1 cell line.Apoptosis of the cells was quantitated,after staining cells with propidium iodide(PI),by both accounting nuclear condensation and flow cytometry.When the cells were treated with ATRA at or higher than 1μM for more than 24h,significant amount of the apoptotic cells was observed.Induction of apoptosis of Ana-1 cells by ATRA was in time-and dose-dependent manners,exhibiting the similar pattern as the apoptosis induced by actinomycin D (ACTD).ATRA treatment of Ana-1 cells also caused the changes of the mRNA levels of apoptosis-associated gene bcl-2,as detected by Northern blot analysis.The temporal changes of bcl-2 expression by ATRA was also parallel to that by ACTD.In conclusion,ATRA can induce apoptosis in macrophage cells,which may be helpful in understanding of immunological functions retinoids.     

mad—overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Mad protein has been shown as an antagonist of cMyc protein in some cell lines.The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally.An eukarryotic vector pCDNA Ⅲ containing full ORF fragment of mad cDNA was transfected into targeted cells.Under G418 selection,stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells.DNA synthesis,cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cells were partially inhibited in comparison to control cells.Flos cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase,resulting in the retardation of cell proliferation.RT-PCR detected a marked inhibition of the expression of cdc25A,an important regulator gene of G0/G1 to S phase in cell cycle.It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-74040M1 cells in the absence of serume.Thus,Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.     

Bcl-2 down modulation in WEHI-3B/CTRES cells resistant to Cholera Toxin （CT）-induced apoptosis

The very different effects of Cholera Toxin （CT） on cell growth and proliferation may depend on the type of ganglioside receptors in cell membranes and different signal transduction mechanisms triggered, but other functions related to the drug resistance mechanisms can not be excluded. The effect of CT treatment on the ＂in vitro＂ clonogenicity, the Population Doubling Time （PDT）, apoptosis, PKA activation and Bax and Bcl-2 expression was evaluated in WEHI-3B cell line and its CT-resistant subclone （WEHI-3B/CTRES）. In WEHI-3B parental cells the dramatic accumulation of cAMP induced by CT correlated well with PKA activation, increased PDT value, inhibition of clonogenicity and apoptosis. H-89 treatment inhibited PKA activation by CT but did not protect the cells from apoptosis and growth inhibition. In WEHI-3B/CTRES no significant CT-dependent accumulation of cAMP occurred with any increase of PKA activity and PDT. In CT resistant cells （WEHI-3B/CTRES）, Bcl-2 expression was down regulated by both CT or drug treatment （eg, ciprofloxacin, CPX） although these cells were protected from CT-dependent apoptosis but not from drug-induced apoptosis. Differently from other cell models described, down regulation of Bcl-2 is proved to be independent on cAMP accumulation and PKA activation. Our observations support the implication of cAMP dependent kinase （PKA） in the inhibition of WEHI-3B cells growth and suggest that, in WEHI-3B/CTRES, Bcl-2 expression could be modulated by CT in the absence of cAMP accumulation. Also in consideration of many contradictory data reported in literature, our cell models （of one sensitive parental cell strain and two clones with different uncrossed specific resistance to CT and CPX） provides a new and interesting tool for better investigating the relationship between the CT signal transduction mechanisms and Bcl-2 expression and function.     

Hydrogen peroxide predisposes neonatal rat ventricular myocytes to Fas-mediated apoptosis

Neonatal rat ventricular myocytes (NRVM) grown in normoxic environment are not susceptible to Fas-induced apoptosis. In the present work, we tested the hypothesis that free radical injury represented by transient exposure to H2O2 sensitizes NRVM to Fas-mediated apoptosis. NRVM were treated with H2O2 (0.5 mM) for 2-4 h and thereafter exposed for 7 h to recombinant Fas ligand (rFasL, 10 ng/ml) plus an enhancing antibody (1 microg/ml). Apoptotic cardiomyocytes were counted and apoptosis-related proteins were measured by Western blot. H2O2 alone induced apoptosis (9.4+/-1.0%) that was preceded by activation of caspases-8 and -3, and PARP degradation. Incubation of NRVM with H2O2, followed by exposure to rFasL, increased the apoptotic index to 13.8+/-2.0%, but did not change caspase-8 or PARP activation. To investigate the mechanism underlying the sensitizing affect of H2O2 towards Fas-induced apoptosis, we studied the effects of H2O2 on the expression of key apoptosis signaling proteins. Incubation with H2O2 for 2-4 h decreased Fas expression and the expression of the Fas-related antiapoptotic proteins FLIP(L) and ARC, and increased the expression of the antiapoptotic proteins bcl-2 and xIAP. FADD expression was unchanged. Next, we tested the effect of H2O2 on the apoptosis-inducing, Fas-dependent Daxx-ASK-1-JUN kinase pathway. H2O2 dramatically increased ASK-1 expression and JUN kinase activation, but did not effect Daxx expression. Based on these findings we concluded that H2O2 sensitizes NRVM to Fas-mediated apoptosis by activating the Daxx-ASK-1-JUN kinase pathway, and by shifting the balance between proapoptotic and antiapoptotic proteins towards the former.     

The CD99 signal enhances Fas-mediated apoptosis in the human leukemic cell line, Jurkat

The CD99 antigen has been implicated in various cellular processes, including apoptosis in T cells. Previously, we reported two monoclonal antibodies that recognize different epitopes of the CD99 molecule, named DN16 and YG32. In this study, we investigated the role of each CD99 epitope in T cell apoptosis. Unlike the DN16 epitope, CD99 ligation via the YG32 epitope failed to induce T cell death. Surprisingly, however, the YG32 signal enhanced Fas-mediated apoptosis in Jurkat T cells. Augmentation of Fas-mediated apoptosis by YG32 ligation was inhibited by treatment with either of the caspase inhibitors z-VAD-fmk or z-IETD-fmk, and YG32 ligation appeared to induce Fas oligomerization. These results suggest that each CD99 epitope plays a distinct role in T cell biology, especially in T cell apoptosis.     

Resistance to mitochondrial- and Fas-mediated apoptosis in human leukemic cells with acquired resistance to 9-beta-D-arabinofuranosylguanosine

We have previously reported that in a MOLT-4 leukemia cell line the acquired resistance to 9-beta-D-arabinofuranosylguanine (Ara-G) is due to deficiency of the activating enzymes deoxyguanosine kinase and deoxycytidine kinase [Biochem. Biophys. Res. Commun. 293 (5) (2002) 1489]. In this study we investigated whether apoptotic pathways are affected in two human T-cell lymphoblastic MOLT-4 cell lines with acquired resistance to Ara-G. In contrast to the MOLT-4 wild type cells, Ara-G resistant cells displayed no increase in caspase-3 or caspase-9 activity, DNA fragmentation, cytochrome c release or a drop in the mitochondrial membrane potential (DeltaPsi(mito)) upon Ara-G treatment. A drop in the DeltaPsi(mito) was induced in wild type cells after treatment with tributyltin, an inducer of mitochondrial permeability transition, and with carbonyl cyanide m-chlorophenylhydrazone, an uncoupling agent that reduces the DeltaPsi(mito), although not in Ara-G resistant cells. Ara-G resistant cells displayed higher levels of the anti-apoptotic protein Bcl-xL in immunoblots. A recent study indicates that Ara-G-induced apoptosis is mediated in part via the Fas pathway [Cancer Res. 43 (2047) (2002) 411]. When cells were treated with anti-Fas antibody, the wild type cell line exhibited increased caspase-3-like activity but the Ara-G resistant cells did not. Using FACS analysis and semi-quantitative PCR, 3-6-fold decreased protein levels and almost no detectable mRNA levels of Fas in the resistant cells were recorded. These data indicate that the inability to induce apoptosis via both the apoptosome pathway and the Fas pathway, due to increased levels of Bcl-xL and a lack of Fas, contributes to Ara-G resistance. This resistance to apoptosis in Ara-G resistant cells may serve to explain the overall resistance to a variety of anti-neoplastic drugs.     

Daxx enhances Fas-mediated apoptosis in a murine pro-B cell line,BAF3

Daxx has been shown to play an essential in type I interferon (IFN-/β)-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and nuclear translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the mechanism of Daxx-mediated apoptosis signaling in B lymphocyte progenitors, here we introduced an efficient suicide switch in a murine pro-B cell line, BAF3, by expressing FK506-binding protein-fused Fas intracellular domain (FKBP-Fas) and Daxx. It allows us to monitor Fas/Daxx-mediated signal by induction of Fas dimerization with the dimerizer drug AP20187. AP20187-mediated Fas dimerization induced not only apoptosis but also Jun N-terminal kinase (JNK) activation. However, AP20187 had no effect on cells expressing either Fas or Daxx only. Furthermore, expression of a JNK inhibitor, the JNK-binding domain of JIP-1, resulted in resistance to AP20187-mediated apoptosis in cells expressing FKBP-Fas and Daxx. These results imply that our novel suicide switch system may provide a powerful tool to delineate or identify the signaling molecules for Daxx-mediated apoptotic machinery in B lymphocyte progenitors through JNK activation.     

Fas-mediated apoptosis in Jurkat cells is suppressed in the pre-G2/M phase

The relationship between the cell cycle and Fas-mediated apoptosis was investigated using Jurkat cells. Analysis of the inducibility of apoptosis by anti-Fas antibody during the cell cycle synchronized by the thymidine double-block method, showed that apoptosis was induced in only 50% of the G2/M phase cells, while most of cells in the other phases underwent apoptosis. These observations indicate that G2/M phase cells are more resistant to Fas-mediated apoptosis than cells in other phases. Furthermore, a detailed analysis of G2/M phase found that only 20–30% of the cells underwent apoptosis 12 h after the removal of the second thymidine block (pre-G2/M phase). This suggests that Fas-mediated apoptosis is potently suppressed during the pre-G2/M phase. A possible explanation for the observation that cells in the pre-G2/M phase are less sensitive to anti-Fas antibody is lower expression level of Fas. To test this possibility, Fas expression levels on the cell surface during the cell cycle were examined. The content of Fas on the cell surface, however, did not change appreciably during the cell cycle. Thus, the suppression of apoptosis in the pre-G2/M phase is determined downstream after the receipt of the apoptotic signal through Fas.     

Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinoma cells

 Cytotoxic lymphocytes may induce apoptosis in their target cells by the FasL (Fas ligand) pathway or the perforin/granzyme B pathway. It has been shown that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-mediated apoptosis. The aims of this study were to determine whether CC cells are also resistant to perforin/granzyme B and whether the FasL resistance lies upstream of caspase-3 activation. The resistance of the Fas-expressing rat CC531s cells to the FasL pathway was confirmed by treating them with recombinant human soluble FasL, using rat hepatocytes as a positive control. The intracellular delivery of granzyme B by sublytic concentrations of perforin, on the other hand, resulted in many features of apoptosis (chromatin condensation, nucleus fragmentation, loss of microvilli and internucleosomal DNA fragmentation) within 3 h. Since both the FasL and perforin/granzyme B pathways converge at caspase-3, we measured caspase-3 activity to learn whether the FasL resistance was due to failure to activate this crucial executioner. Caspase-3 activation occurred in CC531s cells after perforin/granzyme B treatment, but not after the addition of recombinant FasL. Furthermore, we showed that caspase-3 activity is involved in the execution of perforin/granzyme-B-induced apoptosis in CC531s cells, since the cell-permeable caspase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these results suggest that CC cells are sensitive to perforin/granzyme-B-induced apoptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase. Received: 20 November 2000 / Accepted: 8 March 2001     

The CD44 standard/ezrin complex regulates Fas-mediated apoptosis in Jurkat cells

The transmembrane receptor CD44 conveys important signals from the extracellular microenvironment to the cytoplasm, a phenomena known as “outside-in” signaling. CD44 exists as several isoforms that result from alternative splicing, which differ only in the extracellular domain but yet exhibit different activities. CD44 is a binding partner for the membrane-cytoskeleton cross-linker protein ezrin. In this study, we demonstrate that only CD44 standard (CD44s) colocalizes and interacts with the actin cross-linkers ezrin and moesin using well-characterized cell lines engineered to express different CD44 isoforms. Importantly, we also show that the association CD44s-ezrin-actin is an important modulator of Fas-mediated apoptosis. The results highlight a mechanism by which signals from the extracellular milieu regulate intracellular signaling activities involved in programmed cell death. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Soluble CD40 ligand-activated B cells from patients with chronic hepatitis B virus infection as antigen presenting cells to induce hepatitis B virus specific cytotoxic T lymphocytes

Chronic hepatitis B virus (HBV) infection is the result of an inadequate antiviral immune response to the virus. In this study, we aimed to investigate whether the soluble CD40 ligand-activated B (CD40-B) cells could present antigen and induce specific cytotoxic T lymphocytes (CTLs) in patients with chronic HBV infection. We observed that after activated by sCD40L, the expression of CD80, CD86, major histocompatibility complex (MHC) I and II molecules on the CD40-B cells was significantly increased. Cytometry and fluorescence microscopy showed that more than 41.34% CD40-B cells were loaded by the HBcAg peptide. Furthermore, after been activated and HBcAg18–27 antigen peptide pulsed, B cells obtained from patients with chronic HBV infection could induce HBcAg18–27 specific CTLs in vitro. Taken together, our results show that B cells from patients with chronic HBV infection can be activated by sCD40L and may function as antigen presenting cells and induce HBV-specific CTLs.     

Fas-induced apoptosis in B cells

Engagement of the cell surface receptor Fas/APO-1 (CD95) initiates a sequence of intracellular events that leads to apoptotic cell death, and this outcome occurs in B cells as it does in other cell types. Fas signaling for B cell death is of particular interest because the expression and function of Fas is altered by engagement of additional cell surface receptors, leading to marked receptor-specific variation in susceptibility to Fas-induced apoptosis. Evidence suggests that the sensitivity of B cells to Fas-mediated apoptosis is intimately connected to homeostasis in the serological arm of the immune system and plays a role in the dysregulation that occurs in certain autoimmune and malignant dyscrasias.