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As the criteria for liver donation have been extended to include marginal donors, liver grafts are becoming particularly vulnerable to hepatic ischemia-reperfusion injury (IRI). However, no specific measures have been validated to ameliorate hepatic IRI. In this article, we explored whether everolimus has protective effects against hepatic IRI in relation with autophagy. The effects of everolimus were investigated in both in vitro and in vivo hepatic IRI models. Mouse hepatocyte AML12 cells and BALB/c mice were utilized for the establishment of each model. In the IRI-induced AML12 cells, everolimus treatment increased the expressions of autophagic markers (microtubule-associated protein 1 light chain 3 and p62) and decreased pro-apoptotic proteins (cleaved caspase 3 and cleaved poly-ADP ribose polymerase). The blockage of autophagy, using either bafilomycin A1 or si-autophagy-related protein 5, abrogated these anti-apoptosis effects of everolimus. Subsequently, everolimus administration to the hepatic IRI-induced mice provided hepatoprotective effects in terms of (1) decreasing the expressions of pro-apoptotic proteins, (2) inhibiting the release of pro-inflammatory cytokines (IL-6 and tumor necrosis factor-α), (3) reducing elevated liver enzymes (aspartate transaminase, alanine transaminase, and ammonia), and (4) restoring liver histopathology. These findings suggest that everolimus protects the liver against hepatic IRI by way of activating autophagy, and thus could be a potential therapeutic agent for hepatic IRI., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca(2+). Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer's and Parkinson diseases. One key regulator that underlies cell survival and Ca(2+) homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca(2+) dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca(2+) concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca(2+) through the InsP3 receptor (InsP3R). The Ca(2+) efflux in IRE1α-deficient cells correlates with dissociation of the Ca(2+)-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α-TRAF2-ASK1 complex. The increased cytosolic concentration of Ca(2+) induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca(2+) dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca(2+) influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca(2+) homeostasis and cell survival during ER stress and reveal a previously unknown Ca(2+)-mediated cell death signaling between the IRE1α-InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.

Functional role of peripheral benzodiazepine receptor on mitochondrial membrane in apoptosis and insulin secretion from insulinoma cells was studied. A prototypic peripheral benzodiazepine receptor agonist PK11195 induced insulinoma cell apoptosis, while a central benzodiazepine receptor agonist did not. Death of insulinoma cells by PK11195 was inhibited by cyclosporin A, a blocker of mitochondrial permeability transition pore. Caspase inhibitors further inhibited MIN6N8 cell death. PK11195 induced dissipation of mitochondrial potential and cytochrome c translocation to cytoplasm. PK11195 induced an increase in cytoplasmic [Ca(2+)], which was reversed by cyclosporin A. Rhod-2 staining showed decreased mitochondrial [Ca(2+)] after PK11195 treatment. PK11195 potentiated glucose-induced insulin secretion probably due to the increased cytoplasmic [Ca(2+)]. Calpain was activated following Ca(2+) release, and calpain inhibitors attenuated death of insulinoma cells by PK11195. These results suggest that PK11195 induces mitochondrial potential loss, cytochrome c translocation, increased insulin secretion in conjunction with an increase in cytoplasmic [Ca(2+)] and calpain activation, which collectively leads to apoptosis of insulinoma cells.

Inappropriate apoptosis results in the epidermal hyperplasia as in psoriasis and UVB irradiation has been successfully used to treat this kind of skin disorders. Previously, we reported that the novel phytosphingosine derivative, tetraacetyl phytosphingosine (TAPS) induced apoptosis in HaCaT cells. This study examined the effect of UVB irradiation and/or TAPS on the induction of apoptosis in HaCaT. 10 mJ/cm2 of UVB irradiation or 10 microM of TAPS alone exhibited weak cytotoxicity but co-treatment of UVB and TAPS synergistically enhanced the cytotoxicity and apoptosis in HaCaT. The cells treated with UVB and TAPS showed much higher levels of cleaved caspase-3, -8, -9 and Bax than with UVB or TAPS alone, whereas Bcl-2 level was decreased by co-administration of UVB and TAPS. In hairless mice, co-treatment of UVB and TAPS synergistically increased apoptosis, as shown in the HaCaT co-treated with UVB and TAPS. Furthermore, UVB irradiation caused an increase of apoptotic cells in the epidermis and the TAPS-treated mice showed an increase of apoptotic cells in the dermis as well as in the epidermis. These results suggest that the TAPS co-treatment synergistically increases the level of UVB-induced apoptosis via caspase activation by regulating the level of pro-apoptotic Bax and anti-apoptotic Bcl-2., (Copyright 2004 Kluwer Academic Publishers)

LIM kinases (LIMKs) regulate actin polymerization by phosphorylating cofilin and are predominantly expressed in neural tissue. In this study, the effect of LIMK1 overexpression in PC12 cell apoptosis was investigated. PC12 cells overexpressing the wild-type LIMK1 were more resistant to serum-withdrawal-induced cell death and the level of caspase 3 activation in these cells was lower than in the control PC12 cells or than in the PC12 cells expressing a mutant LIMK1 lacking the kinase domain. The inhibition of JNK activation was observed in the PC12 cells overexpressing the wild-type LIMK1 after serum withdrawal. These results suggest that the LIMK1 might allow resistance to apoptosis in PC12 cells by inhibiting JNK activation.

This study describes the involvement of the p38 mitogen-activated protein kinase (MAPK) during interferon-gamma (IFN-gamma) signaling in fetal brain astrocytes. In some pathological conditions of brain, p38 MAPK transduces stress-related signals, increases expression of proinflammatory cytokines, and induces cellular damage or apoptosis. In astrocytes, the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expression level was increased by IFN-gamma. AG490, a JAK inhibitor, blocked TRAIL expression induced by IFN-gamma. SB203580, a specific p38alpha and p38beta2 MAPK inhibitor, decreased the TRAIL expression induced by IFN-gamma. The phosphorylation of the Ser727 site of STAT1, but not the Tyr701 site, was inhibited by SB203580. These results suggest that p38 MAPK modulates STAT1 phosphorylation in IFN-gamma signaling in fetal brain astrocytes., (Copyright 2003 Wiley-Liss, Inc.)

Recent studies have shown that somatostatin (SOM) inhibits interleukin 6 (IL-6) and interferon gamma (IFNgamma) production by lymphocytes and peritoneal macrophages, whereas substance P (SP) enhances these cytokines production. To define the mechanism of the cytokine production enhancements and inhibitions by SOM and SP, we examined the expression of apoptosis modulator, p53, Bcl-2, Bax, inducible nitric oxide synthase (iNOS), Fas, caspase-8 and nitric oxide (NO) in thioglycolate-elicited peritoneal macrophages. SOM caused up-regulation of p53, Bcl-2, Fas and caspase-8 activities, and down-regulation of iNOS expression and NO production. On the other hand, SP slightly induces p53 and highly induces Bcl-2, iNOS expression and NO production. These data suggest that apoptosis by SOM may occur by a Bax- and NO-independent p53 accumulation, and through Fas and caspase-8 activation pathways, and that the inducible expression of Bcl-2 and NO production by SP may contribute to prevent the signals of apoptosis by Bax, and via Fas and caspase-8 activation.

We demonstrated the induction of cell death in a hepatoma cell line by IFN-gamma and its possible mechanism. Among the 2 hepatitis B virus (HBV)-associated hepatoma cell lines, SNU-354 and SNU-368, IFN-gamma induced cell death and increased caspase-3 activity in SNU-368 but not in SNU-354. IFN-gamma induced several changes in the mRNA expression level of apoptosis-regulating genes, e.g., increased expression of Fas, caspase-1 and TNF-related apoptosis-inducing ligand (TRAIL). In particular, IFN-gamma potently increased the mRNA expression of TRAIL in both cell lines. However, it did not change the mRNA expression level of death-mediating TRAIL receptors, e.g., DR4 and DR5, which were constitutively expressed in both cell lines. In contrast, the decoy receptor DcR1 was expressed in SNU-354 but not in SNU-368, and its expression level in SNU-354 was increased by IFN-gamma. Another decoy receptor, DcR2, was constitutively expressed in both cell lines; however, its expression level in SNU-368 was decreased by IFN-gamma. In addition, exogenous recombinant TRAIL reduced viability in SNU-368, but not in SNU-354, cells. From these findings, we speculated that TRAIL up-regulation and the subsequent TRAIL-mediated apoptosis serve as a mechanism of IFN-gamma-induced cell death in SNU-368. To confirm this hypothesis, we demonstrated that soluble DR4-Fc fusion protein, a TRAIL pathway inhibitor, inhibited IFN-gamma-induced cell death in SNU-368. Our results demonstrated that IFN-gamma acts as an inducer of cell death through TRAIL-mediated apoptosis., (Copyright 2001 Wiley-Liss, Inc.)

Although TGF-beta1, a growth inhibitor, is known to also induce apoptosis, the molecular mechanism of this apoptosis is largely undefined. Here, we identify the mechanism of TGF-beta1-induced apoptosis in SNU-16 human gastric cancer cells. Cell cycle and TUNEL analysis showed that, upon TGF-beta1 treatment, cells were initially arrested at the G1 phase and then driven into apoptosis. Of note, caspase-3 was activated in accordance with TGF-beta1-induced G1 arrest. Activated caspase-3 is targeted to cleave p21(cip1), p27(kip1), and Rb, which play important roles in TGF-beta-induced G1 arrest, into inactive fragments. Subsequently, Cdk2 was aberrantly activated due to the cleavage of p21 and p27. We found that the inhibition of Cdk2 activity efficiently blocks TGF-beta1-induced apoptosis, whereas it did not prevent caspase-3 activation or the subsequent cleavage of target proteins. In contrast, the suppression of caspase-3 activity inhibited the cleavage of target proteins, the activation of Cdk2, and the induction of apoptosis. Taken together, our results suggest that activation of caspase-3 by TGF-beta1 may initiate the conversion from G1 cell cycle arrest to apoptosis via the cleavage of p21, p27 and Rb, which in turn causes Cdk2 activation and, most significantly, Cdk2 activation as a downstream effector of caspase is a critical step for the execution of TGF-beta1-induced apoptosis.

Many tumors, including hepatocellular carcinomas (HCCs), resist Fas-mediated cell death, which is one of the effector mechanisms in the host's anti-tumor response; however, this resistance can be abolished by interferon-gamma (IFN-gamma). IFN-gamma may sensitize Fas-mediated cell death in several ways, but the exact mechanism in HCCs is uncertain. In this study, we thoroughly investigated the effect of IFN-gamma on the susceptibility of one human normal liver cell line and 12 HCC cell lines to Fas-mediated cell death. We also investigated the effect of IFN-gamma on the expression of various apoptosis-related genes such as the Fas/TNF-related genes, the bcl-2 family, and the caspase family of genes. Although most cell lines showed considerable constitutive expression of Fas, all tested cell lines resisted Fas-mediated cell death without IFN-gamma. When cells were pretreated with IFN-gamma, only three cell lines were made significantly susceptible to Fas-mediated cell death (SNU-354, SNU-387 and SNU-423); the other 10 cell lines were not affected. IFN-gamma increased the mRNA expression of Fas, TRAIL and caspase-1, and surface Fas was also increased. The strongly sensitized cell lines (SNU-354, SNU-387 and SNU-423) showed a particularly potent increment in surface Fas after IFN-gamma treatment (increase in surface Fas > 1.7-fold). This result enabled us to conclude that a potent increment of surface Fas expression is a major sensitizing mechanism of IFN-gamma. We conclude that IFN-gamma cannot play a sensitizing role in most HCC cell lines and that IFN-gamma makes HCC cells susceptible to Fas-mediated cell death through a marked up-regulation of surface Fas in some HCC cells.

Fas expression increases in perinecrotic areas of glioblastoma. In this study the up-regulation of Fas/FasL by oxidative stress was shown. H(2)O(2) exposure increased Fas expression in two astrocytoma cell lines and cells became sensitive to agonistic anti-Fas antibody. FasL was up-regulated in astrocytoma cells. Apoptosis of Molt-4 cells was augmented by astrocytoma cells pretreated with H(2)O(2). Our findings suggest up-regulation of Fas in astrocytoma cells may lead them to be sensitive to apoptosis when cells are in oxidative stress. Whereas, the up-regulation of FasL may render astrocytoma cells cytotoxic to neighboring brain tissues and infiltrating immune cells.

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.

Here we describe a protein product of the human septin H5/PNUTL2/CDCrel2b gene, which we call ARTS (for apoptosis-related protein in the TGF-beta signalling pathway). ARTS is expressed in many cells and acts to enhance cell death induced by TGF-beta or, to a lesser extent, by other apoptotic agents. Unlike related septin gene products, ARTS is localized to mitochondria and translocates to the nucleus when apoptosis occurs. Mutation of the P-loop of ARTS abrogates its competence to activate caspase 3 and to induce apoptosis. Taken together, these observations expand the functional attributes of septins previously described as having roles in cytokinesis and cellular morphogenesis.

Non-steroidal anti-inflammatory drugs (NSAIDs) have cancer preventive and tumor regressive effects in the human colon, perhaps due to their capability to induce apoptosis of the colon cancer cells. Here, we report that indomethacin induced the expression of Nur77 which has been implicated in activation-induced apoptosis of T-lymphocytes, in a colon cancer cell line, HCT-15. The transcript- and protein-level, the transcriptional activity of Nur77 promoter, and the DNA binding of Nur77 were significantly induced following indomethacin treatment. Among the two potential trans-acting factors that activate Nur77-promoter, indomethacin induced DNA binding and reporter gene activity of AP-1, but not that of related serum response factor (RSRF), suggesting that the transcriptional induction of Nur77 may be mediated through activation of AP-I. Further, we showed that all trans-RA repressed the induction of Nur77 as well as the apoptosis-induced by indomethacin, providing evidence that transcriptional induction of Nur77 may be an important mechanism by which indomethacin induces apoptosis in colon cancer cells.

Objective: Proteins that are phosphorylated during apoptosis are commonly precipitated by autoantibodies found in the sera of patients with systemic lupus erythematosus. We sought to determine whether scleroderma autoantigens such as small nucleolar RNPs (snoRNP) also associate with phosphoproteins in response to various cellular stressors., Methods: We screened a panel of monoclonal antibodies derived from mice exposed to mercury, a well-characterized murine model of the anti-snoRNP autoimmune response, for the ability to selectively precipitate phosphoproteins from radiolabeled lysates prepared from Jurkat T cells subjected to stressful stimuli., Results: Monoclonal antibodies reactive with snoRNPs precipitated a phosphoprotein complex (pp42, pp34, and pp23) from lysates prepared from apoptotic cells. Several novel phosphoproteins (pp62 and pp18) were also observed. The phosphorylation and/or recruitment of these proteins to the snoRNP complex is induced by multiple apoptotic stimuli (e.g., Fas ligation, anisomycin, or ultraviolet irradiation), an effect that is blocked by overexpression of Bcl-2. We were unable to demonstrate an association of the phosphoprotein complex with snoRNPs in cells treated with the xenobiotic agent mercury. The snoRNP-associated phosphoprotein complex is composed of serine/arginine (SR) splicing factors, including SRp40., Conclusion: The association of phosphorylated SR proteins with snoRNPs in cells undergoing apoptosis suggests that the immune response to fibrillarin that characterizes a subset of patients with scleroderma may be related to cell death induced by apoptotic stimuli (e.g., Fas ligation, irradiation, or chemical toxins), or by exposure to mercury.

Nur77 (NGFI-B) is an orphan nuclear receptor that has been implicated in activation-induced T-cell apoptosis. Retinoids, potent immune modulators, were shown to inhibit the activation-induced apoptosis of immature thymocytes and T-cell hybridomas. To illustrate the mechanism of the inhibition, we examined the effects of retinoic acid (RA) on the expression and transactivation functions of Nur77 in the human peripheral blood mononuclear cells and the human T-cell leukemia, Jurkat. All-trans-RA remarkably repressed the DNA binding and transcriptional induction of Nur77. Among the two potential trans-acting factors that activate Nur77 gene promoter, i.e., AP-1 and related serum response factor (RSRF), all-trans-RA repressed DNA binding and reporter gene activity of AP-1 but not that of RSRF, suggesting that the inhibition may be mediated through AP-1. We also demonstrated a posttranscriptional regulation of Nur77 function by retinoid receptors by showing that transactivation activity of Nur77 was significantly inhibited by cotransfection of RARalpha or RXRalpha. Nur77 bound RARalpha or RXRalpha in both yeast and mammalian two-hybrid tests, suggesting that direct protein-protein interaction between these receptors may mediate the inhibition. Taken all together, we demonstrated that RA repressed Nur77 function through multiple mechanisms that may provide the basis for RA inhibition on the apoptosis of activated T-lymphocytes., (Copyright 2000 Academic Press.)

Background: Prostatic epithelial cells are sensitive to the inhibitory effects of TGF-beta. However, TGF-beta signaling in the prostate is dependent on androgenic status. Under the in vivo conditions, it is difficult to dissociate the effect of TGF-beta from that of androgen on the prostate., Methods: The objective of the present study was to create and verify a transgenic mouse system in which epithelial cells of the ventral prostate are insensitive to the actions of TGF-beta. By using a modified prostate-specific promoter, C3(1), the TGF-beta dominant negative receptor is only expressed in the epithelial cells of the ventral prostate, and these cells are resistant to TGF-beta. Morphology of transgenic animal prostates was compared to wild-type animal prostates by immunohistochemistry and microscopy., Results: The prostate of transgenic mice exhibited an abnormal morphology with multiple layers of epithelial cells lining the proximal ducts, in contrast to the simple cuboidal monolayer of cells seen in the normal prostate. This observation was accompanied by a loss of apoptosis in this region, as seen by TUNEL assay. There was no significant difference in serum levels of testosterone between the wild-type and transgenic animals., Conclusions: These results demonstrated that a loss of sensitivity to TGF-beta results in the accumulation of multiple layers of epithelial cells in the proximal region of the ventral prostate. This abnormal growth illustrates that TGF-beta plays an important role in regulating prostate growth. The current transgenic system can be used as an experimental model to study the functional role of TGF-beta in prostatic growth and function., (Copyright 2000 Wiley-Liss, Inc.)

Tumor necrosis factor alpha (TNFalpha) generates a potent cytotoxic effect, however many cancer cells are resistant to TNFalpha-mediated killing and the cause of the differential sensitivity remains to be elucidated. In this study, we demonstrated that TNFalpha induced cell death in four different human colon cancer cell lines. The degree of cytotoxic effect was different in each cell line, in that HCT-15 was relatively sensitive, while DLD-1, HT-29 and WiDr were relatively resistant. TNFalpha induced apoptotic changes such as morphological changes, DNA fragmentation and activation of caspase-3 in HCT-15, but to a lesser degree in the others. Transcriptional expression of TNFR1(p55), as well as that of FLICE, Fas, FADD, DR3, FAF, TRADD, and RIP was similar in these cell lines, indicating that the susceptibility to TNFalpha-induced apoptosis may not be determined by the constitutive expression level of these factors. Interestingly, the cytotoxic effect of TNFalpha was well correlated with the DNA binding activity of NF-kappaB in the colon cancer cell lines. Further, the overexpression of a non-phosphorylated mutant form of IkappaBalpha enhanced the cytotoxicity of TNFalpha in the resistant cell line, DLD-1, indicating that NF-kappaB activity may determine the sensitivity of colon cancer cells to TNFalpha-induced apoptosis. Thus, our results indicate that modulation of NF-kappaB activity may provide a useful tool to sensitize colon cancer cells to TNFalpha treatment.

Retinoids are well known as potential chemopreventive and chemotherapeutic agents against a variety of human cancers. Here, we report that retinoic acid (RA) induced differential growth inhibition in human colon cancer cell lines: while DLD-1, HT-29, and WiDr were relatively resistant, HCT-15 and Colo201 were relatively sensitive. All-trans-retinoic acid caused morphological and biochemical changes such as membrane shrinkage, chromatin condensation, and DNA cleavage, which are typical features of cells undergoing apoptosis in sensitive cell lines. Although retinoic acid receptor (RAR)alpha, beta, gamma and retinoid X receptor alpha were expressed in all cell lines examined, a significant induction of RARbeta by all-trans-RA was observed only in sensitive cell lines, suggesting important roles of RARbeta in RA sensitivity. When a vector containing the RARbeta gene was introduced into a relatively resistant cell line, DLD-1, the cells acquired RA sensitivity. Further, we found that the RARbeta transfectants of DLD-1 expressed an enhanced level of c-Myc and Bax proteins, which may result in the increased susceptibility of the cells to all-trans-RA-induced apoptosis. In summary, our data demonstrated that RA induced growth inhibition and apoptosis in human colon cancer cells and that the induction of RAR3 may mediate the retinoid action.

The signaling pathway leading to TGF-beta1-induced apoptosis was investigated using a TGF-beta1-sensitive hepatoma cell line, FaO. Cell cycle analysis demonstrated that the accumulation of apoptotic cells was preceded by a progressive decrease of the cell population in the G(1) phase concomitant with a slight increase of the cell population in the G(2)/M phase in response to TGF-beta1. TGF-beta1 induced a transient increase in the expression of Cdc2, cyclin A, cyclin B, and cyclin D1 at an early phase of apoptosis. During TGF-beta1-induced apoptosis, the transient increase in cyclin-dependent kinase (Cdk) activities coincides with a dramatic increase in the hyperphosphorylated forms of RB. Treatment with roscovitine or olomoucine, inhibitors of Cdc2 and Cdk2, blocked TGF-beta1-induced apoptosis by inhibiting RB phosphorylation. Overexpression of Bcl-2 or adenovirus E1B 19K suppressed TGF-beta1-induced apoptosis by blocking the induction of Cdc2 mRNA and the subsequent activation of Cdc2 kinase, whereas activation of Cdk2 was not affected, suggesting that Cdc2 plays a more critical role in TGF-beta1-induced apoptosis. In conclusion, we present the evidence that Cdc2 and Cdk2 kinase activity transiently induced by TGF-beta1 phosphorylates RB as a physiological target in FaO cells and that RB hyperphosphorylation may trigger abrupt cell cycle progression, leading to irreversible cell death.

It has been postulated that tumor cells expressing Fas ligand (FasL) can evade immune surveillance by inducing apoptosis in T cells expressing Fas. In this study, we investigated FasL expression in 13 human hepatoma cell lines. Strong FasL expression was detected by reverse transcription-polymerase chain reaction or immunofluorescence in Hep G2.2.15, in which the hepatitis-B-virus (HBV) genome was transfected, and in SNU-354, which showed HBx transcripts. To determine the biological activity of FasL, Hep G2.2. 15 was co-cultured with MOLT-4, T-cell-leukemia cells. Hep G2.2.15 induced apoptosis in MOLT-4 and this was inhibited by the antagonistic anti-Fas antibody, ZB4. For further analysis of the role of HBx in the induction of FasL, PLC/PRF/5 cells were transfected transiently with the HBV genome, or HBx, or the frameshift mutant of HBx. In PLC/PRF/5 cells transfected with the HBV genome or HBx but not in cells transfected with the frameshift mutant of HBx, FasL expression was detected. Our data suggest that HBx plays a role in the induction of FasL in hepatoma cells and in the escape from immune surveillance., (Copyright 1999 Wiley-Liss, Inc.)

Many chemotherapeutic drugs have been found to exert their mode of action via induction of apoptosis in cancer cells. The mechanisms involved in this process are not clear. Recent studies have shown that the Fas/Fas ligand (FasL) system is a key factor controlling apoptotic cell death. In the present study, the involvement of Fas in chemotherapeutic drug-induced apoptosis in hepatoma cell lines was investigated. Five different human hepatoma cell lines, Hep G2, Hep G2.2.15, Hep 3B, SK-Hep-1, and PLC/PRF/5, were used. It was found that they expressed different levels of Fas. However, all five cell lines were susceptible to apoptosis when treated with chemotherapeutic drugs such as 5-fluorouracil (5-FU) or cisplatin. In Hep G2 that constitutively expressed Fas, 5-FU or cisplatin treatment caused an increase in the expression of Fas before the formation of oligonucleosomal DNA fragments, a typical feature of apoptosis. However, in Hep 3B, where Fas is undetectable, apoptosis could also be induced by 5-FU or cisplatin without induction of Fas. The agonistic anti-Fas antibody (CH-11) was capable of inducing apoptosis by itself and promoted drug-induced apoptosis in Hep G2 but not in Hep 3B. The antagonistic anti-Fas antibody (ZB4) inhibited drug-induced apoptosis in Hep G2. Our results suggest that apoptosis can be induced in hepatoma cell lines via both Fas-dependent and Fas-independent pathways.

Proteins cleaved by apoptotic caspases are commonly recognized by autoantibodies found in the serum of patients with rheumatic disease. We report that the 72-kDa signal recognition particle (SRP) protein, a rare target of autoantibodies found in the serum of patients with dermatomyositis and systemic lupus erythematosus, is rapidly cleaved in Jurkat T cells treated with apoptotic (i.e. Fas ligation, treatment with gamma or ultraviolet radiation, or co-culture with anisomycin or staurosporine) but not proliferative (CD3 cross-linking) stimuli. Cleavage of SRP 72 produces a 66-kDa amino-terminal fragment and a 6-kDa carboxyl-terminal fragment that is selectively phosphorylated on serine residues. Cleavage of SRP 72 is prevented by chemical and peptide caspase inhibitors, and by overexpression of bcl-2, an inhibitor of apoptotic cell death. Analysis of the carboxyl terminus of SRP 72 has identified a putative cleavage site (SELD/A) for group III caspases, and carboxyl-terminal serine residues that are highly conserved in phylogeny. Both serine phosphorylation and caspase cleavage of SRP 72 are observed in cells derived from human, dog, rat, and mouse. Canine SRP 72 is cleaved in vitro by recombinant caspase 3 but retains the ability to mediate transport of a signal peptide-containing protein into the endoplasmic reticulum lumen. The 72-kDa component of the SRP joins a growing list of autoantigens that undergo post-translational modifications during programmed cell death.

Hepatocellular carcinoma (HCC) is one of the tumors known to be resistant to Fas-mediated apoptosis. To elucidate the possible mechanisms of this resistance, we examined the mRNA transcripts of genes related to Fas in 10 hepatitis B virus (HBV)-associated HCC cell lines and six HBV-associated HCC tissues. Most of the HBV-associated HCC cell lines showed markedly decreased Fas expression. Some cell lines expressed FAP, a possible inhibitor of apoptosis, or showed downregulated expression of FADD or FLICE, molecules essential to Fas-mediated apoptosis. All six HBV-associated HCC tissues also showed decreased Fas expression compared with their non-malignant counterparts. Our data demonstrate that HBV-associated HCC showed a common defect in the expression of Fas, upregulation of FAP and downregulation of downstream molecules such as FADD and FLICE.

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.

The detergent Triton X-100 was used to establish a model for apoptosis in hepatoma cell lines. The electrophoresis of DNA extracted from 0.01% Triton X-100 treated hepatoma cell lines showed DNA ladder formation, a hallmark of apoptosis. The DNA fragmentation appeared within less than 60 min of the Triton X-100 treatment. Chromatin condensation and apoptotic bodies were observed by hematoxylin and eosin (H & E) stain, and fragmented nucleosome was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) test. Apoptosis was semi-quantitated by measuring the lactate dehydrogenase (LDH) level for cytotoxity. It was found that apoptosis had been induced in more than 90% of the cells treated with Triton X-100 for 150 min. These data show that Triton X-100 efficiently induces the apoptotic cell death in hepatoma cell lines.

Oxidative stress is induced by excess accumulation of reactive oxygen and nitrogen species (RONS). Astrocytes are metabolically active cells in the brain and understanding astrocytic responses to oxidative stress is essential to understand brain pathologies. In addition to direct oxidative stress, exogenous hydrogen peroxide (H2O2) can penetrate biological membranes and enhance formation of other RONS. The present study was carried out to examine the role of insulin in H2O2-induced oxidative stress in rat astrocytic cells. To measure changes in the viability of astrocytes at different concentrations of H2O2 for 3 h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay was used and 500 μM H2O2 was selected to establish a model of H2O2-induced oxidative stress. Further assays showed that 3 h of 500 μM H2O2-induced significant changes in the levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS) and calcium ion (Ca2+) in C6 cells, with insulin able to effectively diminish H2O2-induced oxidative damage to C6 cells. Western blotting studies showed that insulin treatment of astrocytes increased the levels of phosphorylated Akt and magnified the decrease in total Bcl-2 protein. The protective effect of insulin treatment on H2O2-induced oxidative stress in astrocytes by reducing apoptosis may relate to the PI3K/Akt pathway. [ABSTRACT FROM AUTHOR]

The tumor suppressor RASSF1A is inactivated in many human cancers and is implicated in regulation of microtubule stability, cell cycle progression and apoptosis. However, the precise mechanisms of RASSF1A action and their regulation remain unclear. Here we show that Skp2, an oncogenic subunit of the Skp1–Cul1–F–box ubiquitin ligase complex, interacts with, ubiquitinates, and promotes the degradation of RASSF1A at the G1–S transition of the cell cycle. This Skp2-dependent destruction of RASSF1A requires phosphorylation of the latter on serine-203 by cyclin D–cyclin-dependent kinase 4. Interestingly, mutation of RASSF1A-phosphorylation site Ser203 to alanine results in a delay in cell cycle progression from G1 to S phase. Moreover, enforced expression of Skp2 abolishes the inhibitory effect of RASSF1A on cell proliferation. Finally, the delay in G1–S progression after Skp2 removal is normalized by depletion of RASSF1A. These findings suggest that the Skp2-mediated degradation of RASSF1A plays an important role in cell proliferation and survival.Oncogene (2008) 27, 3176–3185; doi:10.1038/sj.onc.1210971; published online 10 December 2007 [ABSTRACT FROM AUTHOR]

Recent studies have shown that the inhibition of casein kinase 2 (CK2) sensitizes many cancer cells to Fas ligand- and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, it has not been demonstrated directly whether CK2 inhibition can also enhance the cytotoxicity of natural killer (NK) cells, which actually use the death ligands to kill cancer cells in vivo. To address whether NK cell-mediated cancer cell death is affected by the inhibition of CK2, we first checked whether the death ligand-induced apoptosis of hepatocellular carcinoma cells (HCCs) and HeLa were affected by CK2 inhibition. We then investigated the effect of CK2 inhibition on NK cytotoxicity against HCCs and HeLa cells and its mechanistic features. Inhibition of CK2 by emodin increased the apoptotic cell death of HepG2, Hep3B and HeLa when the cancer cell lines were treated with a soluble form of recombinant TRAIL or an agonistic antibody of Fas. This phenomenon appeared to be correlated with the expression level of death receptors on the cancer cell surface. More interestingly, the inhibition of CK2 also greatly increased the NK cell-mediated cancer cell killing. The NK cytotoxicity against the cancer cells increased about twofold when the target cells were pretreated with a specific CK2 inhibitor, emodin or 4,5,6,7-tetrabromobenzotriazole. Furthermore, the increase of the NK cytotoxicity against cancer cells by CK2 inhibition was granule-independent and mediated possibly by the death ligands on the NK cell surface. This suggests that CK2 inhibitors could be used to enhance the cytotoxicity of NK cells and consequently increase host tumour immunity. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the potential of tumor-necrosis-factor-related apoptosis-inducing ligand TRAIL to eradicate leukemia cell lines, while sparing normal hematopoietic stem cells. Human Jurkat and Molt-4 cell lines were used to optimize the purging process in umbilical cord blood (UCB) mononuclear cells. The Jurkat cell line was TRAIL sensitive and TRAIL-resistant Molt-4 cell line became sensitive after being treated with TRAIL and a low dose of doxorubicin (0.1 μM), but UCB mononuclear cells remained resistant. DR4 expression was increased when Jurkat cells were treated with TRAIL, and DR5 expression increased after exposing Molt-4 cells to TRAIL plus a low dose of doxorubicin for 24 h. The expression of DR4 and DR5 in UCB mononuclear cells was unchanged after treatment with TRAIL, a low-dose doxorubicin, or TRAIL plus a low dose of doxorubicin. In TRAIL-sensitive Jurkat cells, caspases 8, 9, 3, and 7 were activated by TRAIL treatment and activation of caspases was augmented by TRAIL plus a low dose of doxorubicin than TRAIL or a low dose of doxorubicin alone in Molt-4 cells. Experiments involving mixture of UCB mononuclear cells and Jurkat or Molt-4 cells showed a marked eradication of leukemia cells and the limiting dilution assay demonstrated an eradication rate of more than 4 logs after 24 h incubation with 100 ng/ml of TRAIL in Jurkat cells. In the case of Molt-4 cells, the eradication rate was about 3 logs when TRAIL was used in combination with a low dose of doxorubicin. No significant decrease in the number of granulocyte-macrophage colony-forming unit) (CFU-GM) colonies was detected when UCB mononuclear cells were treated with TRAIL in combination with a low dose of doxorubicin. These results suggest that TRAIL offers the possibility of being used as an ex vivo purging agent for autologous transplantation in hematologic malignancies. [ABSTRACT FROM AUTHOR]