OBJECTIVE

CD4+CD25+ regulatory T cells (known as Treg cells) suppress unwanted and autoreactive T cell responses. Treg cells express the costimulatory molecule CTLA-4 intracellularly, but the mechanisms by which Treg cells exploit CTLA-4 signaling remain unclear. The present study was undertaken to investigate the role of CTLA-4 in controlling the homeostasis and suppressive function of Treg cells.

METHODS

Murine Treg cells were analyzed by flow cytometry for coexpression of CTLA-4 and typical Treg cell-expressed molecules, and the influence of CTLA-4 on T cell proliferation, suppression, and apoptosis was investigated by in vitro assays. To analyze the importance of CTLA-4 in Treg cell-mediated suppression in vivo, wild-type Treg cells were transferred into CTLA-4-deficient mice displaying lymphoproliferation, and survival was monitored over time.

RESULTS

A strong correlation between expression of forkhead box P3 and ex vivo expression of CTLA-4 in Treg cells was observed. Inhibition of CTLA-4 signaling in Treg cells during in vitro stimulation increased cell cycling and led to enhanced activation-induced cell death (AICD), which was mediated by CD95/CD95 ligand-induced activation of caspases. Blockade of CTLA-4 signaling resulted in impairment of the suppressive capacity of Treg cells. Despite these effects, high amounts of Treg cells persisted in CTLA-4-deficient mice. Results of transfer experiments in CTLA-4-deficient mice showed that the mice had a significantly prolonged lifespan when CTLA-4-competent Treg cells were injected.

CONCLUSIONS

Expression of CTLA-4 on Treg cells serves to control T cell proliferation, to confer resistance against AICD, and to maintain the suppressive function of Treg cells.

The wall of the liver sinusoid is made of highly specialized cells, the hepatic stellate cells (HSC) which together with the sinusoidal endothelial cells represent a loose barrier to the corpusculate part of the blood flowing through the liver. Quiescent stellate cells (quiescent HSC) store Vitamin A; "activated" stellate cells become involved in the reaction to acute or chronic noxae damaging the liver parenchyma. Activated HSC show increased protein synthesis capacity, increased DNA-synthesis and acquire a myofibroblast-like phenotype. Under similar conditions liver myofibroblasts (MF) of the portal field and of the pericentral area may also become "activated" by increasing protein synthesis, DNA synthesis and cell division. They express the fibulin-2 gene and produce large amounts of IL-6. In contrast to "activated" HSC they do not undergo spontaneous apoptosis in vitro and do not express the CD95-ligand gene. So far no definite prove has been found for a "transdifferentiation" of HSC to myofibroblasts. On the contrary an increasing amount of data support the conviction that HSC and MF represent two similar but not identical cell populations the latter being comparable to those of other organs.

A properly functioning immune system is dependent on programmed cell death/apoptosis at virtually every stage of lymphocyte development and activity. Carbon monoxide (CO), an enzymatic product of heme oxyenase-1, has been shown to possess anti-apoptotic effects in a number of different model systems. The purpose of the present study was to expand on this knowledge to determine the role of CO in the well established model of Fas/CD95-induced apoptosis in Jurkat cells, and to determine the mechanism by which CO can modulate T-cell apoptosis. Exposure of Jurkat cells to CO resulted in augmentation in Fas/CD95-induced apoptosis, which correlated with CO-induced up-regulation of the pro-apoptotic protein FADD as well as activation of caspase-8, -9, and -3 while simultaneously down-regulating the anti-apoptotic protein BCL-2. These effects of CO were lost with overexpression of the small interfering RNA of FADD. CO, as demonstrated previously in endothelial cells, was also anti-apoptotic in Jurkat cells against tumor necrosis factor and etoposide. We further demonstrate that this pro-apoptotic effect of CO was independent of reactive oxygen species production and involved inhibition in Fas/CD95-induced activation of the pro-survival ERK MAPK. We conclude that in contrast to other studies showing the anti-apoptotic effects of CO, Fas/CD95-induced cell death in Jurkat cells is augmented by exposure to CO and that this occurs in part via inhibition in the activation of ERK MAPK. These data begin to elucidate specific differences with regard to the effects of CO and cell death pathways and provide important and valuable insight into potential mechanisms of action.

Apoptosis is likely to be an important mechanism of cell loss in neurodegenerative diseases, but the signaling cascades activated before DNA fragmentation have not yet been determined. p53 or CD95 gene up-regulation precedes apoptosis in many cell types, and a potential role for these molecules in apoptosis of neurons and glial cells has already been demonstrated in Alzheimer's disease (AD). To determine whether apoptosis in other neurodegenerative diseases is mediated by similar mechanisms, p53 and CD95 expression were examined in postmortem central nervous system tissues from patients with diffuse Lewy body disease (DLBD), Pick's disease (PkD), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Down's syndrome plus Alzheimer's disease (DN+AD). Quantitative immunoblot analysis demonstrated higher temporal lobe levels of p53 and CD95 proteins in DLBD, PkD, and DN+AD, and higher temporal lobe levels of CD95 only in MSA and PSP relative to PD and aged controls (for all, p < 0.01). In histologic sections, increased p53 immunoreactivity was localized in neuronal and glial cell nuclei, neuronal perikarya, and dystrophic neuritic and glial cell processes in the frontal (Area 1 1) and temporal (Area 21) lobes in DLBD, PkD, and DN+AD, the motor cortex and spinal ventral horns in ALS, and the striatum and midbrain in DLBD, MSA, PD, and PSP. Increased CD95 expression and nuclear DNA fragmentation were present in the same cell types and structures that manifested increased nuclear p53 immunoreactivity. The results suggest that p53- or CD95-associated apoptosis may be a common mechanism of cell loss in several important neurodegenerative diseases. In addition, the presence of abundant p53-immunoreactive neurites and glial cell processes appears to be a novel feature of neurodegeneration shared by these distinct diseases.

Fas (CD95/APO-1) is an important mediator of apoptosis. We show that Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 human breast cancer cells become responsive to anti-Fas (CD95) agonistic antibody-triggered apoptosis after pretreatment or cotreatment with vitamin E succinate (VES; RRR-alpha-tocopheryl succinate). In contrast, no enhancement of anti-Fas agonistic antibody-triggered apoptosis was observed following VES pretreatment or cotreatment with Fas-sensitive primary cultures of human mammary epithelial cells, immortalized MCF-10A cells, or T47D human breast cancer cells. Although VES is itself a potent apoptotic triggering agent, the 6-h pretreatment procedure for Fas sensitization did not initiate VES-mediated apoptosis. The combination of VES plus anti-Fas in pretreatment protocols was synergistic, inducing 2.8-, 3.0-, and 6.3-fold enhanced apoptosis in Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells, respectively. Likewise, cotreatment of Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells with VES plus anti-Fas enhanced apoptosis 1.9-, 2.0-, and 2.6-fold, respectively. Functional knockout of Fas-mediated signaling with either Fas-neutralizing antibody (MCF-7-, MDA-MB-231-, and MDA-MB-435-treated cells) or Fas antisense oligomers (MDA-MB-435-treated cells only), reduced VES-triggered apoptosis by approximately 50%. Analyses of whole cell extracts from Fas-sensitive cells revealed high constitutive expression of Mr 43,000 Fas, whereas Fas-resistant cells expressed low levels that were confined to the cytosolic fraction. VES treatment of the Fas-resistant cells caused a depletion of cytosolic Mr 43,000 Fas with a concomitant increase in Mr 43,000 membrane Fas. These data show that VES can convert Fas-resistant human breast cancer cells to a Fas-sensitive phenotype, perhaps by translocation of cytosolic Mr 43,000 Fas to the membrane and show that VES-mediated apoptosis involves Mr 43,000 Fas signaling.

CTL, both CD4+ and CD8+, are essential in the eradication of intracellular pathogens. Data generated using murine T cells have suggested a critical role for CD95 (Fas, Apo-1) in CD4+ T cell-induced apoptosis of target cells. In contrast, CD8+ CTL predominantly use the perforin/granzyme lytic pathway. At present little is known about the mechanism of CD4+ CTL lytic function during intracellular infection in humans. We have used human CD4+ T cells specific for purified protein derivative (PPD) of Mycobacterium tuberculosis to explore whether CD95 is the dominant cytolytic mechanism. PPD-reactive CD4+ clones efficiently lysed Ag-pulsed autologous monocytes, adherent macrophages, and EBV-transformed B cells. Addition of an antagonistic CD95 Ab had a minimal effect on cytolysis, whereas addition of MgEGTA to block perforin/granzyme resulted in complete inhibition of killing. In contrast, lysis of activated peripheral blood B cells could be partially blocked with the antagonistic CD95 Ab. Supporting these observations, monocytes, macrophages, and EBV-transformed B cells were not lysed by an agonistic CD95 Ab. Activated B cells were readily lysed by the agonistic CD95 Ab. T cell clones triggered through the TCR with anti-CD3 were capable of lysing the CD95-sensitive Jurkat T cell line in a CD95-dependent manner, but were also able to release granzymes. We conclude that human CD4+ T cells are capable of lysing PPD-pulsed targets using both perforin/granzyme and CD95 pathways. The contribution of CD95 is strictly dependent on target cell susceptibility to CD95-mediated killing.

In systemic lupus erythematosus (SLE) serum TNF is increased and correlates with its soluble receptors and with disease activity. We therefore investigated (i) whether the TNF in SLE serum is bioactive, (ii) whether SLE cells react to TNF and (iii) whether there are associations with cell death, which is regarded as pathogenic in SLE. Sera from active SLE patients induced an increase in fibroblast CD54, which was abolished by blocking antibodies against TNF, suggesting TNF bioactivity. SLE lymphocytes had a similar surface expression of TNF-RI as healthy lymphocytes, their expression of TNF-RII was slightly increased. Recombinant TNF induced cell death in PBMC of SLE patients, suggesting functional receptors. Serum levels of sTNF-RII (as a surrogate marker for TNF activity) correlated with sTNF-RI and disease activity, as expected, and also correlated with the percentage of dying lymphocytes and with lymphocytic CD95. SLE sera contain increased amounts of biologically active TNF. Peripheral blood lymphocytes of SLE patients express functional TNF receptors. Finally, associations with cell death and CD95 receptors suggest that TNF may be pathogenic in SLE.

OBJECTIVE

It is predicted that HIV-infected individuals in early HIV disease are the most likely group to achieve immune reconstitution following highly active antiretroviral treatment. We assessed whether suppression of HIV replication in this group would improve immune function.

METHODS

Seventeen antiretroviral-naïve patients in early HIV disease were evaluated for immune function and lymphocyte phenotyping using standard immunological assays.

RESULTS

Absolute CD4+ T-cell number increased from a median of 550 to 800 x 10(6) cells/l while CD8+ T-cell numbers were reduced. The decrease in CD8+ cells correlated with a decrease in the CD8+ memory phenotype. Kinetics of CD4+ naïve and memory T-cell rise indicated that 80% of the maximum CD4+ naïve increase was achieved within 18 weeks whereas maximum CD4+ memory T-cell rise was achieved within 36 weeks. Activation markers (HLA-DR, CD38) and an apoptosis-related marker (CD95) were reduced on CD4+ and CD8+ T cells. Lymphocyte proliferation responses to tetanus toxoid, alloantigen, and anti-CD3/CD28 were restored in patients that were initially unresponsive. At baseline, 31% of the patients responded to HIV p24, which increased to 69% post-therapy. The inducible RANTES response was normalized following therapy whereas inducible interferon-gamma, interleukin (IL)-12, and MIP1beta were elevated. The depressed inducible IL-10 response, however, was not altered after therapy.

CONCLUSIONS

This is one of the first studies to demonstrate the restoration of HIV-1 specific responses in non-acute HIV infection, suggesting early intervention with potent antiretroviral therapy may reverse immune-mediated damage not seen with treated patients who have more advanced disease.

Interferon-alpha (IFN-alpha) is an established treatment for chronic myelogenous leukemia (CML) in chronic phase, but the mechanism of its antileukemic activity is not clear. One possible mechanism of action might include the induction of apoptosis, and especially Fas-mediated cell killing may play an important role in the elimination of malignant cells. We investigated Fas receptor (Fas-R) expression and the consequences of Fas-R triggering in CML patients. Using two-color flow cytometry, we found a significantly higher number of Fas-R-expressing CD34+ cells in the bone marrow (BM) of CML patients compared with normal subjects. We have previously shown that IFN-gamma induces Fas-R expression on CD34+ cells; in this study, we investigated whether IFN-alpha induces Fas-R expression on CML progenitor cells. Dose-dependent induction of Fas-R expression was observed after IFN-alpha stimulation of CD34+ cells from CML BM. In methylcellulose culture, IFN-alpha alone at a therapeutic concentration showed only marginal antiproliferative effects on both normal and CML BM progenitors. In contrast, a Fas-R agonist, the anti-CD95 monoclonal antibody CH11, inhibited colony formation from normal progenitors, and the inhibition was even stronger on CML progenitors. When CML BM cells were cultured in the presence of IFN-alpha, Fas-R-mediated inhibition of colony growth was potentiated in a dose-dependent fashion, consistent with IFN-alpha induction of Fas-R expression. This functional effect did not require the presence of accessory cells, since similar results were obtained with purified CD34+ cells. In suspension cultures, we demonstrated that suppression of CML hematopoiesis by IFN-alpha and Fas-R agonist was exerted through Fas-R-mediated induction of apoptosis. Our findings suggest that the Fas-R/Fas-ligand system might be involved in the immunologic regulation of CML progenitor growth and that its effect can be amplified by IFN-alpha.

OBJECTIVE

Recently, we have shown that micro-metastases, in the hypoxic transition zone surrounding lesions generated by radiofrequency ablation (RFA), display strongly accelerated outgrowth. CD95 is best known for its ability to induce apoptosis but can also promote tumorigenesis in apoptosis-resistant tumor cells. Therefore, we tested whether CD95 signaling plays a role in accelerated outgrowth of colorectal liver metastases following RFA.

METHODS

Hypoxia-induced invasion was assessed in three-dimensional EGFP-expressing C26 tumor cell cultures by confocal microscopy. CD95 localization was tested by immunofluorescence. Invasion and outgrowth of liver metastases following RFA were analyzed by post-mortem confocal microscopy and by morphometric assessment of tumor load. Neutralization of CD95L was performed by using antibody MFL4. CD95 was suppressed by lentiviral RNA interference. The role of host CD95L was assessed using gld mice.

RESULTS

Micro-metastases in the hypoxic transition zone following RFA displayed a highly invasive phenotype and increased expression of CD95 and CD95L. Hypoxia-induced tumor cell invasion in vitro increased the expression of CD95 and CD95L and induced translocation of CD95 to the invasive front. In vitro invasion, metastasis invasion, and accelerated tumor growth in the transition zone were strongly suppressed by neutralizing CD95L or by suppressing tumor cell CD95. In contrast, metastasis invasion and outgrowth were unaffected in gld mice.

CONCLUSIONS

Hypoxia causes autocrine activation of CD95 on colorectal tumor cells, thereby promoting local invasion and accelerated metastasis outgrowth in the hypoxic transition zone following RFA. Further pre-clinical work is needed to assess the role of CD95L neutralization, either alone or in combination with chemotherapy, in limiting aggressive recurrence of liver metastases following RFA.

Anti-inflammatory therapy in asthma is reliant on corticosteroids, particularly in their inhaled form. However, steroids are rather non-specific in their actions and they also raise concerns regarding compliance and side-effect Issues. Furthermore, a small proportion of patients with asthma fail to respond to oral glucocorticoids even at high doses. This Article will review the role that steroids and membrane receptor ligation play in the induction of eosinophil apoptosis together with the mechanisms by which corticosteroids enhance the disposal of apoptotic eosinophils by both professional and non-professional phagocytes. Eosinophils are thought to be the major pro-inflammatory effector cell in asthma and their persistence in the airways is probably enhanced by the presence of several asthma-relevant cytokines that prolong eosinophil survival by inhibition of apoptosis (interleukin (IL)-3, IL-5, granulocyte-macrophage colony-stimulating factor, IL-9, IL-13, IL-15). In contrast, a number of signals have been described that accelerate apoptosis in human eosinophils including corticosteroids or ligation of membrane receptors (CD95, CD45, CD69). Thus, the load of lung eosinophils in asthmatic disease is likely to be related to a balance in the tIssue microenvironment between pro- and anti-apoptotic signals. Furthermore, removal of apoptotic eosinophils by phagocytosis by alveolar macrophages or bronchial epithelial cells in a specific receptor-mediated way is as important as the process of apoptosis induction. Corticosteroids enhance the recognition and engulfment of apoptotic eosinophils by macrophages or bronchial epithelial cells. Caspases are key intracellular molecules in the control of apoptosis and defects in caspase-induced apoptosis in eosinophils from steroid-resistant individuals may contribute to the molecular mechanisms underlying glucocorticoid insensitivity in these cells. These findings point the way to new and more targeted anti-inflammatory therapy for asthma and may provide important clues for the development of alternative therapies for glucocorticoid resistance.

Cleavage of poly-(ADP-ribose) polymerase is a process occurring early during the execution phase of apoptosis. Although in many experimental systems PARP cleavage indicates a point of no return, the significance of this proteolytic step for apoptosis remains unclear. Here we compare the susceptibility of cells from wild-type mice and PARP-/- mice to several inducers of apoptosis. Neither the susceptibility of hepatocytes towards CD95 or TNF-mediated apoptosis nor the activation of PARP-cleaving caspases was modified in PARP-/- liver cells. Thymocytes with either genotype exhibited similar sensitivity to treatments with ceramide, dexamethasone, or etoposide. The sensitivity of primary neurons towards apoptosis induced by staurosporine, colchicine, potassium withdrawal, peroxynitrite, or the neurotoxin MPP+ was also unaltered. These data suggest that neither activation nor cleavage of PARP has a causal role in apoptotic cell death of primary, non-transformed cells.

In order to examine whether or not the expression of the apoptosis-related receptor Fas (CD-95/APO-1) and its ligand (FasL) has relevance for patient survival, immunohistochemistry was used to analyze the proteins of both factors in 164 non-small cell lung carcinomas. Patients with Fas-positive tumors exhibited significantly longer survival times than patients with Fas-negative carcinomas. In contrast, FasL did not significantly influence patient survival time. A multivariate analysis of clinical and biological factors indicated that lymph node status and Fas expression were significant prognostic factors. Carcinoma patients who were negative for both Fas and FasL had a significantly higher incidence of lymph node involvement than did carcinoma patients who were positive for Fas and FasL. Carcinomas that were positive for Fas and FasL demonstrated a greater sensitivity to doxorubicin in vitro.

Immunoregulation of lymphocytes and macrophages in the peripheral immune system is achieved in part by activation-induced cell death. Members of the TNF receptor family including Fas (CD95) are involved in the regulation of activation-induced cell death. To determine whether activation-induced cell death plays a role in regulation of dendritic cells (DCs), we examined interactions between Ag-presenting murine DCs and Ag-specific Th1 CD4+ T cells. Whereas mature bone marrow- or spleen-derived DCs expressed high levels of Fas, these DCs were relatively insensitive to Fas-mediated killing by the agonist mAb, Jo-2, as well as authentic Fas ligand expressed on the CD4+ T cell line, A.E7. The insensitivity to Fas-mediated apoptosis was not affected by priming with IFN-gamma and/or TNF-alpha or by blocking the DC survival signals TNF-related activation-induced cytokine and CD40L. However, apoptosis could be induced with C2-ceramide, suggesting that signals proximal to the generation of ceramide might mediate resistance to Fas. Analysis of protein expression of several anti-apoptotic mediators revealed that expression of the intracellular inhibitor of apoptosis Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein was significantly higher in Fas-resistant DCs than in Fas-sensitive macrophages, suggesting a possible role for Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein in DC resistance to Fas-mediated apoptosis. Our results demonstrate that murine DCs differ significantly from other APC populations in susceptibility to Fas-mediated apoptosis during cognate presentation of Ag. Because DCs are most notable for initiation of an immune response, resistance to apoptosis may contribute to this function.

Prior studies have noted that inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) enhanced geldanamycin lethality in malignant hematopoietic cells by promoting mitochondrial dysfunction. The present studies focused on defining the mechanism(s) by which these agents altered survival in carcinoma cells. MEK1/2 inhibitors [PD184352; AZD6244 (ARRY-142886)] interacted in a synergistic manner with geldanamycins [17-allylamino-17-demethoxygeldanamycin (17AAG) and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin] to kill hepatoma and pancreatic carcinoma cells that correlated with inactivation of extracellular signal-regulated kinase 1/2 and AKT and with activation of p38 MAPK; p38 MAPK activation was reactive oxygen species dependent. Treatment of cells with MEK1/2 inhibitors and 17AAG reduced expression of c-FLIP-s that was mechanistically connected to loss of MEK1/2 and AKT function; inhibition of caspase-8 or overexpression of c-FLIP-s abolished cell killing by MEK1/2 inhibitors and 17AAG. Treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent plasma membrane clustering of CD95 without altering the levels or cleavage of FAS ligand. In parallel, treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent association of caspase-8 with CD95. Inhibition of p38 MAPK or knockdown of BID, FAS-associated death domain, or CD95 expression suppressed MEK1/2 inhibitor and 17AAG lethality. Similar correlative data were obtained using a xenograft flank tumor model system. Our data show that treatment of tumor cells with MEK1/2 inhibitors and 17AAG induces activation of the extrinsic pathway and that suppression of c-FLIP-s expression is [Mol Cancer Ther 2008;7(9):2633-48].

Although Fas (APO-1/CD95) is expressed ubiquitously and induces cell death, it is also known to mediate other responses such as inflammation and angiogenesis in vivo. Previously, we have reported that Fas ligation induces selective expression of chemokines (IL-8 and MCP-1) in human astroglioma cells in vitro. In this study, we investigated whether Fas ligation can induce expression of other cytokines. Expression of IL-1alpha, IL-1beta, IL-6, IL-10, IL-12, IFN-beta, IFN-gamma, LT-beta, TGF-beta, TNF-a and TNF-beta mRNA levels in CRT-MG human astroglioma cells upon Fas ligation was investigated using RNase protection assay (RPA). We found that IL-6 mRNA is selectively induced upon Fas ligation, and IL-6 mRNA and protein expression was further investigated using single probe RPA and ELISA. To investigate the in vivo expression of IL-6, human brain specimens were homogenized and ELISA was performed for IL-6 expression. Herein, we demonstrate that: (1) Among these cytokines, only IL-6 was induced upon Fas ligation in a dose- and time-dependent manner; (2) A selective p38 MAP kinase inhibitor, SB202190, and a MEK inhibitor, U0126, suppressed induction of IL-6 mRNA and protein expression by Fas ligation; and (3) Glioblastoma multiforme samples (n = 11) contain significantly higher levels of IL-6 compared to those of control brains (n = 5), which correlate with increased levels of Fas. These results suggest that the Fas-FasL system may play a role in the regulation of tumor growth and survival by inducing the pleiotropic cytokine IL-6.

The purpose of this study was to evaluate the anticancer potency and mechanism of a novel difluorodiarylidenyl piperidone (H-4073) and its N-hydroxypyrroline modification (HO-3867) in human ovarian cancer. Studies were done using established human ovarian cancer cell lines (A2870, A2780cDDP, OV-4, SKOV3, PA-1, and OVCAR3) as well as in a murine xenograft tumor (A2780) model. Both compounds were comparably and significantly cytotoxic to A2780 cells. However, HO-3867 showed a preferential toxicity toward ovarian cancer cells while sparing healthy cells. HO-3867 induced G(2)-M cell cycle arrest in A2780 cells by modulating cell cycle regulatory molecules p53, p21, p27, cyclin-dependent kinase 2, and cyclin, and promoted apoptosis by caspase-8 and caspase-3 activation. It also caused an increase in the expression of functional Fas/CD95 and decreases in signal transducers and activators of transcription 3 (STAT3; Tyr705) and JAK1 phosphorylation. There was a significant reduction in STAT3 downstream target protein levels including Bcl-xL, Bcl-2, survivin, and vascular endothelial growth factor, suggesting that HO-3867 exposure disrupted the JAK/STAT3 signaling pathway. In addition, HO-3867 significantly inhibited the growth of the ovarian xenografted tumors in a dosage-dependent manner without any apparent toxicity. Western blot analysis of the xenograft tumor tissues showed that HO-3867 inhibited pSTAT3 (Tyr705 and Ser727) and JAK1 and increased apoptotic markers cleaved caspase-3 and poly ADP ribose polymerase. HO-3867 exhibited significant cytotoxicity toward ovarian cancer cells by inhibition of the JAK/STAT3 signaling pathway. The study suggested that HO-3867 may be useful as a safe and effective anticancer agent for ovarian cancer therapy.

Taurine is an abundant organic osmolyte with antioxidant and immunomodulatory properties. Its role in the pathogenesis of chronic liver disease is unknown. The liver phenotype was studied in taurine transporter knockout (taut-/-) mice. Hepatic taurine levels were ~21, 15 and 6 mumol/g liver wet weight in adult wild-type, heterozygous (taut+/-) and homozygous (taut-/-) mice, respectively. Immunoelectronmicroscopy revealed an almost complete depletion of taurine in Kupffer and sinusoidal endothelial cells, but not in parenchymal cells of (taut-/-) mice. Compared with wild-type mice, (taut-/-) and (taut+/-) mice developed moderate unspecific hepatitis and liver fibrosis with increased frequency of neoplastic lesions beyond 1 year of age. Liver disease in (taut-/-) mice was characterized by hepatocyte apoptosis, activation of the CD95 system, elevated plasma TNF-alpha levels, hepatic stellate cell and oval cell proliferation, and severe mitochondrial abnormalities in liver parenchymal cells. Mitochondrial dysfunction was suggested by a significantly lower respiratory control ratio in isolated mitochondria from (taut-/-) mice. Taut knockout had no effect on taurine-conjugated bile acids in bile; however, the relative amount of cholate-conjugates acid was decreased at the expense of 7-keto-cholate-conjugates. In conclusion, taurine deficiency due to defective taurine transport triggers chronic liver disease, which may involve mitochondrial dysfunction.

OBJECTIVE

Cantharidin, a natural toxin, is the active substance of mylabris and has antitumor effects in man. Norcantharidin, the demethylated analogue of cantharidin, has been used in the treatment of patients with primary hepatoma and those with leukopenia in China. The present study was designed to investigate whether norcantharidin exerts cytotoxic activity against colorectal cancer cells by inducing apoptosis and to examine the possible mechanism in the phenomenon.

METHODS

Inhibition of proliferation of norcantharidin on Colo205, HT-29, and SW480 colorectal cancer cells was determined by the trypan blue dye exclusion test. Apoptosis of norcantharidin-treated cells was determined by morphological analysis, agarose gel DNA electrophoresis, and quantitated by flow cytometry after staining with propidium iodide. Cell cycle and the cell surface expression of the CD95/CD95 ligand were evaluated by flow cytometry. Caspase 8-like protease and protein phosphatase 1 and 2A activities were also analyzed.

RESULTS

Treatment with norcantharidin of colorectal cancer cells not only inhibited cell proliferation, but also induced apoptosis. Norcantharidin induced apoptosis mainly in two phases: rapid apoptosis in S-phase cells and delayed apoptosis in G2/M arrested cells. Treatment with norcantharidin resulted in an upregulation of the CD95 receptor and CD95 ligand on the cell surface. Furthermore, stimulation with anti-CD95 monoclonal antibody (mAb) resulted in further induction of apoptosis after treatment with norcantharidin. In addition, the apoptosis-inducing effect of norcantharidin was almost completely inhibited by anti-CD95 ligand mAb. Norcantharidin-treated cells showed the activation of caspase 8. Both zVAD-FMK (a broad range caspase inhibitor) and IETD-FMK (a caspase-8 inhibitor) showed apparent inhibition of the apoptosis-inducing effect. Norcantharidin did not show an inhibitory effect on protein phosphatase.

CONCLUSIONS

These results suggest that norcantharidin triggers apoptosis in colorectal cancer cell lines via the activation of the CD95 receptor/ligand system, and that this agent may be useful for developing new therapeutic regimens for the treatment of colorectal carcinoma.

Ceramide, a product of sphingolipid metabolism, is generated in response to various stress stimuli, such as tumor necrosis factor-alpha, CD95/Fas, chemotherapeutic agents, and irradiation. Ceramide may modulate the biochemical and cellular processes that lead to apoptosis. However, the mechanisms by which ceramide regulates apoptotic events are not fully defined. It is believed that the biological effect of ceramide depends on its concentration, the activation or differentiation status of the cell, and the time frame of action. Here, we discuss the metabolism and cell apoptotic signaling of ceramide. The involvement of protein kinases (i.e. PI3K/Akt and GSK-3beta) and protein phosphatases (i.e. PP1 and PP2A), Bcl-2 family proteins, mitochondrial damage, and caspase cascade activation are demonstrated. Further, ceramide and its derivatives have recently been incorporated into strategies for anticancer therapies. An understanding of the apoptotic signaling pathways mediated by ceramide may shed light on its potential for therapeutic intervention.

Activation of apoptosis introduces a site-specific break within intron 11 of the MLL gene. Using the CD95 apoptotic signaling pathway in human lymphoblastoid cells, the 5' fragment of MLL undergoes translocation to intron 4 of AF9 and the proleukemogenic MLL-AF9 fusion gene created is transcribed. Both the breaks in MLL and transcription of the MLL-AF9 fusion gene are suppressed in the presence of the broad spectrum caspase inhibitor, zVAD.fmk. Duplicate cells containing sequence identical MLL-AF9 fusion junctions were identified within a cell population that had recovered from apoptosis. This indicated that cells harboring a translocation initiated by apoptotic cleavage had divided. These data are consistent with a novel pathogenic role for the apoptotic program where translocations with leukemogenic potential are created within cells that have the capacity to divide.

Synthetic ether-linked analogues of phosphatidylcholine and lysophosphatidylcholine, collectively named as antitumour lipids (ATLs), were initially synthesized in the late 60s, but have attracted a renewed interest since the finding that the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, edelfosine), a synthetic analogue of 2-lysophosphatidylcholine considered the ATL prototype, induces a selective apoptotic response in tumour cells, sparing normal cells. Unlike most chemotherapeutic agents currently used, ET-18-OCH3 does not interact with DNA, but act at the cell membrane, and thereby its effects seem to be independent of the proliferative state of target cells. Each part of the molecular structure of ET-18-OCH3 is important for its optimal proapoptotic activity. Recent progress has unveiled the molecular mechanism underlying the apoptotic action of ET-18-OCH3, involving membrane rafts and Fas/CD95 death receptor, and has led to the proposal of a two-step model for the ET-18-OCH3 selective action on cancer cells, namely: a) ET-18-OCH3 uptake into the tumour cell, but not in normal cells; b) intracellular activation of Fas/CD95 through its translocation and capping into membrane rafts. ET-18-OCH3 constitutes the first antitumour drug acting through the intracellular activation of the Fas/CD95 death receptor. Computational docking studies have allowed us to propose a molecular model for the putative interaction of ET-18-OCH3 with the intracellular Fas/CD95 death domain. This novel mechanism of action represents a new way to target tumour cells in cancer chemotherapy and can be of interest as a new framework in designing novel and more selective proapoptotic antitumour drugs.

Alopecia areata (AA) is a suspected hair follicle specific autoimmune disease. The potential for cell transfer of AA using the C3H/HeJ mouse model was examined. Cells isolated from lymph nodes and spleens of AA-affected mice using magnetic bead conjugated monoclonal antibodies were subcutaneously injected into normal C3H/HeJ recipients. Within 5 wk, all CD8(+) cell-injected mice exhibited localized hair loss exclusively at the site of injection that persisted until necropsy. In contrast, some CD4(+) and CD4(+)/CD25(-) cell-injected mice developed extensive, systemic AA, and a combination of CD8(+) and CD4(+)/CD25(-) cells injected yielded the highest frequency of systemic AA induction. CD4(+)/CD25(+) cells were less able to transfer the disease phenotype, partially blockaded systemic AA induction by CD4(+)/CD25(-) cells, and prevented CD8(+) cell-induced, injection site-localized hair loss. CD11c(+) and CD19(+) cells failed to promote significant phenotype changes. Increases in co-stimulatory ligands CD40 and CD80, plus increased leukocyte apoptosis resistance with reduced CD95, CD95L, and CD120b expression, were associated with successful alopecia induction. The results suggest that CD8(+) cells may be the primary instigators of the hair loss phenotype. However, systemic disease expression fate is, apparently determined by CD4(+)/CD25(-) cells, while CD4(+)/CD25(+) lymphocytes may play a predominantly regulatory role.

An apoptotic signal triggered by cell surface death receptors is disseminated to intracellular compartments through protein-protein interactions mediated by conserved domains such as the death effector domain (DED). A unique family of single DED-containing proteins, including DEDD and DEDD2, is targeted to the nucleolus. However, the role of DEDD/DEDD2 in apoptosis remains less understood. Here we show that DEDD and DEDD2 are highly conserved in diverse species, and that they are potent inducers of apoptosis in various cell types. Deletion analysis indicates that both the N-terminal DED domain and the C-terminal region of DEDD2 can induce apoptosis. The cell death activity of this family appears to be related to their nuclear localization. DEDD and DEDD2 bind to two tandem DED-containing caspases, caspase -8 and -10, that are engaged by death receptors. Consistent with the nuclear localization of this family, caspase-8 translocates to the nucleus during CD95-induced apoptosis. DEDD and DEDD2 also readily associate with themselves and with each other. These results suggest that DEDD and DEDD2 may be important mediators for death receptors and that they may target caspases to the nucleus.