Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis

We report here that anticancer drugs such as doxorubicin lead to induction of the CD95 (APO-1/Fas) system of apoptosis and the cellular stress pathway which includes JNK/SAPKs. Ceramide, which accumulates in response to different types of cellular stress such as chemo- and radiotherapy, strongly induced expression of CD95-L, cleavage of caspases and apoptosis. Antisense CD95-L as well as dominant-negative FADD inhibited ceramide- and cellular stress-induced apoptosis. Fibroblasts from type A Niemann-Pick patients (NPA), genetically deficient in ceramide synthesis, failed to up-regulate CD95-L expression and to undergo apoptosis after gamma-irradiation or doxorubicin treatment. In contrast, JNK/SAPK activity was still inducible by doxorubicin in the NPA cells, suggesting that activation of JNK/SAPKs alone is not sufficient for induction of the CD95 system and apoptosis. CD95-L expression and apoptosis in NPA fibroblasts were restorable by exogenously added ceramide. In addition, NPA fibroblasts undergo apoptosis after triggering of CD95 with an agonistic antibody. These data demonstrate that ceramide links cellular stress responses induced by gamma-irradiation or anticancer drugs to the CD95 pathway of apoptosis.     

Fas/APO-1 (CD95) expression in myelodysplastic syndromes

Increased apoptosis of myeloid precursors appears to contribute to the pathophysiology of cytopenias in myelodysplastic syndromes (MDS). Fas /APO-1(CD95) is a cell surface protein inducing an apoptotic signal after its binding to Fas ligand or to a functional anti-Fas antibody. Here we studied Fas expression by immunocytochemistry on marrow slides from 30 cases of MDS. Increased Fas expression in erythroblasts and/or immature granulocytes, compared to controls, was seen in 12 (40%) of the cases. In addition, in 16 of the 18 cases with > or = 5% marrow blasts, a variable proportion of blasts expressed Fas. Increased apoptosis was found by morphological analysis and/or TUNEL technique in marrow cells from 8 of the 26 cases analyzed (31%) The ability of Fas antigen to trigger apoptosis was studied after addition of a functional anti Fas antibody in 5 of the patients with Fas overexpression. Addition of this antibody, however, only lead to mild increase of apoptosis in immature granulocytes (but not other myeloid cells) in 2 of the 5 cases. Thus, increased Fas expression is seen in myeloid and/or blast cells in the majority of MDS cases. However, the relationship between this finding and increased apoptosis in MDS still remains to be established.     

Protein kinase C regulates Fas (CD95/APO-1) expression

Fas (CD95/APO-1) is a transmembrane protein of the TNF/neuron growth factor receptor family. Ligation of Fas by specific Abs or Fas ligand (FasL/CD95 ligand) induces rapid apoptotic cell death in a variety of cell types. Despite progress in understanding the death signals transduced from Fas, very little is known with regard to the mechanisms by which Fas expression is regulated. Using our previously established murine T cell hybridoma model A1.1, we show that specific protein kinase C (PKC) inhibitors could block activation-induced Fas expression and apoptosis. The activation of PKC with PMA or 1-oleoyl-2-acetyl-sn-glycerol could mimic the TCR signal by inducing the expression of Fas but not FasL. PKC-dependent Fas expression was also observed in several murine and human tumor cell lines. Since the inhibition of Ca2+ redistribution by an inhibitor of intracellular Ca2+ mobilization, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride, inhibited TCR-induced FasL but not Fas, the expression of Fas appears to be independent of Ca2+ mobilization. Significantly, expression of the newly identified Fas-regulatory gene, TDAG51, was found to be dependent upon the activity of PKC. PKC activation only induced Fas expression in cells expressing wild-type TDAG51. Thus, Fas expression is likely mediated by PKC through TDAG51.     

The role of p53 and the CD95 (APO-1/Fas) death system in chemotherapy-induced apoptosis

To explore the pathway of p53 dependent cell death, we investigated if p53 dependent apoptosis following DNA damage is mediated by the CD95 (APO-1/Fas) receptor/ligand system. We investigated cell lines of solid human tumors upon treatment with clinically relevant chemotherapeutic drugs known to act via p53 accumulation. Treatment with these cytotoxic drugs led to an upregulation of both, the CD95 receptor (CD95) and the CD95L (CD95L). Induction of the CD95L occurred in p53 wild-type (wt), p53 mutant (mt) and in cell lines lacking p53 altogether (p53-/-). Thus, the regulation of the CD95L in response to chemotherapeutic drugs clearly involves p53 independent mechanisms. Most importantly, upregulation of CD95 occurred only in cell lines with wild-type p53, thereby strongly increasing the responsiveness towards CD95 mediated apoptosis. Thus, upregulation of the CD95 receptor seems to be dependent on intact wild-type p53. Apoptosis was mediated by cleavage of the receptor proximal caspase, caspase-8 (FLICE/MACH). Caspase-8 cleavage was observed, independent of the p53 status of the tumor cells and irrespective whether or not apoptosis was dependent on the CD95 system. Hence, additional effector pathways besides CD95/CD95L signaling are likely to contribute to drug-induced apoptosis.     

CD95 (APO-1/Fas) mutations in childhood T-lineage acute lymphoblastic leukemia

CD95 (APO-1/Fas)-mediated apoptosis is pivotal in normal lymphocyte homeostasis and mutations of CD95 cause a benign autoimmune lymphoproliferation syndrome (ALPS) in humans and mice. However, tumors only rarely develop in these patients, and no CD95 mutations have yet been directly implicated in tumorigenesis. We therefore examined 81 de novo childhood T-lineage acute lymphoblastic leukemias (T-ALL) including 54 steroid-poor responders, 10 relapsed T-ALL, and 10 leukemic T-cell lines, for the presence of CD95 mutations using single-strand confirmation polymorphism and sequence analysis. In leukemic blasts and normal T cells of one patient, a heterozygous mutation in exon 3 of CD95 causing a 68Pro --> 68Leu change associated with decreased CD95-mediated apoptosis was found. In leukemic blasts and normal T cells of a second patient, a homozygous mutation in the promoter of CD95 causing disruption of a consensus sequence for AP-2 binding without decreasing constitutive CD95 expression was detected. No large intragenic alterations of CD95 were found, no homozygous loss was detected in the cell lines, and no CD95 mutations were detected in the relapses. The data presented here show that CD95 mutations occur in some T-ALL and may be of biological importance.     

Hepatic failure and liver cell damage in acute Wilson''s disease involve CD95 (APO-1/Fas) mediated apoptosis

We gave auditory examples of two semantic categories through headphones to 100 surgical patients anaesthetized with propofol and enflurane. This presentation was made during certain stages of the procedure, potentially associated with arousal, and during steady-state anaesthesia. Postoperative review using category generation tests showed successful priming in a pre-induction group but no evidence of implicit memory in the anaesthetized groups. These results suggest that timing an auditory input to coincide with surgical stimulation does not increase the probability of retrieval of information by this type of testing.     

Fas (APO-1, CD95)-mediated apoptosis in thyroid cells is regulated by a labile protein inhibitor

To determine whether thyroid cell apoptosis observed in autoimmune thyroid disease could be related to activation of the Fas pathway, we examined the expression and function of Fas on thyroid follicular cells in vitro. Fas messenger RNA was found to be present using two different techniques and was expressed at equal levels in thyrocytes cultured either in the presence or absence of TSH. Fas antigen protein expression was demonstrated by Western blot of thyroid cell lysates and by immunohistochemical staining of thyrocytes, and the amount of Fas protein present did not appear to vary regardless of culture conditions. Despite expressing substantial amounts of Fas protein, thyrocytes treated with anti-Fas monoclonal antibody failed to undergo apoptosis. The addition of either interferon-gamma or interleukin-1beta to the anti-Fas-treated cell cultures also did not promote apoptotic signaling through this pathway. In contrast, the concomitant administration of cycloheximide allowed the induction of apoptosis through the activation of Fas in thyrocytes. These results suggest that Fas is constitutively expressed in thyrocytes, but that the induction of apoptosis through the Fas pathway is blocked by a labile protein inhibitor.     

Mutation analysis of CD95 (APO-1/Fas) in childhood B-lineage acute lymphoblastic leukaemia

The delta-type phospholipase C (PLC) is thought to be evolutionally the most basal form in the mammalian PLC family. One of the delta-type isoforms, PLC-delta 1, binds to both phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) with a high affinity via its pleckstrin homology (PH) domain. We report here a missense mutation in the region encoding the C-terminal PH domain of the human PLC-delta 1. This is also the first report of a mutation in the human PLC genes. A single base substitution (G to A) causes the amino acid replacement, Arg105 to His. Site-directed mutagenesis of the glutathione-S-transferase (GST)/PLC-delta 1 fusion protein changing Arg105 to His resulted in a fourfold decrease in the affinity of specific Ins(1,4,5)P3 binding and a reduction in PtdIns(4,5)P2 hydrolysing activity to about 40% of that of the wild-type enzyme. This remarkable loss of function can be interpreted in terms of a conformational change in the PH domain.     

Synergy between T cell Receptor and Fas (CD95/APO-1) signaling in mouse thymocyte death

Both extracellular and intracellular calcium (Ca2+) play important roles in hypoxic pulmonary vasoconstriction (HPV) and the vasoconstrictor responses to endogenous pulmonary vasoconstrictor substances, as evidenced by the effect of calcium-channel blockers on these vasoconstrictor responses and the measurement of changes in Ca2+ flux or intracellular Ca2+ concentrations in isolated cells. The more vasoselective the calcium-channel blocker, the greater its effect on pulmonary vasoconstriction. However, these drugs are not selective for the pulmonary vascular bed and are not as potent as pulmonary vasodilators when compared with other vasodilator drugs, including prostaglandin E1, isoproterenol, prostacyclin, or nitroglycerin. Moreover, the primary effect of vasoselective calcium-channel blockers on pulmonary vascular resistance is secondary to the effects of these agents on systemic vascular resistance and cardiac output. Although there is improvement in oxygen delivery, exercise tolerance, and survival in patients with primary pulmonary hypertension who respond to calcium-channel blockers, the response of individual patients to these drugs is difficult to predict because the extent of reversible versus irreversible changes in the pulmonary vasculature is not known. The use of these drugs in patients with chronic hypoxia-induced pulmonary vasoconstriction may be associated with a worsening of ventilation-perfusion mismatching secondary to inhibition of HPV.     

Fas/APO-1/CD95 in health and autoimmune disease: thymic and peripheral aspects

OBJECTIVE: To examine the association between blood transfusion and bacterial infective complications after resection for colorectal adenocarcinoma. DESIGN: Retrospective cohort study. SETTING: District hospital; Norway. SUBJECTS: 446 consecutive patients having resection of colorectal adenocarcinoma. MAIN OUTCOME MEASURES: Postoperative bacterial infective morbidity in hospital. RESULTS: 112 patients (25%) developed postoperative infections in hospital. Univariate analysis showed that the development of infection was significantly associated with increasing age (p=0.02), rectal compared with colonic cancer (p=0.002), preoperative radiotherapy (p=0.005), blood loss during operation (p=0.001), the extent of the primary tumour (T stage): T4 compared with T1-T3 (p=0.004), the presence of regional lymph node metastasis (N stage): N1-N3 compared with N0 (p=0.01), operating surgeon 1 (p=0.009), operating surgeon 2 (p=0.03), and blood transfusion (p < 0.001). Multivariate logistic regression analysis showed that the following variables were independent predictors of infection: age, rectal compared with colonic cancer, T stage, N stage, and blood transfusion. The corrected odds ratios for infection were 1.5 (95% CI 0.8 to 2.8) when 1-3 units of blood were given and 3.1 (95% CI 1.6 to 6.0) when more than three units were given. Storage time did not affect the rate of postoperative infections in patients given transfusions. CONCLUSION: Transfusion of non-filtered stored allogeneic blood suspended in saline-adenine-glucose-mannitol is an independent risk factor for the development of postoperative infections in hospital in patients having a resection of colorectal cancer.     

Caspase activation is required for nitric oxide-mediated, CD95(APO-1/Fas)-dependent and independent apoptosis in human neoplastic lymphoid cells

Nitric oxide (NO), an important effector molecule involved in immune regulation and host defense, was shown to induce apoptosis in lymphoma cells. In the present report the NO donor glycerol trinitrate was found to induce apoptosis in Jurkat cells that are sensitive to CD95-mediated kill. In contrast, a CD95-resistant Jurkat subclone showed substantial protection from apoptosis after exposure to NO. NO induced mRNA expression of CD95 (APO-1/Fas) and TRAIL/APO-2 ligands. Moreover, NO triggered apoptosis in freshly isolated human leukemic lymphocytes which were also sensitive to anti-CD95 treatment. The ability of NO to induce apoptosis was completely blocked by a broad-spectrum ICE (interleukin-1beta converting enzyme)-protease/caspase inhibitor and correlated with FLICE/caspase-8 activation. This activation was abrogated in some neoplastic lymphoid cells but not in others by the inhibitor of protein synthesis cycloheximide. Our results were confirmed using an in vitro experimental model of coculture of human lymphoid target cells with activated bovine endothelial cells generating NO as effectors. Furthermore, the inhibition of endogenous NO production with the inducible NO synthase inhibitor NG-monomethyl-L-arginine caused a complete abrogation of the apoptotic effect. Our data provide evidence that NO-induced apoptosis in human neoplastic lymphoid cells strictly requires activation of caspases, in particular FLICE, the most CD95 receptor-proximal caspase. Depending on the cell line tested this activation required or was independent of the CD95 receptor/ligand system.     

Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal

The early signals generated following cross-linking of Fas/APO-1, a transmembrane receptor whose engagement by ligand results in apoptosis induction, were investigated in human HuT78 lymphoma cells. Fas/APO-1 cross-linking by mAbs resulted in membrane sphingomyelin hydrolysis and ceramide generation by the action of both neutral and acidic sphingomyelinases. Activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) was also detected which appeared to be a requirement for subsequent acidic sphingomyelinase (aSMase) activation, since PC-PLC inhibitor D609 blocked Fas/APO-1-induced aSMase activation, but not Fas/APO-1-induced neutral sphingomyelinase (nSMase) activation. Fas/APO-1 cross-linking resulted also in ERK-2 activation and in phospholipase A2 (PLA2) induction, independently of the PC-PLC/aSMase pathway. Evidence for the existence of a pathway directly involved in apoptosis was obtained by selecting HuT78 mutant clones spontaneously expressing a newly identified death domain-defective Fas/APO-1 splice isoform which blocks Fas/APO-1 apoptotic signalling in a dominant negative fashion. Fas/APO-1 cross-linking in these clones fails to activate PC-PLC and aSMase, while nSMase, ERK-2 and PLA2 activates are induced. These results strongly suggest that a PC-PLC/aSMase pathway contributes directly to the propagation of Fas/APO-1-generated apoptotic signal in lymphoid cells.     

Stimulation of CD40 in human bladder carcinoma cells inhibits anti-Fas/APO-1 (CD95)-induced apoptosis

Genetic predisposition and androgen dependence are important characteristics of the common patterned loss of scalp hair known as male pattern baldness. The involvement of the 5alpha-reductase enzyme in male pattern baldness has been postulated due to its role in the metabolism of testosterone to dihydrotestosterone. There are two known isozymes of 5alpha-reductase. Type I has been predominantly localized to the skin and scalp. Type II, also present on the scalp, is the target of finasteride, a promising treatment for male pattern baldness. We conducted genetic association studies of the 5alpha-reductase enzyme genes (SRD5A1 on chromosome 5 and SRD5A2 on chromosome 2) using dimorphic intragenic restriction fragment length polymorphisms. From a population survey of 828 healthy families comprising 3000 individuals, we identified 58 young bald men (aged 18-30 y) and 114 older nonbald men (aged 50-70 y) for a case control comparison. No significant differences were found between cases and controls in allele, genotype, or haplotype frequencies for restriction fragment length polymorphisms of either gene. These findings suggest that the genes encoding the two 5alpha-reductase isoenzymes are not associated with male pattern baldness. Finally, no clear inheritance pattern of male pattern baldness was observed. The relatively strong concordance for baldness between fathers and sons in this study was not consistent with a simple Mendelian autosomal dominant inheritance. A polygenic etiology should be considered.     

In vitro expansion of hematopoietic progenitor cells induces functional expression of Fas antigen (CD95)

Fas antigen (Fas Ag; CD95) is a cell surface molecule that can mediate apoptosis. Bcl-2 is a cytoplasmic molecule that prolongs cellular survival by inhibiting apoptosis. To investigate the role of both molecules in hematopoiesis, we evaluated the expression of Fas Ag and Bcl-2 on CD34+ hematopoietic progenitor cells expanded in vitro. CD34+ cells isolated from bone marrow were cultured in iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 1% bovine serum albumin, 50 ng/mL stem cell factor, 50 ng/mL interleukin-3 (IL-3), 50 ng/mL IL-6, 100 ng/mL granulocyte colony-stimulating factor, and 3 U/mL erythropoietin for 7 days. Colony-forming unit of granulocytes/macrophages (CFU-GM) and burst-forming unit of erythroids (BFU-E) were expanded 6.9-fold and 8.8-fold in number at day 5 of culture, respectively. Freshly isolated CD34+ cells did not express Fas Ag, whereas approximately half of them expressed Bcl-2. CD34+ cells cultured with hematopoietic growth factors gradually became positive for Fas Ag and rapidly lost Bcl-2 expression. Furthermore, apoptosis was induced in the cultured CD34+ population when anti-Fan antibody (IgM; 1 microgram/mL) was added, as shown by significant decrease in the number of viable cells, morphologic changes, induction of DNA fragmentation, and significant decrease in the number of clonogenic progenitor cells including CFU. GM and BFU-E. These results indicate that functional expression of Fas Ag is induced on CD34+ cells expanded in vitro in the presence of hematopoietic growth factors. Induction of Fas Ag and downregulation of Bcl-2 may be expressed as part of the differentiation program of hematopoietic cells and may be involved in the regulation of hematopoiesis.     

Tetrapeptide DEVD-aldehyde or YVAD-chloromethylketone inhibits Fas/Apo-1(CD95)-mediated apoptosis in renal-cell-cancer cells

The apoptotic machinery has been intensively investigated, and interleukin-1-beta-converting enzyme (ICE) and its homologs directly mediate apoptosis by means of their unique protease activity. Fas/Apo1 (CD95), a member of the TNF-receptor family, mediates apoptosis by binding to its ligand, which is mainly expressed on lymphocytes. Here, we investigated the expression and function of both molecules in renal-cell cancer (RCC). The expression of Fas was examined in 6 RCC cell lines by immunoblotting and all of them expressed Fas. ICE and CPP32/YAMA were also identified among the cell lines. We earlier examined ACHN cells expressing low levels of BCL-2, as well as KRC/Y cells with high levels of BCL-2. Here, we found that the anti-Fas monoclonal antibody, CH-11, induced apoptosis in a dose-dependent fashion more remarkably in ACHN cells. Pre-incubation with the tetrapeptide YVAD-chloromethyl-ketone or DEVD-aldehyde inhibited Fas-mediated apoptosis. These findings suggest that, in RCC, apoptosis is induced by lymphocytes bearing Fas-L, and that it is achieved through the proteolytic action of CPP32/YAMA and/or ICE, or another member of the ICE/ced-3 protease family.