Fas Antigen/APO-1 (CD95) Expression on Myeloma Cells

Many factors involved in the proliferation of myelomas have been reported, and the relationship between these factors and the pathogenesis of multiple myeloma has been discussed. We found that most myeloma cells express Fas antigen/APO-1 (CD95), a cell surface antigen that mediates apoptosis. However only some cells are sensitive to anti-Fas antibody and undergo apoptosis. These data indicate that some multiple myelomas are generated not only by cell proliferation but also by cell immortalization. The mechanism by which myelomas are immortalized is still unclear, but Bcl-2, Bcl-x_L, adult T cell leukemia derived factor (ADF), soluble Fas are all candidate factors for this mechanism. The possibility also exists that inducers of apoptosis, e.g. tumor necrosis factor(TNF), interleukin-lβ-converting enzyme(lCE), Bcl-x_S, or Bax, do not have a lethal effect. In this review, we focus on the system that immortalizes myeloma cells, and suggest the possibility that multiple myeloma constitutes one group of cells which cannot undergo apoptosis in the bone marrow.     

Significance of serum-soluble CD95 (Fas/APO-1) on prognosis in renal cell cancer patients.

Serum-soluble CD95 (sCD95) levels for 72 renal cell cancer patients were significantly higher than those of 17 healthy donors. Twenty-one of 72 patients had elevated (defined as more than mean of healthy donors + 2 s.d.) sCD95. The disease-specific survival rate was significantly lower in the elevated sCD95 group. Serum sCD95 level was shown to be an independent prognostic factor by univariate and multivariate analysis, indicating a possible significant role in determining treatment strategies.     

Deficient activation of CD95 (APO-1/Fas)-mediated apoptosis: a potential factor of multidrug resistance in human renal cell carcinoma

The pronounced resistance of human renal cell carcinoma (RCC) to anticancer-induced apoptosis has primarily been related to the expression of P-glycoprotein and effective drug detoxification mechanisms. Because the CD95 system has recently been identified as a key mediator of anticancer drug-induced apoptosis, we analysed the contribution of the CD95 system to chemotherapy-induced apoptosis in four newly established RCC cell lines. Here, we demonstrate that all RCC cell lines expressed CD95-receptor and -ligand. Exposure to agonistic anti-CD95 antibodies resulted in induction of apoptosis and significant (P < 0.05) reduction of cell number in three out of four cell lines, indicating that the essential components for CD95-mediated apoptosis were present and functionally intact in the majority of these RCC cell lines. Moreover, treatment of cultures with bleomycin or topotecan, a novel topoisomerase I inhibitor with little substrate affinity for P-glycoprotein, led to induction of apoptosis and significant (P < 0.05) dose-dependent reduction of cell number in all RCC cell lines. Both anticancer drugs also induced upregulation of CD95 ligand expression in all cell lines. Additionally, augmentation of CD95 receptor expression was found in three RCC cell lines, including one p53-mutated cell line, whereas another p53-mutated cell line showed no or only a weak CD95 receptor upregulation after exposure to topotecan or bleomycin, respectively. Despite this upregulation of CD95 receptor and ligand, antagonistic antibodies directed against CD95 receptors or ligands could not inhibit induction of apoptosis by topotecan and bleomycin in any cell line. Thus, although a functionally intact CD95 signalling cascade is present in most RCC cell lines, the anticancer drugs topotecan and bleomycin that induce upregulation of CD95 receptor and ligand fail to effectively activate CD95-mediated apoptosis. This deficient activation of CD95-mediated apoptosis might be an important additional factor for the multidrug resistance phenotype of human RCCs.     

Fas/Apo-1 (CD95)-Mediated and CD95-Independent Apoptosis of Malignant Plasma Cells

CD95 (FaslApo-1) mediates apoptosis in cells of various types. Expression of CD95 and its function were investigated in myeloma cells and most plasma cell lines have been found to be CD95-positive. Anti-CD95 antibody induced apoptosis in these cell lines in a manner dependent on intensity of CD95 expression. Bcl-2 expression levels in these cell lines were not correlated with sensitivity to CD95-induced apoptosis. Myeloma cells from approximately 70% of cases analyzed expressed CD95, suggesting a high frequency of CD95 expression of myeloma cells. The incidence of CD95 expression in samples from extramedullary lesions were higher than those of bone marrow myeloma cells.

Anti-CD95 antibody did not induce apoptosis in some freshly isolated myeloma cells despite the expression of CD95 induced apoptosis in myeloma cell lines in a manner dependent on intensity of CD95 expression. In fresh samples, intensity of CD95 expression was not correlated with apoptosis evoked by anti-CD95 antibody. Interestingly, isolated myeloma cells with an high-level expression of CD95 showed spontaneous apoptosis during in vitro incubation. This was observed in samples from cases with high serum levels of lactate dehydrogenase (LDH), suggesting that the LDH was derived from cells undergoing spontaneous apoptosis in vivo.

Taken together, CD95 is expressed in most myeloma cell lines and in many freshly isolated myeloma cells. However, some of the latter escape from CD95-dependent apoptosis despite the expression of CD95. Moreover, some myeloma cells from cases with aggressive disease may undergo apoptosis in a CD95-independent manner. In this review, the significance of CD95 and apoptosis of myeloma cells are discussed.     

Programmed cell death: the influence of CD40, CD95 (Fas or Apo-I) and their ligands

Programmed cell death (PCD) or apoptosis is a process whereby developmental or environmental stimuli activate a specific series of events that culminate in cell death. PCD is essential for normal development and abnormality in the process can lead to defects ranging from embryonic lethality and tissue-specific perturbation of postnatal development to a high susceptibility to malignancy. Therapeutics that modulate the regulation of PCD may provide a new opportunity for the treatment of the PCD related diseases and cancer. CD40 and CD95 (Fas/Apo-I) are transmembrane proteins of the nerve growth factor/tumour necrosis factor α receptor superfamily. The death signal of PCD occurs when the CD95 receptor on the cell surface binds to the CD95 ligand (CD95L) or to the anti-CD95 monoclonal antibody (mAb). In contrast, PCD could be inhibited by the survival signal mediated from the binding of the CD40 receptor to the CD40 ligand (CD40L) or to the anti-CD40 mAb. In this review, the interaction of CD40/CD40L and CD95/CD95L on PCD in normal and malignant cells is discussed.     

Expression and prognostic relevance of the death receptor CD95 (Fas/APO1) in renal cell carcinomas

CD95 (Fas/APO1) is one of the best known members of the death receptor family which can either mediate apoptosis or activate tumor-promoting pathways. Using a tissue microarray we investigated the association between the expression of CD95 and prognosis in 617 patients with renal cell carcinomas (RCCs). CD95 was expressed in the vast majority of RCCs. High CD95 expression was associated with lymph node metastasis and correlated negatively with disease-specific survival. Multivariate Cox regression analysis confirmed CD95 expression as an independent prognostic factor. In conclusion, high CD95 expression is a negative independent prognostic factor in RCCs which could be used to identify high-risk patients with a poor clinical prognosis.     

Soluble Fas (APO-1/CD95) Isoform in Adult T-Cell Leukemia

Fas (APO-1/CD95) consists mainly of 2 isoforms, membrane-anchored (mFas) and soluble (sFas), both of which can mediate apoptosis through the Fas-signalling process, not only in normal but also in leukemi T-cells. This suggests that aberrant expression of either mFas or sFas may affect the natural history of T-cell neoplasms, such as adult T-cell leukemia (ATL). For studying the tumor biology related to Fas-mediated apoptosis, ATL cells with up-regulated Fas proteins and its mRNAs are convenient and useful for understanding apoptotic oncology as it occurs in nature. Most attention, so far, has been focused on mFas, and little is known about neoplasms from the viewpoint of sFas. Accordingly, we herein review and discuss the biological and clinical implications of sFas in ATL.     

Cytotoxic Effect through Fas/APO-1 Expression due to Vitamin K in Human Glioma Cells

Congeners of vitamin K have been found to inhibit growth in various rodent and human tumor cells, but the mechanisms of the inhibitory action are still not well understood. To investigate the modes of actions of vitamin K, we used several vitamin K analogs and examined their cytotoxic effect for human glioma cell lines RBR17T and U251. The analogs included vitamin K₁ (VK₁), vitamin K₂ (VK₂), vitamin K₃ (VK₃), and geranylgeraniol (GGO) which form an unsaturated side chain of VK₂. Cell viability was estimated by MTT assay. DNA fragmentation was demonstrated by gel electrophoresis and flow cytometry. In order to study the mechanism of apoptosis, we measured the changes of intracellular reactive oxygen intermediates (ROI) and Fas/APO-1 expression by flow cytometry. The results showed: (1) VK₂, VK₃, and GGO inhibited cell growth; (2) VK₃ had a more potent cytotoxic effect than VK₂, and VK₃ enhanced the cytotoxic effect of antitumor agents (ACNU and IFN-beta) in RBR17T cells; (3) VK₂, VK₃, and GGO induce apoptosis; (4) VK₃ increased the expression of Fas/APO-1 although VK₂ and GGO did not increase its expression in glioma cells; (5) VK₃ increased the production of intracellular ROI. Catalase and reduced glutathione (GSH) inhibited production of intracellular ROI and antagonized inhibition of cell-growth induced by VK₂, but failed to antagonize that of VK₂ and GGO. We hypothesize that VK₃ induces apoptosis by promoting the generation of intracellular ROI and Fas/APO-1 expression. On the other hand, VK₂ and GGO induce apoptosis but most likely by some other unknown pathway.     

CD95 Antigen Mutations in Hematopoietic Malignancies

The CD95 receptor, also known as Fas/Apo-1, is a member of the Tumor Necrosis Factor receptor (TNF-R) family of death receptors. Apoptosis mediated by CD95 plays a central role in maintaining homeostasis of the immune system. Dysregulation of the CD95 apoptotic pathway has been proposed as a mechanism of oncogenesis by providing a survival advantage to potentially malignant cells. This extended lifespan could allow the accumulation of further mutations leading to malignant transformation. Several mechanisms of resistance to CD95 mediated apoptosis have been identified, including reduced surface expression of the receptor, overexpression of anti-apoptotic molecules, and loss of function mutations. This review will focus on the potential role of the CD95-CD95 ligand system in the pathogenesis of hematological malignancies, with particular emphasis on recent work from our laboratory examining the expression of CD95 in B cell lymphomas. We demonstrate that CD95 mutations occur at low frequency in NHL tumors, however, surface expression of the CD95 protein varies with the subtype of lymphoma. Loss of surface CD95 is more likely to occur in lymphomas of aggressive histology, and is unrelated to the detection of CD95 mutations.     

Frequency and resistance of CD95 (Fas/Apo-1) gene-transfected tumor cells to CD95-mediated apoptosis by the elimination and methylation of integrated DNA

It is important for more effective gene therapies to clarify the mechanisms by which cDNA integrated into cells can maintain or lose its function in vivo. We evaluated genetic and epigenetic events leading to alternation of the introduced CD95 (Fas/Apo-1) gene as a model of gene therapy. Solid tumors formed by CD95 cDNA-transfected hepatoma cells (F6b) were almost completely cured by a single treatment of anti-CD95 monoclonal antibody (mAb) but recurred in gld/gld lpr/lpr mice after initial complete response. Recurred tumors were resistant to repeated mAb treatment. The ratio of resistant cells in tumors was estimated as 4.2 cells per 10(6) cells. The CD95-resistant tumor contained CD95-vanished and CD95-decreased cells. CD95-vanished cells were due to the deletion of CD95cDNA. However, CD95-decreased cells retained CD95cDNA, which was highly methylated when determined with methylation-dependent enzymes and a demethylation reagent, indicating that DNA methylation was responsible for the reduced CD95 expression and resistance to mAb. CD95-decreased cells reduced the CD95 expression further but did not delete cDNA after a second in vivo treatment with anti-CD95 mAb, suggesting that the elimination of cDNA is not induced after its methylation and that cells containing methylated genes became more resistant by further methylation. Thus, the elimination and methylation of integrated cDNA appear to occur through different mechanisms. Our study of resistant tumor cells, which arose by both mutational and epigenetic modifications of the introduced CD95 plasmid, provides important and fundamental information about the fate of introduced cDNA, augmenting the efficiency of gene therapy.     

Apoptosis and Survival in High-grade Astrocytomas as Related to Tumor Fas (APO-1/CD95) Expression

Although a majority of high-grade gliomas express the apoptosis-inducing receptor Fas, little is known about the extent of apoptosis or prognostic significance of Fas expression in these tumors. In situ labeling of apoptotic cells and Ki-67 immunohistochemistry were performed on 51 high-grade human astrocytomas previously characterized for Fas expression. Survival data was compiled from patient records and correlated with tumor grade, apoptotic index (AI) and Fas expression. A significant correlation was found between tumor grade and the AI and Ki-67 labeling index (LI); however, only the AI increased significantly with Fas expression. The AI increased from 0.39 ± 0.12% to 0.82 ± 0.10% in grade III vs. IV astrocytomas (P = 0.003). The Ki-67-LI increased from 3.64 ± 1.5% to 11.35 ± 2.1% in grade III vs. IV astrocytomas (P = 0.004). Additionally, tumors expressing higher Fas levels had a greater AI than those expressing lower levels (0.81 ± 0.11% vs. 0.43 ± 0.11%) (P = 0.017). Despite longer median survivals for patients with tumors exhibiting high Fas expression, statistical significance was not achieved. Patients with grade III astrocytomas demonstrated a median survival of 20 vs. 18 months for tumors with high vs. low Fas expression (P = 0.51). Patients with grade IV astrocytomas demonstrated a median survival of 9 vs. 7.4 months for tumors with high vs. low Fas expression, respectively (P = 0.77). Although the degree of Fas expression in high-grade astrocytomas appears to correlate with the apoptotic rate, no overall differences in survival could be demonstrated between tumors expressing high vs. low Fas levels.     

Arsenic trioxide sensitizes CD95/Fas-induced apoptosis through ROS-mediated upregulation of CD95/Fas by NF-kappaB activation

CD95/Fas is a cell surface protein that belongs to the tumor necrosis factor receptor family. Signals through CD95/Fas are able to induce apoptosis in sensitive cells. Therefore, modalities to regulate the CD95/Fas expression level in tumor cells are called for. In the present study, we show that sublethal doses of arsenic trioxide (As2O3) sensitized CD95/Fas-induced apoptosis in human cervical cancer cells, and the sensitizing effects resulted from As2O3-mediated increase in the expression of the CD95/Fas. N-acetyl-L-cysteine, a specific scavenger of reactive oxygen species, abrogated As2O3-induced upregulation of CD95/Fas and enhancement of CD95/Fas-mediated apoptosis. Furthermore, inhibition of NF-kappaB by transient transfection of IkappaBalpha supersurppessor blocked the increase of CD95/Fas expression following As2O2 treatment. Antisense oligonucleotide of CD95/Fas and ZB4, an antibody that blocks the binding of CD95/Fas ligand to CD95/Fas, reduced the amount of As2O3-sensitized CD95/Fas-induced apoptosis, demonstrating the specificity of CD95/Fas-binding ligands in the As2O3-sensitized CD95/Fas-induced apoptosis. These findings demonstrate that sensitization of human cervical cancer cells to CD95/Fas-mediated apoptosis by As2O3 can be partly due to induction of ROS and subsequent upregulation of CD95/Fas gene expression by NF-kappaB activation.     

乳腺癌MCF-7/Adr耐药细胞CD95分子的多态性表达和功能缺陷

目的：观察ＭＣＦ－１／Ａｄｔ耐药细胞ＣＤ９５分子的表达和功能及其与药敏细胞的差异，初步探讨ＣＤ９５缺陷与多药耐药的潜在关系。方法：对乳腺癌ＭＣＦ－７／Ａｄｔ耐药细胞的ＭＣＦ－７药物敏感细胞进行对比研究。流式细胞仪检测ＣＤ９５蛋白水平，ＲＴ－ＰＣＲ分析ＣＤ９５ ｍＲＮＡ转录体的多态性。碘化内啶染色和ＤＮＡ琼脂糖凝胶电泳观测抗ＣＤ９５单克隆抗体对ＭＣＦ－７／Ａｄｔ耐药细胞的诱导凋亡效应。结果：ＭＣＦ－     

Roles of CXC chemokines and macrophages in the recruitment of inflammatory cells and tumor rejection induced by Fas/Apo-1 (CD95) ligand-expressing tumor

The role of CD95 ligand (FasL/Apo-1L)-expressing tumors in immunosuppression or immunopotentiation is controversial. CD95L-transfected tumors induce immunopotentiation after vigorous neutrophil infiltration. Thus, the induction of neutrophil infiltration by CD95L seems to play an important role in tumor rejection. The mechanism by which CD95L-expressing tumors cause neutrophil infiltration and antitumor immunity has not been well understood. CXC chemokine receptor 2 (CXCR2) knockout (KO) mice are a powerful tool for studying CXC chemokine-mediated neutrophil infiltration. We investigated the roles of CD95L and chemokines in CD95L-induced antitumor activity by using CXCR2 KO mice and CD95LcDNA-transfected MethA (MethA + CD95L) fibrosarcoma. MethA + CD95L cells were completely rejected in wild-type (WT) and even in KO mice. MethA + CD95L cells injected intraperitoneally (i.p.) induced the recruitment of both neutrophils and macrophages in WT but only macrophages in KO mice, although CXC and CC chemokines were released in both mice. Macrophages incubated with MethA + CD95L cells released CXC and CC chemokines. Macrophages derived from WT and KO but not neutrophils from WT mice induced the recruitment of neutrophils when adoptively i.p. transferred with MethA + CD95L cells into CD95L/CD95-deficient mice. The different recruitment of inflammatory cells between WT and KO mice was attributed to bone marrow (BM) cells by BM transfer experiment. Our results demonstrated that CXC chemokines are essential for neutrophil recruitment and that macrophages but not neutrophils play a critical role in the CD95L-induced infiltration of inflammatory cells and the eradication of CD95L-expressing tumor cells.     

Alterations in Fas (CD 95/Apo-1) and Fas ligand (CD178) expression in acute promyelocytic leukemia during treatment with ATRA

Over the recent years, treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) has become a widely accepted therapeutic regimen and is considered a model of differentiation therapy in malignant diseases. However, the role of ATRA treatment beyond that of the induction of differentiation of leukemic blasts is still far from being fully understood. Data from in vivo and in vitro studies have demonstrated that during ATRA treatment of APL there are significant changes not only in the expression of the apoptotic molecules Fas and Fas ligand, but also in the expression of other molecules involved both in the regulation of apoptosis and in interactions between host immune and leukemia cells. These effects may thus contribute, at least in part, to the beneficial effects of ATRA therapy in APL. In this report we review the current status of studies that contribute to our understanding of treatment with ATRA. We focus on ATRA-induced changes in apoptotic pathways, particularly as it relates to the Fas/Fas ligand system.     

Altered Expression of Fas (APO-1, CD95) and Fas Ligand in the Liver of Mice Infected with Schistosoma Japonicum and Schistosoma Mansoni: Implications for Liver Carcinogenesis

Epidemiological studies have associated infection with Schistosoma japonicum and S. mansoni with increased ‍risk of cancers of the liver and colon, although the mechanism of carcinogenesis remains unestablished. The livers of ‍mice experimentally infected with S. japonicum or S. mansoni were analysed for expression of Fas (CD95/APO-1) ‍and Fas ligand immunohistochemically and for apoptotic cell death by the TUNEL method. Fas expression was ‍significantly decreased in hepatocytes around the inflammatory area of egg granulomas; this decrease was most ‍prominent during the chronic phase of infection. Fas ligand was expressed in hepatocytes inside and outside ‍granulomas, especially from mice at the early stage of infection, but not in hepatocytes from uninfected mice. Abnormal ‍proliferation and Fas ligand expression were also observed in capillary bile ducts inside egg granulomas. Apoptotic ‍cell death of inflammatory cells and, less frequently, of hepatocytes was found both inside and around granulomas. ‍The rate of apoptotic cell death was high during the early stage of infection with S. mansoni, but decreased during ‍the chronic phase of infection. Our results suggest that schistosome infection causes alterations of the Fas-Fas ‍ligand system, one of the major apoptotic pathways, in mouse liver.     

Soluble Fas (APO-1/CD95) isoform in adult T-cell leukemia

Fas (APO-1/CD95) consists mainly of 2 isoforms, membrane-anchored (mFas) and soluble (sFas), both of which can mediate apoptosis through the Fas-signalling process, not only in normal but also in leukemia T-cells. This suggests that aberrant expression of either mFas or sFas may affect the natural history of T-cell neoplasms, such as adult T-cell leukemia (ATL). For studying the tumor biology related to Fas-mediated apoptosis, ATL cells with up-regulated Fas proteins and its mRNAs are convenient and useful for understanding apoptotic oncology as it occurs in nature. Most attention, so far, has been focused on mFas, and little is known about neoplasms from the viewpoint of sFas. Accordingly, we herein review and discuss the biological and clinical implications of sFas in ATL.     

Immunohistochemical detection of CD 95 (Fas) & Fas ligand (Fas-L) in plasma cells of multiple myeloma and its correlation with survival

Multiple myeloma (MM) is a malignant disease resulting from an uncontrolled proliferation of a neoplastic plasma cell clone in the bone marrow, which might also be induced by the loss of control on apoptosis. Fas ligand (Fas-L), a member of the tumor necrosis factor family, induces apoptosis mediated via its transmembrane death receptor Fas (Apo-1/CD95) antigen. In the present study, immunostaining was performed on the initial diagnostic bone marrow biopsies of 36 MM patients (1 stage I, 5 stage II, 30 stage III), to evaluate the distribution of Fas receptor and Fas-L on malignant plasma cells. Both Fas and Fas-L were positive in 13 cases and negative in 3, whereas 10 cases were Fas-negative, Fas-L-positive and 10 were Fas-positive, Fas-L-negative. Although no association was found between the expression of Fas receptor or Fas-L and overall survival, Fas-L positivity was significantly associated with a shorter event-free survival (p = 0.0335). In this study, it has been shown that the expression of Fas-L, in malignant plasma cells of myeloma patients significantly shortens the event-free survival, indicating that the defect in apoptosis might be associated with disease progression in MM.     

Expression of Fas (CD95/APO-1) and Fas ligand in lung cancer, its prognostic and predictive relevance.

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.