CC531s colon carcinoma cells induce apoptosis in rat hepatic endothelial cells by the Fas/FasL‐mediated pathway

The mechanisms involved in colorectal carcinoma with liver metastasis are not well known. Metastasizing colon carcinoma cells express more FasL than primary colon carcinoma cells and cancer cells induce apoptosis in hepatocytes by the Fas/FasL pathway. Therefore, this study focused on Fas/FasL expression and functionality in rat liver sinusoidal endothelial cells (LSECs) and CC531s colon carcinoma cells in vitro and in vivo. RT‐PCR and immunochemistry revealed Fas and FasL in LSECs and CC531s, respectively. Functionality of Fas was assessed in vitro by incubation with human recombinant FasL (1–100 ng/ml) with or without enhancer. At concentrations of 10 and 100 ng/ml with enhancer, respectively 21% and 44% of endothelial cells showed signs of apoptosis using Hoechst 33342/propidium iodide staining and electron microscopy. In co‐cultures, apoptosis could be detected in endothelial cells neighboring the CC531s and could be inhibited by an antagonistic FasL antibody. Moreover, 18 h after mesenteric injection of CC531s, the sinusoidal endothelium revealed disruption. In conclusion, (i) CC531s cells induce apoptosis in LSECs in vitro by using Fas/FasL; (ii) CC531s cells damage the sinusoidal endothelial lining in vivo; and (iii) this might provide FasL‐positive tumor cells a gateway towards the hepatocytes.     

Fas ligand down-regulates cytokine-induced Fas receptor expression on insulinoma (NIT-1), but not islet cells, from autoimmune nonobese diabetic mice

In the pathogenesis of autoimmune type 1 diabetes, the apoptosis receptor Fas appears de novo on the surface of insulin-producing beta-cells. Fas expression is thought to be induced by proinflammatory cytokines, such as IL-1beta, interferon-gamma (IFNgamma), and TNFalpha, released by islet-infiltrating mononuclear cells. To determine whether beta-cells can modulate their sensitivity to apoptosis at the level of Fas, we investigated the effect of Fas ligand (FasL) on surface expression of Fas in NIT-1 insulinoma cells from nonobese diabetic (NOD) mice prone to autoimmune diabetes and islet cells from NOD and nonautoimmune BALB/c mice. In NIT-1 insulinoma cells, Fas expression induced by the cytokine combination IL-1beta and IFNgamma was reduced in the presence of FasL, whereas in islet cells Fas expression was unaffected by FasL. The effect of FasL on NIT-1 cells was evident during and after the induction of Fas expression by IL-1beta and IFNgamma. Thus, FasL down-regulates cytokine-induced Fas expression in NOD mouse-derived NIT-1 cells, but not in NOD or BALB/c mouse islets. The ability of NIT-1 cells to down-regulate Fas receptor in response to ligation is similar to that of a variety of tumor cells, which may use this mechanism to escape destruction by cytotoxic T cells. Islets apparently cannot protect themselves against FasL-induced apoptosis by down-regulating the Fas receptor. Understanding how NIT-1 insulinoma cells down-regulate Fas receptor in response to ligation by FasL has therapeutic implications for protecting normal beta-cells in autoimmune type 1 diabetes.     

Stem cell factor increases the expression of FLIP that inhibits IFNgamma -induced apoptosis in human erythroid progenitor cells

Interferon gamma (IFNgamma) acts on human erythroid colony-forming cells (ECFCs) to up-regulate Fas, without a demonstrable change of Fas ligand (FasL) or Fas-associated DD-containing protein (FADD) expression and activates caspase-8 plus caspase-3, which produce apoptosis. Our previous data showed that stem cell factor (SCF) reduced the inhibitory effect of IFNgamma on human ECFCs when both factors were present in the cultures. However, the mechanism by which SCF prevents IFNgamma-induced apoptosis in ECFCs is unclear. In this study we used highly purified human ECFCs to investigate the mechanism of the effect of SCF on IFNgamma-induced apoptosis. Because the binding of FasL to Fas is the first step of the apoptosis cascade and IFNgamma strongly up-regulates Fas expression, we added FasL (50 ng/mL) to the cultures with IFNgamma to accentuate the IFNgamma-induced activation of caspase-8 and caspase-3 plus subsequent apoptosis. SCF (100 ng/mL) clearly inhibited the activation of caspase-8 and caspase-3 induced by IFNgamma and/or FasL, and it also reduced apoptosis as measured by the terminal dUTP nick-end labeling (TUNEL) assay. SCF did not decrease the surface expression of Fas on the ECFCs. FADD-like interleukin 1 beta (IL-1beta)-converting enzyme (FLICE)-inhibitory protein (FLIP) has been reported to interact with FADD and/or caspase-8 at the death-inducing signaling complex (DISC) level following Fas stimulation and acts as a dominant-negative caspase-8. SCF increased FLIP mRNA and protein expression, concomitant with reduced apoptosis, whereas IFNgamma and/or FasL did not change FLIP expression. Reduction of FLIP expression with antisense oligonucleotides decreased the capacity of SCF to inhibit IFNgamma-induced apoptosis, demonstrating a definite role for FLIP in the SCF-induced protection of ECFCs from IFNgamma-initiated apoptosis.     

Suppression of FasL expression in tumor cells and preventing TNF-induced apoptosis was better for immune cells survival

OBJECTIVE: To elucidate if Fas/FasL signal pathway participates in the immune escape of tumor cells, and if contemporarily preventing Fas/FasL and TNF-induced apoptosis is better for immune cells survival than just blocking Fas/FasL-induced apoptotic signal. METHODS: Suppression of FasL expression in mouse H22 hepatocellular cancer cells by siRNA technique. Wild-type Ad5 14.7K gene was amplified by PCR and transduced into Jurkat T cells. Detecting apoptosis of target Jurkat cells by Flow Cytometry. Detection of TNF-alpha in the culture supernatant of H22 cells by ELISA. FasL and 14.7K gene expression in stably transfected or transduced clones were determined by western blotting. RESULTS: FasL expression in H22 cells was down-regulated following stable transfection with a plasmid encoding antisense FasL cDNA. Down-regulation of FasL expression in H22 cells had no effect on tumor growth in vitro. There was an apparent decrease in the number of apoptotic Jurkat T cells following coculture with transfected H22 cells, relative to coculture with FasL-expressing untransfected cells. Compared with untransduced Jurkat cells, apoptotic rates in 14.7K transduced Jurkat cells were significantly reduced in three different E/T ratios (P < 0.01), respectively. CONCLUSIONS: Fas/FasL signal pathway participated in the immune escape of tumor cells by inducing immune cells apoptosis. Reducing the expression of FasL in tumor cells can decrease the apoptotic rate of immune cells. Further blocking of apoptotic signal pathway of immune cells by preventing TNF-induced apoptosis can increase the survival of immune cells.     

大肠癌患者外周血及肿瘤浸润淋巴细胞Fas/FasL的表达

目的　探讨Fas及其配体FasL与大肠癌患者肿瘤局部及全身免疫反应的关系。方法　用流式细胞仪 (FCM )对 40例大肠癌患者外周血淋巴细胞 (PBL)及肿瘤浸润淋巴细胞 (TIL)的Fas及其配体FasL表达进行定性、定量分析 ,以 40例正常人的PBL作对照。结果　大肠癌患者TIL上Fas分子表达率为 16 8% ,PBL上Fas分子表达率为 8 0 % ,正常人PBL上Fas分子表达率为12 6% ,前者表达率明显高于后两者 (P <0 0 5 ) ,大肠癌患者PBL上Fas分子表达明显低于正常人PBL上Fas分子表达 (P <0 0 5 )。结论　Fas分子在大肠癌患者PBL与TIL中的表达不同 ;Fas/FasL在大肠癌患者肿瘤微环境和全身水平均参与了免疫系统与肿瘤的相互作用     

Interferon gamma induces upregulation and activation of caspases 1, 3, and 8 to produce apoptosis in human erythroid progenitor cells

Interferon gamma (IFNgamma) induces apoptosis in purified human erythroid colony-forming cells (ECFC) and inhibits cell growth. Fas (APO-1; CD95) and Fas ligand (FasL) mediate apoptosis induced by IFNgamma, because Fas is significantly upregulated by IFNgamma, whereas Fas ligand is constitutively present in the ECFC and neutralization of FasL greatly reduces the apoptosis. Because conversion of caspases from their dormant proenzyme forms to active enzymes has a critical role in transducing a cascade leading to apoptosis, we performed further studies of the expression and activation of caspases in normal human and IFNgamma-treated day-6 ECFC to better understand the mechanism of IFNgamma action in producing this cell death. RNase protection assays showed that the caspase-1, -2, -6, -8, and -9 mRNAs were upregulated by IFNgamma, whereas the caspase-5 and -7 mRNAs were not increased. Western blots showed that FLICE/caspase-8 was upregulated and activated by 24 hours of incubation with IFNgamma. FADD was not similarly altered by incubation with IFNgamma. Western blots of ICE/caspase-1, which might be required for amplification of the initial FLICE activation signal, showed that pro-ICE expression significantly increased after treatment with IFNgamma for 24 hours and cleavage of pro-ICE also increased. CPP32/apopain/caspase-3, responsible for the proteolytic cleavage of poly (ADP) ribose polymerase (PARP), was also studied and treatment of ECFC with IFNgamma resulted in an increased concentration of caspase-3 by 24 hours and a clear induction of enzyme activation by 48 hours, which was identified by the appearance of its p17-kD peptide fragment. The cleavage of PARP was demonstrated by an obvious increase of the 89-kD PARP cleavage product, which was observed at almost the same time as caspase-3 activation in the IFNgamma-treated cells, whereas untreated ECFC showed little change. Peptide inhibitors of the caspase proteins, DEVD-fmk, DEVD-cho, YVAD-cho, and IETD-fmk, were incubated with the ECFC to obtain further evidence for the involvement of caspases in IFNgamma-induced apoptosis. The activation of FLICE/caspase-8 and CPP32/caspase-3 and cleavage of PARP clearly were inhibited, but the reduction of cell growth due to apoptosis, induced by IFNgamma, was only partially blocked by the presence of the inhibitors. These results indicate that IFNgamma acts on ECFC not only to upregulate Fas, but also to selectively upregulate caspases-1, -3, and -8, which are activated and produce apoptosis, whereas the concentrations of FasL and FADD are not demonstrably changed.     

Role of Fas/FasL pathway in the activation of infiltrating cells in murine acute myocarditis caused by Coxsackievirus B3

OBJECTIVES: This study was designed to investigate the roles of Fas/FasL pathway in myocardial damage in murine acute myocarditis caused by Coxsackie virus B3 (CVB3). BACKGROUND: Cardiac myocyte apoptosis rarely occurs in murine acute myocarditis caused by CVB3. Fas/FasL belong to the tumor necrosis factor receptor/ligand superfamily of costimulatory molecules and are known to play a critical role in the induction of apoptosis, as well as in the cytotoxicty mediated by T-cells and natural killer cells. METHODS: We first analyzed the expression of Fas on cardiac myocytes in vivo and in vitro. Second, we examined the development of myocardial damage, in C3H/He mice treated with an anti-FasL monoclonal antibody (mAb), and in C3H/He-lpr/lpr mice and C3H/He-gld/gld mice infected with CVB3. Third, to investigate the effects of anti-FasL mAb treatment on the activation of the infiltrating cells, we examined the expression of interferon (IFN)-gamma and interleukin (IL)-2 as activation markers in the heart of mice by semiquantitative polymerase chain reaction. RESULTS: Fas was markedly induced on cardiac myocytes with acute myocarditis. Myocardial inflammation was decreased in mice treated with anti-Fas L mAb, C3H/He-lpr/lpr mice and C3H/He-gld/gld mice. Anti-FasL mAb-treatment also decreased the expression of IFN-gamma, IL-2, inducible nitric oxide synthase and CVB3 genomes in myocardial tissue. CONCLUSIONS: Our findings strongly suggested that the Fas/FasL pathway played a critical role in the development of massive myocardial necrosis through activation of infiltrating cells, and raise the possibility of immunotherapy by blocking the Fas/FasL pathway to prevent myocardial damage and improve the prognosis of patients with viral myocarditis.     

Involvement of LFA-1 in hepatic NK cell (pit cell)-mediated cytolysis and apoptosis of colon carcinoma cells.

BACKGROUND/AIMS: Previous studies have shown that hepatic natural killer (NK) cells, also called pit cells, have a higher cytotoxicity against certain tumor cells and have a higher expression of the cell adhesion molecule CD11a as compared with blood NK cells. We further investigated the involvement of the adhesion molecules, reported to be involved in target cell killing by blood NK cells, in pit cell-mediated colon carcinoma cell killing. METHODS: 51Cr-release and DNA fragmentation were used to quantify target cell lysis and apoptosis, respectively. Adhesion of pit cells to CC531s monolayers was quantitated. RESULTS: Flow cytometric analysis showed that pit cells expressed CD2, CD11a, CD18 and CD54. CC531s cells expressed only CD54. Treatment of freshly isolated pit cells with monoclonal antibodies (mAbs) to CD11a and CD18 inhibited not only the pit cell-mediated CC531s cytolysis but also the pit cell-induced apoptosis of CC531s cells. The combination of mAbs to CD11a, CD18 and CD54 further increased the inhibition of pit cell-mediated CC531s cytolysis and apoptosis. Anti-CD2 mAb did not affect these processes. The binding of pit cells to CC531s cells was also inhibited by anti-CD11a, and CD18 mAbs, but not by anti-CD2 mAb. Anti-CD54 mAb reduced the target cell killing and the binding only slightly. CONCLUSIONS: These results indicate that CD11a/CD18 (LFA-1) present on pit cells plays an important role in pit cell-mediated target cell adhesion, lysis and apoptosis. This finding might explain why pit cells, which have a higher expression of LFA-1 as compared to blood NK cells, are more cytotoxic against tumor cells as compared to blood NK cells.     

Growth of FasL-bearing tumor cells in syngeneic murine host induces apoptosis and toxicity in Fas(+) organs

In the current study, we investigated whether the growth of FasL-bearing tumor cells would induce apoptosis and toxicity in organs that express high level of Fas. Sera from C57BL/6 +/+ (wild-type) mice injected with syngeneic FasL(+) tumors, LSA, or EL-4, showed significantly higher levels of soluble FasL than that from the nontumor-bearing mice. Furthermore, the soluble FasL was functional inasmuch as the sera from tumor-bearing mice were able to induce apoptosis in Fas(+) but not Fas(-) targets. Histopathologic studies and in situ TUNEL assay to detect apoptosis were carried out in C57BL/6 +/+ (Fas(+)) or C57BL/6 lpr/lpr (Fas(-)) mice injected with syngeneic LSA and EL-4 tumor cells. The morphology of the liver and thymus from tumor bearing C57BL/6 +/+ mice showed marked damage and tissue destruction. In contrast, the liver and thymus from tumor-bearing C57BL/6 lpr/lpr mice showed minimal damage. Furthermore, the tumor-bearing C57BL/6 +/+, but not the C57BL/6 lpr/lpr, mice exhibited significant apoptosis in the liver and thymus. The FasL responsible for toxicity was tumor derived rather than host derived; tumor-bearing C57BL/6 gld/gld (FasL-defective) mice also exhibited significant apoptosis in the liver and thymus. Together, these data suggested that the in vivo growth of FasL-bearing tumor cells can induce significant apoptosis and toxicity in Fas(+) tissues of the host. Such toxicity may be mediated by the soluble FasL produced by tumor cells. (Blood. 2000;95:2111-2117)     

幽门螺杆菌感染时Fas/Fas Ligand介导的T细胞致胃上皮细胞损伤

目的：评价幽门螺杆菌（Hp)感染时胃细胞Fas/Fas Lingand的表达,功能及其对胃上皮细胞的损伤作用,以了解Hp的免疫致病机制,方法：免疫组化方法检测Hp阳性及阴性胃黏膜FasLigand的表达情况,RT－PCR检测胃T细胞FasLigand mRNA的表达,生物检测技术（JAM）评价胃T细胞通过Fas/Fas Ligand作用杀伤靶细胞的活性,并对Fas/Fas Ligand介导的胃T细胞及培养上清致胃型上皮凋亡进行了检测,结果：Hp感染胃组织中单核细胞Fas Ligand阳性而Hp阴性者无表达,Hp阳性胃黏膜T细胞系表达Fas Ligand mRNA,而Hp阴性者则不表达或弱表达,胃T细胞的Fas Ligand具有生物学活性,能杀伤Fas阳笥的Jurkat细胞并特异杀伤胃上皮细胞系,结论：通过激活胃浸润T细胞的Fas Ligand表达而损伤胃型上皮是Hp免疫致病的机制之一,其在Hp感染时黏膜免疫选择中的作用尚需进一步探讨。     

Dual response to Fas ligation in human endothelial cells: apoptosis and induction of chemokines,interleukin-8 and monocyte chemoattractant protein-1

BACKGROUND: To maintain the integrity of tissues, endothelial cells play critical roles. Fas ligand (FasL) is well known to deliver a death signal through its receptor, Fas. The Fas/FasL system may concomitantly induce expressions of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) besides triggering apoptosis in endothelial cells. We also investigated whether an inhibitor of caspase-8 (Z-IETD-FMK) does modulate IL-8 and MCP-1 secretion. METHODS AND RESULTS: After treatment with interferon-gamma (IFN-gamma), human recombinant FasL (hr FasL) or Fas agonistic antibody (CH-11) was added to cultured human endothelial cells. IFN-gamma up-regulated Fas mRNA levels. Fas ligation promoted apoptosis assessed by fluorescent-activated cell sorter (FACS) analysis in a dose-dependent manner and induced prominent DNA fragmentation. Simultaneously, IL-8 and MCP-1 were secreted from the endothelial cells in response to hr FasL or CH-11 in a dose-dependent manner (P < 0.01). Fas-neutralizing agent (Fas-Fc) suppressed the Fas-mediated secretions of IL-8 and MCP-1 (P < 0.01) both as well as the Fas-mediated apoptosis. On the other hand, whereas Z-IETD-FMK suppressed apoptosis, the inhibitor enhanced the Fas-mediated secretions of both IL-8 and MCP-1 beyond the value of the Fas stimulation alone (P < 0.01), suggesting an enhanced signalling for the chemokine expression. CONCLUSION: In human endothelial cells, the Fas/FasL system induces both IL-8 and MCP-1 secretions probably via a caspase-8 independent pathway. The Fas/FasL system may amplify the inflammatory cascade in the vascular injury and atherogenesis by recruiting leukocytes at the region of apoptotic endothelial damage.     

HDV/HBV感染树鼩肝组织Fas/FasL表达与肝细胞凋亡

目的 探讨HDV感染树鼩肝组织中Fas/FasL表达与HDV感染之间的关系,以及Fas/FasL在丁型肝炎肝细胞凋亡中的作用。 方法 采用免疫组化和原位杂交技术对45份HDV感染树鼩肝组织中HDAg,Fas/FasL和Fas/FasL mRNA的表达进行了检测;应用原位末端标记技术对肝细胞凋亡进行了检测;并应用免疫组化双重染色对HDAg,Fas/FasL的表达以及肝细胞凋亡进行了检测。 结果 45份肝组织中有39份可检出Fas/FasL(阳性率87％),有41份可检出凋亡细胞(阳性率91％),HDAg表达与Fas/FasL表达之间有显著相关性(X_1~2=29.2,X_2~2=27.9,P<0.05),HDAg表达越强,Fas和FasL表达也越强,凋亡在HDAg阳性和阴性细胞中均可发生,以HDAg阳性细胞发生为主,Fas/FasL表达与肝细胞凋亡之间有显著相关性(X_1~2=35.1,X_2~2=40.2,p<0.05),Fas和FasL表达越强,凋亡阳性细胞越多。 结论 丁型肝炎病毒感染和未感染的肝细胞均可发生凋亡,但凋亡只在少数细胞发生;肝细胞内的病毒抗原表达可诱导Fas/FasL的表达;Fas/FasL肝细胞凋亡中起重要作用。     

Cross-Talk between Fas/Fas ligand system and nitric oxide in the pathway subserving granulosa cell apoptosis: a possible regulatory mechanism for ovarian follicle atresia

Recent studies have shown the involvement of Fas/Fas ligand (FasL) system and nitric oxide (NO) in ovarian follicle atresia. Here we asked whether Fas/Fas ligand system interacts with NO using rat granulosa cell culture. Soluble recombinant Fas ligand (rFasL), at 100 ng/ml, significantly decreased cell viability, as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, in the presence of 200 U/ml interferon-gamma, whereas the concurrent addition of a caspase inhibitor, Z-VAD-FMK, at 20 microm, significantly inhibited rFasL-induced cytotoxicity. Hoechst 33342 staining and flow cytometric analysis confirmed the induction of apoptosis in granulosa cells by 100 ng/ml rFasL in the presence of interferon-gamma, which was blocked by the concomitant addition of an NO donor, S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated that rFasL significantly up-regulated caspase-3, -8, and -9 activities in granulosa cells, which were attenuated by concurrent treatment with S-nitroso-N-acetylpenicillamine. Real-time quantitative RT-PCR revealed a significant decrease in inducible NO synthase mRNA levels in rFasL-induced apoptotic granulosa cells. In conclusion, we demonstrated the involvement of Fas/FasL system in inducing apoptosis through activation of a caspase-mediated cascade in rat granulosa cells, which is coupled with a decrease in inducible NO synthase expression. We further showed that NO inhibited Fas/FasL system-induced apoptosis by suppressing activation of the caspases, pointing to a cross-talk between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in ovarian follicle atresia.     

血吸虫病小鼠肝纤维化肝组织中Fas/FasL的表达

目的 探讨Fas／FasL在血吸虫病肝纤维化肝组织中的表达及意义。 方法 采用免疫组化技术检测血吸虫病小鼠肝纤维化肝组织中Fas／FasL的表达,并对照经吡喹酮、已酮可可碱治疗前后其表达的变化,分析其意义。 结果Fas以肝细胞浆表达为主,FasL主要在浸润淋巴细胞浆、肝细胞核表达。经吡喹酮及高剂量已酮可可碱治疗后其肝组织Fas／FasL表达明显减少(P<0.01)。 结论 血吸虫感染可诱导肝细胞Fas／FasL的表达,增强Fas／FasL途径介导的肝细胞凋亡,这一机制在血吸虫病肝纤维化的发病中可能起重要作用。吡喹酮及高剂量已酮可可碱可明显下调Fas／FasL的表达,抑制肝细胞凋亡,改善肝功能。     

Fas ligand/Fas-mediated apoptosis in human coronary artery smooth muscle cells: therapeutic implications of fratricidal mode of action

OBJECTIVE: This study aimed to determine the mode of action of Fas ligand (FasL)/Fas at mediating apoptosis so as to evaluate the potential of FasL in gene therapy for restenosis. METHODS: Passaged human coronary artery smooth muscle (HCASM) cells were infected with recombinant adenoviral vectors expressing murine FasL. Various parameters of FasL expression and apoptosis were measured using FACS, immunofluorescence, calorimetric, and cytotoxicity assays. RESULTS: Most HCASM cells under normal growth conditions expressed Fas and were shown to be susceptible to membrane bound but not soluble FasL. However, some FasL expressing cells survived for up to 7 days. These surviving cells were observed to be spatially distributed and were not in direct physical contact with each other. Upon examination, it was determined that although the majority of the surviving cells expressed FasL, only 30% expressed both Fas and FasL. These cells were capable of inducing apoptosis of target cells and some were also susceptible to FasL expressing cells, provided that the effector and target cells were in close physical contact. FasL/Fas-mediated apoptosis was inhibited by p35, a baculovirus gene that inhibits caspases. Additionally, in contrast to HCASM cells, neither membrane-bound nor soluble FasL induced apoptosis in coronary artery endothelial cells. CONCLUSIONS: FasL expressing HCASM cells do not undergo FasL/Fas mediated "suicide" but kill neighboring cells bearing Fas in a "fratricidal" manner. A small population of HCASM cells expresses no surface Fas. These results imply that HCASM cells transduced in vivo with FasL may serve as "scavengers" and exert a bystander effect on surrounding cells that may be enhanced by co-expression of p35. As FasL-mediated apoptosis occurs in coronary arterial smooth muscle but not endothelial cells, FasL may also offer an advantage over other genes for use in restenosis since the latter may indiscriminately delay re-endothelialization at the sites of gene.     

Fas/Fas配体在大肠癌发生和免疫逃逸及反击中的作用

目的　探讨Fas、Fas配体 (FasL)在大肠癌发生和免疫逃逸及反击中的作用。方法　应用免疫组织化学染色的方法 ,对 5 2例大肠癌、2 7例大肠腺瘤、2 8例溃疡性结肠炎、30例正常大肠组织及 2 3例大肠癌淋巴结转移癌组织进行Fas和FasL的检测。对 5 2例大肠癌用TUNEL法 ,检测不同FasL表达区肿瘤细胞的凋亡情况。结果　 (1)Fas在不同大肠组织中的表达规律是 :大肠癌中表达最低 ,其次是大肠腺瘤、溃疡性结肠炎 ,表达水平最高的是正常大肠组织。FasL在不同大肠组织中的表达规律与Fas正好相反 ,大肠癌组织中表达最高 ,其次是大肠腺瘤、溃疡性结肠炎 ,正常大肠组织中几乎不表达FasL。 (2 )Fas、FasL的表达与大肠癌的组织学类型无关 (P >0 0 5 ) ,与Dukes分期、分化程度、有无淋巴结转移有关 (P <0 0 5 )。 (3)在同一大肠癌组织切片中 ,FasL的表达不均匀 ;FasL表达阳性区(n =2 5 )肿瘤细胞的凋亡率 (81 2 % )比FasL阴性区 (n =2 5 ) (47 4 % )高 (P <0 0 1)。 (4)在 2 3例大肠癌淋巴结转移癌组织中 ,发现 2 3例转移癌FasL均强阳性表达 ,阳性细胞率均大于 75 %。结论　Fas、FasL在大肠癌发生和免疫逃逸及反击中起重要的作用     

Thymineless death in colon carcinoma cells is mediated via Fas signaling

Fas is expressed constitutively in colonic epithelial cells and is also expressed in colon carcinomas and in cultured colon carcinoma cell lines. However, the potential role of Fas signaling in mediating apoptosis in cells of this type remains unknown. We have developed human colon carcinoma cell models deficient in thymidylate synthase that demonstrate acute (TS⁻ cells) or delayed (Thy4 cells) apoptosis following DNA damage induced by thymineless stress. Complete protection of cells from acute apoptosis and prolongation of delayed apoptosis was obtained following exposure to the NOK-1 monoclonal antibody (inhibitory to Fas signaling) during the period of dThd deprivation. These results suggested that apoptosis induced by thymineless stress was regulated by autocrine signaling via Fas–FasL interactions. Fas expression was high in both TS⁻ and Thy4 cells. However, FasL, undetectable in synchronous cultures, was up-regulated in TS⁻ cells at 48 hr, when cells were undergoing acute apoptosis, and in Thy4 cells at 96 hr, correlating with the delayed onset of thymineless death. FasL expression also correlated with acute apoptosis induced in parental GC₃/cl cells, commencing at 48 hr, following thymidylate synthase inhibition by 5-fluorouracil/leucovorin exposure. Fas-mediated apoptosis induced by the cytotoxic anti-Fas monoclonal antibody CH-11 was inhibited following adenoviral delivery of a Bcl-2 cDNA, and Bcl-2 also protected cells from acute apoptosis induced by dThd deprivation. Taken together, these data demonstrate a functional Fas system in these cultured colon carcinoma cell models, and they demonstrate that Fas–FasL interactions can link DNA damage induced by thymineless stress to the apoptotic machinery of colon carcinoma cells.