重组人可溶性TRAIL的制备及其联合硼替佐米诱导肿瘤细胞的凋亡作用

目的:构建重组人肿瘤坏死因子相关的凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)原核表达质粒p ET-28a(+)-TRAIL114-281,优化蛋白表达和纯化条件,制备重组人可溶性TRAIL并鉴定其活性。方法:使用CCK-8初步验证TRAIL是否具有抑制肿瘤细胞生长的生物活性;将制备的TRAIL单独或联合50 nmol/L硼替佐米应用于H460细胞(对TRAIL敏感)和K562细胞(对TRAIL抵抗)24 h,流式细胞术检测细胞凋亡率,比色法检测caspase-8、-9、-3的活化程度,Western blot分析细胞中Bax、Bcl-2和c FLIP蛋白的表达。流式细胞术检测硼替佐米处理H460细胞和K562细胞24 h后DR4和DR5的表达量变化。结果:制备了具有生物学活性且性质稳定的重组人可溶性TRAIL,且成功诱导H460和K562细胞凋亡。不同浓度TRAIL处理H460细胞后其凋亡率随着TRAIL浓度升高而显著升高(P0.05),但K562细胞凋亡率并未随着TRAIL浓度明显升高。联合用药组的H460和K562细胞凋亡率均显著高于单独用药组(P0.05),凋亡过程中caspase-8、-9、-3均被活化,药物处理组的Bcl-2和c FLIP表达量均比对照组下降,尤其联合用药组表达量下降最为显著(P0.05),而Bax表达量无明显变化。硼替佐米处理H460和K562细胞后DR4和DR5表达量均上调(P0.05)。结论:硼替佐米能协同TRAIL启动内源性凋亡途径诱导H460和K562细胞凋亡,其可能机制是通过上调死亡受体DR4和DR5的表达量、下调抗凋亡蛋白Bcl-2和c FLIP的表达量来实现的。     

Defective antigen presentation by macrophages from mice genetically selected for low antibody response

Mice, selected for high (H) and low (L) antibody responses to xenogeneic erythro-cytes (Biozzi mice, selection I), have been tested for their immune responsiveness to lysozymes. The anti-ring-necked pheasant egg-white lysozyme (REL) and anti-hen egg-white lysozyme (HEL) primary plaque-forming cell responses are 20 to 40-fold lower in L than in H mice. The heterogeneity of anti-HEL antibodies in the secondary response, as analyzed by isoelectric focusing, is highly reduced in L as compared to H mice. The in vitro anti-HEL plaque-forming cell secondary response of (H × L)F₁ lymph node (LN) cells is induced by HEL-pulsed macrophages (M?) from H, but not from L, mice. Therefore, genetic differences affecting antibody responsiveness in H and L mice are expressed at the level of antigen-presenting cells. This genetic defect observed in L cells has been studied in more detail using an antigen-specific, T cell-dependent LN cell proliferative assay. After HEL-complete Freund's adjuvant (CFA) priming, LN cells from H or (H × L)F₁ mice respond to HEL, REL (which is cross-reactive with HEL) and purified protein derivative of tuberculin (PPD), whereas LN cells from L mice do not respond to HEL or REL but proliferate in the presence of PPD. Proliferation of HEL-CFA primed (H × L)F₁ LN cells is induced by HEL- or REL-pulsed M? from H but not from L mice, whereas PPD-pulsed M? from H or L mice give similar proliferative responses. By increasing 50-fold the concentration of HEL used to pulse M? from H and L mice, a comparable proliferative response is obtained when HEL-CFA-primed (H × L)F₁ LN cells are stimulated with HEL-pulsed M? from either line. This finding indicates that the genetic defect at the antigen-presenting cell level in L mice is not absolute, but rather reflects a different antigen handling by M? in the two lines.     

人类肾上皮细胞氧化损伤模型的建立

目的: 采用过氧化氢(H₂O₂)对人类肾脏近端小管上皮细胞系(HKC)进行氧化损伤,建立损伤的细胞模型。方法: 通过检测细胞活力、培养基中的丙二醛(MDA)含量和细胞内超氧化物歧化酶(SOD)活性的变化研究细胞损伤的程度;利用激光共聚焦显微镜观察细胞损伤前后骨桥蛋白(OPN)表达水平的变化;采用扫描电子显微镜(SEM)观察细胞损伤前后形貌和膜表面微结构的变化。结果: 用1 mmol/L H₂O₂作用HKC不同时间后,细胞活力和SOD活性逐渐下降,MDA释放量增加,OPN表达量显著增加并在作用时间为1 h时达到最大值;经损伤后的细胞变得干瘪收缩,且膜表面粗糙,鞭毛、突触大部分断裂脱落。结论: H₂O₂能够明显损伤HKC,细胞损伤后,其活力和膜表面超微结构发生变化,并在膜上表达能黏附草酸钙晶体的OPN。因此,可用1 mmol/L H₂O₂作用HKC 1 h建立损伤模型。     

Involvement of tumor necrosis factor-related apoptosis-inducing ligand and tumor necrosis factor-related apoptosis-inducing ligand receptors in viral hepatic diseases

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor cells, but not in most normal cells. The role of TRAIL in hepatic cell death and hepatic diseases is not well understood. The present study investigated the expression of TRAIL and TRAIL receptors (TRAIL-Rs) in patients with hepatitis C virus infection using immunohistochemistry and examined physiological roles under viral infection in the HepG2 cell line. Staining of TRAIL or TRAIL-Rs was prominent in the cytoplasm and membrane of hepatocytes in the periportal area. Some liver-infiltrating lymphocytes also displayed positive staining for TRAIL. Staining intensity was significantly increased with disease progression, particularly in the periportal area. AdCMVLacZ (Q-BIOgene, Carisbad, Calif) infection was also found to induce apoptosis in HepG2 cells and significantly augment TRAIL-induced apoptosis. Anti-TRAIL antibody significantly inhibited apoptosis induced by AdCMVLacZ infection. Flow cytometry analysis revealed that both TRAIL-R2 and TRAIL were up-regulated on the cell surface of HepG2 cells with AdCMVLacZ infection. Transforming growth factor-beta1 also enhanced TRAIL expression in HepG2 cells. These results indicate that TRAIL/TRAIL-R apoptotic pathways play important roles in the hepatic cell death during viral infection.     

人端粒酶逆转录酶核心启动子调控的人TRAIL蛋白在人卵巢癌细胞中的表达

目的：构建人端粒酶逆转录酶基因核心启动子（hTERT）调控的肿瘤坏死因子相关凋亡诱导配体真核表达载体，并检测TRAIL在转染的卵巢癌细胞SKOV3中的表达情况。方法：利用基因重组方法将TRAIL基因克隆人带有hTERT基因核心启动子pIRES2-EGFP真核表达载体中。获得由hTERT基因核心启动子调控的绿色荧光蛋白和带有效应型TRAIL基因的真核表达载体hTERTpromoter-pIRES2-EGFP-TRAIL。采用脂质体介导的方法将hTERT调控的TRAIL基因真核表达载体转染人卵巢癌SKOV3细胞，采用G418筛选获得阳性克隆；应用免疫细胞化学法、蛋白质印迹分析、流式细胞术（FCM）结合间接免疫荧光、RT—PCR法检测转染前后卵巢癌细胞中外源基因的表达。结果：经酶切鉴定及测序结果证实所构建载体正确。转染细胞中的TRAIL表达明显高于对照组细胞。结论：成功地构建了hTRET基因核心启动子调控的TRAIL基因真核表达载体，并在人卵巢癌细胞系SKOV3中得到稳定表达，为进一步研究其对卵巢癌细胞生物学行为的影响奠定了实验基础。     

TNF相关凋亡诱导配体对肝癌细胞系生物学活性的影响

 目的：探讨新型凋亡分子TNF相关凋亡诱导配体(TRAIL)对人肝癌细胞系的作用及生物学活性的影响。 方法： 免疫细胞化学法检测HepG2细胞表面膜结合型TRAIL的表达，ELISA法检测HepG2细胞分泌型TRAIL的表达。MTT和TUNEL法分别进行细胞毒实验和凋亡的检测。HepG2细胞内端粒酶的活性由TRAP-PCR法检测，端粒酶催化亚单位hTERT的表达由流式细胞术进行检测。 结果： HepG2肝癌细胞系表面组成性表达TRAIL分子，并有适量的sTRAIL呈分泌表达。细胞毒实验显示TRAIL能够显著抑制肝癌细胞的生长，TUNEL检测结果显示TRAIL具有诱导肝癌细胞凋亡的效应。端粒酶的活性检测显示，TRAIL能够有效抑制肝癌细胞系内端粒酶的活性以及端粒酶催化亚单位的表达。 结论： TRAIL是一个有效的抑癌分子，能够通过诱导细胞凋亡、抑制端粒酶活性等途径控制肿瘤细胞的生长和进一步杀伤肿瘤细胞。     

The cyclin-dependent kinase inhibitor flavopiridol sensitizes human hepatocellular carcinoma cells to TRAIL-induced apoptosis

Flavopiridol was one of the first cyclin-dependent kinase inhibitors demonstrated to have an antitumor effect in several cancer types. Here, we investigated the effects of flavopiridol on TNF-related apoptosis-inducing ligand (TRAIL) in the human hepatocellular carcinoma (HCC) cell lines HLE and HepG2, and evaluated the role of flavopiridol in apoptosis. To better understand the mechanism of increased TRAIL sensitivity in HCC cells, we determined the effect of flavopiridol on cell surface expression of TRAIL and TRAIL receptors using flow cytometry analysis. The levels of survivin, FLIP, Bcl-xL and X-chromosome-linked IAP (XIAP) in treated and untreated cells was also determined. Flavopiridol decreased cell viability in a dose-dependent manner in the two HCC cell lines tested. The pan-caspase inhibitor z-VAD-FMK did not inhibit the effect. However, subtoxic levels of flavopiridol dramatically enhanced TRAIL-induced apoptosis in both cells. Flavopiridol up-regulated TRAIL, TRAIL-R1 and TRAIL-R2 in both cell lines. In addition, flavopiridol down-regulated expression of survivin in both cell lines, and expression of FLIP and Bcl-xL were down-regulated in HLE cells. In summary, flavopiridol augmented TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of survivin, FLIP and Bcl-xL. Thus, combining flavopiridol with a TRAIL agonist may prove to be an effective new strategy for treatment of HCC.     

脂联素抗人脐静脉内皮细胞氧化损伤的作用研究

目的:研究脂联素（APN）对过氧化氢(H2O2)诱导人脐静脉内皮细胞 (HUVECs) 脂质过氧化的影响。方法：采用Annexin V-FITC标记后流式细胞检测方法观测H2O2引起的细胞凋亡情况。用硫代巴比妥酸微量法测定丙二醛(MDA),黄嘌呤氧化酶法测总超氧化物歧化酶(SOD),可见光法测过氧化氢酶(CAT）。体外自由基捕捉实验检测APN对超氧阴离子和羟自由基的清除率。结果：APN能明显抑制H2O2引起的细胞凋亡。APN预处理HUVECs后,H2O2（200 μmol/L,6 h）所致的细胞膜脂质过氧化反应明显减弱,MDA产生减少,CAT和SOD活性增加,但仅以APN孵育HUVECs对SOD和CAT活性没有明显的影响。结论：结果表明APN对超氧阴离子和羟自由基有明显的清除效果。APN通过抗脂质过氧化发挥抑制细胞凋亡,保护血管内皮细胞抵抗损伤的作用。     

Heavy water labeling of DNA for measurement of cell proliferation and recruitment during primary murine lymph node responses against model antigens

Lymph node (LN) responses to antigens involve inflammatory lymphocyte recruitment and proliferation of rare antigen-specific precursors; the relative contributions of these processes have not been well quantified. The popliteal LN assay (PLNA), used for immunotoxicity screening, measures LN swelling as a surrogate of antigen-specific immunity, but nonspecific irritants cause false-positive results. Quantification of proliferating cells may improve specificity, but commonly-used biosynthetic labels (e.g., BrdU) have limitations. In vivo labeling with heavy water ((2)H(2)O) is nontoxic and (2)H incorporation into the DNA of dividing cells highly consistent, even in apoptotic microenvironments such as the thymus. Here, we have used continuous (2)H(2)O labeling and GC/MS analysis to quantify the cumulative fraction of recently divided cells (f) in draining LN of mice. Priming of BALB/c mice with model antigens (KLH, DNCB) increased both LN cell counts and f in responding lymphocyte subsets, whereas lymphocyte recruitment to LN by irritants (IFA, DMSO) increased cell counts but had little effect on f. Thus, antigen-driven proliferation (possibly including a bystander component) was reflected in f, whereas LN cellularity was primarily increased by recruitment. Cell counts responded differentially to changes in Ag dose and immunization with IFA, whereas f was unaffected by these variables. GC/MS analysis of (2)H(2)O-labeled lymphocyte DNA affords sensitive, precise measurements of fractional lymphoproliferation. Dissection of proliferation and cell recruitment by this approach may be useful for preclinical in vivo screening of novel adjuvants and immunomodulatory agents, for studying their mechanism of action, and for immunotoxicity screening in the PLNA.     

Protein kinase C mediates the signal for interferon-gamma mRNA expression in cytotoxic T cells after their adhesion to laminin.

A cytotoxic T-cell line, CTLL-2 cells, showed spreading after adhering to extracellular matrix proteins such as fibronectin (FN), laminin (LN) and hyarulonic acid (HA). The adhesion of CTLL-2 cells to LN was mediated by very late activation antigen-6 (VLA-6). Expression of interferon-gamma (IFN-gamma) mRNA was enhanced in CTLL-2 cells, also when they adhered to extracellular matrix proteins; and the enhanced IFN-gamma mRNA expression by adhering to LN was blocked by anti-alpha 6 antibody. Calphostin C, a protein kinase C (PKC) inhibitor, markedly inhibited the enhancement of IFN-gamma mRNA expression in a dose-dependent manner, which suggested that PKC acted as a second messenger in the IFN-gamma mRNA expression mediated by the interaction of VLA-6 with LN in CTLL-2 cells. Furthermore, confocal laser-microscopic analysis and Western blot analysis revealed that PKC-alpha was activated after CTLL-2 cells adhered to LN. PKC activity translocated from the cytosol fraction to the particulate fraction, after CTLL-2 cells adhered to LN. Altogether, we suggest that PKC plays an important role in the signal transduction for IFN-gamma mRNA expression after cytotoxic T cells adhere to LN.     

COX-2 inhibitors sensitize human hepatocellular carcinoma cells to TRAIL-induced apoptosis

Cyclooxygenase (COX)-2 is upregulated in a variety of human cancers, including in hepatocellular carcinoma (HCC), whereas it is undetectable in most normal tissue. Evidence suggests that COX-2 is likely to be involved in hepatocarcinogenesis and, thus, COX-2 may be involved in an early process in carcinogenesis, dedifferentiation. To address this possibility, we investigated the effect of COX-2 inhibitors on TNF-related apoptosis, inducing ligand (TRAIL) sensitivity and its molecular mechanisms, with special attention to anti-apoptotic proteins. We used the highly selective COX-2 inhibitors, NS398 and CAY10404. We also used the MTT assay and cytological analysis of DAPI-stained DNA to assess viability and apoptosis in two HCC cells (SK-Hep1 and HLE). In order to ask what led to increased sensitivity to TRAIL in HCC cells, cell surface expression of TRAIL and TRAIL-receptors was investigated using flow cytometry analysis. Expression of survivin, X-chromosome-linked IAP (XIAP), Bcl-xL, AKT and phospho-AKT was also investigated using immunoblotting. COX-2 inhibitors resulted in a concentration-dependent decrease in cell viability in the two HCC cell lines tested. Subtoxic levels of COX-2 inhibitors did not significantly augment TNFalpha-induced apoptosis but did dramatically enhance TRAIL-induced apoptosis in both cell lines. TRAIL receptor 2/death receptor 5 (TRAIL-R2/DR5) expression was significantly up-regulated in SH-Hep1 and HLE cells. TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) expression was up-regulated only in SK-Hep1. Expression of survivin and Bcl-xL was down-regulated in SK-Hep1 and HLE cells in the presence of CAY10404 but XIAP was not affected. Expression of survivin, Bcl-xL and XIAP was down-regulated in SK-Hep1 cells in the presence of NS398. Survivin expression was also down-regulated in the presence of NS398 in HLE cells. Finally, NS398 also decreased phospho-AKT in SK-Hep1 cells. These results demonstrate that COX-2 inhibitors can induce apoptosis and augment TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of both survivin and AKT signaling.     

Establishment of lymph node metastatic model for human gastric cancer in nude mice and analysis of factors associated with metastasis

The actual mechanisms responsible for lymph node metastasis in gastric cancer are still unclear. To investigate the mechanisms of lymph node metastasis in gastric cancer, we established a lymph node metastatic model for human scirrhous gastric carcinoma. Lymph node metastasis had frequently developed after orthotopic implantation of OCUM-2M LN derived from a scirrhous gastric cancer cell line, OCUM-2M, which had low capacity for lymph node metastasis. We elucidated the different characteristics including binding ability, migratory capacity and immunoresponses induced by the cell surface molecules of these two cell lines. The binding ability to Matrigel and migratory capacity of OCUM-2M LN cells were significantly greater than those of OCUM-2M cells. On flow cytometric analysis, both OCUM-2M and OCUM-2M LN cells strongly expressed HLA-I (99.5 and 97.1%) and LFA-3 (76.6 and 99.2%) in level of expression between the two cell lines, but neither cell line expressed HLA-II (0.0 and 0 .0%), B7-1 (0.0 and 0.0%) or B7-2 (0.4 and 0.3%). ICAM-1 expression in OCUM-2M LN cells was weaker (0.7%) than that in OCUM-2M cells (36.8%). Strong adhesiveness and cytotoxicity of mononuclear lymphocytes for OCUM-2M cells were observed in adhesion and cytotoxic assays, both of which were significantly decreased by the addition of anti-ICAM-1 antibodies. On the other hand, the adhesiveness and cytotoxicity of OCUM-2M LN cells were significantly less than those of OCUM-2M cells, and were not affected by the addition of anti-ICAM-1 antibodies. These findings suggest that decreased ICAM-1 expression in a new gastric cancer cell line with a high rate of lymph node metasta-sis may in turn decrease immune responses mediated through LFA-1-dependent effector cell adhesion, and that this escape from the immunosurveillance system may be one of the factors inducing lymph node metas-tasis. In conclusion, we established a gastric cancer cell line, OCUM-2M LN, with a high rate of lymph node metasta sis. An in vivo lymph node-metastatic model with this cell line should be useful for analysing the mech-anism and therapeutic approach of lymph node metastasis. © Rapid Science Ltd.     

Post-thymectomy autoimmune gastritis: fine specificity and pathogenicity of anti-H/K ATPase-reactive T cells

Thymectomy at day 3 of life (d3Tx) results in the development of organ-specific autoimmunity. We have recently shown that d3Tx BALB/c mice which develop autoimmune gastritis contain CD4+ T cells specific for the gastric parietal cell proton pump, H/K ATPase. Here, we demonstrate that freshly explanted gastric lymph node (LN) cells from d3Tx mice react significantly to the H/K ATPase alpha chain, but only marginally to the beta chain. Two H/K ATPase-reactive T cell lines were derived from the gastric LN of d3Tx mice. Both are CD4+, TCR alpha/beta-, and I-Ad restricted, and recognize distinct peptides from the H/K ATPase alpha chain. One cell line secretes Th1 and the other Th2 cytokines, but both are equally potent in inducing gastritis with distinct profiles of cellular infiltration in nu/nu recipient animals. Neither of the cell lines induced disease in normal BALB/c recipients and transfer of disease to nu/nu recipients was blocked by co-transfer of normal BALB/c spleen cells containing CD4+ CD25+ cells. Although CD4+ CD25+ T cells are thought to emigrate from the thymus after day 3 of life, they could be identified in LN of 2-day-old animals. The capacity of CD4+ CD25+ T cells to abrogate the pathogenic activity in vivo of both activated Th1/Th2 lines strongly suggests that this suppressor T cell population may have a therapeutic role in other models of established autoimmunity. The availability of well-characterized lines of autoantigen-specific T cells should greatly facilitate the analysis of the mechanism of action and target of the CD4+ CD25+ immunoregulatory cells.     

阿司匹林增强TRAIL诱导的HepG-2细胞凋亡

目的:研究阿司匹林能否增强肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导的HepG-2细胞凋亡,并初步探讨其作用机制。方法:HepG-2细胞放入含15%胎牛血清的DMEM培养液中培养,改良苯酚-品红染色观察细胞形态变化,MTT法检测细胞增殖程度,TUNEL法检测细胞凋亡指数,RT-PCR检测细胞中survivinmRNA的表达水平,FCM检测细胞凋亡率和细胞周期。结果:不同浓度的阿司匹林联合TRAIL实验组细胞增殖抑制率明显高于空白对照组及TRAIL和阿司匹林单独用药组,且细胞凋亡指数也明显提高,凋亡率与阿司匹林的浓度呈现剂量依赖关系,联合用药组凋亡相关基因survivin的表达与空白对照组和TRAIL和阿司匹林单用组相比明显下调。结论:阿司匹林能够增强TRAIL诱导的HepG-2细胞凋亡,其作用机制可能与survivin基因的表达下调有关。     

Functional expression of tumor necrosis factor-related apoptosis-inducing ligand in human colonic adenocarcinoma cells

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in various transformed cell lines. Therefore, we investigated TRAIL sensitivity, TRAIL-induced nuclear factor-kappaB (NF-kappaB) activation, and expression of TRAIL in human colonic adenocarcinoma cell lines (HT-29, LS180, SK-CO-1). All four TRAIL receptors (TRAIL-R1 through TRAIL-R4) are expressed in these cell lines. TRAIL sensitivity was assessed by assay of cell viability. Cancer cell viabilities were 83 +/- 3.1% (HT-29), 90 +/- 4.3% (LS180), and 88 +/- 6.3% (SK-CO-1) at 24 hours after the addition of 100 ng/ml TRAIL, indicating that these cell lines were relatively resistant to TRAIL. Activation of NF-kappaB was variably influenced by TRAIL administration, with no consistent tendency among the cell lines, indicating that TRAIL-induced NF-kappaB activation might be cell-type dependent. In contrast, TRAIL was expressed in the human colonic adenocarcinoma cell lines by Western blotting and RT-PCR. Increased expression of TRAIL on tumor cells was observed by flow cytometry after cytokine stimulation (IFN-gamma, TNF-alpha) or the addition of chemotherapeutic agents (camptothecin, doxolubicin hydrochloride). TRAIL on HT-29 cells was functional and able to induce apoptosis in Jurkat cells. Jurkat cell viability was increased by the addition of TRAILR1-R4-Fc. In the presence of various cytokines or chemotherapeutic agents, functional TRAIL is expressed on the surface of tumor cells, and this expressed TRAIL might contribute to tumor immune privilege by inducing apoptosis of activated human lymphocytes.     

Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-α pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-α pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.