5-Aminolevulinic acid-based photodynamic therapy suppressed survival factors and activated proteases for apoptosis in human glioblastoma U87MG cells

Glioblastoma is the most common astrocytic brain tumor in humans. Current therapies for this malignancy are mostly ineffective. Photodynamic therapy (PDT), an exciting treatment strategy based on activation of a photosensitizer, has not yet been extensively explored for treating glioblastoma. We used 5-aminolevulinic acid (5-ALA) as a photosensitizer for PDT to induce apoptosis in human malignant glioblastoma U87MG cells and to understand the underlying molecular mechanisms. Trypan blue dye exclusion test showed a decrease in cell viability after exposure to increasing doses of 5-ALA for 4h followed by PDT with a broad spectrum blue light (400-550 nm) at a dose of 18J/cm(2) for 1h and then incubation at 37 degrees C for 4h. Following 0.5 and 1mM 5-ALA-based PDT (5-ALA-PDT), Wright staining and ApopTag assay showed occurrence of apoptosis morphologically and biochemically, respectively. After 5-ALA-PDT, down regulation of nuclear factor kappa B (NFkappaB) and baculovirus inhibitor-of-apoptosis repeat containing-3 (BIRC-3) protein indicated inhibition of survival signals. Besides, 5-ALA-PDT caused increase in Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF). Activation of calpain, caspase-9, and caspase-3 occurred in course of apoptosis. Calpain and caspase-3 activities cleaved alpha-spectrin at specific sites generating 145kD spectrin breakdown product (SBDP) and 120kD SBDP, respectively. The results suggested that 5-ALA-PDT induced apoptosis in U87MG cells by suppression of survival signals and activation of proteolytic pathways. Thus, 5-ALA-PDT can be an effective strategy for inducing apoptosis in glioblastoma.     

Downregulation of Skp2 and p27/Kip1 synergistically induces apoptosis in T98G glioblastoma cells

S-Phase kinase associated protein (Skp) 2 is an F-box protein required for substrate recognition of the SCF^Skp2 ubiquitin ligase complex. Skp2 is often overexpressed in transformed cells and in various types of tumors. Downregulation or inhibition of Skp2 inhibits growth of breast cancer cells and small-cell lung carcinoma cells. We downregulated Skp2 in T98G glioblastoma cells using small interfering RNA (siRNA). Downregulation induced p27 and caused growth arrest and apoptosis. Downregulation of both Skp2 and p27 increased apoptosis synergistically. Cyclin E levels and cyclin E-CDK2 kinase activity increased dramatically when both Skp2 and p27 were downregulated. Coincidently, Bcl-2 but not Bcl-xL expression decreased, and caspase-3 was activated. Inhibition of cyclin E-CDK2 kinase activity by forced expression of p21 reversed these effects. Moreover, stable expression of Bcl-2 also abrogated apoptosis induced by downregulation of Skp2 and p27. We suggest that Skp2 in tumor cells suppresses apoptosis through Bcl-2 expression, potentially through regulation of cyclin E-CDK2 activity.     

Curcumin induces apoptosis in HCT-116 human colon cancer cells in a p21-independent manner

Several micronutrients present in fruits and vegetables exhibit anticancer activity as a result of their actions on molecular targets involved in carcinogenesis and tumor progression. Curcumin, a phenolic phytochemical derived from the rhizome of Curcuma longa, exhibits both cancer-preventative activity and growth inhibitory effects on neoplastic cells. Several studies report that curcumin inhibits cancer cell proliferation and induces apoptosis in cancer cells through p21-mediated cell cycle arrest. Cancer cells that are deficient in p21 are also reported to be more prone to undergo apoptosis in response to a variety of cytotoxic agents. In this study, we determined whether curcumin-induced cytotoxicity in cultures of HCT-116 human colon cancer cells was dependent on p21 status. Curcumin killed wild-type HCT-116 cells in a dose- and time-dependent manner, as measured in an MTT cell viability assay. Moreover, an equivalent cytotoxic effect by curcumin was observed in both p21^+/+ and p21^−/−HCT-116 cells, indicating that curcumin-induced cytotoxicity was p21-independent. Primary cultures of human dermal fibroblasts were less sensitive than HCT-116 colon cancer cells to lower doses of curcumin, suggesting a degree of selectivity for neoplastic cells. Western blot analysis showed that cell death in curcumin-treated cultures of p21^+/+ and p21^−/− HCT-116 cells was associated with a reduction in pro-caspase-3 and PARP-1 cleavage, which are indicative of apoptosis. We conclude that curcumin-induced apoptosis in HCT-116 colon cancer cells does not depend on p21 status.     

姜黄素类似物B67诱导细胞凋亡和G2/M期 阻滞增强对鼻咽癌辐射抗拒细胞的抑制作用

目的: 研究姜黄素类似物B67对鼻咽癌(NPC)辐射抗拒细胞(CNE-2R)活性和增殖的作用,及其对细胞周期和凋亡的影响。方法: 采用MTT和平板克隆形成实验检测B67对CNE-2R细胞及其母细胞CNE-2活性和增殖的抑制作用,采用荧光显微镜和流式细胞仪观测凋亡细胞的形态学变化、细胞凋亡百分率、细胞周期分布和线粒体膜电位的改变,采用裸鼠皮下成瘤实验检验B67作用于CNE-2R细胞后的成瘤性。 结果: B67作用于NPC细胞24、48和72 h后,MTT检测细胞活性的IC₅₀分别为3.96、2.59和0.89 μmol/L(CNE-2R)以及8.84、3.55和1.10 μmol/L(CNE-2),B67作用48 h后平板克隆形成实验检测NPC细胞增殖的IC₅₀分别为0.55 μmol/L(CNE-2R)和0.73 μmol/L(CNE-2),B67作用24 h后NPC细胞的G₂/M期比例的上升分别为5.32%上升到40.01%(CNE-2R)和9.07%上升到15.73%(CNE-2),B67作用48 h后NPC细胞凋亡率的改变分别为5.49%上升到38.06%(CNE-2R)和4.99%上升到35.74%(CNE-2),B67作用48 h后NPC细胞线粒体膜电位的下降分别为66.76%(CNE-2R)和72.09%(NE-2),B67作用24 h后NPC细胞在裸鼠皮下的成瘤率分别为0%(CNE-2R)以及低浓度组100%、高浓度组0%(CNE-2)。 结论: 姜黄素类似物B67可较特异地诱导CNE-2R细胞G₂/M期阻滞和促进细胞凋亡和线粒体膜电位的改变,引起裸鼠皮下成瘤性的差异,以增强其对鼻咽癌辐射抗拒细胞的抑制作用。     

Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.     

Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane

Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca2+ was detected by fura-2 assay, suggesting activation of Ca2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in 'second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.' Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.     

miR-195过表达促进人神经胶质瘤细胞T98G凋亡

目的检测过表达miR-195对人神经胶质瘤细胞T98G增殖的影响,探讨miRNA-195在胶质瘤发病过程中可能机制。方法用合成的miR-195模拟物,转染miR-195表达水平较低的人神经胶质瘤细胞T98G,用MTT、流式细胞术和TUNEL法分别检测T98G细胞增殖、细胞周期和凋亡,用Western blot检测细胞BCL-2的表达。结果 miR-195模拟物转染T98G后,成熟miR-195明显升高(P<0.05)。在T98G中过表达miR-195后,可明显抑制细胞增殖,过表达miR-195能促进T98G细胞的凋亡和下调细胞BCL-2的表达。结论过表达miR-195能显著促进T98G细胞凋亡,提示miR-195可能对胶质瘤的治疗起作用。     

Morphological and cytochemical properties of the human glioblastoma T98G cells during the monolayer culture

The study was carried out with the aim of determination of morphological and cytochemical properties of human glioblastoma T98G cells in monolayer culture. Using light microscopic and immunocytochemical techniques, the glioblastoma cell line studied was shown to have low susceptibility to the effects of environmental factors and changes in culture conditions as well as high proliferative activity. All T98G cells were demonstrated to have positive nuclear reaction for p53 protein. An ability of T98G cells for spontaneous caspase 3-mediated apoptosis was shown using the antibodies against activated caspase 3. The data obtained suggest that glioblastoma T98G cell line could be used as a convenient model for testing the capacity of natural substances and of pharmaceutical agents that are being developed for the selective induction of caspase 3-mediated apoptosis.     

Death and destruction of activated T lymphocytes

The massive clonal expansion that occurs during an antigen-specific immune response results in the flooding of immune organs with activated T lymphocytes. At the end of a specific response, the vast majority of these activated T cells are cleared from the immune system. The T cells receive signals through specific death receptors that are expressed as a result of activation. Death receptors transmit their apoptotic signals through the activation of caspases. Function of the death receptors is intimately linked to cell-cycle control, and many cell-cycle control proteins are caspase substrates. Among CD8⁺ T cells, apoptotic death occurs at a specific site, the sinusoids of the liver. The liver appears to contain a mechanism for the trapping and killing of activated T cells, rendering it an immunologically privileged site.     

牵张应变诱导人牙周膜细胞凋亡过程中caspase蛋白酶表达的初步研究

目的研究在牵张应变诱导人牙周膜细胞凋亡过程中caspase蛋白酶表达的变化。方法对体外培养的人牙周膜细胞分别施加20%牵张应变6、24 h,采用流式细胞术测定细胞凋亡率,采用免疫印迹技术检测caspase-3、-5、-7、-8、-9的蛋白表达,并采用分光光度比色法测定caspase-3、-5、-8、-9的蛋白酶活性变化。结果 20%牵张应变加载6、24 h可诱导人牙周膜细胞发生凋亡。caspase-3的蛋白表达及蛋白酶活性和caspase-7的蛋白表达在24 h牵张应变作用下较对照组增加,caspase-5、-8、-9的蛋白表达及蛋白酶活性在6、24 h牵张应变作用下较对照组增加。结论 20%牵张应变能诱导体外培养的人牙周膜细胞凋亡,此过程中伴随发生了caspase-3、-5、-7、-8、-9蛋白酶的活化。     

Raloxifene,a selective estrogen receptor modulator,induces mitochondria-mediated apoptosis in human endometrial carcinoma cells

Raloxifene is a nonsteroidal benzothiophene that has also been classified as a selective estrogen receptor modulator (SERM) on the basis of studies in which it produced both estrogen-agonistic effects on bone and lipid metabolism and estrogen-antagonistic effects on uterine endometrium and breast tissue. We investigated apoptotic cell death and the apoptotic pathway in human endometrial carcinoma cells (Ishikawa cells) expressing estrogen receptor treated with raloxifene. Cell viability was significantly decreased in Ishikawa cells treated with raloxifene at 20 microM and higher levels. Raloxifene at 20 microM induced 54% inhibition of cell viability after 48 h treatment. Apoptotic parameters were analyzed for determination of apoptotic pathway in Ishikawa cells treated with 20 microM or 40 microM raloxifene for 48 h. The numbers of apoptotic cells were significantly increased in cells treated with raloxifene as compared with control cells. Activities of caspase-3,-8, and-9 were significantly elevated in Ishikawa cells treated with raloxifene. A significant decrease in mitochondrial membrane potential was observed in this treatment. In addition, the levels of cytosolic cytochrome c were significantly elevated in raloxifene-treated cells. Expression of Bid was detected in both control and raloxifene-treated cells, but Bid cleavage was not observed. In caspase inhibitor experiments, cell viability was significantly increased by the caspase-9 inhibitor z-LEHD-fmk and by the caspase-3 inhibitor z-DEVD-fmk. However, cell viability was unaffected by addition of the caspase-8 inhibitor z-IETD-fmk. Thus, raloxifene induced mitochondria-mediated apoptosis in endometrial cancer cells but not via the Bid-mitochondria pathway. It is possibly that raloxifene may be useful as an adjuvant to current chemotherapies for endometrial cancer and possibly is useful as a chemopreventive agent.     

姜黄素通过ATK/FoxM1信号通路调控胃癌干细胞增殖及凋亡

BACKGROUND:Curcumin has crucial inhibitory effects on various cancer cells and cancer stem cells. However, its effect on gastric cancer stem cells and the underlying mechanism of this effect are unclear. OBJECTIVE:To explore the effect of curcumin on gastric cancer stem cells and the underlying mechanism. METHODS:Tumor sphere-forming assay and gastric cancer stem cell markers (EpCAM and CD44) were used to separate gastric cancer stem cells from gastric cancer SGC7901 cell lines. Effects of curcumin on the proliferation and apoptosis of gastric cancer stem cells were determined by MTT and flow cytometry analysis, respectively. Western blot analysis was used to detect the expression levels of FoxM1, p-AKT, and AKT. LY294002, an inhibitor of the PI3K/AKT pathway, was used to determine the regulatory relationship between AKT and FoxM1 signaling pathways. RESULTS AND CONCLUSION:The EpCAM+/CD44+ gastric cancer stem cells were successfully isolated from SGC7901 cells. MTT assay showed that curcumin inhibited the proliferation of gastric cancer stem cells, while flow cytometry analysis showed that curcumin induced apoptosis in gastric cancer stem cells. In addition, the expression levels of p-AKT and FoxM1 were decreased by curcumin treatment. After being treated by LY294002, the expression levels of p-AKT and FoxM1 were down-regulated markedly. In conclusion, curcumin can inhibit cell proliferation and induce apoptosis in gastric cancer stem cells via the ATK/FoxM1 signaling pathway.     

CD44 promotes resistance to apoptosis in human colon cancer cells

Overexpression of CD44, especially its variant isoforms, occurs consistently in colon cancer, as compared to autologous normal colon, and this change occurs also in most other types of cancer. One of the basic features of malignant transformation is the acquisition of resistance to apoptosis. In this study, we asked whether the expression of CD44 and some of its variant isoforms commonly found in colon cancer participate in resistance to apoptosis and what are the mechanisms involved. A human colon cancer cell line, SW620, which does not express CD44 was stably transfected with standard, v3-10, and v8-10 containing isoforms of CD44. Mock-transfected and CD44-transfected cells were exposed to etoposide to induce apoptosis. Apoptotic and concomitant changes relevant to the mechanisms of apoptosis were monitored by flow cytometry, DNA fragmentation, and immunoblot analyses. It was observed that resistance to apoptosis induced by etoposide is promoted by CD44 expression in SW620, and this resistance is better sustained by the full variant isoform, v3-10. Concomitant alterations in caspase 9, caspase 3, Bcl-xl, and Bak indicated that the resistance to apoptosis in this model involved the mitochondrial pathway. The differential response of CD44 transfectants was associated with a downregulation of pRb and phosphorylated AKT. The results of this study are consistent with the conclusion that expression of variant CD44 isoforms which is characteristic of colon cancer, and most other types of cancer, confers a selective advantage to resist apoptosis, thereby promoting cell transformation into a malignant phenotype, in conjunction with other anti-apoptotic factors.     

Caspase抑制剂联合Cocktail蛋白酶抑制剂对胰岛细胞凋亡的保护

背景：目前关于联合应用Caspase抑制剂及Cocktail蛋白酶抑制剂在胰岛细胞分离纯化过程中对胰岛细胞的影响的研究还较少有报道。 目的：比较Caspase抑制剂及Cocktail蛋白酶抑制剂对胰岛细胞在分离纯化及培养过程中的保护作用。 方法：取新生猪,体外分离、纯化和培养猪胰岛,培养24 h后分为3组：①空白对照组。②消化时加抑制剂组仅在消化过程中加入Caspase抑制剂及Cocktail蛋白酶抑制剂。③消化及培养时加抑制剂组在消化及培养的过程中均加入Caspase抑制剂及Cocktail蛋白酶抑制剂。AO-EB染色定性观察细胞形态及凋亡情况,流式细胞术定量检测细胞活力及凋亡。 结果与结论：空白对照组β细胞所占百分比为66.91%,消化时加抑制剂组为84.58%,消化及培养时加抑制剂组为87.15%;空白对照组活细胞、凋亡细胞及死亡细胞的比例分别为56.52%、16.15%、21.25%,消化时加抑制剂组分别为62.27%、14.66%、14.47%,消化及培养时加抑制剂组分比为73.09%、6.83%、10.28%。结果表明,在消化以及体外培养的过程中均加入Caspase抑制剂及Cocktail蛋白酶抑制剂可明显减少细胞的损失,并且可以增加胰岛β细胞的百分比。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Curcumin induces apoptosis in breast cancer cells and inhibits tumor growth in vitro and in vivo

Curcumin has shown therapeutic and/or adjuvant therapeutic effects on the treatment of some patients with breast cancer. However, its mechanisms of action are largely unknown. This study was designed to investigate its antitumor effect and underlying mechanisms in human breast cancer MDA-MB-231 and MCF-7 cells. The MTT assay was used to evaluate cell viability, and flow cytometry, acridine orange staining and transmission electron microscopy were used to detect apoptosis for cultured cells. The protein expression in cells was evaluated by western blot analysis. Breast tumors were established by subcutaneous injection of MDA-MB-231 cells in nude BALB/c mice, and curcumin was administered to the mice. The size of tumors was monitored and the weight of tumors was examined. The exposure of breast cancer cells to curcumin resulted in growth inhibition and the induction of apoptosis in a dose-dependent manner. We also found that the expression of Bcl-2 protein decreased and the expression of Bax protein increased which lead to an increase of the Bax/Bcl-2 ratio. In mice bearing MDA-MB-231 xenograft tumors, administration of curcumin showed a significant decrease of tumor volumes and tumor weight compared with the control. Our results showed that curcumin exhibited antitumor effects in breast cancer cells with an induction of apoptosis.     

TRAIL/Apo-2-ligand-induced apoptosis in human T cells

Members of the tumor necrosis factor (TNF) family such as CD95 (APO-1/Fas) ligand (L) trigger apoptosis in lymphoid cells. Recently, a new member of apoptosis-inducing ligands, TRAIL (TNF-related-apoptosis-inducing-ligand)/Apo-2 ligand, was identified that might act in a similar way. We compared TRAIL and CD95L-induced apoptosis in human lymphoid cells. Expression of TRAIL was found in CD4+ and CD8+ T cells following activation, suggesting that TRAIL participates in T cell-mediated induction of apoptosis. Similar to CD95L, TRAIL-induced apoptosis in target cells is mediated by activation of caspases (ICE/Ced-3 proteases). However, different human lymphoid cell lines and peripheral T cells differ in sensitivity towards induction of apoptosis by TRAIL and CD95L. In addition, T cells are highly sensitive towards CD95L-induced apoptosis after prolonged activation in vitro, but remain completely resistant to TRAIL-induced apoptosis. In contrast, T cells from HIV-1-infected patients previously shown to exhibit increased CD95 sensitivity are even more susceptible towards TRAIL-induced cell death. These data suggest that TRAIL might participate in CD95-independent apoptosis of lymphoid cells and might be involved in deregulated apoptosis in diseases such as leukemias and HIV-1 infection.     

Dengue infection of monocytic cells activates ER stress pathways, but apoptosis is induced through both extrinsic and intrinsic pathways

Monocytic cells are believed to be an important mediator of the pathology of dengue disease in cases of secondary infection where pre-existing antibodies from a first infection facilitate virus entry to Fc receptor bearing cells. In this study we assessed the induction of the ER stress in response to infection of monocytic U937 cells with all four DENV serotypes as well as the induction of apoptosis. Clear evidence of ER stress and the production of pro-apoptotic signals were observed, together with activation of caspase 9. Surprisingly, caspase 8 was also activated, independently of caspase 9, and this was accompanied by an increase in the expression of TNF-α, suggesting the simultaneous but independent activation of both extrinsic and intrinsic apoptosis pathways. Both the induction of ER stress and apoptosis were shown to be serotype independent.     

Fas-mediated apoptosis is modulated by intracellular glutathione in human T cells

Fas antigen is a member of the tumor necrosis factor receptor family that transduces a lethal signal to the Fas-sensitive cells. We previously established the Fas-resistant variant cell lines LAC2D1R and JKT2D1R from the parental Fas-sensitive cell lines, SUPT13 and Jurkat, respectively. Recently, we isolated the Fas-resistant variant CEM2D1R from CCRF-CEM. All of the variants were Fas⁺ but resistant to Fas-mediated apoptosis. Further biochemical analysis revealed that the intracellular gluthathione (GSH) content of the Fas-resistant variants was higher than in the original cells. When the Fas-resistant variants were incubated with buthionine sulfoximine (BSO) or in GSH-free/cysteine-free medium to deplete GSH, Fas resistance was reversed. Incubation of the cells with cycloheximide also decreased intracellular GSH and reversed the Fas resistance. Furthermore, incubation of activated peripheral blood lymphocytes with BSO enhanced Fas-mediated apoptosis. When the Fas-sensitive cells were incubated with N-acetylcysteine (NAC), intracellular GSH was increased and Fas-mediated apoptosis was blocked. In contrast, Fas-resistant variants, as well as Fas-sensitive cells pre-treated with NAC remained susceptible to allogeneic lymphokine-activated killer cells, most likely due to perforin-dependent killing. The results suggest that Fas-mediated apoptosis, but not perforin-dependent killing, is modulated by intracellular GSH in human T lymphocytes.     

c-FLIP(S) reduces activation of caspase and NF-kappaB pathways and decreases T cell survival

Effective stimulation of NF-kappaB in T cells following TCR ligation requires the activity of caspase-8. The active caspase-8 complex includes the paracaspase, MALT1, and Bcl-10, which connect to the NF-kappaB pathway. It has been less clear what regulates the level of caspase-8 activity during T cell activation. A likely candidate is cellular FLIP (c-FLIP), an enzymatically inert caspase-8 homologue. Two alternatively spliced forms of c-FLIP exist, a long form (c-FLIP(L)) and a short-form (c-FLIP(S)). The latter lacks the C-terminal caspase-like domain. c-FLIP(L) can heterodimerize with and activate caspase-8 through an activation loop in the C terminus of c-FLIP(L). Here we show that, in contrast to c-FLIP(L), c-FLIP(S) inhibits activation of caspase-8 in T cells, and consequently reduces recruitment of MALT1 and Bcl-10 to the active caspase complex. This results in reduced activity of NF-kappaB. Consequently, T cells from c-FLIP(S)-transgenic mice undergo more rapid cell death both spontaneously and after activation. The findings suggest that c-FLIP(S) functions to reduce the expansion of T cells during an immune response.