Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumour activity upon systemic administration in mice without showing the deleterious side effects observed with other apoptosisinducing members of the TNF family such as TNF and CD95L. TRAIL may, thus, have great potential in the treatment of human cancer. However, about 60% of tumour cell lines are not sensitive to TRAIL. To evaluate the mechanisms of tumour resistance to TRAIL, we investigated hepatocellular carcinoma (HCC) cell lines that exhibit differential sensitivity to TRAIL. Pretreatment with chemotherapeutic drugs, for example, 5-fluorouracil (5-FU), rendered the TRAIL-resistant HCC cell lines sensitive to TRAIL-induced apoptosis. Analysis of the TRAIL death-inducing signalling complex (DISC) revealed upregulation of TRAIL-R2. Caspase-8 recruitment to and its activation at the DISC were substantially increased after 5-FU sensitisation, while FADD recruitment remained essentially unchanged. 5-FU pretreatment downregulated cellular FLICE-inhibitory protein (cFLIP) and specific cFLIP downregulation by small interfering RNA was sufficient to sensitise TRAIL-resistant HCC cell lines for TRAIL-induced apoptosis. Thus, a potential mechanism for TRAIL sensitisation by 5-FU is the increased effectiveness of caspase-8 recruitment to and activation at the DISC facilitated by the downregulation of cFLIP and the consequent shift in the ratio of caspase-8 to cFLIP at the DISC.

Apoptosis of activated peripheral T cells during the termination phase of an immune response is critical to maintain T-cell homeostasis. Activated T cells can be removed by two mechanisms: activation-induced cell death (AICD) and death by neglect. AICD is triggered by death receptors, whereas death by neglect is induced by cytokine withdrawal. CD95 (APO-1/Fas) belongs to the subfamily of death receptors and plays a major role in AICD. In this review, we focus on the molecular mechanisms of AICD, in particular those involving the CD95 system. Moreover, we discuss the relative contribution of AICD and death by neglect to terminate a T-cell immune response. In order to become fully activated, T cells require a second signal provided by antigen-presenting cells. We discuss how these costimulatory signals counteract pro-apoptotic signals and, finally, which signals might protect T cells from death to generate a pool of memory T cells.

IntroductionInduction of apoptosis in cancer cells is one of the strategies of anticancer therapy. Apoptotic cell death can be induced through the extrinsic or the intrinsic signaling pathways that are ultimately coupled to the activation of effector caspases. 1,2 The extrinsic pathway involves ligation of death receptors followed by the formation of the death-inducing-signaling-complex (DISC) and activation of, for example, pro-caspase-8. Caspase-8 activates caspase-3, which cleaves target proteins leading to apoptosis. Intrinsic death stimuli directly or indirectly activate the mitochondrial pathway by inducing release of cytochrome c and formation of a cytosolic multiprotein complex, the apoptosome, composed of Apaf-1 and pro-caspase-9. Caspase-9 is activated at the apoptosome and, in turn, activates pro-caspase-3. This death pathway is largely controlled by the proapoptotic (eg, Bax, Bad, Bid, and Bak) and antiapoptotic (eg Bcl-2 and Bcl-x L ) Bcl-2 family proteins. 1,2 Caspase-8 may also induce cleavage of Bid, which induces the translocation of the proapoptotic Bcl-2 family proteins Bax and/or Bak to the mitochondrial membrane. 3 Many stimuli, such as growth factor deprivation, ionizing radiation, and reactive oxygen species (ROS), may trigger the intrinsic death pathway. Recently, Ca 2ϩ signals have been implicated to play an important role in regulation of cell death and survival. 1 Certain apoptotic stimuli induce release of Ca 2ϩ from stores in the endoplasmic reticulum (ER), which causes Ca 2ϩ overload of the mitochondria leading to the release of cytochrome c as part of a stress response. ROS, such as . O 2 Ϫ and its reduced product H 2 O 2 , have been considered as cytotoxic byproducts of cellular metabolism. Recent evidence indicates that H 2 O 2 can also serve as a signaling molecule to modulate various physiologic functions, including mobilization of intracellular Ca 2ϩ through activation of phospholipase C␥1 (PLC␥1), a key enzyme involved in Ca 2ϩ signaling. [4][5][6] Over the past decades, much effort has been invested into the search for agents that can differentially induce apoptotic death in cancer cells. In recent years, traditional Chinese herbal remedies have gradually gained considerable attention as a new source of anticancer drugs. Although their curative mechanisms are still largely unknown, some of the drugs have been used to treat cancer. 7 Extracts of the radix of the traditional Chinese herb Huang-Qin (Scutellaria baicalensis Georgi) are among the most popular herbal remedies used in China and several oriental countries for clinical treatment of hyperlipemia, atherosclerosis, hypertension, dysentery, common cold, and inflammatory diseases, such as atopic dermatitis. Huang-Qin extracts show low toxicity in different animals (The grand dictionary of Chinese herbs, 1977). The active components of Huang-Qin are confirmed to be flavonoids. Wogonin (5,7-dihydroxy-8-methoxyflavone) is one of the major bioactive flavonoids of Scutellaria baicalensis Georgi, which has been shown to have an...

Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroidderived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodaminewortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.In the post-genomic area, there has been increasing recognition of the need for reagents and tools that can analyze protein expression and activity in complex biological samples. Activitybased protein profiling (ABPP) 1 has emerged as a promising new approach that allows the simultaneous analysis of many proteins, frequently from a common enzymatic family, within the whole proteome (1-4). This approach relies upon the development of bifunctional chemical probes that act in a covalent and site-directed manner with some subset of the proteome and contain a second functional group for either visualization and/or isolation of the probe-labeled proteins. Among the probes that have been developed for ABPP are molecules that target the serine hydrolyases (1, 3, 5, 6), cysteine proteases (7-9), oxidoreductases (10, 11), protein phosphatases (12), and metalloproteases (13). However, probes for many classes of enzymes remain to be identified.Protein and lipid kinases are two important classes of biomedically relevant enzymes for which no cell-permeable ABPP probes exist. Because the expression and activity of many kinases are known to be dysregulated in a variety of diseases (14, 15), proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Moreover, many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization. Thus, these mechanisms can only be detected by examining protein activity, sometimes within the context of a living cell.One important class of kinases involv...

In the early phase of an immune response, T cells are activated and acquire effector functions. Whereas these short term activated T cells are resistant to CD95-mediated apoptosis, activated T cells in prolonged culture are readily sensitive, leading to activation-induced cell death and termination of the immune response. The translation inhibitor, cycloheximide, partially overcomes the apoptosis resistance of short term activated primary human T cells. Using this model we show in this study that sensitization of T cells to apoptosis occurs upstream of mitochondria. Neither death-inducing signaling complex formation nor expression of Bcl-2 proteins is altered in sensitized T cells. Although the caspase-8 inhibitor c-FLIPlong was only slightly down-regulated in sensitized T cells, c-FLIPshort became almost undetectable. This correlated with caspase-8 activation and apoptosis. These data suggest that c-FLIPshort, rather than c-FLIPlong, confers resistance of T cells to CD95-mediated apoptosis in the context of immune responses.

C D95 (APO-1/Fas) belongs to the subfamily of death receptors among the tumor necrosis factor/ nerve growth factor receptor superfamily. 1 CD95 plays an important role in liver homeostasis. 2 Hepatocytes express high amounts of CD95 and are very sensitive toward CD95 triggering. 3,4 Mice injected with agonistic anti-CD95 antibody rapidly die of liver failure. 5 CD95-mediated apoptosis is involved in a broad spectrum of human liver diseases, including acute liver failure. 6 In vivo silencing of the CD95 gene via small, interfering RNA protects mice from liver failure as well as from fibrosis in a model of autoimmune hepatitis. 7 A key event of CD95 signaling is the formation of a multimeric complex of proteins called death-inducing signaling complex (DISC). Two different pathways are described downstream of CD95. In type I cells, the death signal is propagated by a cascade that is initiated by the activation of large amounts of caspase-8 at the DISC and subsequent activation of downstream caspases. 8 In type II cells, including hepatocytes, DISC formation is weak, and the Heidelberg (M.M. and P.K.).

TNF␣ has previously been used in anticancer therapy. However, the therapeutic application of TNF␣ was largely limited due to its general toxicity and the fact that it activates the NF-B-family transcription factors, which are proinflammatory and antiapoptotic. To overcome this problem in vitro, specific NF-B inhibitors or transcription or protein synthesis inhibitors such as actinomycin D and cycloheximide are usually used in combination to increase TNF␣ killing of tumor cells. However, these agents also cause harmful side effects in vivo. We show here that wogonin, derived from the popular Chinese herb Huang-Qin, attenuates NF-B activity by shifting TNF␣-induced free radical ⅐O 2 ؊ to a more reduced nonradical product, H 2 O 2 , and thereby sensitizes TNF␣-resistant leukemia cells to TNF␣-induced apoptosis. Importantly, wogonin does not affect the viability of normal IntroductionTumor necrosis factor-alpha (TNF␣) was isolated in 1985 as the first mammalian protein with cytotoxicity to tumor cells and that induced tumor regression in mice. 1 It is now known that TNF signaling may either induce cellular activation, apoptosis, or necrosis. 2 TNF␣ was thought to be a potent anticancer agent due to its cytotoxicity against a number of tumor cell lines. However, the clinical use of TNF␣ is limited because of its systemic toxicity largely due to activation of the proinflammatory NF-B-family transcription factors. 3-5 NF-B, apart from its proinflammatory activity, is also a negative regulator that antagonizes TNF␣-induced killing. 4,6 Clinically, the only success in TNF␣ therapy has occurred with isolated limb perfusion for a limited subset of susceptible tumors, such as melanoma and sarcoma. 7 In general, TNF␣ is considered a key inducer of proinflammatory genes, and its primary role is to stimulate innate inflammatory responses to fight infections, whereas the function of its proapoptotic capability remains mysterious. 8 Apoptosis involves 2 main pathways: the extrinsic pathway, which is initiated by binding of ligands to specific death receptors on the cell surface, and the intrinsic pathway, which is initiated at the mitochondria. TNF␣ is an inducer of the extrinsic pathway. In mammalians, TNF␣ signals through 2 distinct receptors, TNF receptor 1 (TNF-R1), the primary receptor for soluble TNF␣, and TNF-R2, the main receptor for membrane-associated TNF␣. 5,9 The TNF-R1 signaling pathway can either trigger activation of NF-Bcrucial for TNF␣-mediated immunity, inflammation, and proliferation-or activation of apical caspases as well as the c-Jun-N-terminal kinase (JNK) cascade, which leads to programmed cell death. 2 TNF-R1 signaling can be divided into 2 distinct stages that sequentially activate NF-B and apical caspases. Within a few minutes after binding of TNF␣ to TNF-R1 (stage 1), a signaling complex containing the receptor itself and the adaptor proteins TRADD, TRAF2, and RIP1 but lacking the FAS/APO-1-associated death domain adaptor protein FADD forms and transduces signals that lead to activation of NF-B and t...

The HTLV-1 transactivator protein Tax is essential for malignant transformation of CD4 T cells, ultimately leading to adult T-cell leukemia/lymphoma (ATL). Malignant transformation may involve development of apoptosis resistance. In this study we investigated the molecular mechanisms by which HTLV-1 Tax confers resistance toward CD95-mediated apoptosis. We show that Tax