CD95-mediated Apoptosis Research Articles

610 POSTER Synergistic cytotoxicity, inhibition of Akt and c-Kit phosphorylation and modulation of gene expression by sorafenib and gemcitabine in human pancreatic cancer cells

This study investigates the role of caspase-8 and DN-FADD, an inhibitor of CD95-dependent caspase-8 activation, in gemcitabine-induced apoptosis of Colo357 pancreatic cancer cells. Gemcitabine-mediated apoptosis was monitored by the kinetics of caspase-8 activation and cytochrome c release. Gemcitabine treatment of Colo357 cells increased CD95 surface expression, raising the possibility of the involvement of CD95 in gemcitabine-mediated caspase-8 activation. However, ectopic expression of DN-FADD and treatment of cells with the antagonistic anti-CD95 antibody ZB4 both failed to suppress gemcitabine-induced apoptosis but substantially inhibited CD95-mediated apoptosis. DN-FADD, which surprisingly accumulated in nuclei of Colo357 cells, was unable to block caspase-8 activation mediated by either gemcitabine or CD95. These observations argue against a role of CD95 in gemcitabine-induced caspase-8 activation and reveal that the anti-apoptotic function of DN-FADD differs from caspase-8 inhibition in Colo357 cells.

Loss of Caspase-9 Provides Genetic Evidence for the Type I/II Concept of CD95-mediated Apoptosis

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.

The CD40-induced protection against CD95-mediated apoptosis is associated with a rapid upregulation of anti-apoptotic c-FLIP

CD95/Fas and CD40 receptors are important regulators of cell survival during germinal center reaction. In this study we used a human follicular lymphoma cell line, HF1A3, to study molecular mechanisms of CD95-mediated apoptosis and CD40-induced rescue from apoptosis. CD95 stimulation induced activation of caspase-8 and -3, collapse of mitochondrial membrane potential (Δ Ψm), release of cytochrome c and fragmentation of nuclear DNA. All these apoptotic events were abrogated, when cells were pretreated with CD40 antibodies before CD95 stimulation. CD40 induced a rapid up regulation of both short and long isoforms of c-FLIP, as these proteins were detectable 4 h after receptor stimulation. The induction of c-FLIP as well as the anti-apoptotic function of CD40 was completely abolished when NF-κB activity was inhibited by a selective inhibitor PDTC. We conclude that the anti-apoptotic signaling of CD40 involves NF-κB-mediated induction of c-FLIP proteins which can interfere with caspase-8 activation. However, it remains to be seen whether c-FLIP proteins are the only one ones involved in CD40-mediated protection.

A CD40–CD95L fusion protein interferes with CD40L-induced prosurvival signaling and allows membrane CD40L-restricted activation of CD95

We analyzed a novel bifunctional fusion protein, CD40ed-CD95Led, consisting amino-terminally of the extracellular domain of CD40 and carboxy-terminally of the extracellular domain of CD95L. On cells lacking CD40L, this fusion protein is poorly active with respect to CD95 activation [median effective dose (ED50)>1 microg/ml], but it stimulates CD95 signaling with high efficiency upon binding to membrane-expressed CD40L (ED50<1 ng/ml). Thus, cell surface immobilization mediated by the CD40 part of the molecule unmasks the high-latent, CD95-stimulating capacity of the otherwise poorly active CD95L fusion protein. Moreover, interaction of the CD40 part of CD40ed-CD95Led with CD40L prevents the activation of cellular CD40. The CD40ed-CD95Led fusion protein therefore simultaneously blocks antiapoptotic CD40 activation and induces CD95-mediated apoptosis. Indeed, T47D cells displaying an antiapoptotic autocrine CD40-CD40L signaling loop were significantly more sensitive toward CD40ed-CD95Led than toward soluble CD95L artificially activated by crosslinking. Fusion proteins of RANK and CD95L (RANKed-CD95Led) and CD40 and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) (CD40ed-TRAILed), with domain architectures similar to CD40ed-Cd95Led, displayed RANKL-dependent CD95 and CD40L-dependent TRAILR2 activation, respectively, indicating the principle feasibility of this fusion protein design.

HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP)

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-expressing T-cell lines derived from HTLV-1-infected patients express elevated levels of c-FLIP(L) and c-FLIP(S). The levels of c-FLIP correlated with resistance toward CD95-mediated apoptosis. Using an inducible system we demonstrated that both resistance toward CD95-mediated apoptosis and induction of c-FLIP are dependent on Tax. In addition, analysis of early cleavage of the BH3-only Bcl-2 family member Bid, a direct caspase-8 substrate, revealed that apoptosis is inhibited at a CD95 death receptor proximal level in Tax-expressing cells. Finally, using siRNA we directly showed that c-FLIP confers Tax-mediated resistance toward CD95-mediated apoptosis. In conclusion, our data suggest an important mechanism by which expression of HTLV-1 Tax may lead to immune escape of infected T cells and, thus, to persistent infection and transformation.

Constitutive Activation of Extracellular Signal-Regulated Kinase Predisposes Diffuse Large B-Cell Lymphoma Cell Lines to CD40-Mediated Cell Death

CD40 promotes survival, proliferation, and differentiation of normal B cells but can cause activation-induced cell death in malignant B lymphocytes. CD40 ligand and anti-CD40 antibodies have been used successfully to induce apoptosis in lymphoma lines both in vitro and in xenograft tumor models. Although this makes CD40 an attractive target for antitumor therapies, the response of malignant B cells to CD40 signaling is variable, and CD40 stimulation can enhance proliferation and can increase chemoresistance in some cell lines. It would therefore be useful to identify markers that predict whether a specific cell line or tumor will undergo apoptosis when stimulated with CD40 and to identify targets downstream of CD40 that affect only the apoptotic arm of CD40 signaling. We have analyzed gene expression patterns in CD40-sensitive and CD40-resistant diffuse large B-cell lymphoma (DLBCL) cell lines to identify signaling pathways that are involved in CD40-mediated apoptosis. CD40-resistant lines expressed pre-B-cell markers, including RAG and VPREB, whereas CD40-sensitive cells resembled mature B cells and expressed higher levels of transcripts encoding several members of the CD40 signaling pathway, including LCK and VAV. In addition, CD40-sensitive DLBCL cell lines also displayed constitutive activation of extracellular signal-regulated kinase (ERK) and failed to undergo apoptosis when ERK phosphorylation was inhibited. In contrast, CD40-resistant lines showed no constitutive activation of ERK and no increase in ERK activity in response to CD40 stimulation. Our results suggest that constitutive activation of ERK may be required for death signaling by CD40.

Nitric Oxide Promotes Resistance to Tumor Suppression by CTLs

Many human tumors express inducible NO synthetase (NOS2), but the roles of NO in tumor development are not fully elucidated. An important step during tumor development is the acquisition of apoptosis resistance. We investigated the dose-dependent effects of endogenously produced NO on apoptosis using ecdysone-inducible NOS2 cell lines. Our results show that short-term NOS2 expression enhances CD95-mediated apoptosis and T cell cytotoxicity dose dependently. Furthermore, we could show that during chronic exposure to NO, besides the primary cytotoxic NO effect, there is selection of cell clones resistant to NO that show cross-resistance to CD95-induced apoptosis and the killing by CTLs. We propose that NO production could initially act as an autocrine suicide or paracrine killing mechanism in cells undergoing malignant transformation. However, once failed, the outcome is fatal. NO promotes tumor formation by enhancing the selection of cells that can evade immune attack by acquiring apoptosis resistance.

EBV latency III immortalization program sensitizes B cells to induction of CD95-mediated apoptosis via LMP1: role of NF-κB, STAT1, and p53

Epstein-Barr virus (EBV) induces CD95 expression and the CD95 gene (FAS) is regulated by NF-kappaB, STAT1, and/or p53. To understand the contribution of these factors in the regulation of CD95 by EBV in lymphoblastoid cell lines (LCLs), we cloned dominant-active IkappaBalpha, active (STAT1alpha) and inactive (STAT1beta) forms of STAT1, p53, a dominant-negative mutant of LMP1, and wild-type LMP1 into a novel double-inducible episomal vector, pRT-1. These plasmids were stably transfected either into wild-type LCLs or EREB2-5 cells, an LCL with an estrogen-regulatable EBNA2 protein. Inhibition of LMP1 signaling decreased expression of CD95, whereas overexpression of LMP1 markedly increased it. Induction of the latency III program in EREB2-5 cells correlated with activation of NF-kappaB, STAT1, and p53. CD95 expression was regulated by these 3 transcriptional systems. STAT1 and p53 activation were secondary to NF-kappaB activation. CD95 surface expression sensitized EBV-infected B cells to the induction of CD95-mediated apoptosis. In vitro inhibition of CD95-CD95 ligand interaction was found to reverse T-cell killing of EBV-infected B cells. Therefore, LMP1 activation of NF-kappaB sensitizes infected B cells to CD95-mediated apoptosis and renders EBV latency III-immortalized B cells susceptible to elimination by the immune system, contributing to the establishment of a host/virus equilibrium.

The role of receptor internalization in CD95 signaling

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-kappaB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.

HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP)

HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP)

Autocrine Production of Interleukin-4 and Interleukin-10 Is Required for Survival and Growth of Thyroid Cancer Cells

Although CD95 and its ligand are expressed in thyroid cancer, the tumor cell mass does not seem to be affected by such expression. We have recently shown that thyroid carcinomas produce interleukin (IL)-4 and IL-10, which promote resistance to chemotherapy through the up-regulation of Bcl-xL. Here, we show that freshly purified thyroid cancer cells were completely refractory to CD95-induced apoptosis despite the consistent expression of Fas-associated death domain and caspase-8. The analysis of potential molecules able to prevent caspase-8 activation in thyroid cancer cells revealed a remarkable up-regulation of cellular FLIP(L) (cFLIP(L)) and PED/PEA-15, two antiapoptotic proteins whose exogenous expression in normal thyrocytes inhibited the death-inducing signaling complex of CD95. Additionally, small interfering RNA FLIP and PED antisense sensitized thyroid cancer cells to CD95-mediated apoptosis. Exposure of normal thyrocytes to IL-4 and IL-10 potently up-regulated cFLIP and PED/PEA-15, suggesting that these cytokines are responsible for thyroid cancer cell resistance to CD95 stimulation. Moreover, treatment with neutralizing antibodies against IL-4 and IL-10 or exogenous expression of suppressor of cytokine signaling-1 of thyroid cancer cells resulted in cFLIP and PED/PEA-15 down-regulation and CD95 sensitization. More importantly, prolonged IL-4 and IL-10 neutralization induced cancer cell growth inhibition and apoptosis, which were prevented by blocking antibodies against CD95 ligand. Altogether, autocrine production of IL-4 and IL-10 neutralizes CD95-generated signals and allows survival and growth of thyroid cancer cells. Thus, IL-4 and IL-10 may represent key targets for the treatment of thyroid cancer.

A Functional Role of Flip in Conferring Resistance of Crohn’s Disease Lamina Propria Lymphocytes to FAS-Mediated Apoptosis

There is evidence that, in Crohn's disease (CD), lamina propria T lymphocytes (LPLs) are resistant to FAS-mediated apoptosis and that this defect contributes to the mucosal T-cell accumulation. In this study we examined the functional role of Flip, a Flice inhibitor protein, in the resistance of CD LPL to FAS-mediated apoptosis. Biopsy specimens and LPLs were taken from CD and ulcerative colitis (UC) patients and normal controls and analyzed for Flip by Western blotting. We also examined whether inhibition of Flip by antisense oligonucleotide restored the susceptibility of CD LPLs to FAS-induced apoptosis. LPL apoptosis was assessed by flow cytometry. After FAS stimulation, the rate of apoptosis of CD3+ LPLs was higher in normal controls and patients with UC than in patients with CD. Enhanced expression of both long and short Flip isoforms was seen in biopsy specimens and purified CD3+ and CD45RO+ LPLs of CD patients in comparison with UC patients and normal controls. No increase in Flip was documented in untreated celiac disease mucosa, thus suggesting the possibility that induction of Flip in the gut does not simply rely on the ongoing inflammation. Finally, we showed that inhibition of Flip by antisense oligonucleotide reverted the resistance of CD LPLs to FAS-induced apoptosis. Data suggest a role for Flip in the resistance of CD LPLs to FAS-mediated apoptosis.

A novel mechanism of CD40-induced apoptosis of carcinoma cells involving TRAF3 and JNK/AP-1 activation

Membrane-presented CD40 agonists can induce apoptosis in carcinoma, but not normal homologous epithelial cells, whereas soluble agonists are growth inhibitory but not proapoptotic unless protein synthesis is blocked. Here we demonstrate that membrane-presented CD40 ligand (CD154) (mCD40L), but not soluble agonists, triggers cell death in malignant human urothelial cells via a direct mechanism involving rapid upregulation of TNFR-associated factor (TRAF)3 protein, without concomitant upregulation of TRAF3 mRNA, followed by activation of the c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway and induction of the caspase-9/caspase-3-associated intrinsic apoptotic machinery. TRAF3 knockdown abrogated JNK/AP-1 activation and prevented CD40-mediated apoptosis, whereas restoration of CD40 expression in CD40-negative carcinoma cells restored apoptotic susceptibility via the TRAF3/AP-1-dependent mechanism. In normal human urothelial cells, mCD40L did not trigger apoptosis, but induced rapid downregulation of TRAF2 and 3, thereby paralleling the situation in B-lymphocytes. Thus, TRAF3 stabilization, JNK activation and caspase-9 induction define a novel pathway of CD40-mediated apoptosis in carcinoma cells.

Inhibition of CD95-Mediated Apoptosis through β1 Integrin in the HSG Epithelial Cell Line

The HSG cell line serves as a model for salivary gland epithelial progenitor cell differentiation. In order for a progenitor cell to differentiate, the cell must maintain viability within its niche. Studies were designed to elucidate the mechanism for integrin-mediated HSG cell survival. HSG cells, grown on Matrigel, were resistant to CD95-mediated apoptosis. Western blot analysis showed that Matrigel induced the expression of bcl-2, bcl-xL, p63, and DeltaNp63. This induction occurred by as early as 2 hrs and remained for 24 hrs. CD95-mediated apoptosis resistance was dependent, however, upon the expression of the bcl-2 family. Furthermore, Matrigel induced bcl-2 family expression was dependent on the transactivation of the EGF receptor pathway since PD98059 and AG1478 inhibited Matrigel induced bcl-2 family expression and caused HSG cells to be sensitive to CD95-mediated apoptosis. Activation of the EGF receptor pathway, by itself, however, was not sufficient to inhibit apoptosis. Blocking antibody showed that bcl-2 family expression was mediated through beta1 integrin. These studies show that salivary progenitor epithelial cell survival is integrin dependent and involves the transactivation of the EGF receptor pathway.

Histone deacetylase inhibition by valproic acid down-regulates c-FLIP/CASH and sensitizes hepatoma cells towards CD95- and TRAIL receptor-mediated apoptosis and chemotherapy

Hepatocellular carcinoma (HCC) is highly resistant to chemotherapy, leading to a poor prognosis of advanced disease. Inhibitors of histone deacetylase (HDACi) induce re-differentiation in tumor cells and thereby re-establish sensitivity towards apoptotic stimuli. HDACi are entering the clinical stage of tumor treatment, and several substances are currently being tested in clinical trials to prove their efficacy in the treatment of leukemias and solid tumors. In this study, we investigated the impact of the HDACi valproic acid (VA) on TRAIL- and CD95-mediated apoptosis in hepatoma cells, as well as its sensitizing effect on a chemotherapeutic agent. Treatment of HepG2 cells with VA increased sensitivity to CD95-mediated apoptosis (4% apoptosis vs. 42%), and treatment with epirubicin (74% vs. 90% viability). Caspase-3 activity was significantly enhanced in cells treated with VA plus anti-CD95 antibodies compared to cells treated with antibodies alone. In parallel, VA strongly augmented the effect of TNF-related apoptosis-inducing ligand (TRAIL or Apo2 ligand) on HepG2 cells (10% vs. 58% apoptosis). VA induced down-regulation of cellular FLICE-inhibitory protein (c-FLIP/CASH, also known as Casper/iFLICE/FLAME-1/CLARP/MRIT/usurpin), providing a possible molecular mechanism underlying the increased sensitivity towards cell death-mediated apoptosis. HDAC inhibitors are a promising class for the treatment of leukemias. In addition, among other class members, VA deserves further evaluation as a treatment option for patients with advanced HCC.

MP-600 Establishment and Characterization of a Novel Human bladder tumor cell line : Analysis of CD95 mediated apoptosis and survivin expression

MP-600 Establishment and Characterization of a Novel Human bladder tumor cell line : Analysis of CD95 mediated apoptosis and survivin expression

Inhibitors of XIAP sensitize CD40-activated chronic lymphocytic leukemia cells to CD95-mediated apoptosis

AbstractPatients with chronic lymphocytic leukemia (CLL) treated with adenovirus CD154 (Ad-CD154, CD40 ligand [CD40L]) gene therapy experienced rapid reductions in leukemia cell counts and lymph node size associated with the induced expression of Fas (CD95). However, CLL cells initially resist CD95-mediated apoptosis within the first 3 days after CD40 ligation in vitro. Thereafter, they become sensitive, which is associated with the CD40-induced expression of the proapoptotic protein B-cell leukemia 2 homology 3 (BH3) interacting domain death agonist (Bid). We hypothesized that the initial resistance to CD95-mediated apoptosis may be due to the high-level expression of X-linked inhibitor of apoptosis protein (XIAP) by CLL cells. Consistent with this, CLL cells from patients 1 day after treatment with autologous Ad-CD154-transduced CLL cells became sensitive to CD95-mediated apoptosis following treatment with a novel XIAP inhibitor, 1540-14. Similarly, 1540-14 specifically enhanced CD95-mediated apoptosis of CLL cells following CD40 ligation in vitro. Immunoblot analyses demonstrated that treatment with 1540-14 allowed CD40-stimulated CLL cells to experience high-level activation of caspases-8 and -3 and cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase following CD95 ligation. This study demonstrates that distal apoptosis regulators contribute to the initial resistance of CD40-activated CLL cells to CD95-mediated apoptosis and suggests that XIAP inhibitors might enhance the effectiveness of immune-based treatment strategies that target CD40, such as CD154 gene therapy. (Blood. 2005;106:1742-1748)

Thiopental-induced Apoptosis in Lymphocytes Is Independent of CD95 Activation

Barbiturate coma is used in patients with traumatic brain injury whenever increases in intracranial pressure remain unresponsive to less aggressive therapeutic regimens. However, barbiturate-mediated neuroprotection correlates with lymphopenia, which increases the risk of infection. The mechanisms by which barbiturates lead to lymphopenia remain to be determined. Freshly isolated human lymphocytes and Jurkat cells were incubated with the barbiturate thiopental for 24 and 48 h. Apoptosis was measured by fluorescein isothiocyanate-Annexin and propidium iodide staining, rhodamine 123 staining, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Caspase-3 activity was detected by Western blot and substrate cleavage assay. Thiopental dose-dependently (5-500 microg/ml) increased apoptosis in Jurkat cells from basal levels (4.4 +/- 1.9%) to 29.7 +/- 2.8% after 24 h and 39.7 +/- 3.2% after 48 h, whereas in lymphocytes, thiopental-induced necrosis was observed. Parallel to apoptosis, thiopental dose-dependently increased caspase-3-like activity in Jurkat cells. However, the pan-caspase inhibitor z-VAD-fmk (20 microm) only marginally reduced thiopental-induced (250 microg/ml) apoptosis in Jurkat cells (20.2 +/- 2.5 to 17.2 +/- 2.5%) and necrosis in lymphocytes (39.2 +/- 7.5 to 30.7 +/- 14%). In contrast, anti-CD95-induced apoptosis in Jurkat cells (27.0 +/- 2.0%) was completely blocked by z-VAD-fmk (8.1 +/- 1.8%). Neither expression of CD95 on Jurkat cells nor pretreatment with a neutralizing anti-CD95 antibody influenced thiopental-induced apoptosis, indicating that thiopental induces apoptosis independently of the CD95 system. The nuclear factor kappaB inhibitor gliotoxin accelerated both thiopental- and CD95-mediated apoptosis, indicating a mutual control mechanism of thiopental- and CD95-induced apoptosis. Thiopental directly induces cell death in lymphocytes and Jurkat cells by a CD95-independent mechanism.

CD21/CD19 Coreceptor Signaling Promotes B Cell Survival during Primary Immune Responses

The adaptive immune response is tightly regulated to limit responding cells in an Ag-specific manner. On B cells, coreceptors CD21/CD19 modulate the strength of BCR signals, potentially influencing cell fate. The importance of the CD95 pathway was examined in response of B cells to moderate affinity Ag using an adoptive transfer model of lysozyme-specific Ig transgenic (HEL immunoglobulin transgene (MD4) strain) B cells. Although adoptively transferred Cr2+/+ MD4 B cells are activated and persist within splenic follicles of duck egg lysozyme-immunized mice, Cr2-/- MD4 B cells do not. In contrast, Cr2-/- MD4 lpr B cells persist after transfer, suggesting that lack of CD21/CD35 signaling results in CD95-mediated elimination. Cr2 deficiency did not affect CD95 levels, but cellular FLIP (c-FLIP) protein and mRNA levels were reduced 2-fold compared with levels in Cr2+/+ MD4 B cells. In vitro culture with Cr2+/+ MD4 B cells demonstrated that equimolar amounts of rHEL-C3d3 were more effective than hen egg lysozyme alone in up-regulating c-FLIP levels and for protection against CD95-mediated apoptosis. Collectively, this study implies a mechanism for regulating B cell survival in vivo whereby the strength of BCR signaling (including coreceptor) determines c-FLIP levels and protection from CD95-induced death.

Inhibition of c-Jun-N-terminal-Kinase Sensitizes Tumor Cells to CD95-Induced Apoptosis and Induces G2/M Cell Cycle Arrest

Loss of susceptibility to apoptosis signals is a crucial step in carcinogenesis. Therefore, sensitization of tumor cells to apoptosis is a promising therapeutic strategy. c-Jun-N-terminal-kinases (JNK) have been implicated in stress-induced apoptosis, but may also contribute to survival signaling. Here we show that CD95-induced apoptosis is augmented by the JNK inhibitor SP600125 and small interfering RNA directed against JNK1/2. SP600125 potently inhibited methyl methane sulfonate-induced phosphorylation of c-Jun, but had minimal effect on apoptosis alone. In contrast, it strongly enhanced CD95-mediated apoptosis in six of eight tumor cell lines and led to a G2/M phase arrest in all cell lines. SP600125 enhanced cleavage of caspase 3 and caspase 8, the most upstream caspase in the CD95 pathway. JNK inhibition up-regulates p53 and its target genes p21Cip1/Waf1 and CD95. However, although HCT116 p53-/- cells and p21+/+ cells were less sensitive to CD95 stimulation than their p53+/+ and p21-/- counterparts, p53 and p21 were not involved in the JNK-mediated effect. JunD, which was described to be protective in tumor necrosis factor-induced apoptosis, was not regulated by JNK inhibition on the protein level. When transcription was blocked by actinomycin D, JNK inhibition still enhanced apoptosis to a comparable extent. We conclude that JNK inhibition has antitumor activity by inducing growth arrest and enhancing CD95-mediated apoptosis by a transcription-independent mechanism.