Cancer control by adaptive immunity involves a number of defined death and clearance mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation. Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence, we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control. When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo, Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1(-/-)Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.

No abstract

The activation of caspase-8, a crucial upstream mediator of death receptor signaling, was investigated in epirubicinand Taxol IntroductionApoptosis, a morphologically and biochemically defined form of cell death, 1 plays a role in a wide variety of biologic systems, including tissue homeostasis and regulation of the immune system. 2,3 The process is a highly orchestrated cellular pathway leading to activation of the downstream death machinery. The central mediator and executioner of the death machinery is a proteolytic system involving a family of cysteinyl proteases, called caspases (for review, see Thornberry and Lazebnik 4 ). Triggering of the apoptotic cascade by different death stimuli, such as ionizing radiation 5 and chemotherapeutic drugs, 6 culminates in caspasedependent cleavage of a set of regulatory proteins, degradation of cellular DNA, and complete disassembly of the cell. Thus far, 14 caspase family members have been identified, and some of them, such as caspase-8, mediate apoptotic signals after the activation of death receptors. 7,8 Others, such as caspase-9, are part of the apoptosome and play a role in signal transduction after mitochondrial damage. 9 Recently, an endoplasmic-reticulum-specific pathway of apoptosis has been described that is mediated by caspase-12. 10 Previous data suggest that cytotoxic drugs induce cell death through CD95/Fas-CD95 ligand interaction, 11 and the relevance of this particular death pathway has been shown, for instance, in doxorubicin-induced apoptosis of leukemic T cells. 12 However, other groups were unable to confirm these findings and showed CD95/Fas-independent induction of apoptosis by chemotherapeutic drugs-for example, doxorubicin and etoposide-in Tlymphoma cells. 13,14 The finding that CD95/Fas-signaling and DNA damage induce different apoptosis-signaling pathways has also been shown in a murine and a human B-lymphoma cell line. 15 Furthermore, experimental evidence has been provided that neither FADD 16 nor caspase-8 17 is required for drug-induced apoptosis.In B cells, the induction of apoptosis plays important roles in humoral immunity. In this context, it has been shown that the antigen receptor BCR and the CD95/Fas receptor transduce pro-apoptotic signals in mature B cells (for review, see Tsubata 18 ). We previously showed that activation-induced apoptosis of normal and malignant B lymphocytes upon antigen-receptor ligation is independent of CD95/Fas and CD95/Fas ligand. 19 This has been confirmed by another study demonstrating that B-cell receptormediated apoptosis, in contrast to activation-induced T-cell apoptosis, is not mediated through known death receptor systems, nor does it involve the initial activation of caspase-8. 20 We nevertheless For personal use only. on May 12, 2018. by guest www.bloodjournal.org From observed that drug treatment leads to processing and activation of procaspase-8. We therefore focused our interest on the specific mechanisms leading to apoptotic death after the treatment of immature and mature B-lymphoma cells wi...

Patients with high-grade serous ovarian cancer (HGSC) frequently receive platinum-based chemotherapeutics, such as cisplatin. Cisplatin binds to DNA and induces DNA-damage culminating in mitochondria-mediated apoptosis. Interestingly, mitochondrial DNA is critically affected by cisplatin but its relevance in cell death induction is scarcely investigated. We find that cisplatin sensitive HGSC cell lines contain higher mitochondrial content and higher levels of mitochondrial ROS (mtROS) than cells resistant to cisplatin induced cell death. In clonal sub-lines from OVCAR-3 mitochondrial content and basal oxygen consumption rate correlate with sensitivity to cisplatin induced apoptosis. Mitochondria are in two ways pivotal for cisplatin sensitivity because not only knock-down of BAX and BAK but also the ROS scavenger glutathione diminish cisplatin induced apoptosis. Mitochondrial ROS correlates with mitochondrial content and reduction of mitochondrial biogenesis by knock-down of transcription factors PGC1α or TFAM attenuates both mtROS induction and cisplatin induced apoptosis. Increasing mitochondrial ROS by inhibition or knock-down of the ROS-protective uncoupling protein UCP2 enhances cisplatin induced apoptosis. Similarly, enhancing ROS by high-dose ascorbic acid or H2O2 augments cisplatin induced apoptosis. In summary, mitochondrial content and the resulting mitochondrial capacity to produce ROS critically determine HGSC cell sensitivity to cisplatin induced apoptosis. In line with this observation, data from the human protein atlas (www.proteinatlas.org) indicates that high expression of mitochondrial marker proteins (TFAM and TIMM23) is a favorable prognostic factor in ovarian cancer patients. Thus, we propose mitochondrial content as a biomarker for the response to platinum-based therapies. Functionally, this might be exploited by increasing mitochondrial content or mitochondrial ROS production to enhance sensitivity to cisplatin based anti-cancer therapies.

Caspase-8 is a key effector of death-receptor-triggered apoptosis. In a previous study, we demonstrated, however, that caspase-8 can also be activated in a death receptorindependent manner via the mitochondrial apoptosis pathway, downstream of caspase-3. Here, we show that caspases-3 and -8 mediate a mitochondrial amplification loop that is required for the optimal release of cytochrome c, mitochondrial permeability shift transition, and cell death during apoptosis induced by treatment with the microtubule-damaging agent paclitaxel (Taxol). In contrast, Smac release from mitochondria followed a different pattern, and therefore seems to be regulated independently from cytochrome c release. Taxol-induced cell death was inhibited by the use of synthetic, cellpermeable caspase-3-(zDEVD-fmk) or caspase-8-specific (zIETD-fmk) inhibitors. Apoptosis signaling was not affected by a dominant-negative FADD mutant (FADD-DN), thereby excluding a role of death receptor signaling in the amplification loop and drug-induced apoptosis. The inhibitor experiments were corroborated by the use of BJAB cells overexpressing the natural serpin protease inhibitor, cytokine response modifier A. These data demonstrate that the complete activation of mitochondria, release of cytochrome c, and execution of drug-induced apoptosis require a mitochondrial amplification loop that depends on caspases-3 and -8 activation. In addition, this is the first report to demonstrate death receptor-independent caspase-8 autoprocessing in vivo.

Nbk/Bik (natural born killer/Bcl-2-interacting killer) is a tissue-speci®c BH3-only protein whose molecular function is still largely unknown. To investigate the mechanism of Nbk action, we established a singlevector adenoviral system based on the Tet-off conditional expression of Nbk. Upon Nbk expression, only Bax-positive, but not Bax-de®cient cells were found to undergo apoptosis. Interestingly, Nbk failed to induce apoptosis in the absence of Bax, even despite expression of the related molecule Bak. Re-expression of Bax restored the sensitivity to Nbk. Similarly, Bax wildtype HCT116 cells were highly susceptible, whereas HCT116 Bax knock-out cells remained resistant to Nbk-induced apoptosis. In Bax-positive cells, Nbk induced a conformational switch in the Bax N-terminus coinciding with cytochrome c release, mitochondrial permeability transition and caspase-9 processing. Immunoprecipitation studies revealed that Nbk interacts with Bcl-x L and Bcl-2 but not with Bax. Since, in addition, Nbk did not localize to the mitochondria, our data suggest a model in which Nbk acts as an indirect killer to trigger Bax-dependent apoptosis, whereas Bak is not suf®cient to confer sensitivity to Nbk.

All commonly used local anaesthetics induce neuronal apoptosis in clinically used concentrations. The neurotoxicity correlates with lipid solubility and thus with the conduction blocking potency of the local anaesthetic, but is independent of the chemical class (ester/amide).

Dysfunction of the p53/Bax/caspase-3 apoptosis signaling pathway has been shown to play a role in tumorigenesis and tumor progression, ie the development of acquired drug resistance. Low expression of the apoptosis inducer Bax correlates with poor response to therapy and shorter overall survival in solid tumors. In the present study, we analyzed the p53/Bax/caspase-3 pathway in a paired and an unpaired sample series of children with acute lymphoblastic leukemia (ALL) at initial diagnosis and relapse. The data demonstrate that both Bax expression levels and the Bax/Bcl-2 ratio are significantly lower in samples at relapse as compared with samples at initial diagnosis (P = 0.013, Wilcoxon signed rank test (paired samples); P = 0.0039, Mann-Whitney U test (unpaired samples)). The loss of Bax protein expression was not a consequence of Bax frameshift mutations of the G 8 tract and could not be attributed to mutations of the p53 coding sequence (exons 5 to 8) which were detected to a similar extent in de novo ALL samples and at relapse. Analysis of the downstream effector caspase-3 showed loss of spontaneous caspase-3 processing at relapse. Whereas nine out of 14 (64%, paired samples) or 37 out of 77 (48%, unpaired samples) ALL patients at initial diagnosis displayed spontaneous in vivo processing of caspase-3, this was completely absent in patients at relapse (paired samples) or detected in only one out of 34 patients at relapse (2.9%, unpaired samples). We therefore conclude that in ALL relapse a severe disturbance of apoptotic pathways occurs, both at the level of Bax expression and caspase-3 activation. Leukemia (2000) 14, 1606-1613.