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The targeted elimination of radio- or chemotherapy-induced senescent cells by so-called senolytic substances represents a promising approach to reduce tumor relapse as well as therapeutic side effects such as fibrosis. We screened an in-house library of 178 substances derived from marine sponges, endophytic fungi, and higher plants, and determined their senolytic activities towards DNA damage-induced senescent HCT116 colon carcinoma cells. The Pan-PI3K-inhibitor wortmannin and its clinical derivative, PX-866, were identified to act as senolytics. PX-866 potently induced apoptotic cell death in senescent HCT116, MCF-7 mammary carcinoma, and A549 lung carcinoma cells, independently of whether senescence was induced by ionizing radiation or by chemotherapeutics, but not in proliferating cells. Other Pan-PI3K inhibitors, such as the FDA-approved drug BAY80-6946 (Copanlisib, Aliqopa®), also efficiently and specifically eliminated senescent cells. Interestingly, only the simultaneous inhibition of both PI3K class I alpha (with BYL-719 (Alpelisib, Piqray®)) and delta (with CAL-101 (Idelalisib, Zydelig®)) isoforms was sufficient to induce senolysis, whereas single application of these inhibitors had no effect. On the molecular level, inhibition of PI3Ks resulted in an increased proteasomal degradation of the CDK inhibitor p21 WAF1/CIP1 in all tumor cell lines analyzed. This led to a timely induction of apoptosis in senescent tumor cells. Taken together, the senolytic properties of PI3K-inhibitors reveal a novel dimension of these promising compounds, which holds particular potential when employed alongside DNA damaging agents in combination tumor therapies., (© 2024. The Author(s).)

This study investigates whether a chronotherapeutic treatment of hepatocellular carcinoma (HCC) may improve treatment efficacy and mitigate side effects on non-tumoral liver (NTL). HCC was induced in Per2::luc mice which were irradiated at four time points of the day. Proliferation and DNA-double strand breaks were analyzed in irradiated and nonirradiated animals by detection of Ki67 and γ-H2AX. Prior to whole animal experiments, organotypic slice cultures were investigated to determine the dosage to be used in whole animal experiments. Irradiation was most effective at the proliferation peaks in HCC at ZT02 (early inactivity phase) and ZT20 (late activity phase). Irradiation effects on NTL were minimal at ZT20. As compared with NTL, nonirradiated HCC revealed disruption in daily variation and downregulation of all investigated clock genes except Per1. Irradiation affected rhythmic clock gene expression in NTL and HCC at all ZTs except at ZT20 (late activity phase). Irradiation at ZT20 had no effect on total leukocyte numbers. Our results indicate ZT20 as the optimal time point for irradiation of HCC in mice at which the ratio between efficacy of tumor treatment and toxic side effects was maximal. Translational studies are now needed to evaluate whether the late activity phase is the optimal time point for irradiation of HCC in man., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Cancer-related fatigue (CRF) and stress are common symptoms in cancer patients and represent early side effects of cancer treatment which affect the life quality of the patients. CRF may partly depend on disruption of the circadian rhythm. Locomotor activity and corticosterone rhythms are two important circadian outputs which can be used to analyze possible effects on the circadian function during cancer development and treatment. The present study analyzes the relationship between locomotor activity rhythm, corticosterone levels, hepatocellular carcinoma (HCC) development, and radiotherapy treatment in a mouse model. HCC was induced in mice by single injection of diethylnitrosamine (DEN) and chronic treatment of phenobarbital in drinking water. Another group received chronic phenobarbital treatment only. Tumor bearing animals were divided randomly into four groups irradiated at four different Zeitgeber time points. Spontaneous locomotor activity was recorded continuously; serum corticosterone levels and p-ERK immunoreaction in the suprachiasmatic nucleus (SCN) were investigated. Phenobarbital treated mice showed damped corticosterone levels and a less stable 24 hours activity rhythm as well as an increase in activity during the light phase, reminiscent of sleep disruption. The tumor mice showed an increase in corticosterone level during the inactive phase and decreased activity during the dark phase, reminiscent of CRF. After irradiation, corticosterone levels were further increased and locomotor activity rhythms were disrupted. Lowest corticosterone levels were observed after irradiation during the early light phase; thus, this time might be the best to apply radiotherapy in order to minimize side effects., (© 2021 The Authors. Journal of Pineal Research published by John Wiley & Sons Ltd.)

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD + -dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G 2 and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.

In recent years it has become more and more apparent that the regulation of gene expression by RNA-binding proteins (RBPs) is of utmost importance for most cellular signaling pathways. RBPs control several aspects of RNA biogenesis including splicing, localization, stability, and translation efficiency. One of these RBPs is RBM47 that recently has been suggested to function as a tumor suppressor as it was shown to suppress breast and colon cancer progression. Here we demonstrate that RBM47 is an important regulator of basal and DNA damage-induced p53 and p21 WAF1/CIP1 protein expression. Knockdown of RBM47 by siRNAs results in a strong reduction in p53 mRNA and protein levels due to an impaired p53 promoter activity. Accordingly, overexpression of Flag-RBM47 enhances p53 promoter activity demonstrating that RBM47 regulates p53 at the transcriptional level. By controlling p53, knockdown of RBM47 concomitantly decreases also p21 expression at the transcriptional level, driving irradiated carcinoma cell lines from different entities into cell death rather than into senescence. Thus, RBM47 represents a novel molecular switch of cell fate decisions that functions as a regulator of the p53/p21-signaling axis.

Microtubule-targeting agents (MTAs) are the most effective chemotherapeutics used in cancer therapy to date, but their clinical use is often hampered by the acquisition of resistance. Thereby, elucidation of the molecular signaling pathways activated by novel FDA-approved MTAs such as eribulin is important for future therapeutic applications. In contrast to several reports, we show here that regardless of the presence of caspase-3, clinically relevant concentrations of eribulin and the classical MTA paclitaxel predominantly induce caspase-independent cell death in MCF-7 breast carcinoma cells. On the molecular level, several key proteins involved in apoptosis such as p53, Plk1, caspase-2, and Bim as well as the two MAPKs ERK and JNK were activated by both compounds to a similar extent. However, none of them proved to be important for eribulin- and paclitaxel-induced cytotoxicity, as their siRNA-mediated knockdown or inactivation by small molecule inhibitors did not alter cell death rates. In contrast, knockdown of the anti-apoptotic Bcl-2 protein, which becomes heavily phosphorylated at Ser70 during MTA treatment, resulted surprisingly in a reduction of MTA-mediated cell death. This phenomenon can be most likely explained by our observation that the absence of Bcl-2 slowed down cell cycle progression resulting in fewer cells entering mitosis, thereby delaying the mitotic capability of these MTAs to induce cell death. Taken together, although eribulin and paclitaxel disturb the mitotic spindle differently, they exhibit no functional differences in downstream molecular cell death signaling in MCF-7 breast cancer cells.

The homeobox gene HLXB9 encodes for the transcription factor HB9, which is essential for pancreatic as well as motor neuronal development. Beside its physiological expression pattern, aberrant HB9 expression has been observed in several neoplasias. Especially in infant translocation t(7;12) acute myeloid leukemia, aberrant HB9 expression is the only known molecular hallmark and is assumed to be a key factor in leukemic transformation. However, so far, only poor functional data exist addressing the oncogenic potential of HB9 or its influence on hematopoiesis. We investigated the influence of HB9 on cell proliferation and cell cycle in vitro , as well as on hematopoietic stem cell differentiation in vivo using murine and human model systems. In vitro , HB9 expression led to premature senescence in human HT1080 and murine NIH3T3 cells, providing for the first time evidence for an oncogenic potential of HB9. Onset of senescence was characterized by induction of the p53-p21 tumor suppressor network, resulting in growth arrest, accompanied by morphological transformation and expression of senescence-associated β-galactosidase. In vivo , HB9-transduced primary murine hematopoietic stem and progenitor cells underwent a profound differentiation arrest and accumulated at the megakaryocyte/erythrocyte progenitor stage. In line, gene expression analyses revealed de novo expression of erythropoiesis-related genes in human CD34 + hematopoietic stem and progenitor cells upon HB9 expression. In summary, the novel findings of HB9-dependent premature senescence and myeloid-biased perturbed hematopoietic differentiation, for the first time shed light on the oncogenic properties of HB9 in translocation t(7;12) acute myeloid leukemia., (Copyright© 2019 Ferrata Storti Foundation.)

The cyclin-dependent kinase inhibitor p21 is an important player in stress pathways exhibiting both tumor-suppressive and oncogenic functions. Thus, expression of p21 has to be tightly controlled, which is achieved by numerous mechanisms at the transcriptional, translational, and posttranslational level. Performing immunoprecipitation of bromouridine-labeled p21 mRNAs that had been incubated before with cytoplasmic extracts of untreated HCT116 colon carcinoma cells, we identified the DEAD-box RNA helicase DDX41 as a novel regulator of p21 expression. DDX41 specifically precipitates with the 3'UTR, but not with the 5'UTR, of p21 mRNA. Knockdown of DDX41 increases basal and γ irradiation-induced p21 protein levels without affecting p21 mRNA expression. Conversely, overexpression of DDX41 strongly inhibits expression of a FLAG-p21 and a luciferase construct, but only in the presence of the p21 3'UTR. Together, these data suggest that this helicase regulates p21 expression at the translational level independent of the transcriptional activity of p53. However, knockdown of DDX41 completely fails to increase p21 protein levels in p53-deficient HCT116 cells. Moreover, posttranslational up-regulation of p21 achieved in both p53 +/+ and p53 -/- HCT116 cells in response to pharmaceutical inhibition of the proteasome (by MG-132) or p90 ribosomal S6 kinases (by BI-D1870) is further increased by knockdown of DDX41 only in p53-proficient but not in p53-deficient cells. Although our data demonstrate that DDX41 suppresses p21 translation without disturbing the function of p53 to directly induce p21 mRNA expression, this process indirectly requires p53, perhaps in the form of another p53 target gene or as a still undefined posttranscriptional function of p53., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

This study addresses the viability of the lichen Xanthoria elegans after high-dose ionizing irradiation in the frame of the STARLIFE campaign. The first set of experiments was intended to resemble several types of galactic cosmic radiation (GCR) as present beyond the magnetic shield of Earth. In the second set of experiments, γ radiation up to 113 kGy was applied to test the limit of lichen resistance to ionizing radiation. Entire thalli of Xanthoria elegans were irradiated in the anhydrobiotic state. After STARLIFE 1, the metabolic activity of both symbionts was quantified by live/dead staining with confocal laser scanning microscopy. The photosynthetic activity was measured after the respective irradiation to assess the ability of the symbiotic green algae to restore photosynthesis after irradiation. The STARLIFE campaign complements the results of the LIFE experiments at the EXPOSE-E facility on the International Space Station by testing the model organism Xanthoria elegans on its resistance to hazardous radiation that might be accumulated during long-term space exposure. In addition, the photosynthetic activity of metabolically active lichen was investigated after X-ray irradiation up to 100 Gy (3.3 Gy/min). Since previous astrobiological experiments were mostly performed with anhydrobiotic lichen, these experiments will broaden our knowledge on the correlation of physiological state and astrobiological stressors. Key Words: Astrobiology-Extremotolerance-Gamma rays-Ionizing radiation-Lichens-Viability. Astrobiology 17, 136-144.

Background: The mechanisms of skin aging have not been completely elucidated. Anecdotal data suggests that EGFR inhibition accelerates aging-like skin changes., Objective: The objective of the study was to evaluate the clinical characteristics and investigate the cellular and molecular mechanisms underlying skin changes associated with the use of EFGRIs., Patients and Methods: Patients during prolonged treatment with EGFRIs (>3 months) were analyzed for aging-like skin changes. Baseline EGFR expression was compared in young (<25 years old) vs. old (> 65 years old) skin. In addition, the regulation of extracellular matrix, senescence-associated genes, and cell cycle status was measured in primary human keratinocytes treated with erlotinib in vitro., Results: There were progressive signs of skin aging, including xerosis cutis, atrophy, rhytide formation, and/or actinic purpura in 12 patients. Keratinocytes treated with erlotinib in vitro showed a significant down-modulation of hyaluronan synthases (HAS2 and HAS3), whereas senescence-associated genes (p21, p53, IL-6, maspin) were upregulated, along with a G1 cell cycle arrest and stronger SA β-Gal activity. There was significantly decreased baseline expression in EGFR density in aged skin, when compared to young controls., Conclusions: EGFR inhibition results in molecular alterations in keratinocytes that may contribute to the observed skin aging of patients treated with respective targeted agents.

MicroRNAs (miRNAs), a class of small non-coding RNAs that usually cause gene silencing by translational repression or degradation of mRNAs, are implicated in DNA damage-induced stress responses. To identify senescence-associated miRNAs, we performed microarray analyses using wild-type and p53-deficient HCT116 colon carcinoma cells that following gamma-irradiation (γIR) are driven into senescence and apoptosis, respectively. Several miRNAs including miR-30e were found upregulated in a p53-dependent manner specifically in senescent cells, but not in apoptotic cells. Overexpression of miR-30e in HCT116 cells not only inhibited γIR-, etoposide- or miR-34a-induced caspase-3-like DEVDase activities and cell death, but greatly accelerated and augmented their senescent phenotype. Consistently, procaspase-3 protein, but not mRNA decreased in the presence of miR-30e, whereas expression of the cyclin-dependent kinase inhibitor p21 increased both at the mRNA and protein level. Performing luciferase reporter gene assays, we identified the 3'-UTR of the caspase-3 mRNA as a direct miR-30e target. In contrast, although miR-30e was unable to bind to the p21 mRNA, it increased expression of a luciferase construct containing the p21 promoter, suggesting that the miR-30e-mediated upregulation of p21 occurs indirectly at the transcriptional level. Interestingly, despite suppressing procaspase-3 expression, miR-30e was unable to protect RKO colon carcinoma cells from DNA damage-induced death or to induce senescence, as miR-30e completely fails to upregulate p21 in these cells. These data suggest that miR-30e functions in a cell type-dependent manner as an important molecular switch for DNA damage-induced stress responses and may thus represent a target of therapeutic value.

The p14(ARF) tumor suppressor triggers cell death or cell cycle arrest upon oncogenic stress. In MCF-7 breast carcinoma cells, expression of the tumor suppressor gene p14(ARF) fails to trigger apoptosis but induces an arrest in the G1 and, to a lesser extent, in the G2 phase in the cell division cycle. Here, inhibition of cell cycle arrest resulted in apoptosis induction in caspase-3 proficient MCF-7 cells upon expression of p14(ARF) . This occurred in the absence of S-phase progression or mitotic entry. In contrast, syngeneic, caspase-3-deficient MCF-7 cells remained entirely resistant to p14(ARF) -induced apoptosis. Thus, cell cycle checkpoint abrogation overcomes resistance to p14(ARF) -induced cell death and promotes cell death via a caspase-3-dependent pathway. Cell death coincided with dissipation of the mitochondrial membrane potential, release of cytochrome c, and was inhibitable by pan-caspase inhibitors and the caspase-3/7 inhibitor zDEVD-fmk. Of note, mitochondrial events of apoptosis execution depended entirely on caspase-3 proficiency indicating that caspase-3 either acts "up-stream" of the mitochondria in a "non-canonical" pathway or mediates a mitochondrial feedback loop to amplify the apoptotic caspase signal in p14(ARF) -induced stress signaling., (Copyright © 2013 UICC.)

The p90 ribosomal S6 kinase (RSK) family is a group of highly conserved Ser/Thr kinases that promote cell proliferation, growth, motility and survival. As they are almost exclusively activated downstream of extracellular signal-regulated kinases 1 and 2 (ERK1/2), therapeutic intervention by RSK inhibition is less likely to produce such severe side effects as those observed following inhibition of the upstream master regulators Raf, MEK and ERK1/2. Here, we report that BI-D1870, a potent small molecule inhibitor of RSKs, induces apoptosis, although preferentially, in a p21-deficient background. On the other hand, BI-D1870 also induces a strong transcription- and p53-independent accumulation of p21 protein and protects cells from gamma irradiation (γIR)-induced apoptosis, driving them into senescence even in the absence of γIR. Although we identified p21 in in vitro kinase assays as a novel RSK substrate that specifically becomes phosphorylated by RSK1-3 at Ser116 and Ser146, RNA-interference, overexpression and co-immunoprecipitation studies as well as the use of SL0101, another specific RSK inhibitor, revealed that BI-D1870 mediates p21 accumulation via a yet unknown pathway that, besides its off-site targets polo-like kinase-1 and AuroraB, also does also not involve RSKs. Thus, this novel off-target effect of BI-D1870 should be taken into serious consideration in future studies investigating the role of RSKs in cellular signaling and tumorigenesis.

Proteasome inhibitors (PIs) potently induce apoptosis in a variety of tumor cells, but the underlying mechanisms are not fully elucidated. Comparing PI-induced apoptosis susceptibilities of various mouse embryonic fibroblast (MEF) lines differing in their c-jun N-terminal kinase (JNK) 1 and 2 status, we show that several hallmarks of apoptosis were most rapidly detectable in JNK2-/- cells, whereas they appeared only delayed and severely reduced in their intensities in cells expressing JNK2. Consistent with our finding that PI-induced apoptosis requires de novo protein synthesis, the proteasomal inhibitor MG-132 induced expression of the BH3-only protein Noxa at the transcriptional level in a JNK1-dependent, but JNK2-opposing manner. As the knockdown of Noxa blocked only the rapid PI-induced apoptosis of JNK2-/- cells, but not the delayed death occurring in JNK1-/- and JNK1+/+ cells, our data uncover a novel PI-induced apoptosis pathway that is regulated by the JNK1/2-dependent expression of Noxa. Furthermore, several transcription factors known to modulate Noxa expression including ATF3, ATF4, c-Jun, c-Myc, HIF1α, and p53 were found upregulated following MG-132 exposure. From those, only knockdown of c-Myc rescued JNK2-/- cells from PI-induced apoptosis, however, without affecting expression of Noxa. Together, our data not only show that a rapid execution of PI-induced apoptosis requires JNK1 for upregulation of Noxa via an as yet unknown transcription factor, but also that JNK2 controls this event in an oppositional manner.

Non-homologous end-joining (NHEJ) is one major pathway for the repair of double-stranded DNA breaks in mammals. Following break recognition, alignment and processing, broken DNA ends are finally rejoined by the essential DNA Ligase IV. In the cell, Ligase IV is unable to function without its constitutive interaction partner XRCC4 and becomes unstable when it is missing, and it has been assumed that XRCC4 may also be required for recruitment of Ligase IV to repair sites. To investigate the function of complex formation between both proteins directly in the living cell, we stably expressed them as bio-fluorescent fusion proteins in human HT-1080 cell clones. Ligase IV or XRCC4 were expressed either alone or both were co-expressed at a roughly equimolar ratio. Labelled proteins were overexpressed manifold in comparison to endogenously expressed proteins. We show that over-expressed Ligase IV was only partially imported into the nucleus and showed a diffuse distribution there, whereas XRCC4 expressed alone was entirely nuclear with a distinct exclusion from nucleoli. When Ligase IV was co-expressed with XRCC4, both proteins formed the natural complex, and Ligase IV was not only efficiently imported but also resembled the sub-nuclear distribution of XRCC4. In addition, Ligase IV, when in complex with XRCC4, acquired a delayed nuclear reimport after mitotic cell division of XRCC4. We further determined by photobleaching the kinetics with which the proteins exchange at UVA laser-irradiated nuclear sites between damage-bound and diffusing states. We found that the dynamic exchange rate of the Ligase IV/XRCC4 complex at micro-irradiated sites was faster than that of XRCC4 expressed alone. In summary, our findings demonstrate a novel function of XRCC4 in controlling nuclear import and sub-nuclear distribution of Ligase IV, and they suggest that XRCC4 modulates the dynamic interaction of the Ligase IV/XRCC4 complex with the NHEJ machinery at double-stranded DNA breaks., (2011 Elsevier B.V. All rights reserved.)

Although caspase-2 represents the most conserved caspase across species and was the second caspase identified, its precise function remains enigmatic. In several cell types we show that knockdown of caspase-2 specifically impaired DNA damage-induced p21 expression, whereas overexpression of a caspase-2 mutant increased p21 levels. Caspase-2 did not influence p21 mRNA transcription; moreover, various inhibitors targeting proteasomal or non-proteasomal proteases, including caspases, could not restore p21 protein levels following knockdown of caspase-2. As, however, silencing of caspase-2 impaired exogenous expression of p21 constructs containing 3'-UTR sequences, our results strongly indicate that caspase-2 regulates p21 expression at the translational level. Intriguingly, unlike depletion of caspase-2, which prevented p21 expression and thereby reverted the γ-IR-induced senescent phenotype of wild-type HCT116 colon carcinoma cells into apoptosis, knockdown of none of the caspase-2-interacting components RAIDD, RIP or DNA-PKcs was able to mimic these processes. Together, our data suggest that this novel role of caspase-2 as a translational regulator of p21 expression occurs not only independently of its enzymatic activity but also does not require known caspase-2-activating platforms.

Microtubule-targeting cancer drugs such as paclitaxel block cell-cycle progression at mitosis by prolonged activation of the mitotic checkpoint. Cells can spontaneously escape mitotic arrest and enter interphase without chromosome segregation by a process termed mitotic slippage that involves the degradation of cyclin B1 without mitotic checkpoint inactivation. Inducing mitotic slippage with chemicals causes cells to die after multiple rounds of DNA replication without cell division, which may enhance the antitumor activity of microtubule-targeting drugs. Here, we explore pathways leading to mitotic slippage by using SU6656 and geraldol, two recently identified chemical inducers of mitotic slippage. Mitotic slippage induced by SU6656 or geraldol was blocked by the proteasome inhibitor MG-132 and involved proteasome-dependent degradation of cyclin B1 and the mitotic checkpoint proteins budding uninhibited by benzimidazole related 1 (BubR1) and cell division cycle 20 (Cdc20) in T98G cells. Mitotic slippage and the degradation of BubR1 and Cdc20 were also inhibited by the caspase-3 and -7 inhibitor DEVD-CHO. MCF-7 cells lacking caspase-3 expression could not degrade BubR1 or undergo mitotic slippage in response to SU6656 or geraldol. Introduction of caspase-3 completely restored the ability of MCF-7 cells to degrade BubR1 and undergo mitotic slippage. However, lack of expression of caspase-3 did not affect cell death after exposure to paclitaxel, with or without mitotic slippage induction. The requirement for caspase-3 for chemically induced mitotic slippage reveals a new mechanism for mitotic exit and a link between mitosis and apoptosis that has implications for the outcome of cancer chemotherapy.

Here we identified an evolutionarily highly conserved and ubiquitously expressed protein (C9orf82) that shows structural similarities to the death effector domain of apoptosis-related proteins. RNAi knockdown of C9orf82 induced apoptosis in A-549 and MCF7/casp3-10b lung and breast carcinoma cells, respectively, but not in cells lacking caspase-3, caspase-10 or both. Apoptosis was associated with activated caspases-3, -8, -9 and -10, and inactivation of caspases 10 or 3 was sufficient to block apoptosis in this pathway. Apoptosis upon knockdown of C9orf82 was associated with increased caspase-10 expression and activation, which was required for the generation of an 11 kDa tBid fragment and activation of Caspase-9. These data suggest that C9orf82 functions as an anti-apoptotic protein that modulates a caspase-10 dependent mitochondrial caspase-3/9 feedback amplification loop. We designate this ubiquitously expressed and evolutionarily conserved anti-apoptotic protein Conserved Anti-Apoptotic Protein (CAAP). We also demonstrated that treatment of MCF7/casp3-10b cells with staurosporine and etoposides induced apoptosis and knockdown of CAAP expression. This implies that the CAAP protein could be a target for chemotherapeutic agents.

Microtubule-interfering cancer drugs such as paclitaxel (PTX) often cause chemoresistance and severe side effects, including neurotoxicity. To explore potentially novel antineoplastic molecular targets, we investigated the cellular response of breast carcinoma cells to short hairpin(sh)RNA-mediated depletion of the centrosomal protein transforming acidic coiled coil (TACC) 3, an Aurora A kinase target expressed during mitosis. Unlike PTX, knockdown of TACC3 did not trigger a cell death response, but instead resulted in a progressive loss of the pro-apoptotic Bcl-2 protein Bim that links microtubule integrity to spindle poison-induced cell death. Interestingly, TACC3-depleted cells arrested in G₁ through a cellular senescence program characterized by the upregulation of nuclear p21(WAF), downregulation of the retinoblastoma protein and extracellular signal-regulated kinase 1/2, formation of HP1γ (phospho-Ser83)-positive senescence-associated heterochromatic foci and increased senescence-associated β-galactosidase activity. Remarkably, the onset of senescence following TACC3 knockdown was strongly accelerated in the presence of non-toxic PTX concentrations. Thus, we conclude that mitotic spindle stress is a major trigger of premature senescence and propose that the combined targeting of the centrosomal Aurora A-TACC3 axis together with drugs interfering with microtubule dynamics may efficiently improve the chemosensitivity of cancer cells.

Altered cell division is associated with overproliferation and tumorigenesis, however, mitotic aberrations can also trigger antiproliferative responses leading to postmitotic cell cycle exit. Here, we focus on the role of the centrosome and in particular of centrosomal TACC (transforming acidic coiled coil) proteins in tumorigenesis and cellular senescence. We have complied recent evidence that inhibition or depletion of various mitotic proteins which take over key in centrosome and kinetochore integrity and mitotic checkpoint function in sufficient to activate a p53-p21(WAF) driven premature senescence phenotype. These findings have direct implications for proliferative tissue homeostasis as well as for cellular and organismal aging.

Although initially described as a regulator of cell cycle progression, the cyclin-dependent kinase inhibitor p21 is now known to also modulate various other biological processes including transcription, differentiation and apoptosis. These versatile activities of p21 are mainly mediated via direct binding to various transcription factors, pro-apoptotic proteins and kinases that are usually inhibited by this interaction. Here we provide in vitro evidence that p21 not only inhibits, but also activates certain kinases in a remarkable substrate-dependent manner. Whereas phosphorylation of the tumor suppressor p53 by several isoforms of the cJun N-terminal kinases (JNKs) was greatly attenuated in the presence of p21, phosphorylation of cJun remained either unaffected or was even enhanced. Furthermore, p21 strongly increased phosphorylation of cFos and MBP by ERK1 and ERK2, while p53 phosphorylation was increased and inhibited, respectively. Also p38α and glycogen synthase kinase-3 beta (GSK-3β) were found differentially regulated by p21 in a substrate-dependent manner, while casein kinase-1 epsilon (CK1ε) was not affected. Together with our finding that the stress-induced p53 phosphorylation pattern differs greatly between p21-proficient and -deficient HCT116 colon carcinoma cells, our results suggest that p21 is able to influence kinase activities both in a negative and positive manner.

Upon DNA damage and other stresses, the transcription factor p53 elicits numerous responses including DNA repair, cell cycle arrest and apoptosis, properties that make p53 the prototype tumor suppressor. In addition, p53 also transactivates genes whose products act in an anti-apoptotic manner providing strong evidence that p53 exhibits both tumor suppressive and tumorigenic functions. Although several events were postulated to contribute to the p53-mediated decision process, the precise mechanism(s) that governs p53 activities is still elusive. Recently, it was found that the p53 gene allows expression of at least nine different isoforms that arise from multiple splicing events and the usage of alternative promoters. Several of these isoforms were shown to critically interfere with the function of the full-length p53 mainly by acting in a dominant-negative manner. However, an isoform-dependent selective activation of p53 target genes was also observed. Furthermore, certain p53 isoforms are aberrantly expressed in various tumors strongly implying their involvement in tumorigenic events. Thus, p53 isoforms may represent crucial determinants in p53-mediated decision processes whose precise functions (their do's and don'ts) are only beginning to emerge.

Pifithrin-alpha (PFT-alpha) was shown to specifically block transcriptional activity of the tumor suppressor p53 and was therefore proposed to be useful in preventing the severe side effects often associated with chemo- and radiotherapy. We report here that although PFT-alpha efficiently protected different cell types from DNA damage-induced apoptosis, it mediated this effect regardless of the presence or absence of p53. Interestingly, PFT-alpha blocked the apoptosome-mediated processing and activation of caspase-9 and -3 without interfering with the activation of mitochondria. Neither the DNA damage-induced activation of Bax or Bak nor the loss of the mitochondrial membrane potential or the final release of cytochrome c were inhibited by this compound. Instead, the ability of PFT-alpha to protect p53-deficient cells from DNA damage-induced caspase activation and apoptosis was greatly diminished after siRNA-mediated downregulation of cyclin-D1 expression. In contrast, downregulation of other proteins involved in cell-cycle progression, such as the retinoblastoma protein, cyclin D3, as well as the cyclin-dependent kinases, 2, 4 and 6, could not abolish this protection. Thus, our data show that PFT-alpha protects cells from DNA damage-induced apoptosis also by a p53-independent mechanism that takes place downstream of mitochondria and that might involve cyclin D1.

The p53 gene encodes several isoforms that can interfere with stress responses by modulating p53 wild-type (wt) function. Recently, a C-terminally truncated splice variant, p53beta, has been implicated in the regulation of p53-dependent apoptosis, whereas the function of similarly spliced p53gamma was not investigated before. Therefore, we studied the impact of these isoforms on the function of endogenous p53wt and compared it with that of exogenously expressed p53wt. We demonstrate that despite an efficient nuclear expression of all isoforms, only p53wt modulates apoptosis induction. Furthermore, only p53wt assembles into a transcription-competent oligomeric complex or translocates to mitochondria upon stress induction. Both C-terminally truncated isoforms fail to modulate the apoptotic function of p53wt because they are unable to associate with p53wt and hence do not bind to p53 DNA recognition sequences. Consistently, the dominant-negative function of transactivation-deficient Delta133p53 is completely lost in the Delta133p53beta variant. Intriguingly, the alternatively spliced C-terminus protects p53beta and p53gamma not only from MDM2-mediated proteasomal degradation, but strongly impairs their binding to this negative regulator. Thus, our data demonstrate the necessity of the regularly spliced C-terminal tail for multiple layers of p53 regulation and function.

Depending on multiple factors DNA damage leads either to cell cycle arrest or apoptosis. One of the main players deciding the fate of a cell is the tumor suppressor p53 that modulates these responses in a transcription-dependent and -independent manner. Over the past few years, however, strong evidence accumulated that p53 engages also powerful pro-survival pathways by transcriptionally activating a multitude of genes whose products efficiently counteract apoptosis. Our review summarizes the current knowledge concerning approximately forty p53-regulated proteins that exert their anti-apoptotic potential by interfering with diverse cellular processes. These activities are surely essential for normal development and maintenance of a healthy organism, but may easily turn into the dark side of the tumor suppressor p53 contributing to tumorigenesis.

Among the members of the Bcl-2 family, the multidomain proteins Bax and Bak are crucial for the activation of mitochondria. However, it is still unclear whether they act in a unique and distinct manner or whether they exhibit redundant functions. To systematically investigate their activation on a single-cell level, we established MCF-7 cell lines stably expressing GFP-fusion variants of these proteins. We found that MCF-7/GFP-Bak cells showed an increased sensitivity to apoptosis induction by staurosporine, actinomycin D, TRAIL and overexpression of Puma compared to GFP-Bax-expressing cells. Independently of the death stimulus used, oligomerization of endogenous and exogenous Bak was mostly detected prior to an activation of Bax, whereas cells displaying oligomerized Bax in the absence of Bak clusters were not observed. In addition, activation of Bax but not Bak was attenuated by a caspase inhibitor. Consistent with this, caspase-3-deficient MCF-7 cells displayed a significantly reduced activation of endogenous Bax than caspase-3-proficient MCF-7 cells. Thus, our data strongly suggest that diverse apoptotic stimuli preferentially engage the Bak pathway, whereas the triggering of Bax occurs, at least partially, downstream of mitochondrial caspase activation, most likely constituting a positive feedback loop for the amplification of the death signal.

Taxane derivatives such as paclitaxel elicit their antitumor effects at least in part by induction of apoptosis, but the underlying mechanisms are incompletely understood. Here, we used different cellular models with deficiencies in key regulators of apoptosis to elucidate the mechanism of paclitaxel-induced cell death. Apoptosis by paclitaxel was reported to depend on the activation of the initiator caspase-10; however, we clearly demonstrate that paclitaxel kills murine embryonic fibroblasts (MEFs) devoid of caspase-10 as well as human tumor cell lines deficient in caspase-10, caspase-8, or Fas-associating protein with death domain. In contrast, the lack of Apaf-1 or caspase-9, key regulators of the mitochondrial pathway, not only entirely protected against paclitaxel-induced apoptosis but could even confer clonogenic survival, depending on the cell type and drug concentration. Thus, paclitaxel triggers apoptosis not through caspase-10, but via caspase-9 activation at the apoptosome. This conclusion is supported by the fact that Bcl-2-overexpressing cells and Bax/Bak doubly-deficient MEFs were entirely resistant to paclitaxel-induced apoptosis. Interestingly, also the single knockout of Bim or Bax, but not that of Bak or Bid, conferred partial resistance, suggesting a particular role of these mediators in the cell-death pathway activated by paclitaxel.

Until recently, the p53 transcriptional target p21, a member of the Cip/Kip family, was almost solely viewed as a nuclear protein with a principal function of inhibiting cyclin-dependent kinase (CDK) activity and hence, cell cycle progression. However, emerging evidence now suggests additional functions for p21 in diverse cellular processes including a role as a modulator of apoptosis. Several mechanisms are suggested by which p21 interferes with the apoptotic machinery that encompass either CDK-independent events such as transcriptional regulation and direct binding to pro-apoptotic gene products in the cytoplasm, or that were based on inhibition of CDKs that in several studies were required downstream of caspases for the generation of characteristic apoptotic alterations. Very recently, we have shown that p21 protects cells from irradiation-induced apoptosis by suppression of CDK activity that appears to be required downstream of the mitochondria for an efficient activation of the caspase cascade. Together with other reports, our results not only demonstrate the close connection of events leading to either cell cycle arrest or apoptosis, but also indicate that the CDK inhibitor p21 battles apoptosis at multiple frontiers.

The role of the cyclin-dependent kinase (CDK) inhibitor p21 as a mediator of p53-induced growth arrest is well established. In addition, recent data provide strong evidence for new emerging functions of p21, including a role as a modulator of apoptosis. The mechanisms, however, by which p21 interferes with the death machinery, especially following ionizing radiation (IR), are largely unknown. Here, we report that IR induced caspase-9 and caspase-3 activation and subsequent apoptosis only in p21-deficient colon carcinoma cells, whereas similar treated wild-type cells were permanently arrested in the G(2)-M phase, correlating with the induction of cellular senescence. Interestingly, activation of the mitochondrial pathway, including caspase-2 processing, depolarization of the outer mitochondrial membrane, and cytochrome c release, was achieved by IR in both cell lines, indicating that p21 inhibits an event downstream of mitochondria but preceding caspase-9 activation. IR-induced p21 protein expression was restricted to the nucleus, and no evidence for a mitochondrial or cytoplasmic association was found. In addition, p21 did neither interact with caspase-3 or caspase-9, suggesting that these events are not required for the observed protection. Consistent with this assumption, we found that CDK inhibitors potently abrogated IR-induced caspase processing and activation without affecting mitochondrial events. In addition, in vitro caspase activation assays yielded higher caspase-3 activities in extracts of irradiated p21-deficient cells compared with extracts of similar treated wild-type cells. Thus, our results strongly indicate that p21 protects cells from IR-induced apoptosis by suppression of CDK activity that seems to be required for activation of the caspase cascade downstream of the mitochondria.

The choroid plexus epithelium constitutes the structural basis of the blood-cerebrospinal fluid barrier. We previously demonstrated that Streptococcus suis (S. suis), a relevant cause of bacterial meningitis in pigs and humans, affects porcine choroid plexus epithelial cell (PCPEC) barrier function and integrity. We now characterized PCPEC cell death and investigated whether apoptosis or necrosis is responsible for the cytotoxicity after infection with different S. suis isolates. We found S. suis strain-dependent histone associated DNA-fragments quantified by ELISA. This response could partially be inhibited by cylcoheximide, cytochalasin D, dexamethasone, herbimycin A, but most effectively by the pan-caspase inhibitor zVAD-fmk. We further detected caspase-3 and -9 activation after infection with all tested S. suis isolates that could also be blocked by zVAD-fmk. However, we found a significantly stronger caspase activity with the protein kinase inhibitor staurosporine. All tested S. suis isolates induced loss of cell viability in PCPEC as shown with the Live/Dead assay, but strain dependent lactate dehydrogenase-release. Both parameters could not be influenced by zVAD-fmk. Immunostaining showed release of high-mobility group box 1 (HMGB1) protein from the nucleus, indicative of necrosis. Transmission electron microscopy showed cell swelling, cytoplasmic vacuolization, loss of membrane integrity, nuclear fermentation but no nuclear condensation, indices for a primarily necrotic cell morphology. Taken together, our findings indicate that S. suis causes cell death in PCPEC by different mechanisms. Although apoptosis may be involved in the process of PCPEC cell death, necrosis seems to be the predominant mechanism. Through inflammation in the choroid plexus during bacterial meningitis, the blood-cerebrospinal fluid barrier function will be compromised.

A novel member of the IkappaB family, human IkappaB-zeta, was identified by a differential screening approach of apoptosis-sensitive and -resistant tumor cells. The protein consists of 6 ankyrin repeats at its COOH terminus and shares about 30% identity with other IkappaB members. IkappaB-zeta associates with both the p65 and p50 subunit of NF-kappaB and inhibits the transcriptional activity as well as the DNA binding of the transcription factor. Interestingly, IkappaB-zeta is localized in the nucleus where it aggregates in matrix-associated deacetylase bodies, indicating that IkappaB-zeta regulates nuclear NF-kappaB activity rather than its nuclear translocation from the cytoplasm. IkappaB-zeta expression itself was regulated by NF-kappaB, suggesting that its activity is controlled in a negative feedback loop. Unlike classical IkappaB proteins, IkappaB-zeta was not degraded upon cell stimulation. Treatment with tumor necrosis factor-alpha, interleukin-1beta, and lipopolysaccharide induced a strong induction of IkappaB-zeta transcripts. Expression of IkappaB-zeta was detected in different tissues including lung, liver, and in leukocytes but not in the brain. Suppression of endogenous IkappaB-zeta by RNA interference rendered cells more resistant to apoptosis, whereas overexpression of IkappaB-zeta was sufficient to induce cell death. Our results, therefore, suggest that IkappaB-zeta functions as an additional regulator of NF-kappaB activity and, hence, provides another control level for the activation of NF-kappaB-dependent target genes.

The proteasome is a multicatalytic enzyme complex that is responsible for degradation of the vast majority of intracellular proteins. Thus, it is involved in diverse cellular processes such as proliferation, differentiation and apoptosis. Especially its latter function yielded in the development of specific proteasomal inhibitors which have recently entered clinical trials due to their tremendous apoptosis-inducing capability. However, several recent studies including ours provided substantial evidence that a combined treatment of tumors with apoptosis-inducing agents and proteasomal inhibitors might even cause adverse effects leading to a prolonged survival of tumor cells. Based on our model of a biphasic role for the proteasome in apoptosis, we believe that a successful combat of tumors that relies on a combinational therapy with proteasomal inhibitors requires careful evaluation of several critical aspects in order to avoid a friend becoming a foe.

Due to their tremendous apoptosis-inducing potential, proteasomal inhibitors (PIs) have recently entered clinical trials. Here we show, however, that various PIs rescued proliferating tumor cells from death receptor-induced apoptosis. This protection correlated with the stabilization of X-linked IAP (XIAP) and c-FLIP and the inhibition of caspase activation. Together with the observation that PIs could not protect cells expressing XIAP or c-FLIP short interfering RNAs (siRNAs) from death receptor-induced apoptosis, our results demonstrate that PIs mediate their protective effect via the stabilization of these antiapoptotic proteins. Furthermore, we show that once these proteins were eliminated, either by long-term treatment with death receptor ligands or by siRNA-mediated suppression, active caspases accumulated to an even larger extent in the presence of PIs. Together, our data support a biphasic role for the proteasome in apoptosis, as they show that its constitutive activity is crucial for the rapid initiation of the death program by eliminating antiapoptotic proteins, whereas at later stages, the proteasome acts in an antiapoptotic manner due to the proteolysis of caspases. Thus, for a successful PI-based tumor therapy, it is crucial to carefully evaluate basal proteasomal activity and the status of antiapoptotic proteins, as their PI-mediated prolonged stability might even cause adverse effects, leading to the survival of a tumor.

The tumor suppressor protein p53 promotes apoptosis in response to death stimuli by transactivation of target genes and by transcription-independent mechanisms. Recently, it was shown that during apoptosis p53 can specifically translocate to mitochondria, where it physically interacts with and inactivates prosurvival Bcl-2 proteins. In the present study, we therefore investigated the role of mitochondrial translocation of p53 for the stress response of tumor cells. In various cell lines, DNA damage induced by either ionizing irradiation or topoisomerase inhibitors triggered a robust translocation of a fraction of p53 to mitochondria to a similar extent. Nevertheless, the cells succumbed to apoptosis only in response to topoisomerase inhibitors, but remained resistant to apoptosis induced by ionizing radiation. Irradiated cells became senescent, although irradiation triggered a functional p53 response and induced expression of p21, Bax, and Puma. Interestingly, even the targeted expression of p53 to mitochondria was insufficient to launch apoptosis, whereas overexpression of wild-type p53 induced Bax activation and apoptotic alterations. Together, these results suggest that, in contrast to previous reports, mitochondrial translocation of p53 does not per se lead to cell death and that this might constitute a mechanism that contributes to the resistance of tumor cells to ionizing radiation-induced apoptosis.

Although signaling by death receptors involves the recruitment of common components into their death-inducing signaling complexes (DISCs), apoptosis susceptibility of various tumor cells to each individual receptor differs quite dramatically. Recently it was shown that, besides caspase-8, caspase-10 is also recruited to the DISCs, but its function in death receptor signaling remains unknown. Here we show that expression of caspase-10 sensitizes MCF-7 breast carcinoma cells to TRAIL- but not tumor necrosis factor (TNF)-induced apoptosis. This sensitization is most obvious at low TRAIL concentrations or when apoptosis is assessed at early time points. Caspase-10-mediated sensitization for TRAIL-induced apoptosis appears to be dependent on caspase-3, as expression of caspase-10 in MCF-7/casp-3 cells but not in caspase-3-deficient MCF-7 cells overcomes TRAIL resistance. Interestingly, neutralization of TRAIL receptor 2 (TRAIL-R2), but not TRAIL-R1, impaired apoptosis in a caspase-10-dependent manner, indicating that caspase-10 enhances TRAIL-R2-induced cell death. Furthermore, whereas processing of caspase-10 was delayed in TNF-treated cells, TRAIL triggered a very rapid activation of caspase-10 and -3. Therefore, we propose a model in which caspase-10 is a crucial component during TRAIL-mediated apoptosis that in addition actively requires caspase-3. This might be especially important in systems where only low TRAIL concentrations are supplied that are not sufficient for the fast recruitment of caspase-8 to the DISC.

Proteases of the caspase family are thought to be activated by proteolytic processing of their inactive zymogens. However, although proteolytic cleavage is sufficient for executioner caspases, a different mechanism has been recently proposed for initiator caspases, such as caspase-8, which are believed to be activated by proximity-induced dimerization. According to this model, dimerization rather than proteolytic processing is considered as the critical event for caspase-8 activation. Such a mechanism would suggest that in the absence of a dimerization platform such as the death-inducing signaling complex, caspase-8 proteolytic cleavage would result in an inactive enzyme. As several studies have described caspase-8 cleavage during mitochondrial apoptosis, we now investigated whether caspase-8 becomes indeed catalytically active in this pathway. Using an in vivo affinity labeling approach, we demonstrate that caspase-8 is activated in etoposide-treated cells in vivo in the absence of the receptor-induced death-inducing signaling complex formation. Furthermore, we show that both caspase-3 and -6 are required for the efficient activation of caspase-8. Our data therefore indicate that interchain cleavage of caspase-8 in the mitochondrial pathway is sufficient to produce an active enzyme even in the absence of receptor-driven procaspase-8 dimerization.

We have shown previously that ionizing radiation (IR) induces a persistent G(2)-M arrest but not cell death in MCF-7 breast carcinoma cells that harbor functional p53 but lack caspase-3. In the present study, we investigated the mechanisms of apoptosis resistance and the roles of p53, caspase-3, and cell cycle arrest in IR-induced apoptosis. The methylxanthine caffeine and the staurosporine analog UCN-01, which can inhibit ATM and Chk kinases, efficiently abrogated the IR-induced G(2)-M arrest and induced mitochondrial activation as judged by the loss of the mitochondrial membrane potential and the release of cytochrome c and Smac/Diablo. However, despite these proapoptotic alterations, cell death and activation of the initiator caspase-9 were not induced in MCF-7 cells but were interestingly only observed after reexpression of caspase-3. Sensitization to IR-induced apoptosis by caffeine or UCN-01 was abrogated neither by cycloheximide nor by pifithrin-alpha, an inhibitor of the transcriptional activity of p53. Furthermore, suppression of p53 by RNA interference could not prevent caffeine- and IR-induced mitochondrial alterations and apoptosis but resulted in an even more pronounced G(2)-M arrest. Collectively, our results clearly show that the resistance of MCF-7 cells to IR-induced apoptosis is caused by two independent events; one of them is a caffeine- or UCN-01-inhibitable event that does not depend on p53 or a release of the G(2)-M arrest. The second event is the loss of caspase-3 that surprisingly seems essential for a fully functional caspase-9 pathway, even despite the previous release of mitochondrial proapoptotic proteins.

Recent data suggest that alpha-toxin, the major hemolysin of Staphylococcus aureus, induces cell death via the classical apoptotic pathway. Here we demonstrate, however, that although zVAD-fmk or overexpression of Bcl-2 completely abrogated caspase activation and internucleosomal DNA fragmentation, they did not significantly affect alpha-toxin-induced death of Jurkat T or MCF-7 breast carcinoma cells. Caspase inhibition had also no effect on alpha-toxin-induced lactate dehydrogenase release and ATP depletion. Furthermore, whereas early assessment of apoptosis induction by CD95 resulted solely in the generation of cells positive for active caspases that were, however, not yet permeable for propidium iodide, a substantial proportion of alpha-toxin-treated cells were positive for both active caspases and PI. Finally, electron microscopy demonstrated that even in the presence of active caspases, alpha-toxin-treated cells displayed a necrotic morphology characterized by cell swelling and cytoplasmic vacuolation. Together, our data suggest that alpha-toxin-induced cell death proceeds even in the presence of activated caspases, at least partially, in a caspase-independent, necrotic-like manner.

Cyclooxygenase-2 (COX-2) is involved in diverse processes such as inflammation, carcinogenesis and apoptosis. As COX-2 inhibitors interfere with these processes, inhibition of COX-2 has been suggested as a promising anticancer treatment. However, the role of COX-2 in modulation of apoptosis as well as the death pathways affected by COX-2 inhibitors are poorly characterized. Here we demonstrate that the selective COX-2 inhibitors NS-398 and nimesulide increased TNF sensitivity of TNF-resistant HeLa H21 and TNF-sensitive HeLa D98 cells, although this cytokine induced significant COX-2 activity, as judged by prostaglandin E(2) (PGE(2)) production, only in H21 cells. TNF did also not induce PGE(2) production in MCF-7/casp-3 cells stably expressing COX-2; however, nimesulide strongly enhanced TNF-induced apoptosis in these cells. Furthermore, COX-2 activity in HeLa H21 cells could be inhibited by NS-398 concentrations that were 10 000-fold lower compared to those required for the induction of cell death. Most intriguingly, sensibilization to apoptosis was specifically observed in response to activation of death receptors. Not only TNF-induced cell death but also apoptosis triggered by the CD95 and TRAIL receptors was enhanced by nimesulide. In contrast, apoptosis induced by the anticancer drugs doxorubicine and etoposide that target the mitochondrial death pathway remained unaffected. Together, our data suggest that COX-2 inhibitors overcome apoptosis resistance and selectively sensitize tumor cells to the extrinsic death receptor-induced apoptotic pathway independently of COX-2.

Radioactive assays are commonly employed to monitor protein or peptide phosphorylation. They not only have all the disadvantages related to radioactivity, but also require large amounts of sample. An alternative is the use of mass spectrometric peptide mapping with sensitivities in the fmole range. We demonstrate here that desalting is a requirement for reproducible results, and we optimized the method for very hydrophilic peptide substrates. The method is very efficient with respect to time and effort.

Activation of caspases has been demonstrated to be involved in thrombocytopenia and prolonged storage of platelet concentrates. Platelets represent enucleate cells that comprise all elements of the mitochondrial apoptosis pathway. However, no apoptotic stimuli capable of activating the endogenous caspase cascade have been identified so far. Using tributyltin (TBT) we could identify a compound that is capable of activating caspase-9 and -3 in platelets. Recent studies implicate that TBT induces apoptosis via the mitochondrial signaling pathway that is characterized by the formation of a high-molecular-weight complex (apoptosome) containing the adapter protein Apaf-1 and active caspase-9. Interestingly, addition of TBT induced the activation of caspase-9 in an ultra-rapid kinetic within the first 2 min. In addition, size exclusion chromatography revealed that TBT-mediated processing of caspase-9 occurs in the absence of the apoptosome. Thus, these data implicate that TBT induces the activation of caspase-9 by a mechanism not involving the formation of the apoptosome.

Apoptotic cell death is executed by the caspase-mediated cleavage of various vital proteins. Elucidating the consequences of this endoproteolytic cleavage is crucial for our understanding of cell death and other biological processes. Many caspase substrates are just cleaved as bystanders, because they happen to contain a caspase cleavage site in their sequence. Several targets, however, have a discrete function in propagation of the cell death process. Many structural and regulatory proteins are inactivated by caspases, while other substrates can be activated. In most cases, the consequences of this gain-of-function are poorly understood. Caspase substrates can regulate the key morphological changes in apoptosis. Several caspase substrates also act as transducers and amplifiers that determine the apoptotic threshold and cell fate. This review summarizes the known caspase substrates comprising a bewildering list of more than 280 different proteins. We highlight some recent aspects inferred by the cleavage of certain proteins in apoptosis. We also discuss emerging themes of caspase cleavage in other forms of cell death and, in particular, in apparently unrelated processes, such as cell cycle regulation and cellular differentiation.

Infections with Staphylococcus aureus, a common inducer of septic and toxic shock, often result in tissue damage and death of various cell types. Although S. aureus was suggested to induce apoptosis, the underlying signal transduction pathways remained elusive. We show that caspase activation and DNA fragmentation were induced not only when Jurkat T cells were infected with intact bacteria, but also after treatment with supernatants of various S. aureus strains. We also demonstrate that S. aureus-induced cell death and caspase activation were mediated by alpha-toxin, a major cytotoxin of S. aureus, since both events were abrogated by two different anti-alpha-toxin antibodies and could not be induced with supernatants of an alpha-toxin-deficient S. aureus strain. Furthermore, alpha-toxin-induced caspase activation in CD95-resistant Jurkat sublines lacking CD95, Fas-activated death domain, or caspase-8 but not in cells stably expressing the antiapoptotic protein Bcl-2. Together with our finding that alpha-toxin induces cytochrome c release in intact cells and, interestingly, also from isolated mitochondria in a Bcl-2-controlled manner, our results demonstrate that S. aureus alpha-toxin triggers caspase activation via the intrinsic death pathway independently of death receptors. Hence, our findings clearly define a signaling pathway used in S. aureus-induced cytotoxicity and may provide a molecular rationale for future therapeutic interventions in bacterial infections.

There is considerable evidence that ionizing radiation (IR) and chemotherapeutic drugs mediate apoptosis through the intrinsic death pathway via the release of mitochondrial cytochrome c and activation of caspases -9 and -3. Here we show that MCF-7 cells that lack caspase-3 undergo a caspase-dependent apoptotic cell death in the absence of DNA fragmentation and alpha-fodrin cleavage following treatment with etoposide or doxorubicin, but not after exposure to IR. Re-expression of caspase-3 restored DNA fragmentation and alpha-fodrin cleavage following drug treatment, but it did not alter the radiation-resistant phenotype of these cells. In contrast to the anticancer drugs, IR failed to induce the intrinsic death pathway in MCF-7/casp-3 cells, an event readily observed in IR-induced apoptosis of HeLa cells. Although IR-induced DNA double-strand breaks were repaired with similar efficiencies in all cell lines, cell cycle analyses revealed a persistent G2/M arrest in the two MCF-7 cell lines, but not in HeLa cells. Together, our data demonstrate that caspase-3 is required for DNA fragmentation and alpha-fodrin cleavage in drug-induced apoptosis and that the intrinsic death pathway is fully functional in MCF-7 cells. Furthermore, they show that the radiation-resistant phenotype of MCF-7 cells is not due to the lack of caspase-3, but is caused by the failure of IR to activate the intrinsic death pathway. We propose (1) different signaling pathways are induced by anticancer drugs and IR, and (2) IR-induced G2/M arrest prevents the generation of an apoptotic signal required for the activation of the intrinsic death pathway.

Proteases of the caspase family constitute the central executioners of apoptosis. Several recent observations suggest that caspases and apoptosis-regulatory molecules exert important functions beyond that of cell death, including the control of T-cell proliferation and cell-cycle progression. Here, Los and colleagues propose a model that directly connects cell suicide mechanisms to the regulation of cell-cycle progression.

Caspase-8 plays an essential role in apoptosis triggered by death receptors. Through the cleavage of Bid, a proapoptotic Bcl-2 member, it further activates the mitochondrial cytochrome c/Apaf-1 pathway. Because caspase-8 can be processed also by anticancer drugs independently of death receptors, we investigated its exact role and order in the caspase cascade. We show that in Jurkat cells either deficient for caspase-8 or overexpressing its inhibitor c-FLIP apoptosis mediated by CD95, but not by anticancer drugs was inhibited. In the absence of active caspase-8, anticancer drugs still induced the processing of caspase-9, -3 and Bid, indicating that Bid cleavage does not require caspase-8. Overexpression of Bcl-x(L) prevented the processing of caspase-8 as well as caspase-9, -6 and Bid in response to drugs, but was less effective in CD95-induced apoptosis. Similar responses were observed by overexpression of a dominant-negative caspase-9 mutant. To further determine the order of caspase-8 activation, we employed MCF7 cells lacking caspase-3. In contrast to caspase-9 that was cleaved in these cells, anticancer drugs induced caspase-8 activation only in caspase-3 transfected MCF7 cells. Thus, our data indicate that, unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 rather functions as an amplifying executioner caspase.

In this study, we sought to investigate in more detail the role of caspase-3 in apoptotic processes in cultured cells and in cell-free extracts of breast cancer cells. We present evidence that apoptosis of caspase-3-deficient MCF-7 breast cancer cells is defective in response to cisplatin treatment, as determined by chromatin condensation, nuclear fragmentation, DNA fragmentation, and release of cytochrome c from the mitochondria. Reconstitution of MCF-7 cells by stable transfection of CASP-3 cDNA restores all these defects and results in an extensive apoptosis after cisplatin treatment. We further show that in extracts from caspase-3-deficient MCF-7 cells, procaspase-9 processing is strongly impaired after stimulation with either cytochrome c or recombinant caspase-8. Reconstitution of MCF-7 cell extracts with procaspase-3 corrects this defect, resulting in an efficient and complete processing of procaspase-9. Together, our data define caspase-3 as an important integrator of the apoptotic process in MCF-7 breast cancer cells and reveal an essential function of caspase-3 for procaspase-9 processing.