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Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live- cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.

Inhibition of the mitochondrial metabolism offers a promising therapeutic approach for the treatment of cancer. Here, we identify the mycotoxin viriditoxin (VDT), derived from the endophytic fungus Cladosporium cladosporioides, as an interesting candidate for leukemia and lymphoma treatment. VDT displayed a high cytotoxic potential and rapid kinetics of caspase activation in Jurkat leukemia and Ramos lymphoma cells in contrast to solid tumor cells that were affected to a much lesser extent. Most remarkably, human hematopoietic stem and progenitor cells and peripheral blood mononuclear cells derived from healthy donors were profoundly resilient to VDT-induced cytotoxicity. Likewise, the colony-forming capacity was affected only at very high concentrations, which provides a therapeutic window for cancer treatment. Intriguingly, VDT could directly activate the mitochondrial apoptosis pathway in leukemia cells in the presence of antiapoptotic Bcl-2 proteins. The mitochondrial toxicity of VDT was further confirmed by inhibition of mitochondrial respiration, breakdown of the mitochondrial membrane potential (ΔΨm), the release of mitochondrial cytochrome c, generation of reactive oxygen species (ROS), processing of the dynamin-like GTPase OPA1 and subsequent fission of mitochondria. Thus, VDT-mediated targeting of mitochondrial oxidative phosphorylation (OXPHOS) might represent a promising therapeutic approach for the treatment of leukemia and lymphoma without affecting hematopoietic stem and progenitor cells.

The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an ‘assembly chaperone’ by building up a stable precursor Sm protein ring structure. Here, we show that—apart from its autophagic role—the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.

Five sesterterpenes (1–5) including two new compounds (1 and 2), as well as a new (6) and a known macrolide (7) were isolated from the endophytic fungus Aplosporella javeedii. The structures of the new compounds were elucidated by analysis of their 1D and 2D NMR and HRMS data as well as by comparison with the literature. Compound 4 and its acetyl derivatives 4a, 4b, 4c which were prepared by acetylation of 4 exhibited moderate cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values ranging from 6.2 to 12.8 μM, respectively. Moreover, 4a and 4c exhibited also cytotoxicity against human leukemia (Jurkat J16) and lymphoma (Ramos) cell lines. Compound 7 showed strong cytotoxicity against the L5178Y cell line, as well as against human Jurkat J16 and Ramos cells with IC50 values of 0.4, 5.8, and 4.4 μM, respectively. Mechanistic studies indicated that 7 induces apoptotic cell death. In addition, compounds 3, 4 and 7 showed low antibacterial activities against Mycobacterium tuberculosis H37Rv and compound 6 against Staphylococcus aureus, respectively, with MICs of 100 μM. Preliminary structure-activity relationships are discussed.

Phomoxanthone A, a bioactive xanthone dimer isolated from the endophytic fungus Phomopsis sp., is a mitochondrial toxin weakening cellular respiration and electron transport chain activity by a fast breakup of the mitochondrial assembly. Here, a multi-disciplinary strategy has been developed and applied for identifying phomoxanthone A target(s) to fully address its mechanism of action, based on drug affinity response target stability and targeted limited proteolysis. Both approaches point to the identification of carbamoyl-phosphate synthase 1 as a major phomoxanthone A target in mitochondria cell lysates, giving also detailed insights into the ligand/target interaction sites by molecular docking and assessing an interesting phomoxanthone A stimulating activity on carbamoyl-phosphate synthase 1. Thus, phomoxanthone A can be regarded as an inspiring molecule for the development of new leads in counteracting hyperammonemia states.

Mitochondria are cellular organelles with crucial functions in the generation and distribution of ATP, the buffering of cytosolic Ca2+ and the initiation of apoptosis. Compounds that interfere with these functions are termed mitochondrial toxins, many of which are derived from microbes, such as antimycin A, oligomycin A, and ionomycin. Here, we identify the mycotoxin phomoxanthone A (PXA), derived from the endophytic fungus Phomopsis longicolla, as a mitochondrial toxin. We show that PXA elicits a strong release of Ca2+ from the mitochondria but not from the ER. In addition, PXA depolarises the mitochondria similarly to protonophoric uncouplers such as CCCP, yet unlike these, it does not increase but rather inhibits cellular respiration and electron transport chain activity. The respiration-dependent mitochondrial network structure rapidly collapses into fragments upon PXA treatment. Surprisingly, this fragmentation is independent from the canonical mitochondrial fission and fusion mediators DRP1 and OPA1, and exclusively affects the inner mitochondrial membrane, leading to cristae disruption, release of pro-apoptotic proteins, and apoptosis. Taken together, our results suggest that PXA is a mitochondrial toxin with a novel mode of action that might prove a useful tool for the study of mitochondrial ion homoeostasis and membrane dynamics.

There is a variety of antineoplastic drugs that are based on natural compounds from ecological niches with high evolutionary pressure. We used two cell lines (Jurkat J16 and Ramos) in a screening to assess 300 different naturally occurring compounds with regard to their antineoplastic activity. The results of the compounds 4,6-dibromo-2-(2&prime, 4&prime, dibromophenoxy)phenol (P01F03), 4,5,6-tribromo-2-(2&prime, dibromophenoxy)phenol (P01F08), and 5-epi-nakijinone Q (P03F03) prompted us to perform further research. Using viability and apoptosis assays on the cell lines of primary human leukemic and normal hematopoietic cells, we found that P01F08 induced apoptosis in the cell lines at IC50 values between 1.61 and 2.95 &mu, M after 72 h. IC50 values of peripheral blood mononuclear cells (PBMNCs) from healthy donors were higher, demonstrating that the cytotoxicity in the cell lines reached 50%, while normal PBMNCs were hardly affected. The colony-forming unit assay showed that the hematopoietic progenitor cells were not significantly affected in their growth by P01F08 at a concentration of 3 &mu, M. P01F08 showed a 3.2-fold lower IC50 value in primary leukemic cells [acute myeloid leukemia (AML)] compared to the PBMNC of healthy donors. We could confirm the antineoplastic effect of 5-epi-nakijinone Q (P03F03) on the cell lines via the induction of apoptosis but noted a similarly strong cytotoxic effect on normal PBMNCs.

A new epidithiodiketopiperazine (ETP), pretrichodermamide G (

Two azaphilone pigments (1 and 2), two dihydrobenzofurans (3 and 4), two macrodiolides (5 and 6), and a dimeric alkyl aromatic constituent (7) were isolated from the goose dung-derived fungus Coniella fragariae. Compounds 1–3 proved to be new natural products. Coniellins H and I (1 and 2) feature a tetracyclic core and an aldehyde group at C-5, which is unusual for azaphilone derivatives. The X-ray structure of pyrenophorin (5) is reported for the first time. Pyrenophorin (5) showed strong cytotoxicity against several cancer cell lines with IC50 values ranging from 0.07 to 7.8 μM.

3-(Hetero)aryl substituted 7-azaindoles possessing multikinase inhibitor activity are readily accessed in a one-pot Masuda borylation-Suzuki coupling sequence. Several promising derivatives were identified as apoptosis inducers and, emphasizing the multikinase inhibition potential, as sphingosine kinase 2 inhibitors. Our measurements provide additional insights into the structure-activity relationship of meriolin derivatives, suggesting derivatives bearing a pyridine moiety with amino groups in 2-position as most active anticancer compounds and thus as highly promising candidates for future in vivo studies.

Polybrominated diphenyl ethers (PBDEs) are a group of molecules with an ambiguous background in literature. PBDEs were first isolated from marine sponges of Dysidea species in 1981 and have been under continuous research to the present day. This article summarizes the two research aspects, (i) the marine compound chemistry research dealing with naturally produced PBDEs and (ii) the environmental toxicology research dealing with synthetically-produced brominated flame-retardant PBDEs. The different bioactivity patterns are set in relation to the structural similarities and dissimilarities between both groups. In addition, this article gives a first structure–activity relationship analysis comparing both groups of PBDEs. Moreover, we provide novel data of a promising anticancer therapeutic PBDE (i.e., 4,5,6-tribromo-2-(2′,4′-dibromophenoxy)phenol, termed P01F08). It has been known since 1995 that P01F08 exhibits anticancer activity, but the detailed mechanism remains poorly understood. Only recently, Mayer and colleagues identified a therapeutic window for P01F08, specifically targeting primary malignant cells in a low µM range. To elucidate the mechanistic pathway of cell death induction, we verified and compared its cytotoxicity and apoptosis induction capacity in Ramos and Jurkat lymphoma cells. Moreover, using Jurkat cells overexpressing antiapoptotic Bcl-2, we were able to show that P01F08 induces apoptosis mainly through the intrinsic mitochondrial pathway.

Targeting of oncogene-driven replicative stress as therapeutic option for high-risk medullobastoma was assessed using a panel of medulloblastoma cells differing in their c-Myc expression [i.e. group SHH (c-Myc low) vs. group 3 (c-Myc high)]. High c-Myc levels were associated with hypersensitivity to pharmacological Chk1 and ATR inhibition but not to CDK inhibition nor to conventional (genotoxic) anticancer therapeutics. The enhanced sensitivity of group 3 medulloblastoma cells to Chk1 inhibitors likely results from enhanced damage to intracellular organelles, elevated replicative stress and DNA damage and activation of apoptosis/necrosis. Furthermore, Chk1 inhibition differentially affected c-Myc expression and functions. In c-Myc high cells, Chk1 blockage decreased c-Myc and p-GSK3α protein and increased p21 and GADD45A mRNA expression. By contrast, c-Myc low cells revealed increased p-GSK3β protein and CHOP and DUSP1 mRNA levels. Inhibition of Chk1 sensitized medulloblastoma cells to additional replication stress evoked by cisplatin independent of c-Myc. Importantly, Chk1 inhibition only caused minor toxicity in primary rat neurons in vitro. Collectively, targeting of ATR/Chk1 effectively triggers death in high-risk medulloblastoma, potentiates the anticancer efficacy of cisplatin and is well tolerated in non-cancerous neuronal cells.

Mangrove associated endophytes are treasure chests for bioprospecting especially in light of the need for new anticancer leads that are necessary to overcome drug resistance of cancer cells. This review highlights the potent anti-tumour compound phomoxanthone A (PXA), which represents a tetrahydroxanthone atropisomer derived from the mangrove-associated fungus Phomopsis longicolla. PXA displayed strong anti-tumour activity when tested against a panel of solid (including cisplatin resistant) tumour cell lines or of blood cancer cell lines with IC 50 values in the submicromolar range whereas it was up to 100 folds less active against peripheral blood mononuclear cells (PBMC) from healthy donors. The anti-tumour activity of PXA was demonstrated to be due to an induction of caspase 3 dependent apoptosis. At the same time PXA was shown to activate immune cells such as murine T-lymphocytes, NK cells and macrophages which might help in fighting resistance during cancer chemotherapy. Structure activity studies that involved several naturally occurring as well as semisynthetic derivatives of PXA indicated the position of the biaryl linkage and the acetyl substituents as structural features that are important for the activity of this natural product.

We investigated cytotoxic mechanisms evoked by the spongean alkaloids aaptamine (Aa) and aeroplysinin-1 (Ap), applied alone and in combination with daunorubicin, employing acute myeloid leukemia (AML) cells. Aa and Ap reduced the viability of AML cells in a dose dependent manner with IC50 of 10-20 µM. Ap triggered apoptotic cell death more efficiently than Aa. Both alkaloids increased the protein level of S139-phosphorylated H2AX (γH2AX), which however was independent of the induction of DNA damage. Expression of the senescence markers p21 and p16 was increased, while the phosphorylation level of p-Chk-2 was reduced following Aa treatment. As a function of dose, Aa and Ap protected or sensitized AML cells against daunorubicin. Protection by Aa was paralleled by reduced formation of ROS and lower level of DNA damage. Both Aa and Ap attenuated daunorubicin-stimulated activation of the DNA damage response (DDR) as reflected on the levels of γH2AX, p-Kap-1 and p-Chk-1. Specifically Ap restored the decrease in S10 phosphorylation of histone H3 resulting from daunorubicin treatment. The cytoprotective effects of Aa and Ap were independent of daunorubicin import/export. Both Aa and Ap abrogated daunorubicin-induced accumulation of cells in S-phase. Inhibition of DNA synthesis was specific for Ap. The data show that Aa and Ap have both congruent and agent-specific pleiotropic effects that are preferential for anticancer drugs. Since Ap showed a broader spectrum of anticancer activities, this compound is suggested as novel lead compound for forthcoming in vivo studies elucidating the usefulness of spongean alkaloids in AML therapy.

Excessive neutrophil activation accompanied by delayed apoptotic cell death in inflammatory conditions causes progressive damage of cells and tissues, leading to life-threatening multiple organ dysfunction syndrome. Previous work suggested that circulating serum factors during inflammation are critically involved in the suppression of neutrophil cell death although the identity of these antiapoptotic mediators remained elusive. In this study, we identified the acute phase protein ?-1 Antitrypsin (AAT) as a potent suppressor of staurosporine (STS)-induced apoptosis in human neutrophils through a mechanism implicating caspases-independent pathways. We show here that serum levels of AAT, potentially in part released by stimulated neutrophils, are markedly elevated in major trauma patients suffering from systemic inflammatory response syndrome (SIRS). Notably, AAT depletion from serum increased sensitivity of human neutrophils for STS-induced cell death. In fact, AAT was demonstrated to confer intrinsic apoptosis resistance by preventing PKC/Akt inactivation and subsequent proteasomal degradation of antiapoptotic Mcl-1 protein in response to STS treatment. Neither MAP kinase ERK1/2 nor caspases were found to be involved in AAT-triggered antiapoptotic pathways in neutrophils. In summary, these results establish a novel pivotal role of circulating AAT in mediating survival by antagonizing the proapoptotic action of the PKC inhibitor STS and should be considered for AAT augmentation therapies in future. CA extern

A novel macrolide, callyspongiolide, whose structure was determined by comprehensive analysis of the NMR and HRMS spectra, was isolated from the marine sponge Callyspongia sp. collected in Indonesia. The compound features a carbamate-substituted 14-membered macrocyclic lactone ring with a conjugated structurally unprecedented diene-ynic side chain terminating at a brominated benzene ring. Callyspongiolide showed strong cytotoxicity against human Jurkat J16 T and Ramos B lymphocytes.

The PI3K/PDK1/Akt signaling axis is centrally involved in cellular homeostasis and controls cell growth and proliferation. Due to its key function as regulator of cell survival and metabolism, the dysregulation of this pathway is manifested in several human pathologies including cancers and immunological diseases. Thus, current therapeutic strategies target the components of this signaling cascade. In recent years, numerous feedback loops have been identified that attenuate PI3K/PDK1/Akt-dependent signaling. Here, we report the identification of an additional level of feedback regulation that depends on the negative transcriptional control of phosphatidylinositol 3-kinase (PI3K) class IA subunits. Genetic deletion of 3-phosphoinositide-dependent protein kinase 1 (PDK1) or the pharmacological inhibition of its downstream effectors, that is, Akt and mammalian target of rapamycin (mTOR), relieves this suppression and leads to the upregulation of PI3K subunits, resulting in enhanced generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3). Apparently, this transcriptional induction is mediated by the concerted action of different transcription factor families, including the transcription factors cAMP-responsive element-binding protein and forkhead box O. Collectively, we propose that PDK1 functions as a cellular sensor that balances basal PIP3 generation at levels sufficient for survival but below a threshold being harmful to the cell. Our study suggests that the efficiency of therapies targeting the aberrantly activated PI3K/PDK1/Akt pathway might be increased by the parallel blockade of feedback circuits.

The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF factor 8 (MFG-E8) is a major opsonin for apoptotic cells, and MFG-E8(-/-) mice spontaneously develop a lupus-like disease. Similar to human systemic lupus erythematosus (SLE), the murine disease is associated with an impaired clearance of apoptotic cells. SLE is routinely treated with glucocorticoids (GCs), whose anti-inflammatory effects are consentaneously attributed to the transrepression of pro-inflammatory cytokines. Here, we show that the GC-mediated transactivation of MFG-E8 expression and the concomitantly enhanced elimination of apoptotic cells constitute a novel aspect in this context. Patients with chronic inflammation receiving high-dose prednisone therapy displayed substantially increased MFG-E8 mRNA levels in circulating monocytes. MFG-E8 induction was dependent on the GC receptor and several GC response elements within the MFG-E8 promoter. Most intriguingly, the inhibition of MFG-E8 induction by RNA interference or genetic knockout strongly reduced or completely abolished the phagocytosis-enhancing effect of GCs in vitro and in vivo. Thus, MFG-E8-dependent promotion of apoptotic cell clearance is a novel anti-inflammatory facet of GC treatment and renders MFG-E8 a prospective target for future therapeutic interventions in SLE.

Aim: To develop a safe and efficient method for targeted, anti-apoptotic gene therapy of corneal endothelial cells (CECs). Materials & methods: Magnetofection (MF), a combination of lipofection with magnetic nanoparticles (MNPs; PEI-Mag2, SO-Mag5, PalD1-Mag1), was tested in human CECs and in explanted human corneas. Effects on cell viability and function were investigated. Immunocompatibility was assessed in human peripheral blood mononuclear cells. Results: Silica iron-oxide MNPs (SO-Mag5) combined with X-tremeGENE-HP achieved high transfection efficiency in human CECs and explanted human corneas, without altering cell viability or function. Magnetofection caused no immunomodulatory effects in human peripheral blood mononuclear cells. Magnetofection with anti-apoptotic P35 gene effectively blocked apoptosis in CECs. Conclusion: Magnetofection is a promising tool for gene therapy of corneal endothelial cells with potential for targeted on-site delivery.

Efficient engulfment of apoptotic cells is essential in multi-cellular organisms in order to prevent inflammatory responses. Apoptotic cells secure this process by releasing 'find-me' signals for the attraction of phagocytes. A major 'find-me' signal liberated from apoptotic cells is lysophosphatidylcholine (LPC). So far, however, the mechanisms underlying LPC release are poorly understood. In this study, we demonstrate that pharmacological inhibition and RNAi-mediated knock-down of the lipid transporter ABCA1 in apoptotic cells completely abolished phagocyte attraction. Moreover, ectopic expression of ABCA1 significantly enhanced monocyte migration to supernatants of apoptotic cells. Hence, ABCA1 represents a novel regulator of LPC release during apoptosis.

Annexin A1 is an intracellular calcium/phospholipid-binding protein that is involved in membrane organization and the regulation of the immune system. It has been attributed an anti-inflammatory role at various control levels, and recently we could show that annexin A1 externalization during secondary necrosis provides an important fail-safe mechanism counteracting inflammatory responses when the timely clearance of apoptotic cells has failed. As such, annexin A1 promotes the engulfment of dying cells and dampens the postphagocytic production of proinflammatory cytokines. In our current follow-up study, we report that exposure of annexin A1 during secondary necrosis coincided with proteolytic processing within its unique N-terminal domain by ADAM10. Most importantly, we demonstrate that the released peptide and culture supernatants of secondary necrotic, annexin A1-externalizing cells induced chemoattraction of monocytes, which was clearly reduced in annexin A1- or ADAM10-knockdown cells. Thus, altogether our findings indicate that annexin A1 externalization and its proteolytic processing into a chemotactic peptide represent final events during apoptosis, which after the transition to secondary necrosis contribute to the recruitment of monocytes and the prevention of inflammation.

Unc-51-like kinase 1 (Ulk1) plays a central role in autophagy induction. It forms a stable complex with Atg13 and focal adhesion kinase (FAK) family interacting protein of 200 kDa (FIP 200). This complex is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1) in a nutrient-dependent way. AMP-activated protein kinase (AMPK), which is activated by LKB1/Strad/Mo25 upon high AMP levels, stimulates autophagy by inhibiting mTORC1. Recently, it has been described that AMPK and Ulk1 interact and that the latter is phosphorylated by AMPK. This phosphorylation leads to the direct activation of Ulk1 by AMPK bypassing mTOR-inhibition. Here we report that Ulk1/2 in turn phosphorylates all three subunits of AMPK and thereby negatively regulates its activity. Thus, we propose that Ulk1 is not only involved in the induction of autophagy, but also in terminating signaling events that trigger autophagy. In our model, phosphorylation of AMPK by Ulk1 represents a negative feedback circuit.

Background The cytokine tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown promising anticancer activity in early clinical settings by selectively inducing apoptosis in different tumour types. However, some tumour entities such as hepatocellular carcinoma (HCC) display an inherent resistance to TRAIL. A huge effort has been made to unravel strategies for a clinically applicable sensitisation of resistant cancer cells to TRAIL. Reversible epigenetic alterations such as DNA methylation play a major role in development, maintenance and resistance phenomena of tumour cells. Currently, several clinical trials are exploiting the potential of epigenetic drugs, such as 5-azacytidine (5-aza-CR) or 5-aza-2′-deoxycytidine (5-aza-dC) to break primary or secondary resistance phenomena of cancer cells. Therefore, 5-aza-CR and 5-aza-dC were investigated in the context of TRAIL resistance. Methods Alterations in proliferation, apoptosis, regulatory proteins and toxicity were investigated in TRAIL-resistant hepatoma, and also in renal, colon and pancreatic cancer cells as well as non-transformed human-derived primary hepatocytes, tissue slices isolated from human liver and non-malignant colon cells, all of which had been exposed to demethylating drugs and/or TRAIL. Results Within hours, 5-aza-CR but not 5-aza-dC sensitised in vitro cultured tumour cells to TRAIL, first by activating caspases, followed by a subsequent induction of apoptosis. This surprisingly rapid sensitisation was confirmed in vivo employing a chorioallantoic membrane assay. As a major mechanism, a 5-aza-CR-induced inhibition of cellular protein synthesis was found which led to a breakdown of tumour-protecting factors such as the antiapoptotic factor FLICE inhibitory protein (FLIP). Importantly, TRAIL and 5-aza-CR did not induce relevant toxicity or apoptosis in primary hepatocytes, liver slices from different human donors and in normal colon cells. Conclusions Molecular evidence is provided for a novel 5-aza-CR-based translational approach enabling a twofold treatment of apoptosis-resistant tumour entities, not only by an epigenetic reversion of the malignancy-associated phenotype but also by an efficient resensitisation to apoptosis-inducing substances such as TRAIL.

In multicellular organisms apoptotic cells are rapidly and efficiently removed by professional or semi-professional phagocytes. The molecular mechanisms and the key players involved in this highly coordinate process, as well as its immunological outcome constitute a vividly expanding field of scientific interest. A plethora of studies provided a detailed understanding of the interaction site between the dying cell and the phagocyte, as well as to the current concept that apoptotic cell removal leads to a non- or anti-inflammatory response, whereas necrotic cell removal stimulates a pro-inflammatory reaction. In contrast, our current knowledge about the soluble factors released from apoptotic cells is rather limited, although meanwhile it is generally accepted that not only the dying cell itself but also the substances, which are liberated during cell death, contribute to the process of corpse clearance and the subsequent immune response. This review is intended to summarize the up-to-date knowledge about apoptotic cell-derived attraction signals, their function as phagocytic chemoattractants, their influence on the immune system, and the receptors, which are engaged in this scenario.

Tissue homeostasis in metazoa requires the rapid and efficient clearance of dying cells by professional or semi-professional phagocytes. Impairment of this finely regulated, fundamental process has been implicated in the development of autoimmune diseases, such as systemic lupus erythematosus. Various studies have provided us a detailed understanding of the interaction between dying cells and phagocytes as well as the current concept that apoptotic cell removal leads to a non- or anti-inflammatory response, whereas necrotic cell removal stimulates a pro-inflammatory reaction. In contrast, our knowledge about the soluble factors released from dying cells is rather limited, although meanwhile it is generally accepted that not only the dying cell itself but also the substances liberated during cell death contribute to the process of corpse clearance and the subsequent immune response. This review article is intended as an up-to-date survey over attraction and danger signals of apoptotic, primary and secondary necrotic cells, their function as chemoattractants in phagocyte recruitment, additional effects on the immune system, and the receptors, which are engaged in this scenario.

The course of malaria does not only depend on the virulence of the parasite Plasmodium but also on properties of host erythrocytes. Here, we show that infection of erythrocytes from human sickle cell trait (HbA/S) carriers with ring stages of P. falciparum led to significantly enhanced PGE(2) formation, Ca(2+) permeability, annexin-A7 degradation, phosphatidylserine (PS) exposure at the cell surface, and clearance by macrophages. P. berghei-infected erythrocytes from annexin-A7-deficient (annexin-A7(-/-)) mice were more rapidly cleared than infected wildtype cells. Accordingly, P. berghei-infected annexin-A7(-/-) mice developed less parasitemia than wildtype mice. The cyclooxygenase inhibitor aspirin decreased erythrocyte PS exposure in infected annexin-A7(-/-) mice and abolished the differences of parasitemia and survival between the genotypes. Conversely, the PGE(2)-agonist sulprostone decreased parasitemia and increased survival of wild type mice. In conclusion, PS exposure on erythrocytes results in accelerated clearance of Plasmodium ring stage-infected HbA/S or annexin-A7(-/-) erythrocytes and thus confers partial protection against malaria in vivo.

Rapid advance in the cancer stem cell field warrants optimism for the development of more reliable cancer therapies within the next 2-3 decades. Below, we characterize and compare the specific markers that are present on stem cells, cancer cells and cancer stem cells (CSC) in selected tissues (colon, breast, liver, pancreas, and prostate). It is becomingevident that successful cancer therapies have to eradicate CSC. Thus, strategies aimed at efficient targeting of CSC are becoming vital for monitoring the progress of cancer therapy and evaluating new therapeutic approaches. Therefore, the last part of the review discusses future directions of this intriguing new research field in the context of new diagnostic and therapeutic opportunities.

Phagocytosis of apoptotic cells is fundamentally important throughout life, because non-cleared cells become secondarily necrotic and release intracellular contents, thus instigating inflammatory and autoimmune responses. Secreted "find-me" and exposed "eat-me" signals displayed by the dying cell in concert with the phagocyte receptors comprise the phagocytic synapse of apoptotic cell clearance. In this scenario, lysophospholipids (lysoPLs) are assumed to act as find-me signals for the attraction of phagocytes. However, both the identity of the lyso-PLs released from apoptotic cells and the nature of the phagocyte receptor are largely unknown. By a detailed analysis of the structural requirements we show here that lysophosphatidylcholine (lysoPC), but none of the lysoPC metabolites or other lysoPLs, represents the essential apoptotic attraction signal able to trigger a phagocyte chemotactic response. Furthermore, using RNA interference and expression studies, we demonstrate that the G-protein-coupled receptor G2A, unlike its relative GPR4, is involved in the chemotaxis of monocytic cells. Thus, our study identifies lysoPC and G2A as the crucial receptor/ligand system for the attraction of phagocytes to apoptotic cells and the prevention of autoimmunity.

There is growing evidence that the onset of autoimmune disorders can be linked to the inefficient removal of apoptotic cells. Since defects in the elimination of apoptotic cells lead to secondary necrosis and subsequent release of intracellular components, this might explain the generation of autoantibodies against intracellular antigens. Accordingly, we wanted to investigate, whether antibodies from patients with the autoimmune liver disease primary biliary cirrhosis (PBC) recognize self-proteins generated and released during apoptosis. Using Western blot analyses we could detect intracellular antigens with serum IgG from PBC patients but not with serum IgG from healthy donors in lysates of Jurkat T-leukemia, HepG2 hepatoma, and HT-29 colon-carcinoma cells. Interestingly, PBC serum IgG also recognized caspase substrates in cells undergoing apoptosis induced by staurosporine or TRAIL (TNF-related apoptosis inducing ligand). In addition to intracellular antigens, serum IgG from PBC patients detected caspase-dependent antigens in the supernatants of apoptotic (secondary necrotic) cells and antigens on the surface of apoptotic Jurkat cells. Among the caspase substrates recognized by PBC serum IgG we could identify the components PDC-E2 and -E1beta of the known autoantigen PDC (pyruvate dehydrogenase complex). Thus, caspase-mediated processing of intracellular proteins might generate de novo autoantigens that upon release contribute to the generation of autoantibodies and autoimmune diseases as PBC.

Apoptosis resistance in activated neutrophils is known to be associated with collateral damage of surrounding tissue, as well as immune and organ dysfunction. Thus, the safe removal of neutrophils by apoptosis induction represents a prerequisite for the resolution of inflammation. Here, we report that intrinsic apoptosis resistance in human neutrophils, isolated from severely injured patients, is based on enhanced stabilization of antiapoptotic myeloid cell leukemia 1 and subsequent impairment of downstream apoptotic pathways. Whereas extrinsic apoptosis induction by the activation of Fas death receptor on inflammatory neutrophils was accompanied by caspase- and proteasome-mediated myeloid cell leukemia 1 degradation, intrinsic apoptosis induction by staurosporine led to a significant stabilization of myeloid cell leukemia 1 protein, which impeded on truncated forms of B cell lymphoma 2-associated X protein and B cell lymphoma 2 homology domain 3-interacting domain death translocation and subsequent cytochrome c release from the mitochondria. We show further that profound inhibition of myeloid cell leukemia 1 degradation is based on the inhibition of caspases and sustained activation of kinases involved in cell survival, such as Akt. Accordingly, impeded myeloid cell leukemia 1 phosphorylation on Ser159 by glycogen synthase kinase 3 and protein ubiquitination has been demonstrated. Inhibition of myeloid cell leukemia 1 activity markedly increased sensitivity to staurosporine-induced cell death. Altogether, these results provide new insights into the mechanisms underlying myeloid cell leukemia 1-mediated apoptosis resistance to staurosporine under inflammatory situations and should be considered for the development of novel therapeutic strategies.

In addition to cell shrinkage, membrane blebbing, DNA fragmentation and phosphatidylserine exposure, intracellular acidification represents a hallmark of apoptosis. Although the mechanisms underlying cytosolic acidification during apoptosis remained largely elusive, a pivotal role of mitochondria has been proposed. In order to investigate the involvement of mitochondria in cytosolic acidification during apoptosis, we blocked the mitochondrial death pathway by overexpression of Bcl-2 and subsequently activated the death receptor pathway by anti-CD95 or TRAIL or the mitochondrial pathway by staurosporine. We show that Bcl-2 but not caspase inhibition prevented staurosporine-induced intracellular acidification. Thus, intracellular acidification in mitochondrial apoptosis is a Bcl-2-inhibitable, but caspase-independent process. In contrast, Bcl-2 only slightly delayed, but did not prevent intracellular acidification upon triggering of death receptors. The Na(+)/H(+) exchanger NHE1 was partially degraded during apoptosis but only to a small extent and and at a delayed time point when cytosolic acidification was almost completed. We therefore conclude that cytosolic acidification is mitochondrially controlled in response to mitochondria-dependent death stimuli, but requires additional caspase-dependent mechanisms during death receptor-mediated apoptosis.

Background: Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen. Patients and methods: M4 proteins were purified by affinity chromatography using IgG fractions of PBC marker sera being CFT positive (n=5) or negative (n=5) and identified by Western blotting, silver staining and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 and pyruvate dehydrogenase complex (PDC). Results: Two AMA patterns of the marker sera were visualized: CFT-positive sera were defined as PDC-E2+/E1+ and the CFT-negative sera as PDC-E2+/E1−. The major proteins in the M4 fraction could be related to the PDC-E1 subunits. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could also be documented in the cohort of 57 PBC patients showing anti-PDC-E1α and E1β antibodies at a frequency of 74% and 67%. Conclusions: CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1. As PDC-E1 subunits as compared with PDC-E2 lack lipoyl-binding sites, they probably have to be considered as an independent and important target.

PKCalpha and Ets1 are both associated with breast cancer progression. Our previous studies suggested that these proteins are likely to functionally interact with one another. Here, we show that attenuation of endogenous PKCalpha expression (siPalpha) by RNA interference leads to reduced Ets1 protein expression in a variety of cancer cells. Pulse-chase experiments and treatment with proteasome inhibitor MG-132 revealed that siPalpha interferes with both Ets1 protein synthesis and stability. The effect of siPalpha on Ets1 expression could be partially prevented by KN-93, suggesting that calcium/calmodulin-dependent kinase II (CaMKII), a modulator of Ets1 activity, may play a role in PKCalpha-dependent Ets1 regulation. In contrast, Ets1-regulating kinases ERK1/2 were not found to be involved in this process. To assess the importance of the PKCalpha/Ets1 interaction, we compared the biological responses of MDA-MB-231 cells to PKCalpha- and Ets1-specific siRNAs (siE1). While only siPalpha induced changes in cellular morphology and anchorage-independent growth, both siRNAs similarly affected cellular responses to the antitumor drug mithramycin A and to UV light. Microarray analyses further showed that the expression of a certain set of genes was equally affected by siPalpha and siE1. The data suggest that Ets1 serves as an effector for PKCalpha to fulfil certain functions in cancer cells.

Death receptor-induced apoptosis is paradigmatically mediated via the recruitment of FADD adapter molecule to the ligand/receptor complex and subsequent activation of caspase-8. However, several observations provided evidence that components of the mitochondrial apoptosis pathway are involved in death receptor-mediated apoptosis. In this regard, caspase-8-mediated activation of Bid induces the release of cytochrome c from the mitochondria, which, in turn, triggers the formation of the apoptosome protein complex, resulting in the activation of caspase-9. Whereas Bax or Bak were shown to be required for the proapoptotic effect of Bid, Bcl-2 was described to interfere with its action. Up to now, contradictory results regarding the role of Bcl-2 in TRAIL-induced apoptosis have been published. In order to study the influence of Bcl-2 on TRAIL-induced cell death more detailed, we utilized a tetracycline-regulated Bcl-2 expression system in Jurkat T cells. After having analysed the dose response for TRAIL-induced activation of caspase-8, -9, -3, breakdown of the mitochondrial membrane potential, and changes in the apoptotic morphology in cells expressing different Bcl-2 levels, we conclude that overexpression of Bcl-2 mediates a partial resistance towards lower doses of TRAIL that can be overcome when higher doses of TRAIL are applied. Thus, the requirement of the mitochondrial pathway for death receptor-induced apoptosis in type II cells should be reconsidered, since the protective effect of Bcl-2 is limited to lower TRAIL doses or early observation time points.

Purpose: Overexpression of antiapoptotic Bcl-2 family members has recently been related to resistance to chemo/radiotherapy in several human malignancies, particularly lymphomas. Hence, innovative approaches bypassing this resistance mechanism are required in the therapeutic approach. This study evaluated whether chemoresistance associated with Bcl-2 and Bcl-xL overexpression would be overcome by activating the death receptor pathway by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the Jurkat cell model Experimental Design: We made use of genetically modified Jurkat cells to evaluate the effect of Bcl-2 or Bcl-xL overexpression on the cytotoxic effect produced by the anticancer drugs doxorubicin, etoposide, and oxaliplatin and TRAIL. Caspase activation was detected by cleavage of caspase-8 and -3. The mitochondrial transmambrane potential was assessed by staining with DiOC6 and flow cytometry. Caspase activity was blocked by the broad-spectrum caspase inhibitor zVAD-fmk. Results: Bcl-2 and Bcl-xL overexpression but not lack of caspase-8 protects the Jurkat cells from the anticancer drug-induced cytolysis. However, Bcl-2/Bcl-xL Jurkat cells retained some susceptibility to TRAIL-induced cytolysis. A highly synergistic cytotoxic effect of the combination of TRAIL with any of the antiblastic used in this study was detected in the chemoresistant cells. This effect was associated with mitochondrial disassemblage and dependent on caspase activation Conclusions: The combination of TRAIL with conventional anticancer drugs may prove to be useful in the treatment of antiapoptotic Bcl-2 family proteins-expressing malignancies.

Efficient engulfment of the intact cell corpse is a critical end point of apoptosis, required to prevent secondary necrosis and inflammation. The presentation of “eat-me” signals on the dying cell is an important part of this process of recognition and engulfment by professional phagocytes. Here, we present evidence that apoptotic cells secrete chemotactic factor(s) that stimulate the attraction of monocytic cells and primary macrophages. The activation of caspase-3 in the apoptotic cell was found to be required for the release of this chemotactic factor(s). The putative chemoattractant was identified as the phospholipid, lysophosphatidylcholine. Further analysis showed that lysophosphatidylcholine was released from apoptotic cells due to the caspase-3 mediated activation of the calcium-independent phospholipase A2. These data suggest that in addition to eat-me signals, apoptotic cells display attraction signals to ensure the efficient removal of apoptotic cells and prevent postapoptotic necrosis.

The molecular mechanisms underlying the clinical effects of alpha interferon (IFN) and ribavirin are not understood. Elimination of infected cells occurs in part by cytotoxic T lymphocytes (CTLs) expressing CD95 ligand and thereby attacking target cells which are positive for the death receptor CD95. Since many viruses have evolved mechanisms to inhibit apoptosis, the opposite, namely, promotion of apoptosis, could be a strategy to strengthen the host antiviral response. In the present study, we have asked whether the antiviral substances IFN and ribavirin could support CD95-mediated apoptosis by interfering with the activation of caspases, a family of proteases known for their essential role in apoptosis. HepG2 cells, stimulated with the agonistic anti-CD95 antibody, served as a minimal model to mimic the CD95 stimulation ocurring during a CTL attack of target cells in vivo. Apoptosis was quantitated by flow cytometric detection of hypodiploid nuclei. Caspase activity was measured by cytofluorometry, immunocytochemistry, and immunoblot analysis. IFN and ribavirin sensitized HepG2 cells for CD95-mediated apoptosis. This effect was correlated with an increase in CD95-mediated caspase activation and enhanced cleavage of the caspase substrate poly(ADP-ribose) polymerase. Furthermore, the positive effect on CD95-mediated caspase activation by IFN and ribavirin was confirmed by immunocytochemistry for activated caspase-3 and by immunoblot detection of activated caspase-3, caspase-7, and caspase-8. Our data demonstrate that the antiviral substances IFN and ribavirin are able to sensitize for CD95-mediated apoptosis. IFN and ribavirin also enhance CD95-mediated caspase activation, which might in part be responsible for the apoptosis-promoting effect of these antiviral compounds.

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.

The induction of apoptosis requires the activation of a highly coordinated signaling network ultimately leading to the activation of caspases. In previous experiments we and others have shown that the tyrosine kinase Lck is required for adequate apoptosis induction in response to ionizing radiation, ceramide incubation and overexpression of the HIV-TAT protein. However, the position of Lck within given apoptotic signaling cascades remains unclear. We therefore aimed to define the role of Lck during radiation-induced apoptosis. Apoptosis induction in response to ionizing radiation, CD95 or TRAIL receptor stimulation was determined in Jurkat T-cells, the Lck-deficient Jurkat clone JCaM1.6- and Lck-retransfected JCaM1.6/Lck. No apoptosis, release of cytochrome c, breakdown of the mitochondrial potential were detectable during the first 48 h after irradiation of JCaM1.6 cells. In parallel, no activation of caspase-9, -8 and -3 was detectable. Since mitochondrial apoptosis pathways act within a feedback mechanism during death-receptor-mediated apoptosis, the influence of the Lck defect on CD95/Fas/Apo-1-L or TRAIL-induced apoptosis was also tested. Both stimuli induced apoptosis in Lck-deficient cells. However, the kinetics of apoptosis induction determined by caspase-8, -9 and -3 activation as well as deltapsi(m) breakdown was slowed. We conclude that the Lck deficiency influences early steps during radiation-induced mitochondrial alterations.

Legionella pneumophila has been shown to induce apoptosis within macrophages, monocytic cell lines and alveolar epithelial cells. The mechanisms and significance of L. pneumophila-associated apoptosis are not well understood. It has been speculated that L. pneumophila may induce apoptosis through ligation of death receptors by bacterial surface components or by secreted bacterial factors. Translocation of apoptotic factor(s) through the Dot/Icm secretion machinery followed by direct activation of caspases within the cytosol is discussed as another possible mechanism of apoptosis induction by L. pneumophila. Here, it is shown that L. pneumophila induced the mitochondrial release of cytochrome c in CD95 (Fas/Apo-1)-negative monocytic Mono Mac 6 cells, indicating that Legionella-induced apoptosis is mediated via the mitochondrial signalling pathway. In addition, blocking of the death receptor pathway at distinct stages using CD95-, FADD- or caspase-8-deficient Jurkat cells did not affect induction of apoptosis by L. pneumophila. Conversely, inhibition of the mitochondrial death pathway by overexpression of the anti-apoptotic protein Bcl-2 potently inhibited the processing of caspases and the induction of apoptosis. Therefore, these findings support a model in which the induction of apoptosis by L. pneumophila is mediated by activation of the intrinsic mitochondrial death pathway in the absence of external death receptor signalling.

Objective: Secondary failure due to late restenosis continues to occur in 30–50% of individuals after PTCA. β-Blockers play an important role in the treatment of CAD. The aim of this study was to investigate the effects of the new β-blocker nebivolol on cell proliferation of human coronary smooth muscle cells (haCSMCs) and endothelial cells (haECs) in comparison to traditional β-blockers. Methods: The effect of nebivolol and other β-blockers on proliferation of HaECs and HaCSMCs was analyzed by bromodeoxyuridine incorporation. Apoptosis was measured by determination of hypodiploid DNA in both cell types. Additionally, in HaECs NO formation, endothelin-1 transcription and secretion were determined. Results: Incubation for 1, 2, 4, 7 or 14 days resulted in a concentration- and time-dependent reduction of proliferation up to 80% in HaECs and HaCSMCs. β-Blockers such as propranolol, metoprolol or bisoprolol did not exert this effect. Nebivolol inhibited accelerated haCSMC proliferation even in the presence of growth factors such as TGFβ1 and PDGF-BB. Nebivolol concentration-dependently induced a moderate apoptosis (10−5 mol/l: 23%) and a decrease of haCSMCs in the S-phase by 66%. HaECs showed comparable results. During nebivolol incubation NO formation of HaCEs increased, while endothelin-1 transcription and secretion were suppressed. Conclusion: Whereas classical β-blockers do not affect cell growth, only nebivolol inhibits haCSMC or haEC proliferation and induces a moderate rate of apoptosis. Furthermore, in HaCEs NO formation increases and endothelin-1 secretion decreases suggesting that nebivolol may represent a β-blocker with great promises in CAD therapy.

After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL(△m/△m)) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre(+) FasL(fl/fl) mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis.

Four tetrahydroxanthone dimers (1-4) and four biogenetically related monomers (5-8), including the new derivatives 4-6, were isolated from the endophyte Phomopsis longicolla. The absolute configurations of 2-4 were established for the first time by TDDFT electronic circular dichroism calculations, and that of phomoxanthone A (1) was revised by X-ray crystallography. Phomoxanthone A (1) showed the strongest pro-apoptotic activity when tested against a panel of human cancer cell lines, including cisplatin-resistant cells, whereas it was up to 100-fold less active against healthy blood cells. It was also the most potent activator of murine T lymphocytes, NK cells, and macrophages, suggesting an activation of the immune system in parallel to its pro-apoptotic activity. This dual effect in combating cancer cells could help in fighting resistance during chemotherapy. Preliminary structure-activity studies of isolated compounds and derivatives obtained by semisynthesis (9a-11) hinted at the location of the biaryl axis and the presence of acetyl groups as important structural elements for the biological activity of the studied tetrahydroxanthones.

Cofilin, an actin-depolymerizing protein, is essential for the functional dynamics of the actin cytoskeleton and for cell viability. In unstimulated human peripheral blood T lymphocytes cofilin is phosphorylated and localized in the cytoplasm. Following co-stimulation through accessory receptors (e.g. CD2 or CD28) - however, not following TCR/CD3 stimulation alone - cofilin undergoes dephosphorylation. The subcellular localization as well as the actin-binding activity of cofilin are regulated by the phosphorylation state of serine-3. Thus, only the dephosphorylated form of cofilin associates with the actin cytoskeleton and possesses the capability to translocate into the nucleus. Recently, LIM-kinase 1 was shown to inactivate cofilin through phosphorylation. Here, we have identified the functional counterparts of LIM-kinase 1: the serine/threonine phosphatases of type 1 and type 2A not only associate with cofilin but also dephosphorylate this 19-kDa protein and thereby mediate cofilin activation. In malignant T lymphoma cells, activation of these phosphatases occurs spontaneously, independent of external stimuli. In untransformed human peripheral blood T lymphocytes, these phosphatases function through a cyclosporin A/FK506-resistant co-stimulatory signaling pathway which is common for the accessory receptors CD2 and CD28. This co-stimulatory signaling pathway is also not affected by a series of other clinically established immunosuppressive drugs (i.e. rapamycin, dexamethasone, leflunomide or mycophenolic acid).

Dying cells actively attract professional phagocytes by sending out soluble 'find-me' signals. The characterization and identification of the corresponding molecular entities have revealed that 'find-me' signals come in different flavors, including peptides, proteins, lipids and complex structures, such as apoptotic microblebs. With the very recent report by Elliott et al., nucleotides as a novel nuance enter the menu.

Apoptosis is induced by different stimuli, among them triggering of the death receptor CD95, staurosporine, and chemotherapeutic drugs. In all cases, apoptosis is mediated by caspases, although it is unclear how these diverse apoptotic stimuli cause protease activation. Two regulatory pathways have been recently identified, but it remains unknown whether they are functionally independent or linked to each other. One is mediated by recruitment of the proximal regulator caspase-8 to the death receptor complex. The other pathway is controlled by the release of cytochrome c from mitochondria and the subsequent ATP-dependent activation of the death regulator apoptotic protease-activating factor 1 (Apaf-1). Here, we report that both pathways can be dissected by depletion of intracellular ATP. Prevention of ATP production completely inhibited caspase activation and apoptosis in response to chemotherapeutic drugs and staurosporine. Interestingly, caspase-8, whose function appeared to be restricted to death receptors, was also activated by these drugs under normal conditions, but not after ATP depletion. In contrast, inhibition of ATP production did not affect caspase activation after triggering of CD95. These results suggest that chemotherapeutic drug–induced caspase activation is entirely controlled by a receptor-independent mitochondrial pathway, whereas CD95-induced apoptosis can be regulated by a separate pathway not requiring Apaf-1 function.

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.

The V600E mutation in the kinase BRAF is frequently detected in melanomas and results in constitutive activation of BRAF, which then promotes cell proliferation by the mitogen-activated protein kinase signaling pathway. Although the BRAFV600E kinase inhibitor vemurafenib has remarkable antitumor activity in patients with BRAFV600E-mutated melanoma, its effects are limited by the onset of drug resistance. We found that exposure of melanoma cell lines with the BRAFV600E mutation to vemurafenib decreased the abundance of antiapoptotic proteins and induced intrinsic mitochondrial apoptosis. Vemurafenib-treated melanoma cells showed increased cytosolic concentration of calcium, a potential trigger for endoplasmic reticulum (ER) stress, which can lead to apoptosis. Consistent with an ER stress-induced response, vemurafenib decreased the abundance of the ER chaperone protein glucose-regulated protein 78, increased the abundance of the spliced isoform of the transcription factor X-box binding protein 1 (XBP1) (which transcriptionally activates genes involved in ER stress responses), increased the phosphorylation of the translation initiation factor eIF2α (which would be expected to inhibit protein synthesis), and induced the expression of ER stress-related genes. Knockdown of the ER stress response protein activating transcription factor 4 (ATF4) significantly reduced vemurafenib-induced apoptosis. Moreover, the ER stress inducer thapsigargin prevented invasive growth of tumors formed from vemurafenib-sensitive melanoma cells in vivo. In melanoma cells with low sensitivity or resistance to vemurafenib, combination treatment with thapsigargin augmented or induced apoptosis. Thus, thapsigargin or other inducers of ER stress may be useful in combination therapies to overcome vemurafenib resistance.

The elimination of apoptotic cells, called efferocytosis, is fundamentally important for tissue homeostasis and prevents the onset of inflammation and autoimmunity. Serum proteins are known to assist in this complex process. In the current study, we performed a multistep chromatographic fractionation of human serum and identified plasminogen, a protein involved in fibrinolysis, wound healing, and tissue remodeling, as a novel serum-derived factor promoting apoptotic cell removal. Even at levels significantly lower than its serum concentration, purified plasminogen strongly enhanced apoptotic prey cell internalization by macrophages. Plasminogen acted mainly on prey cells, whereas on macrophages no enhancement of the engulfment process was observed. We further demonstrate that the efferocytosis-promoting activity essentially required the proteolytic activation of plasminogen and was completely abrogated by the urokinase plasminogen activator inhibitor-1 and serine protease inhibitor aprotinin. Thus, our study assigns a new function to plasminogen and plasmin in apoptotic cell clearance.

Resting T lymphocytes that have recognized antigen bound to a major histocompatibility complex molecule with the T-cell receptor require costimulatory signals through accessory receptors, including CD2, CD4, CD8, and CD28, for their clonal growth and expression of their functional repertoires. Absence of costimulation, in contrast, can induce clonal anergy in vitro and selective tolerance in vivo. Here we have defined a potential intracellular messenger for T-cell activation which is strictly regulated by costimulatory signals mediated through accessory receptors: pp19/cofilin, a small actin-binding protein, undergoes dephosphorylation and subsequent translocation from the cytosol into the nucleus. In untransformed T cells this process correlates with functional responses essential for the induction of T-cell proliferation (i.e., production of interleukin 2). Moreover, spontaneous dephosphorylation as well as nuclear translocation of pp19/cofilin occur in the autonomously proliferating T-lymphoma cell line Jurkat.