Triggering of a novel intrinsic apoptosis pathway by the kinase inhibitor staurosporine: activation of caspase-9 in the absence of Apaf-1.

The protein kinase inhibitor staurosporine is one of the most potent and frequently used proapoptotic stimuli, although its mechanism of action is poorly understood. Here, we show that staurosporine as well as its analog 7-hydroxystaurosporine (UCN-01) not only trigger the classical mitochondrial apoptosis pathway but, moreover, activate an additional novel intrinsic apoptosis pathway. Unlike conventional anticancer drugs, staurosporine and UCN-01 induced apoptosis in a variety of tumor cells overexpressing the apoptosis inhibitors Bcl-2 and Bcl-x(L). Furthermore, activation of this novel intrinsic apoptosis pathway by staurosporine did not rely on Apaf-1 and apoptosome formation, an essential requirement for the mitochondrial pathway. Nevertheless, as demonstrated in caspase-9-deficient murine embryonic fibroblasts, human lymphoma cells, and chicken DT40 cells, staurosporine-induced apoptosis was essentially mediated by caspase-9. Our results therefore suggest that, in addition to the classical cytochrome c/Apaf-1-dependent pathway of caspase-9 activation, staurosporine can induce caspase-9 activation and apoptosis independently of the apoptosome. Since staurosporine derivatives have proven efficacy in clinical trials, activation of this novel pathway might represent a powerful target to induce apoptosis in multidrug-resistant tumor cells.

AMPK-independent induction of autophagy by cytosolic Ca2+ increase.

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca(2+) can induce autophagy by inhibition of mTOR via the CaMKK-alpha/beta-mediated activation of AMPK. Here we demonstrate that Ca(2+) signaling can additionally induce autophagy independently of the Ca(2+)-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca(2+) induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca(2+)-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca(2+) by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca(2+)-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca(2+) flux modifications. Importantly, using AMPK-alpha1(-/-)alpha2(-/-) MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-alpha/beta inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca(2+)-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.

The Akt inhibitor triciribine sensitizes prostate carcinoma cells to TRAIL-induced apoptosis.

Aberrant PI3K/Akt signaling has been implicated in many human cancers, including prostate carcinomas. Currently different therapeutic strategies target the inhibition of this survival pathway. The nucleoside analog triciribine (TCN), which was initially described as a DNA synthesis inhibitor, has recently been shown to function as an inhibitor of Akt. Here, we demonstrate that TCN inhibits Akt phosphorylation at Thr308 and Ser473 and Akt activity in the human prostate cancer cell line PC-3. In addition, TCN sensitized PC-3 cells to TRAIL- and anti-CD95-induced apoptosis, whereas the cells remained resistant to DNA damaging chemotherapeutics. The observed sensitization essentially depended on the phosphorylation status of Akt. Thus, prostate cancer cell lines displaying constitutively active Akt, e.g. PC-3 or LNCaP, were sensitized to death receptor-induced apoptosis. Most importantly with respect to therapeutic application, derivatives of both TCN and TRAIL are already tested in current clinical trials. Therefore, this combinatorial treatment might open a promising therapeutic approach for the elimination of hormone-refractory prostate cancers, which are largely resistant to conventional DNA damaging anticancer drugs or irradiation.

Effects of bacterial N-acyl homoserine lactones on human Jurkat T lymphocytes-OdDHL induces apoptosis via the mitochondrial pathway.

Diverse Gram-negative bacteria communicate with each other by using diffusible N-acyl-homoserine lactone (AHL) signaling molecules to coordinate gene expression with cell population density. This mechanism termed 'quorum sensing' is involved in the regulation of physiological functions as well as multiple virulence determinants. It becomes more and more evident, that bacteria communicate not only with each other but also with their host. Up to now, little is known about this interkingdom communication. The AHL quorum sensing molecule N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL) from Pseudomonas aeruginosa has been shown to influence the immune system of the host. The role and potential influence of other AHL molecules from other bacteria have so far not been determined. In this paper, we investigated the role of 7 different AHLs on apoptosis of human Jurkat T lymphocytes. We found, that among all homoserine lactones tested, only OdDHL rapidly induced apoptosis which was accompanied by the breakdown of the mitochondrial transmembrane potential (DeltaPsi(m)). Since overexpression of anti-apoptotic Bcl-2 completely abrogated the apoptotic effect, we presume that OdDHL induces apoptosis by activation of the intrinsic mitochondrial apoptosis pathway. The reason that bacteria induce apoptosis is largely unknown. We suspect that through apoptosis an anti-inflammatory response is triggered.

Identification of a basolateral sorting signal within the cytoplasmic domain of the interleukin-6 signal transducer gp130.

Interleukin-6-type cytokine receptors are expressed in polarized cells such as hepatocytes and intestinal cells. For the interleukin-6-receptor gp80 and its signal transducer gp130, a preferential basolateral localization was demonstrated in Madin-Darby canine kidney (MDCK) cells and two basolateral sorting signals were identified within the cytoplasmic domain of gp80. The cytoplasmic tail of gp130 is responsible for signaling via the Janus kinase/signal transducer and activator of transcription pathway. In addition, it mediates the internalization of the receptor complex which is dependent on a di-leucine motif. Truncated gp130 lacking the cytoplasmic domain is sorted apically in MDCK cells. For identification of the basolateral sorting signal(s) of gp130, a series of deletion mutants in the cytoplasmic domain of gp130 have been generated and stably expressed in MDCK cells. Biotinylation analyses of these mutants show that a ten amino acids sequence between amino acids 782 and 792 which contains the di-leucine internalization motif is also essential for a basolateral sorting. Accordingly, we detect apical delivery of a gp130 mutant in which the di-leucine motif has been exchanged by two alanines (gp130LL/AA). These findings indicate that the di-leucine motif which directs the internalization of the IL-6 receptor complex also mediates the basolateral sorting of the signal transducer gp130.