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Reiner U Jänicke

Publications and source records attributed to Reiner U Jänicke.

13 recordsLinked to original sources
PubMed2006

p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation.

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.

Apoptosis↗
PubMed2006

Cell death, caspase activation, and HMGB1 release of porcine choroid plexus epithelial cells during Streptococcus suis infection in vitro.

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.

Animals↗
PubMed2006

Friend or foe? The proteasome in combined cancer therapy.

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.

Animals↗
PubMed2006

A novel member of the IkappaB family, human IkappaB-zeta, inhibits transactivation of p65 and its DNA binding.

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.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The proteasome is required for rapid initiation of death receptor-induced apoptosis.

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.

Apoptosis↗
PubMed2005

Irradiation-induced translocation of p53 to mitochondria in the absence of apoptosis.

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.

Animals↗
PubMed2005

Caspase-10 sensitizes breast carcinoma cells to TRAIL-induced but not tumor necrosis factor-induced apoptosis in a caspase-3-dependent manner.

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.

Apoptosis↗
PubMed2004

Caspase-8 can be activated by interchain proteolysis without receptor-triggered dimerization during drug-induced apoptosis.

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.

Apoptosis↗
PubMed2004

Apoptosis resistance of MCF-7 breast carcinoma cells to ionizing radiation is independent of p53 and cell cycle control but caused by the lack of caspase-3 and a caffeine-inhibitable event.

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.

Apoptosis↗
PubMed2003

Cyclooxygenase-2 (COX-2) inhibitors sensitize tumor cells specifically to death receptor-induced apoptosis independently of COX-2 inhibition.

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.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2003

Tributyltin (TBT) induces ultra-rapid caspase activation independent of apoptosome formation in human platelets.

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.

Apoptosis↗
PubMedDate not supplied

A rapid nonradioactive peptide phosphorylation assay.

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.

Biochemistry↗