Gudrun Totzke

Staphylococcus aureus alpha-toxin-induced cell death: predominant necrosis despite apoptotic caspase activation

AbstractRecent data suggest that α-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 α-toxin-induced death of Jurkat T or MCF-7 breast carcinoma cells. Caspase inhibition had also no effect on α-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 α-toxin-treated cells were positive for both active caspases and PI. Finally, electron microscopy demonstrated that even in the presence of active caspases, α-toxin-treated cells displayed a necrotic morphology characterized by cell swelling and cytoplasmic vacuolation. Together, our data suggest that α-toxin-induced cell death proceeds even in the presence of activated caspases, at least partially, in a caspase-independent, necrotic-like manner.

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 G2-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 G2-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-α, 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 G2-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 G2-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.

A Novel Member of the IκB Family, Human IκB-ζ, Inhibits Transactivation of p65 and Its DNA Binding

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

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 E2 (PGE2) production, only in H21 cells. TNF did also not induce PGE2 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.

Stromal cell‐derived factor 1α (SDF‐1α) induces gene‐expression of early growth response‐1 (Egr‐1) and VEGF in human arterial endothelial cells and enhances VEGF induced cell proliferation

Abstract. Stromal cell‐derived factor‐1 (SDF‐1), mainly known as a chemotactic factor for haematopoietic progenitor cells, also provides angiogenetic potency. Since the intracellular signalling of SDF‐1‐induced neovascularization remains unclear, we studied in human umbilical arterial endothelial cells (HUAEC) the influence of SDF‐1α on induction of the genes of early growth response‐1 (Egr‐1) and VEGF, as well as the activation of extracellular regulated kinases (ERK) 1/2, which are all known to be involved in endothelial cell proliferation. We found a time‐dependent induction of Egr‐1 and VEGF mRNA expression and phosphorylation of ERK1/2 by SDF‐1α. Furthermore, we demonstrated that Egr‐1 expression is dependent on ERK 1/2 activation. Finally, we tried to confirm the relevance of the induced gene expression by detecting the [3H]thymidine incorporation as a marker for cell proliferation in HUAEC after stimulation with SDF‐1α alone or together with VEGF. This particular test showed, that SDF‐1α alone has no effect, but is able to significantly enhance VEGF induced DNA synthesis. In summary, SDF‐1α is involved in different steps of endothelial cell proliferation, but, since Egr‐1 and VEGF offer different functions, it may also play a so far undefined role on other conditions of the endothelium.

High Percentage of False-Positive Results of Cytokeratin 19 RT-PCR in Blood: A Model for the Analysis of Illegitimate Gene Expression

Cytokeratin 19 (CK19) RT-PCR is widely used in order to detect circulating tumor cells in peripheral blood and bone marrow. However, increasing amounts of information support the fact that it is also associated with a high percentage of false-positive results. In our study, we not only managed to demonstrate the significant limitations of this method, but were also able to clarify the reasons behind these limitations. We developed a completely novel RT-PCR for CK19 and sequenced an intron at nucleotide (nt) 980 of the CK19 mRNA to exclude DNA contamination. Tumor dilution experiments were performed in order to analyze the specificity and sensitivity of the method. Control experiments using the blood of healthy donors were performed. Tumor cell dilution experiments gave a detection limit of one tumor cell. If tumor cells were mixed with an equal volume of pure mononuclear cells, the detection limit was 1 tumor cell in 105 mononuclear cells. RT-PCR of mononuclear cells from healthy blood donors gave false-positive results in 29% of the cases. We conclude that a significant decrease in the sensitivity of CK19 RT-PCR occurs if it is performed in blood cells and that the illegitimate CK19 gene expression in normal cells can lead to false-positive results. These limitations have to be taken into account if RT-PCR is to be used for the detection of tumor cells either in blood or in bone marrow in clinical practice.

In vitro modulation of inducible nitric oxide synthase gene expression and nitric oxide synthesis by procalcitonin.

ObjectiveSerum procalcitonin (PCT) concentration was recently introduced as valuable diagnostic marker for systemic bacterial infection and sepsis. At present, the cellular sources and biological properties of PCT are unclear. During sepsis and septic shock, inducible nitric oxide synthase (iNOS) gene expression is stimulated followed by the release of large amounts of nitric oxide (NO). We investigated the possible association between PCT and iNOS gene expression in an in vitro cell culture model. DesignProspective, controlled in vitro cell culture study. SettingUniversity research laboratories. InterventionsConfluent rat vascular smooth muscle cells (VSMC) were incubated for 24 hrs and 48 hrs with PCT (1 ng/mL, 10 ng/mL, 100 ng/mL, 1000 ng/mL, 5000 ng/mL) alone or with the combination of tumor necrosis factor-alpha (TNF-&agr;, 500 U/mL) plus interferon-gamma (IFN-&ggr;, 100 U/mL). iNOS gene expression was measured by qualitative as well as quantitative polymerase chain reaction analysis, NO release was estimated by the modified Griess method. Measurements and Main Results PCT in increasing concentrations had no effect on iNOS gene expression and nitrite/nitrate release for 24 hrs and 48 hrs, respectively. However, PCT ameliorated TNF-&agr;/IFN-&ggr;–induced iNOS gene expression in a dose-dependent manner (maximal inhibition at PCT 100 ng/mL by −66% for 24 hrs and −80% for 48 hrs). This was accompanied by a significantly reduced release of nitrite/nitrate into the cell culture supernatant (maximal reduction at PCT 100 ng/mL by −56% and −45% for 24 hrs and 48 hrs, respectively). ConclusionsWe conclude that recombinant PCT inhibits the iNOS-inducing effects of the proinflammatory cytokines TNF-&agr;/IFN-&ggr; in a dose-dependent manner. This might be a counter-regulatory mechanism directed against the large production of NO and the concomitant systemic hypotension in severe sepsis and septic shock.

Identification of syntenin and other TNF-inducible genes in human umbilical arterial endothelial cells by suppression subtractive hybridization

Endothelial cells play an important regulatory role in inflammatory responses by upregulating various proinflammatory gene products including cytokines and adhesion molecules. A highly potent mediator of this process is tumor necrosis factor‐α (TNF). In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF‐inducible genes in human umbilical arterial endothelial cells. Following mRNA isolation of non‐stimulated and TNF‐stimulated cells, cDNAs of both populations were prepared and subtracted by suppression PCR. Sequencing of the enriched cDNAs identified 12 genes differentially expressed including vascular cell adhesion molecule‐1, monocyte chemoattractant protein‐1, interleukin‐8 and IκBα, an inhibitor of the transcription factor nuclear factor‐κB. Interestingly, also syntenin, a PDZ motif‐containing protein which binds to the cytoplasmic domain of syndecans, was identified by SSH. Time course studies using RT‐PCR analysis confirmed that all genes were differentially expressed and rapidly induced by TNF. Our data reveal that SSH is a powerful technique of high sensitivity for the detection of differential gene expression in primary arterial endothelial cells.

The Proteasome Is Required for Rapid Initiation of Death Receptor-Induced Apoptosis

ABSTRACT 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.

Caspase-10 Sensitizes Breast Carcinoma Cells to TRAIL-Induced but Not Tumor Necrosis Factor-Induced Apoptosis in a Caspase- 3-Dependent Manner

ABSTRACT 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.