[Cytokeratin 18 as marker for non-invasive diagnosis and prognosis of acute and chronic liver diseases]. / Zytokeratin-18 als Marker zur nichtinvasiven Diagnostik und Prognose akuter und chronischer Lebererkrankungen.

INTRODUCTION: Currently liver biopsy represents the gold standard to assess severity and fibrosis grade in liver diseases. Since this laborious, costly, and invasive procedure is associated with possible complications, non-invasive methods and biomarkers, which allow for an easy, reliable, and repeatable assessment of liver disease are warranted. Cytokeratin (CK) 18 is an intermediary filament protein, expressed in hepatocytes, which is proteolytically cleaved during liver damage. The resultant CK-18 fragments are released by hepatocytes and can be detected in serum. METHODS: A selective literature search in PubMed for original publications about the detection of CK-18 cell death markers in liver diseases was undertaken. RESULTS: Assessment of CK-18 cell death biomarkers allows for the early detection of liver damage in acute and chronic liver diseases. This is even feasible when transaminases are in the normal ranges. Detection of CK-18 biomarkers can also hint at disease activity and severity. For example, patients with non-alcoholic steatohepatitis exhibit elevated serum cell-death markers compared to those with simple steatosis. Furthermore, in patients with relevant fibrosis higher CK-18 values are found as compared to those with low fibrosis. In acute liver failure, cell death biomarkers may assist decision finding for the necessity of liver transplantation. DISCUSSION: Due to promising results of various studies, CK-18 cell death markers could be applied in clinical routine soon.

Differential regulation and ATP requirement for caspase-8 and caspase-3 activation during CD95- and anticancer drug-induced apoptosis.

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 immunosuppressive fungal metabolite gliotoxin specifically inhibits transcription factor NF-kappaB.

Opportunistic infections, such as aspergillosis, are among the most serious complications suffered by immunocompromised patients. Aspergillus fumigatus and other pathogenic fungi synthesize a toxic epipolythiodioxopiperazine metabolite called gliotoxin. Gliotoxin exhibits profound immunosuppressive activity in vivo. It induces apoptosis in thymocytes, splenocytes, and mesenteric lymph node cells and can selectively deplete bone marrow of mature lymphocytes. The molecular mechanism by which gliotoxin exerts these effects remains unknown. Here, we report that nanomolar concentrations of gliotoxin inhibited the activation of transcription factor NF-kappaB in response to a variety of stimuli in T and B cells. The effect of gliotoxin was specific because, at the same concentrations, the toxin did not affect activation of the transcription factor NF-AT or of interferon-responsive signal transducers and activators of transcription. Likewise, the activity of the constitutively DNA-binding transcription factors Oct-1 and cyclic AMP response element binding protein (CREB), as well as the activation of protein tyrosine kinases p56lck and p59fyn, was not altered by gliotoxin. Very high concentrations of gliotoxin prevented NF-kappaB DNA binding in vitro. However, in intact cells, inhibition of NF-kappaB did not occur at the level of DNA binding; rather, the toxin appeared to prevent degradation of IkappaB-alpha, NF-kappaB's inhibitory subunit. Our data raise the possibility that the immunosuppression observed during aspergillosis results in part from gliotoxin-mediated NF-kappaB inhibition.

The dark side of a tumor suppressor: anti-apoptotic p53.

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

Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment.

Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP(long), c-FLIP(short) and c-FLIP(R), the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP(R) and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP(R). Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP(R) revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP(R) uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and self-oligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP(R) on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms.

Inhibition of the NF-kappaB survival pathway via caspase-dependent cleavage of the IKK complex scaffold protein and NF-kappaB essential modulator NEMO.

Apoptosis is mediated by cysteine-dependent, aspartate-directed proteases of the caspase family that proteolyse strategic intracellular substrates to induce cell suicide. We describe here that engagement of apoptotic processes by Fas triggering or by staurosporine stimulation leads to the caspase-dependent inactivation of the nuclear factor kappa B (NF-kappaB) pathway after cleavage of IKK1 (IkappaB kinase 1) and NEMO (NF-kappaB essential modulator), which are needed to transduce NF-kappaB activation signals. In this study, we have analyzed in more detail, the role of NEMO cleavage, as NEMO, but not IKK1, is important for the pro-survival actions of NF-kappaB. We demonstrate that NEMO is cleaved after Asp355 to remove the last 64 C-terminal amino acids. This short form was unable to rescue NF-kappaB activation by tumor necrosis factor-alpha (TNF-alpha) when transfected in NEMO-deficient cells. Consequently, inactivation of NEMO resulted in an inhibition of the expression of antiapoptotic NF-kappaB-target genes coding for caspase inhibitors (cIAP-1, cIAP-2) or adaptors of the TNF receptor family. NEMO-deficient Jurkat cells transiently expressing a non-cleavable mutant of NEMO were less sensitive to TNF-alpha-induced apoptosis. Therefore, downmodulation of NF-kappaB activation via the proteolytic cleavage of NEMO could represent an amplification loop for apoptosis.

The Fas/APO-1 receptor and its deadly ligand.

The cell surface receptor Fas/APO-1 and its ligand have recently been identified as important mediators of apoptosis. Both molecules are crucial for the maintenance of a sound immune system, and when defective they give rise to severe autoimmune disorders. Understanding the mechanism and regulation of Fas/APO-1-triggered cell death promises important insights for the pathogenesis of AIDS, cancer and autoimmune diseases.

Cell nucleus and DNA fragmentation are not required for apoptosis.

Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death.

Extracellular ATP activates transcription factor NF-kappaB through the P2Z purinoreceptor by selectively targeting NF-kappaB p65.

Cells of the macrophage lineage express a peculiar surface receptor for extracellular ATP, designated P2Z/P2X7 purinergic receptor, that induces pore formation and collapse of the plasma membrane potential. Although the function of the P2Z receptor is largely unknown, accumulating evidence implicates its role in cell signaling and immune reactions. Here, we investigated the effect of P2Z receptor ligation on the activation of NF-kappaB, a transcription factor controlling cytokine expression and apoptosis. Exposure of microglial cells to ATP but not other nucleotides resulted in potent NF-kappaB activation. This effect was specifically mediated by the P2Z receptor, because selective receptor antagonists prevented NF-kappaB activation. NF-kappaB activation required reactive oxygen intermediates and proteases of the caspase family, because it was abolished by antioxidants and specific protease inhibitors. The subunit composition of the ATP-induced NF- kappaB-DNA complex was rather unusual. Whereas exposure to LPS-induced prototypical NF-kappaB p50 homo- and p65 (RelA)/p50 heterodimers, ATP stimulation resulted in the sole appearance of a p65 homodimer. This is the first demonstration that a certain stimulus activates a particular NF-kappaB subunit. Because different NF-kappaB complexes exhibit distinct transcriptional and DNA-binding activities, ATP may control the expression of a subset of NF-kappaB target genes distinct from those activated by classical proinflammatory mediators.

Bcl-2 down-regulates the activity of transcription factor NF-kappaB induced upon apoptosis.

Among the many target genes of the transcription factor NF-kappaB are p53 and c-myc, both of which are involved in apoptosis. This prompted us to investigate the role of NF-kappaB in this process. We report that NF-kappaB is potently activated upon serum starvation, a condition leading to apoptosis in 293 cells. Similar to Bcl-2, a transdominant-negative mutant of the NF-kappaB p65 subunit partially inhibited apoptosis, indicating a direct involvement of the transcription factor in induction of cell death. As expected, the p65 mutant suppresses kappaB-dependent gene expression. Surprisingly, transiently or stably overexpressed Bcl-2 had the same effect. The transcription inhibitory activity of the two proteins correlated with their cell death protective potential. Like Bcl-2, the related protein Bcl-xL but not Bcl-xS was able to suppress kB-dependent transcription. Bcl-2 inhibited NF-kappaB activity by an unusual mechanism. It did not prevent the release of IkappaB in the cytoplasm but down-modulated the transactivating potential of nuclear p65. These data show that NF-kappaB can participate in apoptosis. We suggest that at least part of the anti-apoptotic potential of Bcl-2 may be explained from a hitherto undiscovered activity of Bcl-2 in controlling nuclear gene expression.

alpha-Toxin is a mediator of Staphylococcus aureus-induced cell death and activates caspases via the intrinsic death pathway independently of death receptor signaling.

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

Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells.

The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca(2+)-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca(2+) homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca(2+) leak (while leaving Ca(2+) accumulation unaffected), hence reducing the steady-state [Ca(2+)] levels. As a direct consequence, the [Ca(2+)] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca(2+) influx activated by Ca(2+) store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca(2+)-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca(2+) homeostasis may contribute to the protective role of this oncogene against programmed cell death.

Apoptin is modified by SUMO conjugation and targeted to promyelocytic leukemia protein nuclear bodies.

Apoptin, a protein of the chicken anemia virus (CAV), represents a novel potential anticancer therapeutic, because it induces apoptotic death specifically in tumor but not normal cells. The cellular localization appears to be crucial for apoptin's selective toxicity. In normal cells apoptin remains in the cytoplasm, whereas in transformed cells it migrates into the nucleus and kills the cell. However, the manner by which apoptin is able to distinguish between tumor and normal cells is unknown. Here, we report for the first time that apoptin interacts directly with the promyelocytic leukemia protein (PML) in tumor cells and accumulates in PML nuclear bodies (NBs), which are involved in apoptosis induction and viral replication. We also demonstrate that apoptin is sumoylated and that a sumoylation-deficient apoptin mutant is no longer recruited to PML-NBs, but localizes in the nuclear matrix. This mutant fails to bind PML, but can still induce apoptosis as efficiently as wild-type apoptin. Moreover, apoptin kills also PML-/- cells and promyelocytic leukemia cells with defective PML expression. Our results therefore suggest that apoptin kills tumor cells independently of PML and sumoylation, however, the interaction of apoptin with PML and small ubiquitin-like modifier (SUMO) proteins might be relevant for CAV replication.

Senescence mirrors the extent of liver fibrosis in chronic hepatitis C virus infection.

BACKGROUND: Chronic viral hepatitis is linked to fibrotic liver injury that can progress to liver cirrhosis with its associated complications. Recent evidence suggests a role of senescence in liver fibrosis, although the senescence regulators contributing to fibrosis progression remain unclear. AIM: To investigate the role of senescence and different senescence markers for fibrosis progression in patients with chronic hepatitis C virus (HCV) infection. METHODS: The expression of the cell cycle inhibitors p21, p27 and p16 as well as the senescence markers p-HP1γ and γ-H2AX was analysed in liver tissue with different fibrosis stages. Senescence-associated chitotriosidase activity was measured in sera of HCV patients (n = 61) and age-matched healthy individuals (n = 22). RESULTS: We found a remarkable up-regulation of the cell cycle inhibitors and senescence markers in chronic HCV infection compared to healthy liver tissue. Liver tissue with relevant fibrosis stages (F2-3) or cirrhosis (F4) revealed a significant increase in senescent cells compared to livers with no or minimal fibrosis (F0-1). In cirrhotic livers, a significantly higher number of p-HP1γ, p21 and p27 positive cells was detected compared to liver tissue with F2-3 fibrosis. Importantly, we identified T-cells as the dominant cell type contributing to increased senescence during fibrosis progression. Compared to healthy individuals, serum chitotriosidase was significantly elevated and correlated with histological fibrosis stages and liver stiffness as assessed by transient elastography. CONCLUSIONS: Senescence of hepatic T-cells is enhanced in chronic viral hepatitis and increases with fibrosis progression. Serological detection of senescence-associated chitotriosidase might allow for the non-invasive detection of relevant fibrosis stages.

Unique and overlapping substrate specificities of caspase-8 and caspase-10.

Although caspase-8 has an established role as an initiator of death receptor-mediated apoptosis, the function of its closest homolog, caspase-10, is almost completely unknown. To gain a closer insight into the physiological function of caspase-10, we compared the cleavage of known caspase-8 substrates by both initiator caspases. We demonstrate that caspase-10 and -8 have overlapping cleavage preferences for several substrates such as the kinases RIP and PAK2. Interestingly, in other substrates, such as the Bcl-2 protein Bid, we found additional and distinct cleavage sites for both caspases, which might have important consequences for mitochondrial targeting and propagation of the death signal. Caspase-8 and -10 also caused different interchain cleavage patterns of their enzyme precursors. Together, these results suggest that caspase-8 and -10, despite having overlapping functions, also have selective substrate cleavage specificities and might thereby exert nonredundant roles in apoptosis signaling.

CD95 ligand mediates T-cell receptor-induced apoptosis of a CD4+ CD8+ double positive thymic lymphoma.

Tumors in the thymus can be of different cellular origin. Among the most common tumors are thymoma and lymphoma, which are derived from transformed thymic epithelial cells and transformed lymphocytes, respectively. Thymic lymphoma and their response to apoptotic stimuli are poorly characterized. Here, we analyse apoptosis events in the thymic lymphoma cell line Thy278, which expresses cell surface antigens characteristic of immature double positive thymocytes. Upon T-cell receptor (TCR)/CD3 stimulation, Thy278 cells die by apoptosis, similar as primary thymocytes during negative selection. Caspases are crucial for deletion of both Thy278 cells and normal thymocytes. Moreover, we show that deletion of primary thymocytes and Thy278 cells upon CD3 stimulation is considerably impaired by neutralizing CD95L antibody. Thus, our results not only demonstrate that TCR-induced apoptosis is still functional in transformed thymocytes, but also suggest that Thy278 cells are a helpful model for the molecular analysis of negative selection.

The tyrosine kinase Lck is a positive regulator of the mitochondrial apoptosis pathway by controlling Bak expression.

Tyrosine kinases of the Src family have been implicated in key biological processes. Here, we provide evidence that p56(Lck), a lymphoid-specific Src kinase, is involved in the activation of the mitochondrial apoptosis pathway. Lck-deficient T cells were completely resistant to anticancer drugs. In contrast, apoptosis sensitivity to death receptors was not altered, indicating a specific interference of Lck with the mitochondrial pathway. Re-expression of Lck restored sensitivity to drug-induced apoptosis and triggered mitochondrial cytochrome c release and caspase activation. Further analysis identified that the sensitization by Lck was independent of classical mediators of T-cell signaling, but essentially involved the Bcl-2 protein Bak. Expression of Bak was completely absent in Lck-deficient cells, while re-expression of Lck transcriptionally triggered Bak expression and conferred sensitivity to apoptosis, associated with a proapoptotic conformational change of Bak. Furthermore, in vitro the truncated fragment of Bid specifically activated Bak and cytochrome c release only from mitochondria of Lck-expressing cells. These results do not only demonstrate a sentinel role of Lck in drug resistance but also delineate a hitherto unknown pathway of Src kinases in regulation of Bcl-2 proteins.

Apoptin-induced cell death is modulated by Bcl-2 family members and is Apaf-1 dependent.

Apoptin, a chicken anemia virus-derived protein, selectively induces apoptosis in transformed but not in normal cells, thus making it a promising candidate as a novel anticancer therapeutic. The mechanism of apoptin-induced apoptosis is largely unknown. Here, we report that contrary to previous assumptions, Bcl-2 and Bcl-xL inhibit apoptin-induced cell death in several tumor cell lines. In contrast, deficiency of Bax conferred resistance, whereas Bax expression sensitized cells to apoptin-induced death. Cell death induction by apoptin was associated with cytochrome c release from mitochondria as well as with caspase-3 and -7 activation. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad spectrum caspase inhibitor, was highly protective against apoptin-induced cell death. Apoptosis induced by apoptin required Apaf-1, as immortalized Apaf-1-deficient fibroblasts as well as tumor cells devoid of Apaf-1 were strongly protected. Thus, our data indicate that apoptin-induced apoptosis is not only Bcl-2- and caspase dependent, but also engages an Apaf-1 apoptosome-mediated mitochondrial death pathway.

Induction of p21CIP/WAF-1 and G2 arrest by ionizing irradiation impedes caspase-3-mediated apoptosis in human carcinoma cells.

There is an ongoing controversy regarding the relevance of apoptosis induction by ionizing irradiation as compared with other end points including transient or permanent cell cycle arrest of damaged cells. Here, we show that such permanent cell cycle arrest and apoptosis represent two sides of the same coin. MCF-7 cells fail to express procaspase-3, which results in resistance to apoptosis induced by anticancer drugs. Conversely, restoration of procaspase-3 sensitizes MCF-7 cells to chemotherapeutics including epirubicine, etoposide and taxol. In contrast, irradiation does not trigger apoptotic cell death but results in prolonged arrest in the G2 phase of the cell division cycle regardless of procaspase-3 expression. This suggested that the propensity of MCF-7 cells to arrest at the G2 checkpoint results in resistance to apoptosis upon gamma-irradiation. This G2 arrest was associated with upregulation of p21CIP/WAF-1. Inhibition of DNA-damage-induced stress kinases and p21CIP/WAF-1 expression by caffeine abrogated G2 arrest and induced apoptosis of the irradiated cells in a caspase-3-dependent manner. Inhibition of cell cycle progression by adenoviral expression of the cyclin dependent kinase inhibitor p21CIP/WAF-1 prevented apoptosis upon caffeine treatment indicating that cell cycle progression, that is, G2-release, is required for induction of apoptosis. Likewise, cells homozygously deleted for p21CIP/WAF-1 (HCT116 p21-/-) display enhanced irradiation-induced apoptosis via a caspase-3-dependent mechanism. These data indicate that the disruption of G2 checkpoint control overcomes cell cycle arrest and resistance to gamma-irradiation-induced cell death. Thus, DNA damage may trigger a permanent G2 arrest as an initial inactivation step of tumor cells where the phenomenon of apoptosis is hidden unless cell cycle arrest is overcome. The efficient induction of apoptosis upon G2 release thereby depends on the propensity to activate the key executioner caspase-3. This finding is of crucial importance for the understanding of molecular steps underlying the efficacy of ionizing radiation to delete tumor cells.

Apoptosis-based therapies and drug targets.

The pathogenesis of many diseases is most closely connected with aberrantly regulated apoptotic cell death. The past 15 years have witnessed an explosion in the basic knowledge of mechanisms that regulate apoptosis and the mediators that either trigger or inhibit cell death. Consequently, great interest has emerged in devising therapeutic strategies for modulating the key molecules of life-and-death decisions. Numerous novel approaches are currently being followed employing gene therapy and antisense strategies, recombinant biologics or classical organic and combinatorial chemistry in order to target specific apoptotic regulators. Although drug development is still in its infancy, several therapeutics have progressed to clinical testing or have even been approved in record time. This review outlines the recent advances in the field of apoptosis-based therapies and explores some highlights of a very active field of drug development.