The role of apoptosis repressor with a CARD domain (ARC) in the therapeutic resistance of renal cell carcinoma (RCC): the crucial role of ARC in the inhibition of extrinsic and intrinsic apoptotic signalling.

BACKGROUND: Renal cell carcinomas (RCCs) display broad resistance against conventional radio- and chemotherapies, which is due at least in part to impairments in both extrinsic and intrinsic apoptotic pathways. One important anti-apoptotic factor that is strongly overexpressed in RCCs and known to inhibit both apoptotic pathways is ARC (apoptosis repressor with a CARD domain). METHODS: Expression and subcellular distribution of ARC in RCC tissue samples and RCC cell lines were determined by immunohistochemistry and fluorescent immunohistochemistry, respectively. Extrinsic and intrinsic apoptosis signalling were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT-263 or topotecan. ARC knock-down was performed in clearCa-12 cells using lentiviral transduction of pGIPZ. shRNAmir constructs. Extrinsic respectively intrinsic apoptosis were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT263 or topotecan. Potential synergistic effects were tested by pre-treatment with topotecan and subsequent treatment with ABT263. Activation of different caspases and mitochondrial depolarisation (JC-1 staining) were analysed by flow cytometry. Protein expression of Bcl-2 family members and ARC in RCC cell lines was measured by Western blotting. Statistical analysis was performed by Student's t-test. RESULTS: Regarding the extrinsic pathway, ARC knockdown strongly enhanced TRAIL-induced apoptosis by increasing the activation level of caspase-8. Regarding the intrinsic pathway, ARC, which was only weakly expressed in the nuclei of RCCs in vivo, exerted its anti-apoptotic effect by impairing mitochondrial activation rather than inhibiting p53. Topotecan- and ABT-263-induced apoptosis was strongly enhanced following ARC knockdown in RCC cell lines. In addition, topotecan pre-treatment enhanced ABT-263-induced apoptosis and this effect was amplified in ARC-knockdown cells. CONCLUSION: Taken together, our results are the first to demonstrate the importance of ARC protein in the inhibition of both the extrinsic and intrinsic pathways of apoptosis in RCCs. In this context, ARC cooperates with anti-apoptotic Bcl-2 family members to exert its strong anti-apoptotic effects and is therefore an important factor not only in the therapeutic resistance but also in future therapy strategies (i.e., Bcl-2 inhibitors) in RCC. In sum, targeting of ARC may enhance the therapeutic response in combination therapy protocols.

Expression of TRAIL-splice variants in gastric carcinomas: identification of TRAIL-γ as a prognostic marker.

BACKGROUND: TNF-related apoptosis inducing ligand (TRAIL) belongs to the TNF-superfamily that induces apoptotic cell death in a wide range of neoplastic cells in vivo as well as in vitro. We identified two alternative TRAIL-splice variants, i.e. TRAIL-ß and TRAIL-γ that are characterized by the loss of their proapoptotic properties. Herein, we investigated the expression and the prognostic values of the TRAIL-splice variants in gastric carcinomas. METHODS: Real time PCR for amplification of the TRAIL-splice variants was performed in tumour tissue specimens and corresponding normal tissues of 41 consecutive patients with gastric carcinoma. Differences on mRNA-expression levels of the TRAIL-isoforms were compared to histo-pathological variables and correlated with survival data. RESULTS: All three TRAIL-splice variants could be detected in both non-malignant and malignant tissues, irrespective of their histological staging, grading or tumour types. However, TRAIL-ß exhibited a higher expression in normal gastric tissue. The proapoptotic TRAIL-α expression was increased in gastric carcinomas when compared to TRAIL-ß and TRAIL-γ. In addition, overexpression of TRAIL-γ was associated with a significant higher survival rate. CONCLUSIONS: This is the first study that investigated the expression of TRAIL-splice variants in gastric carcinoma tissue samples. Thus, we provide first data that indicate a prognostic value for TRAIL-γ overexpression in this tumour entity.

Daxx-beta and Daxx-gamma, two novel splice variants of the transcriptional co-repressor Daxx.

Daxx is involved in transcriptional control and apoptosis. It comprises several domains, including a regulatory C terminus that is responsible for the interaction with numerous proteins such as p53, promyelocytic leukemia protein (PML), and Hsp27. Here, we describe the identification and characterization of two novel variants of Daxx termed Daxx-ß and Daxx-γ, which are generated by alternative splicing. Alternative splicing results in a truncated regulatory C terminus in both proteins. As a consequence, Daxx-ß and Daxx-γ show a markedly decreased affinity to PML, which in turn is associated with a different subnuclear localization of these proteins compared with Daxx. Although Daxx is localized mainly in PML-oncogenic domains (PODs) Daxx-ß and Daxx-γ display a distinct distribution pattern. Furthermore, Daxx-ß and Daxx-γ show a decreased affinity to p53 also due to the truncated C terminus. We provide evidence that the p53 recruitment into PODs is Daxx isoform-dependent. The decreased affinity of Daxx-ß/-γ to p53 and PML results in a diffuse localization of p53 throughout the nucleus. In contrast to Daxx, Daxx-ß and Daxx-γ are unable to repress p53-mediated transcription. Therefore, alternative splicing of Daxx might indicate an additional level in the cellular apoptosis network.

Caspase-8 and its inhibitors in RCCs in vivo: the prominent role of ARC.

Activation of the initiator-caspase, caspase-8 is under tight control of multiple antiapoptotic regulators including ARC, cFlip(S), cFlip(L) and PED/PEA-15. Since there is little data regarding the expression of caspase-8 and its antiapoptotic regulators in human tumours in vivo, we analysed their expression in renal cell carcinomas (RCCs) to identify which of these genes might be crucial for the well known impaired apoptosis and--as a result--resistance towards chemotherapy and ionizing radiation of RCCs. Caspase-8, cFlip(S), cFlip(L) and PED/PEA-15 mRNA expression was significantly increased only in early stages of RCCs compared to non-neoplastic renal tissue. In contrast, ARC mRNA expression was significantly increased in RCCs of all stages without differences between the tumour stages and grades. Importantly, the relative mRNA expression ratio between ARC and caspase-8 was significantly increased during carcinogenesis and tumour progression. In contrast, the relative mRNA expression ratio between cFlip(S), cFlip(L) or PED/PEA-15 and caspase-8 remained constant during all tumour stages. In conclusion, our analysis revealed that ARC is the only caspase-8 inhibiting regulator being constantly overexpressed in RCCs. Furthermore, the balance between antiapoptotic ARC and proapoptotic caspase-8 is the only one to be disturbed during carcinogenesis and tumour progression of RCCs. This inhibition of Caspase-8 might therefore be one example for the multiple antiapoptotic functions of ARC in RCCs possibly contributing to the marked resistance of RCCs towards radio- and chemotherapy and reflects a shift of gene expression towards a more antiapoptotic context in RCCs.

Paclitaxel (Taxol)-induced apoptosis in human epithelioid sarcoma cell lines is enhanced by upregulation of CD95 ligand (FasL/Apo-1L).

PURPOSE: Metastasizing epithelioid sarcoma (ES) is an extremely aggressive tumor, because conventional chemotherapy and irradiation are largely ineffective. Here, we analyzed the impact of the CD95-mediated drug-induced apoptosis in ES cell lines. METHODS: The effects of paclitaxel (Taxol) and 5-FU were determined by MTT assay. The extent of apoptosis was analyzed by light microscopy and Annexin V staining (flow cytometry). The expression of death receptors and ligands was defined by RT-PCR, Western blotting and flow cytometry. RESULTS: All cell lines expressed CD95, but not the CD95 ligand. The CD95 activation resulted in apoptosis and cell death in all cell lines. Both paclitaxel and 5-FU are able to trigger apoptosis, and furthermore, to upregulate CD95, whereas only paclitaxel increases CD95 ligand expression. Neutralizing antibodies directed against CD95 ligand effectively inhibited paclitaxel-induced cell death, thereby providing evidence for a direct involvement of the CD95 system in paclitaxel-induced apoptosis. CONCLUSIONS: Concomitant upregulation of CD95 receptor and ligand may significantly enhance the response of ES to anticancer drugs. As evident from the differential response of our clonal ES subpopulations to paclitaxel and 5-FU, effective activation of the CD95 system depends on intrinsic properties of both the chemotherapeutic agent and target cell population.

HA14-1 is able to reconstitute the impaired mitochondrial pathway of apoptosis in renal cell carcinoma cell lines.

Renal cell carcinomas (RCCs) exhibit a marked resistance towards apoptosis. Although most apoptotic stimuli converge at the level of the mitochondria, little is known about the mitochondrial apoptosis pathway in renal cell carcinomas. The aim of the present study, therefore, was to investigate the functionality of the mitochondrial apoptosis pathway in renal cell carcinoma cell lines by exposure to TRAIL, etoposide, HA14-1 and betulinic acid activating the mitochondria by different mechanisms. Sensitivity to TRAIL-induced apoptosis correlated with cleavage of the initiator caspase-8, but the mitochondrial apoptosis pathway was not induced. Similarly, etoposide and betulinic acid could not induce mitochondrial damage. In contrast, HA14-1 was able to activate mitochondrial apoptosis, thereby demonstrating functionally inducible signalling pathways downstream of the mitochondria. The intactness of the pathways upstream of the mitochondria was shown by pretreatment of TRAIL-sensitive cell lines with HA14-1, which could reconstitute TRAIL-induced mitochondrial damage and resulted in a synergistic apoptosis induction. Our results demonstrate that the apoptotic pathways upstream and downstream of the mitochondria are intact and inducible in renal cell carcinoma cell lines. However, resistance towards mitochondrial apoptosis is located on the level of the mitochondria themselves.

The impact of nitrite and antioxidants on ultraviolet-A-induced cell death of human skin fibroblasts.

Nitrite (NO(2)(-)) occurs ubiquitously in biological fluids such as blood and sweat. Ultraviolet A-induced nitric oxide formation via decomposition of cutaneous nitrite, accompanied by the production of reactive oxygen (ROS) or nitrogen species (RNS), represents an important source for NO in human skin physiology. Examining the impact of nitrite and the antioxidants glutathione (GSH), Trolox (TRL), and ascorbic acid (ASC) on UVA-induced toxicity of human skin fibroblasts (FB) we found that NO(2)(-) concentration-dependently enhances the susceptibility of FB to the toxic effects of UVA by a mechanism comprising enhanced induction of lipid peroxidation. While ASC completely protects FB cultures from UVA/NO(2)(-)-induced cell damage, GSH or TRL excessively enhances UVA/NO(2)(-)-induced cell death by a mechanism comprising nitrite concentration-dependent TRL radical formation or GSH-derived oxidative stress. Simultaneously, in the presence of GSH or TRL the mode of UVA/NO(2)(-)-induced cell death changes from apoptosis to necrosis. In summary, during photodecomposition of nitrite, ROS or RNS formation may act as strong toxic insults. Although inhibition of oxidative stress by NO and other antioxidants represents a successful strategy for protection from UVA/NO(2)(-)-induced injuries, GSH and TRL may nitrite-dependently aggravate the injurious impact by TRL or GSH radical formation, respectively.

Survivin and its prognostic significance in pediatric acute B-cell precursor lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: Impaired apoptosis, mediated by members of the inhibitor of apoptosis proteins (IAP) family such as survivin, is thought to contribute to leukemic cell survival. In contrast to low expression of survivin in normal differentiated adult tissues, very high levels of survivin have been described in a number of different tumors. Overexpression of survivin was found to correlate with poor prognosis in a variety of cancers including hematologic malignancies. To date, however, there is no information available on the prognostic role of survivin in pediatric precursor B-cell acute lymphocytic leukemia (BCP-ALL), the most frequent malignancy in childhood. DESIGN AND METHODS: In a retrospective study including 66 pediatric patients we analyzed the impact of survivin protein levels on outcome in BCP-ALL. RESULTS: Survivin overexpression, with an up to ten-fold increase of the normal level, was detected in 65% of the leukemic samples in contrast to negligible expression in non-malignant hematopoietic cells. Despite considerable variety of expression levels in ALL cells, there was no association of survivin levels with established risk factors. However, patients suffering relapse of disease or death had significantly higher survivin expression than those with a favorable outcome. Overexpression of survivin is a significant prognostic marker for 3 year relapse free, event-free and overall survival, again independent of the established prognostic factors in ALL, such as age and leukocyte count at diagnosis as assessed in multivariate analysis. INTERPRETATION AND CONCLUSIONS: Overexpression of survivin in BCP-ALL identifies patients with a high risk of early relapse. Upon confirmation in a prospective analysis, survivin expression may, in the future, serve to further refine treatment stratification with intensification of therapy in those patients prone to relapse.

Disturbed expression of the apoptosis regulators XIAP, XAF1, and Smac/DIABLO in gastric adenocarcinomas.

Dysregulation of apoptosis plays an important role in carcinogenesis and tumor progression. Whereas x-linked inhibitor of apoptosis (XIAP) is a potent inhibitor of apoptosis, its antagonists second mitochondria-derived activator of caspases/direct IAP binding protein with low PI (Smac/DIABLO), and XIAP-associated factor 1 (XAF1) promote apoptosis. To explore the relevance of XIAP, Smac/DIABLO, and XAF1 for carcinogenesis and tumor progression, we analyzed 46 primary gastric adenocarcinomas and non-neoplastic gastric mucosa samples by quantitative real-time polymerase chain reaction. XIAP, Smac/DIABLO, and XAF1 expression was found in all non-neoplastic gastric mucosa samples and all adenocarcinomas. XIAP expression levels did not change between non-neoplastic gastric mucosa and adenocarcinomas or between carcinomas of early and advanced stages. Although Smac/DIABLO expression was significantly (P=0.01) higher in carcinomas, the ratio of XIAP to Smac/DIABLO expression remained stable between non-neoplastic mucosa and carcinomas. XAF1 expression had the tendency to decrease from non-neoplastic mucosa to advanced adenocarcinomas. Importantly, the ratio of XIAP to XAF1 expression significantly (P=0.03) increased from non-neoplastic mucosa to adenocarcinomas and the increase was even higher in carcinomas of advanced stage (P=0.01). Moreover, expression of the XAF1 splice variants differing in the zinc-finger domain essential for XIAP-binding was analyzed and revealed a significant higher (P=0.03) variant-2/variant-1 ratio in advanced carcinomas. In conclusion, an increased expression ratio of XIAP to XAF1 in combination with a disturbed expression of the XAF1 splice variants could be shown in gastric adenocarcinomas. These marked imbalances probably result in an impaired ability for XAF1 to antagonize the effects of XIAP thereby contributing to apoptosis-resistance and generating an important growth advantage.

Survivin splice variants regulate the balance between proliferation and cell death.

Survivin is an inhibitor of apoptosis protein that also plays critical roles in regulating the cell cycle and mitosis. Its prominent expression in essentially all human malignancies, and low or absent expression in most normal tissues, suggests that it would be an ideal target for cancer-directed therapy. Impeding development of safe and effective survivin antagonists for clinical use is a lack of understanding of the molecular mechanisms by which survivin differentially affects apoptosis and cell division, in normal and malignant cells. We show that the diverse functional roles of survivin can be explained, in part, by its heterodimerization with survivin splice variants in tumor cells. Survivin and survivin-DeltaEx3 interact within the mitochondria where they may inhibit mitochondrial-dependent apoptosis. If the expression of all survivin forms is eliminated by siRNA transfections, cells undergo both apoptosis and defective cell division. Overall, we provide new insights suggesting that targeting specific survivin isoforms, rather than survivin alone, may selectively and effectively destroy tumor cells. These findings are likely to have a significant impact in the design of biologic agents for clinical therapy.

Expression of death-associated protein kinase during tumour progression of human renal cell carcinomas: hypermethylation-independent mechanisms of inactivation.

Death-associated protein kinase (DAPK) is a pro-apoptotic Ca(2+)/calmodulin-dependent serine/threonine kinase that is widely expressed in tissues but kept silent in growing cells. Downregulation of DAPK transcription by CpG methylation has been demonstrated in a variety of tumours, providing a selective growth advantage during tumour progression. As the in vivo expression of DAPK in human renal cell carcinomas (RCCs) has not previously been analysed, 72 RCCs were investigated using semi-quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). We found that almost 92% (66/72) of all primary RCCs express DAPK mRNA and results obtained from methylation-specific PCR analyses suggest that aberrant CpG methylation of the DAPK promoter is absent even in DAPK non-expressing tumours. Comparison of early/intermediate with advanced tumour stages of clear cell RCCs showed that no significant changes in the expression levels of DAPK were evident. Chromophilic/papillary RCCs display no significantly different expression patterns of DAPK compared with stage-adjusted clear cell RCCs. Furthermore, on analysing the DAPK enzyme activity in RCC cell lines with DAPK mRNA and protein expression, only 1 out of 11 cell lines showed basal DAPK activity in kinase activity assays, suggesting that DAPK, although expressed in RCC, remains largely inactive. Our study demonstrates the in vivo expression of DAPK in RCCs and reveals that, in contrast to other tumour types, RCCs may not downregulate DAPK mRNA expression during tumour progression. Despite persistent DAPK transcription and translation, however, the markedly reduced DAPK enzyme activity in our RCC cell lines suggested a post-translational inactivation of DAPK in RCCs.

Paclitaxel/Taxol sensitivity in human renal cell carcinoma is not determined by the p53 status.

In this study, we analyzed the role of the p53 status for paclitaxel/Taxol sensitivity in renal cell carcinomas (RCCs) of the clear cell type. Using immunohistochemistry, nuclear p53 accumulation could not be correlated to the paclitaxel/Taxol sensitivity. DNA sequencing detected a p53 gene mutation in two out of eight RCC cell lines, i.e. in exon 8 (cell line clearCa-6), and in exon 9 (cell line clearCa-5). No correlation, however, was found between the p53 status of our RCC cell lines and their paclitaxel/Taxol sensitivity as indicated by the IC50 values. However, paclitaxel-induced growth inhibition in paclitaxel-sensitive RCC cell lines was accompanied by an increase in apoptosis, irrespective of their p53 status. Although CD95 up-regulation was observed in renal cell carcinoma with wild-type p53 upon paclitaxel treatment, paclitaxel-induced apoptosis itself is triggered independently from the CD95 system. In conclusion, the p53 status cannot predict paclitaxel/Taxol sensitivity in RCC cell lines of the clear cell type.

The presence of nitrite during UVA irradiation protects from apoptosis.

Nitrite occurs ubiquitously in biological fluids such as blood and sweat, representing an oxidation product of nitric oxide. Nitrite has been associated with a variety of adverse effects such as mutagenicity, carcinogenesis, and toxicity. In contrast, here we demonstrate that the presence of nitrite, but not nitrate, during irradiation of endothelial cells in culture exerts a potent and concentration-dependent protection against UVA-induced apoptotic cell death. Protection is half-maximal at a concentration of 3 mM, and complete rescue is observed at 10 mM. Nitrite-mediated protection is mediated via inhibition of lipid peroxidation in a similar manner as seen with butylated hydroxytoluene, a known inhibitor of lipid peroxidation. Interestingly, nitrite-mediated protection is completely abolished by coincubation with the NO scavenger cPTIO. Using electron paramagnetic resonance (EPR) spectroscopy or Faraday modulation spectroscopy, we directly prove UVA-induced NO formation in solutions containing nitrite. In conclusion, evidence is presented that nitrite represents a protective agent against UVA-induced apoptosis due to photodecomposition of nitrite and subsequent formation of NO.

Discovery of Novel, Potent, and Specific Cell-Death Inducers in the Jurkat Acute Lymphoblastic Leukemia Cell Line.

The limited clinical efficacy of many cancer therapeutics has initiated intense research efforts toward the discovery of novel chemical entities in this field. In this study, 31 hit candidates were selected from nearly 800,000 database compounds in a ligand-based virtual screening campaign. In turn, three of these hits were found to have (sub)micromolar potencies in proliferation assays with the Jurkat acute lymphatic leukemic cell line. In this assay, the three hits were found to exhibit higher potency than clinically tested cell-death inducers (GDC-0152, AT-406, and birinapant). Importantly, antiproliferative activity toward non-cancer peripheral blood mononuclear cells (PBMCs) was found to be marginal. Further biological characterization demonstrated the cell-death-inducing properties of these compounds. Biological testing of hit congeners excluded a nonspecific, toxic effect of the novel structures. Altogether, these findings may have profound relevance for the development of clinical candidates in tumor therapy.

UVB radiation-mediated expression of inducible nitric oxide synthase activity and the augmenting role of co-induced TNF-alpha in human skin endothelial cells.

Nitric oxide (NO) plays a pivotal role in ultraviolet radiation-induced inflammation in human skin. We had earlier reported on the inducible nitric oxide synthase (iNOS) inducing activity of UVA radiation. We now demonstrate that UVB-exposure induces expression of the iNOS in vessel endothelia of normal human skin and in cultured human dermal endothelial cells (HUDEC), although by a molecular mechanism different from UVA-mediated induction. With HUDEC, UVB induces iNOS expression and leads to significant enzyme activities, although at app. 5-fold lower levels than can be achieved with proinflammatory cytokines. In contrast to our earlier observation with UVA, cytokine-challenge combined with simultaneous UVB-exposure had no additive effects on iNOS expression nor activity. Interestingly, a time-delay between UVB-irradiation and cytokine-challenge enhances endothelial iNOS enzyme activity 2.5-fold over cytokines activation only. This time-dependent effect strongly correlates with UVB-induced endothelial TNF-alpha expression. In HUDEC addition of TNF-alpha results in enhanced expression of the inducible arginine transporter system CAT-2 essential for substrate supply and thus iNOS activity. In summary, UVB induces iNOS mRNA and enzyme activity in HUDEC. Moreover, UVB augments CAT-2 expression through a TNF-alpha- dependent mechanism which essentially contributes to increased iNOS activity.

XIAP expression is an independent prognostic marker in clear-cell renal carcinomas.

Deregulation of apoptosis plays an important role in carcinogenesis, tumor progression, and resistance to chemotherapy. XIAP is considered to be the most potent caspase inhibitor of all known IAP (inhibitor of apoptosis) family members. To explore the relevance of XIAP for progression and prognosis in renal cell carcinomas (RCCs) of the clear-cell type, we analyzed XIAP protein expression in formalin-fixed tissue from 145 clear-cell RCCs by immunohistochemistry. XIAP protein expression was found in 95% of clear-cell RCCs. A significant increase of XIAP expression became evident from well (G1) to poorly (G3) differentiated clear-cell RCCs (P < 0.0001) and from low (pT1) to advanced (pT3) tumor stages (P = 0.0016). Log-rank test showed a significant inverse correlation (P = 0.0174) between XIAP expression and tumor aggressiveness as indicated by patients' survival. Most important, multivariate Cox regression analysis revealed that XIAP expression is an independent prognostic parameter (P = 0.018) in clear-cell RCCs. Our results suggest an important role for XIAP-mediated inhibition of apoptosis during progression of clear-cell RCCs and introduce XIAP expression as a new independent prognostic marker in this tumor type.