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Purpose: We developed an effective way to precisely diagnose prostate cancer using a novel prostate biopsy system that integrates pre-interventional magnetic resonance imaging with peri-interventional ultrasound for perineal navigated prostate biopsy., Materials and Methods: A total of 106 men with findings suspicious for prostate cancer (median age 66 years, prostate specific antigen 8.0 ng/ml and prostate volume 47 ml) underwent multiparametric 3 Tesla magnetic resonance imaging. Suspicious lesions were marked and data were transferred to the novel biopsy system. Using a custom-made biplane transrectal ultrasound probe mounted on a stepper we gathered 3-dimensional ultrasound data and fused them with magnetic resonance imaging data. As a result, suspicious magnetic resonance imaging lesions were superimposed over the transrectal ultrasound data. Three-dimensional biopsy planning was done, including systematic biopsies. Perineal biopsies were taken under live ultrasound guidance and the precise site of each biopsy was documented in 3 dimensions. We evaluated feasibility, safety and cancer detection., Results: Prostate cancer was detected in 63 of 106 patients (59.4%). Magnetic resonance imaging findings correlated positively with histopathology in 71 of 103 patients (68.9%). In magnetic resonance imaging lesions marked as highly suspicious, the detection rate was 95.8% (23 of 24 cases). Lesion targeted cores had a significantly higher positivity rate than nontargeted cores. The procedural targeting error of the first 2,461 biopsy cores was 1.7 mm. Regarding adverse effects, 2 patients experienced urinary retention and 1 had a perineal hematoma. Urinary tract infections did not develop., Conclusions: Perineal stereotactic prostate biopsies guided by the combination of magnetic resonance imaging and ultrasound enable effective examination of suspicious magnetic resonance imaging lesions. Each biopsy core taken is documented accurately for its location in 3 dimensions, enabling magnetic resonance imaging validation and tailored treatment planning. The morbidity of the procedure was minimal., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Background: A defective innate immune response may contribute to the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC). Employing a global gene expression analysis, this study was aimed at identifying specifically regulated genes within the epithelial compartment in inflammatory bowel disease (IBD)., Methods: The epithelial fraction of human ileal mucosa samples from surgical specimens was obtained by laser microdissection. Gene expression was examined by global expression profiling (n = 18, Affymetrix), quantitative reverse-transcription polymerase chain reaction (RT-PCR) (n = 35), immunoblot analysis (n = 9), and immunohistochemistry (n = 25)., Results: Global expression profiling revealed a pronounced downregulation of the retinoic acid-inducible gene I (RIG-I) within the epithelial layer of the ileum in patients with CD but not with UC. The downregulation of RIG-I was confirmed by quantitative RT-PCR, immunoblot analysis, and immunohistochemistry., Conclusions: Epithelial downregulation of RIG-I, a known pattern recognition receptor for viral components, might contribute to alterations of the innate mucosal immune response, particularly in CD., (Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.)

Tumor necrosis factor α (TNF-α) signaling pathways play important roles during tumorigenesis and inflammation. Ubiquitin-dependent processes are central to the regulation of TNF-α and nuclear factor κB (NF-κB) signaling. We performed a targeted siRNA screen for ubiquitin-specific proteases (USPs) and identified USP2 as a modulator of TNF-α-induced NF-κB signaling. We showed that USP2 is required for the phosphorylation of IκB, nuclear translocation of NF-κB and expression of NF-κB-dependent target genes and IL-8 secretion. Our study also provides evidence for isoform-specific functions of USP2. The immunohistochemical analysis of breast carcinomas revealed that USP2 expression is frequently downregulated. Together, our results implicate USP2 as a novel positive regulator of TNF-α-induced NF-κB signaling and show that its expression is altered in tumor cells., (Copyright © 2011 UICC.)

Introduction: Pulmonary blastoma (PB) is a rare malignant lung tumour with an immature mesenchymal and epithelial component resembling fetal lung. In order to define potential therapeutic targets in PB, the authors analysed the status and possible role of EGFR, HER2 and c-KIT in the pathogenesis of this tumour type, and the diagnostic value of β-catenin mutation analysis in PB., Methods: 5 PBs were analysed for EGFR, HER2, c-KIT, and β-catenin expression, as well as for mutations in EGFR, c-KIT, k-ras and the β-catenin gene (CTNNB1)., Results: EGFR expression was observed in all PBs. An EGFR mutation was found in one of the tumours. No overexpression of c-KIT or HER2 was seen. No mutations were found in k-ras or c-KIT. 3 of 5 PBs displayed CTNNB1 mutations. Nuclear translocation of β-catenin was seen in 2 of these tumours., Conclusions: Detection of EGFR expression and mutation in PB suggest EGFR inhibition as a potential therapeutic option in the treatment of advanced PB. Moreover, the data confirm a crucial role of CTNNB1 mutations in the pathogenesis of PB, and indicate that CTNNB1 gene sequencing may be a useful in distinguishing PB from other types of lung cancer.

The main cause of death from novel (swine origin) influenza A/H1N1 infection is acute respiratory distress syndrome. Most fatal cases are immunocompromised patients or patients with a severe underlying disease. Here, we report a fatal case of acute interstitial myocarditis associated with novel influenza A/H1N1 infection in an immunocompetent young woman. A previously healthy 18-year-old woman experienced malaise, diarrhea, and fever for several days prior to a sudden collapse at home. Autopsy revealed a predominantly lymphocytic myocarditis in the absence of a significant respiratory tract infection. Infection with novel (swine origin) influenza A/H1N1 was confirmed by PCR analysis of blood as well as myocardial tissue. Influenza-caused diarrhea with consecutive hypokalemia potentially contributed to the fatal outcome of the myocarditis, characterized by ventricular fibrillation. In conclusion, sudden death by myocarditis may be a rare complication of novel influenza A/H1N1 infection in otherwise healthy individuals, even in the absence of significant respiratory tract infection.

CD95 (Fas/APO1) is one of the best known members of the death receptor family which can either mediate apoptosis or activate tumor-promoting pathways. Using a tissue microarray we investigated the association between the expression of CD95 and prognosis in 617 patients with renal cell carcinomas (RCCs). CD95 was expressed in the vast majority of RCCs. High CD95 expression was associated with lymph node metastasis and correlated negatively with disease-specific survival. Multivariate Cox regression analysis confirmed CD95 expression as an independent prognostic factor. In conclusion, high CD95 expression is a negative independent prognostic factor in RCCs which could be used to identify high-risk patients with a poor clinical prognosis., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Cells dying by necrosis release the high-mobility group box 1 (HMGB1) protein, which has immunostimulatory effects. However, little is known about the direct actions of extracellular HMGB1 protein on cancer cells. Here, we show that recombinant human HMGB1 (rhHMGB1) exerts strong cytotoxic effects on malignant tumor cells. The rhHMGB1-induced cytotoxicity depends on the presence of mitochondria and leads to fast depletion of mitochondrial DNA, severe damage of the mitochondrial proteome by toxic malondialdehyde adducts, and formation of giant mitochondria. The formation of giant mitochondria is independent of direct nuclear signaling events, because giant mitochondria are also observed in cytoplasts lacking nuclei. Further, the reactive oxygen species scavenger N-acetylcysteine as well as c-Jun NH(2)-terminal kinase blockade inhibited the cytotoxic effect of rhHMGB1. Importantly, glioblastoma cells, but not normal astrocytes, were highly susceptible to rhHMGB1-induced cell death. Systemic treatment with rhHMGB1 results in significant growth inhibition of xenografted tumors in vivo. In summary, rhHMGB1 induces a distinct form of cell death in cancer cells, which differs from the known forms of apoptosis, autophagy, and senescence, possibly representing an important novel mechanism of specialized necrosis. Further, our findings suggest that rhHMGB1 may offer therapeutic applications in treatment of patients with malignant brain tumors., (©2010 AACR.)

PEA-15/PED (phosphoprotein enriched in astrocytes 15 kDa/phosphoprotein enriched in diabetes) is a death effector domain-containing protein which is known to modulate apoptotic cell death. The mechanism by which PEA-15 inhibits caspase activation and increases ERK (extracellular-regulated kinase) activity is well characterized. Here, we demonstrate that PEA-15 is not only pivotal in the activation of the ERK pathway but also modulates JNK (c-Jun N-terminal kinase) signaling. Upon overexpression of PEA-15 in malignant glioma cells, JNK is potently activated. The PEA-15-induced JNK activation depends on the phosphorylation of PEA-15 at both phosphorylation sites (serine 104 and serine 116). The activation of JNK is substantially inhibited by siRNA-mediated down-regulation of endogenous PEA-15. Moreover, we demonstrate that glioma cells overexpressing PEA-15 show increased signs of autophagy in response to classical autophagic stimuli such as ionizing irradiation, serum deprivation, or rapamycin treatment. In contrast, the non-phosphorylatable mutants of PEA-15 are not capable of promoting autophagy. The inhibition of JNK abrogates the PEA-15-mediated increase in autophagy. In conclusion, our data show that PEA-15 promotes autophagy in glioma cells in a JNK-dependent manner. This might render glioma cells more resistant to adverse stimuli such as starvation or ionizing irradiation.

Purpose: Stem-like tumor cells comprise a highly tumorigenic and therapy-resistant tumor subpopulation, which is believed to substantially influence tumor initiation and therapy resistance in glioma. Currently, therapeutic, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population; retinoic acid is well known as a potent modulator of differentiation and proliferation in normal stem cells. In glioma, knowledge about the efficacy of retinoic acid-induced differentiation to target the stem-like tumor cell pool could have therapeutic implications., Experimental Design: Stem-like glioma cells (SLGC) were differentiated with all-trans retinoic acid-containing medium to study the effect of differentiation on angiogenesis, invasive growth, as well as radioresistance and chemoresistance of SLGCs. In vivo effects were studied using live microscopy in a cranial window model., Results: Our data suggest that in vitro differentiation of SLGCs induces therapy-sensitizing effects, impairs the secretion of angiogenic cytokines, and disrupts SLGCs motility. Further, ex vivo differentiation reduces tumorigenicity of SLGCs. Finally, we show that all-trans retinoic acid treatment alone can induce antitumor effects in vivo., Conclusions: Altogether, these results highlight the potential of differentiation treatment to target the stem-like cell population in glioblastoma., (Copyright (c) 2010 AACR.)

Approximately 15% of small intestinal adenocarcinomas show inactivation of DNA-mismatch repair (MMR) and display high-level microsatellite instability (MSI-H). MSI-H tumors progress as a result of mutations affecting coding microsatellites (coding microsatellite instability, cMSI) that may result in a functional inactivation of the encoded proteins and provide a selective growth advantage for the affected cell. To investigate the cMSI selection in small intestinal carcinogenesis 56 adenocarcinomas were tested for MSI. Eleven MSI-H carcinomas (19.6%) were identified and subjected to cMSI analysis in 24 potentially tumor relevant genes. Mutation frequencies were similar to those observed in colorectal cancer (CRC). Beside high frequencies of cMSI in TGFbetaR2, ACVR2, and AIM2 we detected MARCKS mutations in 10 out of 11 (91%) tumors with a 30% share of biallelic mutations. Since little is known about MARCKS expression in the intestine, we analyzed MARCKS protein expression in 31 carcinomas. In non-neoplastic mucosa, MARCKS was found to be expressed with a concentration gradient along the crypt-villus axis. In line with cMSI induced functional inactivation of MARCKS, 8 out of 11 MSI-H adenocarcinomas showed regional or complete loss of the protein. In microsatellite stable (MSS) small bowel adenocarcinoma, loss of MARCKS expression was seen in 2 out of 20 tumors (10%). In conclusion, we herein present a cMSI profile of MSI-H small intestinal adenocarcinomas identifying MARCKS as a frequent target of mutation. Loss of MARCKS protein expression suggests a significant role of MARCKS inactivation in the pathogenesis of small intestinal adenocarcinomas.

Chemotherapy of non-Hodgkin's lymphoma is frequently hampered by drug resistance. The monoclonal antibody rituximab specifically targets the CD20 antigen and sensitizes B-cell lymphoma cells to standard anticancer drugs. In the present investigation, we analyzed, whether a combination of rituximab and artesunate may act in a complementary manner and eventually synergize in tumor cell killing. Artesunate is an anti-malarial drug, which also exerts profound activity towards cancer cells. While rituximab alone was minimally cytotoxic, rituximab increased cytotoxicity to artesunate in Ramos cells. Artesunate induced apoptosis, induced Fas/CD95 expression and the formation of reactive oxygen species (ROS) and resulted in a breakdown of mitochondrial membrane potential. This argues for the involvement of both receptor-driven extrinsic and mitochondrial intrinsic routes of apoptosis. Rituximab increased Fas/CD95 expression and ROS formation and decreased mitochondrial membrane potential ultimately leading to increased apoptosis induced by artesunate. The transcription factors YY1 and Sp1 are upstream regulators of apoptosis by controlling the expression of apoptosis-regulating genes. YY1 and Sp1 were down-regulated and Fas/CD95 was up-regulated by rituximab and artesunate indicating that artesunate activated the Fas/CD95 pathway and that rituximab increased the susceptibility of tumor cells to artesunate-induced apoptosis. Furthermore, rituximab affected the expression of antioxidant genes. The antibody decreased artesunate-induced up-regulation of catalase expression and increased artesunate-induced down-regulation of glutathione S-transferase-phi expression. Manganese-dependent superoxide dismutase expression was not changed by artesunate. Antioxidant proteins may help to detoxify artesunate-induced ROS. Rituximab reversed the artesunate-induced expression changes of antioxidant genes and, hence, reduced the detoxification capacity of Ramos cells. The effects of rituximab on antioxidant genes represent a novel mechanism of rituximab for chemosensitization.

Purpose: The death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R) are involved in immune surveillance and tumor development. Here, we studied a possible association between the expression of TRAIL/TRAIL-Rs and the prognosis in patients with renal cell carcinomas (RCC)., Experimental Design: A tissue microarray containing RCC tumor tissue samples and corresponding normal tissue samples from 838 patients was generated. Expression of TRAIL and TRAIL-Rs was examined by immunohistochemistry and the effect of TRAIL and TRAIL-R expression on disease-specific survival was assessed., Results: High TRAIL-R2 expression levels were associated with high-grade RCCs (P < 0.001) and correlated negatively with disease-specific survival (P = 0.01). Similarly, high TRAIL expression was associated with a shorter disease-specific survival (P = 0.01). In contrast, low TRAIL-R4 expression was associated with high-stage RCCs (P < 0.001) as well as with the incidence of distant metastasis (P = 0.03) and correlated negatively with disease-specific survival (P = 0.02). In patients without distant metastasis, multivariate Cox regression analyses revealed that TRAIL-R2 and TRAIL are independent prognostic factors for cancer-specific survival (in addition to tumor extent, regional lymph node metastasis, grade of malignancy, and type of surgery)., Conclusion: High TRAIL-R2, high TRAIL, and low TRAIL-R4 expression levels are associated with a worse disease-specific survival in patients with RCCs. Therefore, the assessment of TRAIL/TRAIL-R expression offers valuable prognostic information that could be used to select patients for adjuvant therapy studies. Moreover, our findings are of relevance for a potential experimental therapeutic administration of TRAIL-R agonists in patients with RCCs.

Resistance to apoptosis is one reason for the poor response of malignant brain tumors to therapy. The PPARgamma-modulating drug Troglitazone downregulates the anti-apoptotic FLIP protein and sensitizes glioblastoma cells to apoptosis induced by the death ligand TRAIL. To investigate the molecular basis of an experimental combination therapy for malignant gliomas with TRAIL and Troglitazone, we investigated the Troglitazone-induced signaling cascades and the expression of TRAIL receptors and FLIP in malignant gliomas. Troglitazone downregulated the FLIP protein through accelerated ubiquitin/proteasome-dependent degradation, which might be mediated by a Troglitazone-induced increase in reactive oxygen species. Moreover, Troglitazone induced the phosphorylation of the MAP kinase ERK1/2 as well as of the BAD protein. Inhibition of either PPARgamma or MEK1/2 blocked the Troglitazone-mediated phosphorylation of BAD and further increased the synergistic induction of glioma cell death by TRAIL and Troglitazone. Immunohistochemical analysis demonstrated that FLIP and TRAIL-R2 were significantly higher expressed in anaplastic (WHO grade III) than in diffuse (WHO grade II) gliomas. High FLIP and low TRAIL-R2 expression levels were associated with a poor prognosis of patients. Our findings warrant a further pre-clinical evaluation of an experimental anti-glioma therapy with TRAIL and Troglitazone, potentially in conjunction with a MAP kinase inhibitor.

Background: Decoy receptor 3 (DcR3) is a soluble protein that binds to and inactivates the death ligand CD95L. Here, we studied a possible association between DcR3 expression and prognosis in patients with renal cell carcinomas (RCCs)., Methods: A tissue microarray containing RCC tumor tissue samples and corresponding normal tissue samples was generated. Decoy receptor 3 expression in tumors of 560 patients was examined by immunohistochemistry. The effect of DcR3 expression on disease-specific survival and progression-free survival was assessed using univariate analysis and multivariate Cox regression analysis. Decoy receptor 3 serum levels were determined by ELISA., Findings: High DcR3 expression was associated with high-grade (P = .005) and high-stage (P = .048) RCCs. The incidence of distant metastasis (P = .03) and lymph node metastasis (P = .002) was significantly higher in the group with high DcR3 expression. Decoy receptor 3 expression correlated negatively with disease-specific survival (P < .001) and progression-free survival (P < .001) in univariate analyses. A multivariate Cox regression analysis retained DcR3 expression as an independent prognostic factor that outperformed the Karnofsky performance status. In patients with high-stage RCCs expressing DcR3, the 2-year survival probability was 25%, whereas in patients with DcR3-negative tumors, the survival probability was 65% (P < .001). Moreover, DcR3 serum levels were significantly higher in patients with high-stage localized disease (P = .007) and metastatic disease (P = .001)., Interpretation: DcR3 expression is an independent prognostic factor of RCC progression and mortality. Therefore, the assessment of DcR3 expression levels offers valuable prognostic information that could be used to select patients for adjuvant therapy studies.

The HIPPI (HIP-1 protein interactor) protein is a multifunctional protein that is involved in the regulation of apoptosis. The interaction partners of HIPPI include HIP-1 (Huntingtin-interacting protein-1), Apoptin, Homer1c, Rybp/DEDAF, and BAR (bifunctional apoptosis regulator). In search for other binding partners of HIPPI, we performed a yeast two hybrid screen and identified BLOC1S2 (Biogenesis of lysosome-related organelles complex-1 subunit 2) as a novel HIPPI-interacting protein. In co-immunoprecipitation assays, BLOC1S2 specifically associates with HIPPI, but not with HIP-1. To study the expression of BLOC1S2 on the protein level, we generated a mouse monoclonal antibody specific for BLOC1S2 and a multiple tissue array comprising 70 normal and cancer tissue samples of diverse origin. BLOC1S2 protein is widely expressed in normal tissue as well as in malignant tumors with a tendency towards lower expression levels in certain subtypes of tumors. On the subcellular level, BLOC1S2 is expressed in an organellar-like pattern and co-localizes with mitochondria. Over-expression of BLOC1S2 in the presence or absence of HIPPI does not induce apoptosis. However, BLOC1S2 and HIPPI sensitize NCH89 glioblastoma cells to the pro-apoptotic actions of staurosporine and the death ligand TRAIL by enhancing caspase activation, cytochrome c release, and disruption of the mitochondrial membrane potential. Given its interaction with HIPPI and its pro-apoptotic activity, BLOC1S2 might play an important functional role in cancer and neurodegenerative diseases.

Glioblastomas, the most malignant of all brain tumors, are characterized by cellular resistance to apoptosis and a highly invasive growth pattern. These factors contribute to the poor response of glioblastomas to radiochemotherapy and prevent their complete neurosurgical resection. However, the driving force behind the distinct motility of glioma cells is only partly understood. Here, we report that in the absence of cellular stress and proapoptotic stimuli, human glioblastoma cells exhibit a constitutive activation of caspases in vivo and in vitro. The inhibition of caspases by various peptide inhibitors decreases the migration of cells in scrape motility assays and the invasiveness of cells in spheroid assays. Similarly, specific small interfering RNA- or antisense-mediated down-regulation of caspase-3 and caspase-8 results in an inhibition of the migratory potential of glioma cells. The constitutive caspase-dependent motility of glioblastoma cells is independent of CD95 activation and it is not mediated by mitogen-activated protein/extracellular signal-regulated kinase kinase signaling. The basal caspase activity is accompanied by a constant cleavage of the motility-associated gelsolin protein, which may contribute to the caspase-mediated promotion of migration and invasiveness in glioblastoma cells. Our results suggest that the administration of low doses of caspase inhibitors that block glioma cell motility without affecting the execution of apoptotic cell death may be exploited as a novel strategy for the treatment of glioblastomas.

Background and Aims: The constant renewal of enterocytes along the crypt-villus axis (CVA) of human small intestine is due to cell-inherent changes resulting in the apoptotic cell death of senescent enterocytes. The aim of the present study was to examine underlying molecular mechanisms of the cell death at the villus tip., Methods: Characterization of human acyl-coenzyme A (CoA) synthetase 5 (ACSL5) was performed by cloning, recombinant protein expression, biochemical approaches, and several functional and in situ analyses., Results: Our data show that different amounts of acyl-CoA synthetase 5-full length (ACSL5-fl) and a so far unknown splice variant lacking exon 20 (ACSL5-Delta 20) are found in human enterocytes. In contrast with the splice variant ACSL5-Delta 20, recombinant and purified ACSL5-fl protein is active at a highly alkaline pH. Over expression of ACSL5-fl protein is associated with a decrease of the anti-apoptotic FLIP protein in a ceramide-dependent manner and an increased cell-surface expression of the death receptor TRAIL-R1. Expression analyses revealed that the ACSL5-fl/ACSL5-Delta 20 ratio increases along the CVA, thereby sensitizing ACSL5-fl-dominated cells at the villus tip to the death ligand TRAIL, which is corroborated by functional studies with human small intestinal mucosal samples and an immortalized human small intestinal cell line., Conclusions: Our results suggest an ACSL5-dependent regulatory mechanism that contributes to the cellular renewal along the CVA in human small intestine. Deregulation of the ACSL5-fl/ACSL5-Delta 20 homeostasis in the maturation and shedding of cells along the CVA might also be of relevance for the development of intestinal neoplasia.

Aim: To determine the role of two antiapoptotic proteins of the inhibitor of apoptosis protein family, cellular inhibitor of apoptosis protein 1 (cIAP1) and cellular inhibitor of apoptosis protein 2 (cIAP2), in human pancreatic carcinogenesis., Methods: mRNA levels were measured in pancreatic tissues and pancreatic cancer cell lines by quantitative reverse transcriptase PCR. Protein expression was assessed in pancreatic cancer cell lines by immunoblotting and in pancreatic tissues by immunohistochemistry, and correlated with pathological and survival data., Results: cIAP1 expression was constantly high in non-neoplastic pancreatic tissues, in pancreatic intraepithelial neoplasia (PanIN) lesions, as well as in a subset of primary and metastatic pancreatic ductal adenocarcinomas (PDAC), and a preferential cytoplasmatic localisation was observed in the tumour tissues. cIAP1 expression was rare in a cohort of cystic tumours. cIAP2 mRNA levels were significantly higher (2.4 fold) in PDAC than in normal tissues. cIAP2 protein was overexpressed in PDAC, and was detectable in low- and high-grade PanIN lesions. Moreover, cIAP2 was often expressed in pancreatic cystic tumours. cIAP1 and cIAP2 mRNA and protein were detected in all the examined cell lines. Survival analysis revealed a shorter survival in patients with cIAP1/cIAP2-positive tumours., Conclusions: cIAP1 might contribute to the regulation of the apoptotic process in the normal and in the neoplastic pancreas, depending on its subcellular localisation. Overexpression of cIAP2 is a common and early event in the progression of pancreatic cancer, and could therefore potentially influence the important pathophysiological aspects of PDAC, such as anoikis or chemoresistance.

Background: The majority of glioblastoma multiforme (GBM) cells express the epidermal growth factor receptor (EGFR). The present study evaluates the combination of temozolomide (TMZ), EGFR inhibition, and radiotherapy (RT) in GBM cell lines., Methods and Materials: Human GBM cell lines U87, LN229, LN18, NCH 82, and NCH 89 were treated with various combinations of TMZ, RT, and the monoclonal EGFR antibody cetuximab. Responsiveness of glioma cells to the combination treatment was measured by clonogenic survival., Results: Overall, double and triple combinations of RT, TMZ, and cetuximab lead to additive cytotoxic effects (independent toxicity). A notable exception was observed for U87 and LN 18 cell lines, where the combination of TMZ and cetuximab showed substantial antagonism. Interestingly, in these two cell lines, the combination of RT with cetuximab resulted in a substantial increase in cell killing over that expected for independent toxicity. The triple combination with RT, cetuximab, and TMZ was nearly able to overcome the antagonism for the TMZ/cetuximab combination in U87, however only marginally in LN18, GBM cell lines., Conclusion: It appears that EGFR expression is not correlated with cytotoxic effects exerted by cetuximab. Combination treatment with TMZ, cetuximab and radiation resulted in independent toxicity in three out of five cell lines evaluated, the antagonistic effect of the TMZ/cetuximab combination in two cell lines could indicate that TMZ preferentially kills cetuximab-resistant cells, suggesting for some cross-talk between toxicity mechanisms. Expression of EGFR was no surrogate marker for responsiveness to cetuximab, alone or in combination with RT and TMZ.

About 15% of sporadic gastrointestinal and endometrial tumors show the microsatellite instability (MSI) phenotype because of loss of DNA mismatch repair (MMR) function. The incidence of MSI in tumors of the central nervous system still remains controversial. Previous studies reported a particular high frequency of MSI (approximately 25%) in young patients suffering from high-grade gliomas. Based on these data and the fact that in different tumor entities MMR deficiency defines a subgroup of tumors with distinct pathogenesis and particular clinicopathological features that may have impact on prognosis and therapy, we screened 624 gliomas from 71 young and 553 adult patients for MMR deficiency by MSI analysis using three highly sensitive diagnostic markers. Alterations of MMR protein expression was examined by immunohistochemistry. A malignant glioma from an adult patient displayed MSI and concomitant loss of nuclear MSH2 and MSH6 protein expression (0.16%; 1/619). No evidence for MSI or loss of MMR protein expression was observed in 71 gliomas from young patients (0%; 0/71) including 41 high-grade astrocytic tumors. Overall, we observed a much lower incidence of MSI among high-grade pediatric gliomas than initially reported and suggest that MMR deficiency does not play a major role in the pathogenesis of glial neoplasms.

Induction of apoptosis by the death ligand TRAIL might be a promising therapeutic approach in cancer therapy. However, since not all tumor cells are sensitive to TRAIL, there is a need for the development of strategies to overcome TRAIL-resistance. The results of the present study show that the anti-diabetic drug troglitazone sensitizes human glioma and neuroblastoma cells to TRAIL-induced apoptosis. This process is accompanied by a substantial increase of active caspase 8 and active caspase 3, but it is independent of troglitazone's effects on the nuclear receptor PPAR-gamma. Troglitazone induces a pronounced reduction in protein expression levels of the anti-apoptotic FLICE-inhibitory protein (FLIP) without affecting FLIP mRNA levels. Further, protein and mRNA expression levels of the anti-apoptotic protein Survivin significantly decrease upon treatment with troglitazone. Moreover, sensitization to TRAIL is partly accompanied by an up-regulation of the TRAIL receptor, TRAIL-R2. A combined treatment with troglitazone and TRAIL might be a promising experimental therapy because troglitazone sensitizes tumor cells to TRAIL-induced apoptosis via various mechanisms, thereby minimizing the risk of acquired tumor cell resistance.

Misalignment of pulmonary vessels (MPV) with alveolar capillary dysplasia (ACD) is a complex congenital vascular abnormality in newborn infants. This condition is often associated with persistent pulmonary hypertension, which leads to severe respiratory distress and finally to the death of the newborn. Here, two cases of MPV with ACD are reported that were associated with atrioventricular septal defects. Both cases showed a combination of a large atrial septal defect and a smaller subvalvular ventricular septal defect. In one newborn, the cardiac malformation was accompanied by a quadricuspid pulmonary valve. These cases emphasize the importance of considering MPV with ACD a possible cause for pulmonary hypertension in the newborn, especially when a concomitant atrioventricular septal defect could represent an alternative explanation for the pulmonary hypertension.

Death ligands (such as Fas/CD95 ligand and TRAIL?Apo2L) and death receptors (such as Fas/CD95, TRAIL-R1?DR4, and TRAIL-R2/DR5) are involved in immune-mediated neutralization of activated or autoreactive lymphocytes, virus-infected cells, and tumor cells. Consequently, dysregulation of death receptor-dependent apoptotic signaling pathways has been implicated in the development of autoimmune diseases, immunodeficiency, and cancer. Moreover, the death ligand TRAIL has gained considerable interest as a potential anticancer agent, given its ability to induce apoptosis of tumor cells without affecting most types of untransformed cells. The FLICE-inhibitory protein (FLIP) potently blocks TRAIL-mediated cell death by interfering with caspase-8 activation. Pharmacologic down-regulation of FLIP might serve as a therapeutic means to sensitize tumor cells to apoptosis induction by TRAIL.

NADE, a p75NTR (low-affinity neurotrophin receptor p75) -associated cell death executor, was initially cloned from a human ovarian granulosa cell cDNA library, as an unknown protein with the name, pHGR74. It was reported to mediate nerve growth factor-induced apoptosis. We independently isolated human NADE (pHGR74) from breast cancer cell lines. Expression of NADE in various human cancer cell lines, and human and murine tissues was examined. NADE was highly expressed in human endocrine-related organs and embryotic murine tissues. Forced expression of NADE in CHO (Chinese hamster ovary) cells and MDA-MB-231 human breast cancer cells had little effect on the growth of the cells in vitro, while it dramatically suppressed cellular growth in vivo. We used the yeast two-hybrid system to search for NADE binding protein. Dynactin was identified as a candidate. The p75NTR was not found in this assay and did not co-immunoprecipitate with human NADE. Furthermore, the cells stably transfected with NADE did not respond to NGF or TNF. Thus, human and murine NADE appear to have different functions.

Apoptosis (programmed cell death) plays important roles in many facets of normal mammalian physiology. Host-pathogen interactions have provided evolutionary pressure for apoptosis as a defense mechanism against viruses and microbes, sometimes linking apoptosis mechanisms with inflammatory responses through NFkappaB induction. Proteins involved in apoptosis and NFkappaB induction commonly contain evolutionarily conserved domains that can serve as signatures for identification by bioinformatics methods. Using a combination of public (NCBI) and private (RIKEN) databases, we compared the repertoire of apoptosis and NFkappaB-inducing genes in humans and mice from cDNA/EST/genomic data, focusing on the following domain families: (1) Caspase proteases; (2) Caspase recruitment domains (CARD); (3) Death Domains (DD); (4) Death Effector Domains (DED); (5) BIR domains of Inhibitor of Apoptosis Proteins (IAPs); (6) Bcl-2 homology (BH) domains of Bcl-2 family proteins; (7) Tumor Necrosis Factor (TNF)-family ligands; (8) TNF receptors (TNFR); (9) TIR domains; (10) PAAD (PYRIN; PYD, DAPIN); (11) nucleotide-binding NACHT domains; (12) TRAFs; (13) Hsp70-binding BAG domains; (14) endonuclease-associated CIDE domains; and (15) miscellaneous additional proteins. After excluding redundancy due to alternative splice forms, sequencing errors, and other considerations, we identified cDNAs derived from a total of 227 human genes among these domain families. Orthologous murine genes were found for 219 (96%); in addition, several unique murine genes were found, which appear not to have human orthologs. This mismatch may be due to the still fragmentary information about the mouse genome or genuine differences between mouse and human repertoires of apoptotic genes. With this caveat, we discuss similarities and differences in human and murine genes from these domain families.

A novel Death Effector Domain-containing protein was identified, DEDD2, which is closest in amino acid sequence homology to death effector domain-containing DNA-binding protein, DEDD. DEDD2 mRNA is expressed widely in adult human tissues with highest levels in liver, kidney, and peripheral blood leukocytes. DEDD2 interacts with FLIP, but not with Fas-associated death domain (FADD) or caspase-8. Overexpression of DEDD2 induces moderate apoptosis and results in substantial sensitization to apoptosis induced by Fas (CD95/APO-1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L), or FADD. In contrast, Bax- or staurosporine-mediated cell death is not affected by expression of DEDD2. Fluorescence microscopy showed that overexpressed DEDD2 translocates to the nucleus, which is dependent on the presence of a bipartite nuclear localization signal in the DEDD2 protein. Mutagenesis studies revealed that the translocation of the DED of DEDD2 to the nucleus is essential for its pro-apoptotic activity. These findings suggest that DEDD2 is involved in the regulation of nuclear events mediated by the extrinsic apoptosis pathway.