Increased binding of anti-dsDNA antibodies to short oligonucleotides modified with topoisomerase I reveals a potential new enzyme function independent from DNA relaxation.

OBJECTIVE: Topoisomerase I (topo I) is a highly conserved enzyme which is known to reduce torsional stress at double-stranded (ds) DNA. Torsional stress induced by supercoiling of dsDNA requires either very long dsDNA existing in genomic DNA or circulation as presented in plasmid DNA. To enable DNA relaxation, topo I induce a transient single-strand break followed by stress-relieving rotation of the released DNA strand. Our group found by serendipity that the topo I inhibitor irinotecan is able to suppress murine systemic lupus erythematosus (SLE), an autoimmune disease which is characterized by the existence of pathogenic anti-dsDNA antibodies (abs). As a possible mechanism we demonstrated in the absence of immunosuppression an increased binding of anti-dsDNA abs to long genomic or circulated plasmid dsDNA modified with topo I. RESULTS: Here we show that this effect requires active site tyrosine of topo I which is known to facilitate DNA relaxation activity. Moreover, topo I enhanced anti-dsDNA abs binding to short linear oligonucleotides down to a size of 42 bp. Since oligonucleotides of such length are devoid of torsional stress and relaxation respectively, our results suggest a new and unknown function for the enzyme topo I.

Ten recommendations for sarcoma surgery: consensus of the surgical societies based on the German S3 guideline "Adult Soft Tissue Sarcomas".

PURPOSE: The evidence-based (S3) guideline "Adult Soft Tissue Sarcomas" (AWMF Registry No. 032/044OL) published by the German Guideline Program in Oncology (GGPO) covers all aspects of sarcoma treatment with 229 recommendations. Representatives of all medical specialties involved in sarcoma treatment contributed to the guideline. This paper compiles the most important recommendations for surgeons selected by delegates from the surgical societies. METHODS: A Delphi process was used. Delegates from the surgical societies involved in guideline process selected the 15 recommendations that were most important to them. Votes for similar recommendations were tallied. From the resulting ranked list, the 10 most frequently voted recommendations were selected and confirmed by consensus in the next step. RESULTS: The statement "Resection of primary soft tissue sarcomas of the extremities should be performed as a wide resection. The goal is an R0 resection" was selected as the most important term. The next highest ranked recommendations were the need for a preoperative biopsy, performing preoperative MRI imaging with contrast, and discussing all cases before surgery in a multidisciplinary sarcoma committee. CONCLUSION: The evidence-based guideline "Adult Soft Tissue Sarcomas" is a milestone to improve the care of sarcoma patients in Germany. The selection of the top ten recommendations by surgeons for surgeons has the potential to improve the dissemination and acceptance of the guideline and thus improve the overall outcome of sarcoma patients.

Prospective Evaluation of Quantitative F-18-FDG-PET/CT for Pre-Operative Thoracic Lymph Node Staging in Patients with Lung Cancer as a Target for Computer-Aided Diagnosis.

PURPOSE: Pre-operative assessment of thoracic lymphonodal (LN) involvement in patients with lung cancer (LC) is crucial when choosing the treatment modality. Visual assessment of F-18-FDG-PET/CT (PET/CT) is well established, however, there is still a need for prospective quantitative data to differentiate benign from malignant lesions which would simplify staging and guide the further implementation of computer-aided diagnosis (CAD). METHODS: In this prospective study, 37 patients with confirmed lung cancer (m/f = 24/13; age: 70 [52-83] years) were analyzed. All patients underwent PET/CT and quantitative data (standardized uptake values) were obtained. Histological results were available for 101 thoracic lymph nodes. Quantitative data were matched to determine cut-off values for delineation between benign vs. malignant lymph nodes. Furthermore, a scoring system derived from these cut-off values was established. Statistical analyses were performed through ROC analysis. RESULTS: Quantitative analysis revealed the optimal cut-off values (p < 0.01) for the differentiation between benign and malignant thoracic lymph nodes in patients suffering from lung cancer. The respective areas under the curve (AUC) ranged from 0.86 to 0.94. The highest AUC for a ratio of lymph node to healthy lung tissue was 0.94. The resulting accuracy ranged from 78.2% to 89.1%. A dedicated scoring system led to an AUC of 0.93 with a negative predictive value of 95.4%. CONCLUSION: Quantitative analysis of F-18-FDG-PET/CT data provides reliable results for delineation between benign and malignant thoracic lymph nodes. Thus, quantitative parameters can improve diagnostic accuracy and reliability and can also facilitate the handling of the steadily increasing number of clinical examinations.

Epigenomic profiling of non-small cell lung cancer xenografts uncover LRP12 DNA methylation as predictive biomarker for carboplatin resistance.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide and is primarily treated with radiation, surgery, and platinum-based drugs like cisplatin and carboplatin. The major challenge in the treatment of NSCLC patients is intrinsic or acquired resistance to chemotherapy. Molecular markers predicting the outcome of the patients are urgently needed. METHODS: Here, we employed patient-derived xenografts (PDXs) to detect predictive methylation biomarkers for platin-based therapies. We used MeDIP-Seq to generate genome-wide DNA methylation profiles of 22 PDXs, their parental primary NSCLC, and their corresponding normal tissues and complemented the data with gene expression analyses of the same tissues. Candidate biomarkers were validated with quantitative methylation-specific PCRs (qMSP) in an independent cohort. RESULTS: Comprehensive analyses revealed that differential methylation patterns are highly similar, enriched in PDXs and lung tumor-specific when comparing differences in methylation between PDXs versus primary NSCLC. We identified a set of 40 candidate regions with methylation correlated to carboplatin response and corresponding inverse gene expression pattern even before therapy. This analysis led to the identification of a promoter CpG island methylation of LDL receptor-related protein 12 (LRP12) associated with increased resistance to carboplatin. Validation in an independent patient cohort (n = 35) confirmed that LRP12 methylation status is predictive for therapeutic response of NSCLC patients to platin therapy with a sensitivity of 80% and a specificity of 84% (p < 0.01). Similarly, we find a shorter survival time for patients with LRP12 hypermethylation in the TCGA data set for NSCLC (lung adenocarcinoma). CONCLUSIONS: Using an epigenome-wide sequencing approach, we find differential methylation patterns from primary lung cancer and PDX-derived cancers to be very similar, albeit with a lower degree of differential methylation in primary tumors. We identify LRP12 DNA methylation as a powerful predictive marker for carboplatin resistance. These findings outline a platform for the identification of epigenetic therapy resistance biomarkers based on PDX NSCLC models.

PU.1 supports TRAIL-induced cell death by inhibiting NF-κB-mediated cell survival and inducing DR5 expression.

The hematopoietic Ets-domain transcription factor PU.1/SPI1 orchestrates myeloid, B- and T-cell development, and also supports hematopoietic stem cell maintenance. Although PU.1 is a renowned tumor suppressor in acute myeloid leukemia (AML), a disease characterized by an accumulation of immature blast cells, comprehensive studies analyzing the role of PU.1 during cell death responses in AML treatment are missing. Modulating PU.1 expression in AML cells, we found that PU.1 supports tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis via two mechanisms: (a) by repressing NF-κB activity via a novel direct PU.1-RelA/p65 protein-protein interaction, and (b) by directly inducing TRAIL receptor DR5 expression. Thus, expression of NF-κB-regulated antiapoptotic genes was sustained in PU.1-depleted AML cells upon TRAIL treatment and DR5 levels were decreased. Last, PU.1 deficiency significantly increased AML cell resistance to anthracycline treatment. Altogether, these results reveal a new facet of PU.1's tumor suppressor function during antileukemic therapies.

QSEA-modelling of genome-wide DNA methylation from sequencing enrichment experiments.

Genome-wide enrichment of methylated DNA followed by sequencing (MeDIP-seq) offers a reasonable compromise between experimental costs and genomic coverage. However, the computational analysis of these experiments is complex, and quantification of the enrichment signals in terms of absolute levels of methylation requires specific transformation. In this work, we present QSEA, Quantitative Sequence Enrichment Analysis, a comprehensive workflow for the modelling and subsequent quantification of MeDIP-seq data. As the central part of the workflow we have developed a Bayesian statistical model that transforms the enrichment read counts to absolute levels of methylation and, thus, enhances interpretability and facilitates comparison with other methylation assays. We suggest several calibration strategies for the critical parameters of the model, either using additional data or fairly general assumptions. By comparing the results with bisulfite sequencing (BS) validation data, we show the improvement of QSEA over existing methods. Additionally, we generated a clinically relevant benchmark data set consisting of methylation enrichment experiments (MeDIP-seq), BS-based validation experiments (Methyl-seq) as well as gene expression experiments (RNA-seq) derived from non-small cell lung cancer patients, and show that the workflow retrieves well-known lung tumour methylation markers that are causative for gene expression changes, demonstrating the applicability of QSEA for clinical studies. QSEA is implemented in R and available from the Bioconductor repository 3.4 (www.bioconductor.org/packages/qsea).

Transoral endoscopic mediastinal surgery (TOEMS)-results of a first clinical study for scarless mediastinal lymph node biopsies.

BACKGROUND: Video-assisted mediastinoscopy (VAM) represents the standard procedure for mediastinal lymph node biopsies. This operation results in a scar at prominent position at the anterior neck. Since there is a trend to less invasive procedures, natural orifice transluminal endoscopic surgery (NOTES) was introduced to different fields of surgery. Based on NOTES we developed a new approach for mediastinoscopy: transoral endoscopic mediastinal surgery (TOEMS). In previous studies using human cadavers and living pigs the feasibility of TOEMS was shown. It was unclear whether TOEMS could be safely applied in patients requiring mediastinal lymph node biopsies. METHODS: We conducted a clinical phase I study recruiting ten patients with unclear mediastinal lymphadenopathy not resolved by prior bronchoscopy. All patients underwent TOEMS for mediastinal lymph node biopsy. The duration of the procedure and complications were monitored. In addition, all patients were examined for pain, swallowing dysfunction and sensation disturbance. RESULTS: TOEMS was accomplished in eight patients. In two patients operation was converted to VAM due to technical problems. Mediastinal lymph nodes were dissected in all patients who finished with TOEMS. On average, two separate lymph stations were reached by TOEMS. Duration of the procedure was 159±22 min. Permanent palsy of the right recurrent laryngeal nerve was noticed in one patient postoperatively. CONCLUSIONS: This is the first report for a human application of NOTES in thoracic surgery. In fact, transoral endoscopic surgery seems to be a feasible approach for mediastinal lymph node biopsies. Further studies are needed to show whether this procedure has an advantage over VAM in terms of pain, complications and accessibility of mediastinal lymph node stations.

Comparison of volatile organic compounds from lung cancer patients and healthy controls-challenges and limitations of an observational study.

This paper outlines the design and performance of an observational study on the profiles of volatile organic compounds (VOCs) in the breath of 37 lung cancer patients and 23 healthy controls of similar age. The need to quantify each VOC considered as a potential disease marker on the basis of individual calibration is elaborated, and the quality control measures required to maintain reproducibility in breath sampling and subsequent instrumental trace VOC analysis using solid phase microextraction-gas chromatography-mass spectrometry over a study period of 14 months are described. Twenty-four VOCs were quantified on the basis of their previously suggested potential as cancer markers. The concentration of aromatic compounds in the breath was increased, as expected, in smokers, while lung cancer patients displayed significantly increased levels of oxygenated VOCs such as aldehydes, 2-butanone and 1-butanol. Although sets of selected oxygenated VOCs displayed sensitivities and specificities between 80% and 90% using linear discriminant analysis (LDA) with leave-one-out cross validation, the effective selectivity of the breath VOC approach with regard to cancer detection is clearly limited. Results are discussed against the background of the literature on volatile cancer marker investigations and the prospects of linking increased VOC levels in patients' breath with approaches that employ sniffer dogs. Experience from this study and the literature suggests that the currently available methodology is not able to use breath VOCs to reliably discriminate between cancer patients and healthy controls. Observational studies often tend to note significant differences in levels of certain oxygenated VOCs, but without the resolution required for practical application. Any step towards the exploitation of differences in VOC profiles for illness detection would have to solve current restrictions set by the low and variable VOC concentrations. Further challenges are the technical complexity of studies involving breath sampling and possibly the limited capability of current analytical procedures to detect unstable marker candidates.

Suppression of lupus nephritis and skin lesions in MRL/lpr mice by administration of the topoisomerase I inhibitor irinotecan.

BACKGROUND: Since the precise mechanism for the pathogenesis of systemic lupus erythematosus (SLE) is unknown, no targeted therapies in addition to immunosuppression are available so far. We recently demonstrated that administration of the topoisomerase I (topo I) inhibitor irinotecan at extremely low concentrations reversed established lupus nephritis in NZB/NZW mice. While profound immunosuppression was absent, we proposed changes in DNA relaxation and anti-double-stranded (ds)DNA antibody binding as the underlying mechanism. To exclude that these effects were restricted to NZB/NZW mice, irinotecan was used in a genetically different strain of lupus-prone mice. METHODS: MRL/lpr mice were treated with high- and low-dose irinotecan beginning at 8 weeks of age. Treatment was repeated every fourth week. In vitro, DNA was relaxed by recombinant topo I, and altered anti-dsDNA antibody binding was measured by enzyme-linked immunosorbent assay. RESULTS: Administration of both high- and low-dose irinotecan prevented proteinuria and prolonged survival in MRL/lpr mice. Moreover, both concentrations of irinotecan significantly improved histopathology of the skin at 18 weeks of age. While only high-dose irinotecan diminished the numbers of plasmablasts and double-negative T cells, no changes in IgG-secreting cells or anti-dsDNA IgG were observed. In vitro, relaxation of DNA by topo I increased the binding of anti-dsDNA IgG but not the binding of anti-dsDNA IgM derived from the plasma of MRL/lpr mice. CONCLUSION: The beneficial effects of topo I inhibition in a second, genetically different strain of lupus-prone mice strongly implicate irinotecan as a new therapeutic option for human SLE.

The Topoisomerase I Inhibitor Irinotecan and the Tyrosyl-DNA Phosphodiesterase 1 Inhibitor Furamidine Synergistically Suppress Murine Lupus Nephritis.

OBJECTIVE: The treatment of lupus nephritis is still an unmet medical need requiring new therapeutic approaches. Our group found recently that irinotecan, an inhibitor of topoisomerase I (topo I), reversed proteinuria and prolonged survival in mice with advanced lupus nephritis. While irinotecan is known to stabilize the complex of topo I and DNA, the enzyme tyrosyl-DNA phosphodiesterase 1 (TDP-1) functions in an opposing manner by releasing topo I from DNA. Therefore, we undertook this study to test whether the TDP-1 inhibitor furamidine has an additional effect on lupus nephritis when used in combination with irinotecan. METHODS: NZB/NZW mice were treated with low-dose irinotecan and furamidine either alone or in combination beginning at age 26 weeks. DNA relaxation was visualized using gel electrophoresis. Binding of anti-double-stranded DNA (anti-dsDNA) antibodies to DNA modified by topo I, TDP-1, and the topo I inhibitor camptothecin was determined by enzyme-linked immunosorbent assay. RESULTS: Compared to treatment with either agent alone, simultaneous treatment with low-dose irinotecan and furamidine significantly improved survival of NZB/NZW mice. Similar to what has been previously shown for irinotecan alone, the combination treatment did not change the levels of anti-dsDNA antibodies. In vitro, recombinant TDP-1 increased topo I-mediated DNA relaxation, resulting in enhanced binding of anti-dsDNA antibodies. In combination with topo I and camptothecin, TDP-1 reversed the inhibitory effects of camptothecin on DNA relaxation and anti-dsDNA binding. CONCLUSION: Affecting DNA relaxation by the enzymes topo I and TDP-1 and their inhibitors may be a promising approach for the development of new targeted therapies for systemic lupus erythematosus.

Low-dose irinotecan improves advanced lupus nephritis in mice potentially by changing DNA relaxation and anti-double-stranded DNA binding.

OBJECTIVE: Despite clear advances in the treatment of systemic lupus erythematosus (SLE), many patients still present with refractory lupus nephritis, requiring new treatment strategies for this disease. This study was undertaken to determine whether reduced doses of the topoisomerase I (topo I) inhibitor irinotecan, which is known as a chemotherapeutic agent, suppress SLE in (NZB × NZW)F1 (NZB/NZW) mice, and to evaluate the potential mechanism by which irinotecan influences the course of SLE. METHODS: NZB/NZW mice were treated with low-dose irinotecan beginning at either 24 weeks of age or established glomerulonephritis, defined as proteinuria of grade ≥3+. Binding of anti-double-stranded DNA (anti-dsDNA) antibodies was measured by enzyme-linked immunosorbent assay (ELISA), and DNA relaxation was visualized by gel electrophoresis. RESULTS: Significantly reduced irinotecan doses improved lupus nephritis and prolonged survival in NZB/NZW mice. The lowest dose successfully used for the treatment of established murine lupus nephritis was >50 times lower than the dose usually used for chemotherapy in humans. As a mechanism, low-dose irinotecan reduced B cell activity. However, the levels of B cell activity in irinotecan-treated mice were similar to those in BALB/c mice of the same age, suggesting that irinotecan did not induce clear immunosuppression. In addition, incubation of dsDNA with topo I increased binding of murine and human anti-dsDNA antibodies, showing for the first time that relaxed DNA is more susceptible to anti-dsDNA antibody binding. This effect was reversed by addition of the topo I inhibitor camptothecin. CONCLUSION: Our findings indicate that topo I inhibition may be a novel and targeted therapy for SLE.

Influence of natural killer cells and perforin­mediated cytolysis on the development of chemically induced lung cancer in A/J mice.

One alternative approach for the treatment of lung cancer might be the activation of the immune system using vaccination strategies. However, most of clinical vaccination trials for lung cancer did not reach their primary end points, suggesting that lung cancer is of low immunogenicity. To provide additional experimental information about this important issue, we investigated which type of immune cells contributes to the protection from lung cancer development. Therefore, A/J mice induced for lung adenomas/ adenocarcinomas by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were depleted of CD4+ or CD8+ T cells, CD11b+ macrophages, Gr-1+ neutrophils and asialo GM1+ natural killer (NK) cells. Subsequent analysis of tumour growth showed an increase in tumour number only in mice depleted of NK cells. Further asking by which mechanism NK cells suppressed tumour development, we neutralized several death ligands of the tumour necrosis factor (TNF) family known to be involved in NK cell-mediated cytotoxicity. However neither depletion of TNF-α, TNF-related apoptosis-inducing ligand, TNF-like weak inducer of apoptosis or FasL alone nor in combination induced an augmentation of tumour burden. To show whether an alternative cell death pathway is involved, we next generated A/J mice deficient for perforin. After challenging with NNK, mice deficient for perforin showed an increase in tumour number and volume compared to wild-type A/J mice. In summary, our data suggest that NK cells and perforin-mediated cytolysis are critically involved in the protection from lung cancer giving promise for further immunotherapeutic strategies for this disease.

Structural modification of DNA--a therapeutic option in SLE?

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs, with glomerulonephritis representing a frequent and serious manifestation. SLE is characterized by the presence of various autoantibodies, including anti-DNA antibodies that occur in approximately 70% of patients with SLE and which contribute to disease pathogenesis. Consequently, immunosuppressive therapies are applied in the treatment of SLE to reduce autoantibody levels. However, increasing evidence suggests that DNA--especially double--stranded DNA-constitutes an important pathogenic factor that is able to activate inflammatory responses by itself in autoimmune diseases. Therefore, modifying the structure of DNA to reduce its pathogenicity might be a more targeted approach for the treatment of SLE than immunosuppression. This article presents information in support of this strategy, and discusses the potential methods of DNA structure manipulation--in light of data obtained from mouse models of SLE--including topoisomerase I inhibition, administration of DNase I, or modification of histones using heparin or histone deacetylase inhibitors.

Superior vena cava syndrome in thoracic malignancies.

The superior vena cava syndrome (SVCS) comprises various symptoms due to occlusion of the SVC, which can be easily obstructed by pathological conditions (eg, lung cancer, due to the low internal venous pressure within rigid structures of the thorax [trachea, right bronchus, aorta]). The resulting increased venous pressure in the upper body may cause edema of the head, neck, and upper extremities, often associated with cyanosis, plethora, and distended subcutaneous vessels. Despite the often striking clinical presentation, SVCS itself is usually not a life-threatening condition. Currently, randomized controlled trials on many clinically important aspects of SVCS are lacking. This review gives an interdisciplinary overview of the pathophysiology, etiology, clinical manifestations, diagnosis, and treatment of malignant SVCS.

Inhibition of tissue transglutaminase sensitizes TRAIL-resistant lung cancer cells through upregulation of death receptor 5.

Tissue transglutaminase (TG2) is implicated in cellular processes such as apoptosis and cell migration. Its acyl transferase activity cross-links certain proteins, among them transcription factors were described. We show here that the TG2 inhibitor KCC009 reversed resistance to tumor necrosis factor-related apoptosis-inducing factor (TRAIL) in lung cancer cells. Sensitization required upregulation of death receptor 5 (DR5) but not of death receptor 4. Upregulation of DR5 involved the first intron of the DR5 gene albeit it was independent from p53 and nuclear factor kappa B. In conclusion, inhibition of tissue transglutaminase provides an interesting strategy for sensitization to TRAIL-induced apoptosis in p53-deficient lung cancer cells.

Reversal of established lupus nephritis and prolonged survival of New Zealand black x New Zealand white mice treated with the topoisomerase I inhibitor irinotecan.

Systemic lupus erythematosus is a chronic autoimmune disorder that predominantly affects women of childbearing age. Lupus-associated glomerulonephritis is a major cause of mortality in these patients. Current treatment protocols for systemic lupus erythematosus include cyclophosphamide, prednisolone, azathioprine, and mycophenolate mofetil. However, in mice none of these agents alone or in combination were shown to reverse established proteinuria. Using New Zealand Black x New Zealand White F1 mice, we report that administration of the topoisomerase I inhibitor irinotecan from week 13 completely prevented the onset of proteinuria and prolonged survival up to at least 90 wk without detectable side effects. Furthermore, application of irinotecan to mice with established lupus nephritis, as indicated by grade 3+ (> or =300 mg/dl) and grade 4+ (> or =2000 mg/dl) proteinuria and, according to a median age of 35 wk, resulted in remission rates of 75% and 55%, respectively. Survival was significantly prolonged with 73 wk (grade 3+ and 4+ combined) versus 40 wk for control animals. Although total IgG and anti-dsDNA Abs in the serum and mesangial IgG deposits in the kidneys were not reduced in irinotecan-treated mice, subendothelial immune deposits were considerably diminished, suggesting a prevention of glomerular basement membrane disruption. This effect was accompanied by increased rates of ssDNA breaks and inhibition of renal cell apoptosis being different to what is known about irinotecan in anticancer therapy. In conclusion, our data provide evidence that irinotecan might represent an entirely new strategy for the treatment of systemic lupus erythematosus.

Cytotoxic effects of camptothecin and cisplatin combined with tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in a model of primary culture of non-small cell lung cancer.

BACKGROUND: The cytokine tumor-necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been shown to preferentially induce apoptosis in cancer cells. A previous study of our group demonstrated that non-small cell lung cancer cell lines can be sensitized to Apo2L/TRAIL-induced apoptosis by chemotherapeutic agents. The aim of the present study was the evaluation of these results in a model of primary culture of non-small cell lung cancer. MATERIALS AND METHODS: Lung cancer tissue and normal lung tissue obtained from 8 patients who underwent operation were cultured and treated with Apo2L/TRAIL alone and in combination with cisplatin and the topoisomerase I inhibitor camptothecin for different periods. Metabolic activity of the tissue was measured by alamar blue. Markers for apoptosis were determined by immunohistochemistry and Western blot. Expression of Apo2L/TRAIL receptors in primary lung cancer and normal lung tissue was evaluated by semi-quantitative RT-PCR. Statistics were performed using nonparametric repeated measures Anova with Dunnett's correction. RESULTS: Treatment with cisplatin, camptothecin and the combination of camptothecin and Apo2L/TRAIL demonstrated significant reduction of metabolic activity in tumor and normal lung tissue. In addition, the combination of camptothecin plus Apo2L/TRAIL revealed greater cytotoxic activity in cancer tissue compared with normal lung tissue. Importantly, no toxic activity of Apo2L/TRAIL alone in normal lung tissue was observed. CONCLUSION: Our results obtained in a model of primary culture of lung cancer suggest that the combination of Apo2L/TRAIL and camptothecin might present an effective strategy for the treatment of non-small cell lung cancer.

Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease.

Chronic cholestasis often results in premature death from liver failure with fibrosis; however, the molecular mechanisms contributing to biliary cirrhosis are not demonstrated. In this article, we show that the death signal mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) may be a key regulator of cholestatic liver injury. Agonistic anti-DR5 monoclonal antibody treatment triggered cholangiocyte apoptosis, and subsequently induced cholangitis and cholestatic liver injury in a mouse strain-specific manner. TRAIL- or DR5-deficient mice were relatively resistant to common bile duct ligation-induced cholestasis, and common bile duct ligation augmented DR5 expression on cholangiocytes, sensitizing mice to DR5-mediated cholangitis. Notably, anti-DR5 monoclonal antibody-induced cholangitis exhibited the typical histological appearance, reminiscent of human primary sclerosing cholangitis. Human cholangiocytes constitutively expressed DR5, and TRAIL expression and apoptosis were significantly elevated in cholangiocytes of human primary sclerosing cholangitis and primary biliary cirrhosis patients. Thus, TRAIL/DR5-mediated apoptosis may substantially contribute to chronic cholestatic disease, particularly primary sclerosing cholangitis.

Activation of non-ischemic, hypoxia-inducible signalling pathways up-regulate cytoprotective genes in the murine liver.

BACKGROUND/AIMS: We investigated the molecular response of a non-ischemic hypoxic stress in the liver, in particular, to distinguish its hepatoprotective potential. METHODS: The livers of mice were subjected to non-ischemic hypoxia by clamping the hepatic-artery (HA) for 2h while maintaining portal circulation. Hypoxia was defined by a decrease in oxygen saturation, the activation of hypoxia-inducible factor (HIF)-1 and the mRNA up-regulation of responsive genes. To demonstrate that the molecular response to hypoxia may in part be hepatoprotective, pre-conditioned animals were injected with an antibody against Fas (Jo2) to induce acute liver failure. Hepatocyte apoptosis was monitored by caspase-3 activity, cleavage of lamin A and animal survival. RESULTS: Clamping the HA induced a hypoxic stress in the liver in the absence of severe metabolic distress or tissue damage. The hypoxic stimulus was sufficient to activate the HIF-1 signalling pathway and up-regulate hepatoprotective genes. Pre-conditioning the liver with hypoxia was able to delay the onset of Fas-mediated apoptosis and prolong animal survival. CONCLUSIONS: Our data reveal that hepatic cells can sense and respond to a decrease in tissue oxygenation, and furthermore, that activation of hypoxia-inducible signalling pathways function in part to promote liver cell survival.

TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.