Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation

Pairing of a given E3 ubiquitin ligase with different E2s allows synthesis of ubiquitin conjugates of different topologies. While this phenomenon contributes to functional diversity, it remains largely unknown how a single E3 ubiquitin ligase recognizes multiple E2s, and whether identical structural requirements determine their respective interactions. The E3 ubiquitin ligase RNF8 that plays a critically important role in transducing DNA damage signals, interacts with E2s UBCH8 and UBC13, and catalyzes both K48- and K63-linked ubiquitin chains. Interestingly, we report here that a single-point mutation (I405A) on the RNF8 polypeptide uncouples its ability in catalyzing K48- and K63-linked ubiquitin chain formation. Accordingly, while RNF8 interacted with E2s UBCH8 and UBC13, its I405A mutation selectively disrupted its functional interaction with UBCH8, and impaired K48-based poly-ubiquitylation reactions. In contrast, RNF8 I405A preserved its interaction with UBC13, synthesized K63-linked ubiquitin chains, and assembled BRCA1 and 53BP1 at sites of DNA breaks. Together, our data suggest that RNF8 regulates K48- and K63-linked poly-ubiquitylation via differential RING-dependent interactions with its E2s UBCH8 and UBC13, respectively.published_or_final_versio

Nicotinic acetylcholine receptor expression in human airway correlates with lung function

Nicotine and its derivatives, by binding to nicotinic acetylcholine receptors (nAChRs) on bronchial epithelial cells, can regulate cellular signaling and inflammatory processes. Delineation of nAChR subtypes and their responses to nicotine stimulation in bronchial epithelium may provide information for therapeutic targeting in smoking-related inflammation in the airway. Expression of nAChR subunit genes in 60 bronchial epithelial biopsies and immunohistochemical staining for the subcellular locations of nAChR subunit expression were evaluated. Seven human bronchial epithelial cell lines (HBECs) were exposed to nicotine in vitro for their response in nAChR subunit gene expression to nicotine exposure and removal. The relative normalized amount of expression of nAChR α4, α5, and α7 and immunohistochemical staining intensity of nAChR α4, α5, and β3 expression showed significant correlation with lung function parameters. Nicotine stimulation in HBECs resulted in transient increase in the levels of nAChR α5 and α6 but more sustained increase in nAChR α7 expression. nAChR expression in bronchial epithelium was found to correlate with lung function. Nicotine exposure in HBECs resulted in both short and longer term responses in nAChR subunit gene expression. These results gave insight into the potential of targeting nAChRs for therapy in smoking-related inflammation in the airway.postprin

Oncogenic mutation profiling in new lung cancer and mesothelioma cell lines

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Aberrant large tumor suppressor 2 (LATS2) gene expression correlates with EGFR mutation and survival in lung adenocarcinomas

BACKGROUND: Large tumor suppressor 2 (LATS2) gene is a putative tumor suppressor gene with potential roles in regulation of cell proliferation and apoptosis in lung cancer. The aim of this study is to explore the association of aberrant LATS2 expression with EGFR mutation and survival in lung adenocarcinoma (AD), and the effects of LATS2 silencing in both lung AD cell lines. METHODS: LATS2 mRNA and protein expression in resected lung AD were correlated with demographic characteristics, EGFR mutation and survival. LATS2-specific siRNA was transfected into four EGFR wild-type (WT) and three EGFR mutant AD cell lines and the changes in LATS2 expression and relevant signaling molecules before and after LATS2 knockdown were assayed. RESULTS: Fifty resected lung AD were included (M:F=23:27, smokers:non-smokers=19:31, EGFR mutant:wild-type=21:29) with LATS2 mRNA levels showed no significant difference between gender, age, smoking and pathological stages while LATS2 immunohistochemical staining on an independent set of 79 lung AD showed similar trend. LATS2 mRNA level was found to be a significant independent predictor for survival status (disease-free survival RR=0.217; p=0.003; Overall survival RR=0.238; p=0.036). siRNA-mediated suppression of LATS2 expression resulted in augmentation of ERK phosphorylation in EGFR wild-type AD cell lines with high basal LATS2 expression, discriminatory modulation of Akt signaling between EGFR wild-type and mutant cells, and induction of p53 accumulation in AD cell lines with low baseline p53 levels. CONCLUSIONS: LATS2 expression level is predictive of survival in patients with resected lung AD. LATS2 may modulate and contribute to tumor growth via different signaling pathways in EGFR mutant and wild-type tumors.postprin

Poly(I:C) induces intense expression of c-IAP2 and cooperates with an IAP inhibitor in induction of apoptosis in cancer cells

<p>Abstract</p> <p>Background</p> <p>There is increasing evidence that the toll-like receptor 3 (TLR3) is an interesting target for anti-cancer therapy. Unfortunately, most laboratory investigations about the impact of TLR3 stimulation on human malignant cells have been performed with very high concentrations - 5 to 100 μg/ml - of the prototype TLR3 ligand, poly(I:C). In a previous study focused on a specific type of human carcinoma - nasopharyngeal carcinoma - we have shown that concentrations of poly(I:C) as low as 100 ng/ml are sufficient to induce apoptosis of malignant cells when combined to a pharmacological antagonist of the IAP family based on Smac mimicry.</p> <p>Methods</p> <p>This observation prompted us to investigate the contribution of the IAP family in cell response to poly(I:C) in a variety of human malignant cell types.</p> <p>Results</p> <p>We report a rapid, intense and selective increase in c-IAP2 protein expression observed under stimulation by poly(I:C)(500 ng/ml) in all types of human malignant cells. In most cell types, this change in protein expression is underlain by an increase in c-IAP2 transcripts and dependent on the TLR3/TRIF pathway. When poly(I:C) is combined to the IAP inhibitor RMT 5265, a cooperative effect in apoptosis induction and/or inhibition of clonogenic growth is obtained in a large fraction of carcinoma and melanoma cell lines.</p> <p>Conclusions</p> <p>Currently, IAP inhibitors like RMT 5265 and poly(I:C) are the subject of separate therapeutic trials. In light of our observations, combined use of both types of compounds should be considered for treatment of human malignancies including carcinomas and melanomas.</p

Counter-current chromatography for the separation of terpenoids: A comprehensive review with respect to the solvent systems employed

Copyright @ 2014 The Authors.This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.Natural products extracts are commonly highly complex mixtures of active compounds and consequently their purification becomes a particularly challenging task. The development of a purification protocol to extract a single active component from the many hundreds that are often present in the mixture is something that can take months or even years to achieve, thus it is important for the natural product chemist to have, at their disposal, a broad range of diverse purification techniques. Counter-current chromatography (CCC) is one such separation technique utilising two immiscible phases, one as the stationary phase (retained in a spinning coil by centrifugal forces) and the second as the mobile phase. The method benefits from a number of advantages when compared with the more traditional liquid-solid separation methods, such as no irreversible adsorption, total recovery of the injected sample, minimal tailing of peaks, low risk of sample denaturation, the ability to accept particulates, and a low solvent consumption. The selection of an appropriate two-phase solvent system is critical to the running of CCC since this is both the mobile and the stationary phase of the system. However, this is also by far the most time consuming aspect of the technique and the one that most inhibits its general take-up. In recent years, numerous natural product purifications have been published using CCC from almost every country across the globe. Many of these papers are devoted to terpenoids-one of the most diverse groups. Naturally occurring terpenoids provide opportunities to discover new drugs but many of them are available at very low levels in nature and a huge number of them still remain unexplored. The collective knowledge on performing successful CCC separations of terpenoids has been gathered and reviewed by the authors, in order to create a comprehensive document that will be of great assistance in performing future purifications. © 2014 The Author(s)

Population genetics of cancer cell clones: possible implications of cancer stem cells

Abstract Background The population dynamics of the various clones of cancer cells existing within a tumour is complex and still poorly understood. Cancer cell clones can be conceptualized as sympatric asexual species, and as such, the application of theoretical population genetics as it pertains to asexual species may provide additional insights. Results The number of generations of tumour cells within a cancer has been estimated at a minimum of 40, but high cancer cell mortality rates suggest that the number of cell generations may actually be in the hundreds. Such a large number of generations would easily allow natural selection to drive clonal evolution assuming that selective advantages of individual clones are within the range reported for free-living animal species. Tumour cell clonal evolution could also be driven by variation in the intrinsic rates of increase of different clones or by genetic drift. In every scenario examined, the presence of cancer stem cells would require lower selection pressure or less variation in intrinsic rates of increase. Conclusions The presence of cancer stem cells may result in more rapid clonal evolution. Specific predictions from theoretical population genetics may lead to a greater understanding of this process.</p

Performance of Detecting IgM Antibodies against Enterovirus 71 for Early Diagnosis

Enterovirus 71 (EV71) infection is more likely to induce severe complications and mortality than other enteroviruses. Methods for detection of IgM antibody against EV71 had been established for years, however, the performance of the methods in the very early diagnosis of EV71 infection had not been fully evaluated, which is especially meaningful because of the short incubation period of EV71 infection. In this report, the performance of an IgM anti-EV71 assay was evaluated using acute sera collected from 165 EV71 infected patients, 165 patients infected with other enteroviruses, and more than 2,000 sera from healthy children or children with other infected diseases. The results showed a 90% sensitivity in 20 patients who were in their first illness day, and similar sensitivity remained till 4 days after onset. After then the sensitivity increased to 95% to 100% for more than one month. The specificity of the assay in non-HFMD children is 99.1% (95% CI: 98.6–99.4), similar as the 99.9% specificity in healthy adults. The cross-reaction rate in patients infected with other non-EV71 enteroviruses was 11.4%. In conclusion, the data here presented show that the detection of IgM anti-EV71 by ELISA affords a reliable, convenient, and prompt diagnosis of EV71 infection

The dynamics of E1A in regulating networks and canonical pathways in quiescent cells

<p>Abstract</p> <p>Background</p> <p>Adenoviruses force quiescent cells to re-enter the cell cycle to replicate their DNA, and for the most part, this is accomplished after they express the E1A protein immediately after infection. In this context, E1A is believed to inactivate cellular proteins (e.g., p130) that are known to be involved in the silencing of E2F-dependent genes that are required for cell cycle entry. However, the potential perturbation of these types of genes by E1A relative to their functions in regulatory networks and canonical pathways remains poorly understood.</p> <p>Findings</p> <p>We have used DNA microarrays analyzed with Bayesian ANOVA for microarray (BAM) to assess changes in gene expression after E1A alone was introduced into quiescent cells from a regulated promoter. Approximately 2,401 genes were significantly modulated by E1A, and of these, 385 and 1033 met the criteria for generating networks and functional and canonical pathway analysis respectively, as determined by using Ingenuity Pathway Analysis software. After focusing on the highest-ranking cellular processes and regulatory networks that were responsive to E1A in quiescent cells, we observed that many of the up-regulated genes were associated with DNA replication, the cell cycle and cellular compromise. We also identified a cadre of up regulated genes with no previous connection to E1A; including genes that encode components of global DNA repair systems and DNA damage checkpoints. Among the down-regulated genes, we found that many were involved in cell signalling, cell movement, and cellular proliferation. Remarkably, a subset of these was also associated with p53-independent apoptosis, and the putative suppression of this pathway may be necessary in the viral life cycle until sufficient progeny have been produced.</p> <p>Conclusions</p> <p>These studies have identified for the first time a large number of genes that are relevant to E1A's activities in promoting quiescent cells to re-enter the cell cycle in order to create an optimum environment for adenoviral replication.</p

Significance of Aurora B overexpression in hepatocellular carcinoma. Aurora B Overexpression in HCC

<p>Abstract</p> <p>Background</p> <p>To investigate the significance of Aurora B expression in hepatocellular carcinoma (HCC).</p> <p>Methods</p> <p>The <it>Aurora B </it>and <it>Aurora A </it>mRNA level was measured in 160 HCCs and the paired nontumorous liver tissues by reverse transcription-polymerase chain reaction. Mutations of the <it>p53 </it>and <it>β-catenin </it>genes were analyzed in 134 and 150 tumors, respectively, by direct sequencing of exon 2 to exon 11 of <it>p53 </it>and exon 3 of <it>β-catenin</it>. Anticancer effects of AZD1152-HQPA, an Aurora B kinase selective inhibitor, were examined in Huh-7 and Hep3B cell lines.</p> <p>Results</p> <p><it>Aurora B </it>was overexpressed in 98 (61%) of 160 HCCs and in all 7 HCC cell lines examined. The overexpression of <it>Aurora B </it>was associated with <it>Aurora A </it>overexpression (<it>P </it>= 0.0003) and <it>p53 </it>mutation (<it>P </it>= 0.002) and was inversely associated with <it>β</it>-<it>catenin </it>mutation (<it>P </it>= 0.002). <it>Aurora B </it>overexpression correlated with worse clinicopathologic characteristics. Multivariate analysis confirmed that <it>Aurora B </it>overexpression was an independent poor prognostic factor, despite its interaction with Aurora A overexpression and mutations of <it>p53 </it>and <it>β</it>-<it>catenin</it>. In Huh-7 and Hep3B cells, AZD1152-HQPA induced proliferation blockade, histone H3 (Ser10) dephosphorylation, cell cycle disturbance, and apoptosis.</p> <p>Conclusion</p> <p><it>Aurora B </it>overexpression is an independent molecular marker predicting tumor invasiveness and poor prognosis of HCC. Aurora B kinase selective inhibitors are potential therapeutic agents for HCC treatment.</p