The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity

Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer

Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2′-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC

Important prognostic factors for the long-term survival of lung cancer subjects in Taiwan

<p>Abstract</p> <p>Background</p> <p>This study used a large-scale cancer database in determination of prognostic factors for the survival of lung cancer subjects in Taiwan.</p> <p>Methods</p> <p>Total of 24,910 subjects diagnosed with lung cancer was analysed. Survival estimates by Kaplan-Meier methods. Cox proportional-hazards model estimated the death risk (hazard ratio (HR)) for various prognostic factors.</p> <p>Results</p> <p>The prognostic indicators associated with a higher risk of lung cancer deaths are male gender (males versus females; HR = 1.07, 95% confidence intervals (CI): 1.03–1.11), males diagnosed in later periods (shown in 1991–1994 versus 1987–1990; HR = 1.13), older age at diagnosis, large cell carcinoma (LCC)/small cell carcinoma (SCC), and supportive care therapy over chemotherapy. The overall 5-year survival rate for lung cancer death was significantly poorer for males (21.3%) than females (23.6%). Subjects with squamous cell carcinoma (SQCC) and treatment by surgical resection alone had better prognosis. We find surgical resections to markedly increase 5-year survival rate from LCC, decreased risk of death from LCC, and no improved survival from SCC.</p> <p>Conclusion</p> <p>Gender and clinical characteristics (i.e. diagnostic period, diagnostic age, histological type and treatment modality) play important roles in determining lung cancer survival.</p

The associated expression of Maspin and Bax proteins as a potential prognostic factor in intrahepatic cholangiocarcinoma

BACKGROUND: Maspin, a member of the serpin family, is a suppressor of tumor growth, an inhibitor of angiogenesis and an inducer of apoptosis. Maspin induces apoptosis by increasing Bax, a member of the Bcl-2 family of apoptosis-regulating proteins. In this exploratory study, we investigated the associated expression of Maspin and Bax proteins as a potential prognostic factor in intrahepatic cholangiocarcinoma (IHCCA). METHODS: Twenty-two paraffin-embedded samples were analyzed by immunohistochemical methods using Maspin, Bax and CD34 antibodies. Maspin was scored semiquantitatively (HSCORE). Apoptosis was assessed using an antibody against cleaved caspase-3. RESULTS: The strong relationship observed between the expression of Maspin and Bax, indicates that Bax is likely to be the key effector of Maspin-mediated induction of apoptosis as indicated by the activation of cleaved caspase-3. We categorized Maspin HSCORE by calculating the optimal cutpoint. A Maspin HSCORE above the cutpoint was inversely related with tumor dimension, depth of tumor and vascular invasion. Uni/multivariate analysis suggests that a Maspin HSCORE below the cutpoint significantly worsens the patients' prognosis. Tumors with Maspin HSCORE below the cutpoint had a shorter survival (11+/-5 months) than did patients with Maspin HSCORE above the cutpoint (27+/-4 months), whereas Kaplan-Meier analysis and logrank test showed no significant difference in overall survival between the patients. CONCLUSION: The associated expression of Maspin and Bax might delay tumor progression in IHCCA. Maspin above the cutpoint might counteract tumor development by increasing cell apoptosis, and by decreasing tumor mass and cell invasion. The combined expression of Maspin and Bax appears to influence the susceptibility of tumor cholangiocytes to apoptosis and thus may be involved in delaying IHCCA progression

Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners, Hierarchy and Physiological Significance

BACKGROUND: While protease-activated-receptor 1 (PAR(1)) plays a central role in tumor progression, little is known about the cell signaling involved. METHODOLOGY/PRINCIPAL FINDINGS: We show here the impact of PAR(1) cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR(1) activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR(1) C-tail did not prevent Shc-PAR(1) association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR(1) C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR(1)-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR(1) -C-tail, hPar1 oncogenic properties are abrogated. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration that a cytoplasmic portion of the PAR(1) C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR(1)-associating region in the breast cancer signaling niche

Tumor-Associated Macrophages (TAMs) Form an Interconnected Cellular Supportive Network in Anaplastic Thyroid Carcinoma

BACKGROUND: A relationship between the increased density of tumor-associated macrophages (TAMs) and decreased survival was recently reported in thyroid cancer patients. Among these tumors, anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumors in humans. TAMs (type M2) have been recognized as promoting tumor growth. The purpose of our study was to analyze with immunohistochemistry the presence of TAMs in a series of 27 ATC. METHODOLOGY/PRINCIPAL FINDINGS: Several macrophages markers such as NADPH oxidase complex NOX2-p22phox, CD163 and CD 68 were used. Immunostainings showed that TAMs represent more than 50% of nucleated cells in all ATCs. Moreover, these markers allowed the identification of elongated thin ramified cytoplasmic extensions, bestowing a "microglia-like" appearance on these cells which we termed "Ramified TAMs" (RTAMs). In contrast, cancer cells were totally negative. Cellular stroma was highly simplified since apart from cancer cells and blood vessels, RTAMs were the only other cellular component. RTAMs were evenly distributed and intermingled with cancer cells, and were in direct contact with other RTAMs via their ramifications. Moreover, RTAMs displayed strong immunostaining for connexin Cx43. Long chains of interconnected RTAMs arose from perivascular clusters and were dispersed within the tumor parenchyma. When expressed, the glucose transporter Glut1 was found in RTAMs and blood vessels, but rarely in cancer cells. CONCLUSION: ATCs display a very dense network of interconnected RTAMs in direct contact with intermingled cancer cells. To our knowledge this is the first time that such a network is described in a malignant tumor. This network was found in all our studied cases and appeared specific to ATC, since it was not found in differentiated thyroid cancers specimens. Taken together, these results suggest that RTAMs network is directly related to the aggressiveness of the disease via metabolic and trophic functions which remain to be determined

Shift Work in Nurses: Contribution of Phenotypes and Genotypes to Adaptation

Daily cycles of sleep/wake, hormones, and physiological processes are often misaligned with behavioral patterns during shift work, leading to an increased risk of developing cardiovascular/metabolic/gastrointestinal disorders, some types of cancer, and mental disorders including depression and anxiety. It is unclear how sleep timing, chronotype, and circadian clock gene variation contribute to adaptation to shift work.Newly defined sleep strategies, chronotype, and genotype for polymorphisms in circadian clock genes were assessed in 388 hospital day- and night-shift nurses.Night-shift nurses who used sleep deprivation as a means to switch to and from diurnal sleep on work days (∼25%) were the most poorly adapted to their work schedule. Chronotype also influenced efficacy of adaptation. In addition, polymorphisms in CLOCK, NPAS2, PER2, and PER3 were significantly associated with outcomes such as alcohol/caffeine consumption and sleepiness, as well as sleep phase, inertia and duration in both single- and multi-locus models. Many of these results were specific to shift type suggesting an interaction between genotype and environment (in this case, shift work).Sleep strategy, chronotype, and genotype contribute to the adaptation of the circadian system to an environment that switches frequently and/or irregularly between different schedules of the light-dark cycle and social/workplace time. This study of shift work nurses illustrates how an environmental "stress" to the temporal organization of physiology and metabolism can have behavioral and health-related consequences. Because nurses are a key component of health care, these findings could have important implications for health-care policy

Intensive care of the cancer patient: recent achievements and remaining challenges

A few decades have passed since intensive care unit (ICU) beds have been available for critically ill patients with cancer. Although the initial reports showed dismal prognosis, recent data suggest that an increased number of patients with solid and hematological malignancies benefit from intensive care support, with dramatically decreased mortality rates. Advances in the management of the underlying malignancies and support of organ dysfunctions have led to survival gains in patients with life-threatening complications from the malignancy itself, as well as infectious and toxic adverse effects related to the oncological treatments. In this review, we will appraise the prognostic factors and discuss the overall perspective related to the management of critically ill patients with cancer. The prognostic significance of certain factors has changed over time. For example, neutropenia or autologous bone marrow transplantation (BMT) have less adverse prognostic implications than two decades ago. Similarly, because hematologists and oncologists select patients for ICU admission based on the characteristics of the malignancy, the underlying malignancy rarely influences short-term survival after ICU admission. Since the recent data do not clearly support the benefit of ICU support to unselected critically ill allogeneic BMT recipients, more outcome research is needed in this subgroup. Because of the overall increased survival that has been reported in critically ill patients with cancer, we outline an easy-to-use and evidence-based ICU admission triage criteria that may help avoid depriving life support to patients with cancer who can benefit. Lastly, we propose a research agenda to address unanswered questions

Intermittent Hypoxia-Induced Cognitive Deficits Are Mediated by NADPH Oxidase Activity in a Murine Model of Sleep Apnea

Background: In rodents, exposure to intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Excessive NADPH oxidase activity may play a role in IH-induced CNS dysfunction. Methods and Findings: The effect of IH during light period on two forms of spatial learning in the water maze and well as markers of oxidative stress was assessed in mice lacking NADPH oxidase activity (gp91phox _/Y) and wild-type littermates. On a standard place training task, gp91phox _/Y displayed normal learning, and were protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to IH as compared to room air (RA) controls, while no changes emerged in gp91phox _/Y mice. Additionally, wild-type mice, but not gp91phox _/Y mice had significantly elevated levels of NADPH oxidase expression and activity, as well as MDA and 8-OHDG in cortical and hippocampal lysates following IH exposures. Conclusions: The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by excessive NADPH oxidase activity, and thus pharmacological agents targeting NADPH oxidase may provid

Interplay between n-3 and n-6 long-chain polyunsaturated fatty acids and the endocannabinoid system in brain protection and repair.

The brain is enriched in arachidonic acid (ARA) and docosahexaenoic acid (DHA), long-chain polyunsaturated fatty acids (LCPUFA) of the n-6 and n-3 series, respectively. Both are essential for optimal brain development and function. Dietary enrichment with DHA and other long-chain n-3 PUFA, such as eicosapentaenoic acid (EPA) have shown beneficial effects on learning and memory, neuroinflammatory processes and synaptic plasticity and neurogenesis. ARA, DHA and EPA are precursors to a diverse repertoire of bioactive lipid mediators, including endocannabinoids. The endocannabinoid system comprises cannabinoid receptors, their endogenous ligands, the endocannabinoids, and their biosynthetic and degradation enzymes. Anandamide (AEA) and 2-archidonoylglycerol (2-AG) are the most widely studied endocannabinoids, and are both derived from phospholipid-bound ARA. The endocannabinoid system also has well established roles in neuroinflammation, synaptic plasticity and neurogenesis, suggesting an overlap in the neuroprotective effects observed with these different classes of lipids. Indeed, growing evidence suggests a complex interplay between n-3 and n-6 LCPUFA and the endocannabinoid system. For example, long-term DHA and EPA supplementation reduces AEA and 2-AG levels, with reciprocal increases in levels of the analogous endocannabinoid-like DHA and EPA-derived molecules. This review summarises current evidence of this interplay and discusses the therapeutic potential for brain protection and repair