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

The 9p21 susceptibility locus for coronary artery disease and the severity of coronary atherosclerosis

<p>Abstract</p> <p>Background</p> <p>Case-control Genome-Wide Association Studies (GWAS) have identified single nucleotide polymorphisms (SNPs) at the 9p21 locus as risk factors for coronary artery disease (CAD). The locus does not contain a clear candidate gene. Hence, the results of GWAS have raised an intense interest in delineating the basis for the observed association. We analyzed association of 4 SNPs at the 9p21 locus with the severity and progression of coronary atherosclerosis, as determined by serial quantitative coronary angiograms (QCA) in the well-characterized Lipoprotein Coronary Atherosclerosis Study (LCAS) population. The LCAS is a randomized placebo-control longitudinal follow-up study in patients with CAD conducted to test the effects of fluvastatin on progression or regression of coronary atherosclerosis.</p> <p>Methods</p> <p>Extensive plasma lipid levels were measured at the baseline and 2 1/2 years after randomization. Likewise serial QCA was performed at the baseline and upon completion of the study. We genotyped the population for 4 SNPs, previously identified as the susceptibility SNPs for CAD in GWAS, using fluorogenic 5' nuclease assays. We reconstructed the haplotypes using Phase 2, analyzed SNP and haplotype effects using the Thesias software as well as by the conventional statistical methods.</p> <p>Results</p> <p>Only Caucasians were included since they comprised 90% of the study population (332/371 with available DNA sample). The 4 SNPs at the 9p21 locus were in tight linkage disequilibrium, leading to 3 common haplotypes in the LCAS population. We found no significant association between quantitative indices of severity of coronary atherosclerosis, such as minimal lumen diameter and number of coronary lesions or occlusions and the 9p21 SNPs and haplotypes. Likewise, there was no association between quantitative indices of progression of coronary atherosclerosis and the SNPs or haplotypes. Similarly, we found no significant SNP or haplotype effect on severity and progression of coronary atherosclerosis.</p> <p>Conclusion</p> <p>We conclude the 4 SNPs at the 9p21 locus analyzed in this study do not impart major effects on the severity or progression of coronary atherosclerosis. The effect size may be very modest or the observed association of the CAD with SNPs at the 9p21 locus in the case-control GWAS reflect involvement of vascular mechanisms not directly related to the severity or progression of coronary atherosclerosis.</p

Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B

BACKGROUND: The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines METHODS: We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP). RESULTS: The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes. CONCLUSION: These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied

Whole Blood DNA Aberrant Methylation in Pancreatic Adenocarcinoma Shows Association with the Course of the Disease: A Pilot Study

Pancreatic tumors are usually diagnosed at an advanced stage in the progression of the disease, thus reducing the survival chances of the patients. Non-invasive early detection would greatly enhance therapy and survival rates. Toward this aim, we investigated in a pilot study the power of methylation changes in whole blood as predictive markers for the detection of pancreatic tumors. We investigated methylation levels at selected CpG sites in the CpG rich regions at the promoter regions of p16, RARbeta, TNFRSF10C, APC, ACIN1, DAPK1, 3OST2, BCL2 and CD44 in the blood of 30 pancreatic tumor patients and in the blood of 49 matching controls. In addition, we studied LINE-1 and Alu repeats using degenerate amplification approach as a surrogate marker for genome-wide methylation. The site-specific methylation measurements at selected CpG sites were done by the SIRPH method. Our results show that in the patient’s blood, tumor suppressor genes were slightly but significantly higher methylated at several CpG sites, while repeats were slightly less methylated compared to control blood. This was found to be significantly associated with higher risk for pancreatic ductal adenocarcinoma. Additionally, high methylation levels at TNFRSCF10C were associated with positive perineural spread of tumor cells, while higher methylation levels of TNFRSF10C and ACIN1 were significantly associated with shorter survival. This pilot study shows that methylation changes in blood could provide a promising method for early detection of pancreatic tumors. However, larger studies must be carried out to explore the clinical usefulness of a whole blood methylation based test for non-invasive early detection of pancreatic tumors

Animal Interactions and the Emergence of Territoriality

Inferring the role of interactions in territorial animals relies upon accurate recordings of the behaviour of neighbouring individuals. Such accurate recordings are rarely available from field studies. As a result, quantification of the interaction mechanisms has often relied upon theoretical approaches, which hitherto have been limited to comparisons of macroscopic population-level predictions from un-tested interaction models. Here we present a quantitative framework that possesses a microscopic testable hypothesis on the mechanism of conspecific avoidance mediated by olfactory signals in the form of scent marks. We find that the key parameters controlling territoriality are two: the average territory size, i.e. the inverse of the population density, and the time span during which animal scent marks remain active. Since permanent monitoring of a territorial border is not possible, scent marks need to function in the temporary absence of the resident. As chemical signals carried by the scent only last a finite amount of time, each animal needs to revisit territorial boundaries frequently and refresh its own scent marks in order to deter possible intruders. The size of the territory an animal can maintain is thus proportional to the time necessary for an animal to move between its own territorial boundaries. By using an agent-based model to take into account the possible spatio-temporal movement trajectories of individual animals, we show that the emerging territories are the result of a form of collective animal movement where, different to shoaling, flocking or herding, interactions are highly heterogeneous in space and time. The applicability of our hypothesis has been tested with a prototypical territorial animal, the red fox (Vulpes vulpes)

Cigarette Smoking and p16INK4α Gene Promoter Hypermethylation in Non-Small Cell Lung Carcinoma Patients: A Meta-Analysis

BACKGROUND:Aberrant methylation of promoter DNA and transcriptional repression of specific tumor suppressor genes play an important role in carcinogenesis. Recently, many studies have investigated the association between cigarette smoking and p16(INK4α) gene hypermethylation in lung cancer, but could not reach a unanimous conclusion. METHODS AND FINDINGS:Nineteen cross-sectional studies on the association between cigarette smoking and p16(INK4α) methylation in surgically resected tumor tissues from non-small cell lung carcinoma (NSCLC) patients were identified in PubMed database until June 2011. For each study, a 2×2 cross-table was extracted. In total, 2,037 smoker and 765 nonsmoker patients were pooled with a fixed-effects model weighting for the inverse of the variance. Overall, the frequency of p16(INK4α) hypermethylation was higher in NSCLC patients with smoking habits than that in non-smoking patients (OR = 2.25, 95% CI = 1.81-2.80). The positive association between cigarette smoking and p16(INK4α) hypermethylation was similar in adenocarcinoma and squamous-cell carcinoma. In the stratified analyses, the association was stronger in Asian patients and in the studies with larger sample sizes. CONCLUSION:Cigarette smoking is positively correlated to p16(INK4α) gene hypermethylation in NSCLC patients

Pharmacogenetics: data, concepts and tools to improve drug discovery and drug treatment

Variation in the human genome is a most important cause of variable response to drugs and other xenobiotics. Susceptibility to almost all diseases is determined to some extent by genetic variation. Driven by the advances in molecular biology, pharmacogenetics has evolved within the past 40 years from a niche discipline to a major driving force of clinical pharmacology, and it is currently one of the most actively pursued disciplines in applied biomedical research in general. Nowadays we can assess more than 1,000,000 polymorphisms or the expression of more than 25,000 genes in each participant of a clinical study – at affordable costs. This has not yet significantly changed common therapeutic practices, but a number of physicians are starting to consider polymorphisms, such as those in CYP2C9, CYP2C19, CYP2D6, TPMT and VKORC1, in daily medical practice. More obviously, pharmacogenetics has changed the practices and requirements in preclinical and clinical drug research; large clinical trials without a pharmacogenomic add-on appear to have become the minority. This review is about how the discipline of pharmacogenetics has evolved from the analysis of single proteins to current approaches involving the broad analyses of the entire genome and of all mRNA species or all metabolites and other approaches aimed at trying to understand the entire biological system. Pharmacogenetics and genomics are becoming substantially integrated fields of the profession of clinical pharmacology, and education in the relevant methods, knowledge and concepts form an indispensable part of the clinical pharmacology curriculum and the professional life of pharmacologists from early drug discovery to pharmacovigilance