Paul D. Lewis

A comprehensive survey of Ras mutations in cancer

All mammalian cells express 3 closely related Ras proteins, termed H-Ras, K-Ras, and N-Ras, that promote oncogenesis when they are mutationally activated at codon 12, 13, or 61. Although there is a high degree of similarity among the isoforms, K-Ras mutations are far more frequently observed in cancer, and each isoform displays preferential coupling to particular cancer types. We examined the mutational spectra of Ras isoforms curated from large-scale tumor profiling and found that each isoform exhibits surprisingly distinctive codon mutation and amino-acid substitution biases. These findings were unexpected given that these mutations occur in regions that share 100% amino-acid sequence identity among the 3 isoforms. Of importance, many of these mutational biases were not due to differences in exposure to mutagens, because the patterns were still evident when compared within specific cancer types. We discuss potential genetic and epigenetic mechanisms, as well as isoform-specific differences in protein structure and signaling, that may promote these distinct mutation patterns and differential coupling to specific cancers.

Detection of damage to the mitochondrial genome in the oncocytic cells of Warthin's tumour.

Warthins tumour of the salivary glands is composed of oncocytic cells containing excessive numbers of mitochondria which show frequent structural abnormalities and reduced metabolic function. Recent evidence of a strong association between cigarette smoking and the occurrence of Warthins tumour prompted this study, to look for evidence of damage to mitochondrial DNA (mtDNA) that could be the result of an increase in oxidative stress; two‐colour fluorescence in situ hybridization (FISH) was developed to show the distribution of mitochondria with deleted mtDNA in paraffin wax‐embedded material. Approximately 10% of mtDNA bears the ‘common’ 4977 bp deletion. Using the polymerase chain reaction (PCR), the 4977 bp deletion was further quantified, in Warthins tumour and age‐matched normal parotid control tissue. Whilst the deletion was present in all parotid tissue, its presence was significantly higher in oncocytic tumour cells. In a small number of controls, there was a trend towards higher concentrations of the deletion in smokers. Copyright

Atomic force microscopy comes of age

AFM (atomic force microscopy) analysis, both of fixed cells, and live cells in physiological environments, is set to offer a step change in the research of cellular function. With the ability to map cell topography and morphology, provide structural details of surface proteins and their expression patterns and to detect pico‐Newton force interactions, AFM represents an exciting addition to the arsenal of the cell biologist. With the explosion of new applications, and the advent of combined instrumentation such as AFM—confocal systems, the biological application of AFM has come of age. The use of AFM in the area of biomedical research has been proposed for some time, and is one where a significant impact could be made. Fixed cell analysis provides qualitative and quantitative subcellular and surface data capable of revealing new biomarkers in medical pathologies. Image height and contrast, surface roughness, fractal, volume and force analysis provide a platform for the multiparameter analysis of cell and protein functions. Here, we review the current status of AFM in the field and discuss the important contribution AFM is poised to make in the understanding of biological systems.

A combination drug therapy improves cognition and reverses gene expression changes in a mouse model of Huntington's disease

Huntingtons disease is a genetic disease caused by a single mutation. It is characterized by progressive movement, emotional and cognitive deficits. R6/2 mice transgenic for exon 1 of the HD gene with 150+ CAG repeats have a progressive neurological phenotype, including deterioration in cognitive function. The mechanism underlying the cognitive deficits in R6/2 mice is unknown, but dysregulated gene expression, reduced neurotransmitter levels and abnormal synaptic function are present before the cognitive decline becomes pronounced. Our goal here was to ameliorate the cognitive phenotype in R6/2 mice using a combination drug therapy (tacrine, moclobemide and creatine) aimed at boosting neurotransmitter levels in the brain. Treatment from 5 weeks of age prevented deterioration in two different cognitive tasks until at least 12 weeks. However, motor deterioration continued unabated. Microarray analysis of global gene expression revealed that many genes significantly up‐ or down‐regulated in untreated R6/2 mice had returned towards normal levels after treatment, though a minority were further dysregulated. Thus dysregulated gene expression was reversed by the combination treatment in the R6/2 mice and probably underlies the observed improvements in cognitive function. Our study shows that cognitive decline caused by a genetic mutation can be slowed by a combination drug treatment, and gives hope that cognitive symptoms in HD can be treated.

Staphylococci and staphylococcal superantigens in asthma and rhinitis: a systematic review and meta‐analysis

To cite this article: Pastacaldi C, Lewis P, Howarth P. Staphylococci and staphylococcal superantigens in asthma and rhinitis: a systematic review and meta‐analysis. Allergy 2011; 66: 549–555.

Influence of DNA repair on nonlinear dose-responses for mutation.

Recent evidence has challenged the default assumption that all DNA-reactive alkylating agents exhibit a linear dose-response. Emerging evidence suggests that the model alkylating agents methyl- and ethylmethanesulfonate and methylnitrosourea (MNU) and ethylnitrosourea observe a nonlinear dose-response with a no observed genotoxic effect level (NOGEL). Follow-up mechanistic studies are essential to understand the mechanism of cellular tolerance and biological relevance of such NOGELs. MNU is one of the most mutagenic simple alkylators. Therefore, understanding the mechanism of mutation induction, following low-dose MNU treatment, sets precedence for weaker mutagenic alkylating agents. Here, we tested MNU at 10-fold lower concentrations than a previous study and report a NOGEL of 0.0075 µg/ml (72.8nM) in human lymphoblastoid cells, quantified through the hypoxanthine (guanine) phosphoribosyltransferase assay (OECD 476). Mechanistic studies reveal that the NOGEL is dependent upon repair of O6-methylguanine (O6MeG) by the suicide enzyme O6MeG-DNA methyltransferase (MGMT). Inactivation of MGMT sensitizes cells to MNU-induced mutagenesis and shifts the NOGEL to the left on the dose axis.

L-Selectin ligands in human endometrium: comparison of fertile and infertile subjects

BACKGROUND L-selectin ligands, localized to the luminal epithelium at the time of implantation, may support the early stages of blastocyst attachment. We have assessed the expression of two L-selectin ligands, defined by MECA-79 and HECA-452 monoclonal antibodies, and the sulfotransferase GlcNAc6ST-2, involved in generation of L-selectin ligand epitopes, in the secretory phase of the endometrium from fertile and infertile patients. METHODS Endometrial samples were obtained from 33 fertile, 26 PCOS, 25 endometriosis and 33 patients diagnosed with unexplained infertility. L-selectin ligands and GlcNAc6ST-2 expression was assessed by immunohistochemistry and immunoblotting. RESULTS Immunohistochemical staining of uterine epithelium, from fertile and infertile women, demonstrated differential expression of MECA-79 and HECA-452 epitopes. In fertile women in the secretory phase MECA-79 was more strongly expressed, particularly on the lumen, than in infertile women. HECA-452 staining was significantly stronger in the glands in PCOS and endometriosis patients than in fertile women. GlcNAc6ST-2 expression was reduced in infertile patients, correlating with MECA-79 expression. CONCLUSIONS This study demonstrated significant differences in expression of L-selectin ligands between fertile and infertile women in natural cycles, and could contribute to patient assessment prior to initiating fertility treatment.

The in vitro anti-tumour activity of zoledronic acid and docetaxel at clinically achievable concentrations in prostate cancer

Bisphosphonates and chemotherapy have increasingly gained favour in the treatment of metastatic hormone resistant prostate cancer. We investigated whether zoledronic acid, at a concentration found at the bone, would enhance the anti-tumour activity of docetaxel in the hormone resistant prostate cancer cell line PC-3. Cells were exposed to zoledronic acid (1 mM) in combination or in sequence with docetaxel (3 nM). Cell viability, apoptosis and markers for inhibition of the mevalonate pathway were analyzed 48 or 72 hours after drug treatment. Reduction in cell viability and increased apoptosis levels were most pronounced with single agent zoledronic acid. Western blot analysis showed an overall reduction in the proliferation marker Mini chromosome maintenance protein 2 (MCM2) and reduction in caspase-3 precursor for all drug treatments and a marked reduction in Rho A levels with single agent zoledronic acid and zoledronic acid-docetaxel sequence. This study highlights the potency of zoledronic acid, when used at concentrations similar to those found at the bone, in reducing cell viability and causing apoptosis. Clinically, these findings suggest that in patients with bone metastases due to hormone resistance prostate cancer, who are not fit enough for systemic chemotherapy, single agent zoledronic acid may have a direct effect on viability of prostate cancer epithelial cells.

Progesterone induces nano-scale molecular modifications on endometrial epithelial cell surfaces.

Background information. The endometrial epithelial cell membrane is a key interface in female reproductive biology. Steroid hormones play a predominant role in cyclic changes which occur at this interface during the female menstrual cycle. Specific changes in the morphology of the endometrial epithelial cell surface become apparent with the epithelial transition that drives the switch from a non‐receptive to receptive surface due to the action of progesterone on an oestrogen primed tissue. AFM (atomic force microscopy) allows the high‐resolution characterization of the endometrial epithelial cell surface. Its contact probe mechanism enables a unique imaging method that requires little sample preparation, yielding topographical and morphological characterization. By stiffening the cell membrane, low concentrations of fixatives allow the surface detail of the cell to be resolved while preserving fine ultra‐structural details for analysis.

Putative prognostic epithelial-to-mesenchymal transition biomarkers for aggressive prostate cancer.

Prostate cancer is the second most frequently diagnosed cancer worldwide and is the sixth leading cause of cancer deaths in men, yet it varies greatly in its aggressiveness. Currently, it is not possible to adequately differentiate between patients whose tumors will remain indolent and those patients whose disease will progress, resulting in unnecessary aggressive treatment. Consequently, there is an urgent need to identify markers of prostate cancer progression, invasiveness and metastasis to more accurately predict prognosis. The aim of this study was to assess the ability of key epithelial-to-mesenchymal transition molecules in identifying prostate cancer patients who are likely to develop aggressive tumors. Using 215 archival patient tissue samples, immunohistochemistry was applied to examine the expression and sub-cellular localization of E-Cadherin, Snail, Slug, Twist, Vimentin, BMP-2 and BMP-7. Of the seven markers assessed, a significantly increased expression of Snail protein was observed within the nucleus of prostate cancer cells and was strongly associated with increasing Gleason score and clinical stage. In addition, loss of E-Cadherin expression at the cellular membrane of prostate cancer cells was also significantly associated with increasing Gleason score, clinical stage, and additionally, a reduction in survival.