Investigation of p16(INK4a) as a prognostic biomarker in oral epithelial dysplasia.

BACKGROUND: Human papilloma virus is a risk factor for oropharyngeal cancer. Evidence for a similar aetiological role in the development of oral dysplasia or its transformation to oral cancer is not as clear. Meta-analyses estimate the prevalence of high-risk human papilloma virus (HPV) serotypes to be three times higher in pre-malignant lesions and cancer than in normal oral mucosa. However, this does not imply a causal relationship. Conflicting results are reported from the few studies examining the prognostic significance of HPV positivity in the development of oral cancer. We aimed to examine the ability of p16(INK4a) protein expression, a surrogate marker of HPV infection, to predict malignant progression in a large cohort of oral dysplasia patients. METHODS: One hundred forty eight oral dysplasia cases underwent immunohistochemical analysis using a monoclonal antibody against p16(INK4a) . Clinical factors were also collated on each case. Slides were double scored independently by two trained observers. Univariate analyses using both logistic and Cox regression models were performed. RESULTS: Thirty nine of 148 cases progressed to cancer. Ten of 148 cases (7%) were p16(INK4a) positive. High grade of dysplasia (P = 0.0002) and lesion morphology (P = 0.03) were found to be prognostic of malignant progression. p16(INK4a) score was not prognostic in this cohort (P = 0.29). This did not change with a time to event analysis (P = 0.24). CONCLUSION: Few studies have assessed the aetiological role of HPV in cancer development from dysplastic lesions. Our study, using one of the largest cohorts of oral dysplasia, demonstrated a low rate of p16(INK4a) positivity and was unable to confirm a prognostic ability for this biomarker.

Aberrant spindle dynamics and cytokinesis in Dictyostelium discoideum cells that lack glycogen synthase kinase 3.

Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis. In animals, GSK-3 associates with mitotic spindles and its inhibition causes mis-regulation of chromosome segregation. Two suppressor screens in yeast point to a more general effect of GSK-3 on cell division, however the direct role of GSK-3 in control of mitosis has not been explored outside the animal kingdom. Here we report that the Dictyostelium discoideum GSK-3 orthologue, GskA, associates with the mitotic spindle during cell division, as seen for its mammalian counterparts. Dictyostelium possesses only a single GSK-3 gene that can be deleted to eliminate all GSK-3 activity. We found that gskA-null mutants failed to elongate their mitotic spindle and were unable to divide in shaking culture, but have no chromosome segregation defect. These results suggest further conservation for the role of GSK-3 in the regulation of spindle dynamics during mitosis, but also reveal differences in the mechanisms ensuring accurate chromosome segregation.

Characterization of Anopheles gambiae transglutaminase 3 (AgTG3) and its native substrate Plugin.

Male Anopheles mosquitoes coagulate their seminal fluids via cross-linking of a substrate, called Plugin, by the seminal transglutaminase AgTG3. Formation of the "mating plug" by cross-linking Plugin is necessary for efficient sperm storage by females. AgTG3 has a similar degree of sequence identity (~30%) to both human Factor XIII (FXIII) and tissue transglutaminase 2 (hTG2). Here we report the solution structure and in vitro activity for the cross-linking reaction of AgTG3 and Plugin. AgTG3 is a dimer in solution and exhibits Ca(2+)-dependent nonproteolytic activation analogous to cytoplasmic FXIII. The C-terminal domain of Plugin is predominantly α-helical with extended tertiary structure and oligomerizes in solution. The specific activity of AgTG3 was measured as 4.25 × 10(-2) units mg(-1). AgTG3 is less active than hTG2 assayed using the general substrate TVQQEL but has 8-10× higher relative activity when Plugin is the substrate. Mass spectrometric analysis of cross-linked Plugin detects specific peptides including a predicted consensus motif for cross-linking by AgTG3. These results support the development of AgTG3 inhibitors as specific and effective chemosterilants for A. gambiae.

Prognostic biomarkers of survival in oropharyngeal squamous cell carcinoma: systematic review and meta-analysis.

BACKGROUND: Human papillomavirus (HPV) positivity improves prognosis in patients with oropharyngeal squamous cell carcinoma (OPSCC). Combining HPV status with other biomarkers may improve its prognostic power. METHODS: The approach was a literature search for longitudinal studies of biomarkers in OPSCC, with systematic review and meta-analysis. RESULTS: In all, 3130 articles were identified; 32 satisfied the inclusion and exclusion criteria. On meta-analysis, there was a significant overall survival (OS) benefit for patients with HPV positive and p16 positive tumors. There was some evidence of improved OS of OPSCC patients with raised bcl2; amplification of 11q3 and loss of 16q genes; and low c-met, ki67, IMD, PLK, FHIT, nuclear surviving, or nuclear cyclin D1. However, none of these was suitable for meta-analysis. CONCLUSION: Survival from OPSCC is associated with several biomarkers, which constitute potential targets for research into improving the prognostic power of HPV in OPSCC. Larger trials are needed, with standardization of cut-points and adherence to consensus reporting guidelines.

The binary oral dysplasia grading system: validity testing and suggested improvement.

OBJECTIVES: A binary system is reputed to be superior to the World Health Organization (WHO) system in grading oral dysplasia. We aimed to validate its reproducibility and prognostic ability and examine whether it could be improved. STUDY DESIGN: Three pathologists graded 141 oral epithelial dysplasia biopsies with the use of both systems. Observer variability and prognostic ability were assessed with the use of kappa and logistic regression models. RESULTS: The binary system showed superior agreement to the WHO system (multirater kappa 0.59 vs. 0.49, respectively) but similar prognostic ability (odds ratio [OR] 4.59 [P = .014] vs. OR 2.25 [P = .02], respectively). Adding smoking and alcohol slightly improved the prognostic ability of both systems (OR 5.10 vs. OR 2.42, respectively). Our new binary system with a refined diagnostic threshold demonstrated a slightly greater prognostic ability and improved ability to differentiate between high- and low-risk moderate dysplasia cases. CONCLUSIONS: The binary system has similar prognostic ability but superior reproducibility compared with the WHO system. Prognostication is improved still further by using a new threshold.

Validation of tissue microarrays in oral epithelial dysplasia using a novel virtual-array technique.

BACKGROUND: Malignant transformation risk in oral epithelial dysplasia (OED) is currently determined by histological assessment. The subjectivity of this approach has led to interest in identifying prognostic biomarkers. Tissue microarrays (TMA) can reduce the utilization of the finite resources of a pathological archive. However, the selectivity involved in TMA construction necessitates the need to ensure that individual cores are representative of the overall features of the donor specimen. We aimed to validate, for the first time, the use of the TMA technique in OED by using a novel virtual array technique. METHODS: Sections from 38 cases of OED were stained with H&E and 6 immunohistochemical (IHC) biomarkers. All were then digitally scanned. Virtual cores were generated by image capturing a 0.6mm(2) area of the IHC slide that corresponded to the same dysplastic area marked on the H&E slide. Two trained blinded observers scored both whole slides and virtual cores independently. The degree of reliability in scores between the individual raters and between virtual TMA cores and slides was assessed using both interclass correlation coefficient (ICCC) and weighted κ statistics. RESULTS: Excellent inter-observer reliability was demonstrated with all the immunohistochemical markers. ICCC ranged from 0.67-1.0 and κ scores >0.8. There was also a high reliability in the scores between whole slides and virtual TMAs, with ICCC of between 0.66 and 0.89 for the 6 markers. CONCLUSIONS: This study validates the use of TMAs in OED using a variety of biomarkers. We also report a novel method for achieving this using a novel virtual-array technique.

Squamous cell carcinoma of the oral cavity rarely harbours oncogenic human papillomavirus.

Although it is now well established that a significant proportion of oropharyngeal squamous cell carcinomas (SCC) harbour oncogenic human papillomavirus (HPV) sequences, the frequency with which these sequences are detected in oral SCC (excluding oropharyngeal subsites) is highly variable. In an attempt to establish the true prevalence of HPV-16 and HPV-18 subtypes in oral SCC, we screened 142 consecutive cases from a UK cohort using both conventional PCR with consensus primers and type-specific quantitative PCR (Q-PCR), while at the same time employing a rigorous protocol to avoid sample contamination. Q-PCR revealed HPV sequences in five cases; two contained HPV-16 alone, two HPV-18 alone, and one sample carried both genotypes. However, only two of these cases (both HPV-16-positive) had moderate viral loads (51 and 91 viral copies per 100 cells respectively) and were positive for HPV DNA by conventional PCR. Both cases contained HPV DNA in tumour cells as shown by Q-PCR analysis of micro-dissected tissue and by in situ hybridisation. The remaining three cases had only very low viral loads (between 3 and 7 viral copies per 100 cells), were negative by conventional PCR and lacked HPV DNA in tumour cells. Our data provide strong evidence that oncogenic HPV is uncommon in oral SCC and that routine HPV testing of these tumours cannot be advocated.

Mutant glycyl-tRNA synthetase (Gars) ameliorates SOD1(G93A) motor neuron degeneration phenotype but has little affect on Loa dynein heavy chain mutant mice.

BACKGROUND: In humans, mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system, and clinical phenotypes ranging from Charcot-Marie-Tooth neuropathy to a severe infantile form of spinal muscular atrophy. GARS is ubiquitously expressed and may have functions in addition to its canonical role in protein synthesis through catalyzing the addition of glycine to cognate tRNAs. METHODOLOGY/PRINCIPAL FINDINGS: We have recently described a new mouse model with a point mutation in the Gars gene resulting in a cysteine to arginine change at residue 201. Heterozygous Gars(C201R/+) mice have locomotor and sensory deficits. In an investigation of genetic mutations that lead to death of motor and sensory neurons, we have crossed the Gars(C201R/+) mice to two other mutants: the TgSOD1(G93A) model of human amyotrophic lateral sclerosis and the Legs at odd angles mouse (Dync1h1(Loa)) which has a defect in the heavy chain of the dynein complex. We found the Dync1h1(Loa/+);Gars(C201R/+) double heterozygous mice are more impaired than either parent, and this is may be an additive effect of both mutations. Surprisingly, the Gars(C201R) mutation significantly delayed disease onset in the SOD1(G93A);Gars(C201R/+) double heterozygous mutant mice and increased lifespan by 29% on the genetic background investigated. CONCLUSIONS/SIGNIFICANCE: These findings raise intriguing possibilities for the study of pathogenetic mechanisms in all three mouse mutant strains.

An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot-Marie-Tooth type 2D peripheral neuropathy.

Mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system in humans, described clinically as Charcot-Marie-Tooth type 2D or distal spinal muscular atrophy type V. Here, we characterise a new mouse mutant, Gars(C201R), with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The Gars(C201R) mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons.

GSK3 is a multifunctional regulator of Dictyostelium development.

Glycogen synthase kinase 3 (GSK3) is a central regulator of metazoan development and the Dictyostelium GSK3 homologue, GskA, also controls cellular differentiation. The originally derived gskA-null mutant exhibits a severe pattern formation defect. It forms very large numbers of pre-basal disc cells at the expense of the prespore population. This defect arises early during multicellular development, making it impossible to examine later functions of GskA. We report the analysis of a gskA-null mutant, generated in a different parental strain, that proceeds through development to form mature fruiting bodies. In this strain, Ax2/gskA-, early development is accelerated and slug migration greatly curtailed. In a monolayer assay of stalk cell formation, the Ax2/gskA- strain is hypersensitive to the stalk cell-inducing action of DIF-1 but largely refractory to the repressive effect exerted by extracellular cAMP. During normal development, apically situated prestalk cells express the ecmB gene just as they commit themselves to stalk cell differentiation. In the Ax2/gskA- mutant, ecmB is expressed throughout the prestalk region of the slug, suggesting that GskA forms part of the repressive signalling pathway that prevents premature commitment to stalk cell differentiation. GskA may also play an inductive developmental role, because microarray analysis identifies a large gene family, the 2C family, that require gskA for optimal expression. These observations show that GskA functions throughout Dictyostelium development, to regulate several key aspects of cellular patterning.

Cell polarity and Dictyostelium development.

Cell polarity is essential for unicellular and multicellular stages of Dictyostelium development. Chemotaxis during early development requires each cell to rapidly reorganize its cytoskeleton to point towards a source of cAMP. This involves a balance between local induction of F-actin polymerization and suppression of pseudopods that point in other directions. Both the lipid phosphatidylinositol (3,4,5) trisphosphate and the soluble signal cGMP have been implicated in these processes, in addition to conserved and novel proteins. During later development cells adopt newly discovered, alternative modes of movement and interact through adhesion molecules. Finally, cells polarize secretion to particular regions of their surface.

Identification of the functional domains of yeast sorting nexins Vps5p and Vps17p.

Sorting nexins (Snxs) are a recently discovered family of conserved hydrophilic cytoplasmic proteins that have been found associated with membranes of the endocytic system and that are implicated in the trafficking of many endosomal membrane proteins, including the epidermal growth factor receptor and transferrin receptor. Snx proteins are partly defined by the presence of a p40 phox homology domain that has recently been shown to bind phosphatidylinositol 3-phosphate. Most Snx proteins also contain a predicted coiled-coils domain in the carboxyl-terminal half of the protein and have been shown to form dimers with other members of the Snx family. The yeast sorting nexins Vps5p and Vps17p form a dimer and are also components of the retromer complex that mediates endosome-to-Golgi transport of the carboxypeptidase Y receptor Vps10p. To functionally define the different domains of the yeast sorting nexins Vps5p and Vps17p, we have generated various truncations to examine the role that the different domains of Vps5p/Vps17p play in their respective functions. Herein, we show that the C-terminal halves of Vps5p and Vps17p, which contain the coiled-coils domains, are necessary and sufficient for their interaction. We have also mapped the retromer assembly domain to the N-terminal half of Vps5p and found that binding of Vps5p by Vps17p synergizes the interaction between Vps5p and other retromer components. Additionally, we have examined which domain(s) of Vps5p is necessary for membrane association.