Maria Davies

Induction of an epithelial to mesenchymal transition in human immortal and malignant keratinocytes by TGF‐β1 involves MAPK, Smad and AP‐1 signalling pathways

Recent data indicate that transforming growth factor‐β1 (TGF‐β1) can act to promote tumour progression in the late stages of carcinogenesis. The mechanism by which this occurs is unknown although a ligand‐induced epithelial–mesenchymal transition (EMT) is thought to be important. In this study, we demonstrate that active Ras is required for TGF‐β1‐induced EMT in human keratinocytes and that epidermal growth factor (EGF) can substitute for mutant Ras. EMT was reversed by the removal of TGF‐β1. Under conditions of TGF‐β1‐induced EMT, cells were growth inhibited by the ligand resulting in G1 arrest. In cells containing normal Ras, TGF‐β1‐activated ERK and p38 mitogen‐activated protein kinases (MAPKs), and levels of activation were further increased by co‐treatment with EGF. Inhibition of MAPK pathways and Smad2/3 signalling blocked the induction of EMT by TGF‐β1. Further, inhibition of the AP‐1 transcriptional complex by [6]‐Gingerol, or by the ectopic expression of JDP2, blocked TGF‐β1‐induced EMT and conversely, stimulation of AP‐1 by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) substituted for EGF in the induction of EMT by TGF‐β1 in cells containing normal Ras. The presence of oncogenic Ras, the treatment of cells with EGF, or the treatment of cells with TPA to activate AP‐1, potentiated TGF‐β1‐induced Smad‐dependent transcription, an effect that was attenuated by the inhibition of MAPKs and AP‐1. The results demonstrate that active Ras and TGF‐β1 co‐operate to reversibly induce EMT in human keratinocytes by mechanisms that involve MAPKs, Smad2/3 and AP‐1. Further we demonstrate that MAPK/AP‐1 signalling enhances Smad transcriptional activity under conditions associated with TGF‐β1‐induced EMT.

The Role of TGF-β in Epithelial Malignancy and its Relevance to the Pathogenesis of Oral Cancer (Part II)

The role of transforming growth factor-beta (TGF-beta) in epithelial malignancy is complex, but it is becoming clear that, in the early stages of carcinogenesis, the protein acts as a potent tumor suppressor, while later, TGF-beta can function to advance tumor progression. We review the evidence to show that the pro-oncogenic functions of TGF-beta are associated with (1) a partial loss of response to the ligand, (2) defects of components of the TGF-beta signal transduction pathway, (3) over-expression and/or activation of the latent complex, (4) epithelial-mesenchymal transition, and (5) recruitment of signaling pathways which act in concert with TGF-beta to facilitate the metastatic phenotype. These changes are viewed in the context of what is known about the pathogenesis of oral cancer and whether this knowledge can be translated into the development of new therapeutic modalities.

TGF-β Signal Transduction in Oro-facial Health and Non-malignant Disease (Part I)

The transforming growth factor-beta (TGF-) family of cytokines consists of multi-functional polypeptides that reg- ulate a variety of cell processes, including proliferation, differentiation, apoptosis, extracellular matrix elaboration, angiogenesis, and immune suppression, among others. In so doing, TGF- plays a key role in the control of cell behavior in both health and disease. In this report, we review what is known about the mechanisms of activation of the peptide, together with details of TGF- signal transduction pathways. This review summarizes the evidence implicating TGF- in normal physiological processes of the craniofacial complex—such as palatogenesis, tooth formation, wound healing, and scarring—and then evaluates its role in non-malignant disease processes such as scleroderma, submucous fibrosis, periodontal disease, and lichen planus.

TGF-β1 acts as a tumor suppressor of human malignant keratinocytes independently of Smad 4 expression and ligand-induced G1 arrest

This study examined the role of TGF-β1 in human keratinocyte malignancy. Two carcinoma-derived human oral keratinocyte cell lines, BICR 31 and H314, were selected on the basis of their known resistance to TGF-β1-induced G1 arrest, the presence of wild type TGF-β cell surface receptors and normal Ras. Smad 4 protein was undetectable in both cell lines, but Smad 2 and Smad 3 were expressed at levels comparable with a fully TGF-β responsive cell line, and treatment of the cells with TGF-β1 resulted in the phosphorylation of Smad 2. Treatment with exogenous TGF-β1 resulted in a failure to induce transcription from an artificial Smad-dependent promoter and a failure to down-regulate c-myc, but resulted in an up-regulation of AP-1 associated genes (Fra-1, JunB and fibronectin). Transient transfection of Smad 4 into BICR 31 restored TGF-β1-induced growth inhibition and Smad-dependent transcriptional activation. Protracted treatment of cells with exogenous TGF-β1 resulted in the attenuation of cell growth in vitro. To over-express TGF-β1, both cell lines were transfected with latent TGF-β1 cDNA; neutralization studies of conditioned media demonstrated that whilst the majority of the peptide was in the latent form, a small proportion was present as the active peptide. Cells that over-expressed endogenous TGF-β1 grew more slowly in vitro compared to both the vector-only controls and cells that did not over-express the peptide. Orthotopic transplantation of cells that over-expressed endogenous TGF-β1 to the floor of the mouth in athymic mice resulted in marked inhibition of primary tumor formation compared to controls. Expression of a dominant-negative TGF-β type II receptor in cells that over-expressed endogenous TGF-β1 resulted in enhanced cell growth in vitro and diminished the tumor suppressor effect of the ligand in vivo, indicating that the endogenous TGF-β1 was acting in an autocrine capacity. The results demonstrate that over-expression of endogenous TGF-β1 in human malignant oral keratinocytes leads to growth inhibition in vivo and tumor suppression in vitro by mechanisms that are independent of Smad 4 expression and TGF-β1-induced G1 arrest.

Attenuated type II TGF-beta receptor signalling in human malignant oral keratinocytes induces a less differentiated and more aggressive phenotype that is associated with metastatic dissemination.

We examined the effect of stable transfection of dominant negative TβR‐II (dn TβR‐II) cDNA in a human oral carcinoma cell line that contained normal Ras and was growth inhibited by TGF‐β1. Two clonal cell lines containing dn TβR‐II were isolated and compared to the vector‐only control and parent cell line. The treatment of cells with exogenous TGF‐β1 resulted in a decrease in ligand‐induced growth inhibition and loss of c‐myc downregulation in test cells compared to controls; transcriptional activation of certain genes including fra‐1 and collagenase was retained. Cells containing dn TβR‐II grew faster in monolayer culture, expressed less keratin 10 and exhibited increased motility and invasion in vitro compared to control cell lines. Endogenous TGF‐β1 production and the regulation of MMP‐2 and MMP‐9 by TGF‐β1 remained unchanged. After orthotopic transplantation to the floor of the mouth in athymic mice, cells containing dn TβR‐II formed comparable numbers of primary tumours at the site of inoculation as controls but the tumours were less differentiated as demonstrated by the absence of keratin 10 immunostaining. Further, metastatic dissemination to the lungs and lymphatics was more evident in grafts of cells containing dn TβR‐II than controls. Taken together, the results demonstrate that attenuation of TGF‐β signalling through transfection of dn TβR‐II cDNA leads to an enhanced growth rate, a loss of tumour cell differentiation and an increase in migration and invasion, characteristics that corresponded to the development of the metastatic phenotype.

16S rRNA next generation sequencing analysis shows bacteria in Alzheimer’s post-mortem brain

The neurological deterioration associated with Alzheimer’s disease (AD), involving accumulation of amyloid-beta peptides and neurofibrillary tangles, is associated with evident neuroinflammation. This is now seen to be a significant contributor to pathology. Recently the tenet of the privileged status of the brain, regarding microbial compromise, has been questioned, particularly in terms of neurodegenerative diseases. It is now being considered that microbiological incursion into the central nervous system could be either an initiator or significant contributor to these. This is a novel study using 16S ribosomal gene-specific Next generation sequencing (NGS) of extracted brain tissue. A comparison was made of the bacterial species content of both frozen and formaldehyde fixed sections of a small cohort of Alzheimer-affected cases with those of cognitively unimpaired (normal). Our findings suggest an increase in bacterial populations in Alzheimer brain tissue compared with normal.

Management of dentine hypersensitivity: efficacy of professionally and self‐administered agents

CONTEXT The gold standard treatment modality for dentine hypersensitivity has not yet been established. This review examines the effectiveness of self and professionally applied treatments for the reduction in pain from dentine hypersensitivity. MATERIALS AND METHODS Electronic (three databases) and hand searches were performed 14-21 July 2014 to identify randomized controlled trials for the treatment of dentine hypersensitivity. RESULTS This systematic review provided numerous treatment modalities for dentine hypersensitivity. Eleven agents and 105 Randomized Controlled Trials were robust enough to be included. The studies varied considerably in design, observation period, active agents, formulation of the whole agent, negative and positive controls and comparator products investigated. The stimuli used were predominantly airblast and tactile or thermal. Due to the heterogeneity between the studies and lack of direct comparison between agents there was insufficient data to undertake a meta-analysis to compare agents for meaningful conclusions. Best available evidence for each treatment agent has been documented as a narrative. CONCLUSIONS Treatments including stannous fluoride, arginine, calcium sodium phosphosilicate and strontium toothpaste appear to be clinically effective for the treatment of dentine hypersensitivity compared to comparators and controls. There is limited evidence to confirm the relative effectiveness of individual professionally applied agents.

Overexpression of JunB in undifferentiated malignant rat oral keratinocytes enhances the malignant phenotype in vitro without altering cellular differentiation.

Our study examined the expression of AP‐1 family members in keratinocytes derived from the rat‐4NQO model of oral carcinogenesis in which extremes of epithelial differentiation and tumour cell aggressiveness are evident. The constitutive expression of JunB was diminished in the undifferentiated, more aggressive tumour phenotype compared with the well‐differentiated, less aggressive keratinocytes, whereas the expression of other AP‐1 family members (c‐jun, junD, c‐fos, fra1, fra2 and fosB) was either very weak or variable. After transfection of the undifferentiated keratinocytes with junB cDNA, clonal populations were isolated that expressed similar levels of JunB protein as the well‐differentiated cells. Both untransfected and transfected cell lines were keratin negative and vimentin positive. Increased expression of JunB in the transfected cells resulted in up‐regulation of c‐Jun and Fra1 and an enhanced AP‐1 activity as demonstrated by transcriptional activation of the prototypic AP‐1 dependent promoter, MMP‐1. JunB transfected cells grew more quickly than vector‐only controls and were refractory to the growth inhibitory effects of TGF‐β1. Over‐expression of JunB resulted in the elevated expression of the AP‐1 dependent proteinase, MMP‐9, whereas the expression of the AP‐1 independent enzyme, MMP‐2, was unaffected. JunB transfected keratinocytes were highly invasive in an in vitro assay of tumour cell invasion compared with vector controls. The results indicate that increased expression of JunB above baseline levels in undifferentiated rat keratinocytes does not alter epithelial differentiation but enhances the malignant phenotype in vitro, possibly by altering the dynamics of the AP‐1 complex.

Efficacy of desensitizing dentifrices to occlude dentinal tubules.

Dentine hypersensitivity occurs when patent dentinal tubules are subjected to external stimuli, with pain being reduced by products that occlude tubules. This study compared the efficacy of a recently developed arginine-containing dentifrice, two established strontium-based products, and a fluoride control to occlude tubules when subjected to acid challenge. Dentine specimens with patent tubules were divided into four groups that were treated with a slurry consisting of one of the pastes mixed with stimulated human saliva. Treated specimens were further subdivided and soaked in 0.3% citric acid for 10 s, 30 s, 2 min, 5 min or 10 min. Tubule occlusion on representative scanning electron microscopy images was scored by blind review. All three desensitizing pastes offered good tubule occlusion, which was maintained to varying degrees following acidic challenge. After immersion in acid for 10 and 30 s, the strontium acetate- and arginine-containing pastes almost fully occluded tubules, but only the strontium acetate paste retained this level of occlusion after immersion in acid for 2 min, with strong statistical evidence that this paste occluded more tubules than the other pastes after immersion in acid for 2 or 5 min. This suggests that strontium acetate pastes may be the most effective at reducing dentine hypersensitivity.

Dysregulation of autocrine TGF-beta isoform production and ligand responses in human tumour-derived and Ha-ras-transfected keratinocytes and fibroblasts.

This study examined the autocrine production of TGF-beta 1, -beta 2 and -beta 3 in culture supernatants from tumour-derived (H series, n = 7; BICR series, n = 5), Ha-ras-transfected (n = 4) and normal (n = 2) human keratinocytes using a sandwich enzyme-linked immunosorbent assay (ELISA). Detection limits were 39.0 pg ml-1 for TGF-beta 1, 78.0 pg ml-1 for TGF-beta 2 and 1.9 ng ml-1 for TGF-beta 3. Tumour-derived oral keratinocytes predominantly produced less TGF-beta 1 than normal oral epithelial cells; the expression of endogenous TGF-beta 2 was variable. In keratinocytes containing mutant Ha-ras, TGF-beta 1 production was enhanced and TGF-beta 2 was undetectable. TGF-beta 3 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) but the protein was not detected in conditioned media, most probably because of the low detection limits of the ELISA for this isoform. Neutralisation experiments indicated that the latent TGF-beta peptide was secreted in keratinocyte conditioned medium. Seven tumour-derived keratinocyte cell lines (H series) and fibroblasts separated from normal (n = 1) and tumour-derived (n = 2) keratinocyte cultures were examined for their response to exogenous TGF-beta 1, -beta 2 and -beta 3. Six of seven tumour-derived keratinocyte cell lines were inhibited by TGF-beta 1 and TGF-beta 2 (-beta 1 > -beta 2); one cell line was refractory to both TGF-beta 1 and TGF-beta 2. Keratinocytes were inhibited (4 of 7), stimulated (1 of 7) or failed to respond (2 of 7) to TGF-beta 3, TGF-beta 1, -beta 2 and -beta 3 stimulated both normal and tumour-associated fibroblasts, but the tumour-associated fibroblasts showed less response to the ligands than their normal counterparts following prolonged treatment with each isoform. The results demonstrate variable autocrine production of TGF-beta isoforms by malignant keratinocytes, with loss of TGF-beta 1 generally associated with the tumour-derived phenotype and modification of endogenous isoform production dependent on the genetic background of the tumour cells. Further, the variable response of the tumour-derived keratinocytes and contiguous fibroblasts to the TGF-beta isoforms suggests that dysregulation of TGF-beta autocrine and paracrine networks are common characteristics of squamous epithelial malignancy.