Stephen S. Prime

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.

Detection of human papillomavirus DNA in biopsies of human oral tissue

We have employed molecular probes produced from DNA fragments of human papillomavirus, cloned into prokaryotic vectors, to detect virus nucleic acid sequences in extracts of human oral tissues. The study was conducted with duplicate coded snap-frozen tissue biopsies from which frozen sections had been taken to accurately assess the pathology of each particular sample. The results show that a large proportion of the oral biopsies contained DNA which hybridized to the viral DNA probes, even under conditions of high stringency. The presence of virus did not correlate with neoplasia in the tissues examined, but HPV like sequences were found in a high proportion (80%) of biopsies taken from areas of keratosis and lichen planus and also in 41 to 46% of normal and tumour tissues.

Comparative analysis of transforming growth factor-β isoforms 1–3 in human and rabbit dentine matrices

Previous studies have implicated transforming growth factor-beta (s)(TGF-beta) in both development. Here TGF-beta isoforms in dentine extracellular matrix were analysed because these molecules may participate in dental issue repair. EDTA-soluble and collagenase-released fractions were isolated from human crown and root and rabbit incisor dentine samples and analysed for TGF-beta isoforms. TGF-beta(1) was the major isoform detected in all samples and the only isoform detected in human dentine samples. TGF-beta(2) was detected only in the collagenase-released fraction of rabbit incisor dentine and was present at low levels. TGF-beta(3) was detected in both EDTA-soluble and collagenase-released fractions of rabbit dentine. Greater levels of the TGF-beta(1) isoform were detected in the rabbit than human dentine samples and some differences in distribution amongst the two tissue fractions were observed between these species. The presence of these isoforms of TGF-beta in dentine may provide a reservoir of growth factor in the matrix that could participate in processes leading to tissue repair after injury.

Molecular changes in oral cancer may reflect aetiology and ethnic origin

Oral cancer, although uncommon in the Western world, accounts for up to 40% of all malignancies in parts of India and South East Asia. Recognised aetiological agents of oral cancer include tobacco and alcohol. This paper reviews the spectrum of molecular changes found in oral squamous cell carcinomas from Western (U.K., U.S.A., Australia) and Eastern (India, S.E. Asia) countries. p53 mutations are common in tumours from the West (47%) but are infrequent in the East (7%). Tumours from India and South East Asia are characterised by the involvement of ras oncogenes, including mutation, loss of heterozygosity (H-ras) and amplification (K- and N-ras), events which are uncommon in the West. The possibility that these genetic differences reflect aetiology and/or ethnic origin is discussed.

Detection of human papillomavirus genes in human oral tissue biopsies and cultures by polymerase chain reaction

We have used the polymerase chain reaction to detect DNA sequences related to human papillomavirus type 16, by simultaneous priming with oligonucleotides from the E6 and L1/L2 open reading frames of the HPV16 genome. The HPV16-related sequence is present at low levels in normal oral tissue, in addition to biopsies and cell cultures from patients with benign and malignant disease. Ultimate analysis of the amplified sequences from the E6(120bp) and L1/L2(173bp) regions of HPV16 was achieved by gel electrophoresis and comparative nucleotide sequencing. The oral carcinoma biopsies and tissue cultures contained DNA sequences which were identical to the E6 region of HPV16, but only rarely contained sequences closely related to the L1/L2 region. The PCR technology should permit the detection, identification and cloning of latent viruses from extremely small tissue biopsies.

Papillomaviruses: The current status in relation to oral disease

Human papillomaviruses of different types are associated with a variety of benign oral lesions and may be associated with some premalignant and malignant oral lesions. However, since it is now clear that a variant of human papillomavirus 16 is harbored by normal oral mucosa, as well as by premalignant and malignant lesions, such associations may not necessarily always be causal. The rapid progress of recent research in this field is reviewed, with particular reference to oral disease, and the current status is discussed.

Keratinocytes synthesize and activate cortisol.

The bioavailability of circulating and/or endogenous hydrocortisone (cortisol) in epidermal cells is a key determinant in inflammatory disease and chronic wounds. It is not known, however, whether epidermal cells can regulate tissue cortisol and whether they are capable of producing endogenous glucocorticoids. In the present study, we show by microarray analysis that epidermal cells express mRNAs to all the major enzymes involved in the metabolic chain from cholesterol to cortisol, including cytocrome P450 chain, 11β‐hydroxysteroid dehydrogenases (HSD11Bs), adrenocorticotropic hormone (ACTH) receptor (MC2R), and glucocorticoid receptor. The two enzymes mediating activation/deactivation of cortisone to cortisol, namely HSD11B1 and HSD11B2, were expressed at the protein level in cultured keratinocytes as well as human skin samples, as shown by Western blotting and immunohistochemistry, respectively. In functional assays, we show that keratinocytes are not only able to activate cortisone to cortisol in a HSD11B‐dependent manner but also silencing of either HSD11B1 or HSD11B2 specifically modulates the bioavailability of the inactive glucocorticoid and the active steroid, respectively. A further key observation was that keratinocytes responded to stimulation with ACTH by a significant increase in the de novo synthesis of cortisol. Taken together, we provide evidence for a novel non‐adrenal steroideal system in human keratinocytes. J. Cell. Biochem. 112: 1499–1505, 2011.

Decreased expression of TGF‐β cell surface receptors during progression of human oral squamous cell carcinoma

This study examined the immunocytochemical expression of the transforming growth factor‐β (TGF‐β) isoforms TGF‐β1, TGF‐β2, and TGF‐β3, together with the TGF‐β cell surface receptors TβR‐I and TβR‐II, in patient‐matched tissue pairs of normal human oral epithelium, primary squamous cell carcinomas, and metastatic lymph node tumour deposits. There were no significant differences in the intensity of TGF‐β isoform specific staining between the normal oral epithelium, the primary tumours, and the lymph node metastases. By contrast, there was significantly less TβR‐II in the metastases than in the primary tumour and between the primary tumour and the normal oral epithelium. Similar trends were evident with TβR‐I, but not at a statistically significant level. This study also examined the structure of TβR‐I and TβR‐II in normal human oral keratinocytes in vitro and in 14 human oral carcinoma cell lines with known responses to TGF‐β1. No structural abnormalities of TβR‐II were present in the normal keratinocytes or in 13 of 14 malignant cell lines; in one line, there were both normal and mutant forms of TβR‐II, the latter being in the form of a frameshift mutation with the insertion of a single adenine base (bases 709–718, codons 125–128), predicting a truncated receptor having no kinase domain. No defects were present in TβR‐I. The structures of TβR‐I and TβR‐II did not correlate with growth inhibition by TGF‐β1. The data suggest that decreased expression of TGF‐β receptors, rather than structural defects of these genes, may be important in oral epithelial tumour progression. In order to examine the functional significance of a specific decrease in TβR‐II expression, a dominant‐negative TβR‐II construct (dnTβR‐II) was transfected into a human oral carcinoma cell line with a normal TGF‐β receptor profile and known to be markedly inhibited by TGF‐β1. In those clones that overexpressed the dnTβR‐II, growth inhibition and Smad binding activity were decreased, whilst the regulation of Fra‐1 and collagenase‐1 remained unchanged following treatment with TGF‐β1. The results demonstrate that a decrease in TβR‐II relative to TβR‐I leads to selective gene regulation with loss of growth inhibition but continued transcription of AP‐1‐dependent genes that are involved in the regulation of the extracellular matrix. Copyright

Papillomaviruses: their possible role in oral disease.

Papillomaviruses are ubiquitous DNA viruses that are epitheliotropic and produce a range of epithelial neoplasms, both benign and malignant, in animals and man. Human papillomaviruses are associated with a variety of rare and uncommon oral lesions, and there has been increasing suspicion that they may be implicated also in some premalignant and malignant oral lesions.

Fibroblast gene expression profile reflects the stage of tumour progression in oral squamous cell carcinoma

Oral cancer is a highly aggressive malignancy with poor prognosis. This study examined the behaviour of fibroblast strains from normal oral mucosa, dysplastic epithelial tissue, and genetically stable (minimal copy number alterations—CNA; minimal loss of heterozygosity—LOH; wild‐type p53; wild‐type p16