Recent data indicate that transforming growth factor-beta1 (TGF-beta1) 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-beta1-induced EMT in human keratinocytes and that epidermal growth factor (EGF) can substitute for mutant Ras. EMT was reversed by the removal of TGF-beta1. Under conditions of TGF-beta1-induced EMT, cells were growth inhibited by the ligand resulting in G1 arrest. In cells containing normal Ras, TGF-beta1-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-beta1. Further, inhibition of the AP-1 transcriptional complex by [6]-Gingerol, or by the ectopic expression of JDP2, blocked TGF-beta1-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-beta1 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-beta1-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-beta1 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-beta1-induced EMT.

Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.

The expression of p53 protein was studied in odontogenic keratocysts (OKC, 11 solitary, 5 recurrent and 6 NBCCS cysts), radicular (RC, n = 5) and dentigerous (DC, n = 5) cysts, using a panel of antibodies to p53 (clone BP53-12, clone 1801 and polyclonal CM1) and a sensitive biotin-streptavidin method on paraffin embedded sections. Of the three antibodies tested, clone BP53-12 gave the most intense and consistent nuclear staining pattern. Clone 1801 and polyclonal CM1 stained only 38% and 71% OKC linings, respectively, but not RC and DC linings. However, BP53-12+ cells were detected in the epithelial linings of all cyst types. Quantification of BP53-12+ cells was performed by manual counting and by relating cell number to unit length of basement membrane as determined by TV image analysis. BP53-12+ cell counts in solitary OKC linings (25.5 +/- 11.0 cells/mmBM) were significantly greater than those in DC (9.3 +/- 4.9 cells/mmBM, P < 0.01) and RC (6.7 +/- 2.6 cells/mmBM, P < 0.01) linings. The epithelial distribution of positive cells in OKC was predominantly suprabasal, which also varied from that of DC and RC linings (P < 0.005). There were no detectable differences in BP53-12 reactivity between the different subtypes of OKC (i.e., solitary, recurrent and NBCCS-associated OKC; P > 0.1). When data for the NBCCS-related OKC group were excluded, there was a significant correlation (r = 0.55, P < 0.01) between p53 and Ki67 labelling. To detect the presence of p53 gene mutations, genomic DNA, extracted from paraffin sections of OKC (4 solitary, 2 recurrent and 4 NBCCS cysts), RC (n = 3) and normal oral mucosa (n = 1), was subjected to a combination of polymerase chain reaction and single-stranded conformation polymorphism (PCR-SSCP) analysis for exons 5-10 of the p53 gene. Exon 4 was not analysed because of compromised DNA quality. No abnormality in banding patterns was found and all samples gave results similar to DNA from known, sequenced, normal p53 gene controls. Absence of p53 mutations within exons 5-9 was confirmed by the direct sequencing of 2 fresh frozen OKC samples (1 solitary and 1 NBCCS cyst). These results suggest that overexpression of p53 protein in OKC epithelium, detected by immunocytochemistry, is not reflected by alteration of the p53 gene and presumably reflects overproduction and/or stabilisation of normal p53 protein.

Patient error in use of bronchodilator metered aerosols Aerosolised bronchodilator drugs in pressurised canisters delivering metered doses are widely used, easy to carry, and apparently easy to operate. The instructions with each canister are reasonably clear, stating that each puff must be deeply inhaled and the breath held afterwards. Since the drug intake depends mostly on the patient's ability to follow the instructions, we thought it was important to find out whether asthmatics used their aerosols correctly. Patients, methods, and resultsTwenty asthmatic patients (12 men, 8 women) with a mean age (±SD) of 44 8 + 15 1 were studied. All had been treated with bronchodilator sprays and knew how to operate them. They were not examined during an acute attack, although all of them had some bronchial obstruction when tested. They received no medication for 24 hours preccding the first test.Specific airway resistance (SRaw), which is the resistance corrected for lung volume,' was measured in a whole-body constant volume plethysmograph at a thoracic gas volume close to functional residual capacity, using the panting technique.2 Each subject was tested, by different procedures, on two successive days between 2 and 5 pm if the initial value of SRaw was comparable. On both days SRaw was measured before and 5 and 10 minutes after bronchodilator aerosol inhalation (salbutamol 100 jtg/puff in 10 patients and fenoterol 200 jug/puff in 10 others). Bronchodilatation was greatest after 10 minutes, therefore only measurements taken at that time were used.On the first day the patients were told to inhale two puffs of a bronchodilator spray in their customary way. On the second day two puffs of the same bronchodilator spray were administered by the physician. Wearing a nose clip, the patients opened their mouth wide, expired deeply, and then inspired deeply. One puff of drug was delivered at the beginning of the inspiration with the canister about 3 cm from the lips. After fully inspiring the patients closed their mouths and held their breath for four seconds. A second puff was administered in the same way. Repetition of the first-day procedure on different occasions by some patients showed there was no systematic error due to the tests not being randomised. The bronchodilatation achieved by the two procedures was similar in only 5 patients. In the 15 others it was greater when the aerosol was given by the physician. Observation showed that only the 5 patients in whom bronchodilatation was similar by both methods correctly inhaled (that is, at the beginning of a deep inspiration) on self-administration. Drugs administered by inhalation now play a major role in the treatment of bronchial asthma. It has been shown that some patients have difficulty inhaling drug aerosols from pressurised cannisters' and this may, of course, be a reason for treatment failure. The aim of this study was to find the number of patients attending an asthma clinic who were not using their inhalers satisfactorily, in spite of apparently adequate tuition i...

Oral squamous cell carcinoma (OSCC) is a lethal disease and early death usually occurs as a result of local invasion and regional lymph node metastases. Current treatment regimens are, to a certain degree, inadequate, with a 5-year mortality rate of around 50% and novel therapeutic targets are urgently required. Using expression microarrays, we identified the eps8 gene as being overexpressed in OSCC cell lines relative to normal oral keratinocytes, and confirmed these findings using RT-PCR and western blotting. In human tissues, we found that Eps8 was upregulated in OSCC (32% of primary tumors) compared with normal oral mucosa, and that expression correlated significantly with lymph node metastasis (P ¼ 0.032), suggesting a diseasepromoting effect. Using OSCC cell lines, we assessed the functional role of Eps8 in tumor cells. Although suppression of Eps8 produced no effect on cell proliferation, both cell spreading and migration were markedly inhibited. The latter cell functions may be modulated through the small GTP-ase, Rac1 and we used pull-down assays to investigate the role of Eps8 in Rac1 signaling. We found that avb6-and a5b1-integrin-dependent activation of Rac1 was mediated through Eps8. Knockdown of either Eps8 or Rac1, inhibited integrindependent cell migration similarly and transient expression of constitutively active Rac1 restored migration of cells in which Eps8 expression had been suppressed. We also showed that knockdown of Eps8 inhibited tumor cell invasion in an organotypic model of OSCC. These data suggest that Eps8 and Rac1 are part of an integrated signaling pathway modulating integrin-dependent tumour cell motility and identify Eps8 as a possible therapeutic target.

Almost all drugs approved for use in humans possess potentially beneficial ‘off-target’ effects in addition to their principal activity. In some cases this has allowed for the relatively rapid repurposing of drugs for other indications. In this review we focus on the potential for re-purposing FTY720 (also known as fingolimod, Gilenya™), an immunomodulatory drug recently approved for the treatment of multiple sclerosis (MS). The therapeutic benefit of FTY720 in MS is largely attributed to the immunosuppressive effects that result from its modulation of sphingosine 1-phosphate receptor signalling. However, this drug has also been shown to inhibit other cancer-associated signal transduction pathways in part because of its structural similarity to sphingosine, and consequently shows efficacy as an anti-cancer agent both in vitro and in vivo. Here, we review the effects of FTY720 on signal transduction pathways and cancer-related cellular processes, and discuss its potential use as an anti-cancer drug.

No abstract

No abstract