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    ABSTRACT: To investigate tumour progression mechanism in transgenic mouse skin carcinogenesis, inducible PTEN ablation (Δ5PTEN(flx)) was targeted to the epidermis of mice expressing activated ras(Ha)/fos oncogenes (HK1.ras and HK1.fos). RU486-treated HK1.ras/fos-Δ5PTEN(flx) epidermis exhibited significant keratinocyte proliferation resulting in hyperplasia and proliferating cysts. While HK1.ras/fos-Δ5PTEN(flx) papillomatogenesis was accelerated, malignant conversion was delayed and tumours exhibited well-differentiated squamous cell carcinoma (wdSCC) histotypes, suggesting inhibition of early-stage malignant progression. Immediate elevated p53/p21 expression was observed in HK1.ras/fos-Δ5PTEN(flx) hyperplasia, cysts and papillomas, and while malignant conversion required p53 loss, elevated p21 expression persisted in most wdSCCs to limit further progression, unless p21 was also lost and wdSCC progressed to more aggressive carcinomas. In contrast, TPA-promoted (that is, c-fos-activated) bi-genic HK1.ras-Δ5PTEN(flx) cohorts lost p53/p21 expression during early papillomatogenesis and rapidly produced poorly differentiated carcinomas (pdSCCs) with high BrdU-labelling and elevated cyclin D1/E2 expression levels, indicative of a progression mechanism driven by failures in cell-cycle control. Intriguingly, HK1.ras/fos-Δ5PTEN(flx) wdSCCs did not exhibit similar failures, as western and immunofluorescence analysis found downregulated cyclin E2 whenever p21 persisted; further, while westerns detected elevated cyclin D1, immunofluorescence identified reduced expression in proliferative basal layer nuclei and a redistributed expression profile throughout p21-positive wdSCC keratinocytes. These data demonstrate that rapid early epidermal responses to ras(Ha)/fos/ΔPTEN co-operation involve induction of p53/p21 to alter differentiation and divert excessive proliferation into cyst formation. Further, despite three potent oncogenic insults p53 loss was required for malignant conversion, and following p53 loss persistent, p53-independent p21 expression possessed the potency to limit early-stage malignant progression via cyclin D1/E2 inhibition.Oncogene advance online publication, 16 September 2013; doi:10.1038/onc.2013.372.
    No preview · Article · Sep 2013 · Oncogene
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    ABSTRACT: Many conceptual advances in biology have been achieved by experimental studies using planar two-dimensional cell culture systems. Recent adaptations of molecular techniques to three-dimensional model systems are bridging the gap in our understanding of biological events in vitro and in vivo in the study of disease progression. Recently, in vitro studies using Förster resonance energy transfer (FRET) have shown that the prototypical RhoGTPases Cdc42, Rac and RhoA are temporally and spatially synchronized during cell migration, with initial RhoA activity inducing protrusion prior to activation of Rac. This simultaneous FRET approach illustrates the tight control and dynamic regulation of RhoGTPase activity necessary for coordinated cell migration in vitro. Here, we discuss our recent work using FLIM-FRET analysis in a three-dimensional setting to reveal another layer of regulation in which RhoA activity is governed by the extracellular microenvironment. We demonstrate that RhoA is spatially regulated into discrete fractions of activity at the leading edge and rear of cells during invasion in vivo or within three-dimensional matrices. Significantly, this spatial regulation of RhoA was absent in two-dimensional in vitro settings. This distinct sub-cellular regulation of RhoA at the poles of invading cells in three-dimensions sets a precedent that other RhoGTPases or signaling proteins may also be differentially regulated in a con-text-dependent manner during key biological processes such as invasion.
    Full-text · Article · Jul 2011 · Small GTPases
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    ABSTRACT: Scar tissue formation during healing of extensive skin wounds may be related to delayed reepithelialization, while elevated levels of hyaluronan may suppress scar tissue formation. This study investigates the expression of hyaluronan by human skin fibroblasts in response to keratinocyte-conditioned medium (KCM), and attempts to identify any active factors within the conditioned medium. Serum-free KCM was assessed for its ability to stimulate the incorporation of (3) H-glucosamine into fibroblast glycosaminoglycans, and hyaluronan synthesis. Conditioned medium was concentrated with an ultrafiltration membrane with a 30 kDa cutoff. Stratifin was assessed for its ability to stimulate hyaluronan synthesis and its role in KCM. KCM stimulated fibroblast glycosaminoglycan synthesis up to a 3.3-fold increase and a 6.5-fold increase in hyaluronan synthesis compared with serum-free controls. Preliminary characterization of the active factors showed that they are retained by a >30 kDa ultrafiltration membrane, and are protease sensitive and heat resistant. Emmprin and stratifin were shown to stimulate fibroblast hyaluronan synthesis, and the hyaluronan-stimulating activity of KCM was removed following stratifin depletion and to a lesser extent by emmprin depletion. Keratinocytes release soluble factors, including stratifin, that stimulate fibroblast hyaluronan synthesis, and this stimulation of fibroblast hyaluronan may contribute to the suppression of scar tissue formation.
    No preview · Article · May 2011 · Wound Repair and Regeneration
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