Analysis of Activated Platelet-Derived Growth FactorβReceptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

Abstract

Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1) and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR) causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm.

Felis catus papillomavirus type-2 E6 binds to E6AP, promotes E6AP/p53 binding and enhances p53 proteasomal degradation

“…Cells grown for 2 days on coverslips were washed, fixed, permeabilized and subjected to background blocking as previously reported 17 . Anti-HA, anti-p53 and anti-E6AP antibodies were applied for 2 h at room temperature (rt) in a humidified chamber at 1:50 dilution in PBS.…”

Telomerase Reverse Transcriptase (TERT) Expression, Telomerase Activity, and Expression of Matrix Metalloproteinases (MMP)-1/-2/-9 in Feline Oral Squamous Cell Carcinoma Cell Lines Associated With Felis catus Papillomavirus Type-2 Infection

“…Cell pellets were subjected to total protein extraction, protein quantification, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting (WB) as described previously (30). Membranes were incubated with the following primary antibodies overnight (O/N) at 4 • C at 1:1,000 dilution: anti-TERT (rabbit polyclonal, Rockland #600-401-252), anti-cMyc (mouse monoclonal, Santa Cruz Biotechnology, #sc-40, clone 9E10), anti-MMP-1 (mouse monoclonal, Santa Cruz Biotechnology, #sc-21731, clone 3B6) and anti-MMP-2 (rabbit polyclonal, Neomarkers, #RB-1537-P0), anti-MMP-9 (mouse monoclonal, Millipore, #MAB3309, clone 56-2a4), and β-actin (mouse monoclonal, Calbiochem #CP01-1EA, clone JLA20) or tubulin (mouse monoclonal, Santa Cruz Biotechnology, #sc-23948, clone B-5-1-2).…”

“…Cells grown for 48 h on coverslips were washed, fixed, permeabilized, and subjected to background blocking as previously reported (30). Primary antibodies anti-TERT, anti-MMP-2, and anti-MMP-9 at 1:50 dilution in PBS were applied for 2 h at rt in a humid chamber.…”

Effects of Synergistic Massage and Physical Exercise on the Expression of Angiogenic Markers in Rat Tendons

“…Tendons are responsible for transferring the forces exerted by muscles to the bone tissue, thus enabling locomotion. It has been shown that tendons change their metabolism in response to mechanical forces and that, depending on the intensity of the mechanical stimulus, tendon fibroblasts secrete various proteins, such as collagen I and cyclooxygenase [ 1 – 5 ]. Although tendons are regarded as low metabolism tissue, under conditions of stress and during healing they can alter their metabolism significantly, leading to the secretion of various growth factors [ 3 ].…”