L motility and lamellipodia formation [ 16 ]. Inside the nervous system, semaphorin signaling requires homo – and heterodimers of NRP1 / NRP2, plexins, and small GTPases including Rac1 ( for reviews, see Refs. [ 17 21 ]). Other elements affecting SEMA3A signalling include things like collapsin response mediator protein ( CRMP) [ 22 ] as well as the PDZ binding protein NIP [ 23 ]. In lung cancer cell lines expressing varying levels of CRMP, their invasive possible was inversely correlated with CRMP expression [ 24 ], a locating which can be constant with antitumor effects of classAddress all correspondence to: Prof. Joelle Roche, IBMIG, EA 2224, Universite de Poitiers, 40 Av du Recteur Pineau, DNA topoisomerase II Proteins Storage & Stability poitiers Cedex 86022, France. E – mail: joelle.roche@univ – poitiers.fr 1 Present address: Institut de Biotechnologies, 123 avenue Albert Thomas, Limoges Cedex 87060, France. Received 11 July 2002; Accepted 14 August 2002. Copyright # 2003 Neoplasia Press, Inc. All rights reserved 1522-8002/03/ 25.SEMA3F Inhibits Tumor Cell Spreading Nasarre et al.semaphorins. At the cellular level, COS7 cells expressing NRP1 and plexin – 1 Ebola Virus sGP Proteins Storage & Stability contract inside 5 to 30 minutes of SEMA3A exposure [ 25 ]. Similar effects were observed with SEMA3F in COS7 cells expressing NRP2 / plexin – 1. Other investigators have shown that exposure to semaphorins outcomes in fast reorganization of actin filaments, generally present in lamellipodia and filopodia [ 26 ]. Based on the inverse staining patterns of VEGF and SEMA3F on primary lung cancer cells, SEMA3F staining at the top edge of motile cells [ 8 ], plus the reported antagonism in between SEMA3A and VEGF in endothelial cells, we recommended that VEGF and SEMA3F could possibly competitively regulate cell motility and adhesion in epithelial cancers. We have studied this in two breast cancer cell lines, MCF7 and C100, which differentially express NRP receptors. We also identified changes in Rac1 localization following exposure to SEMA3F. These results additional strengthen the notion that secreted semaphorins compete with VEGF for effects on tumor cells themselves. As a result, the standard balance involving VEGF and semaphorins, that is often and substantially disrupted in a variety of epithelial cancers, might have crucial consequences on migration because it does to angiogenesis. Components and Procedures Plasmid Constructions AP – SEMA3F was constructed by cloning SEMA3F cDNA into pSecTagA vector ( Invitrogen, Cergy Pontoise, France) in the 30 end in the alkaline phosphatase gene and was generously provided by Dr. M. Tessier – Lavigne [ 13 ]. AP pSecTag ( AP) expressed alkaline phosphatase as negative manage. Rac1 – GFP was a present from Dr. Fort ( CRBM, Montpellier, France). Cell Lines and Transfections The human mammary epithelial cell lines utilized were MCF7 and C100, a derivative of MDA – MB – 435S [ 27 ]. MCF7 cells had been grown in RPMI – 1640 containing ten fetal calf serum ( FCS) and C100 cells were grown in 50 DMEM / 50 Ham’s F12 containing 10 FCS. COS7 cells had been grown in DMEM plus ten FCS. Cell lines had been transfected with plasmids making use of Effectene ( Qiagen, Courtaboeuf, France) with situations suggested by the manufacturer. For AP – SEMA3F transfections in COS7 cells, the medium was replaced 2 days immediately after transfection by DMEM, containing 0.5 FCS or OPTIMEM media ( Invitrogen). The medium was collected four days soon after transfection and applied to MCF7 and C100 cell cultures in serum – cost-free medium for 14 hours. SEMA3F concentration was estimated by alkaline phosphatase activity ( GenHu.