Subject(s)
Electrocardiography , Syncope , Child, Preschool , Diagnosis, Differential , Humans , Syncope/diagnosis , Syncope/etiologyABSTRACT
Slit is a secreted protein known to function through the Roundabout (Robo) receptor as a chemorepellent in axon guidance and neuronal migration, and as an inhibitor in leukocyte chemotaxis. Here we show Slit2 expression in a large number of solid tumors and Robo1 expression in vascular endothelial cells. Recombinant Slit2 protein attracted endothelial cells and promoted tube formation in a Robo1- and phosphatidylinositol kinase-dependent manner. Neutralization of Robo1 reduced the microvessel density and the tumor mass of human malignant melanoma A375 cells in vivo. These findings demonstrate the angiogenic function of Slit-Robo signaling, reveal a mechanism in mediating the crosstalk between cancer cells and endothelial cells, and indicate the effectiveness of blocking this signaling pathway in treating cancers.
Subject(s)
Melanoma, Experimental/blood supply , Melanoma, Experimental/prevention & control , Neovascularization, Pathologic/metabolism , Nerve Tissue Proteins/physiology , Receptors, Immunologic/physiology , Animals , Cell Division , Cell Movement , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Gene Expression Regulation , Humans , Intercellular Signaling Peptides and Proteins , Mice , Microcirculation , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/immunology , Nerve Tissue Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Rabbits , Rats , Receptors, Immunologic/antagonists & inhibitors , Recombinant Fusion Proteins , Signal Transduction , Tumor Cells, Cultured/transplantation , Roundabout ProteinsABSTRACT
A cDNA for the disintegrin domain (hf279) was isolated by PCR from human testis cDNAs. DNA sequencing indicated that hf279 cDNA encoded 93 amino acid residues, and it was identical with the reported sequence of fertilin beta. An expression plasmid, pGEX hf279, was constructed by inserting hf279 cDNA into plasmid pGEX-4T-2 containing gst gene. The expression plasmid was introduced into E.coli BL21(DE3) cells and a substantial amount of soluble fused protein GST-HF93 was obtained by the expression strain HF93/BL21 induced with IPTG. SDS-PAGE analysis revealed that the GST-HF93 fusion protein had an apparent molecular weight of 38 kD and accumulated up to 50% of bacterial soluble proteins. The fusion protein was purified by glutathione S-transferase (GST) Sepharose 4B column (purity 90%) and digested by thrombin to obtain the purified HF93 peptide (purity 80%). Polyclonal antibodies were obtained from the serum of miceimmunized with purified HF93 which was isolated by GST Sepharose 4B column and SDS-PAGE. ELISA and Western blot analysis showed its specificity to HF93. Therefore this antibody can be used in further studies on the function of HF93.
