The proliferation of normal human fibroblast cells was enhanced by the addition of inorganic polyphosphate (poly(P)) into culture media. The mitogenic activities of acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) were also enhanced by poly(P). A physical interaction between poly(P) and FGF-2 was observed, and FGF-2 was both physically and functionally stabilized by poly(P). Furthermore, poly(P) facilitated the FGF-2 binding to its cell surface receptors. Because poly(P) is widely distributed in mammalian tissues, it may be a spontaneous modulator of FGFs. Inorganic polyphosphates (poly(P))1 are linear polymers of many tens or hundreds of orthophosphate residues linked by high energy phosphoanhydride bonds that have been found in a wide range of organisms including bacteria, fungi, algae, mosses, insects, and protozoa and in the tissues of higher plants and animals (1-4). The biological functions of poly(P) have been investigated mostly in microorganisms, and the following functions have been proposed: (i) storage substance of energy or orthophosphate; (ii) chelator of metal cations; (iii) donor for sugar and adenylate kinase; (iv) buffer against alkaline stress; (v) structural element in competence for DNA entry and transformation; and (vi) a regulatory factor of gene expression (1-4). Although the presence of poly(P) has been demonstrated in the rat brain, rat liver, human peripheral blood mononuclear cells, human erythrocytes, human gingival fibroblasts, human osteoblasts, and human plasma and intracellularly in the nucleus, the mitochondria, lysosomes and plasma membrane (5), little is known regarding the functions of poly(P) and the effects of poly(P) on mammalian cells. Recently, the involvement of poly(P) in apoptosis and in modulation of the mineralization process in bone tissue (5, 6) has been suggested.Because there has been no report concerning the direct effect of poly(P) on mammalian cells and because poly(P) is widely distributed in mammalian tissues and plasma (5), we speculated that poly(P) has some physiological effect on cells. Based on this idea, we first studied in this report the effect of poly(P) on mammalian cell growth or proliferation in vitro and revealed the novel poly(P) functions concerning the modulation of mitogenic activity of fibroblast growth factors (FGF) (8). EXPERIMENTAL PROCEDURESMaterials-Normal dermal fibroblasts (NHDF) isolated from adult human were purchased from BioWhittaker, Inc. Normal human gingival fibroblasts (HGF) isolated from adult human were provided by Dr. Nishimura (Osaka Dental University). Balb/c 3T3 cells were from Riken Cell Bank (Tsukuba, Japan). Poly(P) type 65 (sodium salts with average chain length of 65 phosphate residues) was purchased from Sigma. Concentrations of poly(P) are given in terms of phosphate residues. As the control of poly(P), NaPO 4 buffer (orthophosphate) was used. The pH of the NaPO 4 buffer was adjusted to 7.0 by mixing the same concentrations of Na 2 HPO 4 and NaH 2 PO 4 solution. MTS (3-(4,5-dime...

Tumor angiogenesis is necessary for progression and metastasis of solid tumor. Tumor blood vessels are morphologically different from their normal counterparts. In this study, we isolated tumor endothelial cells (TECs) and revealed their abnormalities. We have compared the gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and found that several genes were upregulated in TECs. Expression of the chemokine receptor CXCR7 mRNA was higher in TECs than in NECs. However, information regarding the expression of CXCR7 in the tumor vessels of renal cell carcinoma is limited. CXCR7 and its ligand CXCL12 have been implicated in tumor cell survival. In this study, the expression of CXCR7 in the tumor vessels of renal cell carcinoma (RCC) was investigated. Real-time PCR revealed higher expression level of CXCR7 in cultured TECs than in cultured NECs. Furthermore, similar to mouse TECs, immunostaining revealed strong expression of CXCR7 in vivo in human tumor vessels. These findings suggest that CXCR7 is a novel TEC marker and a target for antiangiogenic therapy for RCC.

Inorganic polyphosphate [poly(P)] is a biopolymer existing in almost all cells and tissues, although its biological functions in higher eukaryotes have not been completely elucidated. We previously demonstrated that poly(P) enhances the function of fibroblast growth factors (FGFs) by stabilizing them and strengthening the affinity between FGFs and their cell surface receptors. Since FGFs play crucial roles in bone regeneration, we further investigated the effect of poly(P) on the cell differentiation of human stem cells via FGF signaling systems. Human dental pulp cells (HDPCs) isolated from human dental pulp show the characteristics of multipotent mesenchymal stem cells (MSCs). HDPCs secreted FGFs and the proliferation of HDPCs was shown to be enhanced by treatment with poly(P). Cell surface receptor-bound FGF-2 was stably maintained for more than 40 hours in the presence of poly(P). The phosphorylation of ERK1/2 was also enhanced by poly(P). The effect of poly(P) on the osteogenic differentiation of HDPCs and human MSCs (hMSCs) were also investigated. After 5 days of treatment with poly(P), type-I collagen expression of both cell types was enhanced. The C-terminal peptide of type-I collagen was also released at higher levels in poly(P)-treated HDPCs. Microarray analysis showed that expression of matrix metalloproteinase-1 (MMP1), osteopontin (OPN), osteocalcin (OC) and osteoprotegerin was induced in both cell types by poly(P). Furthermore, induced expression of MMP1, OPN and OC genes in both cells was confirmed by real-time PCR. Calcification of both cell types was clearly observed by alizarin red staining following treatment with poly(P). The results suggest that the activation of the FGF signaling pathway by poly(P) induces both proliferation and mineralization of stem cells.

Molecules highly expressed in tumor endothelial cells (TEC) are important for specific targeting of these cells. Previously, using DNA microarray analysis, we found that the prostacyclin receptor (IP receptor) gene was upregulated in TEC compared with normal endothelial cells (NEC). Although prostacyclin is implicated in reendothelialization and angiogenesis, its role remains largely unknown in TEC. Moreover, the effect of the IP receptor on TEC has not been reported. In the present study we investigated the function of the IP receptor in TEC. The TEC were isolated from two types of human tumor xenografts in nude mice, while NEC were isolated from normal counterparts. Prostacyclin secretion levels in TEC were significantly higher than those in NEC, as shown using ELISA. Real-time RT-PCR showed that the IP receptor was upregulated in TEC compared with NEC. Furthermore, migration and tube formation of TEC were suppressed by the IP receptor antagonist RO1138452. Immunohistostaining showed that the IP receptor was specifically expressed in blood vessels of renal cell carcinoma specimens, but not in glomerular vessels of normal renal tissue. These findings suggest that the IP receptor is a TECspecific marker and might be a useful therapeutic target. (Cancer Sci 2012; 103: 1038-1044 A ngiogenesis is essential for tumor growth and metastasis and is an important component of cancer progression. Its inhibition is a valuable new approach to cancer therapy.(1-4) Tumor blood vessels deliver oxygen, nutrients and growth factors to cancer cells and permit their dissemination into the systemic circulation, resulting in metastasis.(5,6) Increased tumor vascularity is associated with poor clinical outcome, and the extent of angiogenesis correlates inversely with patient survival.(6) The inhibition of angiogenesis therefore offers an attractive approach to cancer therapy.The pharmacological targeting of vascular endothelial cells suppresses tumor angiogenesis and growth, and the efficacy of anti-angiogenic therapy has been validated in the clinic. Although a humanized monoclonal antibody against vascular endothelial growth factor (VEGF) prolonged survival in patients with certain types of cancer, some types of tumors appear to be less responsive. The results have been more modest than predicted by most preclinical examinations and improvements in progression-free survival are frequently not accompanied by improvements in overall survival. Furthermore, some side-effects have been reported since VEGF is essential for the survival of normal endothelial cells (NEC).(7-11) A target that is more specific for tumor endothelial cells (TEC) is needed to improve the outcome of antiangiogenic therapy.We previously reported that TEC differ from NEC in gene profile (12,13) and behavior, including enhanced cell survival (14,15) and motility. (16)(17)(18) Using DNA microarray analysis, we previously identified several molecules that were preferentially highly expressed in mouse TEC derived from three different types of human tumor xenografts. ...

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