Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis.

Abstract

Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To The thrombospondins are a multigene family of modular glycoproteins involved in regulation of adhesion, migration, and proliferation of a number of normal and transformed cell types (1-4). Human thrombospondin 1 (TS1) and a fragment of hamster TS1 (gp140) are inhibitors of angiogenesis driven by many different stimuli (5, 6). Synthetic peptides from two adjacent domains of human TS1 (the procollagen-like segment and the type 1 repeats) block angiogenesis in several bioassays and inhibit the migration of endothelial cells (ECs) in vitro (5, 6). Furthermore, TS1 and its proteolytic fragments containing these domains inhibit proliferation of ECs in vitro (5-8). TS1 is expressed and secreted by a variety of cell types, including ECs proliferating in culture (9). Several findings suggest that TS1 is a physiological inhibitor of angiogenesis: TS1 is present in quiescent vessels and absent in actively forming EC sprouts (10); TS1 levels are low in EC involved in cord formation in vitro (10); TS1 expression in a human breast carcinoma line reduces tumor growth, metastasis, and angiogenesis (11); TS1 mRNA is decreased in cultures of ECs forming tubules (12); and TS1 expression is virtually shut off in rapidly growing ECs from hemangiomas (13).The mechanisms by which TS1 regulates EC phenotype are not understood. We have used the polyoma virus middle-sized tumor antigen (mT)-transformed mouse brain capillary ECThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. line (bEND.3) (13,14) to examine the role of TS1 in the regulation of EC phenotype. These transformed cells express little or no TS1 (13), and perhaps as a result, they proliferate more rapidly and to higher saturation densities than normal ECs. However, their proliferation, like that of normal ECs, is inhibited by added TS1 (13). It is not clear whether the inhibition by TS1 is direct or is due to the presence of transforming growth factor 13 (TGF-13), a potent inhibitor of EC proliferation, which is associated with TS1 purified from platelets (15). To eliminate this ambiguity and determine the role of TS1 in regulation of EC phenotype, we have expressed human TS1 in bEND.3 cells. Expression of TS1 in these cells results in a reduced growth rate, lower saturation density, decreased fibrinolytic activity, and restored ability to form cords in vitro. Furthermore, TS1 expression blocks the ability of bEND.3 cells to form hemangiomas in vivo. All of these changes occur independently of increased TGF-,B activity. MATERIALS AND METHODSCell Culture and Transfection. bEND.3 cells (obtained...

Glucose and insulin modify thrombospondin 1 expression and secretion in primary adipocytes from diet-induced obese rats

“…Several functions have been attributed to TSP-1 including antiangiogenic [14,38] and proatherogenic [36,40] properties. More recently, this protein has been studied as a potential link between vasculature mechanisms and hyperglycemia [2,39].…”

Methylation and silencing of the Thrombospondin-1 promoter in human cancer

“…Recent studies have indicated that its expression can be positively regulated by P53 (Dameron et al, 1994), RB (Salnikow et al, 1994), nm-23 and one or more unknown genes on chromosome 10q (Hsu et al, 1996). Several lines of evidence suggest that THBS1 is an inhibitor of angiogenesis: (i) THBS1 is present in quiescent vessels and decreased or absent in actively forming endothelial sprouts or cultured endothelial cells forming tubules (Can®eld and Schor, 1995;DiPietro et al, 1994); (ii) THBS1 expression is shut o in rapidly growing endothelial cells from mouse hemangiomas induced by the polyoma virus middle t antigen (Sheibani and Frazier, 1995); (iii) wild type P53 inhibits angiogenesis by stimulating the production of THBS1 in ®broblast cells (Dameron et al, 1994); (iv) THBS1 inhibits endothelial cell adhesion, growth and motility in response to angiogenic stimuli (Panetti et al, 1997), and it also has the ability to induce apoptosis in endothelial cells (Guo et al, 1997); and (v) downregulation of THBS1 expression in endothelial cells enhances in vitro angiogenesis (DiPietro et al, 1994). Thus, THBS1 appears to be a potent anti-angiogenic factor.…”

“…Several studies have implicated alterations in THBS1 expression in the neovascularization observed in human cancers (Roberts, 1996): Decreased expression of THBS1 has been reported in multiple primary tumors (Grossfeld et al, 1997;Zabrenetzky et al, 1994;Clezardin et al, 1993) and cells transformed in vitro by c-jun (Mettouchi et al, 1994), Nickel (Salnikow et al, 1994), v-src (Slack and Bornstein, 1994), the polyoma virus middle-t antigen (Sheibani and Frazier, 1995), and v-myc (Tikhonenko et al, 1996). Reexpression of THBS1 reduced tumor growth, angiogenesis and metastasis in a hemangioma cell line (Sheibani and Frazier, 1995) and a breast cancer cell line (WeinstatSaslow et al, 1994).…”

“…Reexpression of THBS1 reduced tumor growth, angiogenesis and metastasis in a hemangioma cell line (Sheibani and Frazier, 1995) and a breast cancer cell line (WeinstatSaslow et al, 1994). In addition, several tumorsuppressor genes appear to regulate angiogenesis through THBS1 expression (Hsu et al, 1996;Dameron et al, 1994;Good et al, 1990).…”

The Wilms' tumor gene product represses the transcription of thrombospondin 1 in response to overexpression of c-Jun

“…Many, but not all, transformed cell lines express very low levels of TSP1 (Mettouchi et al, 1994;Salnikow et al, 1994;Slack and Bornstein, 1994;Sheibani and Frazier, 1996;Tikhonenko et al, 1996). In some cases, re-expression of TSP1 in transformed cells suppresses their tumorigenicity (Weinstat et al, 1994;Sheibani and Frazier, 1995).…”