Mechanisms by which tumor cells and monocytes expressing the angiogenic factor thymidine phosphorylase mediate human endothelial cell migration

The angiogenic factor thymidine phosphorylase (TP) is highly expressed in many human solid tumors, and the level of its expression is associated with tumor neovascularization, invasiveness, and metastasis and with shorter patient survival time. TP promotes endothelial cell (EC) migration in vitro and angiogenesis in vivo, and these have been linked to its enzymatic activity. The mechanism by which TP stimulates EC migration was investigated using human umbilical vein ECs (HUVECs). TP induced concentration-dependent HUVEC migration, which required a TP gradient and thymidine and which was abrogated by the TP inhibitor CIMU (5-chloro-6(1-imidazolylmethyl)uracil). The chemotactic actions of TP plus thymidine were duplicated by the TP metabolite, 2-deoxyribose-1-phosphate (dR-1-P), and 10-fold more potently by its subsequent metabolite, 2-deoxyribose (2dR). Migration induced by dR-1-P, but not 2dR, was blocked by an alkaline phosphatase inhibitor, suggesting that the actions of dR-1-P first required its conversion to 2dR. In the migration assay, [5′-³H]dThd was metabolized to dR-1-P (96%) and 2dR (3.8%), and a gradient of both metabolites was maintained between the lower and upper chambers over the entire 5-h assay. TP expression in human solid tumors occurs in both tumor epithelial cells and in tumor-associated macrophages. The migration assay was adapted to use TP-transfected carcinoma cells to stimulate HUVEC migration, and they were found to induce more migration than did control vector-transfected cells. Human monocyte cells U937 and THP1, which constitutively expressed high levels of TP, also strongly induced HUVEC migration in the coculture assay. CIMU inhibited tumor-cell and monocyte-induced migration. In contrast, a neutralizing antibody to TP had no effect on cell-stimulated HUVEC migration, even though it completely blocked the migration mediated by purified TP. Thus, the intracellular actions of TP were sufficient to stimulate HUVEC chemotaxis. In contrast to purified TP, when incubated with [5′-³H]-thymidine, cells expressing TP released up to 20-fold more 2dR into the medium than dR-1-P. These studies demonstrate that TP-expressing cells mediate EC migration via the intracellular metabolism of thymidine and subsequent extracellular release of 2dR, which forms a chemotactic gradient.