High level thrombospondin 1 expression in two NIH 3T3 cloned lines confers serum- and anchorage-independent growth

Thrombospondin (TSP) is a trimeric molecule synthesized by a variety of normal and transformed cells and secreted into the extracellular matrix. A number of studies have shown TSP to be intimately involved in the regulation of cellular proliferation. These include the findings that TSP is a mitogen-inducible immediate-early response gene; it localizes to areas of cellular proliferation in the developing mouse embryo; it augments the proliferative response to epidermal growth factor; anti-TSP monoclonal antibodies block cell proliferation; and TSP levels correlate directly with the growth and invasive phenotype and inversely with the degree of differentiation of squamous carcinoma cells. To determine whether TSP could behave as an oncogene, conferring serum and anchorage independence, NIH 3T3 cells were stably transfected with a TSP expression vector. Clones producing high levels of TSP displayed enhanced viability and a proliferative advantage in serum-reduced media. Moreover, this growth advantage could be specifically negated by treatment of the transfected cells with anti-TSP monoclonal antibodies. While the TSP overexpressing clones were capable of anchorage-independent growth in soft agar, surprisingly, they were incapable of forming tumors in nude mice, possibly due to the in vivo antiangiogenic activity of TSP. Regardless, these studies demonstrate that overexpression of thrombospondin, a constituent of the extracellular matrix, results in serum and anchorage independent growth, attributes normally associated with the transformed phenotype.