We report the effects of thrombospondin (TSP) on the proliferation of four different human hepatocellular carcinoma (HCC) cell lines (HuH-7, KIM-I, KYN-2, and KYN-3) and report the proliferative activity of HuH-7 cell using microspectrophotometry. The addition of anti-TSP neutralizing antibody (150, 1500, or 15000 ng/ml) to cultured medium showed marked suppression of cell proliferation in all cell lines. On the other hand, the addition of exogenous TSP (20 or 40 mu g/ml) to culture medium stimulated cell proliferation, in KYN-2 and KYN-3 but not in HuH-7 or KTM-1. Furthermore, in DNA measurements on HuH-7 cells, the effect of the anti-TSP neutralizing antibody induced growth arrest in the G(0)/G(1) phase of the cell cycle and decreased DNA synthesis. In conclusion, TSP promoted the proliferation of HCC cells, and based on our results it is predicted that TSP might act through an autocrine or paracrine growth-supportive mechanism in the extracellular matrix.

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