Response of cultured hepatocytes to a hepatomitogen after initiation by conditioned medium or other factors

Abstract

Injection of a substantially purified hepatomitogen into recipient rats that had 40% of their liver removed resulted in a significant stimulation of hepatic DNA synthesis as determined by the labeling index and the mitotic index. Normal or sham-operated rats did not respond to the injection of the mitogen. The extraction and partial purification of this hepatomitogen have previously been reported (A. Francavilla et al., Cancer Res., 47:5600-5605, 1987). Addition of the factor to an epithelial-like liver-derived cell line in culture (clone 9) or to a hepatoma cell line (HTC-SR) resulted in a dose-dependent stimulation of DNA synthesis. Hepatocytes in primary culture, on the other hand, were not stimulated by the addition of the factor. However, when the mitogen was added to hepatocytes in primary culture, together with conditioned medium, obtained from the responsive cell lines, a significant stimulation of DNA synthesis could be demonstrated in hepatocytes in culture. The stimulation was dose dependent with respect to the mitogen, was abolished by 10 mM hydroxyurea, and was independent of epidermal growth factor. The conditioned medium could be replaced by a protein factor extracted from the two cell lines as previously reported (P. Ove et al., J. Cell. Physiol., 131: 165-174, 1987). It appears that a cofactor is provided by the conditioned medium or by the cell extract, enabling the hepatomitogen to act on hepatocytes in primary culture.

Fig. 1

Lack of response of hepatocytes in primary culture to HSS-F150. The isolation of hepatocytes from normal or 40% hepatectomized rats and assay conditions have been described in “Materials and Methods.” ■, no additions; □, + HSS-F150, 0.1 μg/ml; , + HSS-F150, 1.0 μg/ml. Bars, SD.

Fig. 2

Stimulation of DNA synthesis in hepatocytes in primary culture by HSS-F150 in the presence of 50% conditioned medium (CM). The isolation of hepatocytes and the assay conditions have been described in “Materials and Methods.” Numbers in columns, amount of HSS-F150 added (μg/ml of medium). Bars, SD.

Fig. 3

Effect of hydroxyurea on the stimulation of DNA synthesis in hepatocytes in primary culture by HSS-F150 in the presence of 50% conditioned medium. The isolation of hepatocytes and the assay conditions have been described in “Materials and Methods.” All wells received MEM plus insulin at 3 h, and at 24 h the medium was changed to 50% conditioned medium plus HSS-F150, 0.1 μg/ml, and the cells left for 48 h. The EGF and hydroxyurea concentrations, also added at 24 h where indicated, were 10 ng/ml and 10 mM, respectively. □, (−) hydroxyurea; , (+) hydroxyurea. Bars, SD.