TGF-beta 1 inhibits DNA synthesis and phosphorylation of the retinoblastoma gene product in a rat liver epithelial cell line.

Abstract

In the rat liver epithelial cell line, WB, the ability of TGF-beta 1 to inhibit DNA synthesis was shown to correlate with its ability to inhibit phosphorylation of the protein product of the retinoblastoma susceptibility gene, pRb. When WB cells were serum-starved, then refed with serum-containing medium, a peak of DNA synthesis occurred at about 18 h. Autoradiographs showed that 43.6% of cell nuclei could be labeled with 3H-thymidine at this time. When TGF-beta 1 was added simultaneously with serum, it blocked DNA synthesis and reduced the number of labeled nucleii to 6.3%. Cells treated with serum alone for 18 h also showed a pronounced increase in the highly phosphorylated form of pRb, as shown by mobility shifts in immunoblots, and in active phosphorylation of pRb, as shown by 32P incorporation. Simultaneous addition of TGF-beta 1 with serum abolished both 32P incorporation into pRb and its mobility shift on immunoblots. The effect of TGF-beta 1 on DNA synthesis measured at 18 h was sharply reduced if the cells were incubated with serum for 8 h (and thus allowed to enter S) before the addition of TGF-beta 1. If TGF-beta 1 was added after 8 h of serum treatment, its ability to inhibit pRb phosphorylation at 18 h was unchanged. If TGF-beta 1 was added after 13 h of serum treatment, its effects on pRb phosphorylation were reduced. Thus, as the cell population moved into S, the ability of TGF-beta 1 to inhibit both pRb phosphorylation and DNA synthesis was lost. In higher passages of WB cells the dose-response for inhibition of DNA synthesis by TGF-beta 1 was shifted to the right. Inhibition of pRb phosphorylation by TGF-beta 1 was also lost in higher passage WB cells. Thus, the passage-dependent loss of sensitivity to inhibition of DNA synthesis accompanied the loss of sensitivity to inhibition of pRb phosphorylation. Since the phosphorylation of pRb is believed to be required for the progression of cells from G1 to S, inhibition of pRb phosphorylation may be either a cause or a consequence of the G1 arrest of WB cells by TGF-beta 1.