Comparative proteomic analysis of cell cycle-dependent apoptosis induced by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) can induce G2/M phase-dependent apoptosis and G1/S phase-dependent epithelial-mesenchymal transition (EMT) in hepatocytes, but the underlying mechanism remains poorly understood. In this study, we investigated alterations in the global proteome using two dimensional gel electrophoresis of AML-12 murine hepatocyte cells after treatment with TGF-beta at several time points after synchronization in the G2/M or G1/S phase. Upon TGF-beta treatment, the expression levels of 44 proteins were found to be significantly changed in cells synchronized in the G2/M phase. These proteins were identified by MALDI-TOF/TOF and classified into seven categories according to function. In addition, TGF-beta induced downregulation of glutamine synthetase in cells in G2/M but not G1/S phase, and this was further confirmed by immunoblotting. Moreover, exogenous glutamine completely blocked TGF-beta-induced apoptosis in G2/M and non-synchronized cells, whereas it had no effect on EMT, suggesting that the downregulation of glutamine synthetase is involved in G2/M phase-dependent apoptosis. These results provide new insight into the mechanism of the multifunctional effects of TGF-beta and how apoptosis and EMT are regulated in the same type of cells.