Unbound E2F modulates TGF-β1-induced apoptosis in HuH-7 cells

Abstract

E2F is an important target of the retinoblastoma protein (pRb) and plays a critical role in G(1)/S progression through the cell cycle. TGF-beta1 arrests HuH-7 cells in G(1) by suppressing phosphorylation of pRb and induces apoptosis by inhibiting its expression. In this study, we examined the downstream effects of TGF-beta1-induced apoptosis and the potential roles for pRb and E2F. The results indicated that greater than 90% of the TGF-beta1-induced preapoptotic cells were arrested in G(1) phase of the cell cycle. This was associated with a significant increase in both E2F-DNA-binding activity and transcription of E2F-responsive reporter constructs. In contrast, no significant changes were observed in E2F mRNA and protein levels, and the overexpression of pRb partially inhibited E2F activation. Gel-shift assays identified more than four E2F complexes from preapoptotic and synchronized G(1) HuH-7 cells, each exhibiting different patterns of E2F-associated proteins. The increased E2F activity did not affect the association patterns with pRb, p107 and p130, but altered the formation of an E2F-DP-1 complex. In contrast, E2F-DP-2 exhibited little change in the preapoptotic cells. Moreover, TGF-beta1 induced apoptosis at G(1) and inhibited entry into S phase irrespective of the increased E2F activity. The release of preapoptotic cells from TGF-beta1 resulted in rapid S phase entry and subsequent apoptosis in 33% of cells over a 72 hour period. In conclusion, the results demonstrate that TGF-beta1-induced apoptosis in HuH-7 cells is associated with a marked increase in activity of transcription factor E2F that is partially inhibited by overexpression of pRb. Preapoptotic changes are, in part, reversible upon removal of TGF-beta1 and the majority of cells re-enter the normal cell cycle. Finally, TGF-beta1-induced apoptosis with the associated increase in E2F activity can occur in both the G(1) and S phases of the cell cycle.