Overexpression of Mad transcription factor inhibits proliferation of cultured human hepatocellular carcinoma cells along with tumor formation in immunodeficient animals

Background Dominant negative regulation of critical cell cycle molecules co uld perturb survival of cancer cells and help develop novel therapies. Methods To perturb the activity of c-Myc, which regulates G0/G1 transitions , we overexpressed Mad1 protein with an adenoviral vector, AdMad. Studies w ere conducted with established cell lines, including HepG2, HuH-7 and PLC/P RF/5 liver cancer cells, RAT-1A embryonic fibroblasts and U373MG astrocytom a cells. Results After AdMad-treatment, transduced cells exhibited decreased prolife ration rates in culture conditions. RAT-1A embryonic fibroblasts and U373MG astrocytoma cells showed accumulations in G0/G1, whereas HepG2 and HuH-7 c ells accumulated in G0/G1, and additionally in G2/M, albeit to a lesser ext ent. An in vitro assay using hepatocyte growth factor to stimulate prolifer ation in HuH-7 cells showed blunting of growth factor responsiveness, along with inhibition of cell cycle progression in AdMad-treated cells. No cytot oxicity was observed in AdMad-treated cells in culture, although cells lost clonogenic capacity in soft agar. In vivo assays using HepG2 cell tumors i n immunodeficient mice showed that overexpression of AdMad prevented tumori genesis. Conclusions These studies indicate roles of Mad in G2/M, as well as the pot ential of manipulating cell cycle controls for treating liver cancer. Copyr ight (C) 2000 John Wiley & Sons, Ltd.