S Gagandeep1, M Ott, P D Nisen, R A DePinho, S Gupta. 1. Marion Bessin Liver Research Center, Comprehensive Cancer Research Center, and Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Abstract
BACKGROUND: Dominant negative regulation of critical cell cycle molecules could 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 were conducted with established cell lines, including HepG2, HuH-7 and PLC/PRF/5 liver cancer cells, RAT-1A embryonic fibroblasts and U373MG astrocytoma cells. RESULTS: After AdMad-treatment, transduced cells exhibited decreased proliferation rates in culture conditions. RAT-1A embryonic fibroblasts and U373MG astrocytoma cells showed accumulations in G0/G1, whereas HepG2 and HuH-7 cells accumulated in G0/G1, and additionally in G2/M, albeit to a lesser extent. An in vitro assay using hepatocyte growth factor to stimulate proliferation in HuH-7 cells showed blunting of growth factor responsiveness, along with inhibition of cell cycle progression in AdMad-treated cells. No cytotoxicity was observed in AdMad-treated cells in culture, although cells lost clonogenic capacity in soft agar. In vivo assays using HepG2 cell tumors in immunodeficient mice showed that overexpression of AdMad prevented tumorigenesis. CONCLUSIONS: These studies indicate roles of Mad in G2/M, as well as the potential of manipulating cell cycle controls for treating liver cancer.
BACKGROUND: Dominant negative regulation of critical cell cycle molecules could 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 were conducted with established cell lines, including HepG2, HuH-7 and PLC/PRF/5 liver cancer cells, RAT-1A embryonic fibroblasts and U373MG astrocytoma cells. RESULTS: After AdMad-treatment, transduced cells exhibited decreased proliferation rates in culture conditions. RAT-1A embryonic fibroblasts and U373MG astrocytoma cells showed accumulations in G0/G1, whereas HepG2 and HuH-7 cells accumulated in G0/G1, and additionally in G2/M, albeit to a lesser extent. An in vitro assay using hepatocyte growth factor to stimulate proliferation in HuH-7 cells showed blunting of growth factor responsiveness, along with inhibition of cell cycle progression in AdMad-treated cells. No cytotoxicity was observed in AdMad-treated cells in culture, although cells lost clonogenic capacity in soft agar. In vivo assays using HepG2 cell tumors in immunodeficientmice showed that overexpression of AdMad prevented tumorigenesis. CONCLUSIONS: These studies indicate roles of Mad in G2/M, as well as the potential of manipulating cell cycle controls for treating liver cancer.