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 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.
Gagandeep
S
S
Marion Bessin Liver Research Center, Comprehensive Cancer Research Center, and Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Ott
M
M
Nisen
P D
PD
DePinho
R A
RA
Gupta
S
S
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D006528:Carcinoma, Hepatocellular / Q000276:immunology / Q000473:pathology*; D002455:Cell Division; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007399:Interphase; D008113:Liver Neoplasms / Q000276:immunology / Q000473:pathology*; D051379:Mice; D016513:Mice, SCID; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C079439:MXD1 protein, human; C499479:Mad protein, mouse; D012097:Repressor Proteins; D014157:Transcription Factors