Inhibition of C-MYC transactl vat I on by mad transcription factor interrupts cycling in hepatocellular carcinoma cells

The Mad and Mxi transcription factors heterodimerize with Max and inhibit c-Myc transactivation potential in a dominant negative manner. As c-Myc plays critical roles in GO/G1 transition, we reasoned that overexpression of Mad would alter cell cycle progression. To examine the consequences of c-Myc antagonism, we used HepG2 (hepatoblastoma origin), Huh-7 (HBV -ve) and PLC/PRF/5 (HBV integration +ve) cell lines, which demonstrated abundant adenoviral receptors with LacZ expression in up to 70%-100% and no cytotoxicity after infection with an Adβgal virus. In control rodent Rat-1 embryonic fibroblasts or human U373MG astrocytoma cells, exposure for up to 7 days to an adenovirus expressing Mad (AdMad) had a cytostatic effect and cell number decreased to 10%±0.4% and 21%±7%, respectively, p<0.01. Cycling was inhibited in Rat-1 and U373MG cells with flow cytometry showing GO/G1 arrest, although [3H]-thymidine incorporation into DNA decreased less to 69%±19% (n.s.) and 20%±4% (p<02), respectively. When HepG2, Huh-7 or PLC/PRF/5 cells were treated with AdMad, cell numbers declined significantly in the first two cell lines, probably related to quantitative differences in adenoviral receptors, whereas [3H]thymidine incorporation was not decreased in any cell line. In contrast with Rat-1 or U373MG cells, AdMad-treated liver cells were arrested in GO/G1 plus G2/M. These findings were reminiscent of liver cell synchronization with butyrate, which causes biphasic arrest in GO/G1 and G2/M but with ongoing [3H]-thymidine incorporation. AdMad-treated liver cells only rarely exhibited apoptosis on acridine orange staining, indicating a cytostatic and not a cytolytic effect. Protein transblots using a specific antibody demonstrated Mad protein in all cells exposed to AdMad. CONCLUSIONS: Mad overexpression altered cell cycle progression, with liver cells showing arrest in Gl/GO or G2/M. Whether c-Myc transactivation potential regulates G2/M transition in liver cells requires further analysis. Overexpression of Mad and related c-Myc antagonists in cells provide novel tools for cell cycle analysis and for treating hepatocellular carcinoma.