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

S. Gagandeep, M. Ott, P. Nisen, R. Depinho, Sanjeev Gupta

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
JournalJournal of Investigative Medicine
Volume44
Issue number3
StatePublished - 1996
Externally publishedYes

Fingerprint

Hepatocellular Carcinoma
Transcription Factors
Liver
Cells
Thymidine
Adenoviridae
Rats
Cytostatic Agents
Programmable logic controllers
Cell Cycle
Acridine Orange
Flow cytometry
Butyrates
Fibroblasts
Cell Line
Cytotoxicity
Transcriptional Activation
Viruses
Rodent Control
Cell Count

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Inhibition of C-MYC transactl vat I on by mad transcription factor interrupts cycling in hepatocellular carcinoma cells. / Gagandeep, S.; Ott, M.; Nisen, P.; Depinho, R.; Gupta, Sanjeev.

In: Journal of Investigative Medicine, Vol. 44, No. 3, 1996.

Research output: Contribution to journalArticle

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title = "Inhibition of C-MYC transactl vat I on by mad transcription factor interrupts cycling in hepatocellular carcinoma cells",
abstract = "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.",
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T1 - Inhibition of C-MYC transactl vat I on by mad transcription factor interrupts cycling in hepatocellular carcinoma cells

AU - Gagandeep, S.

AU - Ott, M.

AU - Nisen, P.

AU - Depinho, R.

AU - Gupta, Sanjeev

PY - 1996

Y1 - 1996

N2 - 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.

AB - 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.

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