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Growth Arrest Specific 1 (Gas1) Gene Overexpression in Liver Reduces the In Vivo Progression of Murine Hepatocellular Carcinoma and Partially Restores Gene Expression Levels.

Sacilotto N, Castillo J, Riffo-Campos ÁL, Flores JM, Hibbitt O, Wade-Martins R, López C, Rodrigo MI, Franco L, López-Rodas G - PLoS ONE (2015)

Bottom Line: Present results suggest that the overexpression of Gas1 (growth arrest specific 1) gene reduces the size, proliferating activity and malignancy of liver tumors.Moreover, the number of carcinoma foci in the liver and the number of lung metastases were reduced.These results are related with the finding that overexpression of Gas1 in Hepa 1-6 cells arrests cell cycle before S phase, with a significant (p < 0.01) and concomitant reduction in the expression of cyclin E2 gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Valencia, Burjassot, Valencia, Spain.

ABSTRACT
The prognosis of hepatocellular carcinoma patients is usually poor, the size of tumors being a limiting factor for surgical treatments. Present results suggest that the overexpression of Gas1 (growth arrest specific 1) gene reduces the size, proliferating activity and malignancy of liver tumors. Mice developing diethylnitrosamine-induced hepatocellular carcinoma were subjected to hydrodynamic gene delivery to overexpress Gas1 in liver. This treatment significantly (p < 0.05) reduced the number of large tumors, while the difference in the total number of lesions was not significant. Moreover, the number of carcinoma foci in the liver and the number of lung metastases were reduced. These results are related with the finding that overexpression of Gas1 in Hepa 1-6 cells arrests cell cycle before S phase, with a significant (p < 0.01) and concomitant reduction in the expression of cyclin E2 gene. In addition, a triangular analysis of microarray data shows that Gas1 overexpression restores the transcription levels of 150 genes whose expression was affected in the diethylnitrosamine-induced tumors, thirteen of which are involved in the hedgehog signaling pathway. Since the in vivo Gas1 gene delivery to livers of mice carrying hepatocellular carcinoma reduces the size and proliferating activity of tumors, partially restoring the transcriptional profile of the liver, the present study opens promising insights towards a therapeutic approach for hepatocellular carcinoma.

No MeSH data available.


Related in: MedlinePlus

Transfection of Hepa 1–6 cells with the growth-arrest specific 1 (Gas1) gene.(A) Anti-HA immunofluorescence of Hepa 1–6 cells transfected with pcDNA3/CAG-HAGas1 in the absence of detergents to preserve the integrity of membranes. Nuclei were counterstained with DAPI. (B) Double immunofluorescent staining anti-HA/anti-BrdU of Hepa 1–6 cells transfected with pcDNA3/CAG-HAGas1. (C) Cell cycle analysis of GFP-positive HEPA 1–6 cells after transfection with pIRES/GFP empty vector. 106 cells (areas indicated in the upper row) were sorted and subjected to cell cycle analysis after propidium iodide staining (lower row). (D) As (C), after transfection with pIRES/HAGas1. (E) Quantitation of the % of cells in the different stages of the cell cycle from the flow cytometry analysis. Experiments were done in triplicate. (F) Analysis of Ccne2 expression in Hepa 1–6 cells overexpressing Gas1. A representative RT-PCR showing Gas1 and Ccne2 expression in cells transfected with either empty pcDNA3/CAG (control) or pcDNA3/CAG-HAGas1 (HAGas1), 15 h after transfection. (G) qRT-PCR to determine CycE2 expression in cells as in (F). qRT-PCR was performed in triplicate from three independent experiments. Values were averaged and normalized to 18S rRNA. **, p<0.01. ***, p<0.001. In A and B the bar represents 11 μm.
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pone.0132477.g001: Transfection of Hepa 1–6 cells with the growth-arrest specific 1 (Gas1) gene.(A) Anti-HA immunofluorescence of Hepa 1–6 cells transfected with pcDNA3/CAG-HAGas1 in the absence of detergents to preserve the integrity of membranes. Nuclei were counterstained with DAPI. (B) Double immunofluorescent staining anti-HA/anti-BrdU of Hepa 1–6 cells transfected with pcDNA3/CAG-HAGas1. (C) Cell cycle analysis of GFP-positive HEPA 1–6 cells after transfection with pIRES/GFP empty vector. 106 cells (areas indicated in the upper row) were sorted and subjected to cell cycle analysis after propidium iodide staining (lower row). (D) As (C), after transfection with pIRES/HAGas1. (E) Quantitation of the % of cells in the different stages of the cell cycle from the flow cytometry analysis. Experiments were done in triplicate. (F) Analysis of Ccne2 expression in Hepa 1–6 cells overexpressing Gas1. A representative RT-PCR showing Gas1 and Ccne2 expression in cells transfected with either empty pcDNA3/CAG (control) or pcDNA3/CAG-HAGas1 (HAGas1), 15 h after transfection. (G) qRT-PCR to determine CycE2 expression in cells as in (F). qRT-PCR was performed in triplicate from three independent experiments. Values were averaged and normalized to 18S rRNA. **, p<0.01. ***, p<0.001. In A and B the bar represents 11 μm.

Mentions: In order to choose the strongest promoter to induce expression of Gas1 in these mammalian cells, we compared the efficiency of the widely used CAG and CMV promoters by a luciferase assay. In this assay, using Hepa 1–6 cells routinely transfected with around 45% efficiency (S1A Fig), the CAG promoter drives an expression 14-fold higher than the CMV promoter (S1B Fig). Therefore, the former was used for the subsequent overexpression experiments. Endogenous GAS1 protein is not detectable in asynchronously growing Hepa 1–6 cells. However, an exogenously expressed HA-tagged GAS1 can be detected with both anti-HA and anti-GAS1 antibodies in these cells (S1C Fig), localizing to the cell membrane (Fig 1A).


Growth Arrest Specific 1 (Gas1) Gene Overexpression in Liver Reduces the In Vivo Progression of Murine Hepatocellular Carcinoma and Partially Restores Gene Expression Levels.

Sacilotto N, Castillo J, Riffo-Campos ÁL, Flores JM, Hibbitt O, Wade-Martins R, López C, Rodrigo MI, Franco L, López-Rodas G - PLoS ONE (2015)

Transfection of Hepa 1–6 cells with the growth-arrest specific 1 (Gas1) gene.(A) Anti-HA immunofluorescence of Hepa 1–6 cells transfected with pcDNA3/CAG-HAGas1 in the absence of detergents to preserve the integrity of membranes. Nuclei were counterstained with DAPI. (B) Double immunofluorescent staining anti-HA/anti-BrdU of Hepa 1–6 cells transfected with pcDNA3/CAG-HAGas1. (C) Cell cycle analysis of GFP-positive HEPA 1–6 cells after transfection with pIRES/GFP empty vector. 106 cells (areas indicated in the upper row) were sorted and subjected to cell cycle analysis after propidium iodide staining (lower row). (D) As (C), after transfection with pIRES/HAGas1. (E) Quantitation of the % of cells in the different stages of the cell cycle from the flow cytometry analysis. Experiments were done in triplicate. (F) Analysis of Ccne2 expression in Hepa 1–6 cells overexpressing Gas1. A representative RT-PCR showing Gas1 and Ccne2 expression in cells transfected with either empty pcDNA3/CAG (control) or pcDNA3/CAG-HAGas1 (HAGas1), 15 h after transfection. (G) qRT-PCR to determine CycE2 expression in cells as in (F). qRT-PCR was performed in triplicate from three independent experiments. Values were averaged and normalized to 18S rRNA. **, p<0.01. ***, p<0.001. In A and B the bar represents 11 μm.
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Related In: Results  -  Collection

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pone.0132477.g001: Transfection of Hepa 1–6 cells with the growth-arrest specific 1 (Gas1) gene.(A) Anti-HA immunofluorescence of Hepa 1–6 cells transfected with pcDNA3/CAG-HAGas1 in the absence of detergents to preserve the integrity of membranes. Nuclei were counterstained with DAPI. (B) Double immunofluorescent staining anti-HA/anti-BrdU of Hepa 1–6 cells transfected with pcDNA3/CAG-HAGas1. (C) Cell cycle analysis of GFP-positive HEPA 1–6 cells after transfection with pIRES/GFP empty vector. 106 cells (areas indicated in the upper row) were sorted and subjected to cell cycle analysis after propidium iodide staining (lower row). (D) As (C), after transfection with pIRES/HAGas1. (E) Quantitation of the % of cells in the different stages of the cell cycle from the flow cytometry analysis. Experiments were done in triplicate. (F) Analysis of Ccne2 expression in Hepa 1–6 cells overexpressing Gas1. A representative RT-PCR showing Gas1 and Ccne2 expression in cells transfected with either empty pcDNA3/CAG (control) or pcDNA3/CAG-HAGas1 (HAGas1), 15 h after transfection. (G) qRT-PCR to determine CycE2 expression in cells as in (F). qRT-PCR was performed in triplicate from three independent experiments. Values were averaged and normalized to 18S rRNA. **, p<0.01. ***, p<0.001. In A and B the bar represents 11 μm.
Mentions: In order to choose the strongest promoter to induce expression of Gas1 in these mammalian cells, we compared the efficiency of the widely used CAG and CMV promoters by a luciferase assay. In this assay, using Hepa 1–6 cells routinely transfected with around 45% efficiency (S1A Fig), the CAG promoter drives an expression 14-fold higher than the CMV promoter (S1B Fig). Therefore, the former was used for the subsequent overexpression experiments. Endogenous GAS1 protein is not detectable in asynchronously growing Hepa 1–6 cells. However, an exogenously expressed HA-tagged GAS1 can be detected with both anti-HA and anti-GAS1 antibodies in these cells (S1C Fig), localizing to the cell membrane (Fig 1A).

Bottom Line: Present results suggest that the overexpression of Gas1 (growth arrest specific 1) gene reduces the size, proliferating activity and malignancy of liver tumors.Moreover, the number of carcinoma foci in the liver and the number of lung metastases were reduced.These results are related with the finding that overexpression of Gas1 in Hepa 1-6 cells arrests cell cycle before S phase, with a significant (p < 0.01) and concomitant reduction in the expression of cyclin E2 gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Valencia, Burjassot, Valencia, Spain.

ABSTRACT
The prognosis of hepatocellular carcinoma patients is usually poor, the size of tumors being a limiting factor for surgical treatments. Present results suggest that the overexpression of Gas1 (growth arrest specific 1) gene reduces the size, proliferating activity and malignancy of liver tumors. Mice developing diethylnitrosamine-induced hepatocellular carcinoma were subjected to hydrodynamic gene delivery to overexpress Gas1 in liver. This treatment significantly (p < 0.05) reduced the number of large tumors, while the difference in the total number of lesions was not significant. Moreover, the number of carcinoma foci in the liver and the number of lung metastases were reduced. These results are related with the finding that overexpression of Gas1 in Hepa 1-6 cells arrests cell cycle before S phase, with a significant (p < 0.01) and concomitant reduction in the expression of cyclin E2 gene. In addition, a triangular analysis of microarray data shows that Gas1 overexpression restores the transcription levels of 150 genes whose expression was affected in the diethylnitrosamine-induced tumors, thirteen of which are involved in the hedgehog signaling pathway. Since the in vivo Gas1 gene delivery to livers of mice carrying hepatocellular carcinoma reduces the size and proliferating activity of tumors, partially restoring the transcriptional profile of the liver, the present study opens promising insights towards a therapeutic approach for hepatocellular carcinoma.

No MeSH data available.


Related in: MedlinePlus