Event Title

Effect of Hedgehog Signaling Pathway on VDUP1 Expression in Human Embryonic Mesenchymal cells (HEPM)

Location

Edwards Hall Lobby

Document Type

Poster Presentation (1 hour)

Description

Vitamin D3 Upregulated protein-1 (VDUP1) is a tumor suppressor gene. Its expression is reduced in tumor versus non-tumor cells although the mechanism for the reduction is not known. Studies on the developing Drosophila nervous system demonstrated that mutants lacking functional Hedgehog (Hh) signaling expressed a higher amount of VDUP1, suggesting that Hh signaling can regulate VDUP1 expression within the brain. The hypothesis is that Hh signaling within tumor cells results in reduced VDUP1 expression. HEPM cell line is known to exhibit activated Hh signaling. Transfection of HEPM cells with plasmid containing 8 copies of the consensus Gli binding site inserted upstream of luciferase (Gli-pluc) and β-galactosidase (β-Gal) was used to assess Hh signaling. Transfection followed by treatment with recombinant Hh protein increased luciferase activity. Transfection with a plasmid containing the human VDUP1 promoter upstream of luciferase (VDUP1-pluc) and β-Gal was used to assess VDUP1 promoter activity. Transfection followed by treatment with recombinant Hh protein resulted in decreased luciferase activity. HEPM cells treated with GANT61, an inhibitor of Gli, and stained with anti-VDUP1 and anti-HDAC showed increased VDUP1 immunofluorescence, primarily in the nucleus. These results confirmed active Hh signaling in HEPM cells with the ability to downregulate VDUP1 expression by inhibiting the pathway.

Start Date

April 2014

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Apr 26th, 1:00 PM

Effect of Hedgehog Signaling Pathway on VDUP1 Expression in Human Embryonic Mesenchymal cells (HEPM)

Edwards Hall Lobby

Vitamin D3 Upregulated protein-1 (VDUP1) is a tumor suppressor gene. Its expression is reduced in tumor versus non-tumor cells although the mechanism for the reduction is not known. Studies on the developing Drosophila nervous system demonstrated that mutants lacking functional Hedgehog (Hh) signaling expressed a higher amount of VDUP1, suggesting that Hh signaling can regulate VDUP1 expression within the brain. The hypothesis is that Hh signaling within tumor cells results in reduced VDUP1 expression. HEPM cell line is known to exhibit activated Hh signaling. Transfection of HEPM cells with plasmid containing 8 copies of the consensus Gli binding site inserted upstream of luciferase (Gli-pluc) and β-galactosidase (β-Gal) was used to assess Hh signaling. Transfection followed by treatment with recombinant Hh protein increased luciferase activity. Transfection with a plasmid containing the human VDUP1 promoter upstream of luciferase (VDUP1-pluc) and β-Gal was used to assess VDUP1 promoter activity. Transfection followed by treatment with recombinant Hh protein resulted in decreased luciferase activity. HEPM cells treated with GANT61, an inhibitor of Gli, and stained with anti-VDUP1 and anti-HDAC showed increased VDUP1 immunofluorescence, primarily in the nucleus. These results confirmed active Hh signaling in HEPM cells with the ability to downregulate VDUP1 expression by inhibiting the pathway.