The Sp1 transcription factor contributes to the tumor necrosis factor-induced expression of the angiogenic factor thymidine phosphorylase in human colon carcinoma cells

Geng Hui Zhu, Michelle Lenzi, Edward L. Schwartz

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

Thymidine phosphorylase (TP; also known as platelet-derived endothelial cell growth factor, PD-ECGF) is an angiogenic factor that is chemotactic for endothelial cells and has been found to induce neovascularization in vivo. TP is frequently overexpressed in human solid tumors, where its expression has been correlated with increased tumor microvessel density, invasion, and metastasis, and shorter patient survival. In this report, TP activity in the WiDr colon carcinoma cell line was found to be induced 100-fold by tumor necrosis factor (TNFα), a secretory product of activated macrophages that has indirect angiogenic activities. Increased TP activity was accompanied by increased TP mRNA levels and without an increase in mRNA stability. TNFα-induced TP mRNA levels were reduced by mithramycin, a DNA-binding transcription inhibitor specific for GC-rich sequences. Transcriptional regulation by TNFα was confirmed by transient transfection of WiDr with upstream TP sequences in a luciferase reporter construct. Deletion analysis of the reporter pinpointed two regions of the TP promoter with regulatory elements for both TNFα-inducible and basal expression, and they contained, respectively, three and one consensus binding sites for the Sp1-family of transcription factors. One additional region contributed only to basal TP expression, and it contained three Sp1 sites. TNFα-induced TP expression decreased when point mutations were made in three of the four Sp1 sites postulated to contribute to both basal and TNFα-inducible expression. Electrophoretic mobility shift assays further demonstrated binding of nuclear Sp1 to these three sites. Sp1-binding activity was also increased in cells treated with TNFα. These studies establish a role for Sp1 in the regulation of expression of the angiogenic factor TP in colon cancer WiDr cells.

Original languageEnglish (US)
Pages (from-to)8477-8485
Number of pages9
JournalOncogene
Volume21
Issue number55
DOIs
StatePublished - Dec 5 2002

Fingerprint

Thymidine Phosphorylase
Sp1 Transcription Factor
Angiogenesis Inducing Agents
Colon
Tumor Necrosis Factor-alpha
Carcinoma
Plicamycin
GC Rich Sequence
Messenger RNA
RNA Stability
Electrophoretic Mobility Shift Assay
Microvessels
Luciferases
Point Mutation
Genetic Promoter Regions
Colonic Neoplasms
Transfection
Neoplasms
Endothelial Cells
Macrophages

Keywords

  • Angiogenic factor
  • Colorectal cancer
  • Thymidine phosphorylase
  • Transcription regulation

ASJC Scopus subject areas

  • Molecular Biology
  • Cancer Research
  • Genetics

Cite this

The Sp1 transcription factor contributes to the tumor necrosis factor-induced expression of the angiogenic factor thymidine phosphorylase in human colon carcinoma cells. / Zhu, Geng Hui; Lenzi, Michelle; Schwartz, Edward L.

In: Oncogene, Vol. 21, No. 55, 05.12.2002, p. 8477-8485.

Research output: Contribution to journalArticle

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abstract = "Thymidine phosphorylase (TP; also known as platelet-derived endothelial cell growth factor, PD-ECGF) is an angiogenic factor that is chemotactic for endothelial cells and has been found to induce neovascularization in vivo. TP is frequently overexpressed in human solid tumors, where its expression has been correlated with increased tumor microvessel density, invasion, and metastasis, and shorter patient survival. In this report, TP activity in the WiDr colon carcinoma cell line was found to be induced 100-fold by tumor necrosis factor (TNFα), a secretory product of activated macrophages that has indirect angiogenic activities. Increased TP activity was accompanied by increased TP mRNA levels and without an increase in mRNA stability. TNFα-induced TP mRNA levels were reduced by mithramycin, a DNA-binding transcription inhibitor specific for GC-rich sequences. Transcriptional regulation by TNFα was confirmed by transient transfection of WiDr with upstream TP sequences in a luciferase reporter construct. Deletion analysis of the reporter pinpointed two regions of the TP promoter with regulatory elements for both TNFα-inducible and basal expression, and they contained, respectively, three and one consensus binding sites for the Sp1-family of transcription factors. One additional region contributed only to basal TP expression, and it contained three Sp1 sites. TNFα-induced TP expression decreased when point mutations were made in three of the four Sp1 sites postulated to contribute to both basal and TNFα-inducible expression. Electrophoretic mobility shift assays further demonstrated binding of nuclear Sp1 to these three sites. Sp1-binding activity was also increased in cells treated with TNFα. These studies establish a role for Sp1 in the regulation of expression of the angiogenic factor TP in colon cancer WiDr cells.",
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AB - Thymidine phosphorylase (TP; also known as platelet-derived endothelial cell growth factor, PD-ECGF) is an angiogenic factor that is chemotactic for endothelial cells and has been found to induce neovascularization in vivo. TP is frequently overexpressed in human solid tumors, where its expression has been correlated with increased tumor microvessel density, invasion, and metastasis, and shorter patient survival. In this report, TP activity in the WiDr colon carcinoma cell line was found to be induced 100-fold by tumor necrosis factor (TNFα), a secretory product of activated macrophages that has indirect angiogenic activities. Increased TP activity was accompanied by increased TP mRNA levels and without an increase in mRNA stability. TNFα-induced TP mRNA levels were reduced by mithramycin, a DNA-binding transcription inhibitor specific for GC-rich sequences. Transcriptional regulation by TNFα was confirmed by transient transfection of WiDr with upstream TP sequences in a luciferase reporter construct. Deletion analysis of the reporter pinpointed two regions of the TP promoter with regulatory elements for both TNFα-inducible and basal expression, and they contained, respectively, three and one consensus binding sites for the Sp1-family of transcription factors. One additional region contributed only to basal TP expression, and it contained three Sp1 sites. TNFα-induced TP expression decreased when point mutations were made in three of the four Sp1 sites postulated to contribute to both basal and TNFα-inducible expression. Electrophoretic mobility shift assays further demonstrated binding of nuclear Sp1 to these three sites. Sp1-binding activity was also increased in cells treated with TNFα. These studies establish a role for Sp1 in the regulation of expression of the angiogenic factor TP in colon cancer WiDr cells.

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