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Suppression of thymidine phosphorylase expression by promoter methylation in human cancer cells lacking enzyme

Vincenzo Guarcello1, Carmelo Blanquicett, Fardos N M Naguib

  • 1Department of Pharmacology and Toxicology, Comprehensive Cancer Center, Center for AIDS Research, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

Cancer Chemotherapy and Pharmacology
|September 7, 2007
PubMed
Abstract

Insights

Thymidine phosphorylase (TP) expression in cancer is regulated by promoter methylation. Inhibiting methylation reactivates TP, suggesting its role in cancer prognosis.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Epigenetics

Background:

  • Thymidine phosphorylase (TP) activity varies across human cancer cell lines.
  • Mechanisms regulating TP expression in cancer remain largely unknown.
  • Promoter methylation is a known mechanism for reversible gene silencing.

Purpose of the Study:

  • Investigate the role of TP promoter methylation in regulating TP expression in human cancer cell lines.
  • Determine the impact of methylation on TP gene silencing.
  • Explore potential therapeutic strategies targeting TP methylation.

Main Methods:

  • TP promoter methylation analysis using bisulfite sequencing.
  • Quantification of TP mRNA levels via real-time quantitative PCR.
  • Assessment of TP promoter activity using Luciferase reporter assays.
  • Evaluation of DNA-protein interactions with electrophoretic mobility shift assays.

Main Results:

  • TP expression varied significantly across cell lines, with DLD-1 showing no expression and SKBR-3 showing high expression.
  • Extensive promoter methylation correlated with absent TP expression in DLD-1 cells.
  • Methylation inhibition (5-aza-2dC) reactivated TP expression in DLD-1 cells, enhanced by histone deacetylase inhibition (Trichostatin-A).
  • Methylation inhibited transcription factor binding, implicating Sp1 and Sp3 in TP regulation.

Conclusions:

  • TP promoter methylation is a key mechanism for down-regulating TP expression in cancer.
  • Reactivation of TP through methylation inhibition shows potential for cancer therapy.
  • TP methylation status may serve as a prognostic biomarker in cancer patients.

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