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Cancer Chemotherapy and Pharmacology
Erschienen in: Cancer Chemotherapy and Pharmacology 1/2008

01.06.2008 | Original Article

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

verfasst von: Vincenzo Guarcello, Carmelo Blanquicett, Fardos N. M. Naguib, Mahmoud H. el Kouni

Erschienen in: Cancer Chemotherapy and Pharmacology | Ausgabe 1/2008

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Abstract

Purpose

Thymidine phosphorylase (TP, EC 2.4.2.4) activity varies in different human cancer cell lines. Nevertheless, little is known about the regulatory mechanisms of TP expression in such cancers. Promoter methylation of dinucleotide cytosine–guanine (CpG) sites is a known mechanism of reversible gene expression silencing.

Methods

TP promoter methylation was investigated in five cancer cell lines (SKBR-3, 786-O, HT-29, MDA-231, DLD-1). TP mRNA levels were determined by real-time quantitative PCR. The degree of methylation was identified by bisulfite sequencing. Minimal TP promoter activity was determined by Luciferase reporter assays. DNA–protein interactions were evaluated by electrophoretic mobility shift assays.

Results

SKBR-3 cells exhibited the highest TP expression, 786-O, HT-29, and MDA-231 cells exhibited intermediate TP expression, while DLD-1 cells did not express TP as demonstrated by TP mRNA, protein, and enzyme activity levels. SKBR-3 lacked methylation in the TP promoter, intron 1 and exon 1 regions, while DLD-1 showed extensive methylation. Treatment of DLD-1 and SKBR-3 with the methylation-inhibitor, 5-aza-2′-deoxycytidine (5-aza-2dC), resulted in a concentration-dependent increase in TP mRNA and protein levels in DLD-1 but not SKBR-3 cells. Trichostatin-A treatment, a histone deacetylase inhibitor, improved the 5-aza-2dC-induced TP re-activation. Electrophoretic mobility shift assays demonstrated that methylation significantly inhibits transcription factor binding. Supershift analyses suggest that the Sp1 and Sp3 (to a lesser degree) transcription factors have a role in the regulation of TP expression.

Conclusions

These findings suggest that TP promoter methylation is a mechanism for down-regulation of TP expression in cancer cells and may have implications in modulating prognosis of cancer patients.
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Metadaten
Titel
Suppression of thymidine phosphorylase expression by promoter methylation in human cancer cells lacking enzyme activity
verfasst von
Vincenzo Guarcello
Carmelo Blanquicett
Fardos N. M. Naguib
Mahmoud H. el Kouni
Publikationsdatum
01.06.2008
Verlag
Springer-Verlag
Erschienen in
Cancer Chemotherapy and Pharmacology / Ausgabe 1/2008
Print ISSN: 0344-5704
Elektronische ISSN: 1432-0843
DOI
https://doi.org/10.1007/s00280-007-0578-5

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