Suppression of thymidine phosphorylase expression by promoter methylation in human cancer cells lacking enzyme activity.
OBJECTIVE
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.
Guarcello
Vincenzo
V
Department of Pharmacology and Toxicology, Comprehensive Cancer Center, Center for AIDS Research, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Blanquicett
Carmelo
C
Naguib
Fardos N M
FN
El Kouni
Mahmoud H
MH
D015153:Blotting, Western; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects*; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000187:drug effects / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007438:Introns / Q000235:genetics; D009363:Neoplasm Proteins / Q000187:drug effects / Q000378:metabolism; D009369:Neoplasms / Q000201:enzymology* / Q000235:genetics*; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013447:Sulfites / Q000378:metabolism; D013939:Thymidine Phosphorylase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics*