Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87.
The expression of many cellular genes is modulated by DNA methylation and histone acetylation. These processes can influence malignant cell transformation and are also responsible for the silencing of DNA constructs introduced into mammalian cells for therapeutic or research purposes. As a better understanding of these biological processes may contribute to the development of novel cancer treatments and to study the complex mechanisms regulating gene silencing, we established a cellular system suitable to dissect the mechanisms regulating DNA methylation and histone acetylation. For this purpose, we stably transfected the neuroblastoma cell line U87 with a cytomegalovirus promoter-driven reporter gene construct whose expression was analyzed following treatment with the DNA methylation inhibitor 5'-aza-2'-deoxycytidine or histone deacetylation inhibitor trichostatin A. Both substances reactivated the silenced cytomegalovirus promoter, but with different reaction kinetics. Furthermore, whereas the kinetics of reactivation by trichostatin A did not substantially change over the time range considered (5 days), reactivation induced by 5'-aza-2'-deoxycytidine showed profound differences between day 1 and longer time points. We showed that this effect is related to the down-regulation of DNA replication by 5'-aza-2'-deoxycytidine. Finally, we have shown that the simultaneous administration of trichostatin A and 5'-aza-2'-deoxycytidine results in reactivation of the CMV promoter according to a cooperative, not synergistic or additive, mechanism. In conclusion, our cellular system should represent a powerful tool to investigate the complex mechanisms regulating gene silencing and to identify new anticancer drugs.
Grassi
G
G
Department of Molecular Pathology, University Hospital of Tübingen, Liebermeisterstrasse 8, D-72076, Tübingen, Germany. ggrassi@units.it
Maccaroni
P
P
Meyer
R
R
Kaiser
H
H
D'Ambrosio
E
E
Pascale
E
E
Grassi
M
M
Kuhn
A
A
Di Nardo
P
P
Kandolf
R
R
Küpper
J-H
JH
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D005909:Glioblastoma; D006655:Histone Deacetylases / Q000494:pharmacology; D006657:Histones / Q000187:drug effects*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008962:Models, Theoretical; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D006657:Histones; D006877:Hydroxamic Acids; D011500:Protein Synthesis Inhibitors; C012589:trichostatin A; D006655:Histone Deacetylases; D001374:Azacitidine