Carcinogenesis Advance Access originally published online on July 17, 2003
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Carcinogenesis, Vol. 24, No. 10, 1625-1635,
October 2003
© 2003 Oxford University Press
CANCER BIOLOGY |
Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87
1 Department of Molecular Pathology, University Hospital of Tübingen, Liebermeisterstrasse 8, D-72076, Tübingen, Germany, 2 Department of Internal Medicine, University Hospital of Trieste, Cattinara 34149, Trieste, Italy, 3 Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma, Italy, 4 Dipartimento di Medicina Molecolare e Patologia, Università di Roma La Sapienza, Roma, Italy, 5 Department of Chemical, Environmental and Raw Materials Engineering – DICAMP, University of Trieste, Piazzale Europa 1, I-34127 Trieste, Italy and 6 Laboratory of Cellular and Molecular Cardiology, Department of Internal Medicine, University of Rome Tor Vergata, Roma, Italy
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.
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