Carcinogenesis Advance Access published online on July 17, 2003
Carcinogenesis, doi:10.1093/carcin/bgg118
© 2003 by Oxford University Press
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CANCER BIOLOGY
1 Department of Molecular Pathology, University Hospital of Tübingen, Liebermeisterstr. 8, D-72076, Tübingen, Germany; Department of Internal Medicine, University Hospital of Trieste, Cattinara 34149, Trieste, Italy
* Corresponding author. E-mail: ggrassi{at}units.it.
Received 18 December 2002
; revised 25 June 2003
; accepted 30 June 2003
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 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 trichostatin A reactivation kinetic did not substantially change over the time range considered (five days), reactivation sustained by 5'-aza-2'deoxycytidine showed profound differences between day one 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 proved that the simultaneous administration of trichostatin A and 5'-aza-2'deoxycytidine resulted in the reactivation of the CMV promoter according to a co-operative but 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 anti-cancer drugs.
Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human gliobastoma cell line U87
2 Department of Molecular Pathology, University Hospital of Tübingen, Liebermeisterstr. 8, D-72076, Tübingen, Germany
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
6 Laboratory of Cellular & Molecular Cardiology, Department of Internal Medicine, University of Rome Tor Vergata, Roma, Italy
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