1. ¹San Raffaele Bio-medical Science Park of Rome, Rome, Italy
2. ²Departments of Histology and Medical Embryology, University 'La Sapienza', Rome, Italy
3. ³Biochemical Sciences, University 'La Sapienza', Rome, Italy
4. ⁴Cellular Biotechnology and Hematology, University 'La Sapienza', Rome, Italy
5. ⁵European Institute of Oncology (IEO), Milan, Italy
6. ⁶FIRC Institute of Molecular Oncology (IFOM), Milan, Italy
7. ⁷Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital and McGill University Department of Oncology and Medicine, Montreal, Quebec, Canada
8. ⁸Department of Biopathology, University Tor Vergata, Rome

Correspondence: C Nervi, San Raffaele Bio-medical Science Park of Rome, University of Rome 'La Sapienza', Via di Castel Romano 100, Rome 00128, Italy. E-mail: clara.nervi@uniroma1.it

Received 26 March 2004; Revised 16 August 2004; Accepted 8 October 2004; Published online 31 January 2005.

Abstract

The acute promyelocytic leukemia (PML)-retinoic acid receptor (RAR) fusion product recruits histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activities on retinoic acid (RA)-target promoters causing their silencing and differentiation block. RA treatment induces epigenetic modifications at its target loci and restores myeloid differentiation of APL blasts. Using RA-sensitive and RA-resistant APL cell lines and primary blasts, we addressed the functional relevance of the aberrant methylation status at the RA-target promoter RAR2 and the mechanism by which methylation is reversed by RA. RA decreased DNMT expression and activity, which correlated with demethylation at specific sites on RAR2 promoter/exon-1, and the ability of APL blasts to differentiate in vitro and in vivo. None of these events occurred in an RA-resistant APL cell line containing a PML-RAR defective for ligand binding. The specific contribution of the HDAC and DNMT pathways to the response of APL cells to RA was also tested by inhibiting these enzymatic activities with TSA and/or 5-azacytidine. In RA-responsive and RA-resistant APL blasts, TSA and 5-azacytidine induced specific changes on the chromatin state at RA-target sites, increased the RA effect on promoter activity, endogenous RA-target gene expression and differentiation. These results extend the rationale for chromatin-targeted treatment in APL and RA-resistant leukemias.