Re-expression of DNA methylation-silenced CD44 gene in a resistant NB4 cell line: rescue of CD44-dependent cell death by cAMP

Abstract

In the acute promyelocytic leukemia cell line, NB4, activation of the CD44 receptor triggers apoptosis. This pathway does not operate in the retinoid-maturation-resistant NB4-LR1 subclone. In this work, we show that the CD44 gene is silenced in these cells. The molecular defect involves DNA methylation of cytosine phosphate guanine (CpG) island and underacetylation of histone H3 at CD44 promoter. The methylating inhibitor 5-aza-CdR and cyclic AMP (cAMP) reverse the CD44 gene silencing. Contrary to 5-aza-CdR, cAMP does not induce DNA demethylation or histone modification at the CD44 promoter, whereas an H3pS10/AcK14 dual modification is observed on a global level. cAMP also induces the expression of c-Jun transcription factor and its recruitment at the CD44 promoter. Chromatin immunoprecipitation assays further show the association of brahma (Brm), a subunit of SWI/SNF chromatinremodelling complex involved in the crosstalk between transcription and RNA polymerase II (RNA Pol II) processing, as well as the binding of phosphorylated RNA Pol II to the proximal promoter region of CD44. Finally, our study reveals that cAMP re-establishes the CD44-mediated cell death signalling. We propose that one of the actions of cAMP in restoring normal cell phenotype of leukaemia cells may consist in a broad trans-reactivation of silenced genes, despite marked hypermethylation of their promoters, as illustrated here with CD44 re-expression.

Epigenetic regulation of CD44in Hodgkin and non-Hodgkin lymphoma

“…In accordance with this view, the CD44 promoter is reportedly stimulated by growth factors, particularly by the Ras-Erk signaling pathway [38]. Interesting in this context is also that hypermethylated CD44 could be reactivated not only by Aza but also by cAMP in an ATRA-resistant acute promyelocytic leukemia cell line [39]. Thus DNA methylation is one important but not exclusive mechanism regulating the expression of CD44 .…”

CD44 in hematological neoplasias

“…The ATRA-resistant subclone NB4-LR1 displayed no CD44 expression due to epigenetic silencing mechanisms, which can be reversed treating the respective line with the DNA methylating inhibitor 5-aza-CdR. A similar effect is seen upon treatment with cyclic AMP and subsequent CD44 ligation by the anti-CD44 specific antibody A3D8, which results in apoptotic cell death [103]. Hertweck …”

cFos Mediates cAMP-Dependent Generation of ROS and Rescue of Maturation Program in Retinoid-Resistant Acute Promyelocytic Leukemia Cell Line NB4-LR1

“…We have demonstrated that cAMP induces post-translational modifications (Ser10-phosphorylation/Lys14-acetylation) of histone H3 in bulk chromatin, known to favor the transcriptional activity of immediate early genes [11], [12], [13], as well as the expression and the dual Ser63/Ser73 phosphorylation of transcription factor cJun. The latter is further recruited concomitantly with chromatin remodeling factors to the AP-1 site of the CD44 promoter, leading to the transcriptional re-expression of CD44 and the restoration of a functional receptor at the surface of NB4-LR1 cells [10]. These results strongly suggest that cAMP may reverse the silencing of genes through chromatin remodeling and transcription factor activation.…”

“…The molecular mechanisms by which cAMP acts to normalize the phenotype of resistant leukemia cells are still poorly understood. Besides the already known mutations in the PML-RAR fusion gene [8], [9], our recent studies have revealed the existence of aberrant epigenetic events in ATRA-resistant NB4-LR1 cells, responsible for the downregulation of genes associated with differentiation [10]. This is the case for the CD44 gene, encoding for a well-known receptor implicated in the maturation of myeloid cells.…”