Abstract
BackgroundGlucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. GCs activate the glucocorticoid receptor (GR), to regulate a complex genetic network, culminating in apoptosis. Normal lymphoblasts and many lymphoid malignancies are sensitive to GC-driven apoptosis. Resistance to GCs can be a significant clinical problem, however, and correlates with resistance to several other major chemotherapeutic agents.MethodsWe analyzed the effect of treatment with the cytosine analogue 5 aza-2’ deoxycytidine (AZA) on GC resistance in two acute lymphoblastic leukemia (T or pre-T ALL) cell lines- CEM and Molt-4- and a (B-cell) myeloma cell line, RPMI 8226. Methods employed included tissue culture, flow cytometry, and assays for clonogenicity, cytosine extension, immunochemical identification of proteins, and gene transactivation. High throughput DNA sequencing was used to confirm DNA methylation status.ConclusionsTreatment of these cells with AZA resulted in altered DNA methylation and restored GC-evoked apoptosis in all 3 cell lines. In CEM cells the altered epigenetic state resulted in site-specific phosphorylation of the GR, increased GR potency, and GC-driven induction of the GR from promoters that lie in CpG islands. In RPMI 8226 cells, expression of relevant coregulators of GR function was altered. Activation of p38 mitogen-activated protein kinase (MAPK), which is central to a feed-forward mechanism of site-specific GR phosphorylation and ultimately, apoptosis, occurred in all 3 cell lines. These data show that in certain malignant hematologic B- and T-cell types, epigenetically controlled GC resistance can be reversed by cell exposure to a compound that causes DNA demethylation. The results encourage studies of application to in vivo systems, looking towards eventual clinical applications.
Highlights
Glucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells
Repression of gene expression based on DNA methylation can be reversed by analogs of cytosine such as 5 aza-2’ deoxycytidine (AZA), which incorporate into the genome of actively dividing cells and can render the cytosine nucleotide sites which they replace incapable of methylation [1,3,6,8,12]
Brief exposure to the genomic DNA demethylating agent AZA restores the GC-dependent apoptotic response in each of three cell lines We evaluated the contribution of the epigenetic state to GC resistance in three commonly used model systems of human lymphoid hematologic malignancies: 1) CEMC1-15, a GC-resistant clone of the pediatric acute lymphoblastic leukemia (ALL) cell line CCRF-CEM; 2) Molt-4, an uncloned T-cell derived pediatric ALL cell line; and 3) RPMI 8226, an uncloned myeloma line (B-cell lineage)
Summary
Glucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. Malignant cells may preempt the natural epigenetic mechanism of cytosine methylation to alter expression of genes so as to escape anti-cancer therapy. Disruption of the normal DNA methylation patterns can result in an aberrant epigenetic state with substantial consequences, such as repression of tumor suppressors, leaving cells susceptible to oncogenetic transformation [1,2,3,4,5,6,7]. Repression of gene expression based on DNA methylation can be reversed by analogs of cytosine such as 5 aza-2’ deoxycytidine (AZA), which incorporate into the genome of actively dividing cells and can render the cytosine nucleotide sites which they replace incapable of methylation [1,3,6,8,12]. AZA alone shows promising activity against acute myeloid leukemia and myelodysplastic syndromes [1,3,12,13]
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