title: Epigenetic alteration by DNA-demethylating treatment restores apoptotic response to glucocorticoids in dexamethasone-resistant human malignant lymphoid cells.
abstract: BACKGROUND
  Glucocorticoids (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.
  
  
  METHODS
  We 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.
  
  
  CONCLUSIONS
  Treatment 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.
authors.affiliation: Department of Biochemistry & Molecular Biology, (ALM present address, Department. of Pediatrics, & Assay Devel. Service Division Galveston National Lab.), University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Aaron L
authors.initials: AL
authors.lastname: Miller
authors.affiliation: Department of Biochemistry & Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, LA CG present address, Depts. of Medicine and of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Chuandong
authors.initials: C
authors.lastname: Geng
authors.affiliation: Department of Biology & Biochemistry, Centers for Biomedical & Environmental Genomics and/or Nuclear Receptors & Cell Signaling, University of Houston, Houston, TX, USA ; Department of Pharmacology & Toxicology, and Sealy Center for Structural Biology & Molecular Biophysics, Univ. of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Georgiy
authors.initials: G
authors.lastname: Golovko
authors.affiliation: Department of Biology & Biochemistry, Centers for Biomedical & Environmental Genomics and/or Nuclear Receptors & Cell Signaling, University of Houston, Houston, TX, USA.
authors.forename: Meenakshi
authors.initials: M
authors.lastname: Sharma
authors.affiliation: Department of Biochemistry & Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, LA CG present address, Depts. of Medicine and of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX, USA ; Present address St. Jude Children's Hospital, Memphis, TN, USA.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Schwartz
authors.affiliation: Department of Pharmacology & Toxicology, and Sealy Center for Structural Biology & Molecular Biophysics, Univ. of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Jiabin
authors.initials: J
authors.lastname: Yan
authors.affiliation: Department of Pharmacology & Toxicology, and Sealy Center for Structural Biology & Molecular Biophysics, Univ. of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Sowers
authors.affiliation: Department of Biology & Biochemistry, Centers for Biomedical & Environmental Genomics and/or Nuclear Receptors & Cell Signaling, University of Houston, Houston, TX, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Widger
authors.affiliation: Department of Biology & Biochemistry, Centers for Biomedical & Environmental Genomics and/or Nuclear Receptors & Cell Signaling, University of Houston, Houston, TX, USA ; Department of Pharmacology & Toxicology, and Sealy Center for Structural Biology & Molecular Biophysics, Univ. of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Yuriy
authors.initials: Y
authors.lastname: Fofanov
authors.affiliation: Department of Biochemistry & Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, LA CG present address, Depts. of Medicine and of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Wayne V
authors.initials: WV
authors.lastname: Vedeckis
authors.affiliation: Department of Biochemistry & Molecular Biology, (ALM present address, Department. of Pediatrics, & Assay Devel. Service Division Galveston National Lab.), University of Texas Medical Branch, Galveston, TX, USA ; Department of Biology & Biochemistry, Centers for Biomedical & Environmental Genomics and/or Nuclear Receptors & Cell Signaling, University of Houston, Houston, TX, USA.
authors.forename: E Brad
authors.initials: EB
authors.lastname: Thompson
keywords: 5 Aza-2’ deoxycytidine; Apoptosis; CEM; Dexamethasone; Epigenetic; Glucocorticoid; Glucocorticoid receptor; High throughput sequencing; Leukemia; Lymphoid; Methylation; Molt-4; Myeloma; Phosphorylation; RPMI 8226; p38