siDNMT1 increases γ-globin expression in chemical inducer of dimerization (CID)-dependent mouse ßYAC bone marrow cells and in baboon erythroid progenitor cell cultures.

OBJECTIVE: These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult-stage erythroid cells. MATERIALS AND METHODS: DNMT1 levels were reduced by nucleofection of small interfering RNA targeting DNMT1 in chemical inducer of dimerization-dependent multipotential mouse bone marrow cells containing the human ß-globin gene locus in the context of a yeast artificial chromosome and in primary cultures of erythroid progenitor cells derived from CD34(+) baboon bone marrow cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real-time polymerase chain reaction and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabeling of globin chains followed by high-performance liquid chromatography analysis. The effect on DNA methylation was determined by bisulfite sequence analysis. RESULTS: Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin messenger RNA, an increased γ/γ+ß chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor. CONCLUSIONS: DNMT1 is required to maintain DNA methylation of the γ-globin gene promoter and repress γ-globin gene expression in adult-stage erythroid cells.

S110, a novel decitabine dinucleotide, increases fetal hemoglobin levels in baboons (P. anubis).

BACKGROUND: S110 is a novel dinucleoside analog that could have advantages over existing DNA methyltransferase (DNMT) inhibitors such as decitabine. A potential therapeutic role for S110 is to increase fetal hemoglobin (HbF) levels to treat ß-hemoglobinopathies. In these experiments the effect of S110 on HbF levels in baboons and its ability to reduce DNA methylation of the γ-globin gene promoter in vivo were evaluated. METHODS: The effect of S110 on HbF and γ-globin promoter DNA methylation was examined in cultured human erythroid progenitors and in vivo in the baboon pre-clinical model. S110 pharmacokinetics was also examined in the baboon model. RESULTS: S110 increased HbF and reduced DNA methylation of the γ-globin promoter in human erythroid progenitors and in baboons when administered subcutaneously. Pharmacokinetic analysis was consistent with rapid conversion of S110 into the deoxycytosine analog decitabine that binds and depletes DNA. CONCLUSION: S110 is rapidly converted into decitabine, hypomethylates DNA, and induces HbF in cultured human erythroid progenitors and the baboon pre-clinical model.

Decitabine increases fetal hemoglobin in Papio anubis by increasing γ-globin gene transcription.

OBJECTIVE: The mechanism responsible for increased fetal hemoglobin levels following decitabine treatment remains controversial. These experiments were performed to evaluate the role of transcriptional vs. translational mechanisms in the ability of decitabine to increase fetal hemoglobin levels in vivo. MATERIALS AND METHODS: Three normal, nonanemic baboons were treated with decitabine subcutaneously (0.5 mg/kg/d) for 10 days. The effect of decitabine on globin chain synthesis and globin messenger RNA levels was measured in pre- and posttreatment bone marrow aspirates by biosynthetic radiolabeling with [(3)H] leucine followed by separation of globin chains by high-performance liquid chromatography, and real-time polymerase chain reaction, respectively. The effect on DNA methylation of the ɛ- and γ-globin gene promoters was determined by bisulfite sequence analysis. RESULTS: Decitabine treatment of normal, nonanemic baboons induced similar increases in the γ/γ+ß chain synthetic ratio and the γ/total ß-like globin RNA ratio and also increased expression of ɛ-globin transcripts. Increased expression of ɛ- and γ-globin was associated with decreased DNA methylation of the ɛ- and γ-globin gene promoters. CONCLUSIONS: Decitabine increases fetal hemoglobin in vivo by transcriptional activation of the γ-globin gene.

Transcriptional activation of the gamma-globin gene in baboons treated with decitabine and in cultured erythroid progenitor cells involves different mechanisms.

OBJECTIVE: To investigate the mechanism(s) responsible for increased gamma-globin expression in vivo in decitabine-treated baboons and in vitro in cultured erythroid progenitor cells (EPC) from adult baboon bone marrow (BM). MATERIALS AND METHODS: Fetal liver, adult BM erythroid cells pre- and post-decitabine, and cultured EPCs were analyzed for distribution of RNA polymerase II, histone acetylation, and histone H3 (lys4) trimethyl throughout the gamma-globin gene complex by chromatin immunoprecipitation. DNA methylation of the gamma-globin promoter was determined by bisulfite sequencing. Expression of the baboon Igamma- and Vgamma-globin chains was determined by high performance liquid chromatography (HPLC). Expression of BCL11A, a recently identified repressor of gamma-globin expression, was analyzed by Western blot. RESULTS: Increased gamma-globin expression in decitabine-treated baboons and cultured EPC correlated with increased levels of RNA polymerase II, histone acetylation, and histone H3 (lys4) trimethyl associated with the gamma-globin gene consistent with a transcriptional activation mechanism. Cultured EPC expressed the Igamma- and Vgamma-globin chains in a pattern characteristic of fetal development. The level of DNA methylation of the gamma-globin gene promoter in EPC cultures was similar to BM erythroid cells from normal adult baboons. Different BCL11A isoforms were observed in BM erythroid cells and cultured EPC. CONCLUSION: The mechanism responsible for increased gamma-globin expression in cultured EPC was unexpectedly not associated with increased DNA hypomethylation of the gamma-globin gene promoter compared to normal BM erythroid cells, in contrast to BM erythroid cells of decitabine-treated baboons. Rather, increased fetal hemoglobin in EPC cultures was associated with a fetal Igamma/Vgamma chain ratio and a difference in the size of the BCL11A protein compared to normal BM erythroid cells.

Antibody microarray-based profiling of complex specimens: systematic evaluation of labeling strategies.

Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined in this study the suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis. N-hydroxysuccinimidyl ester (NHS)- and Universal Linkage System (ULS)-based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labeling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labeling were analyzed and optimized in this study. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labeling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios. Finally, particularly sensitive detection chemistry was applied to monitoring cytokine production of stimulated peripheral T cells. Microarray data were in accord with quantitative cytokine levels measured by ELISA and Luminex, demonstrating comparable reliability and femtomolar range sensitivity of the established microarray approach.