sodium-oxybate and Leukemia--Erythroblastic--Acute

Ghrelin, a ligand of the GH secretagogue receptor (GHS-R 1a), is a 28-amino acid peptide with an unusual octanoyl group on Ser3, crucial for its biological activity. For the first time, ghrelin and GHS-R 1b, a truncated variant of the receptor resulting from alternative splicing, but not GHS-R 1a, mRNAs were detected in the human erythroleukemic cell line HEL. Two antibodies, used for RIA, were directed against octanoylated and total (octanoylated and desoctanoylated) ghrelin, and the recognized epitopes were characterized. Using reverse phase HPLC analysis followed by RIA, we demonstrated that octanoylated and desoctanoylated ghrelins were present in HEL cells and their culture medium, of which more than 90% was octanoylated. The ghrelin levels were not affected after 24 h treatment with sodium butyrate, phorbol 12-myristate 13-acetate, or forskolin, but a significant 3-fold increase in desoctanoylated ghrelin was detected in the culture medium after 48 h treatment with sodium butyrate. The antighrelin SB801 and SB969 antisera inhibited HEL cell proliferation by 24% and 39%, respectively, after 72 h. Taken together, these data suggested that endogenous ghrelin stimulated HEL cell proliferation by an autocrine pathway involving an unidentified receptor, distinct from GHS-R1a, and that the HEL cell line represents a unique model to study the octanoylation of ghrelin.

Topics: Alternative Splicing; Amino Acid Sequence; Antibodies; Binding, Competitive; Butyrylcholinesterase; Carboxylic Ester Hydrolases; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Culture Media; Dose-Response Relationship, Drug; Ghrelin; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Peptide Hormones; Peptides; Radioimmunoassay; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Oxybate; Time Factors

Posttranslational modification of histones is a common means of regulating chromatin structure and thus diverse nuclear processes. Using a hydrophilic interaction liquid chromatographic separation method in combination with mass spectrometric analysis, the present study investigated the alterations in histone H4 methylation/acetylation status and the interplay between H4 methylation and acetylation during in vitro differentiation of mouse erythroleukemia cells and how these modifications affect the chromatin structure. Independently of the type of inducer used (dimethyl sulfoxide, hexamethylenebisacetamide, butyrate, and trichostatin A), we observed a strong increase in non- and monoacetylated H4 lysine 20 (H4-Lys(20)) trimethylation. An increase in H4-Lys(20) trimethylation, however, to a clearly lesser extent, was also found when cells accumulated in the stationary phase. Since we show that trimethylated H4-Lys(20) is localized to heterochromatin, the increase in H4-Lys(20) trimethylation observed indicates an accumulation of chromatin-dense and transcriptionally silent regions during differentiation and during the accumulation of control cells in the stationary phase, respectively. When using the deacetylase inhibitors butyrate or trichostatin A, we found that H4 hyperacetylation prevents H4-Lys(20) trimethylation, but not mono- or dimethylation, and that the nonacetylated unmethylated H4-Lys(20) is therefore the most suitable substrate for H4-Lys(20) trimethylase. Summarizing, histone H4-Lys(20) hypotrimethylation correlates with H4 hyperacetylation and H4-Lys(20) hypertrimethylation correlates with H4 hypoacetylation. The results provide a model for how transcriptionally active euchromatin might be converted to the compacted, transcriptionally silent heterochromatin.

Topics: Acetamides; Acetylation; Amino Acid Sequence; Animals; Antineoplastic Agents; Blotting, Western; Butyrates; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Chromatin; Chromatography, High Pressure Liquid; Chromatography, Liquid; Dimethyl Sulfoxide; Enzyme Inhibitors; Heterochromatin; Histones; Hydroxamic Acids; Leukemia, Erythroblastic, Acute; Lysine; Mass Spectrometry; Metalloendopeptidases; Methylation; Mice; Microscopy, Fluorescence; Molecular Sequence Data; Peptides; Protein Processing, Post-Translational; Sodium Oxybate; Time Factors; Transcription, Genetic