trichostatin-a and beta-Thalassemia

Derivation of embryonic stem cells from patient-specific cloned blastocysts by somatic cell nuclear transfer (SCNT) holds promise for both regenerative medicine and cell-based drug discovery. However, the efficiency of blastocyst formation after human SCNT is very low. The developmental competence of SCNT embryos has been previously demonstrated in several species to be enhanced by treatment with histone deacetylase inhibitors, such as trichostatin A (TSA), to increase histone acetylation. In this study, we report that treatment of SCNT embryos with 5  nM TSA for 10  h following activation incubation increased the developmental competence of human SCNT embryos constructed from β-thalassemia fibroblast cells. The efficiency of blastocyst formation from SCNT human embryos treated with TSA was approximately 2 times greater than that from untreated embryos. Cloned blastocysts were confirmed to be generated through SCNT by DNA and mitochondrial DNA fingerprinting analyses. Further, treatment of SCNT embryos with TSA improved the acetylation of histone H3 at lysine 9 in a manner similar to that observed in in vitro fertilized embryos.

Topics: Acetylation; Base Sequence; beta-Thalassemia; Blastocyst; Blastomeres; Cell Shape; Cells, Cultured; Cloning, Organism; Coculture Techniques; DNA, Mitochondrial; Female; Fibroblasts; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Karyotype; Male; Microsatellite Repeats; Molecular Sequence Data; Nuclear Transfer Techniques; Sequence Analysis, DNA; Sperm Injections, Intracytoplasmic

The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.

Topics: Anemia, Sickle Cell; Animals; beta-Thalassemia; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Enzyme Inhibitors; Fetal Hemoglobin; Hemoglobin A; Hemoglobin, Sickle; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Adhesion Molecule-1; Iron Chelating Agents; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pulmonary Veins; Regional Blood Flow; Thromboplastin; Vascular Cell Adhesion Molecule-1; Venules; Vorinostat

In addition to conventional therapy, current treatment of thalassemia and sickle cell anemia includes inducers of hemoglobin F synthesis (hydroxyurea, erythropoietin, azacytidine and butyrate). However, because of concerns about the dose-limiting myelotoxicity, potential carcinogenicity and high cost of the above agents, an intensive search for less toxic or more effective drugs is ongoing. In this study we tested the effect of valproic acid and trichostatin, alone or in combination with hemin, on gamma chain synthesis in human erythroid liquid cultures.. The agents were tested on erythroid human liquid cultures derived from normal peripheral blood, peripheral blood from beta(s)/beta(thal) patients, normal cord blood and normal bone marrow samples. The effect of the agents was expressed as increase of gamma/gamma+beta m-RNA, measured with competitive reverse transcriptase-polymerase chain recation (RT-PCR), or as increase of HbF, measured by high performance liquid chromatography (HPLC).. Addition of valproic acid or trichostatin to human erythroid cell cultures preferentially enhanced gamma mRNA synthesis in all blood samples (2.9 to 3.5-fold). The addition of hemin enhanced the effect up to 10-fold.. Valproic acid, trichostatin and their combination with hemin (all three FDA-approved drugs) preferentially increase gamma-globin chain synthesis and may be helpful for the treatment of hemoglobinopathies.

Topics: Antifungal Agents; beta-Thalassemia; Cells, Cultured; Drug Interactions; Erythroid Precursor Cells; GABA Agents; Gene Expression; Globins; Hemin; Humans; Hydroxamic Acids; Valproic Acid