| Kinase Assay: |
| Cancer Res, 2002, 62(17), 4916-4921. | | FK228 (depsipeptide) as a natural prodrug that inhibits class I histone deacetylases.[Pubmed: 12208741] | | For the enzyme assay, 10 μL of [3H]acetyl-labeled histones (25,000 cpm/10 μg) are added to 90 μL of the HDAC enzyme fraction extracted from 293T cells overexpressing HDAC1 or HDAC2 in the presence of increasing concentrations of Romidepsin, and the mixture is incubated at 37 °C for 15 minutes. The enzyme reaction is linear for at least 1 hour. The reaction is stopped by the addition of 10 μL of concentrated HCl. The released [3H]acetic acid is extracted with 1 mL of ethylacetate, and 0.9 mL of the solvent layer is taken into 5 mL of aqueous counting scintillant II solution for determination of radioactivity. The IC50 values are determined from at least three independent dose-response curves. |
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| Cell Research: |
| Mol Cancer Ther,2002 Sep;1(11):937-41. | | Histone deacetylase inhibitors all induce p21 but differentially cause tubulin acetylation, mitotic arrest, and cytotoxicity.[Pubmed: 12481415] | Cell lines:HL60, Jurkat, A549, and MCF-7
Concentrations: Dissolved in DMSO, final concentrations ~10 μM
Incubation Time: 72 hours
Method:
Cells are exposed to various concentrations of Romidepsin for 72 hours in 96-well plates. 20 μL of 5 mg/mL MTT solution in PBS is added to each well for 4 hours. After removal of the medium, 170 μL of DMSO is added to each well to dissolve the formazan crystals. The absorbance at 540 nm is determined. In addition, cells are incubated with trypan blue, and the numbers of blue (dead) cells and transparent (live) cells are counted in a hemocytometer. For cell cycle analysis, cells are incubated for 30 minutes in propidium iodide staining solution containing 0.05 mg/mL propidium iodide, 1 mM EDTA, 0.1% Triton X-100, and 1 mg/mL RNase A in PBS. The suspension is then passed through a nylon mesh filter and analyzed on a Becton Dickinson FACScan.
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| Animal Research: |
| Biochem Pharmacol, 2002 Oct 1;64(7):1079-90. | | Effects of FK228, a novel histone deacetylase inhibitor, on human lymphoma U-937 cells in vitro and in vivo.[Pubmed: 12234611] | Animal Models: Male scid mice inoculated i.p. with U-937 cells
Dosages:~1 mg/kg once or twice a week
Administration: Treated i.p.
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