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was added to each well and the plates were reincubated for
3 h. Absorbance was then measured on a 96-well plate
reader at 490 nm. The IC50 values for HDAC inhibitors
were determined by performing dose–response curves with
individual compounds and determining the concentration
of inhibitor producing 50% decrease in maximal signal
(diluent control). For other examples of the use of
HCT116 cells in the evaluation of HDAC inhibitors, see
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15. HDAC inhibitors were screened against recombinant
human HDAC1 produced in Hi5 insect cells. The enzyme
was cloned with a FLAG tag at the C-terminal of the gene
and affinity purified using Anti-FLAG M2 agarose. The
deacetylase assays were carried out in a 50 lL reaction.
HDAC1 (75 ng of enzyme) diluted in 15 lL of reaction
buffer (25 mM Tris–HCl (pH 8), 137 mM NaCl, 2.7 mM
KCl, 1 mM MgCl2) was mixed with either buffer alone
(10 lL) or buffer containing compound (10 lL) for 30 min
at ambient temperature. The reaction was initiated by
addition of 25 lL of acetylated histone H4 peptide
(25 lM) (KI 174 Biomol) diluted in buffer and incubated
for one hour at ambient temperature. The remaining
protocol is as for the pooled enzyme (vide supra).
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12. In vitro enzyme assay of pooled histone deacetylases from
nuclear extracts prepared from the human cervical cancer
cell line HeLa: assays were carried out in a 40 lL reaction.
2.5 lg of nuclear extract diluted in 15 lL of reaction buffer
(25 mM Tris–HCl (pH 8), 137 mM NaCl, 2.7 mM KCl,
1 mM MgCl2) was mixed with either buffer alone (5 lL) or
buffer containing compound (5 lL) for 30 min at ambient
temperature. 25 lM of fluor de lys substrate (Biomol)
diluted in 20 lL of buffer was then added to initiate the
reaction and incubated for 1 h at ambient temperature.
The reaction was stopped by addition of an equal volume
(40 lL) fluor de lys developer (Biomol) containing
trichostatin A at 2 lM. The reaction was allowed to
develop for 30 min at ambient temperature and then
fluorescence measured at an excitation wavelength of
360 nm and an emission wavelength of 465 nm. IC50
values for HDAC enzyme inhibitors were determined by
performing dose–response curves with individual com-
pounds and determining the concentration of inhibitor
producing 50% decrease in the maximal signal (no
inhibitor control).
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20. (DMSO-d6) 1.13 (t, 3H, CH3CH2), 1.73 (m, 4H, piperidine
3-CH2), 2.10 (t, 2H, piperidine 2-axial CH), 2.60 (m, 1H,
piperidine 4-CH), 2.94 (d, 2H, piperidine 2-equiv CH), 3.28
(m, 2H, CH3CH2) 3.55 (s, 2H, phenyl-CH2-N), 4.85 (s, 2H,
NH2), 6.59 (m, 1H, CONHCHCH), 6.77 (d, 1H, NH2CH),
6.96 (m, 1H, NH2CHCH), 7.17 (d, 1H, CONHCH), 7.39
(m, 4H, piperidine-phenyl o-CH and ethylbenzamide
m-CH), 7.80 (d, 2H, ethylbenzamide o-CH), 7.90 (d, 2H,
piperidine-phenyl m-CH), 8.38 (t, 1H, CH3CH2NH), 9.55
(s, 1H, CONH); mass spectrum: M+H+ 457.
13. HCT116 cells are adherent epithelial human colon carci-
noma cell lines. Inhibition of proliferation in whole cells
was assayed using the Promega cell titer 96 aqueous
proliferation assay (Promega #G5421). The HCT116 cells
were seeded in 96-well plates at 1 · 103 cells/well, and
allowed to adhere overnight. They were treated with
inhibitors for 72 h. The 20 lL of the tetrazolium dye MTS