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A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces

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Leukemia (2008) 22, 1026–1034
& 2008 Nature Publishing Group All rights reserved 0887-6924/08 $30.00
www.nature.com/leu
ORIGINAL ARTICLE
A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis
in T-cell lymphomas
S Balasubramanian1, J Ramos1, W Luo2, M Sirisawad1, E Verner2 and JJ Buggy1
1
Department of Cancer Biology, Pharmacyclics Inc., Sunnyvale, CA, USA and 2Department of Chemistry, Pharmacyclics Inc.,
Sunnyvale, CA, USA
We have developed a potent, histone deacetylase 8 (HDAC8)-
specific inhibitor PCI-34051 with 4200-fold selectivity over the
other HDAC isoforms. PCI-34051 induces caspase-dependent
apoptosis in cell lines derived from T-cell lymphomas or
leukemias, but not in other hematopoietic or solid tumor lines.
Unlike broad-spectrum HDAC inhibitors, PCI-34051 does not
cause detectable histone or tubulin acetylation. Cells defective
in T-cell receptor signaling were still sensitive to PCI-34051-
induced apoptosis, whereas a phospholipase C-c1 (PLCc1)-
defective line was resistant. Jurkat cells showed a dose-
dependent decrease in PCI-34051-induced apoptosis upon
treatment with a PLC inhibitor U73122, but not with an inactive
analog. We found that rapid intracellular calcium mobilization
from endoplasmic reticulum (ER) and later cytochrome c
release from mitochondria are essential for the apoptotic
mechanism. The rapid Ca2 þ flux was dependent on PCI-34051
concentration, and was blocked by the PLC inhibitor U73122.
Further, apoptosis was blocked by Ca2 þ chelators (BAPTA) and
enhanced by Ca2 þ effectors (thapsigargin), supporting this
model. These studies show that HDAC8-selective inhibitors
have a unique mechanism of action involving PLCc1 activation
and calcium-induced apoptosis, and could offer benefits
including a greater therapeutic index for treating T-cell
malignancies.
Leukemia (2008) 22, 1026–1034; doi:10.1038/leu.2008.9;
published online 7 February 2008
Keywords: HDAC isoform-specific inhibitors; protein acetylation;
phospholipase C-g1; calcium-induced apoptosis; T-cell signaling
Introduction
Histone deacetylase (HDAC) enzymes represent a family of
proteins with complex, multifunctional roles in vivo, including
transcriptional regulation, regulation of tubulin and cytoskeletal
function, control of cardiac growth, regulation of thymocyte
development and facilitation of DNA repair.1 HDAC enzymes
function in part to control the acetylation state of nucleosomal
histones, thereby regulating transcription, but more recently, it
has been shown that there are many non-histone acetylation
targets as well, including tubulin, heat-shock proteins and
a variety of transcription factors, such as p53 and NF-kB
subunit p65.2,3 There are 11 known isoforms of the classic
Zn2 þ -dependent HDAC family, denoted HDAC 1–11.1,4 In the
past several years, broad-spectrum inhibitors of these HDAC
enzymes have been developed and have shown potential for the
treatment of cancer, particularly hematologic tumors. One such
Correspondence: Dr S Balasubramanian, Department of Cancer
Biology, Pharmacyclics Inc., 995 E. Arques Avenue, Sunnyvale, CA
94085, USA.
E-mail: sriram@pcyc.com
Received 9 October 2007; revised 20 December 2007; accepted 3
January 2008; published online 7 February 2008
broad-spectrum HDAC inhibitor is vorinostat, a hydroxamic
acid-based inhibitor, which was recently approved to treat
patients with cutaneous T-cell lymphoma.5,6 Several other
broad-spectrum inhibitors, including LBH589, PXD101 and
PCI-24781, are currently in clinical trials for various cancer
indications, including both solid and hematologic malignan-
cies.7–10 Although promising, these compounds inhibit several
HDAC isoforms and disrupt multiple cellular processes that
depend on protein acetylation, many of which may not be
involved in the maintenance of tumor progression. Isoform-
selective inhibitors offer the ability to alter distinct pathways,
which are more specifically involved in the tumor phenotype,
and could therefore provide a wider therapeutic index
compared with the compounds currently in the clinic. A specific
inhibitor of the class II isoform HDAC6 has been developed,11,12
and similar attempts have also been made to develop class I
isoform-specific inhibitors, particularly HDAC8.13,14 However,
several factors, including the similarity between their catalytic
sites, the difficulty in obtaining purified active proteins and until
recently, lack of X-ray crystal structures, have hampered the
search for potent isoform-specific inhibitors, and this in turn
has slowed down the progress in delineating the biology of the
individual HDAC isoforms in cancer.15,16
Knockdown experiments of selective HDAC isoforms have
revealed that HDAC8 is essential for cell survival.17 HDAC8 is
a 49 kDa HDAC isoform with deacetylase activity in vitro that is
expressed in multiple tissue types and tumor cell lines.18 On the
basis of sequence homology, HDAC8 is considered to be a class
I enzyme, although phylogenetic analysis has shown it to lie
near the boundary of the class I and class II enzymes.19 HDAC8
is different from the prototypical class I enzyme in several
respects, including its reported cytoplasmicFas opposed to
nuclearFsubcellular localization, the binding of various metals
including Fe(II) and K þ to its active site, and the negative
regulation of its catalytic activity by phosphorylation of Ser39 by
cyclic-AMP-dependent protein kinase (PKA).20–22 The three-
dimensional crystal structure of human HDAC8 was recently
solved by two independent groups.17,23 The structure of HDAC8
not only led to a firmer understanding of how catalysis occurs
within the HDAC family of enzymes but also revealed unique
features of HDAC8, including conformational flexibility prox-
imal to the binding site pocket mediated by the L1 active site
loop and a unique influence of Ser39 phosphorylation on active
site inhibition. All of these observations were integrated into our
screen for novel HDAC8-selective inhibitors that led to the
present work.
Here, we describe the characterization of a novel HDAC8-
selective inhibitor, PCI-34051. PCI-34051 inhibits pure recom-
binant HDAC8 with a Ki of 10 nM with 4200-fold selectivity
over the other HDACs tested, including HDACs 1, 2, 3, 6 and
10. Importantly, PCI-34051 was found to induce apoptosis at

low micromolar concentrations in cell lines derived from T-cell
lymphomas, including Jurkat and HuT78, whereas doses as high
as 20 mM had no effect on B-cell- or myeloid-derived lympho-
mas or solid tumor lines. Investigation of PCI-34051-induced
apoptosis revealed the involvement of PLCg1-induced calcium
mobilization and led to the discovery of a mechanism of action
unique to the HDAC field.
Materials and methods
Cell lines and reagents
Cell lines were obtained from DSMZ (38124 Braunschweig,
Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH, Germany) or ATCC (Manassas, VA, USA). Cells were
grown in RPMI 1640 with 10% fetal bovine serum in a 5% CO2/air
incubator at 37 1C. Thapsigargin and BAPTA-AM were from
Calbiochem (San Diego, CA, USA), phospholipase C inhibitor
U73122 and its inactive analog U73343 were from BioSource
International (Camarillo, CA, USA), caspase inhibitor quinoline-
valine-aspartic-CH2-O-Phenyl (QVD) was from MP Biomedicals
(Solon, OH, USA) and Fas ligand (FasL) was from Upstate (Lake
Placid, NY, USA). Anti-HDAC8, anti-cytochrome c and anti-Hsc70
antibodies were from Santa Cruz Biotechnology nos.sc-11405,
sc-13156 and sc-7298, respectively (Santa Cruz, CA, USA).
Anti-cytochrome c oxidase subunit II (anti-COXII,no.A-6404) and
anti-mouse-AlexaFluor680 were from Molecular Probes (Eugene,
OR, USA) and anti-rabbit secondary antibodies conjugated to
IRdye800 was from Rockland Immunochemicals (Gilbertsville, PA,
USA). PCI-24781 is a broad-spectrum HDAC inhibitor, which was
synthesized as previously described.24 PCI-34051 is a specific
inhibitor of HDAC8, the synthesis and structure of which has
been described.25 Both compounds were determined to be greater
than 99.5% pure by HPLC and dissolved in dimethyl sulfoxide to
obtain stock solutions of 10 mM, which were subsequently diluted
into aqueous buffers.
Histone deacetylase activity
Histone deacetylase activity was measured using a continuous
trypsin-coupled assay, which has been described in detail
previously.26 For inhibitor characterization, measurements were
performed in a reaction volume of 100 ml1 using 96-well assay
plates in a fluorescence plate reader. For each isozyme, the
HDAC protein in reaction buffer (50 mM HEPES, 100 mM KCl,
0.001% Tween-20, 5% dimethyl sulfoxide , pH 7.4, supplemen-
ted with bovine serum albumin at concentrations of 0–0.05%)
was mixed with inhibitor at various concentrations and allowed
to incubate for 15 min. Trypsin was added to a final concentration
of 50 nM, and acetyl-Gly-Ala-(N-acetyl-Lys)-amino-4-methylcou-
marin was added to a final concentration of 25–100 mM to initiate
the reaction. After a 30 min lag time, the fluorescence was
measured over a 30 min time frame using an excitation
wavelength of 355 nm and a detection wavelength of 460 nm.
The increase in fluorescence with time was used as the measure
of the reaction rate. Inhibition constants Ki(app) were obtained
using the program BatchKi (Biokin, Pullman, WA, USA).
Cell proliferation assay
Tumor cell lines and human umbilical vein endothelial cells
were cultured for at least two doubling times, and growth was
monitored at the end of compound exposure using an Alamar
Blue (Biosource) fluorometric cell proliferation assay as recom-
mended by the manufacturer. Compounds were assayed in
HDAC8-specific inhibitor PCI-34051 in T-cell lymphoma
S Balasubramanian et al
1027
triplicate wells in 96-well plates. The concentration required
to inhibit cell growth by 50% (GI50) and 95% confidence
intervals were estimated from nonlinear regression using a four-
parameter logistic equation.
Western blotting
Cells were washed with phosphate-buffered saline and resus-
pended in triple-detergent lysis buffer (50 mM Tris-Cl pH 8.0,
150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1.0% NP-40,
supplemented with 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4,
2 mM b-glycerophosphate and the Complete protease inhibitor
cocktail (Roche Molecular Biochemicals, Indianapolis, IN,
USA)) on ice for 10 min. After centrifugation, equal quantities
of protein were resolved on SDS-polyacrylamide gels (Bio-Rad
Laboratories, Hercules, CA, USA). Gels were transferred to
polyvinylidene difluoride membrane using a semi-dry Transfer
Cell (Bio-Rad Laboratories) and western blotted, using an anti-
Hsc70 antibody to control for loading and transfer. Bands were
imaged and quantified in the linear range and normalized to
Hsc70, using the Odyssey Infrared Imaging System (LICOR,
Lincoln, NE, USA).
Apoptosis assays
Cytotoxicity was evaluated after 2 or 3 days of treatment with
PCI-34051 alone and in combination with QVD, BAPTA-AM,
thapsigargin and phospholipase C inhibitor (as described in the
figure legends) using Annexin-V staining. Annexin-V binding27
was assayed with a FACSCalibur instrument (Becton-Dickinson,
San Jose, CA, USA) using reagents from BioVision (Mountain
View, CA, USA) as per manufacturer’s protocol.
Caspase activation assays
Caspase enzyme activity was measured in Jurkat cells using
the Apotarget Caspase Colorimetric Protease Assay (BioSource)
as per manufacturer’s protocol following treatment with
PCI-34051.
Intracellular calcium measurements
For the spectrofluorimetric measurements, cells (1  106
cells ml1) were incubated for 1 h in Hanks’ balanced salt
solution (HBSS; Invitrogen, Carlsbad, CA, USA) containing 10%
fetal bovine serum and 5 mM Indo1-AM (Invitrogen) at 37 1C in
the dark. Cells were then harvested, centrifuged (200g for 5 min)
and washed three times with HBSS to remove extracellular
Indo1, and readjusted to 1  106 cells ml1 in HBSS. Fluores-
cence was monitored throughout each experiment at 37 1C with
a fluorescent plate reader (Fluoroskan Ascent FL; Thermo
Scientific, Waltham, MA, USA). After a 5 min temperature
equilibration period, samples were excited at 338 nm and
emission was collected at 405 and 485 nm, corresponding to
the Ca2 þ -bound and Ca2 þ -free Indo1 fluorescence emitted,
respectively, at 6-s intervals over a 1-min period. Drug (or
control) was then added, and acquisition was continued for
5 min. Maximal ratio values were determined by the addition of
10 mM ionomycin at the end of the measurements. Intracellular
(Ca2 þ ) changes are shown as changes in the ratio of Ca2 þ -
bound and Ca2 þ -free Indo1.
Cytochrome c release
Jurkat and J.gamma1 cells were harvested at 12 and 24 h after
the drug treatments. Harvested cells were pelleted and washed
Leukemia
 HDAC8-specific inhibitor PCI-34051 in T-cell lymphoma
S Balasubramanian et al
1028
with cold phosphate-buffered saline. The washed cell pellets
were then lysed and mitochondria were isolated according to
the manufacturer’s protocol using the Mitochondria Isolation Kit
for cultured cells (Pierce, Rockford, IL, USA). The subcellular
fractions were then boiled in Laemmli sample buffer and
subjected to western blotting for cytochrome c and cytochrome
c oxidase, as outlined above.
Results
PCI-34051 is a potent and selective inhibitor of HDAC8
The HDAC8 inhibitor PCI-34051 was derived from a low
molecular weight hydroxamic acid scaffold that possessed
promising potency (HDAC8; Ki ¼ 2 mM) and selectivity (approxi-
mately fivefold) for HDAC8 relative to the other class I HDACs.
Modifications to the core scaffold resulted in the potent inhibitor
PCI-34051 (HDAC8 Ki ¼ 10 nM), which was chosen for further
preclinical development. Table 1 compares the inhibition
constants for PCI-34051 with the broad-spectrum inhibitors
PCI-24781 and vorinostat (SAHA) for each HDAC isoform
tested. PCI-34051 showed greater than 200-fold selectivity over
HDAC1 and HDAC6, and greater than 1000-fold selectivity
over HDAC2, HDAC3 and HDAC10.
HDAC8-inhibitor specifically induces cell death in
T-cell-derived lines
To determine if HDAC8 inhibition by PCI-34051 affects the
growth and viability of tumor cells, seven solid tumor cell lines
were treated with various concentrations of inhibitor for 72 h. As
shown in Table 2, most of the solid tumor lines did not show cell
death (as measured by Annexin-V flow cytometry) and minimal
growth inhibition by PCI-34051, while the pan-HDAC inhibitor
PCI-24781 induces cell death and inhibits the growth of all these
Table 1 PCI-34051 is 4200-fold selective for HDAC8
IC50 (mM)
Compound HDAC1 HDAC2 HDAC3 HDAC6
PCI-24781 0.005 0.019 0.008 0.017
SAHA 0.028 0.06 0.044 0.022
PCI-34051 4 450 450 2.9
Abbreviation: HDAC, histone deacetylase.
In vitro inhibition constants for the pan-HDAC inhibitors PCI-24781 and SAHA compared to PCI-34051.
Leukemia
lines. This result suggests that inhibition of HDAC8 contributes
little, if any, to the antitumor activity of pan-HDAC inhibitors in
solid tumor cell lines, with the possible exception of the ovarian
tumor line OVCAR-3, where PCI-34051 showed a GI50 of 6 mM
and 15% cell death. In contrast, analysis of hematologic tumor
cell lines revealed that PCI-34051 induced growth inhibition
(GI50 ¼ 2.4–4 mM) and significant cell death following 72 h of
treatment in four tumor T-cell-derived lines, Jurkat (derived from
a T-cell leukemia), HuT78 (derived from peripheral T-cell
lymphoma), HSB-2 and Molt-4 (both derived from T-ALL)
(Figure 1 and Supplementary Table 1). No cell death was
observed in other hematological tumors, such as those of B-cell
(Ramos, Raji, DHL-4, DB), monocytic (THP-1), myeloid (K562)
or myeloma (RPMI-8226) origins; further we did not observe cell
death or CD69 activation by PCI-34051 in normal resting or
activated (by aCD3/aCD28) T cells (data not shown). The
selective sensitivity of Jurkat and HuT78 cell lines to PCI-34051
could not be explained by differential protein expression levels,
since HDAC8 was expressed in each cell line tested at levels not
correlated with the cellular effects (Figure 2a). In contrast to the
pan-HDAC inhibitor PCI-24781, neither significant tubulin nor
histone acetylation was observed in the sensitive cell lines
treated with PCI-34051 at concentrations o25 mM at 24 h
(Figure 2b) nor at earlier timepoints (data not shown), suggesting
that the mechanism of action cannot be explained by non-
selective inhibition of other HDAC isoforms. Taken together,
these results show that PCI-34051 induces a selective cytotoxic
effect in cell lines derived only from T-cell malignancies.
PCI-34051 induces caspase-dependent apoptosis
To investigate if the cell death induced by PCI-34051 is
dependent on caspase activation, Jurkat cells were treated with
PCI-34051 at 5 mM for 2 days in the presence or absence of the
pancaspase inhibitor, QVD (Figure 3a). Cell death was almost
HDAC8 HDAC10 Structure
0.19 0.024
0.41 0.04
0.01 13

completely blocked by QVD, indicating that caspase cleavage is
essential to tumor cell kill by PCI-34051. Similarly, when
caspase-3 activity was measured at various times after treatment
with 5 mM PCI-34051, increasing levels of activity were observed
from 12 to 24 to 48 h (Figure 3b). PCI-34051 at 5 mM stimulates
poly(ADP-ribose) polymerase cleavage at 48 h, another hall-
mark of apoptosis, consistent with the higher levels of caspase
activity at this timepoint (Figure 3c). Interestingly, PCI-34051
does not stimulate Bid cleavage, a characteristic effect of the
extrinsic apoptotic pathway (Figure 3d), while PCI-24781 does,
as has been observed for other non-selective HDAC inhibi-
tors.28,29 These results suggest that caspase-induced apoptosis is
the major mechanism of HDAC8-inhibitor induced cell death.
Cell death in Jurkat-derived cell lines is dependent on
PLCg1 activity but does not require T-cell receptor
signaling
Since apoptosis by PCI-34051 was only observed in T-cell-
derived cell lines, we wished to determine whether the T-cell
receptor (TCR) signaling pathway is involved in this process. To
this end, Annexin-V binding was measured in several derivatives
Table 2 PCI-34051 is not cytotoxic to solid tumor cell lines unlike
the pan-inhibitor PCI-24781
HDAC8-specific inhibitor PCI-34051 in T-cell lymphoma
S Balasubramanian et al
1029
of the Jurkat cell line known to contain specific mutations in
various components of the TCR signaling cascade. These
included lines with mutations in the TCR itself (J.RT3-T.5, TCR
b-chain deficient),30 or in essential downstream signaling
components such as ZAP-70 (P116) or PLCg1 (J.gamma1).31,32
While P116 and J.RT3-T.5 were sensitive to PCI-34051
(Figure 4a) the PLCg1-deficient J.gamma1 line showed a marked
decrease in the extent of PCI-34051-induced apoptosis,
implying that PLCg1 activity is essential for this process.
PLCg1 plays a critical role in cellular responses to external
(growth factors, neurotransmitters) and internal (PI3K) stimuli in
T cells, hydrolyzing phosphatidylinositol-4,5-bisphosphate
(PIP2) to produce diacylglycerol (DAG) and inositol 1,4,5-
triphosphate (IP3); the latter binds to IP3 receptors on the
endoplasmic reticulum (ER), leading to an increase in intra-
cellular calcium flux.33 To verify that the enzymatic activity of
PLCg1 is essential to the mechanism of apoptosis induction by
PCI-34051, Jurkat and J.gamma1 cells were treated with the
drug in the presence or absence of the phospholipase C inhibitor
U-73122 or the inactive analog U73343.34–36 A dose-dependent
decrease in apoptosis was seen with increasing concentrations
of U-73122 in the Jurkat wild-type cells but not with the inactive
analog U-73343 (Figure 4b). As before, the PLCg1-deficient line
J.gamma1 showed minimal PCI-34051-induced apoptosis, and
this was unaffected by either U-73122 or U-73343. These results
show that the enzymatic activity of PLCg1 is required for
apoptosis induction by PCI-34051 in these cells.

PCI-34051 PCI-24781
PCI-34051 induces PLCg1-dependent calcium flux
Cell line GI50 (mM) Apoptotic GI50 (mM) Apoptotic
cells (%) cells (%) PLCg1-dependent calcium mobilization can result in the uptake
of calcium by mitochondria, in turn releasing cytochrome c into
A549 (lung) 19 0 0.18 47 the cytosol, ultimately leading to caspase activation and cell
HCT116 (colon) 420 3 0.2 70 death.37 To see if changes in calcium levels affect PCI-34051-
HeLa (cervix) 420 NA 0.2 NA induced apoptosis, Jurkat or J.gamma1 cells were treated with
U87 (glioma) 17 0 1.2 NA
thapsigargin (which induces calcium release from ER by binding
RKO (colon) 14 0 1.5 45
MCF-7 (breast) 420 0 0.15 37 to SERCA pumps) or BAPTA-AM (a cell-permeable calcium
Ovcar-3 6 15 0.16 55 chelator)38 followed by the addition of PCI-34051. Jurkat cells
(ovarian) treated with thapsigargin, at a concentration (0.2 mM) that
Abbreviation: NA, not available.
produces little apoptosis by itself in 24 h, showed an enhance-
Cell growth inhibition constants (assessed with Alamar Blue) and ment of PCI-34051-induced Annexin-V binding from approxi-
percentage of apoptosis (measured by Annexin-V flow cytometry) after mately 18% to over 30% of the cells (Figure 5a). Interestingly,
3 days of treatment. J.gamma1 cells are sensitive to thapsigargin, which induces

100
Annexin-V Positive Cells (%)

80 Control
PCI-34051 (5µM)

60

40

20

0
Jurkat HuT78 HSB-2 Molt-4 DHL-4 Ramos Raji DB K562
T-cell lines B-cell lines
Figure 1 The HDAC8-selective inhibitor PCI-34051 is cytotoxic to T-cell derived lines. The percentage of apoptosis measured by Annexin-V flow
cytometry after 2 days of dosing with 5 mM PCI-34051 is shown. Except for T-cell lines, all others tested exhibited very little apoptosis. HDAC8,
histone deacetylase 8.

Leukemia
 HDAC8-specific inhibitor PCI-34051 in T-cell lymphoma
S Balasubramanian et al
1030
a DHL-4 Jurkat HF-1 Ramos Raji
HDAC8
Hsc70
b PCI-24781 PCI-34051
0.2 0 3.13 6.25 12.5 25 50 100
β-tubulin
Acetylated
tubulin
Acetylated
histones
Figure 2 The cytotoxicity of PCI-34051 does not depend upon the
expression level of HDAC8 nor on the acetylation of tubulin or
histones after treatment. (a) HDAC8 protein expression assessed by
western blotting with a polyclonal anti-HDAC8 antibody in several
cell lines. (b) Jurkat cells were treated with PCI-34051 or PCI-24781
for 24 h at the concentrations indicated and western blotted with anti-
tubulin antibody (top) or anti-acetyl-lysine antibody (bottom). PCI-
34051 does not induce acetylation of either tubulin or histones in
Jurkat cells at o25 mM, whereas the pan-HDAC inhibitor PCI-24781
robustly acetylates both at 0.2 mM. HDAC8, histone deacetylase 8.
DB K562 apoptosis in approximately 12% of the cells, but there is only
a slight effect (2%) of added PCI-34051. These results indicate
that the added effect of PCI-34051 in Jurkat cells is likely due to
increased calcium flux because of the IP3 produced by PLCg1
activity, which is lacking in the J.gamma1 cells. Conversely, the
calcium chelator BAPTA-AM, at concentrations that did not
induce any cytotoxicity by itself, was able to produce a dose-
dependent reduction in PCI-34051-induced apoptosis at 24 h in
0 µM Jurkat cells to control levels, but had no effect in J.gamma1 cells
(Figure 5b). Taken together, these results suggest that steady-
state calcium levels strongly influence the apoptosis induced by
PCI-34051, and further, that the calcium-mobilizing activity of
PLCg1 has an important role in this process.
To determine whether HDAC8 inhibition does lead to the
activation of PLCg1-dependent Ca2 þ mobilization, Jurkat cells
were loaded with Indo-1 and treated with PCI-34051. A rapid
and dose-dependent increase in intracellular calcium levels was
observed at the same concentrations that lead to apoptosis
(Figure 6a). When Jurkat cells were treated with thapsigargin
alone, a similar dose–response was obtained (Figure 6b). The
maximum amplitude was approximately the same in both cases,
suggesting that the PCI-34051 induced calcium mobilization
primarily from intracellular stores.39,40 This conclusion was
supported by the increase in calcium levels with the addition of
ionomycin (as indicated by the second arrow) in both cases. In
J.gamma1 cells, this rapid calcium mobilization by PCI-34051
was completely abolished (Figure 6c). However, thapsigargin
could still mobilize calcium in these cells, which could also
respond to ionomycin as shown. PCI-34051-induced calcium

a 50 c
PCI-34051
40 kD
Control 24h 48h CPT
Positive Cells (%)

-150-
Annexin-V

30
PARP
-100-
20
Cleaved
PARP -75-
10
HSC70
-50-
0
Control QVD-OPh PCI-34051 QVD-OPh
+ PCI-34051
Treatment
b 8.0 d
Control
PCI-34051 (1µM)
6.0
Caspase Activity

PCI-34051 (5µM)
Normalized

PCI-34051 (25µM)
Cont 24781 34051 kD
4.0 -50-
Actin
-37-
2.0 -25-
FL-Bid
-20-
FLBid/Actin 14.6 3.1 14.6
0.0
12 Hr 24 Hr 48 Hr
Figure 3 PCI-34051(5 mM ) stimulates caspase-dependent apoptosis and induces caspase-3 activity in Jurkat cells treated for 2 days. (a) Cell death
(measured by Annexin-V flow cytometry) induced by PCI-34051 is blocked by 10 mM QVD (pan-caspase inhibitor). (b) Caspase-3 activity was
measured in Jurkat cells using the Apotarget Colorimetric Assay (BioSource). (c) PARP cleavage measured in Jurkat cells after treatment with 5 mM
PCI-34051 for 24 or 48 h or 0.3 mg ml1 CPT for 16 h. (d) Western blot of full-length Bid (FL-Bid) in Jurkat cells treated with 0.5 mM PCI-24781, 5 mM
PCI-34051 or DMSO control for 24 h. The quantitation of the bands was performed as described in Materials and methods. A decrease in FL-Bid is
seen with PCI-24781 but not PCI-34051 treatment. CPT, camptothecin; DMSO, dimethyl sulfoxide; PARP, poly(ADP-ribose) polymerase.

Leukemia
 HDAC8-specific inhibitor PCI-34051 in T-cell lymphoma
S Balasubramanian et al
1031
100 Thapsigargin-induced calcium flux was unaffected by U73122
in either cell line (not shown). Taken together, the results further
80
support the notion that an early phase of PCI-34051-induced
Positive Cells (%)

calcium release is due to PLCg1 activity, and in turn, that this

Annexin V

60
process is correlated to the induced apoptosis.
40

20 PCI-34051 induces cytochrome c release from

mitochondria
0
Jurkat J.gamma1 P116 J.RT3-T.5 It has been shown that ER Ca2 þ mobilization can induce the
release of cytochrome c from the mitochondria in a biphasic
60 process resulting in caspase-dependent cell death.37,41,42 To
determine if this occurs in response to PCI-34051, the levels of
Positive Cells (%)

cytosolic and mitochondrial cytochrome c were measured after

40
Annexin-V

fractionation at various timepoints. As shown in Figure 7, at 12 h

in Jurkat cells, cytosolic cytochrome c is observed in both the
20
PCI-34051 and FasL-treated samples, and this is increased at
24 h in both samples. Strikingly, there is no release of
cytochrome c from mitochondria in the PLCg1 deficient
0 J.gamma1 cells at either timepoint after treatment with PCI-
- 0.8 0.1 0.2 0.4 - 0.8 0.1 0.2 0.4
34051. The FasL-induced release of cytochrome c is observed,
U- U-73122 (µM) U- U-73122 (µM)
73343 73343 likely due to Bid-induced mitochondrial cytochrome c release
(µM) (µM) under these conditions. These results show that in Jurkat cells,
Controls +PCI-34051 (10µM) the apoptotic cascade is initiated by the action of the HDAC8-
Figure 4 PLCg1 activity is required for apoptosis induction by PCI- specific inhibitor at 12 h, and that this is mediated by the activity
34051. (a) Jurkat-derived PLCg1-deficient J.gamma1 cells are resistant of PLCg1.
to PCI-34051-induced apoptosis. Jurkat and J.gamma1cells were
treated with 5 mM PCI-34051 for 2 days and Annexin-V-positive cells
measured by flow cytometry. (b) PLC inhibitor modulates PCI-34051- Discussion
induced apoptosis in Jurkat cells. Jurkat and J.gamma1 cells were
treated with PLC inhibitor U73122 and inactive analog U73343 with
or without PCI-34051and Annexin-V measured after 2 days. In this study, we present the biological characterization of a
novel and selective small molecule inhibitor of histone
deacetylase 8, PCI-34051. This rationally designed inhibitor of
a 35
HDAC8 enzymatic activity inhibits cell growth and induces
Jurkat apoptosis only in T-cell-derived tumor lines, such as Jurkat and
30 J.gamma1 Hut78, but not in solid tumor lines, or in other hematopoietic
Positive Cells (%)

25 tumors such as those derived from B cells, plasma cells or

Annexin-V

20 monocytes. This selectivity has not been previously observed for

any HDAC inhibitor.
15 The mechanism by which PCI-34051 induces apoptosis in T-cell
10 lines is also novel to the histone deacetylase field, and is shown
5 here to involve in Ca2 þ signaling via PLCg1. In previous work, the
biphasic pathway by which PLCg1-dependent Ca2 þ flux leads to
0
Control PCI-34051 TG PCI-34051 apoptosis was elucidated.37,41 These studies reported that Ca2 þ
+ TG released internally from the ER (by FasL or Ca2 þ ionophores) can
enter mitochondria nearby and release a small amount of
b 20
Jurkat
cytochrome c; this in turn can bind to the IP3R on the ER and
Positive Cells (%)

J.gamma1
16 release more calcium, which effects a mass efflux of cytochrome c
Annexin-V

12 from all mitochondria, initiating the apoptotic cascade. Both

phases of Ca2 þ release are essential for the apoptosis to occur. In
8
this study, we show that both the PLCg1-dependent calcium efflux
4 from ER and cytochrome c release from mitochondria are involved
0 in the mode of action of PCI-34051. The maximal amplitude of
0 0.5 1 0 0.5 1
PCI-34051-induced Ca2 þ flux is similar to that of thapsigargin,
BAPTA-AM (uM) BAPTA-AM (uM) which selectively triggers intracellular Ca2 þ flux from ER by
DMSO PCI-34051
inhibiting SERCA pumps;40 however, PCI-34051-induced calcium
Figure 5 PCI-34051-induced apoptosis is enhanced by Ca2 þ effector flux is dependent on PCLg1 activity, unlike thapsigargin. That the
thapsigargin and inhibited by the Ca2 þ chelator BAPTA-AM. Jurkat Ca2 þ release is primarily intracellular is also supported by the
and J.gamma1 cells were treated for 24 h with (a) PCI-34051 (5 mM), other results that BAPTA-AM, a membrane-permeable Ca2 þ
thapsigargin (0.2 mM) or the combination. (b) PCI-34051 (10 mM), chelator, blocks the PCI-34051-induced Ca2 þ release as well as
BAPTA-AM (0.5 and 1 mM) or the combination. the subsequent apoptosis. Interestingly, it is noted that PLCg1 is the
primary isoform of PLC in T cells, whereas B cells (in which PCI-
release was also affected by the pretreatment of these cells with 34051 does not induce apoptosis) instead contain a second isoform
the PLC inhibitor U73122, resulting in a delay in achieving PLCg2.43,44 It is not clear at this point, however, if this is the reason
steady-state levels, while U73343 had no effect (Figure 6d). underlying the lack of apoptosis induction by PCI-34051 in B cells.

Leukemia
 HDAC8-specific inhibitor PCI-34051 in T-cell lymphoma
S Balasubramanian et al
1032
5.00 5.00

4.00 4.00
PCI-34051
Ratio 405/485

Ratio 405/485
Jurkat TG 1µM
10µM
3.00 3.00 Jurkat
5µM
PCI-34051 10µM
2.5µM
2.00 Control 2.00 J.gamma1 TG 1µM
J.gamma1
1.00 1.00 PCI-34051 10µM
PCI-34051 Ionomycin Drug Inomycin
0.00 0.00
Time (Sec)
4.00 4.00
Thapsigargin

Ratio 405/485
Ratio 405/485

PCI-34051
3.00 1µM 3.00 PCI-34051 + U-73343
0.5µM
0.25µM PCI-34051 + U-73122
2.00 Control 2.00 U-73343
U-73122
1.00 1.00 Control
Drug Ionomy cin Drug
0.00 0.00
0 100 200 300 400 0 100 200 300 400 500
Time (Sec) Time (sec)

Figure 6 PCI-34051 induces rapid calcium mobilization in Jurkat but not in PLC-g1-deficient cells. (a) Cells were preloaded with Indo-1, and
increasing concentrations of PCI-34051 were added at the time marked by the first vertical arrow, followed by ionomycin (10 mM) as indicated.
Calcium levels were measured by fluorescence as described in Materials and methods. (b) Thapsigargin added at increasing doses as shown
induces calcium flux in Jurkat cells. (c) Jurkat and J.gamma1 cells were preloaded with Indo-1 and PCI-34051 (10 mM) or thapsigargin (TG, 1 mM)
was added, followed by ionomycin as indicated. Thapsigargin induced calcium flux in J.gamma1 cells but PCI-34051 did not. (d) Jurkat cells were
loaded with Indo-1 and pretreated with either U73122 (0.4 mM) or U73343 (0.8 mM). PCI-34051 (10 mM) was added when indicated by the arrow.
Neither U73122 nor U73343 alone had any effect on calcium levels (baseline).

Jurkat apoptosis induction in response to PCI-34051 does not require

TCR signaling, as both the TCR-b chain and ZAP-70 kinase
Control PCI-34051 FasL knockouts are not impaired. Therefore, the initiation of Ca2 þ
Mito Cytosol Mito Cytosol Mito Cytosol flux by PCI-34051 via PLCg1 occurs downstream of the antigen
12 Hr receptor complex, and may involve kinases other than ZAP-70.
It is possible that Tec-family kinases could play a role in this
process, as it has been shown that knocking out Itk in T cells
24 Hr reduces PLCg1 tyrosine phosphorylation and IP3 levels, but does
not affect CD3 or ZAP-70 phosphorylation,45 placing Itk at a
J.gamma1 likely intervention point in the mechanism of action of PCI-
34051. Further, it has been shown recently that selective small
Control PCI-34051 FasL
molecule inhibitors of Itk also blocked PLCg1 activation, IP3
Mito Cytosol Mito Cytosol Mito Cytosol production and intracellular Ca2 þ release.46 We are currently
12 Hr exploring this intriguing possibility in this laboratory.
This study provides evidence for the involvement of PLCg1
24 Hr
activation in the action of PCI-34051, delineating the calcium-
mediated downstream pathway leading to apoptosis. But how
Figure 7 Cytochrome c translocation from mitochondria to cytosol does the inhibition of HDAC8 enzymatic activity by PCI-34051
following PCI-34051 treatment in Jurkat but not in J.gamma1 cells. lead to the activation of PLCg1? It is possible that PLCg1 itself is
Jurkat and J.gamma1 cells were treated with 10 mM PCI-34051 or normally deacetylated by HDAC8. Also, as mentioned earlier, in
10 ng ml1 FasL for 12 or 24 h, the mitochondrial and cytosolic T cells, PLCg1 is activated by binding to a complex consisting of
(including ER) fractions separated using the Pierce Mitochondria
several adaptor proteins, including LAT, Gads and SLP76, and
Isolation kit and analyzed by western blotting with anti-cytochrome c
(and anti-cytochrome c oxidase, not shown). ER, endoplasmic phosphorylated by kinases including Syk and Itk.43,47 Any one
reticulum; FasL, Fas Ligand. or more of these could be potential HDAC8 targets, and many
are specific for T cells. This may provide an explanation for the
selectivity of this compound since PLCg1 itself is expressed in
PLCg1 is an essential downstream component of T-cell other cell types,43,48 including those where PCI-34051 is not
receptor signaling, following stimulation with antigens or anti- active (Table 2, Supplementary Table 1 and Figure 1). But very
CD3 antibodies. PLCg1 is activated by translocation from little data exist on potential targets of HDAC8 activity, and
cytoplasm to the plasma membrane, association with LAT and Figure 2b shows no evidence of other hyperacetylated proteins;
SLP76 and phosphorylation, likely on Y783.43 However, we are currently pursuing a candidate approach with some of

Leukemia

these potential targets. It is possible that other non-HDAC8
targets of PCI-34051 activity may exist, although none of the
kinases mentioned nor any closely related proteases are
inhibited by this compound (data not shown). Interestingly,
a recent study49 identified N-thioacetyl-lysine as an in vitro
substrate of HDAC8, but not of HDAC1 or HDAC2, suggesting
that HDAC8 could be an in vivo dethioacetylase. Finally,
another recent paper50 used acetyl-lysine peptide analogs to
probe the substrate-binding pocket of HDAC8, and found
a slightly higher rate of catalysis for propionyl-lysine peptide
compared to the acetyl-lysine peptide. So, we cannot exclude at
present the possibility that the in vivo substrate of HDAC8 is
something other than acetyl-lysine.
In conclusion, we have identified and characterized the most
potent isoform-selective inhibitor of a histone deacetylase to
date, one which is 4200-fold selective for HDAC8 in vitro,
compared to all the other class I and selected class II HDAC
isoforms tested. We have shown that this compound, PCI-
34051, does not induce histone or tubulin acetylation, and
induces apoptosis only in T-cell-derived tumor cells. Further, we
have demonstrated that apoptosis induction occurs at least in
part through calcium flux effected by PLCg1 activation, which,
to the best of our knowledge, is the first time such a mechanism
has been described for any HDAC inhibitor. We have also
shown that this results in cytochrome c release from mitochon-
dria and caspase 3 activation, eventually resulting in apoptosis.
The remarkable selectivity of this compound for T-cell-derived
tumors and its mechanism of action suggest that it may have
significantly less toxicity than the broad-spectrum HDAC
inhibitors, and thus may prove to be of benefit in the treatment
of T-cell-derived malignancies.
References
1 de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB.
Histone deacetylases (HDACs): characterization of the classical HDAC
family. Biochem J 2003; 370: 737–749.
2 Glozak MA, Sengupta N, Zhang X, Seto E. Acetylation and
deacetylation of non-histone proteins. Gene 2005; 363: 15–23.
3 Kim SC, Sprung R, Chen Y, Xu Y, Ball H, Pei J. et al. Substrate and
functional diversity of lysine acetylation revealed by a proteomics
survey. Mol Cell 2006; 23: 607–618.
4 Verdin E, Dequiedt F, Kasler HG. Class II histone deacetylases:
versatile regulators. Trends Genet 2003; 19: 286–293.
5 Kelly WK, Marks PA. Drug insight: histone deacetylase
inhibitorsFdevelopment of the new targeted anticancer agent
suberoylanilide hydroxamic acid. Nat Clin Pract Oncol 2005; 2:
150–157.
6 Marks PA, Breslow R. Dimethyl sulfoxide to vorinostat: develop-
ment of this histone deacetylase inhibitor as an anticancer drug.
Nat Biotechnol 2007; 25: 84–90.
7 Xu WS, Parmigiani RB, Marks PA. Histone deacetylase inhibi-
tors: molecular mechanisms of action. Oncogene 2007; 26:
5541–5552.
8 Garber K. HDAC inhibitors overcome first hurdle. Nat Biotechnol
2007; 25: 17–19.
9 Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone
deacetylase inhibitors. Nat Rev Drug Discov 2006; 5: 769–784.
10 Rosato RR, Grant S. Histone deacetylase inhibitors in clinical
development. Expert Opin Investig Drugs 2004; 13: 21–38.
11 Haggarty SJ, Koeller KM, Wong JC, Grozinger CM, Schreiber SL.
Domain-selective small-molecule inhibitor of histone deacetylase
6 (HDAC6)-mediated tubulin deacetylation. Proc Natl Acad Sci
USA 2003; 100: 4389–4394.
12 Hideshima T, Bradner JE, Wong J, Chauhan D, Richardson P,
Schreiber SL et al. Small-molecule inhibition of proteasome and
aggresome function induces synergistic antitumor activity in
multiple myeloma. Proc Natl Acad Sci USA 2005; 102: 8567–
8572.
HDAC8-specific inhibitor PCI-34051 in T-cell lymphoma
S Balasubramanian et al
1033
13 Hu E, Dul E, Sung CM, Chen Z, Kirkpatrick R, Zhang GF et al.
Identification of novel isoform-selective inhibitors within class I
histone deacetylases. J Pharmacol Exp Ther 2003; 307: 720–728.
14 Krennhrubec K, Marshall BL, Hedglin M, Verdin E, Ulrich SM.
Design and evaluation of ‘Linkerless’ hydroxamic acids as
selective HDAC8 inhibitors. Bioorg Med Chem Lett 2007; 17:
2874–2878.
15 Karagiannis TC, El Osta A. Will broad-spectrum histone deacety-
lase inhibitors be superseded by more specific compounds?
Leukemia 2007; 21: 61–65.
16 Wang DF, Helquist P, Wiech NL, Wiest O. Toward selective
histone deacetylase inhibitor design: homology modeling, docking
studies, and molecular dynamics simulations of human class I
histone deacetylases. J Med Chem 2005; 48: 6936–6947.
17 Vannini A, Volpari C, Filocamo G, Casavola EC, Brunetti M,
Renzoni D et al. Crystal structure of a eukaryotic zinc-dependent
histone deacetylase, human HDAC8, complexed with a hydro-
xamic acid inhibitor. Proc Natl Acad Sci USA 2004; 101:
15064–15069.
18 Buggy JJ, Sideris ML, Mak P, Lorimer DD, McIntosh B, Clark JM.
Cloning and characterization of a novel human histone deacety-
lase, HDAC8. Biochem J 2000; 350 (Pt 1): 199–205.
19 Gregoretti IV, Lee YM, Goodson HV. Molecular evolution of the
histone deacetylase family: functional implications of phylogenetic
analysis. J Mol Biol 2004; 338: 17–31.
20 Waltregny D, Glenisson W, Tran SL, North BJ, Verdin E, Colige A
et al. Histone deacetylase HDAC8 associates with smooth muscle
alpha-actin and is essential for smooth muscle cell contractility.
FASEB J 2005; 19: 966–968.
21 Lee H, Sengupta N, Villagra A, Rezai-Zadeh N, Seto E. Histone
deacetylase 8 safeguards the human ever-shorter telomeres 1B
(hEST1B) protein from ubiquitin-mediated degradation. Mol Cell
Biol 2006; 26: 5259–5269.
22 Lee H, Rezai-Zadeh N, Seto E. Negative regulation of histone
deacetylase 8 activity by cyclic AMP-dependent protein kinase A.
Mol Cell Biol 2004; 24: 765–773.
23 Somoza JR, Skene RJ, Katz BA, Mol C, Ho JD, Jennings AJ et al.
Structural snapshots of human HDAC8 provide insights into the
class I histone deacetylases. Structure 2004; 12: 1325–1334.
24 Buggy JJ, Cao ZA, Bass KE, Verner E, Balasubramanian S, Liu L
et al. CRA-024781: a novel synthetic inhibitor of histone
deacetylase enzymes with antitumor activity in vitro and in vivo.
Mol Cancer Ther 2006; 5: 1309–1317.
25 Tai VW-F, Verner E, Balasubramanian S, Lee CS, Buggy JJ. Indole
derivatives as inhibitors of histone deacetylase. USPTO 2007; PCN
07-39 2007109178/WO-A2.
26 Schultz BE, Misialek S, Wu J, Tang J, Conn MT, Tahilramani R
et al. Kinetics and comparative reactivity of human class I
and class IIb histone deacetylases. Biochemistry 2004; 43:
11083–11091.
27 Overbeeke R, Steffens-Nakken H, Vermes I, Reutelingsperger C,
Haanen C. Early features of apoptosis detected by four different
flow cytometry assays. Apoptosis 1998; 3: 115–121.
28 Ruefli AA, Ausserlechner MJ, Bernhard D, Sutton VR, Tainton KM,
Kofler R et al. The histone deacetylase inhibitor and chemother-
apeutic agent suberoylanilide hydroxamic acid (SAHA) induces a
cell-death pathway characterized by cleavage of Bid and produc-
tion of reactive oxygen species. Proc Natl Acad Sci USA 2001; 98:
10833–10838.
29 Inoue S, Riley J, Gant TW, Dyer MJ, Cohen GM. Apoptosis
induced by histone deacetylase inhibitors in leukemic cells is
mediated by Bim and Noxa. Leukemia 2007; 21: 1773–1782.
30 Ohashi PS, Mak TW, Van den EP, Yanagi Y, Yoshikai Y, Calman AF
et al. Reconstitution of an active surface T3/T-cell antigen receptor
by DNA transfer. Nature 1985; 316: 606–609.
31 Zhang W, Irvin BJ, Trible RP, Abraham RT, Samelson LE.
Functional analysis of LAT in TCR-mediated signaling pathways
using a LAT-deficient Jurkat cell line. Int Immunol 1999; 11:
943–950.
32 Irvin BJ, Williams BL, Nilson AE, Maynor HO, Abraham RT.
Pleiotropic contributions of phospholipase C-gamma1 (PLC-
gamma1) to T-cell antigen receptor-mediated signaling: reconsti-
tution studies of a PLC-gamma1-deficient Jurkat T-cell line. Mol
Cell Biol 2000; 20: 9149–9161.
Leukemia
 HDAC8-specific inhibitor PCI-34051 in T-cell lymphoma
S Balasubramanian et al
1034
33 Patterson RL, van Rossum DB, Nikolaidis N, Gill DL,
Snyder SH. Phospholipase C-gamma: diverse roles in receptor-
mediated calcium signaling. Trends Biochem Sci 2005; 30:
688–697.
34 Smith RJ, Sam LM, Justen JM, Bundy GL, Bala GA, Bleasdale JE.
Receptor-coupled signal transduction in human polymorpho-
nuclear neutrophils: effects of a novel inhibitor of phospholipase
C-dependent processes on cell responsiveness. J Pharmacol Exp
Ther 1990; 253: 688–697.
35 Tu CL, Chang W, Bikle DD. Phospholipase cgamma1 is required
for activation of store-operated channels in human keratinocytes.
J Invest Dermatol 2005; 124: 187–197.
36 Aires V, Adote S, Hichami A, Moutairou K, Boustani ES, Khan NA.
Modulation of intracellular calcium concentrations and T cell
activation by prickly pear polyphenols. Mol Cell Biochem 2004;
260: 103–110.
37 Boehning D, Patterson RL, Sedaghat L, Glebova NO, Kurosaki T,
Snyder SH. Cytochrome c binds to inositol (1, 4, 5) trisphosphate
receptors, amplifying calcium-dependent apoptosis. Nat Cell Biol
2003; 5: 1051–1061.
38 Yoshida I, Monji A, Tashiro K, Nakamura K, Inoue R, Kanba S.
Depletion of intracellular Ca2+ store itself may be a major factor in
thapsigargin-induced ER stress and apoptosis in PC12 cells.
Neurochem Int 2006; 48: 696–702.
39 Navarrete C, Sancho R, Caballero FJ, Pollastro F, Fiebich BL,
Sterner O et al. Basiliolides, a class of tetracyclic C19 dilactones
from Thapsia garganica, release Ca(2+) from the endoplasmic
reticulum and regulate the activity of the transcription factors
nuclear factor of activated T cells, nuclear factor-kappaB, and
activator protein 1 in T lymphocytes. J Pharmacol Exp Ther 2006;
319: 422–430.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
Leukemia
View publication stats
40 Treiman M, Caspersen C, Christensen SB. A tool coming of age:
thapsigargin as an inhibitor of sarco-endoplasmic reticulum
Ca(2+)-ATPases. Trends Pharmacol Sci 1998; 19: 131–135.
41 Wozniak AL, Wang X, Stieren ES, Scarbrough SG, Elferink CJ,
Boehning D. Requirement of biphasic calcium release from the
endoplasmic reticulum for Fas-mediated apoptosis. J Cell Biol
2006; 175: 709–714.
42 Szabadkai G, Rizzuto R. Participation of endoplasmic reticulum
and mitochondrial calcium handling in apoptosis: more than just
neighborhood? FEBS Lett 2004; 567: 111–115.
43 Wilde JI, Watson SP. Regulation of phospholipase C gamma
isoforms in haematopoietic cells: why one, not the other? Cell
Signal 2001; 13: 691–701.
44 Wang D, Feng J, Wen R, Marine JC, Sangster MY, Parganas E et al.
Phospholipase Cgamma2 is essential in the functions of B cell and
several Fc receptors. Immunity 2000; 13: 25–35.
45 Liu KQ, Bunnell SC, Gurniak CB, Berg LJ. T cell receptor-initiated
calcium release is uncoupled from capacitative calcium entry in
Itk-deficient T cells. J Exp Med 1998; 187: 1721–1727.
46 Lin TA, McIntyre KW, Das J, Liu C, O’Day KD, Penhallow B et al.
Selective Itk inhibitors block T-cell activation and murine lung
inflammation. Biochemistry 2004; 43: 11056–11062.
47 Braiman A, Barda-Saad M, Sommers CL, Samelson LE. Recruit-
ment and activation of PLCgamma1 in T cells: a new insight into
old domains. EMBO J 2006; 25: 774–784.
48 Katan M. Families of phosphoinositide-specific phospholipase C:
structure and function. Biochim Biophys Acta 1998; 1436: 5–17.
49 Fatkins DG, Zheng W. A spectrophotometric assay for histone
deacetylase 8. Anal Biochem 2008; 372: 82–88.
50 Smith BC, Denu JM. Acetyl-lysine analog peptides as mechanistic
probes of protein deacetylases. J Biol Chem 2007; 282: 37256–37265.