- Open Access
Histone deacetylase turnover and recovery in sulforaphane-treated colon cancer cells: competing actions of 14-3-3 and Pin1 in HDAC3/SMRT corepressor complex dissociation/reassembly
© Rajendran et al; licensee BioMed Central Ltd. 2011
- Received: 12 February 2011
- Accepted: 30 May 2011
- Published: 30 May 2011
Histone deacetylase (HDAC) inhibitors are currently undergoing clinical evaluation as anti-cancer agents. Dietary constituents share certain properties of HDAC inhibitor drugs, including the ability to induce global histone acetylation, turn-on epigenetically-silenced genes, and trigger cell cycle arrest, apoptosis, or differentiation in cancer cells. One such example is sulforaphane (SFN), an isothiocyanate derived from the glucosinolate precursor glucoraphanin, which is abundant in broccoli. Here, we examined the time-course and reversibility of SFN-induced HDAC changes in human colon cancer cells.
Cells underwent progressive G2/M arrest over the period 6-72 h after SFN treatment, during which time HDAC activity increased in the vehicle-treated controls but not in SFN-treated cells. There was a time-dependent loss of class I and selected class II HDAC proteins, with HDAC3 depletion detected ahead of other HDACs. Mechanism studies revealed no apparent effect of calpain, proteasome, protease or caspase inhibitors, but HDAC3 was rescued by cycloheximide or actinomycin D treatment. Among the protein partners implicated in the HDAC3 turnover mechanism, silencing mediator for retinoid and thyroid hormone receptors (SMRT) was phosphorylated in the nucleus within 6 h of SFN treatment, as was HDAC3 itself. Co-immunoprecipitation assays revealed SFN-induced dissociation of HDAC3/SMRT complexes coinciding with increased binding of HDAC3 to 14-3-3 and peptidyl-prolyl cis/trans isomerase 1 (Pin1). Pin1 knockdown blocked the SFN-induced loss of HDAC3. Finally, SFN treatment for 6 or 24 h followed by SFN removal from the culture media led to complete recovery of HDAC activity and HDAC protein expression, during which time cells were released from G2/M arrest.
The current investigation supports a model in which protein kinase CK2 phosphorylates SMRT and HDAC3 in the nucleus, resulting in dissociation of the corepressor complex and enhanced binding of HDAC3 to 14-3-3 or Pin1. In the cytoplasm, release of HDAC3 from 14-3-3 followed by nuclear import is postulated to compete with a Pin1 pathway that directs HDAC3 for degradation. The latter pathway predominates in colon cancer cells exposed continuously to SFN, whereas the former pathway is likely to be favored when SFN has been removed within 24 h, allowing recovery from cell cycle arrest.
- Colon Cancer Cell
- HCT116 Cell
- Human Colon Cancer Cell
- HDAC Activity
Epigenetic changes play a critical role in cancer development [1–5]. These changes include the dysregulation of histone deacetylases (HDACs) and the altered acetylation status of histone and non-histone proteins [6–8]. Efforts have been directed at reversing aberrant acetylation patterns in cancers through the use of HDAC inhibitors. HDAC inhibitors induce cell cycle arrest, differentiation, and apoptosis in cancer cells, some have anti-inflammatory activities, and a number have progressed to clinical trials [8–12].
HDACs can be overexpressed in colorectal cancers and in several other cancer types [13–18]. Silencing of HDACs, individually or in combination, has provided insights into the associated molecular pathways that regulate cell cycle transition, proliferation, and apoptosis [14, 18–20]. In human colon cancer cells, silencing of HDAC3 resulted in growth inhibition, decreased cell survival, and increased apoptosis . Similar effects were noted for HDAC2 and, to a lesser extent, for HDAC1. Subsequent work  identified a role for HDAC4 in regulating p21WAF1 expression, via a corepressor complex involving HDAC4, HDAC3, and SMRT/N-CoR (silencing mediator for retinoid and thyroid hormone receptors/nuclear receptor co-repressor). Spurling et al. reported that knockdown of HDAC3 increased constitutive, trichostatin A (TSA)-, and tumor necrosis factor (TNF)-α-induced expression of p21WAF1, although HDAC3 silencing alone did not account for all the gene expression changes observed upon general HDAC inhibition. Cells with lowered HDAC3 expression had increased histone H4-K12 acetylation (H4K12ac) and were poised for gene expression changes . Ma et al. observed that recruitment of p300 to the survivin promoter led to the concomitant recruitment of other protein partners, including HDAC6, resulting in transcriptional repression. Thus, there is accumulating evidence for the involvement of multiple HDACs in colon cancer development.
HDAC activity and histone acetylation status can be influenced by dietary factors and their metabolites [21–23]. For example, broccoli and broccoli sprouts are a rich source of glucoraphanin, the glucosinolate precursor of the cancer chemoprotective agent sulforaphane (SFN) [24–28]. SFN has been reported to inhibit HDAC activity in human colon cancer cells , and this was confirmed in prostate and breast cancer cells [30, 31]. A structurally-related isothiocyanate also inhibited HDAC activity in human leukemia cells, resulting in chromatin remodeling and growth arrest . Combining these findings with the changes induced by SFN in NF-E2-related factor 2 (Nrf2) signaling [24–28, 33], a "one-two" chemoprotective model can be proposed. In the first stage, SFN parent compound induces phase 2 detoxification pathways, and in the second stage SFN metabolites alter HDAC activity and histone status, leading to the unsilencing of tumor suppressors such as p21WAF1[34–36]. An unresolved question from our earlier studies  was the fate of individual HDACs in SFN-treated colon cancer cells. If, indeed, SFN metabolites act as weak ligands for HDACs , does this result in de-recruitment and/or turnover of specific HDAC proteins, and is this reversible? These questions were examined in the present investigation, along with the molecular mechanisms involved.
SFN-induced changes in HDAC activity and protein expression
Changes in HDAC protein expression are reversed upon SFN removal
The corresponding whole cell lysates were subjected to immunoblotting (Figure 3B). Expression levels of HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, and HDAC8 were reduced when SFN was added to the assay and not removed, compared with the corresponding vehicle controls at 24 h (lane 2 versus lane 1, Figure 3B). When SFN was removed after 6 h and replaced with fresh media containing no SFN, there was complete recovery of HDAC1 and HDAC2 by 24 h, but no recovery of the other HDACs at this time-point (lane 3, Figure. 3B).
After a further 24 h, the HDAC activity had fully recovered in cells treated with SFN for 6 h (Figure 3A, 48 h, gray bar versus white bar), and there was complete recovery of all HDAC proteins, except HDAC6 (Figure 3B, compare lane 6 versus lane 4). Notably, even in cells exposed to SFN for 24 h followed by SFN removal, partial recovery of HDAC activity was detected by 48 h (Figure 3A, solid black bar). By 72 h, HDAC activity and protein expression had more-or-less fully recovered, except in cells treated continuously with SFN.
Histone acetylation, cell cycle, and apoptosis changes upon SFN removal
SFN-induced loss of HDAC3 is independent of caspase activity
SFN-induced loss of HDAC3 is unaffected by selected proteasome and lysosome inhibitors, but is attenuated by cycloheximide and actinomycin D
Total cell lysates next were probed with an anti-ubiquitin antibody (Figure 6C). High-molecular weight poly-ubiquitylated bands were detected in the vehicle controls (lane 1), and these bands were reduced by SFN treatment (lane 2). In contrast, PYR-41 produced a striking increase in poly-ubiquitylated bands (lane 3), over and above those that accumulated in response to MG132 treatment (lane 5). SFN co-treatment partially overcame the increased poly-ubiquitylation associated with either PYR-41 or MG132 (Figure 6 C, compare lane 4 versus lane 3, and lane 6 versus lane 5).
As noted in the introduction, regulation of p21WAF1 in colon cancer cells has been associated with a corepressor complex involving HDAC3-HDAC4-SMRT/N-CoR . Treatment with cycloheximide (CHX) for 6 h, in the presence or absence of SFN, depleted SMRT, N-Cor and HDAC4, as well as p21WAF1, but had little or no effect on HDAC3 expression (Figure 6D, lanes 3 and 4). Similar results were obtained with Actinomycin D, in the presence or absence or SFN, although the loss of p21WAF1 was less marked (Figure 6D, lanes 5 and 6). These data supported the view that HDAC3 protein was relatively stable in HCT116 cells, whereas SMRT, N-Cor, and HDAC4 (as well as p21WAF1) had a shorter half-life. On the other hand, SFN treatment reduced HDAC3 protein expression at 6 h without attenuating SMRT, N-Cor, or HDAC4. Notably, the SFN-induced loss of HDAC3 protein (lane 2) was fully or partially blocked by CHX (lane 4) and Actinomycin D treatment (lane 6), respectively. These findings implicated one or more protein partner(s) with a relatively short half-life in the HDAC3 turnover mechanism triggered by SFN.
Role of 14-3-3 and Pin1 in the SFN-induced loss of HDAC3
As expected, 14-3-3 levels were higher in the cytoplasm than in the nucleus, but time-course studies indicated a partial shift of 14-3-3 to the nucleus following SFN exposure (Figure 7B). Thus, whereas cytoplasmic 14-3-3 expression remained relatively constant in the -SFN controls (lanes 1-4), SFN treatment led to reductions in cytoplasmic 14-3-3, most notably at 6 h (lane 6), and there was a corresponding increase in nuclear 14-3-3 (lane 14). Two other SMRT partners were decreased in the nucleus (Figure 7C), namely protein kinase CK2 (casein kinase II) and peptidyl-prolyl cis/trans isomerase 1 (Pin1). CK2, which phosphorylates SMRT and has a phospho-acceptor site on HDAC3 [50, 51], was reduced markedly in the nucleus 6-24 h post-SFN treatment (lanes 12-14). Pin1, which negatively regulates SMRT protein stability , increased gradually in the nucleus in -SFN controls (lanes 9-11), but remained relatively low in SFN-treated cells (lanes 12-14). In the cytoplasm, no marked changes were detected for CK2 or Pin1 in the presence or absence of SFN (lanes 1-8).
In co-immunoprecipitation (co-IP) experiments, pulling-down HDAC3 identified SMRT as a binding partner both in the cytoplasm and nucleus (Figure 7D). SFN treatment completely blocked HDAC3/SMRT interactions in the cytoplasm at 6 h (lane 2), and attenuated these associations in the cytoplasm and nucleus at 24 h (lanes 4 and 8). Although nuclear p-SMRT was increased by SFN (Figure 7A), less nuclear p-SMRT was pulled down with HDAC3 at 6 and 24 h post-SFN exposure (lanes 6 and 8, Figure 7D). No HDAC3/p-SMRT interactions were detected in the cytoplasm. Our interpretation of these findings was that increased phosphorylation of HDAC3 and SMRT led to corepressor complex dissociation, with less SMRT and p-SMRT interacting with HDAC3 after SFN treatment. Interestingly, the increased nuclear 14-3-3 at 6 h post-SFN exposure (Figure 7B, lane 14) was paralleled by enhanced binding of 14-3-3 to HDAC3 in the nucleus (Figure 7D, lane 6), which was further augmented both in the cytoplasm and nucleus at 24 h (Figure 7D, lanes 4 and 8, respectively). In the nucleus, CK2 associations with HDAC3 increased at 6 and 24 h post-SFN treatment (lanes 6 and 8, Figure 7D), despite the lower total nuclear CK2 levels in SFN-treated cells (Figure 7C, lanes 12-14). This result suggested that SFN shifted the pool of nuclear CK2 towards HDAC3/SMRT, favoring phosphorylation and complex disassembly.
In addition to the enhanced association of 14-3-3 with HDAC3, SFN treatment also increased Pin1 interactions with HDAC3 in the nucleus at 6 h (Figure 7D, lane 6). Pin1 pull-downs confirmed SMRT and HDAC3 nuclear interactions 6 and 24 h after SFN exposure, as well as HDAC6 binding, whereas little or no HDAC1 and HDAC2 were bound to Pin1 (Additional File 1). Because Pin1 has been implicated in the degradation of several proteins, including SMRT , we knocked-down Pin1 in HCT116 cells (Figure 7E). Following Pin1 knockdown, the SFN-induced loss of HDAC3 was prevented, and there was reduced H4K12ac as compared with Pin1 siRNA control. Induction of p21WAF1 by SFN was unaffected by Pin1 knockdown (Figure 7E).
This is the first investigation to examine the fate of individual HDACs in human colon cancer cells treated with SFN, with the dual aims of clarifying the mechanisms of the observed HDAC protein turnover and the timing of HDAC recovery following SFN removal. Pappa et al. previously performed transient exposure experiments with SFN, observing that G2/M arrest and cytostatic growth inhibition were reversible in the cell line 40-16. In the present study, HCT116 cells were plated at low density so as to allow HDAC changes to be followed for at least 72 h. Under these conditions, 6-24 h of SFN exposure followed by SFN removal resulted in the complete recovery of HDAC activity and HDAC protein expression, along with the normalization of histone acetylation and p21WAF1 status. Although apoptosis induction was detected, most notably at higher SFN concentrations, caspase-3-mediated cleavage of HDAC3  was excluded as a contributing mechanism in the loss of HDAC3 protein. Other HDACs are known to be cleaved by caspases; for example, caspase-8-mediated cleavage of HDAC7 has been reported . HDAC7 and several other class II HDACs were unaffected at the protein level by SFN treatment; however, a formal examination of each caspase and its potential HDAC target(s) may be warranted.
Changes in HDAC6 were of interest because this HDAC has been described as a master regulator of cellular responses to cytotoxic insults . We performed several experiments on HDAC6 and observed the following: (i) HDAC6 protein loss was first detected at around 24 h post-SFN treatment (versus 6 h for HDAC3); (ii) although delayed relative to other HDACs, HDAC6 was fully recovered by 72 h in the SFN reversibility studies; (iii) as with HDAC3, HDAC6 loss was not prevented by a cell-permeable pan caspase inhibitor; (iv) immunoprecipitation of HDAC3 followed by HDAC6 from whole cell lysates accounted for all of the HDAC inhibitory effects of SFN (Additional File 2); and (v) transient overexpression of HDAC6 in HCT116 cells completely blocked the increased tubulin acetylation associated with SFN treatment, as well as the induction of H4K12ac. Gibbs et al. performed ectopic overexpression of HDAC6 in human prostate cancer cells, observing SFN-mediated inhibition of HDAC6 activity, HSP90 hyperacetylation, and destabilization of the androgen receptor. Decreased endogenous HDAC6 and HDAC3 protein expression was recently reported in SFN-treated prostate epithelial cells , although the precise molecular mechanisms were not pursued. We conclude that HDAC6, along with its corepressor partners, is an important target for SFN action in human prostate and colon cancer cells. However, depletion of HDAC3 followed by HDAC6 (Additional File 2), or HDAC6 followed by HDAC3 (data not shown), suggested that HDAC3 accounted for approximately two-thirds and HDAC6 one-third of the SFN actions on HDAC activity in HCT116 cells. This observation coupled with the delayed loss and slower recovery of HDAC6 compared with HDAC3 suggested that HDAC3 plays a pivotal "sentinel" role, although HDAC6 mediating HDAC3 activity probably warrants further investigation.
In the present investigation, co-IP experiments indicated that dissociation of HDAC3/SMRT corepressor complexes occurred within 6 h of SFN treatment. SMRT and N-Cor are known to be regulated by distinct kinase signaling pathways , resulting in corepressor complex disassembly and redistribution from the nucleus to the cytoplasmic compartment. Erk2, a mitogen-activated protein kinase, disrupts SMRT self-dimerization, releasing HDAC3 and other protein partners from the corepressor complex, thereby lowering transcriptional repression . SFN is known to activate kinase signaling pathways [27, 61, 62], and we observed increased p-HDAC3 and p-SMRT in the nucleus within 6 h of SFN exposure, along with increased CK2 binding to HDAC3. In prior studies, phosphorylation of HDAC4 triggered its nuclear export and binding to 14-3-3 . In an analogous fashion, we now report, for the first time, that there was increased binding of 14-3-3 to HDAC3 following SFN treatment. This raises the possibility that 14-3-3 sequesters HDAC3 in the cytosolic compartment, pending the subsequent release and re-entry of HDAC3 into the nucleus (e.g., upon SFN removal).
Supporting this hypothesis were the results using phosphospecific antibodies to 14-3-3. The loss of cytoplasmic and nuclear p-14-3-3(T232) upon SFN treatment is consistent with this phosphorylation impeding interactions with client proteins, such as HDAC3, and indeed no p-14-3-3(T232) was pulled down with HDAC3 in the presence or absence of SFN treatment (Figure 8C). Loss of T232 phosphorylation upon SFN treatment would provide access to the adjacent nuclear export signal in 14-3-3 , facilitating nuclear-cytoplasmic trafficking. On the other hand, phosphorylation of S58 in 14-3-3 shifts the pool of 14-3-3 towards more of the monomeric form, although some interaction of p-14-3-3(S58) with HDAC3 was detected. The current model (Figure 8D) proposes 14-3-3 interacting with HDAC3 phosphorylated at S424; however, other phosphorylation sites in HDAC3 may be involved, such as those associated with glycogen synthase kinase-3β . Based on the findings with class IIa HDACs , 14-3-3 binding to HDAC3 might block the nuclear localization signal and facilitate cytoplasmic retention of HDAC3, leaving the nuclear export signal accessible to proteins such as CRM1 that further traffic HDAC3 from the nucleus to the cytoplasm. Additional work is needed to clarify this model, including the relative contributions of monomeric versus dimeric 14-3-3, and the role of other known phosphorylation sites in 14-3-3 [53–55].
Another interesting and novel observation was that SFN increased the binding of HDAC3 to Pin1. Pin1 knockdown completely blocked the SFN-induced loss of HDAC3, although this did not interfere with the induction of p21WAF1. One explanation may be that HDAC1 and HDAC2 are the primary repressor HDACs of p21WAF1, and neither one interacted with Pin1 before or after SFN treatment (Additional File 1). Importantly, Pin1 binding to p-SMRT has been reported to trigger SMRT degradation . Proteins such as c-Myc and cyclin E use a common Pin1-interacting motif to allow turnover by the Fbw7 E3 ligase , but this motif does not exist in SMRT . This suggests that a novel E3 ligase may be involved in the turnover of SMRT, and possibly HDAC3. There are estimated to be 500-1000 E3 ligases in human cells , and further work is warranted to identify the E3 ligase involved in HDAC3 turnover. Although PYR-41 has been reported as an E1 inhibitor , it also affects sumoylation pathways, which complicated the interpretation of PYR-41 effects on SFN-induced HDAC3 turnover in HCT116 cells. Interestingly, a selective inhibitor of CK2, 4,5,6,7-tetrabromo-2-azabenzimidazole, dose-dependently depleted Pin1 and concomitantly increased HDAC3 protein expression in HCT116 cells, further confirming the inverse association between these two proteins (P. Rajendran, data not presented).
Although the details are far from definitive and several questions remain, a model is proposed for SFN actions in human colon cancer cells (Additional File 3). Following SFN treatment, kinase signaling pathways facilitate CK2 recruitment to nuclear HDAC3/SMRT corepressor complexes resulting in the phosphorylation of HDAC3 and SMRT, complex dissociation, binding to 14-3-3 or Pin1, and trafficking from the nucleus to the cytoplasm. In the cytoplasmic compartment, sequestration of HDAC3 by 14-3-3 competes with a pathway involving Pin1-directed HDAC3 degradation. Upon SFN removal, it is postulated that HDAC3 and SMRT are released from 14-3-3 to re-enter the nucleus, reassembling the corepressor complexes to silence gene activation. Further work is needed to clarify the possible involvement of a unique E3 ligase that targets both HDAC3 and SMRT for simultaneous degradation. This model highlights the role of kinase signaling pathways triggered by SFN, but does not exclude direct actions of SFN or its metabolites on HDACs . For example, entry of SFN metabolites into the HDAC3 pocket might lead to conformational changes and/or altered protein interactions that facilitate CK2 binding. These mechanisms are under further investigation in SFN-treated colon cancer cells, including time-course analyses of histone marks and the phospho-acetyl switch .
This investigation has addressed several mechanistic questions about SFN and the HDAC changes that occur in human colon cancer cells. Despite its reported "pleiotropic" actions as a chemoprotective agent, SFN exhibited a degree of selectivity towards individual HDACs, with several class II HDACs being unaffected at the protein level. Notably, immunodepletion of HDAC3 and HDAC6, along with their corepressor partners, accounted entirely for the SFN-induced changes in HDAC activity, and cells were rescued by forced overexpression of these two HDACs. Thus, HDAC3 and HDAC6 appear to be key mediators of the transcriptional changes that occur following SFN treatment, and likely regulate the acetylation status of non-histone proteins in addition to α-tubulin, HSP90, and the androgen receptor. A novel competing pathway has been proposed involving sequestration by 14-3-3 versus Pin1-mediated degradation of HDAC3, but further details of the model await further study.
Cell culture and reagents
Human HCT116 colon cancer cells (ATCC, Manassas, VA) were cultured at 37°C, 5% CO2 in McCoy's 5A medium (Life Technologies, Carlsbad, CA) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. SFN (Toronto Research Chemicals Inc. North York, ON, Canada) was prepared in DMSO and stored at a stock concentration of 10 mg/mL at -20°C. Chemical inhibitors leupeptin, ALLN, MG-132 (Sigma, St. Louis, MO) and PYR-41 (Calbiochem, San Diego, CA), were dissolved in DMSO (10 mM) and small aliquots (30 μl) were stored at -20°C. Z-VAD (OMe)-FMK was from SM Biochemicals LLC (Anaheim, CA). Cycloheximide and actinomycin D were purchased from Sigma (St. Louis, MO).
Cells in the exponential growth phase were plated at a cell density of 5,000 cells per well in 96-well tissue culture plates. After attachment overnight, cells were treated with 15 μM SFN for selected times i.e., 2, 24, 48 and 72 h. At these time points cell viability was determined using the MTT assay, as described previously , and cell number was counted using a Neubauer chamber.
Cells in the exponential growth phase were plated at a cell density of 0.1 × 106 cells in 60-mm culture dishes and treated with 0 (DMSO) or 15 μM SFN. Adherent and non-adherent cells were collected at different time points i.e., 3, 6, 9, 24, 48 and 72 h in cold PBS, fixed in 70% ethanol, and stored at 4°C for at least 48 h. Fixed cells were washed with PBS once and resuspended in propidium iodide (PI)/Triton X-100 staining solution containing RNaseA. Samples were incubated in the dark for 30 min before cell cycle analysis. DNA content was detected using EPICS XL Beckman Coulter and analyses of cell distribution in the different cell cycle phases were performed using Multicycle Software (Phoenix Flow Systems, San Diego, CA).
Cells in the exponential growth phase were plated at a cell density of 0.1 × 106 cells in 60-mm culture dishes. After overnight incubation cells were treated with either 0 (DMSO) or 15 μM SFN. In some experiments a range of SFN concentrations was used (0, 10, 15, 25, 35 μM). Adherent and non-adherent cells were harvested by trypsinization at different time points, ranging from 2 to 72 h, and then washed with ice-cold PBS. Whole-cell extracts were prepared using lysis buffer containing 20 mM (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, and 1 μg/ml leupeptin. The harvested cell pellet obtained after centrifugation was resuspended in lysis buffer and frozen at -80°C for at least 15 min, thawed on ice, vortexed for 30s and centrifuged at 13,200 × g for 5 min. To study the reversibility of SFN effects, 0.1 × 106 cells in 60-mm culture dishes were treated with DMSO or 15 μM SFN for 6 or 24 h, and the media was replaced with fresh growth medium (containing no SFN) until harvest. Whole-cell extracts were prepared at 6, 24, 48 and 72 h, and samples were frozen at -80°C until further use. Cytoplasmic and nuclear lysates were prepared using NE-PER® Nuclear & cytoplasmic extraction reagent (#78833, Thermo scientific, Rockford, IL). The insoluble fraction was dissolved in SDS lysis buffer containing 65 mM Tris-HCl, pH 8.0, 2% SDS, 50 mM DTT, and 150 mM NaCl. Protease (Roche) and phosphatase (Sigma, St. Louis, MO) inhibitor cocktails were added immediately before use. Protein concentration of cell lysates was determined using the BCA assay (Pierce, Rockford, IL).
In vitro HDAC activity
HDAC activity was measured from whole cell lysates using the Fluor-de-Lys HDAC activity assay kit (Biomol, Plymouth Meeting, PA), as reported before . Incubations were performed at 37°C with 10 μg of whole-cell extracts along with the fluorescent substrate in HDAC assay buffer for 30 min. Assay developer was then added and the samples incubated at 37°C for another 30 min and read using a Spectra MaxGemini XS fluorescence plate reader (Molecular Devices), with excitation at 360 nm and emission at 460 nm. The results were expressed as AFU or AFU/μg protein.
Equal amounts of protein (20 μg/lane) were separated by SDS-PAGE on 4-12% Bis-Tris gel or 3-8% Tris acetate gel for larger proteins (NuPAGE, Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). Membranes were saturated with 2% BSA for 1 h, followed by overnight incubation at 4°C with primary antibodies against β-actin (1:50,000 Sigma, #A5441), casein kinase-IIα (1:200, Santa Cruz, #9030), cleaved caspase-3 (1:1000, Cell Signaling, #9661), acetyl histone H4K12 (1:500, Upstate, #07-595), histone H4 (1:1000, Cell Signaling, #2592), HDAC1 (1:200, Santa Cruz, #7872), HDAC2 (1:200, Santa Cruz, #7899), HDAC3 (1:200, Santa Cruz, #11417), HDAC4 (1:200, Cell Signaling, #2072), HDAC6 (1:200, Santa Cruz, #11420), HDAC8 (1:200, Santa Cruz, #11405), HDAC10 (1:200, Biovision, #3610-100), phosphoHDAC3 (1:1000, Cell Signaling, #3815), HDAC3 N-19 (1:200, Santa Cruz, #8138), N-Cor (1:1000, Abcam, #ab24552), p21WAF1 (1:1000, Cell Signaling, #2947), PARP (1:1000, Cell Signaling, #9542), phosphoSMRT (pS2410, kindly provided by Dr. Marty Mayo, Univ. of Virginia, 1:1000), Pin1 (1:1000, Millipore, #07-091), SMRT (1:600, Millipore, #04-1551), acetyl α-tubulin (1:2000, Sigma, #T6793), α-tubulin (1:1000, Abcam, #ab7291), ubiquitin (1:3000, BD Pharmingen, #550944), pan14-3-3 (1:500, Santa Cruz, #629), p-14-3-3(T232) and p-14-3-3(S58), both used at 1:500 dilution (Epitomics Inc., Burlingame, CA). After washing, membranes were incubated with respective horseradish peroxidase conjugated secondary antibodies (Bio-Rad, Hercules, CA) for 1 h. Immunoreactive bands were visualized via Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate (Perkin Elmer, Inc, Waltham, MA) and detected with FluorChem-8800 Chemiluminescence and Gel Imager (Alpha Innotech).
HCT116 cells were treated with either DMSO or 15 μM SFN with or without pre-treatment for 1 h with PYR-41 (50 nM). Cells were harvested after 6 or 24 h and either whole cell extracts or cytoplasmic and nuclear lysates from adherent and non-adherent cells were prepared as previously described. Protein concentration was determined by BCA assay (Pierce, Rockford, IL). Protein (500 μg) was precleared with Protein A Sepharose CL-4B (Amersham Biosciences) on a rotator at 4°C for 1.5 h. Pre-cleared supernatant was collected and immunoprecipitated overnight with anti-HDAC3 (2 μg, Santa Cruz, #11417) or anti-HDAC6 (2 μg, Santa Cruz, #11420) rabbit polyclonal antibody. Protein A Sepharose beads were collected and washed before immunoblotting with anti-HDAC3 (1:200), anti-SMRT (1:500), anti-phosphoSMRT (1:700), anti-Pin1 (1 μg/ml), anti-14-3-3 (1:500), and anti-casein kinase-IIα (1:100) antibodies. The supernatant depleted of HDAC3 and/or HDAC6 was collected and kept frozen at -80°C until used for HDAC activity assays. In some experiments, HDAC3 pulls-downs were followed by immunoblotting for p-14-3-3(T232) and p-14-3-3(S58), both at 1:250 dilution.
Overexpression and knock-down experiments
HDAC3 and HDAC6, as transfection-ready DNA in pCMV6-XL4 vector, and Pin1 siRNA (Trilencer-27) and control siRNA were from Origene (Rockville, MD). Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) at a ratio of 1:3-1:4 in reduced serum medium (OPTI-MEM, Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. SFN treatment started after 24 h of transfection. Immunoblotting was carried out with whole cell lysates prepared using lysis buffer.
The results of each experiment shown are representative of at least three independent assays. Where indicated, results were expressed as mean ± standard error (mean ± SE), and differences between the groups were determined using Student's t-test. For multiple comparisons, ANOVA followed by the Dunnett's test was performed using GraphPad Prism. A p-value <0.05 was considered as statistically significant, and indicated as such with an asterisk (*) in the corresponding figure.
We thank Andrew Quest (Faculty of Medicine, University of Chile, Santiago, Chile) and Siva Kolluri (Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR) for useful discussions. Marty Mayo (The University of Virginia, Charlottesville, VA) provided phospho-SMRT antibody. Work supported by NIH grants CA090890, CA65525, CA122906, CA122959 and CA80176. Experiments in the Cell Imaging and Analysis Core were supported by Award T32 ES007060 from the National Institute of Environmental Health Sciences. The content of this report is solely the responsibility of the authors and does not necessarily represent the official views of NIH.
- Sharma S, Kelly TK, Jones PA: Epigenetics in cancer. Carcinogenesis. 2010, 31: 27-36. 10.1093/carcin/bgp220PubMed CentralView ArticlePubMedGoogle Scholar
- Chi P, Allis CD, Wang GG: Covalent histone modifications - miswritten, misinterpretation and mis-erased in human cancer. Nat Rev Cancer. 2010, 10: 457-469. 10.1038/nrc2876PubMed CentralView ArticlePubMedGoogle Scholar
- Poke FS, Qadi A, Holloway AF: Reversing aberrant methylation patterns in cancer. Curr Med Chem. 2010, 17: 1246-1254. 10.2174/092986710790936329View ArticlePubMedGoogle Scholar
- Iorio MV, Croce CM: MicroRNAs in cancer: small molecules with a huge impact. J Clin Oncol. 2009, 27: 5848-5856. 10.1200/JCO.2009.24.0317PubMed CentralView ArticlePubMedGoogle Scholar
- Dueñas-González A, Lizano M, Candelaria M, Cetina L, Arce C, Cervera E: Epigenetics of cervical cancer: an overview and therapeutic perspectives. Mol Cancer. 2005, 4: 38- 10.1186/1476-4598-4-38PubMed CentralView ArticlePubMedGoogle Scholar
- Garske AL, Oliver SS, Wagner EK, Musselman CA, LeRoy G, Garcia BA, Kutateladze TG, Denu JM: Combinatorial profiling of chromatin binding modules reveals multisite discrimination. Nat Chem Biol. 2010, 6: 283-290. 10.1038/nchembio.319PubMed CentralView ArticlePubMedGoogle Scholar
- Buchwald M, Kramer OH, Heinzel T: HDACi - targets beyond chromatin. Cancer Lett. 2009, 280: 160-167. 10.1016/j.canlet.2009.02.028View ArticlePubMedGoogle Scholar
- Minucci S, Pelicci PG: Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer. 2006, 6: 38-51. 10.1038/nrc1779View ArticlePubMedGoogle Scholar
- Müller S, Krämer OH: Inhibitors of HDACs - effective drugs against cancer?. Curr Cancer Drug Targets. 2010, 10: 210-228. 10.2174/156800910791054149View ArticlePubMedGoogle Scholar
- Lane AA, Chabner BA: Histone deacetylase inhibitors in cancer therapy. J Clin Oncol. 2009, 27: 5459-5468. 10.1200/JCO.2009.22.1291View ArticlePubMedGoogle Scholar
- Lin Z, Murray PM, Ding Y, Denny WA, Ferguson LR: Quinazolines as novel anti-inflammatory histone deacetylase inhibitors. Mutat Res. 2010, 690: 81-88.View ArticlePubMedGoogle Scholar
- Ma X, Ezzeldin HH, Diasio RB: Histone deacetylase inhibitors: current status and overview of recent clinical trials. Drugs. 2009, 69: 1911-1934. 10.2165/11315680-000000000-00000View ArticlePubMedGoogle Scholar
- Huang BH, Laban M, Leung CH, Lee L, Lee CK, Salto-Tellez M, Raju GC, Hooi SC: Inhibition of histone deacetylase 2 increases apoptosis and p21Cip1/WAF1 expression, independent of histone deacetylase 1. Cell Death Differ. 2005, 12: 395-404. 10.1038/sj.cdd.4401567View ArticlePubMedGoogle Scholar
- Wilson AJ, Byun DS, Popova N, Murray LB, L'Italien K, Sowa Y, Arango D, Velcich A, Augenlicht LH, Mariadason JM: Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer. J Biol Chem. 2006, 281: 13548-13558. 10.1074/jbc.M510023200View ArticlePubMedGoogle Scholar
- Zhu P, Martin E, Mengwasser J, Schlag P, Janssen KP, Gottlicher M: Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis. Cancer Cell. 2004, 5: 455-463. 10.1016/S1535-6108(04)00114-XView ArticlePubMedGoogle Scholar
- Spurling CC, Godman CA, Noonan EJ, Rasmussen TP, Rosenberg DW, Giardina C: HDAC3 overexpression and colon cancer cell proliferation and differentiation. Mol Carcinog. 2007, 47: 137-147.View ArticleGoogle Scholar
- Ashktorab H, Belgrave K, Hosseinkhah F, Brim H, Nouraie M, Takikto M, Hewitt S, Lee EL, Dashwood RH, Smoot D: Global histone H4 acetylation and HDAC2 expression in colon adenoma and carcinoma. Dig Dis Sci. 2009, 54: 2109-2117. 10.1007/s10620-008-0601-7PubMed CentralView ArticlePubMedGoogle Scholar
- Wilson AJ, Byun DS, Nasser S, Murray LB, Ayyanar K, Arango D, Figueroa M, Melnick A, Kao GD, Augenlicht LH, Mariadason JM: HDAC4 promotes growth of colon cancer cells via repression of p21. Mol Biol Cell. 2008, 19: 4062-4075. 10.1091/mbc.E08-02-0139PubMed CentralView ArticlePubMedGoogle Scholar
- Senese S, Zaragoza K, Minardi S, Muradore I, Ronzoni S, Passafaro A, Bernard L, Draetta GF, Alcalay M, Seiser C: Role for histone deacetylase 1 in human tumor proliferation. Mol Cell Biol. 2007, 27: 4784-4795. 10.1128/MCB.00494-07PubMed CentralView ArticlePubMedGoogle Scholar
- Ma H, Nguyen C, Lee KS, Kahn M: Differential roles for the coactivators CBP and p300 on TCF/β-catenin-mediated survivin gene expression. Oncogene. 2005, 24: 3619-3631. 10.1038/sj.onc.1208433View ArticlePubMedGoogle Scholar
- Dashwood RH, Ho E: Dietary histone deacetylase inhibitors: from cells to mice to man. Semin Cancer Biol. 2007, 17: 363-369. 10.1016/j.semcancer.2007.04.001PubMed CentralView ArticlePubMedGoogle Scholar
- Davis CD, Ross SA: Dietary components impact histone modifications and cancer risk. Nutr Rev. 2007, 65: 88-94.View ArticlePubMedGoogle Scholar
- Rajendran P, Williams DE, Ho E, Dashwood RH: Metabolism as a key to histone deacetylase inhibition. Crit Rev Biochem Mol Biol. 2011, 46: 181-199. 10.3109/10409238.2011.557713PubMed CentralView ArticlePubMedGoogle Scholar
- Fahey JW, Zhang Y, Talalay P: Broccoli sprouts: and exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc Natl Acad Sci USA. 1997, 94: 10367-10372. 10.1073/pnas.94.19.10367PubMed CentralView ArticlePubMedGoogle Scholar
- Jeffery EH, Keck AS: Translating knowledge generated by epidemiological and in vitro studies into dietary cancer prevention. Mol Nutr Food Res. 2008, 52 (Suppl 1): S7-S17.PubMedGoogle Scholar
- Lai RH, Keck AS, Wallig MA, West LG, Jeffery EH: Evaluation of the safety and bioactivity of purified and semi-purified glucoraphanin. Food Chem Toxicol. 2008, 46: 195-202. 10.1016/j.fct.2007.07.015View ArticlePubMedGoogle Scholar
- Cheung KL, Kong AN: Molecular targets of dietary phenyl isothiocyanate and sulforaphane for cancer chemoprevention. AAPS J. 2010, 12: 87-97. 10.1208/s12248-009-9162-8PubMed CentralView ArticlePubMedGoogle Scholar
- Traka MH, Spinks CA, Doleman JF, Melchini A, Ball RY, Mills RD, Mithen RF: The dietary isothiocyanate sulforaphane modulates gene expression and alternative gene splicing in a PTEN null preclinical murine model of prostate cancer. Mol Cancer. 2010, 9: 189- 10.1186/1476-4598-9-189PubMed CentralView ArticlePubMedGoogle Scholar
- Myzak MC, Karplus PA, Chung FL, Dashwood RH: A novel mechanism of chemoprotection by sulforaphane: inhibition of histone deacetylase. Cancer Res. 2004, 64: 5767-5774. 10.1158/0008-5472.CAN-04-1326View ArticlePubMedGoogle Scholar
- Myzak MC, Hardin K, Wang R, Dashwood RH, Ho E: Sulforaphane inhibits histone deacetylase activity in BPH-1, LnCaP and PC-3 prostate epithelial cells. Carcinogenesis. 2006, 27: 811-819.PubMed CentralView ArticlePubMedGoogle Scholar
- Pledgie-Tracy A, Sobolewski MD, Davidson NE: Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines. Mol Cancer Ther. 2007, 6: 1013-1021.View ArticlePubMedGoogle Scholar
- Ma X, Fang Y, Beklemisheva A, Dai W, Feng J, Ahmed T, Liu D, Chiao JW: Phenylhexyl isothiocyanate inhibits histone deacetylases and remodels chromatin to induce growth arrest in human leukemia cells. Int J Oncol. 2006, 28: 1287-1293.PubMedGoogle Scholar
- Kwak MK, Kensler TW: Targeting NRF2 signaling for cancer chemoprevention. Toxicol Appl Pharmacol. 2010, 244: 66-76. 10.1016/j.taap.2009.08.028PubMed CentralView ArticlePubMedGoogle Scholar
- Telang U, Brazeau DA, Morris ME: Comparison of the effects of phenethyl isothiocyanate and sulforaphane on gene expression in breast cancer and normal mammary epithelial cells. Exp Bio Med. 2009, 234: 287-295.View ArticleGoogle Scholar
- Herman-Antosiewicz A, Xiao H, Lew KL, Singh SV: Induction of p21 protein protects against sulforaphane-induced mitotic arrest in LNCaP human prostate cancer cell line. Mol Cancer Ther. 2007, 6: 1673-1681. 10.1158/1535-7163.MCT-06-0807View ArticlePubMedGoogle Scholar
- Myzak MC, Dashwood WM, Orner GA, Ho E, Dashwood RH: Sulforaphane inhibits histone deacetylase in vivo and suppresses tumorigenesis in Apcmin mice. FASEB J. 2006, 20: 506-508.PubMed CentralPubMedGoogle Scholar
- Dashwood RH, Myzak MC, Ho E: Dietary HDAC inhibitors: time to rethink weak ligands in cancer chemoprevention?. Carcinogenesis. 2006, 27: 344-349. 10.1093/carcin/bgi253View ArticlePubMedGoogle Scholar
- Singh AV, Xiao D, Lew KL, Dhir R, Singh SV: Sulforaphane induces caspase-mediated apoptosis in cultured PC-3 human prostate cancer cells and retards growth of PC-3 xenografts in vivo. Carcinogenesis. 2004, 25: 83-90.View ArticlePubMedGoogle Scholar
- Escaffit F, Vaute O, Chevillard-Briet M, Segui B, Takami Y, Nakayama T, Troucher D: Cleavage and cytoplasmic relocalization of histone deacetylase 3 are important for apoptosis progression. Mol Cell Biol. 2007, 27: 554-567. 10.1128/MCB.00869-06PubMed CentralView ArticlePubMedGoogle Scholar
- Scott FL, Fuchs GJ, Boyd SE, Denault JB, Hawkins CJ, Dequiedt F, Salvesen GS: Caspase-8 cleaves histone deacetylase 7 and abolishes its transcription repressor function. J Biol Chem. 2008, 283: 19499-19510.PubMed CentralView ArticlePubMedGoogle Scholar
- Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, Yoshida M, Wang XF, Yao TP: HDAC6 is a microtubule-associated deacetylase. Nature. 2002, 417: 455-458. 10.1038/417455aView ArticlePubMedGoogle Scholar
- Matthias P, Yoshida M, Khochbin S: HDAC6 a new cellular stress surveillance factor. Cell Cycle. 2008, 7: 7-10.View ArticlePubMedGoogle Scholar
- Buglio D, Mamidipudi V, Khaskhely NM, Brady H, Heise C, Besterman J, Martell RE, MacBeth K, Younes A: The class-I HDAC inhibitor MGCD0103 induces apoptosis in Hodgkin lymphoma cell lines and synergizes with proteasome inhibitors by an HDAC6-independent mechanism. Br J Haematol. 2010, 151: 387-396. 10.1111/j.1365-2141.2010.08342.xPubMed CentralView ArticlePubMedGoogle Scholar
- Jagannath S, Dimopoulos MA, Lonial S: Combined proteasome and histone deacetylase inhibition: a promising synergy for patients with relapsed/refractory multiple myeloma. Leuk Res. 2010, 34: 1111-1118. 10.1016/j.leukres.2010.04.001View ArticlePubMedGoogle Scholar
- Dasmahapatra G, Lembersky D, Kramer L, Fisher RI, Friedberg J, Dent P, Grant S: The pan-HDAC inhibitor vorinostat potentiates the activity of the proteasome inhibitor carfilzomib in human DLBCL cells in vitro and in vivo. Blood. 2010, 115: 4478-4487. 10.1182/blood-2009-12-257261PubMed CentralView ArticlePubMedGoogle Scholar
- Heider U, Rademacher J, Lamottke B, Mieth M, Moebs M, von Metzler I, Assaf C, Sezer O: Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteasome inhibitor bortezomib in cutaneous T-cell lymphoma. Eur J Haematol. 2009, 82: 440-449. 10.1111/j.1600-0609.2009.01239.xView ArticlePubMedGoogle Scholar
- Yang Y, Kitagaki J, Dai RM, Tsai YC, Lorick KL, Ludwig RL, Pierre SA, Jensen JP, Davydov IV, Oberoi P, Li C-CH, Kenten JH, Beutler JA, Vousden KH, Weissman AM: Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Res. 2007, 67: 9472-9481. 10.1158/0008-5472.CAN-07-0568View ArticlePubMedGoogle Scholar
- Jonas BA, Privalsky ML: SMRT and N-Cor corepressors are regulated by distinct kinase signaling pathways. J Biol Chem. 2004, 279: 54676-54686. 10.1074/jbc.M410128200PubMed CentralView ArticlePubMedGoogle Scholar
- Nebbioso A, Manzo F, Miceli M, Conte M, Manente L, Baldi A, De Luca A, Rotili D, Valente S, Mai A, Usiello A, Gronenmeyer H, Altucci L: Selective class II HDAC inhibitors impair myogenesis by modulating the stability and activity of HDAC-MEF2 complexes. EMBO Reports. 2009, 10: 776-782. 10.1038/embor.2009.88PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou Y, Gross W, Hong SH, Privalsky ML: The SMRT corepressor is a target of phosphorylation by protein kinase CK2 (casein kinase II). Mol Cell Biochem. 2001, 220: 1-13. 10.1023/A:1011087910699PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang X, Ozawa Y, Lee H, Wen YD, Tan TH, Wadzinski BE, Seto E: Histone deacetylase 3 (HDAC3) activity is regulated by interaction with protein serine/threonine phosphatase 4. Genes Dev. 2005, 197: 827-839. 10.1101/gad.1286005.View ArticleGoogle Scholar
- Stanya KJ, Liu Y, Means AR, Kao HY: Cdk2 and Pin1 negatively regulate the transcriptional corepressor SMRT. J Cell Biol. 2008, 183: 49-61. 10.1083/jcb.200806172PubMed CentralView ArticlePubMedGoogle Scholar
- Obsilova V, Silhan J, Boura E, Teisinger J, Obsil T: 14-3-3 proteins: a family of versatile molecular regulators. Physiol Res (Suppl 3). 2008, 57: S11-S21.Google Scholar
- Healy S, Khan DH, Davie JR: Gene expression regulation through 14-3-3 interactions with histones and HDACs. Discov Med. 2011, 59: 349-358.Google Scholar
- Zhou J, Shao Z, Kerkela R, Ichijo H, Muslin AJ, Pombo C, Force T: Serine 58 of 14-3-3ζ is a molecular switch regulating ASK1 and oxidant stress-induced cell death. Mol Cell Biol. 2009, 29: 4167-4176. 10.1128/MCB.01067-08PubMed CentralView ArticlePubMedGoogle Scholar
- Nishino TG, Miyazaki M, Hoshino H, Miwa Y, Horinouchi S, Yoshida M: 14-3-3 regulates the nuclear import of class IIa histone deacetylases. Biochem Biophys Res Commun. 2008, 377: 852-856. 10.1016/j.bbrc.2008.10.079View ArticlePubMedGoogle Scholar
- Pappa G, Bartsch H, Gerhauser C: Biphasic modulation of cell proliferation by sulforaphane at physiologically relevant exposure times in a human colon cancer cell line. Mol Nutr Food Res. 2007, 51: 977-984. 10.1002/mnfr.200700115View ArticlePubMedGoogle Scholar
- Gibbs A, Schwartzman J, Deng V, Alumkal J: Sulforaphane destabilizes the androgen receptor in prostate cancer cells by inactivating histone deacetylase 6. Proc Natl Acad Sci USA. 2009, 106: 16663-16668. 10.1073/pnas.0908908106PubMed CentralView ArticlePubMedGoogle Scholar
- Clarke JD, Dashwood RH, Ho E: Differential effects of sulforaphane on histone deacetylases, cell cycle arrest and apoptosis in normal prostate epithelial cells and cancerous prostate cells (PC3). Mol Nutr Food Res. 2011Google Scholar
- Varlakhanova N, Hahm JB, Privalsky ML: Regulation of SMRT corepressor dimerization and composition by MAP kinase phosphorylation. Mol Cell Endocrinol. 2011, 332: 180-188. 10.1016/j.mce.2010.10.010PubMed CentralView ArticlePubMedGoogle Scholar
- Clarke JD, Dashwood RH, Ho E: Multi-targeted prevention of cancer by sulforaphane. Cancer Lett. 2008, 269: 291-304. 10.1016/j.canlet.2008.04.018PubMed CentralView ArticlePubMedGoogle Scholar
- Myzak MC, Dashwood RH: Chemoprotection by sulforaphane: keep one eye beyond Keap 1. Cancer Lett. 2006, 233: 208-218. 10.1016/j.canlet.2005.02.033PubMed CentralView ArticlePubMedGoogle Scholar
- Kimura MT, Irie S, Shoji-Hoshino S, Mukai J, Nadano D, Oshimura M, Sato TA: 14-3-3 is involved in p75 neutrophin receptor-mediated signal transduction. J Biol Chem. 2001, 276: 17291-17300. 10.1074/jbc.M005453200View ArticlePubMedGoogle Scholar
- Bardai FH, D'Mello SR: Selective toxicity by HDAC3 in neurons: regulation by Akt and GSK3β. J Neurosci. 2011, 31: 1746-1751. 10.1523/JNEUROSCI.5704-10.2011PubMed CentralView ArticlePubMedGoogle Scholar
- Yamaguchi T, Cubizolles F, Zhang Y, Reichert N, Kohler H, Seiser C, Matthias P: Histone deacetylases 1 and 2 act in concert to promote G1-to-S progression. Genes Dev. 2010, 24: 455-469. 10.1101/gad.552310PubMed CentralView ArticlePubMedGoogle Scholar
- Simboeck E, Sawicka A, Zupkovitz G, Senese S, Winter S, Dequiedt F, Ogris E, Di Croce L, Chiocca S, Seiser C: A phosphorylation switch regulates the transcriptional activation of cell cycle regulator p21 by histone deacetylase inhibitors. J Biol Chem. 2010, 285: 41062-41073. 10.1074/jbc.M110.184481PubMed CentralView ArticlePubMedGoogle Scholar
- Nian H, Delage B, Pinto JT, Dashwood RH: Allyl mercaptan, a garlic-derived organosulfur compound, inhibits histone deacetylase and enhances Sp3 binding on the P21WAF promoter. Carcinogenesis. 2008, 29: 1816-1824. 10.1093/carcin/bgn165PubMed CentralView ArticlePubMedGoogle Scholar
- Nian H, Bisson WH, Dashwood WM, Pinto JT, Dashwood RH: Alpha-keto acid metabolites of organoselenium compounds inhibit histone deacetylase activity in human colon cancer cells. Carcinogenesis. 2009, 30: 1416-1423. 10.1093/carcin/bgp147PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.