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Role of Class I and Class II histone deacetylases in carcinoma cells using siRNA
BBRC Biochemical and Biophysical Research Communications 310 (2003) 529–536 www.elsevier.com/locate/ybbrc
Role of Class I and Class II histone deacetylases in carcinoma cells using siRNAq Keith B. Glaser,* Junling Li, Michael J. Staver, Ru-Qi Wei, Daniel H. Albert, and Steven K. Davidsen Cancer Research, R47J-AP9, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064-6121, USA Received 31 July 2003
Abstract The role of the individual histone deacetylases (HDACs) in the regulation of cancer cell proliferation was investigated using siRNA-mediated protein knockdown. The siRNA for HDAC3 and HDAC1 demonstrated significant morphological changes in HeLa S3 consistent with those observed with HDAC inhibitors. SiRNA for HDAC 4 or 7 produced no morphological changes in HeLa S3 cells. HDAC1 and 3 siRNA produced a concentration-dependent inhibition of HeLa cell proliferation; whereas, HDAC4 and 7 siRNA showed no effect. HDAC3 siRNA caused histone hyperacetylation and increased the percent of apoptotic cells. These results demonstrate that the Class I HDACs such as HDACs 1 and 3 are important in the regulation of proliferation and survival in cancer cells. These results and the positive preclinical results with non-specific inhibitors of the HDAC enzymes provide further support for the development of Class I selective HDAC inhibitors as cancer therapeutics. Ó 2003 Elsevier Inc. All rights reserved. Keywords: siRNA; Histone deacetylase (HDAC); Apoptosis; Proliferation; Histone acetylation; Chromatin remodeling; Histone deacetylase inhibitors; Gene knockdown
Acetylation of nucleosomal histones in part regulates gene transcription in most cells. Differential acetylation of nucleosomal histones results in either transcriptional activation (hyperacetylation) or repression (hypoacetylation) [1,2]. This phenomenon, chromatin remodeling, is tightly regulated by the balance of histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities [1,2]. The role of chromatin remodeling in carcinogenesis is based primarily on experiments with HDAC inhibitors, e.g., sodium butyrate and trichostatin A. HDAC inhibitors induce the hyperacetylation of q Abbreviations: HDAC, histone deacetylase; siRNA, small interfering RNA; nt, nucleotide; HAT, histone acetyltransferase; RA, retinoic acid; APL, acute promyelocytic leukemia; TGFb, transforming growth factor b; TSA, trichostatin A; PBS, phosphate-buffered saline; DTT, dithiothrietol; TBS, tris-buffered saline; FACS, fluorescence activated cell sorting; SAHA, suberoylanilide hydroxamic acid; HeLa S3, S3 variant of HeLa cervical carcinoma; T24, transitional cell bladder carcinoma. * Corresponding author. Fax: 1-847-935-3622. E-mail address: [email protected] (K.B. Glaser).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.043
nucleosomal histones, resulting in the expression of repressed genes that produce growth arrest, terminal differentiation, and/or apoptosis in carcinoma cells [2–4]. These pharmacological properties of HDAC inhibitors have generated significant interest in HDACs as targets for anticancer therapy [3–6]. The HDACs belong to a deacetylase superfamily and can be divided into two distinct classes based upon their structure; the Class I (HDACs 1, 2, 3, 8, and 11) and Class II (HDACs 4, 5, 6, 7, 9, and 10) enzymes [7–10]. HDACs do not appear to function independently in the regulation of transcription but in concert with multiprotein complexes that are recruited to specific regions in the genome that in turn generate the unique spectrum of expressed and silenced genes [11]. Known repressors are multiprotein complexes that contain DNA binding proteins (e.g., NCoR, SMRT, MEF, MeCP2, sin3A, etc.) that commonly use histone deacetylases to repress transcription and block the function of some tumor suppressor genes [3,11]. HDAC inhibitors are able to derepress these genes, resulting in antiproliferative effects
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in vitro and antitumor effects in vivo [3,5,12,13]. However, since there are 11 members in the HDAC family, the question as to which of the HDAC(s) are involved in the regulation of genes that lead to the antiproliferative and antitumor phenotype currently remains unanswered. Attempts to address this question have been made in yeast where deletion mutants for the three classes in the deacetylase family were evaluated by transcriptional profiling [14]. To address this question, we used specific small interfering double stranded RNA, siRNA, molecules to selectively knockdown individual human HDACs. In this paper, we describe the selective knockdown of HDACs 1, 2, 3, 4, and 7 in HeLa S3 cells. The knockdown of HDACs 1 and 3 produced a significant morphological phenotype similar to that observed with HDAC inhibitor treatment of these cells; whereas, knockdown of HDACs 4 and 7 produced no changes in cell morphology. The knockdown of HDACs 1 and 3 was antiproliferative; whereas, knockdown of HDACs 4 and 7 was without effect. These data suggest that the Class I HDACs, HDACs 1 and 3, are essential to the proliferation and survival of mammalian carcinoma cells. Mechanistic studies demonstrated that knockdown of HDAC3 resulted in hyperacetylation of histone H3 and apoptosis in HeLa S3 cells, supporting the antiproliferative effects of Class I HDAC knockdown.
Materials and methods Cell culture and reagents. Human cervical carcinoma cells, HeLa S3, were purchased from ATCC (Bethesda, MD), and were grown according to ATCC guidelines. Cells were trypsinized and seeded at 1 106 cells per 100 mm dish, and allowed to grow overnight at 37 °C with 95%/5% air/CO2 and 80% relative humidity. Cells were treated with the HDAC siRNA for 48 h. SiRNAs were designed randomly for HDAC1 (Xeragon, Huntsville, AL) or using the Dharmacon (Colorado Springs, CO) protocol of 19 nucleotides after an AA in the DNA sequence, at least 75 nucleotides from the ATG, and between 30 and 70% GC content. The position of the siRNA on the target gene is designated by the nucleotide (nt) at which the siRNA (AA in most cases) is located on the target gene. SiRNAs were prepared according to the manufacturer’s instructions (stock concentration 20 lM in molecular biology grade water). Oligofectamine was used for all siRNA transfections (Invitrogen, San Diego, CA). Transfection of HeLa S3 cells with siRNA. HeLa S3 cells were plated at 1 106 cells/100 mm tissue culture dish approximately 24 h before transfection and just prior to transfection the medium was exchanged for 3.6 ml OptiMEM. The siRNA was appropriately diluted to a final volume of 200 ll with OptiMEM (Gibco-BRL) and incubated at room temperature for 5 min. Oligofectamine (Invitrogen) amount was optimized for transfection of HeLa S3 cells using a FITClabeled siRNA (100 nM) and fixed at 20 ll per 100 mm dish which gave >90% transfection efficiency without cytotoxicity. The diluted siRNA was then added to the oligofectamine solution (20 ll in 180 ll of Op_ in at room tiMEM), mixed by inversion, and incubated for 30 m temperature. The siRNA:oligofectamine solution was then added to the HeLa S3 cells in a dropwise fashion over the entire plate, 400 ll per 100 mm dish containing 3.6 ml of OptiMEM. The cells were transfected
for 4–6 h at 37 °C in a humidified CO2 incubator. After transfection the cell medium was removed and replaced with complete growth medium and the cells were allowed to grow for an additional 48 h. Cells were rinsed 1 with phosphate buffered saline (PBS), harvested by scraping into PBS, and centrifuged at 700g for 10 min and cell pellets were stored at )80 °C until processing. Cell proliferation assay. To determine the effects of siRNA knockdown on cell proliferation, 48 h post-transfection the cells were collected by trypsinization, counted in a hemacytometer with trypan blue dye, and plated at 2500 viable cells per well into a 96-well tissue culture dish in a final volume of 200 ll. Seventy two hours later the medium was replaced with alamarBlue solution (10% final in PBS with a final volume of 100 ll per well) and incubated on cells for 4 h at 37 °C in a CO2 incubator. The effects on proliferation were determined by fluorescence measurement (544 Ex: 590 Em) in an fmax fluorescence plate reader (Molecular Devices, San Diego, CA). Percent inhibition of proliferation was determined by comparison of treated cells to control untreated cells with the background fluorescence without cells subtracted out. Each experimental point was obtained in triplicate. Electrophoresis and Western blotting. SDS–PAGE was performed using 4–12% Novex NuPAGE gels with the 4-morpholinepropanesulfonic (MES) acid buffer system (Invitrogen, San Diego, CA). After electrophoresis, the proteins were transferred to PVDF membranes (Invitrogen) and blocked for 2 h with 10% non-fat milk in Tris-buffered saline (TBS). After transfer the residual protein in the gels was stained with Coomassie Blue and the major protein bands were used to confirm equivalent protein loading. Blots that demonstrated <20% variation in protein loading based on Coomassie blue stained gels were used in these studies. Primary antibodies were diluted in 10% non-fat milk in TBS (anti-HDAC1 (1 lg/ml) was from UBI (Lake Placid, NY), anti-HDAC3 (1:5000 dilution) was from Abcam (Cambridge, UK), and anti-HDAC 2, 4, and 7 (1:1000 dilution) were from Santa Cruz Biotechnologies (San Diego, CA). All primary antibodies were incubated with membranes overnight at 4 °C. The membranes were then washed 3 with 0.1% Tween 20 in TBS. Secondary (HRP conjugated) antibodies (Biosource International, Camarillo, CA) were diluted in 10% non-fat milk in TBS (1:5000) and incubated for 2 h with shaking. The membranes were then washed 10 5 min in 0.1% Tween 20 in TBS. Proteins were visualized by enhanced chemiluminescence with the Pierce Dura SuperSignal substrate (Pierce, Rockford, IL). FACS analysis of cell cycle and acetylated histone H3. HeLa S3 cells were cultured at 0.5 106 cells per 100 mm dish overnight. Cells were transfected for 4 hr with oligofectamine as described above with the exception that after the transfection, medium was added with 20% serum and the cells were cultured for 48 h, then split 1:6 and cultured for various times. At specific time points the cells were trypsinized, fixed with 80% ethanol, washed two times with PBS, and then incubated with propidium iodide (50 lg/ml). DNA content was determined by fluorescence cell analysis using a FACSCalibur (Becton–Dickinson) flow cytometer and cell cycle distribution was analyzed with CellQuest software. For histone H3 analysis, HeLa S3 cells were treated as for the cell cycle analysis, trypsinized, washed two times with PBS, permeabilized with 0.5% Triton X-100 for 5 min, washed two times with PBS, and incubated with anti-acetyl histone H3 polyclonal antibody (UBI, Lake Placid, NY) at 1 lg per 106 cells for 45 min. The cells were then washed two times with PBS and then incubated with anti-rabbit-biotin conjugated secondary antibody (Jackson Immuno Research) at 1 lg per 106 cells for 45 min. The cells were washed two times with PBS and incubated with streptavidin-PE (BD Biosciences) at 0.5 lg per 106 cells for 45 min. The labeled cells were washed two times with PBS prior to FACS analysis. Hyperacetylation of histone H3 was analyzed on a FACSCalibur (Becton–Dickinson) flow cytometer and the data were analyzed using CellQuest software. Rabbit IgG and no primary antibody were used as negative controls for background fluorescence.
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Results
siRNA silencing of HDACs in HeLa S3 cells
siRNA silencing of HDAC1
siRNAs were obtained for HDACs 1–8 and evaluated in HeLa S3 cells. Western blot analysis detected the presence of HDACs 1, 2, 3, 4, and 7 in HeLa S3 cells. The presence of HDACs 5, 6, and 8 could not be confirmed using the existing anti-HDAC antibodies or were not present in sufficient quantity in HeLa S3 cells and were not studied further. As shown in Fig. 1, dose-responsive knockdown of HDAC protein was observed for HDACs 1 (nt 861), 3 (nt 483), 4 (nt 337), and 7 (nt 186). HDAC2 siRNA (2 out of 4 reduced proteins at 100 nM) did not demonstrate a dose-responsive knockdown and were therefore not evaluated further (data not shown). Densitometry of these blots at the 30 nM siRNA concentrations is shown in Fig. 2. Maximal reduction of the HDAC proteins was between 80% and 97% of the oligofectamine treated controls. This occurred at 30 nM for most siRNA with the exception of HDAC4 where 100 nM siRNA was needed to reduce the protein amount by 89% (data not shown). Reduction in the amount of the targeted HDACs was specific for that isozyme as demonstrated by cross-blotting experiments for the other HDACs present in HeLa S3 whole cell lysates (data not shown). From these knockdown experiments, two of the Class I and two of the Class II HDACs were effectively knocked down and could be used in further studies for the evaluation of cellular phenotype.
Seven siRNAs were designed against the HDAC1 gene to recognize the sequences starting with the ATG (nucleotide, nt 1), nt 89, nt 120, nt 519 (containing the active site histidine), nt 861, nt 949, and nt 1321. Screening of these siRNAs at a concentration of 100 nM, 48 h after transfection into HeLa S3 cells, by Western blot analysis demonstrated that two of the seven siRNAs (nt 861 and nt 1321) effectively knocked down protein (49% and 48% reduction, respectively, based on densitometry). The oligofectamine and the scrambled siRNA controls appeared similar and showed only a slight reduction of HDAC1 compared with the non-treated controls. The reduction caused by HDAC1-nt 861 was subsequently demonstrated to be less variable and dose responsive and was therefore used in all subsequent studies with HDAC1 (Fig. 1).
Morphological changes induced by HDAC siRNAs Morphological assessment of cells after 48 h treatment with siRNAs against HDAC 1 and 3 demonstrated altered cell structure (Fig. 3); whereas, siRNAs against HDACs 4 and 7 did not affect the cell morphology.
Fig. 1. Concentration response for siRNA versus HDACs 1 (nt 861), 3 (nt 483), 4 (nt 337), and 7 (nt 186). A concentration response was evaluated for each siRNA and their respective scrambled siRNA from 10 to 100 nM. Oligo, oligofectamine reagent control. Cells were transfected and processed as described in Materials and methods. Forty eight hours after transfection the cells were processed into whole cell lysates analyzed by SDS–PAGE and protein knockdown was evaluated by Western blot analysis. Each blot is a representative of several showing the same pattern of protein knockdown for the respective siRNA. *Low protein load based on Coomassie Blue staining of gel as described in Materials and methods.
Fig. 2. Densitometry of siRNA knockdown of HDACs. The blots from Fig. 1 were quantitated using UN-SCAN-IT software version 5.1 (Silk Scientific, Oren, UT). The % control values represent the percentage of siRNA-treated sample relative to the oligofectamine-treated sample.
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Again, the scramble siRNA control did not produce a morphological change in the HeLa S3 cells (Fig. 3D). The non-selective HDAC inhibitor TSA (1 lM) produced similar cell shape changes as the knockdown of HDAC3 (Fig. 3E), and SAHA (15 lM) produced a morphological change between TSA and the HDAC 1 siRNA (Fig. 3F). These morphological changes are consistent with the observed alteration in cytoskeletal gene expression, e.g., gelsolin, which occurs with small molecule HDAC inhibitors [15]. Antiproliferative response of HeLa S3 cells to HDAC siRNAs
Fig. 3. Effect of HDAC siRNA and small molecule HDAC inhibitors on HeLa S3 cell morphology. Photomicrographs were taken of cells treated for 48 h with either siRNA (100 nM, B, D), scrambled siRNA (100 nM, A, C) or the HDAC inhibitors TSA (1 lM, E) or SAHA (15 lM, F). Each photomicrograph is a representative of several experiments showing similar morphological changes.
When HDAC1 is knocked down by siRNA the cells take on a flattened morphology with extensive focal contacts (see arrows in Fig. 3A). The scramble siRNA does not produce this phenotype on the cells even at 200 nM (Fig. 3B). Likewise, the siRNA against HDAC3 produced a profound morphological change into spindle like cells with elongation of the filopodia (Fig. 3C).
HeLa S3 cells were transfected with siRNAs for HDAC 1, 3, 4, and 7 for 48 h to allow protein knockdown, the cells were then trypsinized and normalized to 2500 viable cells per well in 96 well tissue culture dishes and allowed to grow for an additional 72 h. This protocol was used to minimize the adverse effects of transfection reagents on cell growth and to ensure the knockdown of the target protein prior to evaluation of the effects on proliferation. Knockdown of HDAC7 did not have an antiproliferative effect on HeLa S3 cells, even at 100 nM; in contrast, knockdown of HDAC1 produced a concentration-dependent inhibition of cell proliferation (Fig. 4A). The scrambled duplexes of the HDAC1 and HDAC7 siRNAs had no effect on proliferation. Neither the HDAC4 siRNA nor the HDAC4 scramble siRNA significantly inhibited proliferation (<10%) at concentrations up to 100 nM; however, knockdown of HDAC3 demonstrated a concentration-dependent inhibition of proliferation between 1 and 30 nM with no increase in inhibition observed between 30 and 100 nM (Fig. 4B). These results demonstrate that cells with reduced amounts of the Class I HDACs 1 and 3 proliferate at a slower rate than control cells or cells with reduced amounts of Class II HDACs 4 and 7.
Fig. 4. Effect of siRNA and scrambled siRNA on the proliferation of HeLa S3 cells. Forty eight hours after transfection, HeLa S3 cells were detached by trypsinization, counted, and plated at 2500 cells/well in a 96-well tissue culture plate. Seventy two hours later cell proliferation was assessed using alamarBlue (modified MTT assay) by adding a 10% solution to each well for 2–4 h at 37 °C in a tissue culture incubator. (A) Effect of HDAC1 and 7 siRNA on proliferation. (B) Effect of HDAC3 and 4 siRNA on proliferation. Each graph is a representative of two separate experiments with each point carried out in triplicate showing similar results.
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Effect of HDAC knockdown on histone H3 acetylation and apoptosis To demonstrate that the knockdown of an HDAC would result in biochemical changes similar to those observed with small molecule inhibitors of HDACs, the effects of HDAC3 siRNA were studied on histone H3 acetylation and induction of apoptosis (sub-G0/G1 population) by FACS analysis. A time course analysis of histone H3 acetylation by FACS demonstrated that the maximal difference between scramble and siRNA effects were observed 72 h post-transfection (Fig. 5A). Relative to untreated and scramble treated control cells, the HDAC3 siRNA produced a concentration-dependent increase in acetylation of histone H3 between 10 and 200 nM. The amount of acetylated histone H3 in the siRNA-treated cells was similar to those produced in SAHA-treated cells. The siRNA for HDAC 1 could not
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be evaluated in the hyperacetylation or apoptosis assays due to a high background activity seen with the HDAC1 scrambled siRNA. HDAC3 siRNA treatment at 72 h post-transfection demonstrated the maximal response of apoptosis, largest fraction of adherent cells undergoing apoptosis, as judged by the accumulation of cells in the sub-G0/G1 population (Fig. 5B). Similar to the results observed for histone H3 acetylation, there was a concentration-dependent induction of apoptosis with the siRNA that was not observed for the scrambled duplex (Fig. 5C). These data agree well with the anti-proliferative response that was measured 48 h past the maximal induction of apoptosis in these cells. As only adherent cells were evaluated for apoptosis, the longer time points (>72 h) demonstrate a decrease in sub-G0/G1 cells possibly due to the loss of late apoptotic cells from the adherent population.
Fig. 5. FACS analysis of accumulation of acetylated histone H3 and induction of apoptosis in HeLa S3 cells by the HDAC3 siRNA. (A) Accumulation of acetylated histone H3 in a concentration responsive manner for HDAC3 siRNA. Cells were taken 72 h post-transfection for the maximal response of HeLa S3 cells. The accumulation of acetylated histone H3 was determined relative to the effect of the scrambled siRNA at each concentration. (B) Effect of HDAC 3 siRNA and scramble on the accumulation of cells in the sub-G0/G1 population, apoptotic population. HeLa S3 cells were stained with propidium iodide (50 lg/ml) prior to analysis of cell cycle distribution by FACS. Fraction of the cell population in each part of the cell cycle was determined using CellQuest software. Each graph is a representative of at least two separate experiments showing similar results.
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Discussion The HDACs are a large family of related proteins that function in part to regulate the transcription of genes through posttranslational modification of histones and possibly other proteins such as transcription factors [3]. HDAC recruitment to specific promoter regions results in repression of transcription and can be used by cancer cells to provide a survival advantage. HDAC inhibitors have been used extensively to study the effects of de-repression of genes on cancer cell survival in vitro and in tumor development in animal models of carcinogenesis [3,5,12,16–18]. These studies have shown substantial antiproliferative and antitumor effects. The genes regulated by HDAC inhibitors are cell line specific and can also be HDAC inhibitor selective [19]. As small molecule HDAC inhibitors have little selectivity for the different isozymes of the HDAC family [13,20], a pharmacological quandary arises as to which of the HDACs are responsible for the anticancer phenotype observed. The goal of these studies was to define the contribution(s) of the different HDAC isozymes, especially Class I versus Class II, to the proliferation and survival of carcinoma cells using siRNA to selectively knockdown individual HDAC enzymes. HDAC inhibitors possess the ability to cause the differentiation of carcinoma cells [4,13,21–24]. In part, this is accomplished by the specific changes in gene, e.g., the actin binding protein gelsolin, transcription that regulate cellular morphology [22]. Gelsolin expression is sometimes lost upon transformation of normal cells and the carcinoma cells then adopt a more undifferentiated phenotype favoring the cell’s ability to continue to divide. In T24 cells, HDAC inhibitors cause the up-regulation of gelsolin at the mRNA and protein level that cause changes in cell morphology consistent with changes in the actin cytoskeleton [22]. HDAC inhibitors, especially TSA, produce a loss of the lamellar structures of T24 cells and a more contracted phenotype. The siRNA against HDAC3 produced a very similar morphological change as that seen with TSA. The siRNA against HDAC1 produced a flattened morphology and punctate spots in the lamellae consistent with the formation of points of focal adhesion. No such morphological changes were observed with the scrambled siRNAs or with siRNA directed against the Class II HDACs, HDAC 4 and 7. At least at the level of cell structure the knockdown of Class I HDACs seems to recapitulate the effects observed with small molecule inhibitors of the HDACs that inhibit all the isozymes within the HDAC family. HDAC inhibitors have demonstrated very little selectivity between the different HDAC isozymes [13,20], with the exception of trapoxin A which weakly inhibits HDAC6 relative to the other HDACs [20]. Therefore, these inhibitors do not aid in the differentiation of the
relevant HDACs that regulate proliferation and apoptosis. The difficulty in the isolation and purification of individual HDACs in recombinant form confounds the ability to use or develop small molecule inhibitors to make this differentiation. The use of siRNA provides a tool to knockdown the individual HDACs to determine their relative contribution to the properties identified using the HDAC inhibitors. TSA and SAHA demonstrate no selectivity for the different isoforms and inhibit all with essentially the same IC50 value [13,20]. The siRNA against HDAC3 produced similar effects to these HDAC inhibitors and most strikingly caused the accumulation of hyperacetylated histones. These results suggest that the siRNA against HDAC3 is functioning by the same mechanism as the HDAC inhibitors. The siRNA against the Class II HDACs, HDAC 4 and 7, did not affect the proliferation of HeLa S3 and this is in agreement with the lack of effect of these siRNAs on cellular morphology. The results presented herein demonstrate that the Class I HDACs (HDACs 1 and 3) are intimately involved in the processes that govern the proliferation and events that regulate programmed cell death of carcinoma cells. Deletion of yeast RPD3 affected the genes that control cell cycle, consistent with the changes we observed with the knockdown of HDACs 1 and 3 on cell proliferation and apoptosis [14]. The results with the siRNA knockdown of HDAC3 also agree with the conditional knockout of HDAC3 in a chicken B cell line which is lethal [25]. The genetic knockout of HDAC9, a Class II HDAC, in mice was without effect on the development or viability of these animals [26] and is consistent with the siRNA knockdown of Class II HDACs lacking an effect on morphology or proliferation. It is also important to note that the expression pattern of the HDACs in a particular cell line may have an influence on the phenotype observed upon knockout of that HDAC; similarly, we have previously demonstrated that the expression profiles induced by HDAC inhibitors is cell line dependent [19]. The Class I HDACs bind directly to transcriptional regulators such as those involved in the regulation of TGFb signaling, c-ski, Sno, and Smads [11,27–29]. In general, this accounts for the discovery of several potent HDAC inhibitors using reporter constructs of the TGFb signaling pathway [30–35]. In some instances, the N-Cor and SMRT corepressors have Class II HDACs as part of their corepressor complexes [36,37]. It has been demonstrated that the HDAC enzymatic activity of the N-CoR and SMRT complexes may be due to an associated HDAC3 with the corepressor complex [38,39]. Therefore, the role of individual HDACs in the overall functioning of these complexes may be difficult to understand in the absence of highly selective small molecule HDAC inhibitors. These siRNA knockdown experiments demonstrate the importance of several
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members of the Class I HDACs in the processes controlled by these corepressor complexes. Likewise, one issue of the knockdown or knockout of a protein that is part of multi-component complexes is that by affecting the amount of protein present it may also affect the integrity of the complex and thus compromise its function as a secondary effect. The experiments using siRNA provide evidence to the importance of the individual HDACs in different intracellular processes and also imply the importance of the corepressor complexes in these processes. In summary, by efficiently knocking down the expression of the Class I HDACs (1 and 3) we were able to demonstrate a pronounced cellular morphological phenotype that was not present when the Class II HDACs (4 and 7) were equivalently knocked down. When the Class I HDACs, 1 and 3, were knocked down there was an antiproliferative effect that was not observed when the Class II HDACs, 4 and 7, were reduced. These effects can be correlated with an increased accumulation of acetylated histone H3 and an increase in apoptotic cells for the HDAC3 siRNA. These data confirm that these siRNAs were acting through a similar mechanism as that observed with non-selective HDAC inhibitors and are consistent with the proposed phenotypes when HDACs are genetically knocked down or out. These knockdown studies provide a defined selectivity profile that establishes the critical role played by the Class I HDACs (HDACs 1 and 3) or the complexes containing these HDACs in carcinoma cell proliferation and survival and further supports the development of selective inhibitors designed against the Class I HDACs for the treatment of cancers.
Acknowledgments We thank Dr. Steve Tahir for his input and assistance in establishing the FACS analysis in our laboratory. We would also like to thank Dr. Steve Fesik for his critical review and support of this work.
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[32] Y. Masuoka, A. Nagai, K. Shin-ya, K. Furihata, K. Nagai, K.-i. Suzuki, Y. Hayakawa, H. Seto, Spiruchostatin A and B, novel gene expression-enhancing substances produced by Pseudomonas sp., Tet. Lett. 42 (2001) 41–44. [33] T. Sohn, G. Su, B. Ryu, C. Yeo, S. Kern, High-throughput drug screening of the DPC4 tumor-suppressor pathway in human pancreatic cancer cells, Ann. Surgery 233 (2001) 696–703. [34] G. Su, T. Sohn, B. Rhyu, S. Kern, A novel histone deacetylase inhibitor identified by high-throughput transcriptional screening of a compound library, Cancer Res. 60 (2000) 3137–3142. [35] K. Glaser, J. Li, M. Aakre, D. Morgan, G. Sheppard, K. Stewart, J. Pollock, P. Lee, C. O’Connor, S. Anderson, D. Mussatto, C. Wegner, H. Moses, Transforming growth factor b mimetics: discovery of 7-[4-(4-cyanophenyl)phenoxy]-heptanohydroxamic acid, a biaryl hydroxamate inhibitor of histone deacetylase, Mol. Cancer Ther. 1 (2002) 759–768. [36] H. Kao, M. Downes, P. Ordentlich, R. Evans, Isolation of a novel histone deacetylase reveals that class I and class II deacetylases promote SMRT-mediated repression, Gene Dev. 14 (2000) 55–66. [37] J. Li, J. Wang, J. Wang, Z. Nawaz, J. Liu, J. Qin, J. Wong, Both corepressor proteins SMRT and N-CoR exist in large protein complexes containing HDAC3, EMBO J. 19 (2000) 4342–4350. [38] W. Fischle, F. Dequiedt, M. Hendzel, M. Guenther, M. Lazar, W. Voelter, E. Verdin, Enzymatic activity associated with class II HDACs is dependent on a multiprotein complex containing HDAC3 and SMRT/N-CoR, Mol. Cell 9 (2002) 45–57. [39] M. Guenther, O. Barak, M. Lazar, The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3, Mol. Cell. Biol. 21 (2001) 6091–6101.
Role of Class I and Class II histone deacetylases in carcinoma cells using siRNAq Keith B. Glaser,* Junling Li, Michael J. Staver, Ru-Qi Wei, Daniel H. Albert, and Steven K. Davidsen Cancer Research, R47J-AP9, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064-6121, USA Received 31 July 2003
Abstract The role of the individual histone deacetylases (HDACs) in the regulation of cancer cell proliferation was investigated using siRNA-mediated protein knockdown. The siRNA for HDAC3 and HDAC1 demonstrated significant morphological changes in HeLa S3 consistent with those observed with HDAC inhibitors. SiRNA for HDAC 4 or 7 produced no morphological changes in HeLa S3 cells. HDAC1 and 3 siRNA produced a concentration-dependent inhibition of HeLa cell proliferation; whereas, HDAC4 and 7 siRNA showed no effect. HDAC3 siRNA caused histone hyperacetylation and increased the percent of apoptotic cells. These results demonstrate that the Class I HDACs such as HDACs 1 and 3 are important in the regulation of proliferation and survival in cancer cells. These results and the positive preclinical results with non-specific inhibitors of the HDAC enzymes provide further support for the development of Class I selective HDAC inhibitors as cancer therapeutics. Ó 2003 Elsevier Inc. All rights reserved. Keywords: siRNA; Histone deacetylase (HDAC); Apoptosis; Proliferation; Histone acetylation; Chromatin remodeling; Histone deacetylase inhibitors; Gene knockdown
Acetylation of nucleosomal histones in part regulates gene transcription in most cells. Differential acetylation of nucleosomal histones results in either transcriptional activation (hyperacetylation) or repression (hypoacetylation) [1,2]. This phenomenon, chromatin remodeling, is tightly regulated by the balance of histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities [1,2]. The role of chromatin remodeling in carcinogenesis is based primarily on experiments with HDAC inhibitors, e.g., sodium butyrate and trichostatin A. HDAC inhibitors induce the hyperacetylation of q Abbreviations: HDAC, histone deacetylase; siRNA, small interfering RNA; nt, nucleotide; HAT, histone acetyltransferase; RA, retinoic acid; APL, acute promyelocytic leukemia; TGFb, transforming growth factor b; TSA, trichostatin A; PBS, phosphate-buffered saline; DTT, dithiothrietol; TBS, tris-buffered saline; FACS, fluorescence activated cell sorting; SAHA, suberoylanilide hydroxamic acid; HeLa S3, S3 variant of HeLa cervical carcinoma; T24, transitional cell bladder carcinoma. * Corresponding author. Fax: 1-847-935-3622. E-mail address: [email protected] (K.B. Glaser).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.043
nucleosomal histones, resulting in the expression of repressed genes that produce growth arrest, terminal differentiation, and/or apoptosis in carcinoma cells [2–4]. These pharmacological properties of HDAC inhibitors have generated significant interest in HDACs as targets for anticancer therapy [3–6]. The HDACs belong to a deacetylase superfamily and can be divided into two distinct classes based upon their structure; the Class I (HDACs 1, 2, 3, 8, and 11) and Class II (HDACs 4, 5, 6, 7, 9, and 10) enzymes [7–10]. HDACs do not appear to function independently in the regulation of transcription but in concert with multiprotein complexes that are recruited to specific regions in the genome that in turn generate the unique spectrum of expressed and silenced genes [11]. Known repressors are multiprotein complexes that contain DNA binding proteins (e.g., NCoR, SMRT, MEF, MeCP2, sin3A, etc.) that commonly use histone deacetylases to repress transcription and block the function of some tumor suppressor genes [3,11]. HDAC inhibitors are able to derepress these genes, resulting in antiproliferative effects
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in vitro and antitumor effects in vivo [3,5,12,13]. However, since there are 11 members in the HDAC family, the question as to which of the HDAC(s) are involved in the regulation of genes that lead to the antiproliferative and antitumor phenotype currently remains unanswered. Attempts to address this question have been made in yeast where deletion mutants for the three classes in the deacetylase family were evaluated by transcriptional profiling [14]. To address this question, we used specific small interfering double stranded RNA, siRNA, molecules to selectively knockdown individual human HDACs. In this paper, we describe the selective knockdown of HDACs 1, 2, 3, 4, and 7 in HeLa S3 cells. The knockdown of HDACs 1 and 3 produced a significant morphological phenotype similar to that observed with HDAC inhibitor treatment of these cells; whereas, knockdown of HDACs 4 and 7 produced no changes in cell morphology. The knockdown of HDACs 1 and 3 was antiproliferative; whereas, knockdown of HDACs 4 and 7 was without effect. These data suggest that the Class I HDACs, HDACs 1 and 3, are essential to the proliferation and survival of mammalian carcinoma cells. Mechanistic studies demonstrated that knockdown of HDAC3 resulted in hyperacetylation of histone H3 and apoptosis in HeLa S3 cells, supporting the antiproliferative effects of Class I HDAC knockdown.
Materials and methods Cell culture and reagents. Human cervical carcinoma cells, HeLa S3, were purchased from ATCC (Bethesda, MD), and were grown according to ATCC guidelines. Cells were trypsinized and seeded at 1 106 cells per 100 mm dish, and allowed to grow overnight at 37 °C with 95%/5% air/CO2 and 80% relative humidity. Cells were treated with the HDAC siRNA for 48 h. SiRNAs were designed randomly for HDAC1 (Xeragon, Huntsville, AL) or using the Dharmacon (Colorado Springs, CO) protocol of 19 nucleotides after an AA in the DNA sequence, at least 75 nucleotides from the ATG, and between 30 and 70% GC content. The position of the siRNA on the target gene is designated by the nucleotide (nt) at which the siRNA (AA in most cases) is located on the target gene. SiRNAs were prepared according to the manufacturer’s instructions (stock concentration 20 lM in molecular biology grade water). Oligofectamine was used for all siRNA transfections (Invitrogen, San Diego, CA). Transfection of HeLa S3 cells with siRNA. HeLa S3 cells were plated at 1 106 cells/100 mm tissue culture dish approximately 24 h before transfection and just prior to transfection the medium was exchanged for 3.6 ml OptiMEM. The siRNA was appropriately diluted to a final volume of 200 ll with OptiMEM (Gibco-BRL) and incubated at room temperature for 5 min. Oligofectamine (Invitrogen) amount was optimized for transfection of HeLa S3 cells using a FITClabeled siRNA (100 nM) and fixed at 20 ll per 100 mm dish which gave >90% transfection efficiency without cytotoxicity. The diluted siRNA was then added to the oligofectamine solution (20 ll in 180 ll of Op_ in at room tiMEM), mixed by inversion, and incubated for 30 m temperature. The siRNA:oligofectamine solution was then added to the HeLa S3 cells in a dropwise fashion over the entire plate, 400 ll per 100 mm dish containing 3.6 ml of OptiMEM. The cells were transfected
for 4–6 h at 37 °C in a humidified CO2 incubator. After transfection the cell medium was removed and replaced with complete growth medium and the cells were allowed to grow for an additional 48 h. Cells were rinsed 1 with phosphate buffered saline (PBS), harvested by scraping into PBS, and centrifuged at 700g for 10 min and cell pellets were stored at )80 °C until processing. Cell proliferation assay. To determine the effects of siRNA knockdown on cell proliferation, 48 h post-transfection the cells were collected by trypsinization, counted in a hemacytometer with trypan blue dye, and plated at 2500 viable cells per well into a 96-well tissue culture dish in a final volume of 200 ll. Seventy two hours later the medium was replaced with alamarBlue solution (10% final in PBS with a final volume of 100 ll per well) and incubated on cells for 4 h at 37 °C in a CO2 incubator. The effects on proliferation were determined by fluorescence measurement (544 Ex: 590 Em) in an fmax fluorescence plate reader (Molecular Devices, San Diego, CA). Percent inhibition of proliferation was determined by comparison of treated cells to control untreated cells with the background fluorescence without cells subtracted out. Each experimental point was obtained in triplicate. Electrophoresis and Western blotting. SDS–PAGE was performed using 4–12% Novex NuPAGE gels with the 4-morpholinepropanesulfonic (MES) acid buffer system (Invitrogen, San Diego, CA). After electrophoresis, the proteins were transferred to PVDF membranes (Invitrogen) and blocked for 2 h with 10% non-fat milk in Tris-buffered saline (TBS). After transfer the residual protein in the gels was stained with Coomassie Blue and the major protein bands were used to confirm equivalent protein loading. Blots that demonstrated <20% variation in protein loading based on Coomassie blue stained gels were used in these studies. Primary antibodies were diluted in 10% non-fat milk in TBS (anti-HDAC1 (1 lg/ml) was from UBI (Lake Placid, NY), anti-HDAC3 (1:5000 dilution) was from Abcam (Cambridge, UK), and anti-HDAC 2, 4, and 7 (1:1000 dilution) were from Santa Cruz Biotechnologies (San Diego, CA). All primary antibodies were incubated with membranes overnight at 4 °C. The membranes were then washed 3 with 0.1% Tween 20 in TBS. Secondary (HRP conjugated) antibodies (Biosource International, Camarillo, CA) were diluted in 10% non-fat milk in TBS (1:5000) and incubated for 2 h with shaking. The membranes were then washed 10 5 min in 0.1% Tween 20 in TBS. Proteins were visualized by enhanced chemiluminescence with the Pierce Dura SuperSignal substrate (Pierce, Rockford, IL). FACS analysis of cell cycle and acetylated histone H3. HeLa S3 cells were cultured at 0.5 106 cells per 100 mm dish overnight. Cells were transfected for 4 hr with oligofectamine as described above with the exception that after the transfection, medium was added with 20% serum and the cells were cultured for 48 h, then split 1:6 and cultured for various times. At specific time points the cells were trypsinized, fixed with 80% ethanol, washed two times with PBS, and then incubated with propidium iodide (50 lg/ml). DNA content was determined by fluorescence cell analysis using a FACSCalibur (Becton–Dickinson) flow cytometer and cell cycle distribution was analyzed with CellQuest software. For histone H3 analysis, HeLa S3 cells were treated as for the cell cycle analysis, trypsinized, washed two times with PBS, permeabilized with 0.5% Triton X-100 for 5 min, washed two times with PBS, and incubated with anti-acetyl histone H3 polyclonal antibody (UBI, Lake Placid, NY) at 1 lg per 106 cells for 45 min. The cells were then washed two times with PBS and then incubated with anti-rabbit-biotin conjugated secondary antibody (Jackson Immuno Research) at 1 lg per 106 cells for 45 min. The cells were washed two times with PBS and incubated with streptavidin-PE (BD Biosciences) at 0.5 lg per 106 cells for 45 min. The labeled cells were washed two times with PBS prior to FACS analysis. Hyperacetylation of histone H3 was analyzed on a FACSCalibur (Becton–Dickinson) flow cytometer and the data were analyzed using CellQuest software. Rabbit IgG and no primary antibody were used as negative controls for background fluorescence.
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Results
siRNA silencing of HDACs in HeLa S3 cells
siRNA silencing of HDAC1
siRNAs were obtained for HDACs 1–8 and evaluated in HeLa S3 cells. Western blot analysis detected the presence of HDACs 1, 2, 3, 4, and 7 in HeLa S3 cells. The presence of HDACs 5, 6, and 8 could not be confirmed using the existing anti-HDAC antibodies or were not present in sufficient quantity in HeLa S3 cells and were not studied further. As shown in Fig. 1, dose-responsive knockdown of HDAC protein was observed for HDACs 1 (nt 861), 3 (nt 483), 4 (nt 337), and 7 (nt 186). HDAC2 siRNA (2 out of 4 reduced proteins at 100 nM) did not demonstrate a dose-responsive knockdown and were therefore not evaluated further (data not shown). Densitometry of these blots at the 30 nM siRNA concentrations is shown in Fig. 2. Maximal reduction of the HDAC proteins was between 80% and 97% of the oligofectamine treated controls. This occurred at 30 nM for most siRNA with the exception of HDAC4 where 100 nM siRNA was needed to reduce the protein amount by 89% (data not shown). Reduction in the amount of the targeted HDACs was specific for that isozyme as demonstrated by cross-blotting experiments for the other HDACs present in HeLa S3 whole cell lysates (data not shown). From these knockdown experiments, two of the Class I and two of the Class II HDACs were effectively knocked down and could be used in further studies for the evaluation of cellular phenotype.
Seven siRNAs were designed against the HDAC1 gene to recognize the sequences starting with the ATG (nucleotide, nt 1), nt 89, nt 120, nt 519 (containing the active site histidine), nt 861, nt 949, and nt 1321. Screening of these siRNAs at a concentration of 100 nM, 48 h after transfection into HeLa S3 cells, by Western blot analysis demonstrated that two of the seven siRNAs (nt 861 and nt 1321) effectively knocked down protein (49% and 48% reduction, respectively, based on densitometry). The oligofectamine and the scrambled siRNA controls appeared similar and showed only a slight reduction of HDAC1 compared with the non-treated controls. The reduction caused by HDAC1-nt 861 was subsequently demonstrated to be less variable and dose responsive and was therefore used in all subsequent studies with HDAC1 (Fig. 1).
Morphological changes induced by HDAC siRNAs Morphological assessment of cells after 48 h treatment with siRNAs against HDAC 1 and 3 demonstrated altered cell structure (Fig. 3); whereas, siRNAs against HDACs 4 and 7 did not affect the cell morphology.
Fig. 1. Concentration response for siRNA versus HDACs 1 (nt 861), 3 (nt 483), 4 (nt 337), and 7 (nt 186). A concentration response was evaluated for each siRNA and their respective scrambled siRNA from 10 to 100 nM. Oligo, oligofectamine reagent control. Cells were transfected and processed as described in Materials and methods. Forty eight hours after transfection the cells were processed into whole cell lysates analyzed by SDS–PAGE and protein knockdown was evaluated by Western blot analysis. Each blot is a representative of several showing the same pattern of protein knockdown for the respective siRNA. *Low protein load based on Coomassie Blue staining of gel as described in Materials and methods.
Fig. 2. Densitometry of siRNA knockdown of HDACs. The blots from Fig. 1 were quantitated using UN-SCAN-IT software version 5.1 (Silk Scientific, Oren, UT). The % control values represent the percentage of siRNA-treated sample relative to the oligofectamine-treated sample.
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Again, the scramble siRNA control did not produce a morphological change in the HeLa S3 cells (Fig. 3D). The non-selective HDAC inhibitor TSA (1 lM) produced similar cell shape changes as the knockdown of HDAC3 (Fig. 3E), and SAHA (15 lM) produced a morphological change between TSA and the HDAC 1 siRNA (Fig. 3F). These morphological changes are consistent with the observed alteration in cytoskeletal gene expression, e.g., gelsolin, which occurs with small molecule HDAC inhibitors [15]. Antiproliferative response of HeLa S3 cells to HDAC siRNAs
Fig. 3. Effect of HDAC siRNA and small molecule HDAC inhibitors on HeLa S3 cell morphology. Photomicrographs were taken of cells treated for 48 h with either siRNA (100 nM, B, D), scrambled siRNA (100 nM, A, C) or the HDAC inhibitors TSA (1 lM, E) or SAHA (15 lM, F). Each photomicrograph is a representative of several experiments showing similar morphological changes.
When HDAC1 is knocked down by siRNA the cells take on a flattened morphology with extensive focal contacts (see arrows in Fig. 3A). The scramble siRNA does not produce this phenotype on the cells even at 200 nM (Fig. 3B). Likewise, the siRNA against HDAC3 produced a profound morphological change into spindle like cells with elongation of the filopodia (Fig. 3C).
HeLa S3 cells were transfected with siRNAs for HDAC 1, 3, 4, and 7 for 48 h to allow protein knockdown, the cells were then trypsinized and normalized to 2500 viable cells per well in 96 well tissue culture dishes and allowed to grow for an additional 72 h. This protocol was used to minimize the adverse effects of transfection reagents on cell growth and to ensure the knockdown of the target protein prior to evaluation of the effects on proliferation. Knockdown of HDAC7 did not have an antiproliferative effect on HeLa S3 cells, even at 100 nM; in contrast, knockdown of HDAC1 produced a concentration-dependent inhibition of cell proliferation (Fig. 4A). The scrambled duplexes of the HDAC1 and HDAC7 siRNAs had no effect on proliferation. Neither the HDAC4 siRNA nor the HDAC4 scramble siRNA significantly inhibited proliferation (<10%) at concentrations up to 100 nM; however, knockdown of HDAC3 demonstrated a concentration-dependent inhibition of proliferation between 1 and 30 nM with no increase in inhibition observed between 30 and 100 nM (Fig. 4B). These results demonstrate that cells with reduced amounts of the Class I HDACs 1 and 3 proliferate at a slower rate than control cells or cells with reduced amounts of Class II HDACs 4 and 7.
Fig. 4. Effect of siRNA and scrambled siRNA on the proliferation of HeLa S3 cells. Forty eight hours after transfection, HeLa S3 cells were detached by trypsinization, counted, and plated at 2500 cells/well in a 96-well tissue culture plate. Seventy two hours later cell proliferation was assessed using alamarBlue (modified MTT assay) by adding a 10% solution to each well for 2–4 h at 37 °C in a tissue culture incubator. (A) Effect of HDAC1 and 7 siRNA on proliferation. (B) Effect of HDAC3 and 4 siRNA on proliferation. Each graph is a representative of two separate experiments with each point carried out in triplicate showing similar results.
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Effect of HDAC knockdown on histone H3 acetylation and apoptosis To demonstrate that the knockdown of an HDAC would result in biochemical changes similar to those observed with small molecule inhibitors of HDACs, the effects of HDAC3 siRNA were studied on histone H3 acetylation and induction of apoptosis (sub-G0/G1 population) by FACS analysis. A time course analysis of histone H3 acetylation by FACS demonstrated that the maximal difference between scramble and siRNA effects were observed 72 h post-transfection (Fig. 5A). Relative to untreated and scramble treated control cells, the HDAC3 siRNA produced a concentration-dependent increase in acetylation of histone H3 between 10 and 200 nM. The amount of acetylated histone H3 in the siRNA-treated cells was similar to those produced in SAHA-treated cells. The siRNA for HDAC 1 could not
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be evaluated in the hyperacetylation or apoptosis assays due to a high background activity seen with the HDAC1 scrambled siRNA. HDAC3 siRNA treatment at 72 h post-transfection demonstrated the maximal response of apoptosis, largest fraction of adherent cells undergoing apoptosis, as judged by the accumulation of cells in the sub-G0/G1 population (Fig. 5B). Similar to the results observed for histone H3 acetylation, there was a concentration-dependent induction of apoptosis with the siRNA that was not observed for the scrambled duplex (Fig. 5C). These data agree well with the anti-proliferative response that was measured 48 h past the maximal induction of apoptosis in these cells. As only adherent cells were evaluated for apoptosis, the longer time points (>72 h) demonstrate a decrease in sub-G0/G1 cells possibly due to the loss of late apoptotic cells from the adherent population.
Fig. 5. FACS analysis of accumulation of acetylated histone H3 and induction of apoptosis in HeLa S3 cells by the HDAC3 siRNA. (A) Accumulation of acetylated histone H3 in a concentration responsive manner for HDAC3 siRNA. Cells were taken 72 h post-transfection for the maximal response of HeLa S3 cells. The accumulation of acetylated histone H3 was determined relative to the effect of the scrambled siRNA at each concentration. (B) Effect of HDAC 3 siRNA and scramble on the accumulation of cells in the sub-G0/G1 population, apoptotic population. HeLa S3 cells were stained with propidium iodide (50 lg/ml) prior to analysis of cell cycle distribution by FACS. Fraction of the cell population in each part of the cell cycle was determined using CellQuest software. Each graph is a representative of at least two separate experiments showing similar results.
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Discussion The HDACs are a large family of related proteins that function in part to regulate the transcription of genes through posttranslational modification of histones and possibly other proteins such as transcription factors [3]. HDAC recruitment to specific promoter regions results in repression of transcription and can be used by cancer cells to provide a survival advantage. HDAC inhibitors have been used extensively to study the effects of de-repression of genes on cancer cell survival in vitro and in tumor development in animal models of carcinogenesis [3,5,12,16–18]. These studies have shown substantial antiproliferative and antitumor effects. The genes regulated by HDAC inhibitors are cell line specific and can also be HDAC inhibitor selective [19]. As small molecule HDAC inhibitors have little selectivity for the different isozymes of the HDAC family [13,20], a pharmacological quandary arises as to which of the HDACs are responsible for the anticancer phenotype observed. The goal of these studies was to define the contribution(s) of the different HDAC isozymes, especially Class I versus Class II, to the proliferation and survival of carcinoma cells using siRNA to selectively knockdown individual HDAC enzymes. HDAC inhibitors possess the ability to cause the differentiation of carcinoma cells [4,13,21–24]. In part, this is accomplished by the specific changes in gene, e.g., the actin binding protein gelsolin, transcription that regulate cellular morphology [22]. Gelsolin expression is sometimes lost upon transformation of normal cells and the carcinoma cells then adopt a more undifferentiated phenotype favoring the cell’s ability to continue to divide. In T24 cells, HDAC inhibitors cause the up-regulation of gelsolin at the mRNA and protein level that cause changes in cell morphology consistent with changes in the actin cytoskeleton [22]. HDAC inhibitors, especially TSA, produce a loss of the lamellar structures of T24 cells and a more contracted phenotype. The siRNA against HDAC3 produced a very similar morphological change as that seen with TSA. The siRNA against HDAC1 produced a flattened morphology and punctate spots in the lamellae consistent with the formation of points of focal adhesion. No such morphological changes were observed with the scrambled siRNAs or with siRNA directed against the Class II HDACs, HDAC 4 and 7. At least at the level of cell structure the knockdown of Class I HDACs seems to recapitulate the effects observed with small molecule inhibitors of the HDACs that inhibit all the isozymes within the HDAC family. HDAC inhibitors have demonstrated very little selectivity between the different HDAC isozymes [13,20], with the exception of trapoxin A which weakly inhibits HDAC6 relative to the other HDACs [20]. Therefore, these inhibitors do not aid in the differentiation of the
relevant HDACs that regulate proliferation and apoptosis. The difficulty in the isolation and purification of individual HDACs in recombinant form confounds the ability to use or develop small molecule inhibitors to make this differentiation. The use of siRNA provides a tool to knockdown the individual HDACs to determine their relative contribution to the properties identified using the HDAC inhibitors. TSA and SAHA demonstrate no selectivity for the different isoforms and inhibit all with essentially the same IC50 value [13,20]. The siRNA against HDAC3 produced similar effects to these HDAC inhibitors and most strikingly caused the accumulation of hyperacetylated histones. These results suggest that the siRNA against HDAC3 is functioning by the same mechanism as the HDAC inhibitors. The siRNA against the Class II HDACs, HDAC 4 and 7, did not affect the proliferation of HeLa S3 and this is in agreement with the lack of effect of these siRNAs on cellular morphology. The results presented herein demonstrate that the Class I HDACs (HDACs 1 and 3) are intimately involved in the processes that govern the proliferation and events that regulate programmed cell death of carcinoma cells. Deletion of yeast RPD3 affected the genes that control cell cycle, consistent with the changes we observed with the knockdown of HDACs 1 and 3 on cell proliferation and apoptosis [14]. The results with the siRNA knockdown of HDAC3 also agree with the conditional knockout of HDAC3 in a chicken B cell line which is lethal [25]. The genetic knockout of HDAC9, a Class II HDAC, in mice was without effect on the development or viability of these animals [26] and is consistent with the siRNA knockdown of Class II HDACs lacking an effect on morphology or proliferation. It is also important to note that the expression pattern of the HDACs in a particular cell line may have an influence on the phenotype observed upon knockout of that HDAC; similarly, we have previously demonstrated that the expression profiles induced by HDAC inhibitors is cell line dependent [19]. The Class I HDACs bind directly to transcriptional regulators such as those involved in the regulation of TGFb signaling, c-ski, Sno, and Smads [11,27–29]. In general, this accounts for the discovery of several potent HDAC inhibitors using reporter constructs of the TGFb signaling pathway [30–35]. In some instances, the N-Cor and SMRT corepressors have Class II HDACs as part of their corepressor complexes [36,37]. It has been demonstrated that the HDAC enzymatic activity of the N-CoR and SMRT complexes may be due to an associated HDAC3 with the corepressor complex [38,39]. Therefore, the role of individual HDACs in the overall functioning of these complexes may be difficult to understand in the absence of highly selective small molecule HDAC inhibitors. These siRNA knockdown experiments demonstrate the importance of several
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members of the Class I HDACs in the processes controlled by these corepressor complexes. Likewise, one issue of the knockdown or knockout of a protein that is part of multi-component complexes is that by affecting the amount of protein present it may also affect the integrity of the complex and thus compromise its function as a secondary effect. The experiments using siRNA provide evidence to the importance of the individual HDACs in different intracellular processes and also imply the importance of the corepressor complexes in these processes. In summary, by efficiently knocking down the expression of the Class I HDACs (1 and 3) we were able to demonstrate a pronounced cellular morphological phenotype that was not present when the Class II HDACs (4 and 7) were equivalently knocked down. When the Class I HDACs, 1 and 3, were knocked down there was an antiproliferative effect that was not observed when the Class II HDACs, 4 and 7, were reduced. These effects can be correlated with an increased accumulation of acetylated histone H3 and an increase in apoptotic cells for the HDAC3 siRNA. These data confirm that these siRNAs were acting through a similar mechanism as that observed with non-selective HDAC inhibitors and are consistent with the proposed phenotypes when HDACs are genetically knocked down or out. These knockdown studies provide a defined selectivity profile that establishes the critical role played by the Class I HDACs (HDACs 1 and 3) or the complexes containing these HDACs in carcinoma cell proliferation and survival and further supports the development of selective inhibitors designed against the Class I HDACs for the treatment of cancers.
Acknowledgments We thank Dr. Steve Tahir for his input and assistance in establishing the FACS analysis in our laboratory. We would also like to thank Dr. Steve Fesik for his critical review and support of this work.
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