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Vol. 2, 401-408, April 2003     Molecular Cancer Therapeutics
© 2003 American Association for Cancer Research

Modulation of Histone Acetylation by [4-(Acetylamino)-N-(2-Amino-phenyl) Benzamide] in HCT-8 Colon Carcinoma

Alan J. Kraker1, Craig A. Mizzen2, Brian G. Hartl, Johnson Miin, C. David Allis and Ronald L. Merriman

Department of Cancer Pharmacology, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105 [A. J. K., B. G. H., J. M., R. L. M.], and Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908 [C. A. M., C. D. A.]

1 To whom requests for reprints should be addressed, at Cancer Pharmacology, Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105. Phone: (734) 622-7759; Fax: (734) 622-7158; E-mail: Alan.Kraker{at}pfizer.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CI-994 or N-acetyldinaline [4-(acetylamino)-N-(2-amino-phenyl) benzamide] is an antitumor cytostatic agent currently undergoing clinical trial. Although several changes in cellular metabolism induced by the drug have been characterized, the primary molecular mechanism of its antitumor activity has been previously unknown. Here, we show that CI-994 is a histone deacetylase (HDAC) inhibitor that causes histone hyperacetylation in living cells. In assays of isolated enzymes, CI-994 inhibited HDAC-1 and HDAC-2 in a concentration-dependent fashion but had no effect on the activity of the prototypical histone acetyltransferase GCN5. Acetylated histone H3-specific Western blots were used to monitor histone acetylation in HCT-8 colon carcinoma cells treated with CI-994 in vitro. CI-994 induced hyperacetylation of H3 in a time- and dose-dependent fashion. H3 hyperacetylation was detectable as early as 30 min after the addition of CI-994 to cells. These data demonstrate that inhibition of HDAC is an early event in cells treated with CI-994 and suggest that this inhibition is mechanistically related to the antitumor activity of this compound.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Four decades of research on the composition of eukaryotic chromatin has identified numerous posttranslationally modified forms of histones (1). Although many details remain to be elucidated, the levels of many modified forms of histones are dynamically regulated, and specific patterns of modification are required for appropriate regulation of gene transcription, chromosome segregation, cell cycle progression, and the maintenance of genome integrity (2–4). Moreover, mutations involving histone-modifying enzymes or factors that recruit them to genetic loci have been implicated in the etiology or pathogenesis of developmental disorders and proliferative diseases (5, 6). Thus, enzymes that regulate the levels of certain histone modifications are attractive potential targets for pharmacological intervention in the control of proliferative diseases.

Recent research into dynamic histone acetylation is particularly significant in this regard. Proteins known to function in transcriptional activation, including GCN5 in yeast and CBP/p300, PCAF, and TAFII250 in humans, possess HAT3 activity (7–9). Acetylation by multiprotein complexes containing these enzymes neutralizes the positive charge associated with the {epsilon}-amino group of conserved lysine residues within the NH2-terminal domains of the core histones H2A, H2B, H3, and H4. In general, this is thought to enhance the accessibility of nucleosomal DNA for transcription by attenuating direct masking by the NH2-terminal domains and also by destabilizing higher order folding of nucleosomal filaments mediated by internucleosomal contacts made by these domains (3, 4). Additionally, recent evidence suggests that recognition of acetylated residues in histones contributes to locus-specific recruitment of transcriptional regulators containing bromodomain motifs (10, 11). It is important to note that although most of the information currently available on HATs focuses on the relationship between histone acetylation and transcriptional activation, evidence that HATs such as Esa1 and SAS2 are required for cell cycle progression and transcriptional silencing, respectively (12, 13), suggests additional roles for acetylation. Moreover, evidence that transcription factors, including EKLF, GATA-1, HMG I(Y), TFIIE, TFIIF, and the tumor suppressor p53, are acetylated by certain HATs suggests that mechanisms using acetylation to regulate transcription and other nuclear processes are not exclusive to histones (8).

Conversely, the action of HDACs, enzymes that restore the positive charge to lysine residues upon hydrolysis of {epsilon}-amino acetyl moieties, is generally associated with repression of transcription, and many of the HDACs characterized, to date, are components of multiprotein complexes containing other proteins known to function in transcriptional repression (4, 14). Isolation of the first protein identified to be an HDAC and many subsequent analyses of HDAC function have exploited the availability of small molecule inhibitors of HDACs (15–17). These compounds have contributed significantly to our understanding of the regulation of histone acetylation and gene transcription. Evidence that some of these compounds also inhibit cell proliferation has led to studies examining their use in antitumor therapy and the relationship of histone acetylation to cell growth (18–23). HDAC inhibitors known to have antiproliferative effects belong to several different chemical classes, including (a) compounds containing hydroxamic acids such as TSA and suberoylanilide hydroxamine acid, (b) short-chain fatty acids such as butyric acid, (c) cyclic tetrapeptides containing an epoxy moiety such as trapoxin, (d) cyclic tetrapeptides without epoxy groups such as apicidin, and (e) benzamides, e.g., MS-27-275 (reviewed in Ref. 24).

A substituted benzamide compound (CI-994; structure shown in Fig. 1) has been the subject of study in clinical trials for a number of neoplastic diseases (25–28), although the mechanism of action of this compound has not been elucidated. Effects of the compound on cellular processes related to cell cycle progression and the phosphorylation and stability of a low molecular weight nuclear protein (possibly a histone) that correlate with growth inhibition by the compound have been described previously (29). Although the identity of this low molecular weight protein remains unknown, the potential significance of modulation of histone modifications by CI-994 led us to examine the effects of CI-994 on histone acetylation. Here, we show that histone H3 is rapidly hyperacetylated in cultured HCT-8 cells after treatment with CI-994. Assays to determine the effect of CI-994 on the HAT activity of recombinant Gcn5 and on the HDAC activity of HDAC-1 and HDAC-2 complexes immunoprecipitated from HCT-8 cells both provide evidence that hyperacetylation in vivo results from inhibition of HDACs by CI-994. Monitoring histone acetylation may provide a useful tool for evaluation of pharmacodynamic and toxicological parameters in CI-994 therapy that could lead to improvements in its clinical application. Furthermore, our evidence that HDAC inhibition is a cellular mechanism will facilitate additional investigation of the mechanism of the antitumor effect of CI-994.



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  		Fig. 1. Structure of CI-994.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents.
Antibodies to acetylated histone H3 were from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies to total histone H3 were from Biodesign International (Kennebunk, ME). Tritium-labeled bovine acetylated histone H4 fragment was from Amersham-Pharmacia (112Ci/mmol, TRQ8527; Oakbrook, IL). TSA was from Wako (Richmond VA) and was dissolved at 10 mM in DMSO as a stock solution. CI-994 was provided by the Compound Management Department of Pfizer and was dissolved at 100 mM in DMSO. The benzamide analogue MS-275 (21, 22) was from the Compound Management Department of Pfizer and was also dissolved at 10 mM in DMSO. All three compounds were stored at -70°C. Sodium butyrate was prepared as a 1.0 M stock in water and kept at -70°C. Before each experiment, aliquots of these stocks were diluted appropriately in water to give a series of working stocks, which were subsequently diluted 1:10 in the final assay mixture to attain the concentrations tested.

Cells.
HCT-8 cells were grown in logarithmic phase in RPMI 1640 supplemented with 10% fetal bovine serum. For growth delay studies, cells were seeded into 96-well plates at 2000 cells/well (180 µl/well) and allowed to attach and enter log phase growth before treatment with the growth inhibitory compounds. Compounds were diluted into growth medium containing not more than 0.5% (vol/vol) DMSO and then added to the cells. Cells were treated continuously for 3 days with compounds, fixed with 10% TCA, incubated for 30 min at 4°C, washed five times with tap water, stained with the protein dye sulfrhodamine B according to published protocols (30), and evaluated for growth in a 96-well plate absorbance reader.

Nuclear Extract.
Partially purified HDAC was prepared from HCT-8 cells following a published method (31). The final nuclear extract containing the deacetylase gave 1.5 mg/ml protein. The nuclear extracts were assayed as previously described (31) with the modification that the assay interval was 20 min rather than 4 h.

Determination of Acetylated Histone.
Cells for evaluation of histone acetylation levels were seeded into 10-cm plates at 2.2 x 106 cells/plate, treated the next day with compound or solvent control, and then processed for histone extraction as follows. Cells were scraped from the culture plates into PBS, centrifuged at 1000 rpm (250 x g) for 10 min, washed with PBS and then resuspended in 1.0 ml of extraction buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 250 µM 4-(2-aminoethyl) benzenesulfonylfluoride (protease inhibitor)]. Sulfuric acid was added to 0.4 N, and the precipitate was incubated on ice for 30 min. The precipitate was centrifuged in a microfuge at 14400 rpm for 10 min at 4°C. The supernatant liquid was transferred into a new tube and made 20% in TCA. The resulting pellet was washed once in acetone containing 0.1% HCl and once in acetone alone. The precipitate (collected by centrifugation) was dried by desiccation. This pellet was then resuspended in Laemmli sample buffer (for tris-glycine gels) or tris-bis sample buffer (for tris-bis gels), electrophoresed through either 4–20% polyacrylamide gradient tris-glycine gels or for later experiments, through 10% polyacrylamide tris-bis gels. The resolved proteins were transferred to nitrocellulose membranes and then Western blotted for total histone or acetylated histone.

Immunoprecipitation Assay for HDAC Activity.
Whole cell lysates of HCT-8 and MX-1 xenografts from untreated animals were prepared by homogenizing tumor pieces in lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na EDTA, 10% glycerol, 0.5% NP40, 1 mM PMSF, 10 µg/ml aprotinin, pepstatin, and leupeptin; 0.5 ml/100 mg tissue). Lysates were clarified by centrifugation (12,000 x g) for 10 min at 4°C. For each HDAC immunoprecipitation assay, 50 µl of clarified homogenate (~200 µg total protein) were diluted with 450 µl TBST [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] containing 1 mM PMSF in a 1.5-ml microcentrifuge tube. Equal amounts (0.5 µg) of purified IgG from rabbit antisera to HDAC-1 (no. 06-720; Upstate Biotechnology) or to HDAC-2 (no. sc-7899; Santa Cruz Biotechnology) were then added and immune complexes allowed to form for 1 h at 4°C. Protein A-Sepharose (10 µl of settled beads) was then added, and the components were mixed on a rotator for 1 h at 4°C. Immune complexes were collected by centrifugation and washed five times with cold TBST. The beads were then washed once in cold assay buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1 mM PMSF, 10 µg/ml aprotinin, pepstatin, and leupeptin] and resuspended in 200 µl of assay buffer. Compounds to be tested for inhibition of deacetylase activity were then added and the mixture incubated for 20 min on ice. An aliquot corresponding to 40,000 dpm of hyperacetylated histones (labeled with 3H-acetate in culture) was then added to each tube and the reactions mixed on a rotator for 1 h at 30°C. Tubes were centrifuged briefly, and the reactions terminated by adding 50 µl of stop buffer (1.0 M HCl, 0.16 M CH3COOH). Each reaction was then extracted with 600 µl of ethyl acetate and 3H-acetate release determined by liquid scintillation counting a 500 µl aliquot of the ethyl acetate phase.

3H-acetate labeled histones were prepared by incubating 108 HeLa cells in 25 ml of DMEM containing 10 mM butyrate and 5 mCi 3H-acetic acid, Na salt (4.1 Ci/mmol; NEN no. NET003H) for 3 h at 37°C. The cells were then washed three times in Tris-buffered saline and nuclei prepared as previously described (32), except that 100 nM TSA was included in the lysis and wash buffers. Histones were extracted with 0.4 N H2SO4 and recovered by TCA precipitation (20% w/v final). The specific activity of the resulting crude histones was ~4000 dpm 3H/µg.

Effect of Deacetylase Inhibitors on Yeast GCN5 in Vitro.
The effects of CI-994 and TSA on the HAT activity of recombinant yeast GCN5 in vitro were assayed using purified GCN5 expressed in bacteria as described previously (33). Reactions containing 10 µg of chicken erythrocyte core histones, ~4 pmol of yGCN5, various amounts of CI-994 and TSA, and 25 µl of 2x HAT assay buffer [100 mM Tris-HCl (pH 8.0), 20% glycerol, 2 mM EDTA, 2 mM DTT, and 1 mM PMSF] in a total volume of 40 µl, were assembled in microtubes on ice. The concentrations of CI-994 and TSA tested were achieved by adding 5 µl of 10x working stocks prepared as described above, and reactions were incubated on ice for 20 min before the addition of 3H-acetylCoA. Reactions were initiated by adding 0.1 µCi of 3H-acetylCoA (1.88 Ci/mmol; NEN no. NET290) in a volume of 10 µl to each tube with incubation at 30°C for 10 min. Duplicate aliquots of 10 µl were then spotted on squares of P81 paper (Whatman) and histone acetylation quantitated by liquid scintillation counting as described previously (34).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In Vitro Growth Delay.
Growth delay experiments in HCT-8 cells were performed to determine a concentration for subsequent mechanistic studies. The 72-h IC50 for growth delay on plastic was 4.7 µM for CI-994. A similar determination for the effects of TSA, a natural product inhibitor of HDAC, gave an IC50 of 20 nM.

Acetylated Histone H3 Levels.
On the basis of earlier work that had shown a time- and concentration-dependent effect of CI-994 on cell cycle progression, thymidine incorporation, levels of a Mr 16,000 phosphoprotein, and cell growth (29), we exposed HCT-8 cells to different concentrations of CI-994 for various intervals. Cells were then lysed and histones prepared by acid extraction to enable analyses of acetylation status. Fig. 2 shows Western blots of acetylated histone H3 and total histone H3 from HCT-8 cells treated with 1 or 10 µM CI-994 for 9, 5, or 2 h. Cultures treated with 50 nM TSA were processed in parallel to serve as a positive control because this treatment has been shown previously to induce histone acetylation (35). After either 5 or 9 h of treatment, both concentrations of CI-994 clearly caused increases in the amount of acetylated histone H3 relative to the untreated control sample, as did the positive control agent TSA. After 2 h of treatment, the levels of acetylated H3 were elevated in cells exposed to 10 µM CI-994, but the effect was less apparent with 1 µM compound. Although the amount of total histone H3 (Fig. 2, bottom panel) analyzed varied somewhat from sample to sample, the fold increase in signal intensity for acetylated H3 between control and treated samples far exceeded the variation in signal for total H3 in nearly all cases. Examination of the time course of H3 hyperacetylation in HCT-8 cells treated with 10 µM CI-994 over a wider range of intervals (Fig. 3) revealed that measurable increases in acetylated histone H3 were seen after as little as 30 min of treatment, with the largest portion of the total increase apparent by 9 h.



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  		Fig. 2. Effect of 2–9 h time course of treatment on acetylated histone H3. HCT-8 cells were seeded at 2.2 x 106/10-cm tissue culture plate and allowed to attach overnight. Cells were treated with the indicated concentration of compound for the times shown. Cells were harvested by scraping into PBS, lysed, and the resultant pellet extracted with 0.4 N H2SO4. The extract was evenly split, resolved via gel electrophoresis as described, and Western blotted for either acetylated histone H3 or total histone H3. The resulting autoluminograph was quantitated using laser densitometry and analyzed with ImageQuant (Molecular Dynamics) software.

 


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  		Fig. 3. Effect of 10 min to 24 h time course of treatment on acetylated histone. HCT-8 cells were treated with 10 µM CI-994 for the indicated times before extraction as described in Fig. 2. Cells were harvested at the times of treatment indicated in the figure. The resulting autoluminograph was quantitated using laser densitometry and analyzed with ImageQuant (Molecular Dynamics) software.

 
Effect of CI-994 on GCN5 HAT Activity.
Because the increased acetylation of H3 seen in cells treated with CI-994 could result from activation of HATs and/or inhibition of HDACs, the effect of CI-994 on the activity of a known HAT was examined in vitro. The prototypical HAT yeast GCN5 was selected for these assays because both the recombinant and native forms of this enzyme preferentially modify H3 over other histones (8, 33). Moreover, this enzyme contains the acetylCoA binding motif common to the majority of HATs identified to date (7). The data from such an assay are shown in Fig. 4. Coincubation with up to 100 µM CI-994 did not activate GCN5 HAT activity nor did up to 400 nM TSA cause activation of this HAT.



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  		Fig. 4. Effect of CI-994 on the histone acetyltransferase GCN5. Yeast GCN5-containing lysate (~4 pmol/tube) was assayed for its ability to transfer labeled acetate from acetyl CoA to chicken erythrocyte histones. Reactions were run for 10 min at 30°C, after which aliquots were taken to spot on P81 phosphocellulose paper for liquid scintillation counting. Data shown are means of duplicate determinations.

 
Inhibition of HDAC Activity in Nuclear Extracts.
The effect of CI-994 on HDAC activity in vitro was evaluated to establish whether this correlated with the hyperacetylation response observed in living cells. Nuclear extract prepared from HCT-8 cells served as the source of crude deacetylase activity in assays that measured hydrolysis of 3H-acetate from a commercially prepared histone H4 peptide substrate as described previously (31). As shown in Fig. 5, CI-994 inhibited crude HDAC activity in a concentration-dependent fashion with 50% inhibition achieved between 25 and 50 µM CI-994. The positive control agent TSA also inhibited crude HDAC activity in a concentration-dependent fashion in accordance with a previous report (36). TSA was significantly more potent than CI-994 in this assay: only ~12% of crude HDAC activity remained in the presence of 6.25 nM TSA, the lowest concentration tested. This potent inhibition of crude HDAC activity by TSA seems to correlate well with the degree of H3 hyperacetylation observed in cells exposed to 50 nM TSA (Figs. 2 and 3). However, the degree of H3 hyperacetylation observed in cells exposed to 10 µM CI-994 (Figs. 2 and 3) exceeds that expected based on the apparent IC50 of 25–50 µM CI-994 observed in this assay.



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  		Fig. 5. Effect of CI-994 on histone deacetylase activity in nuclear extracts. Nuclear protein extract (4.5 µg) from HCT-8 cells was incubated with assay buffer and labeled acetylated histone H4 peptide fragment (final concentration of 1.9 nM) in a total volume of 200 µl for 20 min in the presence of inhibitors as indicated. The reaction was stopped by the addition of acidified ethyl acetate that was also used to extract labeled acetate hydrolyzed from the peptide substrate. Data shown are means of duplicate determinations.

 
Effect of CI-994 on Immunoprecipitated HDACs.
Genes encoding 15 known and putative HDACs have been identified in the human genome to date (14, 37). The protein products of these genes can be arranged into three classes based on sequence homology with the yeast Rpd3, Hda1, and Sir2 HDAC proteins characterized previously. The HDAC proteins characterized thus far in metazoans differ in abundance among tissues, and moreover, differences in sensitivity to TSA and other inhibitors have been described previously (37–39). As an initial step in addressing the issue of which HDAC proteins contribute to the hyperacetylation response of HCT-8 cells exposed to CI-994, we studied the effect of CI-994 on HDAC-1 and HDAC-2 immunoprecipitated from lysates of HCT-8 xenograft tumor tissue. We also compared the effects of MS-275 (21, 22), a structurally related HDAC inhibitor also in clinical trials, to CI-994 in these immunoprecipitate enzyme assays. HDAC-1 and HDAC-2 were selected for this test because preliminary analyses revealed these isoforms were relatively abundant in HCT-8 cells. Furthermore, immunoprecipitated HDAC-1 and HDAC-2 could be used to assay the effect of CI-994 because commercially available antisera to these isoforms did not directly inhibit the activity of this protein (data not shown). As shown in Fig. 6A, coincubation with CI-994 inhibited the activity of immunoprecipitated HDAC-1 in a concentration-dependent fashion with an IC50 of ~100 µM CI-994 apparent under the conditions used. MS-275 exhibited a concentration-dependent inhibition of HDAC-1 with potency similar to that of CI-994. Similar to the findings on crude HDAC activity reported above, TSA was a significantly more potent inhibitor of HDAC-1 with an apparent IC50 falling between 4 and 40 nM in agreement with previously published results (19, 40). The same potency for CI-994 against HDAC-1 isolated from MX-1 mammary tumor xenograft tumor tissue was also seen (data not shown). Fig. 6B shows the equipotent effect of CI-994 and MS-275 against HDAC-2 isolated from HCT-8 cells. As with HDAC-1, TSA is a very potent inhibitor with an IC50 between 4 and 40 nM.



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  		Fig. 6. Effect of CI-994 on immunoprecipitated HDAC. Approximately 200 µg total protein from whole cell lysates of HCT-8 xenograft tumor transplants were used as the source of immunoprecipitated HDACs. Approximately 10 µg (40,000 dpm) acetylated histone isolated from labeled HeLa cells in culture was added to the immunoprecipitates along with the indicated concentrations of inhibitors. The reactions were allowed to progress for 1 h at 30°C and were terminated by the addition of stop buffer and extracted with ethyl acetate. Data shown are means of duplicate determinations. A, immunoprecipitated HDAC-1 was the source of the enzyme. B, HDAC-2 immunoprecipitated with rabbit antiserum was the enzyme used.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CI-994 shows promise as an antitumor agent, but a potential mechanism for its antitumor action was not apparent before these studies. In vitro studies (29), in vivo efficacy, pharmacokinetic studies (41, 42), and clinical studies (25, 26, 27) have all been carried out without a clear picture of the molecular basis of activity.

The results of the experiments presented here provide evidence that the antitumor activity of CI-994 is linked to its ability to inhibit HDACs. Increased acetylation of histone H3 was apparent as early as 30 min after addition of CI-994 to HCT-8 cells, which was the earliest characterized cellular response to the compound. The next most immediate effects known were the loss of a Mr 16,000-phosphorylated nuclear protein after ~6–8 h of treatment with CI-994 followed by the imposition of a G1 cell cycle arrest (29). Other HDAC inhibitors, including MS-275 (N-(2-aminophenyl)-4-[N-(pyridin-3yl-methoxycarbonyl)aminomethyl]benzamide) and trapoxin (a natural cyclotetrapeptide) have been shown to cause a G1 cell cycle block in other cell types (22, 43).

In addition to inhibition of histone acetylation in intact cells, CI-994 also inhibited catalytic activity of HDACs from crude nuclear extracts assayed in vitro with a synthetic substrate but with lower potency than that seen in cells. When the mixture of HDACs present in crude nuclear extract from HCT-8 cells was used for direct determination of enzyme inhibition (Fig. 5), CI-994 was also found to be significantly less potent than TSA. A portion of this difference in potency between CI-994 and TSA may be attributable to the use of a 3H-acetylated H4 synthetic peptide substrate in these experiments. If CI-994 and TSA affect different subsets of HDACs, differences in the substrate preferences of these enzymes could introduce bias in assays performed with a single type of substrate. Because we can restore most or all activity by repeatedly washing anti-HDAC-1 immunoprecipitates after CI-994-treatment, we infer that CI-994 is a reversible inhibitor of HDACs (data not shown).

A compound that is structurally similar to CI-994, designated MS-275, shown previously to be an inhibitor of HDAC (21, 22) showed potency similar to that of CI-994 in isolated enzyme in vitro assays used here (Fig. 6). The concentrations of CI-994 at which inhibition was seen in these two in vitro assays were well above those that resulted in increases in acetylated histone in whole cells. This difference in CI-994 potency between whole cell and in vitro-isolated enzyme assays may be attributable to the different conditions of the assays. Unlike the in vitro assays, subcellular compartmentalization was intact in the cellular assays, and appropriate concentrations of cofactors and other physiological regulatory elements were in place when cells were used to assess the effect of CI-994 on histone acetylation. When immunoprecipitated HDAC-1 or HDAC-2 were used as the source of enzyme (Fig. 6), potencies for enzyme inhibition by CI-994 were obtained that were very similar to one another in each of the isoforms and, furthermore, were similar to potency of CI-994 seen in the crude nuclear preparations of HDAC. Different isoforms of the HDACs (14, 37) may be differentially expressed in cells, may have different substrate specificities, and may be differentially sensitive to inhibitors leading to variable patterns of apparent potency. Sensitivity of HDAC isoforms to small molecule inhibitors is under study by us and others (39, 44, 45). Data in this study suggest that for at least HDAC-1 and HDAC-2, these substituted benzamide inhibitors do not show isoform selectivity. Additional experimentation will be required to fully address this possibility.

Analyses of the composition of the Sin3, NuRD, and Mi2 complexes prepared by chromatographic or affinity methods have shown that HDAC1 and HDAC2 coexist in these complexes in vivo (46–48). This association potentially compromises the ability of immunoprecipitation to determine the relative inhibition of HDAC1 and HDAC2 by CI-994 and other compounds. However, because Western blot analyses demonstrate that anti-HDAC1 immunoprecipitates from whole cell lysates contain much more HDAC1 than HDAC2 and conversely that anti-HDAC2 immunoprecipitates from whole cell lysates contain much more HDAC2 than HDAC1 (49, 50), we infer that the deacetylase activities we have measured derive, for the most part, from the intended form.

In this study overall, the in vitro inhibition of HDAC using two different types of assays combined with similar results seen with a structurally related compound (MS-275) that has been characterized as a direct inhibitor of HDAC supports the characterization of CI-994 as an inhibitor of HDAC.

Another possible explanation of differences in potency of effect of CI-994 between in vitro isolated enzyme or partially purified cellular fractions and effect in whole cells may lie in possible metabolic products of CI-994 that possess greater potency than the parent compound. However, the available evidence suggests only minimal structural modification of the compound takes place in vivo leaving parent compound intact in plasma or in urinary or fecal excretion (41, 51), thus leading to the conclusion that metabolism is not likely an explanation for the observed differences.

Because an increase in acetylated histone in cells could result from an activation of HAT, as well as from an inhibition of HDAC, we evaluated the effects of CI-994 on the in vitro activity of the HAT GCN5 (Fig. 4). The compound showed no activation of HAT activity at concentrations up to 100 µM. This demonstrates, especially in the context of the inhibition of HDAC seen in vitro, that the compound does not affect histone acetylation by activation of HAT under these conditions.

A continuing challenge exists in understanding how HDAC inhibition by CI-994 relates to its antitumor activity. The cell cycle distribution effects of the compound resulting in a primary block at the G1-S boundary (29) may reflect induction of cell cycle inhibitory proteins such as p21waf1/cip1, which is induced by a number of other HDAC inhibitors (22, 36, 43), but this remains to be determined. In addition, induction of apoptosis has been described as a consequence of HDAC inhibition by a variety of small molecule inhibitors (40, 43, 52). Whether CI-994 participates in this induction in cells or tumors also remains to be seen, although a recent in vivo study showing tumor shrinkage of LC12 squamous cell lung carcinoma treated with either CI-994 alone or in combination with gemcitabine suggests that apoptosis may be a mechanism through which the compound is exerting an antitumor effect (53, 54).

A better understanding of the use of CI-994 in ongoing clinical trials may be gained through application of the knowledge of its inhibitory effect on HDAC. In vivo antitumor efficacy studies and continuing clinical investigation of CI-994 done in combination with conventional antitumor agents may shed more light on the optimal use of the compound as a better understanding is gained of the potential benefits associated with appropriate manipulation of histone modifications in cancer therapy.


   Footnotes
 
2 Present address: Department of Cell and Structural Biology, University of Illinois, B107 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801. Back

3 The abbreviations used are: HAT, histone acetyltransferase; HDAC, histone deacetylase; CI-994, 4-(acetylamino)-N-(2-aminophenyl)benzamide; PMSF, phenylmethylsulfonyl fluoride; TCA, trichloroacetic acid; TSA, trichostatin A. Back

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.

Received 12/10/01; revised 11/26/02; accepted 2/ 3/03.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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