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Annals of Clinical & Laboratory Science 35:397-406 (2005)
© 2005 Association of Clinical Scientists

Histone Deacetylase Inhibitor Pharmacodynamic Analysis by Multiparameter Flow Cytometry

Eun Joo Chung1, Sunmin Lee1, Edward A. Sausville2, Qin Ryan2, Judith E. Karp3, Ivana Gojo4, William G. Telford5, Min-Jung Lee1, Hye Sik Kong6 and Jane B. Trepel1
1 Medical Oncology Clinical Research Unit, Center for Cancer Research, NCI, NIH; 2 Clinical Trials Unit, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, NCI, NIH; 3 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD; 4 Greenebaum Cancer Center at the University of Maryland Medical Center, Baltimore, MD; 5 Experimental Transplantation and Immunology Branch; 6 Urologic Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland

Address correspondence to Jane B. Trepel, Medical Oncology Clinical Research Unit, CCR, NCI, NIH, Building 10, Rm 12N230, 10 Center Drive, Bethesda, MD 20892, USA; tel 301 496 1547; fax 301 402 0172; e-mail trepel{at}helix.nih.gov. The current address of E. A. Sausville M.D., Ph.D., is: Greenebaum Cancer Center, University of Maryland Medical Center, 22 S Greene Street, Baltimore, MD 21201, USA.


   Abstract
Top
 Abstract
Introduction
 Methods and Materials
 Results
 Discussion
 References
 
Histone deacetylase (HDAC) inhibitors are a promising new class of anticancer drug. The aim of this study was to develop a versatile and sensitive technique for the pharmacodynamic (PD) assessment of HDAC inhibitor activity as monotherapy and in combination therapy. A multiparameter flow cytometric assay was developed initially in healthy donor lymphocytes and leukemia cell lines, and then tested in peripheral blood of solid tumor patients and in bone marrow aspirates of leukemia patients on phase I trials of the HDAC inhibitor MS-275. A technique was developed that allows highly sensitive single parameter determination of HDAC inhibitor activity in as little as 50 µl of whole blood. Multiparameter analysis enabled correlation on a single cell basis of protein acetylation with biologically relevant markers including cell lineage antigens, an apoptosis marker, and PD markers of other anti-cancer agents. The level of protein acetylation can be readily detected and quantified in peripheral blood or in bone marrow aspirates by flow cytometric analysis. The technique described has significant advantages for the PD assessment of HDAC inhibitors as monotherapy and as a component of combination therapy trials.

Keywords: HDAC, pharmacodynamic analysis, multiparameter, flow cytometry


   Introduction
Top
 Abstract
 Introduction
Methods and Materials
 Results
 Discussion
 References
 
The posttranslational modification of histones by acetylation and the association of acetylated histone with transcriptional activity were initially described 40 yr ago [1–3]. In the last 10 yr there has been a dramatic increase in interest in histone deacetylases (HDACs), both as key components of the transcription-regulatory apparatus and as targets for anticancer drug development. It is now clear that HDACs and histone acetyltransferases (HATs) are critical regulators of gene expression, that these enzymatic functions are frequently subverted in cancer, and that some structurally diverse small molecule HDAC inhibitors have promising activity in preclinical and early clinical trials [3–15].

Although HDAC inhibitors typically induce hyperacetylation of one or more core histones, protein acetylation, and thus the potential realm of HDAC substrates, is far more extensive than originally appreciated. Over 40 proteins have been reported to be modified by acetylation, including many proteins of central importance in cancer cell biology such as the retinoblastoma protein, p53, E2F, c-myc, beta-catenin, tubulin, and Hsp90 [16–18]. To date it is not known which protein or set of proteins is the critical effector of the anticancer activity of small molecule HDAC inhibitors, and the answer is likely to vary depending on the HDAC inhibitor and the tumor molecular subtype. Pharmacodynamic (PD) assays are critical for the assessment of clinical trials of molecular targeted agents. Although tumor biopsies are preferable to surrogates, biopsies are frequently difficult to obtain and many clinical trials have used peripheral blood mononuclear cells (PBMC) for PD analysis. As reported here, we have developed a flow cytometric HDAC inhibitor PD assay. This assay has a number of advantages over previously reported techniques, especially for the analysis of peripheral blood, bone marrow aspirates, malignant effusions, or other sources of cells in suspension. The assay requires as little as 50 µl of whole blood, and can be used to look at an array of questions including correlation of acetylation with cell lineage-specific markers, apoptosis markers, non-histone protein acetylation, and PD markers of agents that may be used in combination with HDAC inhibitor therapy.


   Methods and Materials
Top
 Abstract
 Introduction
 Methods and Materials
Results
 Discussion
 References
 
Patients.  PD studies of MS-275-treated patients were performed on peripheral blood obtained from patients enrolled in the NCI phase I study of MS-275 in advanced and refractory solid tumors or lymphoma. Patient characteristics, study design, and assessment of toxicity and response have been described [19]. Bone marrow aspirates were obtained from patients enrolled in the phase I study of MS-275 in patients with poor-risk hematologic malignancy at the Sidney Kimmel Comprehensive Cancer Center and the Greenebaum Cancer Center. The MS-275 trials were conducted under IRB-approved protocols of an NCI-sponsored IND. The protocol design and conduct have followed all applicable regulations, guidance, and local policies.

Antibodies.  Anti-acetylated lysine polyclonal (catalog #9441) and monoclonal (catalog #9681) antibodies were purchased from Cell Signaling Technology (Beverly, MA); APC-Cy7-conjugated anti-CD14 antibody (catalog #MHCD1414) was purchased from Caltag Laboratories (Burlingame, CA); FITC-conjugated anti-CD15 (catalog #555401), Cy-Chrome-conjugated anti-CD19 (catalog #555414), PE-conjugated anti-CD3 (catalog #347347), and PE-conjugated anti-caspase-3, active form (catalog #550821) antibodies were purchased from BD Transduction Laboratories (San Jose, CA); anti-acetylated tubulin monoclonal antibody (catalog #T 6793) was obtained from Sigma (St. Louis, MO); anti-Hsp70 antibody (catalog #sc-1060) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies, obtained from Caltag Laboratories, were FITC-conjugated goat F(ab’)2 anti-rabbit IgG (H+L) (catalog #L43001), FITC-conjugated goat F(ab’)2 anti-mouse IgG (H+L) (catalog #M35001), and PE-conjugated mouse F(ab’)2 anti-rabbit IgG (H+L) (catalog #M35004).

Antibody conjugation.  To expand the number of colors that could be looked at simultaneously, several antibodies were directly labeled using the Zenon labeling technique and Zenon labeling kits (Molecular Probes, Eugene, OR). Briefly, 1 µg of purified antibody was prepared in a small volume of PBS (<20 µl). The antibody was mixed with 5 µl of the Zenon labeling reagent and incubated for 5 min at room temperature. The Zenon blocking reagent (5 µl) was added, the mixture was incubated 5 min at room temperature, and the antibody was used for cell staining as described below.

Drugs and reagents.  MS-275 was supplied by Schering AG. Imatinib mesylate (imatinib, formerly STI571) was provided by Novartis. Trichostatin A (TSA, catalog #T8552) was obtained from Sigma, and Ficoll-Paque Plus was obtained from Amersham Biosciences (Piscataway, NJ). 17-allylamino-17-demethoxygeldanamycin (17-AAG) was obtained from the Developmental Therapeutics Program, NCI, NIH. Zenon Allophycocyanin Rabbit IgG Labeling Kit (catalog #Z-25351), Alexa Fluor 405 (Cascade Blue) Mouse IgG1 Labeling Kit (catalog #Z-25013), and 4,6 diamidino-2-phenylindole (DAPI, catalog # D21490 [GenBank] ) were purchased from Molecular Probes (Eugene, OR).

Immunocytochemistry.  Cells were pelleted onto glass slides by cytocentrifugation, stained as described below for flow cytometric analysis, counterstained with the fluorescent DNA dye DAPI, and viewed using a Leica DM IRB fluorescence microscope equipped with a Z-axis motor (Ludl Electronics, Hawthorne, NY). Stacks of images (13 to 19 optical sections at a step size of 0.3 µm) were taken with a digital camera (Hamamatsu) and processed using Openlab Volume Deconvolution software (Improvision, Lexington, MA).

Preparation of cells for in vitro studies.  PBMC from healthy donor buffy coats were isolated by centrifugation on Ficoll-Paque Plus, washed 2x with PBS, and incubated in complete medium (RPMI 1640 with 10% fetal bovine serum and antibiotics) containing 10 nM or 1 µM MS-275 for 24 hr at 37°C. The acetylation response to MS-275 was also studied in unfractionated buffy coats from healthy donors. The cells were washed 2x with PBS, resuspended in complete medium, MS-275 was added, and the cells were incubated for 24 hr at 37°C. K562 cells from the American Type Culture Collection (Manassas, VA) were grown in complete medium and incubated with MS-275, imatinib, TSA, or 17-AAG.

Staining for flow cytometry.  Fixation and permeabilization: Staining for flow cytometric analysis was performed on 50-100 µl of whole blood, PBMC (5x106 cells) or K562 (5x106 cells). The cells were washed with PBS, resuspended in fixation buffer (0.4 % paraformaldehyde in PBS), incubated for 5–10 min at 37°C, and washed with wash buffer (PBS containing 0.1% BSA). The fixed cells were resuspended in permeabilization buffer (0.4 % Triton X-100 in wash buffer), incubated for 5 min at room temperature, and washed with wash buffer. The permeabilization, washing, and staining methods resulted in significant RBC lysis and a separate RBC lysis step was not required.

Single or two-color flow cytometric assay.  After fixation and permeabilization, the cells were resuspended in 100 µl of wash buffer and incubated with anti-acetylated-lysine, anti-acetylated-tubulin, or anti-Hsp70 for 1 hr at room temperature and washed with wash buffer. The cells were incubated with secondary antibodies for 1 hr at room temperature and washed with wash buffer. For detection of apoptotic cells, antibody to the activated form of caspase 3 was added at the time of secondary antibody addition. Analysis of labeled cells used CELLQuest software (BDIS, San Jose, CA).

Multicolor flow cytometric assay.  For analysis of >2 colors, after fixation and permeabilization the cells were resuspended in 100 µl of wash buffer, directly-labeled antibodies were added, the cells were incubated for 1 hr at room temperature, washed, and analyzed. For each multicolor experiment, tubes were prepared in which the cells were stained separately for each fluorophore to be analyzed, and these samples were run to establish the compensation parameters.

Flow cytometric analysis.  For analysis of protein acetylation versus forward- or side-scatter cells were run on a FACSCalibur (Becton Dickinson). For analysis of >2 colors per cell, an LSRII (Becton Dickinson) equipped with the following lasers was used: (1) a Coherent Sapphire 488 nm diode-pumped solid state laser with 20 mW power output, (2) a JDS Uniphase helium-neon 633 nm (red) laser with 17 mW power output, (3) a Coherent Vioflame 408 nm violet laser diode with 25 mW power output. The 488 nm laser was used for FITC-conjugated anti-CD15 antibody, PE-conjugated anti-CD3, PE-conjugated antibody to activated caspase 3, and PE-Cy5 (Cychrome)-labeled anti-CD19. The helium-neon laser was used for polyclonal antibody to acetylated lysine, which was labeled with APC using the Zenon kit, or for APC-Cy7-conjugated anti-CD14. The violet laser was used for mono-clonal anti-acetylated lysine labeled with Cascade Blue.


   Results
Top
 Abstract
 Introduction
 Methods and Materials
 Results
Discussion
 References
 
Immunocytochemical analysis of protein acetylation.  To determine if an antibody to acetylated lysine can be used to assess the response to HDAC inhibitors, unfractionated buffy coats of healthy donors were incubated with the HDAC inhibitor MS-275 and examined for the level of protein acetylation by immunocytochemistry. Untreated cells showed a variable level of acetylation that ranged from undetectable to moderate (Fig. 1AGo). In the majority of cells treated with MS-275 (1 µM, 24 hr), protein acetylation was markedly increased (Fig. 1BGo). Examination of MS-275-treated cells by optical sectioning demonstrated that both cytoplasmic and nuclear staining could be visualized, with considerable cell-to-cell heterogeneity in the localization of acetylated proteins. The left image of Fig. 1CGo displays a cell with predominantly nuclear signal and the right image shows a cell with predominantly cytoplasmic signal. Comparison of the concentration-dependent effect of MS-275 on histone H3 acetylation and lysine acetylation by immunocytochemistry demonstrated a similar degree and kinetics of response over a period of 24 hr (data not shown).



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  		Fig. 1. Immunocytochemical analysis of protein acetylation. Healthy donor unfractionated buffy coats were treated with vehicle alone (A) or 1 µM MS-275 for 24 hr (B), labeled with anti-acetylated lysine antibody, and counterstained with DAPI. (C) Subcellular localization of acetylated proteins (left panel, predominantly nuclear staining; right panel, predominantly cytoplasmic staining). Cells were treated and stained as in (B).

 
Flow cytometric analysis of protein acetylation in healthy donor PBMC.  Although immunocytochemical techniques can clearly demonstrate the effects of HDAC inhibitors, a flow cytometric approach would have several advantages, particularly for quantification and multiple parameter analysis. Therefore we tested whether the antibody to acetylated lysine could be used in a flow cytometric assay. The technique was first used to examine healthy donor PBMC incubated in vitro with MS-275. The 2-parameter dot plot of forward- versus side-scatter was consistent with a cell population of predominantly small lymphocytes with a small population of larger, more granular cells (Fig. 2AGo). The negative control of cells incubated with normal rabbit IgG followed by FITC-conjugated secondary antibody showed no background staining (Fig. 2BGo).



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  		Fig. 2. Protein acetylation in healthy donor PBMC incubated in vitro for 24 hr with MS-275. (A) dot plot; (B) gray line depicts cells treated with vehicle and stained with secondary antibody alone. Filled area depicts cells treated with vehicle and stained with normal rabbit IgG followed by secondary antibody. (C) Concentration-dependent increase in acetylation.

 
Incubation in vitro with MS-275 induced a concentration-dependent increase in acetylation that could be seen with as little as 10 nM MS-275 (Fig. 2CGo). Various experiments demonstrated the reproducibility and stability of the protein acetylation assay, including (1) comparison of the same samples stained and analyzed immediately versus samples stained and analyzed after fixation and storage at 4°C for 48 hr, (2) comparison of the same samples stained and analyzed immediately versus samples stained and analyzed after viable cell freezing and thawing, (3) comparison of the same samples viably frozen, thawed, stained, and run in separate experiments over a period of several mo, and (4) analysis of hyperacetylation in samples stored at –80°C for over one yr (data not shown).

Flow cytometric analysis of protein acetylation in PBMC of patients receiving MS-275.  We next tested peripheral blood from patients on the NCI MS-275 phase I protocol. Use of whole, unfractionated peripheral blood is advantageous for several reasons, ie, a very low volume of blood can be used, cell loss is minimized, and processing time is shorter. Furthermore, the response can be followed in all nucleated cells of peripheral blood. Therefore we tested whether we could use whole blood as the starting material for the flow assay. The 2-parameter dot plots of forward- versus-side scatter of patient whole blood, shown in Figs. 3A and BGo, were consistent with populations of lymphocytes, granulocytes, and monocytes. Red blood cells were not seen because the assay fixation and permeabilization protocol was optimized to maximally induce RBC lysis without detectable effect on nucleated cell recovery or staining intensity (data not shown). Approximately 50% of the nucleated blood cells were hyperacetylated at 24 hr after MS-275 administration (Figs. 3C and DGo). There was an increase in the percent of positive cells and in the intensity of the acetylated lysine signal at 48 hr after MS-275 administration (Fig. 3CGo).



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  		Fig. 3. Protein acetylation in whole blood in response to MS-275 in vivo. Two patients on the NCI MS-275 protocol at the 6 mg/m2 dose level. (A & B): dot plots pre-treatment; (C & D): single parameter histograms show the level of acetylated lysine (faintly shaded area, pre-treatment; thin line, 24 hr post-treatment; thick line, 48 hr post-treatment).

 
Flow cytometric analysis of protein acetylation in a lymphocyte subset.  An advantage of the flow assay is the ability to determine the level of protein acetylation in distinct subsets of cells. To exploit this capability, we first took unfractionated whole blood from 2 patients on the NCI MS-275 phase I protocol and stained the cells for expression of acetylated lysine and the T cell marker CD3. In both patients distinct cell subsets were clearly seen, and the T cell population showed a strong hyperacetylation response (Fig. 4Go).



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  		Fig. 4. Protein acetylation versus the lymphocyte subset marker CD3 in response to MS-275 in vivo. Assay performed using whole blood from 2 patients on the NCI MS-275 protocol treated at 10 mg/m2. (A and B) patient 1; (C and D) patient 2 (x-axis, level of acetylated-lysine, y-axis, level of CD3 expression).

 
Five-color, seven-parameter flow cytometry of protein acetylation.  The flow assay for protein acetylation described here can be done using tabletop flow cytometers commonly available in clinical and research laboratories. However, to fully exploit the capabilities of this assay it can be expanded to examine simultaneously many more parameters, limited only by the instrumentation and fluorescent probe technology. As an example of this capability, we examined whole blood samples of patients on the MS-275 phase I protocol for protein acetylation pre- and post-treatment versus forward-scatter, side-scatter and 4 lineage markers, anti-CD19 (B cells), anti-CD15 (myeloid cells), anti-CD3 (T cells), and anti-CD14 (monocytes). Because of the complexity of displaying multiparameter data we used 2 axes to display the data but multiple colors to display the populations. Each of the cell populations had a distinctive pattern of basal protein acetylation and each population responded to the administration of MS-275 in vivo (Fig. 5Go). In panels A and B, the CD19+ (red) population is in the left 2 CD3– quadrants, and the CD3+ (pink) population is in the right 2 quadrants. Both CD19+ and CD3+ populations move up the y-axis in response to treatment. Similarly, increased protein acetylation is seen in response to treatment in CD15+ and CD14+ populations (panels C and D). CD15+ cells have the highest level of staining among the untreated populations and the smallest apparent response.



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  		Fig. 5. Multicolor flow cytometric analysis. Healthy donor unfractionated buffy coats incubated with 1 µM MS-275 for 24 hr. (A and C) untreated cells and (B and D) cells incubated with MS-275. (A and B) protein acetylation versus CD3 expression. (C and D) protein acetylation versus CD15 expression. Each color represents a different cell population.

 
Flow cytometric analysis of protein acetylation in bone marrow aspirates.  In addition to analysis of peripheral blood, this technique can be used to assess other sources of cells in suspension, such as bone marrow aspirates and malignant effusions. Bone marrow aspirates from 2 patients treated at the Greenebaum Cancer Center on the MS-275 phase I trial in hematologic malignancies were analyzed pre-treatment and 7 days after MS-275 administration. Prior to treatment the bone marrow samples contained >80% blasts. There was a uniform, weakly positive shift in patient #1, treated at 6 mg/m2, and in patient #2, treated at 8 mg/m2, there was a bimodal pattern with a population of cells showing a robust response (Fig. 6Go).



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  		Fig. 6. Protein acetylation in bone marrow aspirates in response to MS-275 in vivo. Acetylation was assessed in 2 patients on the University of Maryland MS-275 protocol 7 days after drug administration.

 
Dual pharmacodynamic analysis of protein acetylation and apoptosis in cells treated with MS-275 and imatinib.  The flow cytometric assay presented here could be a powerful tool to monitor pharmacodynamic changes induced by anticancer drugs used in monotherapy or combination therapy protocols. HDAC inhibitors have been reported to promote imatinib-mediated apoptosis in Bcr/Abl+ human myeloid leukemia cells, including imatinib-resistant cells [20,21]. We therefore examined the effect of MS-275 and imatinib, alone and in combination, on apoptosis in the Bcr/Abl+ cell line K562. Because MS-275 and imatinib have both been reported to induce apoptosis associated with caspase 3 activation [21–23], we took advantage of the availability of an antibody that specifically recognizes activated caspase 3 to develop a flow assay that can be used to monitor drug-induced apoptosis and then combined this with the assay for detection of acetylated lysine. It can be seen that both MS-275 and imatinib increased the percent of apoptotic cells, and MS-275 strongly upregulated acetylation in >50% of the cells (Fig. 7Go). A population of cells lost acetylation versus control and these cells were positive for activated caspase 3. Furthermore, a population of non-apoptotic cells was present after MS-275 or imatinib alone, and this population was almost eliminated by treatment with both MS-275 and imatinib.



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  		Fig. 7. Flow cytometric analysis of apoptosis versus protein acetylation. K562 cells were incubated with vehicle alone (A), 1 µM imatinib (B), 1 µM MS-275 (C), or both (D) for 48 hr. Dot plots display acetylated lysine on the x-axis and activated caspase 3 on the y-axis.

 
Multiparameter PD analysis of an HDAC inhibitor in combination with an Hsp90 inhibitor.  The previous experiment demonstrated measurement of apoptosis versus acetylation in response to 2 anti-cancer drugs. An additional capability is to look at specific PD markers simultaneously to monitor combination therapy. It has been reported that 17-AAG, an inhibitor of Hsp90, can synergize with histone deacetylase inhibitors in human leukemia cells [24]. Here we show the use of an antibody specific for acetylated tubulin as a marker of HDAC inhibitor activity versus an antibody to Hsp70, which is a PD marker of Hsp90-directed drugs. As the HDAC inhibitor for this experiment we used TSA, a member of the subgroup of HDAC inhibitors that induce tubulin hyperacetylation [25,26]. As shown by multidimensional dot plots (Figs. 8A and CGo) or by displaying each parameter separately (Figs. 8B and DGo), there is marked increase in Hsp70 expression and tubulin acetylation in response to treatment of K562 cells with 17-AAG and TSA.



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  		Fig. 8 Multiparameter pharmacodynamic analysis. K562 cells were treated with 1 µM 17-AAG and 500 nM TSA for 24 hr. (A and C) Dot plots of Hsp70 versus acetylated tubulin pre- (A) and post-treatment (C). (B and D) Single parameter histograms of Hsp70 (B) and acetylated tubulin (D), pre- and post-treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
References
 
PD analyses are of central importance in clinical trials of molecularly targeted cancer therapeutics. The identification of the best marker in an HDAC inhibitor trial, however, is rather complex. There are at least 19 HDAC isoforms in humans [27]. Little is known about their normal tissue or tumor distribution and data are largely incomplete on the isoform-specific effects of HDAC inhibitors. Hyperacetylation of histone H3 or H4 has been associated with regulated gene expression and has been used for PD analysis in HDAC inhibitor clinical trials. Although regulation of gene expression may be critically important in the therapeutic response to HDAC inhibitors, further work needs to be done to identify which genes should be monitored. This problem is compounded by the frequent necessity of using surrogates for PD analysis. The chromatin environment of a resting lymphocyte is likely to be very different from a proliferating tumor cell, typically of epithelial origin.

We have found that cDNA microarray of the HDAC inhibitors MS-275 and TSA induced upregulation of TGF-beta type II receptor and TGF-beta signaling, and this was confirmed by extensive molecular analysis in vitro [28]. To date we have not seen this in response to MS-275 in PBMC treated in vitro or isolated from solid tumor patients on the NCI phase I MS-275 study (data not shown). Thus it is not known which, if any, genes act as effectors of HDAC inhibitor anticancer activity. Furthermore, the relevant genes are likely to be tumor type-specific and quite possibly are not modulated in surrogate cells.

Recent data suggest that non-transcriptional events should also be considered in analysis of HDAC inhibitor antineoplastic activity [29]. More than 40 proteins, both nuclear and cytoplasmic, are substrates for acetylation [17,18,30]. Rather than measuring acetylation of any one protein, here we have established an assay that detects acetylated lysine, and thus has the potential to detect most of the previously identified acetylated proteins. As shown in Fig. 1Go, this antibody identifies both nuclear and cytoplasmic signals, and the nuclear versus cytoplasmic distribution varies from cell to cell. Several cytoplasmic proteins may be important in the therapeutic response, as may proteins that shuttle between the nucleus and cytoplasm. Of particular interest currently is the observation that Hsp90, an important target for anticancer therapeutics [31–36], can be acetylated in response to HDAC inhibitors, and that this correlates with marked changes in the levels of Hsp90 client proteins [17,37]. There are very few antibodies available for specific acetylated proteins. However, similarly to tyrosine-phosphorylated proteins, the number of antibodies should increase, and when available the specific antibodies can easily be incorporated into the flow assay.

The multiparameter flow assay described greatly facilitates correlative investigations that depend on single cell analysis, such as the level of acetylation versus induction of apoptosis or differentiation, cell subtype versus response, or response to combination therapy. The analyses of whole blood samples from patients pre- and post-treatment with the HDAC inhibitor MS-275 shown in Fig. 5Go demonstrate the power of the multiparameter flow technique to provide PD information on complex mixtures of cells. Each of the cell populations measured, including myeloid cell populations, has a characteristic basal level of acetylation and demonstrates a hyperacetylation response. The feasibility of measuring the protein hyperacetylation response in bone marrow aspirates is shown in Fig. 6Go. Further studies are required to determine the kinetics of response, dose-dependence, and correlation with treatment efficacy.

The multiparameter technique requires only 0.1 ml of whole blood and allows quantification of multiple signals on thousands of cells within min. These single cell correlations are impossible in batch techniques such as western blot, ELISA or micro-array, and would be considerably more difficult and time-consuming using immunocytochemistry and image analysis. The 1-color flow cytometric assay described here can be performed on the amount of blood in a finger stick. For either technique, density gradient centrifugation and RBC lysis are not required, which minimizes cell loss and preparation time and allows the cells to be fixed and stabilized without the need of special laboratory techniques and expertise.

An important direction for the clinical development of HDAC inhibitors is the identification of the most appropriate, molecularly-defined patient populations. The multiparameter flow assay could be advantageous for identification of molecular marker-positive versus negative cells and detection of response in these subpopulations. The assay can be used for blood, bone marrow aspirates, effusions, and lymph node suspensions and could possibly be adapted for tumor biopsies.

The ability of a flow technique to detect and analyze rare cells makes it an attractive approach to PD analysis of circulating epithelial tumor cells, especially as enrichment techniques are improved. A second important direction for HDAC inhibitors is the development of effective combination drug strategies. Two of the drug combinations shown here, an HDAC inhibitor plus imatinib and an HDAC inhibitor plus 17-AAG, have been reported to be effective in preclinical studies. For the agents shown here and for a variety of other molecular targeted drugs, the multiparameter flow technique is well suited to analysis of the efficacy of combination therapy. Thus the multiparameter flow technique can be advantageous for current studies of HDAC inhibitors and for future HDAC inhibitor clinical development.


   Acknowledgments
 
We thank Dr. Susan Leitman and the Department of Transfusion Medicine, Clinical Center, NIH, for help in obtaining and processing peripheral blood leukocytes from healthy donors. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Dr. Jane Trepel received research funding under a Cooperative Research and Development Agreement between the NCI and Schering AG. Dr. Sausville reports that, after leaving the government, he is a consultant to Schering AG.


   References
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
 References
 

  1. Allfrey VG, Faulkner R, Mirsky AE. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. PNAS USA 1964;51:786–794.[Free Full Text]
  2. Kouzarides T. Acetylation: a regulatory modification to rival phosphorylation? Embo J 2000;19:1176–1179.[Medline]
  3. Grozinger CM, Schreiber SL. Deacetylase enzymes: biological functions and the use of small-molecule inhibitors. Chem Biol 2002;9:3–16.[Medline]
  4. Urnov FD, Wolffe AP. Chromatin organisation and human disease. Emerging Theraputic Targets 2000;4: 665–685.
  5. Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov 2002;1:287–299.[Medline]
  6. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3:415–428.[Medline]
  7. Melnick A, Licht JD. Histone deacetylases as therapeutic targets in hematologic malignancies. Curr Opin Hematol 2002;9:322–332.[Medline]
  8. Curtin M, Glaser K. Histone deacetylase inhibitors: the Abbott experience. Curr Med Chem 2003;10:2373–2392.[Medline]
  9. Yoshida M, Matsuyama A, Komatsu Y, Nishino N. From discovery to the coming generation of histone deacetylase inhibitors. Curr Med Chem 2003;10:2351–2358.[Medline]
  10. Gilbert J, Gore SD, Herman JG, Carducci MA. The clinical application of targeting cancer through histone acetylation and hypomethylation. Clin Cancer Res 2004;10:4589–4596.[Abstract/Free Full Text]
  11. Kristeleit R, Stimson L, Workman P, Aherne W. Histone modification enzymes: novel targets for cancer drugs. Expert Opin Emerg Drugs 2004;9:135–154.[Medline]
  12. Lindemann RK, Gabrielli B, Johnstone RW. Histone-deacetylase inhibitors for the treatment of cancer. Cell Cycle 2004;3:779–788.[Medline]
  13. Marks PA, Richon VM, Miller T, Kelly WK. Histone deacetylase inhibitors. Adv Cancer Res 2004;91:137–168.[Medline]
  14. Piekarz R, Bates S. A review of depsipeptide and other histone deacetylase inhibitors in clinical trials. Curr Pharm Des 2004;10:2289–2298.[Medline]
  15. Rosato RR, Grant S. Histone deacetylase inhibitors in clinical development. Expert Opin Investig Drugs 2004; 13:21–38.[Medline]
  16. Sterner DE, Berger SL. Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 2000;64:435–459.[Abstract/Free Full Text]
  17. Yu X, Guo ZS, Marcu MG, Neckers L, Nguyen DM, Chen GA, Schrump DS. Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228. JNCI 2002;94:504–513.[Abstract/Free Full Text]
  18. Oh HJ, Chung EJ, Lee S, Loaiza-Perez A, Sausville EA, Trepel JB, . Targeting histone deacetylase as a strategy for cancer prevention, In: Cancer Chemoprevention. Cancer Drug Discovery and Development (Kelloff GJ, Hawk ET, Sigman CC, Eds):. Humana Press, Totowa, NJ, 2004; pp 659–677.
  19. Ryan QC, Headlee D, Acharya M, Sparreboom A, Trepel JB, Ye J, Figg WD, Hwang K, Chung EJ, Murgo A, Melillo G, Elsayed Y, Monga M, Kalnitskiy M, Zwiebel J, Sausville EA. Phase I and pharmacokinetic study of MS-275, a histone deacetylase inhibitor, in patients with advanced and refractory solid tumors and lymphoma. J Clin Oncol 2005;[Epub ahead of print].
  20. Nimmanapalli R, Fuino L, Bali P, Gasparetto M, Glozak M, Tao J, Moscinski L, Smith C, Wu J, Jove R, Atadja P, Bhalla K. Histone deacetylase inhibitor LAQ824 both lowers expression and promotes proteasomal degradation of Bcr-Abl and induces apoptosis of imatinib mesylate-sensitive or -refractory chronic myelogenous leukemia-blast crisis cells. Cancer Res 2003;63:5126–5135.[Abstract/Free Full Text]
  21. Yu C, Rahmani M, Almenara J, Subler M, Krystal G, Conrad D, Varticovski L, Dent P, Grant S. Histone deacetylase inhibitors promote STI571-mediated apoptosis in STI571-sensitive and -resistant Bcr/Abl+ human myeloid leukemia cells. Cancer Res 2003;63:2118–2126.[Abstract/Free Full Text]
  22. Lucas DM, Davis ME, Parthun MR, Mone AP, Kitada S, Cunningham KD, Flax EL, Wickham J, Reed JC, Byrd JC, Grever MR. The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells. Leukemia 2004; 18:1207–1214.[Medline]
  23. Mow BM, Chandra J, Svingen PA, Hallgren CG, Weisberg E, Kottke TJ, Narayanan VL, Litzow MR, Griffin JD, Sausville EA, Tefferi A, Kaufmann SH. Effects of the Bcr/abl kinase inhibitors STI571 and adaphostin (NSC 680410) on chronic myelogenous leukemia cells in vitro. Blood 2002;99:664–671.[Abstract/Free Full Text]
  24. Rahmani M, Yu C, Dai Y, Reese E, Ahmed W, Dent P, Grant S. Coadministration of the heat shock protein 90 antagonist 17-allylamino-17-demethoxygeldanamycin with suberoylanilide hydroxamic acid or sodium butyrate synergistically induces apoptosis in human leukemia cells. Cancer Res 2003;63:8420–8427.[Abstract/Free Full Text]
  25. Blagosklonny MV, Robey R, Sackett DL, Du L, Traganos F, Darzynkiewicz Z, Fojo T, Bates SE. Histone deacetylase inhibitors all induce p21 but differentially cause tubulin acetylation, mitotic arrest, and cytotoxicity. Mol Cancer Ther 2002;1:937–941.[Abstract/Free Full Text]
  26. Koeller KM, Haggarty SJ, Perkins BD, Leykin I, Wong JC, Kao MC, Schreiber SL. Chemical genetic modifier screens: small molecule trichostatin suppressors as probes of intracellular histone and tubulin acetylation. Chem Biol 2003;10:397–410.[Medline]
  27. Yang XJ, Seto E. Collaborative spirit of histone deacetylases in regulating chromatin structure and gene expression. Curr Opin Genet Dev 2003;13:143–153.[Medline]
  28. Park SH, Lee SR, Kim BC, Cho EA, Patel SP, Kang HB, Sausville EA, Nakanishi O, Trepel JB, Lee BI, Kim SJ. Transcriptional regulation of the transforming growth factor beta type II receptor gene by histone acetyltransferase and deacetylase is mediated by NF-Y in human breast cancer cells. J Biol Chem 2002;277:5168–5174.[Abstract/Free Full Text]
  29. Johnstone RW, Licht JD. Histone deacetylase inhibitors in cancer therapy: is transcription the primary target? Cancer Cell 2003;4:13–18.[Medline]
  30. Sterner DE, Berger SL. Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 2000;64:435–459.
  31. Goetz MP, Toft DO, Ames MM, Erlichman C. The Hsp90 chaperone complex as a novel target for cancer therapy. Ann Oncol 2003;14:1169–1176.[Abstract/Free Full Text]
  32. Neckers L, Ivy SP. Heat shock protein 90. Curr Opin Oncol 2003;15:419–424.[Medline]
  33. Sausville EA, Tomaszewski JE, Ivy P. Clinical development of 17-allylamino,17-demethoxygeldanamycin. Curr Cancer Drug Targets 2003;3:377–383.[Medline]
  34. Solit DB, Scher HI, Rosen N. Hsp90 as a therapeutic target in prostate cancer. Semin Oncol 2003;30:709–716.[Medline]
  35. Workman P. The opportunities and challenges of personalized genome-based molecular therapies for cancer: targets, technologies, and molecular chaperones. Cancer Chemother Pharmacol 2003;1(Suppl 52):S45–S56.
  36. Bagatell R, Whitesell L. Altered Hsp90 function in cancer: a unique therapeutic opportunity. Mol Cancer Ther 2004;3:1021–1030.[Abstract/Free Full Text]
  37. Atadja P, Hsu M, Kwon P, Trogani N, Bhalla K, Remiszewski S. Molecular and cellular basis for the anti-proliferative effects of the HDAC inhibitor LAQ824. Novartis Found Symp 2004;259:249–266; discussion 266–268, 285–288.[Medline]



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