Histone Deacetylase Inhibitors Selectively Suppress Expression of HDAC7

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Histone deacetylase inhibitors selectively suppress expression of HDAC7

Milos Dokmanovic, Gisela Perez, Weisheng Xu, histones or p21 gene expression. Selective down-regulation Lang Ngo, Cathy Clarke, Raphael B. Parmigiani, of HDAC7 protein may serve as a marker of response of and Paul A. Marks tumors to HDACi. [Mol Cancer Ther 2007;6(9):2525–34]

Cell Biology Program; Memorial Sloan-Kettering Cancer Center, New York, New York Introduction Histone deacetylases (HDAC) comprise 18 members in humans and are generally classified into class I and class II Abstract based on homologies to yeast deacetylases RPD3 and There are 18 histone deacetylases (HDAC) generally HDA1,respectively,and class III with homology to yeast divided into four classes based on homology to yeast Sir2 group (1–4). Class I includes HDAC1,HDAC2, HDACs. HDACs have many protein substrates in addition HDAC3,and HDAC8; class II includes HDAC4,HDAC5, to histones that are involved in regulation of gene HDAC6,HDAC7,and HDAC9. HDAC6 and HDAC10 expression, cell proliferation, and cell death. Inhibition of have two catalytic sites and may be called class IIB. HDACs can cause accumulation of acetylated forms of HDAC11 has conserved residues in its catalytic core that these proteins, thus altering their function. HDAC inhib- are shared by both class I and class II enzymes and is itors (HDACi), such as the hydroxamic acid–based sometimes placed in a class IV (2). Classes I,II,and IV vorinostat (suberoylanilide hydroxamic acid), inhibit the HDACs are zinc dependent. In addition to histones, + zinc-containing classes I, II, and IV, but not the NAD - HDACs have many nonhistone protein targets,which dependent class III, enzymes. HDACis are a group of novel are involved in regulation of gene expression,cell proli- anticancer agents. Vorinostat is the first HDACi approved feration,migration,and death pathways (1–4). Further- for clinical use in the treatment of the cancer cutaneous more,phylogenetics evidence indicates that these enzymes T-cell lymphoma. Factors affecting expression of HDACs are present in organisms that do not have histones (5). are not well understood. This study focuses on the effect It seems that these enzymes may more properly be of the HDACi vorinostat on the expression of class I and considered ‘‘lysine deacetylases’’ rather than specifically class II HDACs. We found that vorinostat selectively HDACs (6). down-regulates HDAC7 with little or no effect on the Biological activity of different HDACs are not redundant. expression of other class I or class II HDACs. Fourteen cell Class I HDACs generally have a nuclear localization, lines were examined, including normal, immortalized, whereas class II HDACs may shuttle between cytoplasm genetically transformed, and human cancer-derived cell and nucleus and have biological activities related to specific lines. Down-regulation of HDAC7 by vorinostat is more cell lineages (1–4,6). Class III HDACs have homology pronounced in transformed cells sensitive to inhibitor- to yeast Sir2 group of deacetylases,have an absolute induced cell death than in normal cells or cancer cells + requirement for NAD ,and are not inhibited by com- resistant to induced cell death. Modulation of HDAC7 pounds that inhibit classes I and II enzymes (e.g., levels by small interfering RNA–mediated knockdown trichostatin A or vorinostat). Analysis of HDAC1 knockout or by HDAC7 overexpression is associated with growth phenotype indicates that it is essential for proliferation arrest but without detectable changes in acetylation of and viability of embryos despite compensatory increases in HDAC2 and HDAC3 (7). HDAC9 null mice have defects in cardiac muscle development (8,9). HDAC7 null mouse embryos display defects in blood vessel development and integrity (10). HDAC7 is involved in T-cell differentiation Received 4/9/07; revised 6/29/07; accepted 7/11/07. (11,12). In addition to inhibition of enzymatic activity, Grant support: NIH grant P30CA-08748-41, Jack and Susan Rudin Foundation grant, David H. Koch Foundation grant, Experimental valproic acid is reported to selectively induce proteasomal Therapeutics Center at Memorial Sloan-Kettering Cancer Center Prostate degradation of HDAC2 (13). Vorinostat down-regulates Cancer Research award, and DeWitt Wallace Research fund. HDAC3 and several other proteins in chronic myeloid cell The costs of publication of this article were defrayed in part by the lines (14). payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18U.S.C. Section 1734 solely to The expression of certain HDACs is altered in tumor indicate this fact. compared with normal cells. Increased expression of Requests for reprints: Paul A. Marks, Cell Biology Program, Memorial HDAC1 has been found in gastric cancers,esophageal Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065. Phone: 212-639-2861; Fax: 212-639-2861. squamous cell carcinoma,hormone-refractory prostate E-mail: [email protected] cancer,and colon cancer (15–18). HDAC2 expression is Copyright C 2007 American Association for Cancer Research. increased in human colon cancer,in which there is a loss doi:10.1158/1535-7163.MCT-07-0251 of APC tumor suppressor (19). HDAC3 expression is

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increased in human tumor samples of colon cancer Materials and Methods compared with normal tissue (18,20). HDAC5 is down- Cells and Cell Culture regulated in colon cancer (21). HDAC proteins are The following normal cells were used: normal lung recruited to oncogenic fusion proteins in various leuke- fibroblast (WI38) obtained from American Type Culture mias (22). Collection (ATCC),normal foreskin fibroblast cell line HDAC inhibitors (HDACi) include a group of novel (hFS) obtained from Cell Culture Core Facility-Yale Skin targeted agents with significant anticancer activity in Diseases Research Center,and normal prostate epithelium patients with both hematologic and solid tumors at doses cell line (PrEC) obtained from Clonetics/Cambrex-Cambrex that are well tolerated by patients (1–4,23). Vorinostat BioScience Rockland,Inc. (Table 1). Genetically manipu- (suberoylanilide hydroxamic acid),the leading HDACi in lated cell lines include lung fibroblast (VA13),foreskin clinical development,is a hydroxamic acid that inhibits fibroblast (BJE,BJELB,BJELR),and prostate epithelium catalytic activities of both class I and class II HDACs (23). (PrEC-LE,PRC-LHSR) that were immortalized or trans- Vorinostat has received Food and Drug Administration formed by distinct oncogenic elements [cell lines were approval for the treatment of cutaneous T-cell lymphoma, obtained from ATCC and Weinberg (27) and Hahn (28)]. representing the first of the new HDACi to be approved Human prostate cancer–derived cell lines (LNCaP,DU145, for clinical use (23). PC3),human bladder carcinoma–derived cell lines (T24), Normal cells are relatively resistant to HDACi-induced and multiple myeloma–derived cell lines (ARP-1) were cell death. The difference in sensitivity of normal and obtained from ATCC (Table 1). Normal,immortalized and transformed cells to HDACi seems to be related to ‘‘cell transformed foreskin cells were maintained in culture as context’’ (1–4,6). HDACi can alter the structure and described (27). Human prostate cancer cell lines were function of a broad range of proteins regulating cell maintained in culture as previously described (24). Immor- proliferation,migration,and death that are substrates of talized human prostate epithelial cell (PrEC-LE) and HDACs (1–4,6). Cancer cells generally have multiple transformed human prostate epithelial cells (PrEC-LHSR) defects in proteins regulating cell proliferation,cell migra- were kindly provided by Dr. William Hahn (Dana- tion,and cell death (1–4,6,24). Thus,cancer cell may have Faber Cancer Center; ref. 28). All prostate epithelial cells less capacity to compensate for the HDACi effects than (normal,immortalized,and transformed) were propagated normal cells (6,25,26). in defined medium (PrEGM),as recommended by Clonetics/ This study focuses on the effects of the HDACi on the Cambrex (28). expression of class I and class II HDAC proteins. We Human bladder carcinoma–derived cell line T24 (29), found that vorinostat selectively suppresses HDAC7 with normal lung fibroblast cell line WI-38,and transformed little or no effect on the expression of other class I and lung fibroblast cell line VA-13 were obtained from ATCC class II HDACs examined. Fourteen cell lines were studied,including three normal,three immortalized,and and cultured as previously described (26,29). Multiple eight transformed cell lines,of which six were sensitive myeloma–derived cell line ARP-1 was maintained in and two were resistant to vorinostat-induced cell death. culture as previously described (30). f 6 Down-regulation of HDAC7 protein by vorinostat was For protein analysis,cells were plated at 10 per most pronounced in transformed cells,which are sensitive 150-mm plate and harvested at described time points. to HDACi-induced cell death. In normal cells and in For cell growth and viability curves,cells were plated at cancer cells that are resistant to HDACi-induced cell 25,000 to 50,000 cells per 1-mL well in triplicate. Cells death,there was relatively little or no detectable suppres- were recovered after trypsinization,and cell density and sion of HDAC7 protein. Depsipeptide,a structurally viability were determined (29). different HDACi than vorinostat,had similar effects. The Constructs down-regulation of HDAC7 protein by HDACi is associ- Flag-tagged HDAC7-overexpressing construct in ated with down-regulation of HDAC7 mRNA with no pcDNA3.1 vector was a gift from Dr. Eric Verdin,University alteration in the half-life of HDAC7 protein. Selective of California,San Francisco (31). down-regulation of HDAC7 by small interfering RNA Drugs and Chemicals resulted in growth arrest in normal and transformed cells Vorinostat was synthesized as described (32) and diluted but did not cause other changes associated with vorino- in DMSO,for addition to culture medium at concentrations stat,e.g.,cell death of transformed cells,induction of p21, indicated for each study. Depsipeptide (33) was obtained and accumulation of acetylated histones or of acetylated from National Cancer Institute/NIH and added to the tubulin. Overexpression of HDAC7 protein was associated culture medium at concentrations indicated below. with growth arrest in transformed cells but did not block Western Blot Analysis vorinostat effects on transformed cell death. The present Cell lysates were prepared,and the protein concentration findings indicate that HDACi can selectively and pro- of extracted lysate was determined by a Bradford protein foundly suppress HDAC7 expression in transformed cells assay (29). Protein (30 Ag) was subjected to SDS-PAGE sensitive to HDACi-induced cell death. Expression of (Bio-Rad) and transferred to a Hybond-poly (vinylidene HDAC7 may be a marker of response of tumor cells to difluoride) membrane (Amersham Pharmacia Biosciences). vorinostat therapy. Membranes were then blocked with PBS–0.1% Tween with

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Table 1. Characteristics and sources of cells

Cell line Original cell line Cell type Genetic manipulation Mutations Reference/source hFS Foreskin fibroblast Normal None NA Yale Skin Diseases Research Center BJE Foreskin fibroblast Immortalized* hTERTc NA Weinberg laboratory (27) BJELB Foreskin fibroblast immortalized hTERT and SV40 NA Weinberg laboratory (27) oncogenic products BJELR Foreskin fibroblast Transformedb hTERT,SV40,H-Ras NA Weinberg laboratory (27) PrEC Prostate epithelial Normal none NA Clonetics/Cambrex PrEC-LE Prostate epithelial Immortalized Large T and hTERT NA Hahn laboratory (28) PrEC-LHSR Prostate epithelial Transformed Large T,hTERT, NA Hahn laboratory (28) small T,and H-Ras LNCaP Lymph node Prostate cancerx N/A PTEN-mutated ATCC metastasis p53-wild type Rb-wild type DU-145 Brain metastasis Prostate Cancer NA PTEN-wild type ATCC p53-mutated Rb-mutated PC-3 Bone metastasis Prostate Cancer NA PTEN-mutated ATCC P53-mutated RB-wild type T24 Bladder carcinoma Transformed NA P53-mutated ATCC Rb-positive WI-38 Lung fibroblast Normal NA NA ATCC VA-13 Lung fibroblast Transformed SV40-transformed NA ATCC ARP-1 Multiple myeloma Transformed NA NA J. Hardoc (Arkansans Cancer Research Center)

Abbreviation: NA,not applicable. *Immortalized cells grow indefinitely in suitable media but do not cause tumors on transplant to animal models. cRetroviral infection with indicated gene(s). bTransformed cells will form a tumor when transplanted to animal model. xProstate cancer cell lines derived from biopsies of human prostate cell metastatic site.

5% bovine serum albumin and incubated with specific Small Interfering RNA Transfection primary antibody for 18 to 24 h. Horseradish peroxidase– Small interfering RNA transfections were done by elec- labeled secondary antibody was added and visualized troporating respective cell lines using conditions commer- using the Western Lightening Chemiluminescence reagent cially available from Amaxa,Inc. HDAC7 small interfering (Perkin-Elmer Life Sciences). RNA was purchased from Qiagen (Hs_HDAC7A-2-HP Antibodies small interfering RNA). HDAC7 sense small interfering The primary antibodies used were as follows: polyclonal RNA sequence is (GGCUGGAAACAGAAACCCA) dTdT, rabbit anti-mouse HDAC1 at 1:300 (Upstate Biotechno- and HDAC7 antisense small interfering RNA sequence is logy),polyclonal rabbit anti-human HDAC2 at 1:1000 r(UGGGUUUCUGUUUCCAGCC) dTdT. Control (nonsi- (Calbiochem),polyclonal rabbit anti-human HDAC3 at lencing) small interfering RNA was commercially available 1:300 (Santa Cruz Biotechnology),polyclonal rabbit anti- from Qiagen. Control (mock) transfections were done with human HDAC4,HDAC5,HDAC6,HDAC7,and HDAC8 buffer used for dissolving small interfering RNA. at 1:1,000 (Santa Cruz Biotechnology), polyclonal rabbit Northern Blot Analysis anti-HDAC10 antibody at 1:200 (Bio-Vision),monoclonal mRNA for Northern blot analysis was prepared by mouse antitubulin at 1:2,000 (Calbiochem), anti-p21 mouse harvesting cells and resuspending cellular material in monoclonal antibody CP74 at 1:1,000 (Neo Marker), mouse Trizol according to the standard protocol (34). HDAC7 monoclonal anti–acetylated tubulin antibody at 1:1,000 probe was prepared from commercially available partial (Sigma),polyclonal rabbit anti–acetyl histone H3 at 1:200 cDNA clone. (RZPD-GMBH). (Upstate),and polyclonal rabbit anti–acetyl histone H4 at Measurement of HDAC7 Protein Half-life 1:200 (Upstate). To measure HDAC7 protein half-life,transformed fore- The secondary antibodies used were as follows: donkey skin fibroblast cells BJELR were pulse chased according to a anti-rabbit horseradish peroxidase conjugated at 1:10,000 modified protocol described in Xu et al. (35). Cells were (Amersham Biosciences) and goat anti-mouse peroxidase grown to 50% confluence in 60-mm dishes and preincu- conjugated at 1:10,000 (Jackson Immunoresearch). bated for 30 min in methionine-free 5% FCS DMEM to

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deplete cold methionine and cysteine from the cells. by the addition of excess cold methionine/cysteine. Cells Cells were washed once in PBS,then labeled for 3 h with were rinsed twice in ice-cold PBS and then cultured for 4 h 10 mCi/mL S35 methionine/cysteine in methionine/ in the presence and absence of 5 Amol/L vorinostat. Cells cysteine-free DMEM–10% FCS. Reaction was terminated were washed in cold PBS,recovered,lysed,and frozen in

Figure 1. Effect of vorinostat on cell growth and viability of normal, immortalized, and transformed cells. A, foreskin fibroblast cells; normal (hFS), immortalized (BJE and BJELB), and transformed (BJELR) cells were cultured with different concentrations of vorinostat (as indicated). B, different prostate epithelium cells; normal (PrEC), immortalized (PrEC-LE), and transformed (PrEC-LHSR) were cultured with different concentrations of vorinostat (as indicated).

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Figure 2. Effect of vorinostat on HDAC1, HDAC2, HDAC3, HDAC8and HDAC4, HDAC5, HDAC6 protein levels in normal, immortalized, and transformed cells. A, Western blot analysis of class I HDAC1, HDAC2, HDAC3, HDAC8and class II HDAC4 and HDAC6 in normal (hFS), immortalized (BJE and BJELB), and transformed (BJELR) cells cultured without (no treatment) and with vorinostat (5 Amol/L) for the times indicated. a-Tubulin was used as normalization for Western blot analysis. B, Western blot analysis of class I HDAC1, HDAC2, HDAC3, and HDAC8and class II HDAC4, HDAC5, and HDAC6 of normal (PrEC), immortalized (PrEC-LE), and transformed (PrEC-LHSR) cells cultured without (no treatment) and with vorinostat (5 Amol/L) for times indicated. C, Western blot analysis of class I HDAC1, HDAC2, and HDAC3 and class II HDAC4, HDAC6, and HDAC10 in transformed human prostate cells LNCaP and DU145 sensitive to vorinostat (5 Amol/L) – induced cell death and PC3 resistant to vorinostat (5 Amol/L) – induced cell death.

liquid nitrogen until cells are ready to perform immuno- BJELB,PrEC-LE),and transformed cells (BJELR,PrEC- precipitation. TCA precipitation experiment determined LHSR,ARP-1,VA13,LNCaP,DU-145,PC-3,and T24; labeled incorporation into the cells. HDAC7 immunopre- Fig. 1; Table 1). Data were not shown for LNCaP,DU-145, cipitation with polyclonal anti–HDAC-7 rabbit antibody PC-3,ARP-1,WI-38,VA-13,andT24,as these were was done (35). HDAC7 band was detected by drying the previously published (24,26,29,30,34). Cells differed in gel and placing it overnight in a storage phosphor screen sensitivity to vorinostat-induced cell growth arrest; ARP-1 (Amersham Biosciences). The signal of the amplified bands cell growth was inhibited at 1 Amol/L vorinostat,whereas was quantified with a PhosphoiImager (Molecular Dynamics) 5 Amol/L vorinostat were required to completely inhibit using ImageQant software. the growth of normal (hFS),immortalized (BJE and BJELB), and transformed (BJELR) foreskin cells (Fig. 1A). Vorino- stat at 2.5 Amol/L inhibited growth of normal (PrEC), Results immortalized (PrEC-LE),and transformed (PrEC-LHSR) Vorinostat Inhibits Growth of Normal, Immortalized, prostate epithelial cells (Fig. 1B). The effects of vorinostat and Transformed Cells on normal PrEC cell line growth were only apparent after Vorinostat inhibited the growth of all cells studied: day 3,and this may reflect slow growth of PrEC normal normal cells (hFS,PrEC,WI38),immortalized cells (BJE, prostate cells (Fig. 1B). The cell number in control PrEC

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culture was 2 104 cells per milliliter compared with published data for WI38,VA-13,ARP-1,LNCaP,DU-145; 1.3 104 cells per milliliter in cultures with the inhibitor. refs. 24,26,30,34; Fig. 1A and B). Culture with 5 Amol/L Vorinostat at 2.5 Amol/L inhibited growth of prostate vorinostat caused 10% loss in viability in immortalized cancer–derived cell lines LNCaP,DU-145,and PC-3 (24) BJELB and >20% loss in viability in the transformed cell and WI38 and VA-13 cells (26). Culture with 5 Amol/L line BJELR by day 3 (Fig. 1A). Vorinostat at 5 Amol/L vorinostat completely inhibited the growth of T24 bladder caused no detectable loss in viability in immortalized cell carcinoma–derived cell line (29). line BJE or in normal cell lines hFS and WI38 and normal Vorinostat-Induced Cell Death of Transformed Cells, prostate cells PrEC (data previously published; ref. 26; But Not of Normal Cells Fig. 1). Vorinostat at 10 Amol/L caused an 80% loss in Normal (hFS,WI38, and PrEC) and immortalized (BJE) viability in immortalized PrEC-LE and >90% loss in viability foreskin cell lines were relatively resistant to vorinostat- in transformed cell line PrEC-LHSR by day 3 (Fig. 1B). induced cell death compared with immortalized cell lines Prostate cancer–derived cell lines LNCaP and DU-145 lost BJELB and PrEC-LE and transformed cell lines BJELR, >50% in viability in culture with 5 Amol/L vorinostat, PrEC-LHSR,VA13,ARP1,LNCaP,and DU145 (previously whereas PC-3 cells were resistant to HDACi-induced cell

Figure 3. Effect of vorinostat on HDAC7 protein level in normal, immortalized, and transformed cells. A, Western blot analysis of HDAC7 level in hFS, BJE, BJELB, and BJELR cells cultured with vorinostat for the time and with concentration of inhibitor indicated. B, Western blot analysis of HDAC7 level in prostate epithelium cells (PrEC, PrEC-LE, and PrEC-LHSR) cultured without (no treatment) and with vorinostat. C, Western blot analysis of HDAC7 in LNCaP, DU145, and PC3. D, Western blot analysis of HDAC7 in bladder carcinoma – derived cell line T24 cultured with vorinostat as indicated. Sensitive and resistant refers to vorinostat-induced cell death. Culture of the cell lines with vorinostat was done for the duration and concentration as indicated. HDAC2 was a loading control in series (A) as well as a-tubulin (from Fig. 2B and C).

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death (24). Bladder carcinoma cell line T24 was resistant to vorinostat-induced cell death at 5 Amol/L (29). The relative resistance of the normal cells (foreskin and prostate) to vorinostat-induced cell death,compared with certain transformed cells,is consistent with previous findings (26,36). Effect of Vorinostat on Expression of Class I and Class II HDACs We examined the effect of vorinostat on expression of class I HDAC1,HDAC2,HDAC3,and HDAC8 and class II HDAC4 and HDAC6 in foreskin fibroblast cells hFS (normal fibroblast cell line),BJE (immortalized cell line), BJELB (immortalized cell line),and BJELR (transformed cell line; Fig. 2A): the effect of vorinostat on the expression of class I HDAC1,HDAC2,HDAC3,and HDAC8 and class II HDAC4,HDAC5,and HDAC6 in prostate epithelium- derived cells,PrEC (normal cells),PrEC-LE (immortalized cell line),and PrEC-LHSR (transformed cell line; Fig. 2B) and class I HDAC1,HDAC2,and HDAC3 and class II HDAC4,HDAC6,and HDAC10 in human prostate cancer cells LNCaP,DU-145,and PC-3 (Fig. 2C). There was little or no consistent change in the level of these class I (HDAC1, HDAC2,HDAC3,and HDAC8) and class II (HDAC4, HDAC5,HDAC6,and HDAC10) HDACs in normal,immortalized,or transformed cells cultured with 5 Amol/L vorinostat for 24 or 48 h (Fig. 2A-C). Vorinostat caused a decrease in HDAC7 protein level in a dose- and time-dependent manner in all cells examined (Fig. 3). A decrease in HDAC7 protein was detectable in normal hFS cells only in culture with 5 Amol/L vorinostat for 48 h or 10 Amol/L vorinostat for 24 h. In immortalized Figure 4. Effect of vorinostat on the level of HDAC7 mRNA message in foreskin cells. Northern blot analysis was done on foreskin fibroblast cell cells (BJE and BJELB),the decrease in HDAC7 was A A samples collected from cells cultured with 5 mol/L vorinostat for the detectable at 5 mol/L vorinostat after 24 h of culture, times indicated. 18S mRNA was used as a loading control. whereas in transformed cells (BJELR),a decrease was detectable at 1 Amol/L vorinostat within 24 h of culture (Fig. 3A). similar to vorinostat,caused a selective decrease in HDAC7 PrEC (Fig. 3B) showed relatively little suppression of in LNCaP and DU-145 to a much greater degree than in HDAC7 proteins compared with immortalized (PrEC-LE) PC-3 cells (data not shown). and transformed PrEC-LHSR cells cultured with 5 Amol/L Effect of Vorinostat on HDAC7 mRNA Expression vorinostat for 24 h. A decrease in HDAC7 level was We next determined if the selective decrease in HDAC7 detected in PrEC-LHSR prostate cancer cells with 5 Amol/L protein level induced by vorinostat was associated with a vorinostat at 24 h of culture (Fig. 3B). decrease in HDAC7 mRNA. We used HDAC7 partial In prostate cancer cell lines LNCaP and DU-145,5 Amol/L cDNA as a probe and 18S as a loading control (Fig. 4). vorinostat caused a marked decrease in HDAC7 within Culture with 5 Amol/L vorinostat caused a decrease in 24 h of cultures (Fig. 3C). In PC-3 cell line resistant to HDAC7 mRNA in normal cells at 6 and 14 h,whereas by vorinostat-induced cell death,the decrease in HDAC7 was 6 h,it was undetectable in immortalized foreskin (BJE much less pronounced than in either LNCaP or DU145 and BJELB) and transformed foreskin (BJELR) cells that (Fig. 3C). In the culture T24 cells,a cell line resistant to persisted through 48-h duration of culture (Fig. 4). Vori- vorinostat-induced cell death (29),HDAC7 protein de- nostat at 5 Amol/L caused a profound decrease in HDAC7 crease is pronounced only in culture with 10 Amol/L mRNA in prostate cancer cell lines LNCaP,DU-145,and vorinostat for 48 h (Fig. 3D). ARP-1,a human multiple PC-3 (data not shown). myeloma cell line,is very sensitive to vorinostat-induced Vorinostat did not alter HDAC7 protein stability as cell death (26) and showed a marked decrease in HDAC7 determined in transformed BJELR cells (data not shown). protein in culture with 1.0 Amol/L within 24 h of culture Effect of HDAC7 Small Interfering RNA and Transient (data not shown). Overexpression of HDAC7 Protein We examined the effect of another HDACi,depsipeptide, We next determined the effect of suppressing HDAC7 that is structurally different than vorinostat on HDAC7 protein with HDAC7 small interfering RNA. Mock-treated level in cells sensitive (DU145 and LNCaP) and resistant cells and nonsilencing control oligos were used as controls (PC-3 cells) to induced death by this agent. Depsipeptide, and compared with the effects of down-regulating HDAC7

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protein by small interfering RNA. After electroporation,the of HDAC7 from cytomegalovirus-based construct did not cells were allowed to recover for 24 h and then replated. result in change of p21 expression or the level of acetylated HDAC7 small interfering RNA oligos strongly reduced tubulin in transformed BJELR cells (Fig. 5C). Levels of HDAC7 protein within 24 h after electroporation (day 0) in global histone H3 and histone H4 acetylation were not both normal (hFS) and transformed (BJELR) cells (Fig. 5A). also changed by HDAC7 knockdown (data not shown). To test the effect of HDACi in mock,nonsilencing,and Vorinostat increased expression of p21 and accumulation HDAC7 small interfering RNA–treated BJELR cells,the of acetylated tubulin and acetylated histones in these cells were cultured without and with different concen- transformed cells (Fig. 5C). trations of vorinostat. In cells cultured without inhibitor,a similar level of HDAC7 protein was found in mock and nonsilencing BJELR cells whereas there was no detectable Discussion HDAC7 protein in cells electroporated with HDAC7 small This study has found that HDACis,vorinostat and interfering RNA oligo (Fig. 5B). In BJELR cells cultured depsipeptide,selectively down-regulate HDAC7,a class II with 2 or 5 Amol/L vorinostat for 24 h,as expected,there HDAC. This is the first report that identifies class II was little or no detectable HDAC7 protein. hFS and BJELR HDAC7 protein level as a target for vorinostat and cells treated with small interfering RNA oligos for HDAC7 depsipeptide. We have shown this down-regulation of had a decreased rate of growth but no loss in viability HDAC7 with 14 different cell lines,including a normal compared with control cells. There was no difference in foreskin fibroblast,a normal prostate epithelial and a sensitivity to vorinostat-induced cell death in cells with normal lung fibroblast cell line,two immortalized foreskin HDAC7 knockdown compared with cells expressing fibroblast and immortalized prostate epithelium cell lines, HDAC7 (data not shown). and transformed cell lines derived either by genetic Vorinostat can cause an increase in global histone manipulation of foreskin fibroblast and prostate epithelium acetylation,tubulin acetylation,and p21 level in many or derived from prostate cancer,bladder carcinoma,and transformed cells (29,30). Suppression of HDAC7 by small multiple myeloma. Vorinostat-induced HDAC7 down- interfering RNA oligos,as well as transient overexpression regulation is associated with reduction in HDAC7 mRNA

Figure 5. Effect of small interfering RNA for HDAC7 on HDAC7 expression in normal (hFS) and transformed (BJELR) cells. A, Western blot analysis of HDAC7 level in hFS and BJELR cells after electroporation with mock, nonsilencing, and small interfering RNA for HDAC7 oligos (day 0). B, Western blot analysis of HDAC7 in cells cultured with different concentrations of vorinostat (as indicated) for 24 h. a-Tubulin was used for loading control. C, Western blot analysis of HDAC7, p21, and acetylated tubulin in BJELR cells electroporated with small interfering RNA for HDAC7 to down-regulate HDAC7 and electroporated with HDAC7- expressing vector to overexpress HDAC7. BJELR cells were also cultured with vorinostat at concentrations indicated for 24 h, and HDAC7 levels assayed by Western blot.

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Halkidou K, Gaughan L, Cook S, Leung HY, Neal DE, Robson CN. normal or transformed cells. Although the mechanism of Upregulation and nuclear recruitment of HDAC1 in hormone refractory growth inhibition is not clear,our data indicate that this prostate cancer. Prostate 2004;59:177 – 89. effect is independent of p21. 18. Wilson AJ, Byun DS, Popova N, et al. Histone deacetylase 3 (HDAC3) Vorinostat has been recently approved for clinical and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer. J Biol Chem 2006;281: treatment of cutaneous T-cell lymphoma,a rare T-cell 13548– 58. malignancy (23). There are multiple clinical trials,either 19. Zhu P, Martin E, Mengwasser J, Schlag P, Janssen KP, Gottlicher M. alone or in combination with other agents,that are Induction of HDAC2 expression upon loss of APC in colorectal tumori- genesis. Cancer Cell 2004;5:455 – 63. currently under way with vorinostat. HDAC7 may be a 20. Shebzukhov YV, Koroleva EP, Khlgatian SV, et al. Antibody response useful biomarker predictive of response to therapy with to a non-conserved C-terminal part of human histone deacetylase 3 in vorinostat. colon cancer patients. Int J Cancer 2005;117:800 – 6. 21. Scanlan MJ, Welt S, Gordon CM, et al. Cancer-related serological recognition of human colon cancer: identification of potential diagnostic Acknowledgments and immunotherapeutic targets. Cancer Res 2002;62:4041 – 7. We thank Dr. Eric Verdin (University of California, San Francisco) for the 22. Redner RL, Liu JM. Leukemia fusion proteins and co-repressor gift of HDAC7-overexpressing plasmid and Joann Perrone and Mabel complexes: changing paradigms. J Cell Biochem 2005;94:864 – 9. Miranda for their expertise in literature searches and preparation of this manuscript. Vorinostat and related compounds are the intellectual 23. Marks PA, Breslow R. 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Milos Dokmanovic, Gisela Perez, Weisheng Xu, et al.

Mol Cancer Ther 2007;6:2525-2534.

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