ARTICLE

Modulation of p53, ErbB1, ErbB2, and Raf-1 Expression in Lung Cancer Cells by Depsipeptide FR901228

Xiaodan Yu, Z. Sheng Guo, Monica G. Marcu, Len Neckers, Dao M. Nguyen, G. Aaron Chen, David S. Schrump

Affiliation of authors: X. Yu, Z. S. Guo, D. M. Nguyen, G. A. Chen, D. S. Schrump (Thoracic Oncology Section, Surgery Branch), M. G. Marcu, L. Neckers (Tumor Cell Biology Section), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD.

Correspondence to: David S. Schrump, M.D., Thoracic Oncology Section, Surgery Branch, Bldg. 10, Rm. 2B-07, 10 Center Dr., Bethesda, MD 20892–1502 (e-mail: David_Schrump{at}nih.gov).


    ABSTRACT
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Histone deacetylases (HDACs) modulate chromatin structure by regulating acetylation of core histone proteins. HDAC inhibitors, such as depsipeptide FR901228 (FK228), induce growth arrest and apoptosis in a variety of human cancer cells by mechanisms that cannot be attributed solely to histone acetylation. This study evaluated the mechanisms by which FK228 mediates apoptosis in non-small-cell lung cancer (NSCLC) cells. Methods: Proliferation and apoptosis were assessed in a panel of NSCLC cell lines that vary in the expression of the growth-regulating proteins p53, pRb, and K-Ras treated with a clinically relevant dose of FK228 (25 ng/mL). Western blot and immunoprecipitation techniques were used to analyze expression of cell-cycle proteins (cyclin A, cyclin E, p53, and p21), signaling-related proteins (ErbB1, ErbB2, and Raf-1), activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2), binding of mutant p53 and Raf-1 to heat shock protein (Hsp)90, and acetylation of Hsp90. Results: FK228 treatment inhibited growth and induced apoptosis in NSCLC cells expressing wild-type or mutant p53. FK228 treatment led to altered expression of cyclin A, cyclin E, and p21, and to reduced expression of mutant, but not wild-type, p53. FK228-treated cells also were depleted of ErbB1, ErbB2, and Raf-1 proteins, and exhibited lower ERK1/2 activity. FK228 treatment also inhibited the binding of mutant p53 and Raf-1 to Hsp90; this inhibition was associated with acetylation of Hsp90. Conclusions: FK228 depletes the levels of several oncoproteins that are normally stabilized by binding to Hsp90 in cancer cells. The resulting ability of FK228 to diminish signal transduction via pathways involving Raf-1 and ERK may contribute to the potency and specificity of this novel antitumor agent.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An emerging body of literature supports the role of chromatin structure in regulating cell-cycle progression, differentiation, and apoptosis in normal and malignant cells (13). The basic structure of chromatin (referred to as the nucleosome) is composed of 146 base pairs of DNA coiled around an octamer of histones consisting of two molecules each of histone H2A, H2B, H3, and H4. Nucleosomes, which are separated by variable lengths of linker DNA bound to histone H1, associate with additional proteins to form higher order chromatin [reviewed in (4)]. Specific chromatin-remodeling complexes (such as SWI/SFF, RSF, and WCRF), variations in histone subtypes, and the phosphorylation, methylation, and acetylation status of histone proteins determine the nature and specificity of DNA–histone interactions (5,6). In general, increased DNA–histone interactions condense chromatin and repress transcription, whereas decreased DNA–histone interactions relax chromatin and enhance gene expression (5,7).

The steady-state level of acetylation of core histones is governed by the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs) (4). Several families of HATs have been identified, including PCAF-GCN-5, p300/CBP, TAF-250, SRC-1, and MO2, which exhibit highly restricted substrate specificities [reviewed in (8,9)]. To date, several major classes of HDACs have been defined. Class I HDACs (HDAC-1, -2, -3, and -8) are similar to yeast Rpd3p (7); these HDACs associate with proteins such as sin-3a, NCOR, and MECP-2 to repress transcription (4,7). Class II HDACs (HDAC-4, -5, -6, and -7) are more similar to yeast Hda1p (10). Recent studies indicate that the activation and subcellular localization of these HDACs may be regulated by mitogen-activated protein (MAP) kinases (7). For example, the extracellular signal-regulated kinases 1 and 2 (ERK1/2) associate with and phosphorylate HDAC-4, thereby facilitating its translocation to the nucleus and repression of transcription (11). Similar mechanisms may regulate the activation and localization of other class II HDACs, which also shuttle between the cytoplasm and nucleus and are recruited to distinct regions of chromatin by DNA-specific sequences (12,13). Another class of HDACs exhibits homology to yeast Sir2p (10); limited information is available concerning the role of these HDACs in normal and malignant mammalian cells.

Recently, considerable efforts have focused on identifying novel agents that modulate chromatin structure and elucidating the mechanisms by which these compounds mediate growth arrest and apoptosis in cancer cells. Depsipeptide FR901228 (FK228) is one of several HDAC inhibitors in early clinical development that exhibit in vitro and in vivo cytotoxic activity against a variety of human tumor cells (14,15). Previously we reported that, when administered alone or in sequence with 5 aza-2` deoxycytidine, FK228 induced apoptosis in cultured lung cancer cells but not in normal human bronchial epithelial cells (16); similar findings have been reported by Zhu and colleagues (17). Although FK228 is equipotent to trichostatin A regarding inhibition of HDAC activity in cultured cancer cells (14), the mechanisms regulating FK228-mediated cytotoxicity have not been fully elucidated. Indeed, several recent studies (1820) have indicated that histone acetylation alone is not sufficient to account for the antiproliferative effects of HDAC inhibitors. This study was undertaken to characterize the mechanisms by which FK228 induces growth arrest and apoptosis in lung cancer cells.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Drugs and Chemicals

FK228 and 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) were obtained from the Developmental Therapeutics Program, National Cancer Institute (Bethesda, MD). Trichostatin A and 4,5 dimethyl-2-yl 2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). PD 98059 was obtained from Alexis Biochemicals (San Diego, CA).

Cell Lines

The H460, A549, H358, H2087, H2227, H322, and H596 human non-small-cell lung cancer (NSCLC) lines and the SK-Br-3 breast cancer line were available in tissue banks at the National Cancer Institute. Detailed information regarding the origin and genotype of these lines can be obtained from the American Type Culture Collection website (www.atcc.org). Briefly, H460 and A549 express wild-type p53, pRb, and activated K-Ras and H-Ras, respectively. H358 cells are p53 null and express pRb and activated K-Ras. H2087, H2227, and H322 express mutant p53 and pRb; H322 cells express activated K-Ras. H596 cells express mutant p53 but do not express pRb. All cell lines were maintained in RPMI-1640 medium containing 10% fetal bovine serum and antibiotics and were confirmed to be free of Mycoplasma contamination.

Measurement of Proliferation and Apoptosis

Lung cancer cells were seeded into 96-well plates (1 x 104 cells per well). After 24 hours, cells were treated with FK228 (25 ng/mL) diluted in complete medium for 6 or 24 hours. After drug exposure, medium was aspirated and replenished with complete medium. At appropriate time points, cell proliferation was evaluated by MTT assays according to the manufacturer's recommended protocol. Each experiment was performed three times.

For the analysis of apoptosis, 1 x 106 lung cancer cells were exposed to complete medium or FK228 (25 ng/mL) diluted in complete medium for 6 or 24 hours. After 6 hours of drug exposure, the medium was aspirated and replenished with complete medium. Twenty-four hours after drug exposure, cells were harvested and apoptosis was quantified by ApoBrdU techniques (21) using protocols and reagents contained in the ApoBrdU kit (BD PharMingen, San Diego, CA).

RNA Isolation and Northern Hybridization

A probe for p53 was generated by reverse transcriptase–polymerase chain reaction, gel purified, and radiolabeled with 32[P]d-adenosine triphosphate (32[P]d-ATP) by random primer techniques. Total RNA was isolated from lung cancer lines using an RNeasy kit (QIAGEN, Valencia, CA) and resolved by electrophoresis through 1% agarose formaldehyde gels (20 µg of total RNA per lane). After the gels were denatured in a solution containing 0.05 M NaOH and 1.5 M NaCl for 30 minutes, the gels were renatured in a solution containing 0.5 M Tris (pH 7.5) and 1.5 M NaCl for 20 minutes. The RNA samples were then blotted overnight onto Magnacharge nylon membranes (Micron Separations, Inc., Westboro, MA) by capillary transfer in 20x saline sodium citrate (SSC) buffer. The membranes were hybridized with the 32P-labeled, denatured p53 probe in Hybrisol® I solution (Intergen, Purchase, NY) overnight at 45 °C. The blots were washed twice with 2x SSC containing 0.1% sodium dodecyl sulfate (SDS) for 10 minutes at room temperature: once with 0.2x SSC containing 0.1% SDS at 42 °C for 15 minutes, and once with 0.1x SSC containing 0.1% SDS at 68 °C for 15 minutes. Bound radioactivity was detected by exposing the blots to x-ray films for 2–48 hours at –70 °C.

Immunoprecipitation and Western Blotting

The following primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA): murine monoclonal anti-p53 (DO-1) (dilution 1 : 500) and goat polyclonal anti-p53 (dilution 1 : 200); murine monoclonal anti-p21 (dilution 1 : 200); murine monoclonal anti-MDM2 (dilution 1 : 200); rabbit polyclonal anti-p14/ARF (dilution 1 : 200); goat polyclonal anti-heat shock protein (Hsp) 90 (dilution 1 : 200); and mouse monoclonal anti-Hsp70 (dilution 1 : 200). Another murine monoclonal anti-Hsp90 antibody (dilution 1 : 200) was purchased from Stressgen (Victoria, British Columbia, Canada). Murine monoclonal anti-cyclin A (dilution 1 : 200) and anti-cyclin E (dilution 1 : 200) antibodies were obtained from BD Biosciences (Franklin Lakes, NJ). Murine monoclonal antibodies recognizing c-Raf-1 (dilution 1 : 1000) and epidermal growth factor receptor (EGFR)/ErbB1 (dilution 1 : 1000) were obtained from Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibodies recognizing HER-2/ErbB2, phosphorylated p53(Ser37), phosphorylated p53(Ser15), phosphorylated p53(Ser20), phosphorylated p53(Ser392), acetylated lysine, phospho-p44/42 MAP kinase(Thr202/Tyr204), and p44/42 MAP kinase (all used at dilution 1 : 1000) were purchased from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal antibody recognizing acetylated p53 (dilution 1 : 50) and murine monoclonal anti-{beta}-actin antibody (dilution 1 : 5000) were obtained from Oncogene Research Products (Boston, MA).

For the immunoprecipitation experiments, cellular extracts from approximately 1 x 106 cells were prepared in lysis buffer containing 10 mM Tris (pH 7.5), 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40 (NP-40), 1 mM Na3VO4, and 1mM phenylmethylsulfonyl fluoride, incubated on ice for 10 minutes, and centrifuged at 10 000g to remove cellular debris. Protein concentrations in the lysates were quantified using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Approximately 500 µg of total proteins was incubated with 2 µg of primary antibody at 4 °C for 2 hours, after which 20 µL of protein A/G-Plus-Agarose (Santa Cruz Biotechnology) was added to the mixture and incubated overnight at 4 °C. Agarose–antibody–protein complexes were washed three times with lysis buffer. After discarding the supernatant from the final wash, the antibody–protein complexes were resuspended in 40 µL of Laemmli loading buffer (Bio-Rad Laboratories, Hercules, CA) and boiled for 2 minutes. Twenty microliters of each sample was loaded onto 10% polyacrylamide gels (10% Tris–HCL Ready Gel; Bio-Rad Laboratories), and the immunoprecipitated proteins were separated by gel electrophoresis.

For western blot analysis, approximately 1 x 106 to 1 x 107 cells were lysed in laemmli buffer. Whole-cell lysates were incubated on ice for 10 minutes and centrifuged at 10 000g to remove cellular debris; protein concentrations in the lysates were quantified using a BCA protein assay kit. Approximately 60 µg of total proteins were resolved on 4%–20% SDS-polyacrylamide gels. Proteins were then electroblotted onto Immun-blotTM polyvinylidene difluoride membranes (Bio-Rad Laboratories) and incubated at room temperature for 1 hour with a solution containing 5% nonfat dry milk, 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween 20 (TBST). The membranes were probed with the indicated primary antibodies overnight at 4 °C, washed in TBST buffer, and then incubated for 60 minutes with appropriate horseradish peroxidase-conjugated, species-specific secondary antibodies at room temperature. The membranes were washed in TBST buffer, and antibody-labeled proteins were detected using protocols and reagents contained in the enhanced chemiluminescence (ECLTM) kit (Amersham, Buckinghamshire, England).

ERK1/2 Kinase Assays

ERK1/2 activity was determined by analyzing MAP kinase-induced phosphorylation of Elk-1 using reagents and protocols contained in the p44/42 MAP Kinase Assay Kit (Cell Signaling Technology). Briefly, 2 x 106 H322 lung cancer cells were seeded in six-well plates and treated with complete medium or FK228 (25 ng/mL) diluted in complete medium for 6 or 24 hours. Immediately following drug exposure, cells were lysed in a buffer contained in the kit. Phosphorylated p44/42 (ERK1/2) was immunoprecipitated from approximately 200 µg of total cellular protein using 15 µL of immobilized murine monoclonal anti-phospho-p44/42 (Thr202/Tyr204) MAP kinase antibody. After being washed, the pellet was incubated for 30 minutes at 30 °C with 50 µL of kinase buffer plus 200 µM ATP and 2 µg of glutathione-S-transferase (GST)-Elk-1 fusion protein. The reaction was stopped by the addition of SDS sample buffer and boiled for 5 minutes, and the entire sample was loaded onto an SDS–polyacrylamide gel. The gel was then immunoblotted with a rabbit polyclonal antibody recognizing phosphorylated Elk-1 (dilution 1 : 1000). Phospho-p44/42 and total p44/42 levels were evaluated using western blot techniques and phosphorylation-specific rabbit polyclonal antibodies (dilution 1 : 1000).

Novobiocin–Sepharose, ATP–Sepharose, and Geldanamycin Binding Assays

Novobiocin–sepharose, ATP–sepharose, and geldanamycin–affigel beads were prepared as described (22). 35S-labeled Hsp90 was prepared by in vitro transcription and translation using the TnTTM-coupled Rabbit Reticulocyte Lysate kit (Promega, Madison, WI) (23). For direct competition experiments, radiolabeled Hsp90 was added to novobiocin–sepharose, ATP–sepharose, or geldanamycin–affigel beads in the presence of increasing concentrations of FK228; Hsp90 was eluted and quantified as described (22). For analysis of the indirect inhibition of Hsp90 binding activity by FK228, approximately 5 x 106 SK-Br-3 cancer cells were treated with FK228 (25 ng/mL) for 2, 4, 6, or 24 hours. Immediately after drug exposure for all treatment conditions, cells were lysed in TNESV buffer (50 mM Tris, 2 mM EDTA, 100 nM NaCl, 1 nM sodium vanadate, 1% NP-40, pH 7.5), and total cellular proteins were quantified using the BCA protein assay. Hsp90 was affinity precipitated from 200 µg of total protein using ATP–sepharose beads and detected by western blot analysis as described (22).


    RESULTS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Effects of FK228 on Proliferation and Apoptosis in Lung Cancer Cells

In preliminary experiments, MTT and ApoBrdU assays were used to evaluate the effects of FK228 on proliferation and apoptosis, respectively, in cultured lung cancer cells. NSCLC lines were treated for 6 or 24 hours with FK228 at a dose of 25 ng/mL; this dose of FK228, which was used previously for our gene induction studies (16), corresponds to 50% of the free drug concentration in plasma from lung cancer patients receiving this HDAC inhibitor at the maximum tolerated dose (Schrump DS: unpublished data). FK228 mediated time-dependent growth inhibition and apoptosis in lung cancer cells (representative data pertaining to H322 and H460 cells are depicted in Fig. 1, A and BGo, respectively). Although growth arrest appeared comparable in H322 and H460 cells after FK228 exposure, apoptosis was more pronounced in H322 cells, which express mutant p53 and high levels of ErbB1 and ErbB2, relative to H460 cells, which express wild-type p53 and low levels of ErbB1 and ErbB2 oncoproteins.




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Fig. 1. Time-dependent growth inhibition (A) and apoptosis (B) in H322 and H460 lung cancer cell lines after exposure to FK228 (25 ng/mL). A) H322 and H460 cells were exposed to FK228 for 6 or 24 hours, after which they were grown for the indicated time periods in complete medium without additional drug. Cells maintained in complete normal medium (NM) and not exposed to FK228 were used as a control. Proliferation was determined by 4,5 dimethyl-2-yl 2,5-diphenyl tetrazolium bromide assays according to protocols recommended by the manufacturer (Sigma Chemical Co., St. Louis, MO). Points represent the mean optical density at 570 nm (OD570) and upper 95% confidence interval for triplicate cultures. B) Apoptosis in H322 and H460 cells after FK228 exposure for 6 or 24 hours. The percentage of apoptotic cells was determined 24 hours after initiation of drug treatment by the ApoBrdU technique (21). Bars represent the mean and upper 95% confidence intervals of three independent experiments. Gray bars represent cells treated with NM; black bars represent cells treated with FK228 (25 ng/mL) for 6 hours; open bars represent cells treated with FK228 (25 ng/mL) for 24 hours.

 
Effects of FK228 on Cyclin A, Cyclin E, p21, and p53 Expression

To examine potential mechanisms regulating FK228-mediated growth arrest and apoptosis in lung cancer cells, western blot techniques were used to evaluate the expression of a variety of cell-cycle-related proteins in H358, H460, and H596 lung cancer cell lines after 6 or 24 hours of FK228 (25 ng/mL) exposure. These three cell lines were chosen for this analysis because they exhibit different p53, pRb, and K-Ras expression profiles. Consistent with previously published studies regarding HDAC inhibitors in cancer cells (24,25), FK228 decreased cyclin A protein levels and increased p21 protein levels in all three cell lines. Furthermore, FK228 increased cyclin E protein levels in H358 and H460 cells, which express Rb, but had no effect on cyclin E protein levels in H596 cells, which do not express this tumor suppressor protein. In addition, FK228 appeared to increase wild-type p53 protein levels in H460 cells and to decrease mutant p53 protein levels in H596 cells (Fig. 2, AGo).



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Fig. 2. Effect of FK228 treatment on p21, p53, cyclin A, and cyclin E expression in non-small-cell lung cancer (NSCLC) cells. A) H358, H460, and H596 NSCLC cells were treated with FK228 (25 ng/mL) for 6 or 24 hours. Proteins from whole-cell lysates, obtained 24 hours after initiation of drug treatment, were resolved by polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted using a murine anti-p21 antibody, a murine anti-p53 antibody (DO-1) that recognizes mutant and wild-type p53, a murine anti-cyclin A antibody, and a murine anti-cyclin E antibody. All immunoblots were stripped and probed with a mouse monoclonal anti-{beta}-actin antibody to control for loading. B) Additional NSCLC lines (H2227, H2087, H322, and A549) were treated with FK228 (25 ng/mL) for 6 or 24 hours. p53 expression was assessed by immunoblot analysis 24 hours after initiating drug treatment using a murine monoclonal antibody (DO-1). C) H2087, H596, H460, and A549 NSCLC cell lines were treated with FK228 (25 ng/mL) for 24 hours. Northern analysis was performed to detect p53 messenger RNA (mRNA) levels before and after FK228 treatment. Upper panel, p53 mRNA levels; lower panel, ethidium bromide-stained ribosomal RNA levels to demonstrate equivalent loading of RNA. mt = mutant; wt = wild-type.

 
Because initial data suggested that wild-type and mutant p53 protein levels were differentially affected by FK228, additional western blot experiments were performed to further examine the effects of FK228 on p53 expression in lung cancer cells (Fig. 2, BGo). Wild-type p53 protein levels in A549 cells were essentially stable after FK228 exposure. In contrast, mutant p53 protein levels were decreased in H322, H2087, and H2227 cells after FK228 exposure in a manner analogous to that observed in H596 cells. The differences in p53 protein levels after FK228 exposure could not be attributed to differences in steady-state messenger RNA (mRNA) levels because northern analysis revealed that FK228 treatment decreased steady-state levels of both mutant and wild-type p53 mRNA (Fig. 2, CGo). Collectively, these results indicated that FK228 might preferentially stabilize wild-type p53 protein in lung cancer cells.

Effects of FK228 on MDM2 and p14/ARF Expression

The stability of p53 is regulated to a great extent by its interactions with MDM2 and p14/ARF. MDM2 facilitates nuclear export and degradation of p53 via ubiquitin-mediated proteolysis, whereas p14/ARF interacts with MDM2, thereby inhibiting ubiquitination of p53 (26). Western blot experiments were performed to examine whether FK228-mediated changes in p53 protein levels could be due to alterations in MDM2 or p14/ARF expression. Because our previous experiments indicated that depletion of mutant p53 protein levels occurred after a relatively brief exposure to FK228, these experiments were performed using only 6-hour FK228 exposure conditions. As shown in Fig. 3Go, FK228 treatment decreased expression of MDM2 as well as p14/ARF in H2227 and H2087 lung cancer cells. After FK228 exposure, MDM2 levels were essentially unchanged in H322 and H460 cells, which do not express p14/ARF.



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Fig. 3. Effect of FK228 treatment on the p53-associated proteins MDM2 and p14/ARF. H2227, H2087, H322, and H460 non-small-cell lung cancer cells were treated with FK228 (25 ng/mL) for 6 hours. Proteins from whole-cell lysates, obtained 24 hours after initiation of drug treatment, were resolved by polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted for MDM2 and p14/ARF protein levels using a mouse monoclonal antibody specific for MDM2 and a rabbit polyclonal antibody specific for p14/ARF. The immunoblots were then stripped and probed with a murine monoclonal anti-{beta}-actin antibody to control for loading. H322 and H460 cells do not express p14/ARF.

 
Effects of FK228 on Phosphorylation and Acetylation of p53

Although deceased p14/ARF expression might have contributed to the reduction in mutant p53 protein levels observed in H2227 and H2087 lung cancer cells after FK228 exposure, this mechanism could not account for the depletion of mutant p53 in H322 cells or the stabilization of wild-type p53 levels in H460 cells. Consequently, additional experiments were performed to determine whether FK228 could differentially modulate phosphorylation and/or acetylation of wild-type and mutant p53 because these post-translational modifications are known to influence the stability of p53 (26). Western blot experiments using phosphorylated position-specific antibodies revealed that a 6-hour FK228 exposure did not increase phosphorylation at Ser15, Ser20, or Ser392 (data not shown). However, FK228 increased phosphorylation at position Ser37 of wild-type and mutant p53 in A549 and H596 lung cancer cells, respectively (Fig. 4Go).



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Fig. 4. Effect of FK228 treatment on p53 phosphorylation and acetylation in non-small-cell lung cancer cells. A549 and H596 cells were treated with FK228 (25 ng/mL) for 6 or 24 hours. Analysis was performed 24 hours after the initiation of drug treatment for all treatment groups. Phosphorylation of p53 was determined by western blot analysis of whole-cell lysates using a rabbit polyclonal antibody specific for phosphorylated p53 [phospho-p53(Ser37) IB] or a murine monoclonal antibody (DO-1) recognizing mutant and wild-type p53 (p53 IB). To detect changes in p53 acetylation, p53 was immunoprecipitated from cell lysates with mouse monoclonal antibody DO-1. The immunoprecipitates were then analyzed by western blot analysis with a rabbit polyclonal antibody specific for acetylated p53 (acetylated p53 IB/p53IP) or a goat polyclonal antibody that recognizes wild-type or mutant p53 (p53 IB/p53 IP).

 
Because acetylation of p53 could not be readily detected by western blot techniques, an antibody that recognizes mutant p53 as well as wild-type p53 was used to immunoprecipitate p53 from cell lysates, after which western blot analysis was performed using an antibody specific for acetylated p53. FK228 mediated time-dependent acetylation of wild-type p53 in A549 cells (Fig. 4Go). Acetylation of p53 in H596 lung cancer cells could not be evaluated conclusively using these methods because total mutant p53 levels were markedly depleted after the 6-hour FK228 exposure.

Effects of FK228 on Raf-1, ErbB1, and ErbB2 Protein Levels

The effects of FK228 on p53 expression in lung cancer cells appeared analogous to those mediated by ansamycins isolated from actinomycetes broth (27); these antibiotics stabilize wild-type p53 and deplete a variety of oncoproteins (including mutant p53) in cancer cells (28). To further examine this issue, the effects of FK228 in H322 lung cancer cells were directly compared with those observed after exposure to the ansamycin derivative, 17-AAG (21). In addition, H322 cells were treated with trichostatin A to ascertain whether other HDAC inhibitors might have effects similar to those mediated by FK228. As shown in Fig. 5Go, the effects of FK228 were remarkably comparable to those of 17-AAG regarding depletion of mutant p53, as well as Raf-1, ErbB1, and ErbB2 protein levels. Trichostatin A also had similar effects, suggesting that oncoprotein depletion may be a common mechanism by which HDAC inhibitors mediate antiproliferative effects in cancer cells.



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Fig. 5. Effect of FK228, trichostatin A (TSA), and 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) treatment on p53, Raf-1, ErbB1, and ErbB2 protein levels in non-small-cell lung cancer cells. H322 cells were left untreated (lane 1) or were treated with FK228 (25 ng/mL) for 6 (lane 2) or 24 (lane 3) hours, TSA (300 nM) for 6 (lane 4) or 24 (lane 5) hours, or 17-AAG (1 µM) for 6 (lane 6) or 24 (lane 7) hours. Proteins from whole-cell lysates, obtained 24 hours after initiation of drug treatments for all groups, were resolved by polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted sequentially for mouse anti-p53, mouse anti-Raf-1, rabbit anti-ErbB2, mouse anti-ErbB1, and finally, mouse anti-{beta}-actin to control for protein loading.

 
Effects of FK228 on Binding of Mutant p53 and Raf-1 to Hsp90

Because 17-AAG depletes mutant p53, Raf-1, and ErbB oncoproteins in cancer cells by disrupting their interaction with Hsp90 (27,28), a series of immunoprecipitation experiments were performed to examine whether this mechanism also contributed to FK228-mediated oncoprotein depletion in H322 lung cancer cells. Although FK228 did not affect the level of Hsp90, this HDAC inhibitor clearly inhibited binding of mutant p53 with Hsp90 (Fig. 6, AGo). Under these conditions, no detectable p53–Hsp90 complexes were detectable within 2 hours of FK228 treatment. The fact that p53 levels were not dramatically reduced immediately after FK228 exposure indicated that disruption of p53–Hsp90 binding preceded the depletion of mutant p53 observed 18 hours after completion of the 6-hour FK228 treatment depicted in Figs. 2 and 4GoGo.




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Fig. 6. Effect of FK228 treatment on heat shock protein (Hsp) 90 expression and binding to mutant p53 (A) or Raf-1 (B) in lysates from H322 non-small-cell lung cancer cells. A) Lysates from H322 cells that were either left untreated (lanes 1 and 5) or treated with FK228 (25 ng/mL) for 2 (lanes 2 and 6), 4 (lanes 3 and 7), or 6 (lanes 4 and 8) hours were prepared immediately after drug exposure. For lanes 14, 500 µg of total proteins was immunoprecipitated with murine monoclonal antibody DO-1, which binds to mutant p53; the immunoprecipitates were analyzed by western blot analysis using goat polyclonal anti-Hsp90 (upper panel) or goat polyclonal anti-p53 (lower panel). For lanes 58, cell lysates before immunoprecipitation were subjected to western blot analysis. B) Lysates from H322 cells that were left untreated or treated with FK228 (25 ng/mL) for 2 or 4 hours were prepared immediately after drug treatment. Equivalent amounts (500 µg) of total proteins were immunoprecipitated (IP) with a mouse anti-Raf-1 monoclonal antibody, after which the immunoprecipitates were analyzed by western blot analysis using a murine monoclonal antibody specific for Raf-1 (Raf-1 IP/Raf-1 IB; top panel), a murine antibody specific for Hsp90 (Raf-1 IP/Hsp90 IB; middle panel), or a goat polyclonal antibody specific for Hsp70 (Raf-1 IP/ Hsp70 IB; bottom panel). IB = immunoblot.

 
Because FK228 inhibited binding of the Hsp90 chaperone protein with mutant p53, additional immunoprecipitation experiments were performed to determine whether this HDAC inhibitor also disrupted the interaction between Hsp90 and another client oncoprotein, Raf-1. Fig. 6, BGo, shows that, within 2 hours, FK228 inhibited the binding of Raf-1 to Hsp90 and that, by 4 hours, the decreased interaction between Raf-1 and Hsp90 coincided with increased binding between Raf-1 and Hsp70, an event that precedes oncoprotein degradation within the proteosome (27).

Mitogenic signals emanating from ErbB1 and ErbB2 are mediated via Ras to Raf-1, leading to MEK-mediated phosphorylation of ERK1/2, which in turn activates a variety of downstream targets including the transcription factors Elk-1, Jun, and c-myc [reviewed in (29,30)]. Additional experiments were performed to determine whether depletion of ErbB1, ErbB2, and Raf-1 protein levels by FK228 inhibited signal transduction via the Raf-1/MEK/ERK pathway in H322 lung cancer cells. As shown in Fig. 7Go, FK228 inhibited ERK1/2 activity in these cells, as evidenced by a reduction of phosphorylated ERK1/2 (phospho-p44/42) protein levels, and decreased phosphorylation of Elk-1. Inhibition of ERK1/2 activity after FK228 treatment was comparable with that observed after exposure to the MEK inhibitor PD 98059.



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Fig. 7. Effect of FK228 treatment on p44/42 mitogen-activated protein (MAP) kinase (MAPK; ERK1/2) activity in H322 non-small-cell lung cancer cells. H322 cells were left untreated (lane 1), were treated with FK228 (25 ng/mL) for 6 (lane 2) or 24 (lane 3) hours, or were treated with PD 98059 (100 µM) for 24 hours (lane 4). Twenty-four hours after initiation of drug treatment, the cells were lysed, and approximately 200 µg of total proteins was immunoprecipitated using a murine monoclonal anti-phospho-p44/42 (Thr202/Tyr204) MAP kinase antibody. p44/42 MAPK (ERK1/2) activity in the immunoprecipitate was determined by analyzing phosphorylation of a glutathione-S-transferase (GST)-Elk-1 fusion protein using reagents and protocols in the p44/42 MAP Kinase Assay Kit (Cell Signaling Technology, Beverly, MA). The entire sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and immunoblotted with rabbit polyclonal antibodies that recognize either phosphorylated Elk-1 (phospho-Elk-1), phospho-p44/42 MAPK (Thr202/Tyr204), or p44/42 MAPK (ERK1/2).

 
The ansamycins 17-AAG and geldanamycin, as well as the coumarin antibiotic novobiocin, deplete mutant p53, Raf-1, and ErbB2 by directly inhibiting their interaction with Hsp90 (22,27). Additional experiments were performed to determine whether FK228 could competitively inhibit the binding of Hsp90 to several of these target proteins and to ATP–sepharose because the amino terminus of Hsp90 contains an ATP binding motif within the ansamycin binding pocket (27). In addition to H322 lung cancer cells, SK-Br-3 breast cancer cells were used for these experiments because these cells have been used extensively for examining mechanisms of oncoprotein depletion by agents targeting Hsp90 (28). FK228 did not competitively inhibit binding of mutant p53 to Hsp90 expressed in rabbit reticulocyte lysates (data not shown). Furthermore, co-incubation of FK228 with radiolabeled Hsp90 did not decrease binding of Hsp90 to geldanamycin–sepharose, novobiocin–sepharose, or ATP–sepharose (representative data for ATP–sepharose experiments are shown in Fig. 8Go, A). In contrast, a time-dependent reduction in Hsp90/ATP–sepharose binding was observed in lysates from FK228-treated SK-Br-3 cells, suggesting a more subtle mechanism by which FK228 modulated Hsp90 binding activity (Fig. 8, BGo).



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Fig. 8. Effect of FK228 treatment on the binding of heat shock protein (Hsp) 90 to adenosine triphosphate (ATP)-sepharose in vitro (A) and in vivo (B), and on acetylation of Hsp90 (C). A) Hsp90 was transcribed and translated in vitro using rabbit reticulocyte lysates. Two micrograms of chicken Hsp90 was radiolabeled with 35S in vitro using a TnTTM kit (Promega, Madison, WI). Equal aliquots of Hsp90 were incubated with 20 mg of ATP-sepharose resin in the presence of increasing concentrations (5–100 ng/mL) of FK228. Hsp90 concentrations bound to the ATP–sepharose resin were assessed by western blot analysis using a murine monoclonal anti-Hsp90 antibody. B) SK-Br-3 cells were left untreated or were treated with FK228 (25 ng/mL) for 2, 4, 6, or 24 hours. Lysates were prepared immediately after drug exposure, and Hsp90 was affinity precipitated with ATP–sepharose. The Hsp90 concentration in the precipitates was assessed by western blot analysis with a specific goat polyclonal anti-Hsp90 antibody. C) SK-Br-3 cells were left untreated or were treated with FK228 (25 ng/mL) for 0.5, 1, 1.5, 2, or 3 hours. Hsp90 was immunoprecipitated from 500 µg of total protein from SK-Br-3 cell lysates immediately after FK228 treatment using a murine monoclonal anti-Hsp90 antibody. Acetylated Hsp90 in the precipitates was assessed by western blot analysis using a rabbit polyclonal antibody that recognizes acetylated lysine residues.

 
Because acetylation status can influence protein–protein interactions, and because FK228 mediated the acetylation of p53, additional experiments were performed to determine whether FK228 exposure might also induce acetylation of Hsp90. SK-Br-3 cells were treated with FK228 for various times, and Hsp90 was immunoprecipitated from cell lysates with the use of a murine anti-Hsp90 antibody, followed by western blot analysis using an antibody that recognizes acetylated lysine. As shown in Fig. 8, CGo, FK228 mediated acetylation of Hsp90 in a manner consistent with the time-dependent inhibition of the interaction of this chaperone protein with ATP–sepharose, as well as mutant p53, and Raf-1 client oncoproteins in cancer cells.


    DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Given the importance of histone acetylation in modulating gene expression in normal cells and the potential cross-talk between oncogene signaling and chromatin structure, it is not surprising that considerable efforts have focused on the elucidation of the mechanisms by which HDAC inhibitors mediate cytotoxicity in cancer cells. To date, five structural classes of histone deacetylase inhibitors have been identified, including short-chain fatty acids such as sodium butyrate; hydroxaminic acids, such as suberoylanilide hydroxamic acid (SAHA), oxamflatin, and trichostatin A; cyclic tetrapeptides which contain a 2-amino-8-oxo-9,10-epoxy-decanoyl (AOE) moiety such as trapoxin A; cyclic peptides without the AOE moiety such as FK228 and apicidin; and benzamides such as MS27–275. Several agents from different classes are in early clinical development (15). Potent HDAC inhibitors, including trichostatin A, trapoxin, and FK228, are known to modulate the expression of a variety of cell-cycle-related proteins, including cyclin A, cyclin D, cyclin E, p21, and E2F, and to inhibit cdk2 kinase activity (24,25,31,32). However, the precise mechanisms by which these agents mediate growth inhibition and apoptosis preferentially in cancer cells remains uncertain; histone acetylation alone appears to be insufficient to account for the potent cytotoxic effects of these novel agents (9,14,32,33).

In this study, we sought to define potential mechanisms by which FK228 mediates growth arrest and apoptosis in lung cancer cells. We used a variety of NSCLC cell lines that differed regarding p53 status (i.e., wild-type or mutant p53) and expression of other cell-cycle-related and signaling-related proteins (i.e., pRb, ErbB1, ErB2, and Ras). Our analysis revealed that, in addition to modulating expression of cyclin A, cyclin E, and p21, FK228 stabilizes wild-type p53 protein levels and depletes mutant p53 in NSCLC cells. Although several investigators have observed changes in p53 expression in cancer cells after exposure to HDAC inhibitors (3436), the mechanisms responsible for these phenomena have not been fully defined. Our data indicate that FK228-mediated stabilization of wild-type p53 protein levels in lung cancer cells may be attributable in part to increased phosphorylation and acetylation of this tumor suppressor protein; these post-translational modifications inhibit MDM2-mediated ubiquitination of p53 and enhance sequence-specific DNA binding of this tumor suppressor protein (26,37).

Our findings that FK228 depletes mutant p53, as well as ErbB1, ErbB2, and Raf-1 oncoproteins in lung cancer cells in a manner analogous to that reported for geldanamycin and its derivatives (27,28) are of potentially great interest. In all likelihood, decreased ERK1/2 kinase activity resulting from FK228-mediated depletion of ErbB and Raf-1 oncoproteins contributes to the inhibition of p53 transcription (38) and to the time-dependent growth suppression in lung cancer cells after FK228 exposure (39). Raf-1 is a critical mediator of mitogenic signals emanating from a variety of receptor tyrosine kinases, including ErbB1 and ErbB2 (40). Raf-1 activation of the MEK/ERK pathway inhibits apoptosis in cancer cells, and MEK inhibitors can abrogate the antiapoptotic function of Raf-1 (4144). In addition, Raf-1 can antagonize apoptosis signal-regulating kinase through an MEK/ERK-independent mechanism (45). Like the MEK inhibitor PD 98059, FK228 decreased ERK1/2 kinase activity in H322 lung cancer cells, which contain an activated K-Ras and express high levels of ErbB1 and ErbB2, attesting to the potency of this HDAC inhibitor with regard to abrogating MAP kinase signal transduction. It is interesting that the proapoptotic effects of FK228 were much more pronounced in H322 cells than in H460 cells, which also express an activated K-Ras but do not overexpress ErbB oncoproteins. Given the fact that 17-AAG-mediated inhibition of Ras/Raf/MEK/ERK signaling induces growth arrest and apoptosis in colon cancer cells (39,46), our data strongly suggest that the cytotoxic effects of FK228 in NSCLC cells are attributable at least in part to an inhibition of Raf-1 signaling. These findings could also account for a previous observation that FK228 inhibits the Ras-transformed phenotype (47).

Although the effects of FK228 appeared analogous to those of geldanamycin and its derivatives, our data indicate that the precise mechanisms are not identical. Geldanamycin, 17-AAG, and novobiocin are known to directly bind to Hsp90, inducing a conformational switch that alters the assembly of Hsp90 multichaperone complexes and thereby destabilizes multiple client proteins, including mutant p53, ErbB1, ErbB2, platelet-derived growth factor, Raf-1, and cyclin-dependent kinase 4 (22,27,28). After exposure to geldanamycin, the multichaperone complex shifts from a stable one containing Hsp90 and lacking Hsp70 to an unstable one containing decreased amounts of Hsp90 and increased amounts of Hsp70; this shift is followed rapidly by the degradation of client proteins (27,48). In our study, we observed that FK228 reduced the binding of mutant p53 and Raf-1 to Hsp90, coincident with increased binding to Hsp70 in a manner consistent with the time-dependent depletion of these oncoproteins in lung cancer cells. Preliminary experiments suggest that this mechanism also contributes to FK228-mediated depletion of ErbB1 and ErbB2 in NSCLC cells. Experiments are in progress to determine whether other HDAC inhibitors, such as trichostatin A, can deplete oncoproteins in cancer cells by similar mechanisms.

Unlike geldanamycin and related compounds, which target Hsp90, FK228 appears to mediate its effects by mechanisms more subtle than direct physical association with this chaperone protein. Our experiments clearly indicate that FK228 promotes acetylation of Hsp90 in a manner consistent with time-dependent destabilization of Hsp90–client protein complexes. Whereas the effects of this acetylation have not been fully elucidated, it is conceivable that acetylation of Hsp90 (or perhaps other associated proteins) could dramatically influence the assembly and stability of Hsp90 multiprotein complexes. Acetylation is a common mechanism modulating the stability and function of a variety of proteins involved in cell-cycle regulation (9). For example, acetylation of p53 and E2F1 increases DNA binding and transcriptional activation mediated by these proteins (26,49,50). In contrast, acetylation of high mobility group I (HMGI) transcription factors inhibits their binding to DNA (51). Furthermore, acetylation of pRb inhibits its phosphorylation by cyclin-dependent kinases and enhances its binding to MDM2 (52). Conceivably, acetylation of Hsp90 alters its association with client proteins or other members of the multiprotein chaperone complex, leading to oncoprotein degradation. Experiments are in progress to examine this question.

Although not formally demonstrated in our experiments, it is possible that FK228 mediates its antitumor effects by modulating the activity of class II HDACs. Because activation of ERK1/2 increases transport of class II HDACs into the nucleus (11), inhibition of MAP kinase signaling and ERK1/2 activity by FK228 may alter histone acetylation and gene expression in cancer cells. This mechanism could also account for the ability of some HDAC inhibitors to induce differentiation in cancer cells (15). If this is the case, agents such as 17-AAG or PD 98059 that target the Ras/Raf-1/MEK/ERK pathway might also induce growth arrest and differentiation in cancer cells via modulation of chromatin structure.


    REFERENCES
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received September 8, 2001; revised January 23, 2002; accepted February 4, 2002.


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