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 208921502 (e-mail: David_Schrump{at}nih.gov).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 transcriptasepolymerase 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 248 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--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. Agaroseantibodyprotein complexes were washed three times with lysis buffer. After discarding the supernatant from the final wash, the antibodyprotein 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% TrisHCL 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 TrisHCl (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 SDSpolyacrylamide 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).
NovobiocinSepharose, ATPSepharose, and Geldanamycin Binding Assays
Novobiocinsepharose, ATPsepharose, and geldanamycinaffigel 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 novobiocinsepharose, ATPsepharose, or geldanamycinaffigel 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 ATPsepharose beads and detected by western blot analysis as described (22).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 B, 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.
|
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, A).
|
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. 3, 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.
|
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. 4).
|
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. 5, 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.
|
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, A). Under these conditions, no detectable p53Hsp90 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 p53Hsp90 binding preceded the depletion of mutant p53 observed 18 hours after completion of the 6-hour FK228 treatment depicted in Figs. 2 and 4
.
|
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. 7, 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.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 Hsp90client 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Timmermann S, Lehrmann H, Polesskaya A, Harel-Bellan A. Histone acetylation and disease. Cell Mol Life Sci 2001;58:72836.[Medline]
2
Baylin SB, Esteller M, Rountree MR, Bachman KE, Schuebel K, Herman JG. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet 2001;10:68792.
3 Wang C, Fu M, Mani S, Wadler S, Senderowicz AM, Pestell RG. Histone acetylation and the cell-cycle in cancer. Front Biosci 2001;6:D61029.[Medline]
4 Annunziato AT, Hansen JC. Role of histone acetylation in the assembly and modulation of chromatin structures. Gene Expr 2000;9:3761.[Medline]
5 Rice JC, Allis CD. Histone methylation versus histone acetylation: new insights into epigenetic regulation. Curr Opin Cell Biol 2001;13:26373.[Medline]
6 Nakayama T, Takami Y. Participation of histones and histone-modifying enzymes in cell functions through alterations in chromatin structure. J Biochem (Tokyo) 2001;129:4919.[Abstract]
7
Wade PA. Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin. Hum Mol Genet 2001;10:6938.
8 Kouzarides T. Histone acetylases and deacetylases in cell proliferation. Curr Opin Genet Dev 1999;9:408.[Medline]
9
Kouzarides T. Acetylation: a regulatory modification to rival phosphorylation? EMBO J 2000;19:11769.
10
Grozinger CM, Hassig CA, Schreiber SL. Three proteins define a class of human histone deacetylases related to yeast Hda1p. Proc Natl Acad Sci U S A 1999;96:486873.
11
Zhou X, Richon VM, Wang AH, Yang XJ, Rifkind RA, Marks PA. Histone deacetylase 4 associates with extracellular signal-regulated kinases 1 and 2, and its cellular localization is regulated by oncogenic Ras. Proc Natl Acad Sci U S A 2000;97:1432933.
12
Grozinger CM, Schreiber SL. Regulation of histone deacetylase 4 and 5 and transcriptional activity by 1433-dependent cellular localization. Proc Natl Acad Sci U S A 2000;97:783540.
13
Struhl K. Histone acetylation and transcriptional regulatory mechanisms. Genes Dev 1998;12:599606.
14 Nakajima H, Kim YB, Terano H, Yoshida M, Horinouchi S. FR901228, a potent antitumor antibiotic, is a novel histone deacetylase inhibitor. Exp Cell Res 1998;241:12633.[Medline]
15
Marks PA, Richon VM, Rifkind RA. Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells. J Natl Cancer Inst 2000;92:12106.
16 Weiser TS, Guo ZS, Ohnmacht GA, Parkhurst ML, Tong-On P, Marincola FM, et al. Sequential 5-Aza-2 deoxycytidine-depsipeptide FR901228 treatment induces apoptosis preferentially in cancer cells and facilitates their recognition by cytolytic T lymphocytes specific for NY-ESO-1. J Immunother 2001;24:15161.
17
Zhu WG, Lakshmanan RR, Beal MD, Otterson GA. DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors. Cancer Res 2001;61:132733.
18
Brinkmann H, Dahler AL, Popa C, Serewko MM, Parsons PG, Gabrielli BG, et al. Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts. J Biol Chem 2001;276:224919.
19
Saunders N, Dicker A, Popa C, Jones S, Dahler A. Histone deacetylase inhibitors as potential anti-skin cancer agents. Cancer Res 1999;59:399404.
20
Richon VM, Emiliani S, Verdin E, Webb Y, Breslow R, Rifkind RA, et al. A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases. Proc Natl Acad Sci U S A 1998;95:30037.
21 Li X, Darzynkiewicz Z. Labelling DNA strands breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif 1995;28:5719.[Medline]
22
Marcu MG, Schulte TW, Neckers L. Novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins. J Natl Cancer Inst 2000;92:2428.
23
Blagosklonny MV, Toretsky J, Bohen S, Neckers L. Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90. Proc Natl Acad Sci U S A 1996;93:837983.
24 Eickhoff B, Ruller S, Laue T, Kohler G, Stahl C, Schlaak M, et al. Trichostatin A modulates expression of p21waf1/cip1, Bcl-xL, ID1, ID2, ID3, CRAB2, GATA-2, hsp86 and TFIID/TAFII31 mRNA in human lung adenocarcinoma cells. Biol Chem 2000;381:10712.[Medline]
25 Sandor V, Senderowicz A, Mertins S, Sackett D, Sausville E, Blagosklonny MV, et al. P21-dependent g(1) arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228. Br J Cancer 2000;83:81725.[Medline]
26 Colman MS, Afshari CA, Barrett JC. Regulation of p53 stability and activity in response to genotoxic stress. Mutat Res 2000;462:17988.[Medline]
27 Neckers L, Schulte TW, Mimnaugh E. Geldanamycin as a potential anti-cancer agent: its molecular target and biochemical activity. Invest New Drugs 1999;17:36173.[Medline]
28 Schulte TW, Neckers LM. The benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin binds to HSP90 and shares important biologic activities with geldanamycin. Cancer Chemother Pharmacol 1998;42:2739.[Medline]
29 Shulte TW, Blagosklonny MV, Romanova L, Mushinski JF, Monia BP, Johnston JF, et al. Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1-MEK-mitogen-activated protein kinase signalling pathway. Mol Cell Biol 1996;16:583945.[Abstract]
30
Pearson G, Bumeister R, Henry DO, Cobb MH, White MA. Uncoupling Raf1 from MEK1/2 impairs only a subset of cellular responses to Raf activation. J Biol Chem 2000;275:373036.
31
Sambucetti LC, Fischer DD, Zabludoff S, Kwon PO, Chamberlin H, Trogani N, et al. Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects. J Biol Chem 1999;274:349407.
32 Kim YB, Ki SW, Yoshida M, Horinouchi S. Mechanism of cell cycle arrest caused by histone deacetylase inhibitors in human carcinoma cells. J Antibiot (Tokyo) 2000;53:1191200.[Medline]
33 Qiu L, Kelso MJ, Hansen C, West ML, Fairlie DP, Parsons PG. Anti-tumour activity in vitro and in vivo of selective differentiating agents containing hydroxamate. Br J Cancer 1999;80:12528.[Medline]
34 Suzuki T, Yokozaki H, Kuniyasu H, Hayashi K, Naka K, Ono S, et al. Effect of trichostatin A on cell growth and expression of cell cycle- and apoptosis-related molecules in human gastric and oral carcinoma cell lines. Int J Cancer 2000;88:9927.[Medline]
35 Pellizzaro C, Coradini D, Daniotti A, Abolafio G, Daidone MG. Modulation of cell cycle-related protein expression by sodium butyrate in human non-small cell lung cancer cell lines. Int J Cancer 2001;91:6547.[Medline]
36 Coradini D, Pellizzaro C, Marimpietri D, Abolafio G, Daidone MG. Sodium butyrate modulates cell cycle-related proteins in HT29 human colonic adenocarcinoma cells. Cell Prolif 2000;33:13946.[Medline]
37 Ashcroft M, Vousden KH. Regulation of p53 stability. Oncogene 1999;18:763743.[Medline]
38 Agarwal ML, Ramana CV, Hamilton M, Taylor WR, DePrimo SE, Bean LJ, et al. Regulation of p53 expression by the RAS-MAP kinase pathway. Oncogene 2001;20:252736.[Medline]
39 An WG, Schnur RC, Neckers L, Blagosklonny MV. Depletion of p185erbB2, Raf-1 and mutant p53 proteins by geldanamycin derivatives correlates with antiproliferative activity. Cancer Chemother Pharmacol 1997;40:604.[Medline]
40 Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 2000;351(Pt 2):289305.[Medline]
41 Cleveland JL, Troppmair J, Packham G, Askew DS, Lloyd P, Gonzalez-Garcia M, et al. v-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells. Oncogene 1994;9:221726.[Medline]
42 Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995;270:132631.[Abstract]
43
Erhardt P, Schremser EJ, Cooper GM. B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/Erk pathway. Mol Cell Biol 1999;19:530815.
44
Le Gall M, Chambard JC, Breittmayer JP, Grall D, Pouyssegur J, Van Obberghen-Schilling E. The p42/p44 MAP kinase pathway prevents apoptosis induced by anchorage and serum removal. Mol Biol Cell 2000;11:110312.
45
Chen J, Fujii K, Zhang L, Roberts T, Fu H. Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK-ERK independent mechanism. Proc Natl Acad Sci U S A 2001;98:77838.
46
Hostein I, Robertson D, DiStefano F, Workman P, Clarke PA. Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis. Cancer Res 2001;61:40039.
47 Ueda H, Nakajima H, Hori Y, Fujita T, Nishimura M, Goto T, et al. FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum No. 968. I. Taxonomy, fermentation, isolation, physico-chemical and biological properties, and antitumor activity. J Antibiot (Tokyo) 1994;47:30110.[Medline]
48 Pratt WB. The role of the hsp90-based chaperone system in signal transduction by nuclear receptors and receptors signaling via MAP kinase. Annu Rev Pharmacol Toxicol 1997;37:297326.[Medline]
49 Gu W, Roeder RG. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 1997;90:595606.[Medline]
50
Martinez-Balbas MA, Bauer UM, Nielsen SJ, Brehm A, Kouzarides T. Regulation of E2F1 activity by acetylation. EMBO J 2000;19:66271.
51
Munshi N, Merika M, Yie J, Senger K, Chen G, Thanos D. Acetylation of HMG I(Y) by CBP turns off IFN expression by disrupting the enhanceosome. Mol Cell 1998;2:45767.[Medline]
52 Chan HM, Krstic-Demonacos M, Smith L, Demonacos C, La Thangue NB. Acetylation control of the retinoblastoma tumour-suppressor protein. Nat Cell Biol 2001;3:66774.[Medline]
Manuscript received September 8, 2001; revised January 23, 2002; accepted February 4, 2002.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |