Histone Deacetylase Inhibitors Induce Premature Sister Chromatid Separation and Override the Mitotic Spindle Assembly Checkpoint

Research Article

Laura Magnaghi-Jaulin, Gre´gory Eot-Houllier, Ge´raldine Fulcrand, and Christian Jaulin

INSERM EMI 229, CRLC Val d’Aurelle-Paul Lamarque, and Universite´MONTPELLIER1, Montpellier, France

Abstract differentiation, apoptosis, or growth arrest pathways (7–10). Histone deacetylase inhibitors (HDACI) are powerful anti- Therefore, it has been suggested that nontranscriptional targets proliferative drugs, and are currently undergoing clinical may be equally important for HDACI antitumor activity (11–13). trials as antitumor agents. It would be valuable for both One potential nontranscriptional target is the centromere/kineto- cancer therapy and our knowledge of basic cellular processes chore structure. Indeed, low-dose trichostatin A (TSA) treatment to understand the mechanisms by which HDACIs blockcell destabilizes the higher-order structure of pericentric heterochro- proliferation. Most current models postulate that HDACIs matin, and leads to HP1 spreading and centromere subnuclear allow the reexpression of tumor suppressor genes silenced localization (14). Pericentric heterochromatin plays an important in cancer cells. However, other mechanisms, distinct from role in centromere function, and HDACI treatments may lead to transcription regulation, may participate in HDACI antipro- kinetochore defects and aberrant mitosis. Accordingly, several liferative properties. We report that HDACI treatment induces studies report that HDACI treatment increases chromosomal premature sister chromatid separation in cells in which the instability characterized by chromatin bridges, chromosome mitotic spindle assembly checkpoint (SAC) has already been breaks, and segregation defects (14–16). These features suggest activated. This effect was transcription-independent. In that the nontranscriptional targets of HDACI include mitosis and addition, HDACI-treated mitotic cells displayed SAC inactiva- the mechanisms that regulate its accuracy. Segregation deficiency tion characteristics, including anaphase-promoting complex/ is a common characteristic of HDACI-treated cells and of mitotic cyclosome target degradation, cyclin-dependent kinase 1 spindle assembly checkpoint (SAC) defects. We therefore examined inactivation, histone H3 dephosphorylation, and loss of the whether HDACIs can target the SAC. SAC component MAD2 from the kinetochore. Thus, HDAC The SAC is a cellular mechanism that ensures faithful sister inhibition renders the SAC ineffective. Our findings help chromatid segregation during mitosis by delaying anaphase until elucidate the molecular mechanisms of proliferative cell death all kinetochores have been attached to spindle microtubules (17). induced by HDACI treatment and may allow new HDACI-based A defect in this function results in unbalanced chromosome preclinical and clinical trial protocols to be redesigned so as segregation, and consequently, aneuploid daughter cells. Unat- to target mitosis. [Cancer Res 2007;67(13):6360–7] tached kinetochores generate an inhibitory signal acting on the anaphase-promoting complex/cyclosome (APC/C), a multiprotein complex whose ubiquitin ligase activity is required to target Introduction mitosis-specific factors (such as cyclin B1 and securin) to Histone deacetylase (HDAC) inhibitors (HDACI) exhibit anti- proteasome-dependent degradation. The attachment of the last proliferative activity against a variety of human cancers. They are free kinetochore abrogates the inhibitory signal and thus permits currently undergoing clinical trials and the outlook is promising. the transition from metaphase to anaphase and subsequent exit However, the molecular basis of their antitumor properties remains from mitosis. Inhibition of the SAC by mutation of its components, poorly understood (1, 2). It was first suggested that HDAC haploinsufficiency, or down-regulation of one of its elements inhibition induces the reexpression of silenced tumor suppressor results in premature sister chromatid separation (PSCS) and APC/ genes by affecting the chromatin structure of their promoters (3). C target degradation despite of SAC activation conditions (18–21). For example, treatment of cells with HDACI often induces Furthermore, long-term total SAC inhibition is not viable. Null the expression of the cyclin-dependent kinase (cdk) inhibitor mutations of the SAC component MAD2 is lethal (22) and WAF1/CIP1 p21 in a p53-independent manner (4, 5). The expression of extended inhibition of SAC by RNA interference affecting key SAC a number of genes other than cell cycle regulator genes is also elements leads to cell death within a few generations (23). The altered by HDACI treatment: they include genes involved in the most likely explanation for this lethality is the accumulation of induction of apoptosis, the activation of the immune response, and nonviable aneuploid cells due to premature anaphase entry. the inhibition of angiogenesis (6). However, gene profiling studies Consequently, because the cell toxicity resulting from SAC showed that the expression of a surprisingly small fraction of genes inhibition is expected to be restricted to proliferating cells, the is affected by HDACI treatments and it remains uncertain whether SAC is a putative therapeutic target for anticancer treatments (24). these particular genes are sufficient to drive cancer cells towards However, the only SAC down-regulation techniques currently known are genetic and, in the foreseeable future, unsuitable for application to human therapy. Requests for reprints: Christian Jaulin, INSERM EMI 229, CRLC Val d’Aurelle-Paul To determine whether HDACIs can be used as pharmacologic Lamarque, Parc Eurome´decine,34298 Montpellier Cedex 5, France. Phone: 33-46761- tools to target the SAC, we investigated mitosis exit in HDACI- 3751; Fax: 33-46761-3787; E-mail: [email protected]. I2007 American Association for Cancer Research. treated cells in which the SAC was activated. We found that HDACI doi:10.1158/0008-5472.CAN-06-3012 treatment triggers transcription-independent mitosis exit in a large

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. HDACIs Override the Spindle Checkpoint proportion of prometaphase-arrested cells. We monitored SAC monoclonal anti–cyclin B1 antibodies (Santa Cruz), polyclonal anti-securin inhibition by detection of the major mitosis exit markers, including antibodies (Zymed), or anti-actin antibodies (Sigma) according to standard sister chromatid separation and proteasome-dependent degrada- procedures. Western Lighting Chemiluminescence Plus (Perkin-Elmer) was tion of key APC/C targets. used to visualize binding, according to the instructions of the supplier. Cdk1 kinase assays and immunoprecipitation. Cdk1 immunoprecipitation followed by cdk1 kinase assays were as previously described (26). Materials and Methods Assays were analyzed by autoradiography, and kinase activity was Cell culture and drug treatment. HeLa cells (human cervical determined using a Phosphoimager (BAS-5000, FUJI). Cdk1 immunopreci- carcinoma, obtained from American Type Culture Collection) were cultured pitates were analyzed by immunoblotting using a monoclonal anti-cdk1 in DMEM (Life Technologies) supplemented with L-glutamine, antibiotics, antibody (Santa Cruz). and 10% heat-decomplemented FCS (Invitrogen). In all experiments, Immunofluorescence microscopy. Chromosome spread slides (Fig. 6B) growing cells were incubated in nocodazole (500 ng/mL) for 8 h and were washed twice in PBS for 10 min, incubated with 0.1% Triton X-100, accumulated mitotic cells were harvested by mitotic shake-off (MSO) prior washed thrice in PBS, blocked with 5% FCS, and stained with rabbit to other treatments. Nocodazole was permanently maintained in the polyclonal anti-H3 serine 10 phosphorylated antibodies (UBI) overnight medium when HDACIs or other drugs were added. For transcription at 4jC. Goat anti-rabbit conjugated-FITC antibodies (Biosource Interna- inhibition and proteasome inhibition assays, a-amanitin (2 Ag/mL) or tional) were used for secondary staining for 1 h at room temperature. MG132 (3 Amol/L) were added 1 h before TSA treatment; a-amanitin or For whole cell immunofluorescence (Figs. 5B and 6A), cells were grown on MG132 were maintained in the culture medium after the addition of TSA. polylysine-treated coverslips, fixed in 3% formaldehyde, washed in PBS, Unless otherwise stated, the TSA concentration was 200 ng/mL. All reagents incubated for 15 min with 100 mmol/L of glycine (pH 7), permeabilized were purchased from Sigma-Aldrich, except for MG132 (Biomol Int.). with 0.1% Triton X-100, washed in PBS, blocked with 5% FCS, and incu- Chromosome spreads. Metaphase spreads were done essentially as bated with rabbit polyclonal anti-lamin A/C antibodies (Cell Signaling), described (25) and DNA was stained with 4¶,6-diamidino-2-phenylindole human CREST serum (a kind gift from Dr. C. Johanet, Hoˆpital Saint- (DAPI; 100 ng/mL) using Mowiol as the mounting medium. At least 500 Antoine, Paris), or rabbit polyclonal anti-MAD2 antibodies (Covance). cells were scored for each experimental point. All experiments were done at Goat anti-rabbit conjugated-FITC antibodies (Biosource International) least in duplicate. and goat anti-human conjugated-TRITC antibodies (Jackson Immuno- Immunoblotting. Cells were harvested, counted, lysed directly in Research) were used as secondary antibodies. In all experiments, DNA was Laemmli buffer and boiled for 5 min. Samples corresponding to 6 Â 104 stained with DAPI (100 ng/mL) and Mowiol was used as the mounting cells were separated by SDS-PAGE and transferred to nitrocellulose mem- medium. Samples were examined under an epifluorescence microscope branes (Hybond-C Extra, Amersham); the membranes were probed with (Leica DMRM).

Figure 1. TSA treatment induces both PSCS and partially decondensed chromosomes in nocodazole-arrested mitotic cells. Top, experimental outline. Cells were incubated with nocodazole for 8 h, and mitotic cells were collected by MSOand were incubated with nocodazole in the presence or absence of TSA for 16 h. A, chromosome spreads from control mitotic nocodazole (left) or nocodazole/ TSA–treated (200 ng/mL, 16 h) HeLa cells (right). Brackets, incompletely condensed sister chromatids lying separated and adjacent to each other. B, the frequency of PSCS is TSA-dose dependent. Various doses of TSA (0, 80, 200, or 500 ng/mL) were added to nocodazole-arrested mitotic HeLa cells. C, increasing TSA dose decreases the mitotic index in nocodazole-arrested mitotic HeLa cells.

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Figure 2. The effect of TSA on other cells. Top, experimental outline (see legend to Fig. 1). A, TSA-induced PCSC in nocodazole-arrested MDA-MB-231 breast cancer cell line (MDA) and human untransformed fibroblasts (HDF). B, TSA treatment of nocodazole-arrested MDA-MB-231 and HDF cells results in mitotic index decrease. Columns, means from at least two independent experiments; bars, SD.

Results mortality was <5%. Therefore, specific mitotic cell mortality cannot Prometaphase-arrested cells undergo HDACI-induced PSCS account for the observed decrease in the mitotic index. in a transcription independent manner. In all experiments, PSCS increase and mitotic index decrease induced by TSA were mitotic SAC was activated by pretreatment with the microtubule- also observed in a human breast cancer cell line (MDA-MB-231) destabilizing agent nocodazole. Nocodazole treatment activates the and in human primary fibroblasts and, thus, are not specific to the SAC by preventing microtubule-kinetochore interaction. Once the transformed HeLa cell line (Fig. 2). SAC is activated, cells accumulate in prometaphase and, depending To determine if the effects described above were specific for TSA on the cell type and the nocodazole concentration, remain arrested or are found with other HDACIs, nocodazole-treated cells were between a few hours and several days. Under our culture incubated with various different HDACIs belonging to three A conditions, >60% of HeLa cells were arrested in prometaphase different classes (Fig. 3 ). Sodium butyrate and valproic acid are following an 8-h nocodazole treatment and remained stably short-chain fatty acids, TSA is a hydroxamic acid and apicidin blocked in prometaphase for the following 24 h without significant belongs to the cyclic tetrapeptide class of HDACIs (1). Sodium mitosis exit or cell death. HeLa cells were treated with nocodazole butyrate and apicidin induced PSCS (although to a lesser extent A, top for 8 h and mitosis-arrested cells were collected by MSO. Then, the than TSA) but valproic acid did not (Fig. 3 ); however, all cells were incubated with TSA, a powerful HDACI, for 16 h. HDACIs tested decreased the mitotic index of nocodazole-treated Nocodazole was present in the culture medium during TSA cells (Fig. 3A, bottom). This suggests that HDAC inhibition in SAC- treatment. A proportion of chromosome spreads from nocodazole/ activated mitotic cells causes abnormal mitosis exit. TSA–treated cells displayed a characteristic PSCS phenotype Although gene transcription in metaphase-arrested cells is (Fig. 1A): sister chromatids were no longer attached at the almost completely silenced (27), it is possible that HDACI induced centromere and the constriction point was not visible anymore. aberrant mitotic transcription. To determine whether the TSA- In addition, despite the extended nocodazole treatment (which is induced PSCS was due to the reactivation of transcription at loci known to overcondense metaphasic chromosomes), chromosome normally silenced during mitosis, nocodazole-treated cells were arms in nocodazole/TSA–treated cells displaying PSCS seemed to incubated with a-amanitin (at a concentration known to be longer and less tightly condensed than in the controls. This completely inhibit transcription by RNA polymerase II) prior to PSCS phenomenon, also called ‘‘precocious anaphase,’’ was TSA treatment (28). This inhibition of transcription had no effect observed in f25% of nocodazole/TSA–treated cells, and all on TSA-induced PSCS (Fig. 3B, top), or on the mitotic index chromosomes in any one cell displayed an identical phenotype. (Fig. 3B, bottom). These results suggest that HDAC inhibition No PSCS was detected in control nocodazole-treated HeLa cells. overrides the prometaphase block in mitotic SAC-activated cells in TSA-induced PSCS was dose-dependent (Fig. 1B). In addition, a transcription-independent manner. the mitotic index of control nocodazole-treated cells was >80%, Key targets of APC/C are degraded in a proteasome- whereas that of TSA-treated cells declined in a TSA dose- dependent manner following TSA treatment. SAC activation dependent manner to <40% (Fig. 1C). Cell viability was assessed inhibits the ubiquitin ligase complex APC/C. Activation of APC/C by trypan blue exclusion and cell counting. Under these treatment at the metaphase-anaphase transition leads to polyubiquitination conditions, a few apoptotic cells (condensed fragmented nuclei as of cyclin B1 and securin, such that they are targeted to a depicted on Fig. 5B, cells labeled ‘‘ap’’) were detected although cell proteasome-dependent degradation pathway. These two proteins

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. HDACIs Override the Spindle Checkpoint are believed to be the only essential APC/C targets (29). Under including cdk1 inactivation, histone H3 serine 10 dephosphoryla- conditions of sustained SAC activation (i.e., nocodazole treatment), tion, nuclear membrane reformation, and release of SAC compo- APC/C targets accumulate and reach a high stable concentration. nents from the kinetochore. To determine whether APC/C activity was affected by TSA To determine whether TSA-induced degradation of cyclin B1 led treatment, the stability of APC/C targets was assessed by Western to a decrease of cdk1 activity, cdk1 was immunoprecipitated and blotting using anti-cyclin B1 and anti-securin antibodies as probes. its kinase activity was measured with histone H1 as a substrate. Cyclin B1 accumulated, as expected, in nocodazole-treated cells TSA treatment induced a substantial decrease in the cdk1 activity (Fig. 4, lane 2). TSA treatment of nocodazole-arrested cells resulted in nocodazole-treated cells, although the amounts of cdk1 protein in a sharp decrease in the abundance of cyclin B1 (lane 3). Pre- recovered in the immunoprecipitates from treated and untreated treatment with the proteasome inhibitor MG132 prevented this samples were comparable (Fig. 5A). Coomassie staining of the gel TSA-induced decrease (lane 4). Similar results were obtained for showed that reduced H1 phosphorylation signal following TSA securin. The amount of cyclin B1 or securin detected in TSA- treatment was not due to reduced amounts of histone H1 treated cells was intermediate between those observed in untreated recovered in the assay (Fig. 5A,‘‘H1 Total’’). Proteasome inhibition and nocodazole-treated cells. These data show that TSA treatment in nocodazole/TSA–treated cells did not rescue the cdk1 activity induces a proteasome-dependent degradation of major APC/C (Fig. 5A), although the addition of MG132 blocked TSA-induced targets. cyclin B1 degradation (Fig. 4). This can be explained by the finding TSA treatment induces classical mitosis exit markers. Our that cdk1 and polyubiquitinated cyclin B1 were dissociated by a data suggests that HDAC inhibition leads to SAC dysfunction. To nonproteolytic function of the proteasome (30). Thus, in the confirm that prometaphase-arrested cells indeed exit mitosis in the presence of MG132, polyubiquitinated cyclin B1 is not degraded presence of HDACI, we examined classical markers of mitosis exit and can be detected by immunoblotting, but is not associated with

Figure 3. Different unrelated HDACIs cause PSCS and decrease the mitotic index. TSA-induced PSCS and the mitotic index decrease do not require transcription. Top, experimental outline (see legend to Fig. 1). A, PSCS frequency and decreased mitotic index in the presence of various HDACIs. Nocodazole-arrested mitotic HeLa cells were treated for 16 h with TSA (200 ng/mL), sodium butyrate (NaB; 100 mmol/L), apicidin (Api;5Ag/mL), or valproic acid (VPA; 30 mmol/L). Columns, percentage of PSCS (top) and mitotic index (bottom) observed in chromosome spreads. B, TSA-induced PSCS frequency and decreased mitotic index do not involve transcription. Nocodazole-arrested mitotic HeLa cells were treated or not treated with 2 Ag/mL of a-amanitin for 1 h, then treated with 200 ng/mL of TSA for 16 h, without removing the nocodazole or a-amanitin. Columns, percentage of PSCS (top) and mitotic index (bottom) observed in chromosome spreads. Columns, means from at least two independent experiments; bars, SD. www.aacrjournals.org 6363 Cancer Res 2007; 67: (13). July 1, 2007

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Cancer Research cdk1. Quantification experiments showed that cdk1 activity was three times higher in control nocodazole-treated cells than in nocodazole/TSA–treated cells (Fig. 5A, bottom). Mitotic index measurements (Fig. 1C) showed that TSA treatment was associated with more nuclei-like figures displaying decondensed chromatin but were unable to indicate whether a nuclear envelope was reformed around the chromatin. To resolve this question, nocodazole/TSA–treated cells and control (nocodazole alone) cells were immunolabeled for lamin A/C (Fig. 5B). Most TSA-treated nuclei-like figures were positive for lamin A/C, indicating that a nuclear envelope had reformed in these cells. SAC inhibition and subsequent anaphase entry are characterized by the release of SAC components from the kinetochores. We examined kinetochore localization of MAD2, a major downstream SAC effector, by immunofluorescence. Kinetochores were labeled with a human CREST autoimmune serum, which recognizes the kinetochore proteins CENP-A, CENP-B, and CENP-C, whereas Figure 4. HDAC inhibition by TSA in nocodazole-arrested mitotic HeLa cells MAD2 was detected with a specific antibody. Characteristic CREST- induces proteasome-dependent degradation of APC/C target proteins. Top, positive kinetochore dots were observed in both control and experimental outline (see legend to Fig. 1). Immunoblot analysis of total cellular protein extract obtained from control untreated cycling HeLa cells (lane 1) and nocodazole/TSA–treated mitotic cells, indicating that TSA treat- nocodazole-arrested mitotic HeLa cells (lanes 2–5), treated with 3 Amol/L of ment does not lead to major kinetochore damage, although MAD2 MG132 for 1 h (lanes 4 and 5), and then with 200 ng/mL of TSA for 16 h (lanes 3 and 4), without removing the nocodazole or MG132. Actin was used as was found to be displaced from kinetochores in approximately one an immunoblot loading control. third of nocodazole/TSA–treated mitotic cells (Fig. 6A). MAD2 antibodies failed to label chromosome spreads, so we could not Previous reports suggest a role for HDACs in SAC regulation but determine whether the mitotic cells that had lost MAD2 from the various studies led to apparently contradictory conclusions. For kinetochores were the same as those displaying PSCS. Nevertheless, example, it has been reported that HDAC inhibition in mitosis the respective proportions of cells exhibiting these phenomena prevented sister chromatid centromere separation, suggesting that were similar enough to assume that this was indeed the case. HDACIs trigger the SAC abnormally (15). In contrast, other authors We also tested the loss of histone H3 serine 10 (H3S10) reported that HDACIs induced a down-regulation of cyclin B1– phosphorylation. During normal mitosis, H3S10 becomes phos- dependent kinase activity (32), suggesting that HDACI treatment phorylated in prophase and remains phosphorylated until early leads to checkpoint inhibition. Accordingly, Dowling et al. anaphase. In metaphase-arrested cells (SAC activated), this described premature mitosis exit, H3S10 dephosphorylation, and phosphorylation was maintained. Chromosome spreads from diminished BUBR1 kinetochore localization following TSA treat- control (nocodazole alone) and nocodazole/TSA–treated cells were ment (31). These results clearly plead in favor of the existence of a immunostained for H3S10 phosphorylation (Fig. 6B). All chromo- TSA-induced SAC defect. However, recent data show that a slow some spreads from nocodazole-treated control cells were positive but continuous degradation of cyclin B induces an apparent for H3S10 labeling. In contrast, in nocodazole/TSA–treated cells, mitosis exit in spite of the presence of a functional and active SAC chromosome spreads displaying a normal phenotype (i.e., attached (33). In this particular situation, other APC/C targets are not sister chromatids) were labeled by the H3S10-specific antibody, degraded and MAD2 and BUBR1 remain attached to kinetochores. whereas chromosome spreads displaying PSCS were negative for With the noteworthy exception of BUBR1 partial loss from H3S10 labeling. Figure 6B shows one normal, H3S10-positive, kinetochores, data reported in ref. (31) could be attributed to a spread and, within the same microscope field, a H3S10-negative TSA-induced cyclin B degradation alone. BUBR1 delocalization spread displaying a TSA-induced PSCS (arrow). Loss of H3S10 described in ref. (31) is incomplete, and thus, it is not clear whether phosphorylation following TSA treatment has been previously the remaining kinetochore-bound BUBR1 was sufficient to reported using immunoblot techniques (31). However, our maintain the SAC or not. Conflicting reports exist on the amount chromosome spread immunostaining approach allowed us to of BUBR1 required to activate the SAC. One study reported that show that cells which displayed reduced TSA-induced H3S10 25% of the normal amount of BUBR1 led to SAC defects (34), phosphorylation were the same as those which displayed PSCS. although in another study, heterozygous mice expressing only 29% This indicates that cells presenting a TSA-induced PSCS are of the normal amount of BUBR1 showed no SAC malfunction (35). engaged into exit from mitosis. Taken together, our findings show Therefore, although the data reported by Dowling et al. represent that HDAC inhibition renders the SAC ineffective. the strongest evidence of a TSA-induced SAC malfunction to date, one cannot formally exclude that TSA induced cyclin B depletion– like defects and diminished BUBR1 kinetochore localization Discussion without inactivating the SAC. The finding by Cimini et al. (15) Understanding the molecular mechanisms of the antiprolifer- that TSA treatment induces persistent sister chromatid cohesion in ative properties of HDACIs is a major issue both in terms of anaphase would support this hypothesis. Thus, the primary anticancer therapy and in our knowledge of basic cellular consequences of SAC inhibition being the activation of APC/C functions. We investigated the integrity of the mitotic SAC in cells and the subsequent proteasome-dependent degradation of its only treated with HDACIs and found that HDAC inhibition results in known essential targets: cyclin B and securin, it seems essential to SAC being overridden. examine sister chromatin cohesion and securin status in order to

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. HDACIs Override the Spindle Checkpoint assess definitively whether or not SAC function is altered. Our hyperacetylated form of histone H3 showed that histone H3 was results show that TSA treatment induces securin degradation and hyperacetylated in all cells, ruling out the possibility that TSA did sister chromatid separation. Therefore, the abnormal persistent not act on a particular cell subpopulation (data not shown). chromatid cohesion observed in ref. (15) following TSA treatment However, this ‘‘partial’’ effect is consistent with the finding that TSA was more likely the consequence of a TSA-induced impaired treatment led to only partial proteasome degradation of APC/C key chromatin topological organization (chromatid entanglement) targets. Although the reason for this heterogeneous response is not rather than a failure to cleave cohesins. clear, a similarly incomplete response has also been observed in We found that when nocodazole-treated cells (to induce SAC other SAC inhibition studies. For example, MAD2 knockdown in activation) were subsequently treated with TSA, 25% of the mitotic HeLa and HaCat cells induces PCSC in f50% of the cells (20). cells displayed a characteristic PSCS phenotype which resembles Similarly, mutations of the gene encoding the SAC component that observed when the SAC is inhibited by antibody injection (18), BUBR1 in the mosaic variegated aneuploidy syndrome (36) leads to RNA interference (20, 21), or by genetic means (19). Sister PSCS in only a fraction of the cells (37). chromatids that have separated prematurely are considered a Not all HDACI were as effective in inducing PSCS. TSA was the hallmark of a defective checkpoint (19). We also showed that TSA best inducer, sodium butyrate and apicidin were intermediate, and treatment induced the proteasome-dependent degradation of valproic acid did not induce PSCS. However, all HDACI induced a cyclin B1 and securin, two essential APC/C targets. Finally, we similar decrease in the mitotic index in nocodazole-treated cells. monitored exit from mitosis by different and independent means This apparent discrepancy may be due to the differences in the including testing for loss of cdk1 activity, histone H3 serine 10 spectra of action of these drugs. Although little is known about the dephosphorylation, reformation of the nuclear envelope, and potential for each drug to inhibit a given subset of HDACs, TSA is MAD2 release from kinetochores. All tests done indicated that one of the most potent HDACIs and is believed to act on both class TSA treatment induced exit from mitosis in SAC-activated cells. I and class II HDACs. Thus, we propose that at least two distinct Thus, our results show that HDAC inhibition overrides the SAC. HDACs are required in mitosis: one necessary to maintain the SAC In every experiment done, HDACI treatment induced PSCS in and sensitive to all the drugs tested, and the other required for only part of the cell population. Immunostaining experiments in anaphase progression and sensitive to TSA but insensitive to nocodazole/TSA–treated cells with an antibody specific for a valproic acid (and partially sensitive to the two other drugs tested).

Figure 5. Decreased cyclin B1–associated cdk1 kinase activity and nucleus formation in nocodazole-arrested mitotic HeLa cells following TSA treatment. Top, experimental outline (see legend to Fig. 1). A, cdk1 kinase activity against a histone H1 substrate from nocodazole-arrested mitotic HeLa cells treated with TSA, MG132, and TSA, or neither (nocodazole alone). Top, histone H1 phosphorylation by cdk1 kinase activity (H1 32P), Coomassie staining of the gel showing H1 recovery in the assay (H1 Total), and control immunoblot of immunoprecipitated cdk1 kinase (IP cdk1). Bottom, quantification of cdk1 kinase activity. Columns, means from four independent experiments; bars, SD. B, reformation of nuclear envelope induced by TSA. Immunofluorescence microscopy showing nocodazole-arrested mitotic HeLa cells treated or not treated with TSA for 16 h, in the persistent presence of nocodazole. DAPI staining (DNA) and lamin A/C immunostaining. N, nuclei; m, mitotic figure; ap, apoptotic cell. Nuclear envelope–positive cells are more frequent in the mitotic TSA-treated samples than in the controls. www.aacrjournals.org 6365 Cancer Res 2007; 67: (13). July 1, 2007

Figure 6. Loss of mitosis markers from chromosomes in nocodazole-arrested HeLa cells exposed to TSA. Top, experimental outline (see legend to Fig. 1). A, immunofluorescence microscopy showing DAPI, CREST, and MAD2 staining on nocodazole-arrested mitotic HeLa cells treated or not treated with TSA. B, chromosome spread stained with DAPI and with antihistone H3 phosphorylated on serine 10. Arrow, cell with a PSCS phenotype that has lost H3S10 phosphorylation.

Under this hypothesis, nocodazole/TSA–treated cells escape SAC HDACIs. Histone acetylation is involved in transcription, so it is activation, but a proportion remains blocked in anaphase, allowing plausible that HDAC inhibition leads to the reexpression of the observation of PSCS. Conversely, nocodazole/valproic acid– antiproliferative genes silenced in tumor cells. However, a number treated cells would exit mitosis without pausing in anaphase. In of nonhistone proteins are regulated by acetylation and the this case, PSCS would not be observed but the mitotic index would possibility of an HDACI antiproliferative mechanism independent decrease, in accordance with our observations. from transcription has emerged (11–13). Our results show that In addition to PSCS, TSA treatment led to partial decondensa- HDACI-driven SAC inhibition is transcription-independent, and tion of mitotic chromosomes. This finding was restricted to cells thus, raise the possibility that the targets of HDACIs in anticancer displaying PSCS suggesting that the observed decondensation is a therapy could be, at least in part, nontranscriptional. consequence of mitosis exit rather than a direct effect of chromatin hyperacetylation. Similar chromosome morphologic changes have been observed in HeLa and HaCat cells in which the key SAC Acknowledgments component MAD2 has been knocked down (20). Thus, this Received 8/14/2006; revised 2/26/2007; accepted 4/19/2007. incomplete decondensation phenotype is likely to be a direct Grant support: Association pour la Recherche sur le Cancer (grant no. 4466), consequence of TSA-induced SAC overriding. Ligue Contre le Cancer (Comite´sDe´partementauxde l’He´raultand de l’Aveyron and Comite´Re´gional du Languedoc-Roussillon), and GEFLUC (Languedoc-Roussillon). The precise nature of the antiproliferative mechanism(s) of G. Eot-Houllier is supported by a Ligue Nationale Contre le Cancer fellowship. HDACIs remains a subject of debate. Sustained SAC inhibition leads G. Fulcrand is supported by a Ligue Re´gionale Contre le Cancer Research Ph.D. scholarship (Comite´du Languedoc Roussillon). L. Magnaghi-Jaulin is an investigator to the death of proliferative cells within a few generations (23) and of the Centre National de la Recherche Scientifique. our results show that HDAC inhibition results in SAC inhibition The costs of publication of this article were defrayed in part by the payment of page making HDACIs the only clinically validated pharmacologic SAC charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. inhibitors known thus far. We propose that this SAC-inhibiting We thank C. Johanet and J. Piette for the gift of reagents and J.M. Vanacker, activity may contribute to the antiproliferative properties of J.M. Brondello, and J. Piette for discussions and critical reading of the manuscript.

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