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

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Histone Deacetylase Inhibitors Induce Premature Sister Chromatid Separation and Override the Mitotic Spindle Assembly Checkpoint Research Article Histone Deacetylase Inhibitors Induce Premature Sister Chromatid Separation and Override the Mitotic Spindle Assembly Checkpoint 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 Cancer Res 2007; 67: (13). July 1, 2007 6360 www.aacrjournals.org 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 immunoprecip- itation 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,
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