hdac and hsp90 inhibitors down-regulate pttg1/securin but do not induce aneuploidy

GENES, CHROMOSOMES & CANCER 48:194–201 (2009)

HDAC and Hsp90 Inhibitors Down-RegulatePTTG1/Securin But Do Not Induce Aneuploidy

Agustın Hernandez,1* Guillermo Lopez-Lluch,2 Placido Navas,2 and Jose A. Pintor-Toro1

1Cell Signaling Department,Centro Andaluzde Biolog�|a Molecular y Medicina Regenerativa (CABIMER-CSIC),Avda.Ame¤ ricoVespucio s/n,Seville,Spain2Cell Biologyand Biotechnology Section,Centro Andaluzde Biolog�|a del Desarrollo-CSIC,Universidad Pablo de Olavide-CSIC,CIBERER,Carretera de Utrera Km.1,Seville,Spain

Human securin regulates correct chromatid separation. However, there are conflicting reports on the aneugenic effects of

its gene deletion. Here we show that PTTG1/securin gene expression is dramatically repressed when Hsp90 or histone

deacetylases are inhibited. However, these treatments do not increase the proportion of aneuploid cells. This was also

confirmed using RNAi (silencing of PTTG1/securin �80%). As expected, histone deacetylases arrested cells in both G1 and

G2. However, sec�/� HCT116 cells showed a greater disposition to arrest cells in G2 than secþ/þ cells due to insufficient

induction of CDKN1A. These results indicate that chromatid separation is controlled through redundant mechanisms and

reveal a new aspect of securin in cell cycle regulation. VVC 2008 Wiley-Liss, Inc.

INTRODUCTION

Aneuploidy is considered to be at the root of

transformation (Venkitaraman, 2007) and signifi-

cant alterations in the levels of proteins control-

ling correct chromatid separation are seen as

putative neoplasic promoters. Human securin is

one of such proteins (PTTG1/securin for its gene).

Its best known function is to inhibit sister chro-

matid separation before anaphase: securin binds

to and inhibits separase, the caspase-like protease

that degrades SCC1 cohesins, until securin itself

is degraded after induction of the APC complex

at the onset of anaphase (Pines, 2006). However,

necessity for securin is unresolved, as several

studies show contradictory results. In fission

yeast, deletion of the securin ortholog Cut2 is le-

thal (Funabiki et al., 1996) but in budding yeast,

DPds1 strains are viable under certain growth

conditions (Yamamoto et al., 1996). In both cases,

mutation of yeast orthologs produces massive an-

euploidy under nonpermissive conditions. In

mammals, some studies have proposed securin to

be redundant to hold sister chromatids together

until anaphase (Huang et al., 2005). Cell lines

devoid of both PTTG1/securin alleles have been

produced in HCT116 (Jallepalli et al., 2001; Pfle-

ghaar et al., 2005). However, although a first com-

munication displayed data showing that around

30% of sec�/� HCT116 cells suffered chromosome

losses (Jallepalli et al., 2001), a later article con-

fined that effect to the first few cell divisions after

gene knock-out (Pfleghaar et al., 2005). Studies

with animal models have also yielded paradoxical

results: Pttg1 knock-out mice seem to develop nor-

mally whereas mouse embryonic fibroblasts grow

abnormally in culture (Mei et al., 2001).

PTTG1/securin is overexpressed in many types

of cancer from various origins (Vlotides et al.,

2007). The extent of this overexpression is vari-

able but can be correlated to malignancy and

metastatic proneness (Heaney et al., 2000; Ram-

aswamy et al., 2003). The consequences of

PTTG1/securin overexpression are not fully

understood but include resistance to apoptosis, to

DNA damage or TP53-dependent cell cycle

arrest and, paradoxically, aneuploidy (Bernal and

Hernandez, 2007; Bernal et al., 2002; Kim et al.,

2007). In this context, pharmacological repression

of PTTG1/securin in tumor cells may be instru-

mental in slowing down metastatic processes. On

the other hand, it can mean the induction of fur-

ther aneuploidy, as a side effect. Recently, we

have described that dicoumarol is an unsuspected

Hsp90 inhibitor (Hernandez et al., 2008). Also,

we showed that treatment of cancer cells with

Supported by: The Spanish Ministry of Education, Grantnumber: SAF2005-07713-C03-02; Andalusian Regional Govern-ment, Grant numbers: BIO-177 and CVI-186; AndalusianRegional Government, Spain.

*Correspondence to: Agustın Hernandez, Instituto de Bioguı-mica Vegetal y Fotosıntesis (IBVF-CSIC), Avda. Americo Vespu-cio 49, Seville 41092, Spain. E-mail: [email protected]

Received 4 April 2008; Accepted 3 October 2008

DOI 10.1002/gcc.20630

Published online 11 November 2008 inWiley InterScience (www.interscience.wiley.com).

VVC 2008 Wiley-Liss, Inc.

dicoumarol and other Hsp90 inhibitors reduces

the levels of securin through repression of its

gene. It is important then to ascertain if chemo-

therapeutic drugs that decrease PTTG1/securinlevels could cause increases in aneuploidy with

unknown consequences.

In the present work, we show that HDAC (his-

tone deacetylase) inhibitors repress PTTG1/securin mRNA expression, similarly to Hsp90

inhibitors. Using a novel technique to quantify

aneugenic events in cell cultures (Muehlbauer

and Schuler, 2005), we also show that short-term

pharmacological treatments with Hsp90, HDAC

inhibitors, or RNAi against PTTG1/securin do not

lead to aneuploidy. However, the presence of

PTTG1/securin dictates the profile of cell cycle

arrest induced by these drugs.

MATERIALS AND METHODS

Cell Lines, Reagents, and Treatments

Wild-type and PTTG1/securin-knock-outHCT116 human colon carcinoma cells (secþ/þ

HCT116 and sec�/� HCT116) were kindly pro-

vided by Dr. B. Vogelstein (Johns Hopkins Uni-

versity, Baltimore, MD) and have been described

previously (Jallepalli et al., 2001). HCT116 cells

were maintained in McCoy’s 5A medium plus

10% fetal bovine serum. Unless otherwise stated,

cells were incubated with dicoumarol (150 lM),

17AAG (17-allylaminodemethoxygeldanamycin,

1 lM), novobiocin (0.8 mM), butyrate (3 mM),

valproate (3 mM), nicotinamide (15 mM), or taxol

(paclitaxel, 50 nM) for 24 hr prior determination

of their effects.

Silencing of PTTG1/Securin

Interference of PTTG1/securin gene was done

as in Bernal and Hernandez (2007). Transfection

of RNAi oligonucleotides was done using Oligo-fectamine (Invitrogen, Carlsbad, USA) according to

manufacturer instructions.

Immunoblotting

Cells were lysed and soluble proteins were

harvested in RIPA buffer plus protease in-

hibitors (Roche, Barcelona, Spain) and 1 mM

phenylmethylsuphonylfluoride. Proteins were re-

solved on SDS-PAGE gels and transferred to

nitrocellulose filters using standard procedures.

Immunoblotting was performed using the follow-

ing antibodies: anti-AKT and TP53 (DO-1 mono-

clonal) (Santa Cruz Biotechnologies, Santa Cruz,

USA), Anti b-actin (Sigma-Aldrich, Madrid,

Spain), anti-activated Caspase-3, HER2, and

Serine-15 phosphorylated TP53 (Cell Signaling

Technologies, Danvers, USA), and Anti-PARP1

(Roche, Barcelona, Spain).

Flow Cytometry

Floating and adherent cells were stained with

propidium iodide and processed for flow cytome-

try analysis on a Coulter Epics XL apparatus as

described (Hernandez et al., 2008). Determina-

tion of aneuploidy in cultured cells was estimated

according to Muehlbauer and Schuler (2005).

Briefly, cell samples were fixed in 4.0 ml of 70%

ethanol (�20�C). After overnight storage at

�20�C, cells were hydrated and permeabilized

for 15 min in 5 ml of 0.1% saponin/PBS solution

followed by labeling for 60 min with 500 ll of

phospho-histone H3 (ser 10) 6G3 (H3-P) mono-

clonal antibody (Cell Signaling Technologies,

Danvers, USA). After washing, secondary fluores-

cent antibody was added in PBS [Alexa Fluor

488VVR goat anti-mouse IgG conjugate (Molecular

Probes, Eugene, USA)] for 60 min at 37�C in the

dark. Finally, cells were washed again with PBS

and stained with propidium iodide. Discrimina-

tion to analyze only single cell events was per-

formed during acquisition.

Semiquantitative RT-PCR

One microgram of total RNA was subjected to

cDNA synthesis using a polyT(18) primer and

SuperScript RNAse H Reverse Transcriptase

(Invitrogen) according to manufacturer instruc-

tions. One microliter of the final cDNA was sub-

jected to PCR as in Hernandez et al. (2008).

Primers annealing on the glyceraldehyde-3-phos-

phate dehydrogenase cDNA were used to check

equal cDNA loading onto PCR mixes. Saturation

of the reaction and specificity of primers were

checked using a threefold excess of cDNA

obtained from sec�/� HCT116 cells (data not

shown).

Other Methods

Protein contents were quantified using a Bio-

Rad Protein Assay kit (Bio-Rad) according to the

manufacturer’s instructions and using bovine se-

rum albumin as a standard. Experiments were

typically done in triplicate. Differences were ana-

lyzed by unpaired t tests; a P value of <0.05 was

considered significant.

SECURIN REPRESSION DOES NOT INDUCE ANEUPLOIDY 195

Genes, Chromosomes & Cancer DOI 10.1002/gcc

RESULTS

HDAC Inhibitors Down-Regulate PTTG1/Securin

Gene Expression

PTTG1/securin gene expression depends on

Hsp90 (Hernandez et al., 2008). As HDAC inhib-

itors exert part of its antitumoural effects through

stabilization of the acetylated (inactive) form of

Hsp90 or some of its cochaperones, like Hsp70

(Nimmanapalli et al., 2003; Wang et al., 2007),

we wanted to know if butyrate, valproate, or nico-

tinamide could provoke PTTG1/securin gene

repression. Inhibitors of class I and II HDACs

(butyrate and valproate) provoked a marked

decrease in mRNA levels of PTTG1/securin after

24 hr in HCT116 cells, similar to the action of di-

coumarol (Fig. 1A). Conversely, nicotinamide, an

inhibitor of NADH-dependent deacetylases (class

III HDAC or sirtuins), had only a minor effect on

mRNA levels (Fig. 1A) and no effect on polypep-

tide amounts (data not shown); thus, this study

was no further pursued.

To confirm that HDAC inhibitors acted par-

tially through Hsp90, we evaluated their effects

on two well-known markers of Hsp90-activity

(AKT and HER2) and apoptosis (PARP1 and

Caspase 3 18 kDa fragment). As a comparison,

we included true Hsp90 inhibitors dicoumarol,

17AAG and novobiocin. As expected, all com-

pounds lead to drastic reductions in securin poly-

peptide amounts (Fig. 1B), in accordance to their

effect on PTTG1/securin mRNA. On the whole,

either Hsp90 or HDAC inhibitors affected both

Hsp90-activity and apoptosis markers. Strikingly,

HDAC inhibitors reduced the levels of HER2

without visibly affecting those of AKT but this is

also observed with some Hsp90 inhibitors; for

example, dicoumarol only affects AKT levels

marginally. Regarding apoptosis induction, butyr-

ate was a more potent death inducer than valproate.

Thus, although both produced a decrease in

PARP1, butyrate led to a clearer activation of Cas-

pase 3 than valproate and to a greater percentage of

cells in sub-G1 phase (Fig. 2). Having in mind that

different mechanisms of action can be associated to

different profiles in markers of Hsp90 inhibition

(Rosenhagen et al., 2003), these results agree well

with butyrate and valproate exerting part of their

action through inhibition of Hsp90.

Absence of Securin Promotes G2 Arrest in Hsp90

or HDAC Inhibitor Treated Cells

We also wanted to know if repression of

PTTG1/securin gene had any effects on cell cycle

or if its absence conferred a differential sensitiv-

ity to these compounds. We analyzed the cell

cycle of HCT116 cells treated with Hsp90 and

HDAC inhibitors and compared them with sec�/�

HCT116 cells. As previously reported (Jallepalli

et al., 2001), cell cycle profiles were very similar

for untreated wild-type HCT116 and sec�/�

HCT116 cells (Fig. 2). However, a clear differ-

ence was apparent in the cell cycle arrest pro-

voked by dicoumarol, 17AAG, butyrate and, to

a lesser extent, by valproate: in all these cases,

sec�/� HCT116 cells showed an approximately

twofold greater tendency to arrest at G2 com-

pared with wild-type. It must be noted that dif-

ferences in mitotic index were modest in

comparison with either untreated controls or taxol

treatment (Fig. 4B), indicating a true G2 arrest

rather than a mitotic arrest. Curiously, sec�/�

HCT116 cells showed no differences in cell cycle

arrest pattern when treated with novobiocin. The

reason for this is unknown. With regard to apo-

ptosis, no marked differences were observed in

sub-G1 populations between wild-type and sec�/�

HCT116 cells (Fig. 2).

Joseph et al. (2005) showed that the TP53 sta-

tus could influence cell cycle arrest profiles in

butyrate-treated cells and sec�/� HCT116 present

Figure 1. HDAC inhibitors down-regulate PTTG1/securin geneexpression. A: Semiquantitative RT-PCR of HCT116 wild-type cellstreated with HDAC inhibitors. Concentrations as in Materials andMethods. B: Apoptosis induction and Hsp90-inhibition markers. Cellstreated as in Materials and Methods were subjected to Western blotanalysis with the indicated antibodies.

196 HERNANDEZ ETAL.


both greater levels of wild-type TP53 (Bernal

and Hernandez, 2007) and enhanced TP53 activ-

ity (Bernal, 2002). We compared the effect of

butyrate, as an example of the treatments previ-

ously done, on cell cycle regulators in wild-type

HCT116 cells and in cell lines devoid of securin

or TP53. As expected, levels of phosphorylated

(active) TP53 were greater in sec�/� HCT116

cells compared with wild-type and displayed little

differences on butyrate treatment. Polo-like ki-

nase 1 levels were effectively brought down by

butyrate in all cell lines and so were those of

cyclin B1 (Fig. 3), in accordance with butyrate

being able to induce both G1 and G2 arrest.

However, CDKN1A gene product levels showed

remarkable differences in untreated cells.

Although they were undetectable in p53 KO cells,

there were noticeable levels in wild-type and a clear

band in sec�/� HCT116 cells. When challenged

with butyrate, wild-type HCT116 cells were able to

induce CDKN1A around 8.5-fold, as evaluated by

densitometry of the bands in Figure 3. This induc-

tion was even more dramatic in TP53�/� HCT116

(19.5-fold), reaching end levels around 1.3-fold

greater than those of wild-type. On the contrary,

sec�/� HCT116 cells showed no induction of

CDKN1Awhen treated with butyrate and their lev-

els remained below those of treated wild-type

HCT116. This behavior translated into TP53�/�

showing the smallest proportion of cells arrested in

G2, followed by wild-type and sec�/� HCT116 cells

that displayed the greatest proportion.

Hsp90 and HDAC Inhibition Does Not Lead

to Massive Aneuploidy

Hsp90 and HDAC inhibitors produced a

marked decrease in levels of securin mRNA and

polypeptide in short term (24 hr) treatments.

Under these conditions, cells typically undergo

one or two cell divisions. We then studied if

these compounds could induce aneuploidy associ-

ated to repression of PTTG1/securin. Muehlbauer

and Schuler (2005) recently reported a novel

Figure 2. Cell cycle profiles of cells treated with Hsp90 or HDACinhibitors. HCT116 cells (wild-type or sec�/�) were stained with pro-pidium iodide and analyzed by flow cytometry. Quantitation as per-centages of cells in each phase of the cell cycle is shown in insets.Apopt.: apoptotic cells. Data are from a representative experiment.

Figure 3. Effects of butyrate on proteins governing G1 and G2arrest. Wild-type, sec�/� and TP53�/� HCT116 cells were collectedand split in two aliquots after 24 hr treatment with butyrate. West-ern blot analysis with the indicated antibodies was performed withone of the aliquots and the remaining cells were fixed, stained withpropidium iodide, and subjected to FACS. Data from a representativeexperiment.



quantitative method for the determination of an-

euploidy in cultured cells. Figure 3A shows typi-

cal dot plots obtained with this method.

Mitotically active single cells are stained with an

antibody raised against phosphorylated histone

H3 (areas a and b in Fig. 4A). Aneuploid cells

could have either a greater or a smaller chromo-

some complement. However, we did not attempt

to quantify cells with DNA content smaller than

4n (for cells in mitosis) or 2n (nonmitotic cells) as

aneuploid because, under the conditions tested,

there was a significant proportion of apoptotic

cells undergoing nuclear DNA fragmentation.

Hence, only cells in areas b and c are considered

true aneuploids.

We applied this method to determine aneu-

ploidy induced by Hsp90 and HDAC inhibitors.

As a control, we included a taxol treatment, a

well-known aneuploidy inducer (Muehlbauer and

Schuler, 2005). We measured four parameters:

mitotic index or percentage of cells in mitosis

[which corresponds to areas (a þ b)/(a þ b þ c þd)] as an indicator of mitotic arrest, the ratio of

mitotic aneuploid cells with respect to total cells

in mitosis [b/(a þ b)] as a measure of the propor-

tion of mitoses leading to aneuploidy, the propor-

tion of aneuploid cells in mitosis with respect to

total cycling cells [b/(a þ b þ c þ d)] as a marker

of the proportion of aneuploid cells still able to

progress through the cell cycle, and finally the ratio

of nonmitotic aneuploid cells over total cycling

cells [c/(a þ b þ c þ d)] to measure accumulated

past events of chromosome missorting, because

this often prevents cells to proliferate. Also, as the

number of cells under area b is small in comparison

with those under c, this last parameter can also be

used as a measure of the proportion of total aneu-

ploid cells over the total population. Total of all

non-apoptotic cells (a þ b þ cþ d) is abbreviated as

TCC (total cycling cells).

Noteworthy, taxol had no significant effects on

the levels of securin polypeptide in HCT116

cells (Fig. 5A). Cells treated with inhibitors

showed no differences in mitotic index with

respect to untreated controls, with the exception

of dicoumarol (approximately three-fold reduc-

tion) and taxol-treated cells (around four-fold

increase), in accordance with this last drug arrest-

ing cells at mitosis (Ikui et al., 2005). Dicouma-

rol-treated cells displayed clear increases in the

proportion of aneugenic events per mitosis [b/(aþ b)] (Fig. 4B). However, as dicoumarol also

reduces the mitotic index, the net result is a neg-

ligible increase in the proportion of aneuploid

cells with respect to the total cycling cell popula-

tion measured as b/TCC or as c/TCC (Fig. 4C).

Butyrate had no discernible effects on mitotic

index while having a small but clear effect on the

proportion of aneuploid events per mitosis (Fig.

4B) and, therefore, this translated into a modest

Figure 4. Analysis of aneugenic events in Hsp90 or HDAC inhibi-tor-treated HCT116 wild-type cells by flow cytometry. A: Typicaldot-plots obtained after staining with Histone H3 phosphorylated/anti-mouse-Alexa Fluor 488 antibodies (FL1) and propidium iodide(FL2). Areas on histograms correspond to (a) mitotic cells withDNA 5 4n, (b) mitotic cells with DNA >4n, (c) nonmitotic cellswith DNA >4n, (d) normal cycling cells in G1, S, or G2 phase of thecell cycle. All cells showing DNA contents smaller than a full comple-ment for the phase they are in are considered as undergoing apopto-sis (e.g., ‘‘apoptotic’’ on left panel). Left panel: untreated cells; rightpanel: 50 nM taxol-treated cells. B: Effects on mitosis by differentHsp90 or HDAC inhibitors in HCT116 cells. C: Effects of Hsp90 orHDAC inhibitors on overall aneuploidy. Letters denote events regis-tered on the dot-plot areas shown above (Mitotic Index 5 (a 1 b)/TCC; TCC: total cycling cells, i.e., a 1 b 1 c 1 d). Data are averages6 SEM. Asterisks denote statistically significant differences (P � 0.05)with respect to data obtained for corresponding controls (vehicle), asevaluated by unpaired t tests.

198 HERNANDEZ ETAL.


increase in the proportion of aneuploid cells in

the TCC (Fig. 4C). Under the conditions tested,

taxol did not increase the proportion of aneuploid

cells per mitosis. However, as it provoked accu-

mulation of cells in mitosis, taxol treated cells

showed an approximately fourfold increase in

cells with abnormally high DNA contents when

the total cycling cell population was considered

(Fig. 4C). Then, Hsp90 inhibitors did not

increase the number of aneuploid cells in culture

and HDAC inhibitors only modestly increased

those of nonmitotically active cells.

Silencing of PTTG1/Securin Does Not

Provoke Aneuploidy

Faint levels of securin were still detectable in

treated cells and might, in theory, suffice to in-

hibit aneuploidy. To confirm the results obtained

with inhibitors, we silenced PTTG1/securin in

HCT116 wild-type cells by transfecting RNAi ol-

igonucleotides. Silencing can be accomplished in

the same time scale as inhibitor treatments and,

therefore, should clarify if repression of PTTG1/securin can provoke abnormal chromosome sort-

ing while cells divide once or twice. We com-

pared the levels of aneuploid cells in RNA-

vehicle (mock) treated cells and silenced cells.

Figure 5B shows that posttranscriptional silencing

of PTTG1/securin was achieved with an efficiency

of approximately �90%. Mitotic indices were

similar and no significant differences were

observed in the proportion of aneugenic events in

HCT116 cells silenced for 48 hr when compared

with mock-treated controls (Table 1).

Effects of HDAC and Hsp90 Inhibitors on

Aneuploidy Induction in Sec2/2 HCT116 Cells

Although short-term pharmacological or post-

transcriptional reductions of securin levels

seemed to have no effect on aneuploidy induc-

tion, a full long-term ablation of PTTG1/securin

gene could still be a factor favoring aneugenic

events through other mechanisms described for

Hsp90 and HDAC inhibitors. sec�/� HCT116

cells were treated with Hsp90 and HDAC inhibi-

tors for 24 hr and their effects on chromosome

segregation efficiency estimated as earlier. Again,

dicoumarol reduced significantly the mitotic

index, albeit to a lesser extent than in wild-type

HCT116 (Figs. 4A and 6A). Strikingly, butyrate

and valproate treated cells showed divergent

effects: whereas butyrate reduced modestly the

mitotic index and increased the proportion of

aneuploid events per mitosis, valproate led to a

greater mitotic index but no differences in aneu-

genic events per mitosis. However, when the

whole cycling cell population was considered, a

consistent three-fold greater percentage of aneu-

genic events in both mitotic and nonmitotic cells

was observed with both HDAC inhibitors. Com-

pared with those levels found in wild-type cells,

HDAC inhibitors had an approximately two-fold

greater effect in sec�/� HCT116 cells. On their

turn, cells treated with Hsp90 inhibitors showed

no deviations from controls and modest differen-

ces in comparison with wild-type HCT116.

DISCUSSION

Hsp90 and HDAC inhibitors are considered

among the most promising antitumoural drugs for

their pleiotropic effect. We recently showed that

Hsp90 inhibitors repressed PTTG1/securin gene

expression (Hernandez et al., 2008) and have

shown here that inhibition of class I or II HDACs

act similarly. Therefore, we were interested to

know if these types of drugs produced chromo-

somal instability because of their effect on

PTTG1/securin. However, despite the fact that

treated cells showed remarkably low levels of

securin and that they were still proliferating, no

TABLE 1. Silencing of PTTG1/Securin Does NotInduce Aneuploidy

M. I. b/(a þ b) b/TCC c/TCC

Mock 1.93 � 0.01 4.17 � 2.25 0.08 � 0.03 1.12 � 0.15RNAi 1.86 � 0.13 7.07 � 4.28 0.14 � 0.07 1.30 � 0.14

Percentages of cells in mitosis (Mitotic index, M. I.), aneuploid cells in

mitosis with respect to total mitotic cells [b/(a þ b)], aneuploid cells

in mitosis with respect to total cycling cells (b/TCC) or aneuploid

cells in other stages of the cell cycle with respect to total cycling cells

(c/TCC) were quantified by flow cytometry as in Materials and Meth-

ods. Data are averages � S.E.M.

Figure 5. Effect of taxol and RNAi on securin polypeptide levels.A: HCT116 wild-type cells were subjected to Western blot after24 hr treatment with vehicle (untreated), 100 lM dicoumarol or50 nM taxol. B: HCT116 cells were transfected with RNAi oligonu-cleotides targeting PTTG1/securin and subjected to Western blot after48 hr.



increase in aneugenic events could be observed

(Fig. 4). These results indicate that securin levels

can be safely reduced without increasing risks of

aneuploidy. Moreover, these results also indicate

that securin functions are redundant in human

cells and gives weight to previous studies propos-

ing that separase phosphorylation governs effec-

tively sister chromatid separation even in the

absence of PTTG1/securin (Gorr et al., 2005;

Huang et al., 2005).

Along with securin involvement in chromatid

cohesion, our interest was spurred by reports stat-

ing that inhibition of Hsp90 was associated with

misalignment of chromosomes and aneuploidy

(Niikura et al., 2006). Also, HDAC inhibition

with butyrate has been long associated to numeri-

cal chromosome changes (Larsen et al., 1995;

Gomez-Vargas and Vig, 2002), although the exact

mechanism remains obscure. If repression of

PTTG1/securin was accountable in part for the

effects observed or if its absence aggravated them

was an important question to answer. HDACs are

associated with pericentric heterochromatin for-

mation and, hence, chromosome segregation dur-

ing mitosis (David et al., 2003) but other

mechanisms may also be at play (Kimata et al.,

2008). Butyrate and valproate showed a clearer

effect on aneuploidy induction on sec�/� HCT116

cells. This indicates that securin is a sensitizing

factor toward aneuploidy induction by HDACs

but that the mode of action of these compounds

probably does not involve securin. Noticeably,

both HDAC inhibitors provoked somewhat differ-

ent cellular responses, indicative of differences in

their mechanisms. On the other hand, lack of a

clear difference on apoptosis or aneuploidy induc-

tion with Hsp90 inhibitors between wild-type

and sec�/� HCT116 cells treated with Hsp90

inhibitors prevent assigning any relevance to

securin in the citotoxic effects observed using

these compounds.

At any rate, PTTG1/securin deletion does not

come without a price for the cell: Hsp90 or

HDAC inhibition provoked a differential G2 cell

cycle arrest in these cells (Fig. 2). It is tempting

to associate DNA-damage with this phenomenon:

G2 arrest is often associated with genotoxic

stress, securin has been shown to associate with

Ku-heterodimers of the NHEJ pathway of DNA

repair (Romero et al., 2001; Kim et al., 2007), its

deletion hampers repair of DNA-damage and

induces gross chromosomal rearrangements on

DNA-damage (Bernal et al., 2008). Also, DNA-

damaged sec�/� HCT116 cells present an unyield-

ing G2 arrest. However, there is no reason to

believe that lack of securin induces DNA-damage

on its own (Bernal and Hernandez, 2007) and this

is reflected in sec�/� HCT116 showing no altera-

tions in cell cycle profiles under unstressed condi-

tions. Moreover, the profile of cell cycle arrest

produced by adriamycin-induced DNA-damage

in sec�/� HCT116 cells is not different from that

in wild-type ones, albeit it is more persistent

(Bernal et al., 2008). This latter effect may be

linked to sec�/� HCT116 displaying enhanced

TP53 functions due to greater levels of this tran-

scription factor and it not being inhibited by

securin (Bernal and Hernandez, 2007; Bernal

et al., 2002). Indeed, greater levels of TP53,

including those of its active (phosphorylated)

form, influence background levels of CDKN1A

gene product, one of its most sensitive targets

and a cyclin inhibitor governing G1 arrest (Bernal

and Hernandez, 2007). Butyrate-dependent

Figure 6. Analysis of aneugenic events in Hsp90 or HDAC inhibi-tor-treated sec�/� HCT116 cells by flow cytometry. A: Effects on mi-tosis by different Hsp90 or HDAC inhibitors in HCT116-KO cells. B:Effects of Hsp90 or HDAC inhibitors on overall aneuploidy. Seelegend to Figure 3 for details.

200 HERNANDEZ ETAL.


CDKN1A gene expression is induced through

Sp1 and ZBP89 transcription factors and is essen-

tially independent of TP53 (Sowa and Sakai,

2000). However, as seen in Figure 3, TP53 status

can influence CDKN1A response to HDAC

inhibitors: increased levels of TP53 impaired

CDKN1A induction whereas its absence pro-

moted a greater response. The mechanism is ulti-

mately unknown, but it seems plausible that

TP53 in sec�/� HCT116 cells binds in excess to

CDKN1A promoter; this, in turn, would prevent

binding of Sp1 and/or ZBP-89 when activated by

butyrate treatment, maybe through steric hin-

drance. As a result, G1 arrest is inefficient in sec�/

� HCT116 and cells progress until they arrest in

G2. Further work is in progress to delineate the

influence of TP53 amounts in CDKN1A regula-

tion. Also, further investigation needs to be done

to ascertain the role and influence of securin in

cell cycle control.

ACKNOWLEDGMENTS

The authors wish to thank Dr. M. A. Moreno,

Ms. S. Santa-Cruz, and Ms. G. Serrano for their

invaluable technical assistance.

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hdac and hsp90 inhibitors down-regulate pttg1/securin but do not induce aneuploidy

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