Table Of ContentOncogene(2011)30,2679–2690
&2011MacmillanPublishersLimited Allrightsreserved0950-9232/11
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ORIGINAL ARTICLE
EAPP: Gatekeeper at the crossroad of apoptosis and p21-mediated
cell-cycle arrest
P Andorfer and H Rotheneder
MaxF.PerutzLaboratories, Department ofMedicalBiochemistry, MedicalUniversity ofVienna, Vienna,Austria
We previously identified and characterized E2F-asso- inhibitors interact with CDKs and inhibit their activity
ciatedphospho-protein(EAPP),anuclearphosphoprotein (Chen etal.,1996). Thetumor suppressorp53isacom-
that interacts with the activating members of the E2F ponent of several checkpoints (Giono and Manfredi,
transcription factor family. EAPP levels are frequently 2006) and controls the expression of various growth-
elevated in transformed human cells. To examine the inhibitory or apoptotic genes (Rozan and El-Deiry,
biological relevance of EAPP, we studied its properties 2007).AmongtheseistheCDKN1Agenethatcodesfor
in stressed and unstressed cells.Overexpression of EAPP p21 (or p21WAF1/Cip1), which induces growth arrest by
in U2OS cells increased the fraction of G1 cells and binding and inhibiting CDK complexes (el-Deiry et al.,
lead to heightened resistance against DNA damage- or 1993; Chen et al., 1995; Goubin and Ducommun, 1995)
E2F1-induced apoptosis in a p21-dependent manner. and by sequestering away proliferating cell nuclear
EAPP itself becomes upregulated in confluent cells and antigen from DNA polymerase d (Flores-Rozas et al.,
after DNA damage and stimulates the expression of p21 1994;Wagaetal.,1994).Thereby,butalsobyitsability
independently of p53. It binds to the p21 promoter and to promote cellular senescence and differentiation,
seems to be required for the assembly of the transcrip- p21 prevents the development of tumors (for reviews
tion initiation complex. RNAi-mediated knockdown of see (Abbas and Dutta, 2009; Cazzalini et al., 2010).
EAPP expression brought about increased sensitivity In addition, p21 influences gene expression via certain
towards DNA damage and resulted in apoptosis even in promoter elements (Zhu et al., 2002) and by its inter-
the absence of stress. Our results indicate that the level action with transcription factors (Delavaine and La
of EAPP is critical for cellular homeostasis. Too much Thangue,1999;Kitauraetal.,2000).Paradoxically,p21
of it results in G1 arrest and resistance to apoptosis, also prevents apoptosis (Roninson, 2002) on the one
which,paradoxically,mightfavorcellulartransformation. hand because the initiation of apoptosis often requires
Too little EAPP seems to retard the expression not only an active cell cycle, on the other hand by interference
of the p21 gene, but also of a number of other genes and withapoptoticsignaling(Asadaetal.,1999)andcaspase
ultimately results in apoptosis. activation(Suzukietal.,1999;Baptiste-Okohetal.,2008)
Oncogene(2011)30,2679–2690;doi:10.1038/onc.2010.639; Some of these antiapoptotic activities depend on the
published online 24 January 2011 cytoplasmic localization of p21 (Blagosklonny, 2002).
We have previously identified and characterized an
Keywords: EAPP;p21;apoptosis;DNAdamage;cell-cycle E2F-binding protein, which we termed E2F-associated
arrest phospho-protein (EAPP) (Novy et al., 2005) and which
wefoundoverexpressedinmanytumorcelllines.EAPP
interactswithE2F1-3,comprisingtheactivatorgroupof
E2F proteins, and modulates E2F-dependent transcrip-
Introduction
tion. Its expression is regulated by the transcription
factors Sp1, Sp3 and Egr-1 binding to the TATA-less
The cell cycle of all eukaryotes is controlled by cyclin-
promoteroftheEAPPgene(Schwarzmayretal.,2008).
dependent kinases (CDKs), the catalytic partners of
Very recently it has been shown that EAPP negatively
cyclins (Sherr and Roberts, 2004). A variety of
regulates the expression of monoaminoxidase B via Sp1
checkpoint mechanisms have evolved that monitor
binding sites (Chen et al., 2010).
cell-cycle progression and respond to perturbations
WeshowinthisstudythatEAPPlevelsincreaseupon
like replication errors or DNA damage by modifying
DNA damage and higher EAPP levels seem to protect
the activity of CDKs (Hartwell and Weinert, 1989;
cells from apoptosis. This protection can also be
Pietenpol and Stewart, 2002). Cyclin-dependent kinase
achieved by ectopic expression of EAPP and correlates
with an increased number of cells in G1 phase and an
Correspondence: Dr H Rotheneder, Max F. Perutz Laboratories, upregulationofp21.RNAi-mediatedknockdownofp21
DepartmentofMedicalBiochemistry,MedicalUniversityofVienna, abrogatesthiseffectofEAPP.KnockdownofEAPPon
DrBohr-Gasse9/2,A-1030Vienna,Austria.
the other hand results in apoptosis. We analyzed the
E-mail:[email protected]
expressionofp21bywestern,reversetranscription–PCR
Received 16 August 2010; revised 1 December 2010; accepted 20
December2010;publishedonline24January2011 (RT–PCR) and reporter gene assays and could show
EAPP:Atthecrossroadofapoptosisandarrest
PAndorferandHRotheneder
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that the knockdown of EAPP reduces p21 mRNA and et al., 2009). As overexpression of EAPP resulted in
protein, whereas overexpression of EAPP results in slower growth, we checked whether EAPP also goes up
elevated p21 levels. Chromatin immunoprecipitation with increasing cell density. Figure 2a shows that this
(ChIP) experiments demonstrated that EAPP binds to is indeed the case, suggesting a possible connection
the p21 promoter in the vicinity of the TATA box and between the two proteins. To examine this we expres-
might be involved in transcription initiation. sed either GFP or GFP-EAPP with or without a
p21-specific small interfering RNA in U2OS cells
(Figure 2b). The GFP-positive cells were then analyzed
by flow cytometry to determine and compare the frac-
Results
tion of cells in G1-, S- or G2-phase (Figures 2c and d).
The comparison of GFP-positive and GFP-negative
Overexpression of EAPP alters cell-cycle progression
cells on the one hand and of GFP- and GFP-EAPP-
We have shown earlier that EAPPcan influencethe cell
expressing cells on the other hand demonstrated again
cycle and many transformed cell lines exhibit elevated
the cell cycle slowing property of EAPP. The knock-
levelsofEAPP(Novyetal.,2005).Toexaminewhether
down of p21 abrogated this growth-retarding effect,
EAPPisinvolvedintheresponsetostress,wecompared
suggesting that it is exclusively or at least primarily
the effects of certain drugs known to inhibit cell-cycle
mediated by p21.
progression in cells with different EAPP levels. We
transfected U2OS cells with either an expression vector
EAPPregulatesp21expressionviaSp1sitesandbindsto
for EAPP or an empty vector and treated these cells
the p21 promoter
with nocodazole and colchicine that arrest cells in
Western analysis showed that increasing the level of
mitosis by interfering with the assembly of the mitotic
EAPP results in higher p21, whereas a knockdown of
spindle, or with etoposide and methyl-methanesulfo-
EAPP lowers p21 (Figure 3a). To find out whether
nate, which cause DNA damage and cell-cycle arrest,
EAPP influences the expression or the stability of p21,
preferentially in S or G2 phase. Subsequently the
we first performed RT–PCR and compared the p21
cells were analyzed by flow cytometry. In all four cases
mRNAlevelsincellswithlowered,normalandelevated
the ectopic expression of EAPP brought about a
EAPP. Figure 3b shows that knockingdown EAPP
prominent G1 peak not present in mock-transfected
resulted in a concomitant reduction of p21 mRNA,
cells (Figure 1a). As it is very improbable that EAPP
whereas overexpression of EAPP caused a modest rise
can overcome the effects of four different drugs, the
of p21 mRNA. To determine whether EAPP influences
most likely explanation seemed to be an EAPP-induced
p21 mRNA levels by affecting promoter activity, we
slowing-downofthecellcycleresultinginanaccumula-
carried out reporter assays. It turned out that the
tion of cells in G1. Gating of U2OS cells expressing a
knockdown of EAPP reduced promoter activity,
greenfluorescentprotein(GFP)-taggedversionofEAPP
whereas its ectopic expression stimulated the promoter
revealed that the cells that expressed GFP-EAPP had a
(Figure 3c). Conversely, the cyclin A promoter was not
higher G1 fraction than those that did not (Figure 1b).
inhibited by the knockdown of EAPP but even slightly
stimulated (Supplementary Figure 2). A major determi-
The EAPP-induced G1 arrest is mediated by p21 nant for the transcription rate of a protein-coding gene
Cells approaching confluency usually grow slower and is its occupancy by RNA polymerase II (Pol II).
this is at least partially caused by elevated p21 (Perucca To examine whether EAPP can influence the activity
Nocodazole 1μ M Colchicine 5μ M
GFP
G1SG2 G1SG2 G1SG2 G1SG2 G1SG2 G1SG2 G1SG2 G1SG2 G1SG2
Log Mock HA-EAPP Mock HA-EAPP Log No GFP Low levels High levels
of GFP of GFP
MMS 0,2mM Etoposide 20 μ M
GFP-
EAPP
G1SG2 G1SG2 G1SG2 G1SG2 G1SG2 G1SG2 G1SG2 G1SG2
Mock HA-EAPP Mock HA-EAPP
Figure1 EAPPoverexpressionincreasestheG1fractionofU2OScells.(a)U2OScellsweretransfectedwithanexpressionvectorfor
HA-EAPP or a control vector and treated with different drugs for 16h. Nocodazole and colchicine arrest cells during mitosis,
etoposideandmethyl-methanesulfonatecauseDNAdamage,hencestallcellsinS-phase.Harvestedcellswereanalyzedforcell-cycle
distribution.(b)ToinvestigateuntreatedcellseitheraGFP-EAPPoraGFPcontrolvectorweretransfectedintoU2OScells,gatedfor
theirGFPlevelsandseparatelyanalyzed.
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No-GFP GFP-gated
Cell density
EAPP→
p21→ GFP
p53→
β−Actin→
p21→
β−Actin→
GFP-EAPP
80
* GFP
70 GFP-EAPP
e
e Stag 5600 GGFFPP+-EsAhRPPN+As-ph2R1NA-p21 pSGupFePr -+p21
cl
Cy 40
ell 30
of C 20 *
%
10 GFP-EAPP +
0 pSuper-p21
G1 S G2
Figure2 p21hasamajorroleintheEAPP-triggeredG1cell-cyclearrest.Itsknockdownabrogatesthegrowth-inhibitingeffectof
EAPP. (a) Rising cell density results in higher EAPP and p21 levels as shown on a western blot (upper panel) and increases the
fraction of G1 cells as shown by FACS analysis (lower panel). (b) U2OS cells were transfected with expression vectors for GFP
orGFP-EAPPwithorwithoutanexpressionvectorforsmallhairpinRNAtargetingp21mRNAandanalyzedbywesternblottingand
immunostaining with an anti-p21 antibody. (c) The same cells were gated for GFP expression and separately analyzed by flow
cytometry. (d) The cell-cycle distribution of GFP-positive cells of three independent experiments were quantified with FlowJo
(Tree Star Inc, Ashland, OR, USA) and averaged. All data are presented as the mean±s.d. from at least three independent
experiments.*Po0.05.
of Pol II on the p21 gene, we performed ChIP with an H4, nor the specific K9 acetylation on H3, nor the
anti-PolIIantibody.Theseexperimentsdemonstratedthat phosphorylationofS10onH3showedanyalterationupon
the presence of Pol II on the exon 1 of the p21 gene is EAPPknockdown(SupplementaryFigure3).Thebinding
severely reduced upon knockdown of EAPP (Figure 3d). of p53 to its cognate binding sites further upstream on
To determine the sequence requirements for the observed the p21 promoter also remained unchanged (Figure 4c).
effects,wecarriedoutluciferaseassayswiththefull-length Interestingly, a fraction of p53 can also be found at the
p21 promoter and several truncations thereof (Figure 3e) TATA region. It might represent Sp1-associated p53
and compared their activity under normal and EAPP (Laggeretal.,2003)and/ormightbebroughttotheTATA
knockdown conditions. The proximal promoter compris- boxbyloopingoftheDNA(Lietal.,2007a).Thisfraction
ing four of the six Sp1 (nucleotides (cid:2)101 to þ16 relative of p53 was reduced upon EAPP depletion (Figure 4c).
to the transcription start site) is sufficient to confer Binding of TBP at the TATA box and bending of the
regulation by EAPP (Figure 3f). Mutation of either one DNA is considered the first step in the assembly of the
of the two distal of these sites (Sp1 binding sites 3 and 4) preinitiationcomplexonTATAboxcontainingpromoters.
abolished the influence of EAPP (Figure 3g). This is followed by the recruitment of TAFs (TBP
To figure out whether EAPP is associated with the associated factors) to constitute TFIID, the binding of the
p21promoter,we conductedChIP assays andamplified othercomponentsofthebasaltranscriptionmachineryand
precipitated DNA from five different regions of the p21 finally the association with RNA pol II (reviewed in
gene(Figure4a).Appreciableprecipitationwithananti- (Kornberg, 2007; Sikorski and Buratowski, 2009)). To
EAPP antibody could only be achieved with the region examine whether reducing EAPP interferes with the build
comprising the TATA box (Figure 4b). upofthetranscriptioninitiationcomplex,weagaincarried
out ChIP assays in EAPP knockdown and control cells.
Reduction of EAPP did not influence the binding of TBP
EAPPseemstoinfluencetheassemblyofthepreinitiation
butthepresenceofTAF1andTAF4,PolIIandcdk9was
complex
clearly reduced in these cells. In contrast, the binding of
Histone modification is one of the key mechanisms in transcriptionfactorswaseithernotaffected(E2F1)oreven
transcriptional regulation. The protruding N-terminal
increased(Sp1)bylowerEAPPlevels(Figures4c,dande).
domainsofhistonesaresubjecttomethylation,acetylation
andphosphorylation(Lietal.,2007b).WeperformedChIP
assays with modification-specific anti-histone antibodies EAPP is involved in the DNA damage-induced
to investigate whether EAPP alters the histone modifi- upregulation of p21
cation status of the promoter and thereby influences p21 DNA damage results in the induction of p53, which
transcription. Neither the general acetylation of H3 and in turn stimulates the expression of p21. We used
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y
vit 3 **
cti2.5
A
e 2
s
GFP-EApP2P1→→ p21 mRNA→ cifera1.15
endogβ. −EAAcPtiPn →→→ GAPDH mRNA→ Rel. Lu0.05 MockshRNA-EAP**P HA-EAPP
1.8
1.6 POL II
% of Input 00011.....146824 -2301 -1394 Sp-11 0B1inding Sites +1
0.02 p21-wt
shRNA-Scrambled shRNA-EAPP p53 p53 1 2 3 4 5 6 TATA
p21 Exon 1 p21-BstX
p21-Pst
p21-101
p21-101mt3
shRNA-Scrambled→ p21-101mt4
shRNA-EAPP→ p21-Pstmt3
p21 Exon 1
1.2 1.2
shRNA-Scrambled shRNA-Scrambled
vity 1 shRNA-EAPP vity 1 shRNA-EAPP
e Acti 0.8 ** s Acti 0.8 **
ucifreas 00..46 Lucifera 00..46
el. L 0.2 ** Rel. 0.2 **
R * ** 0
0 p21-101 p21-101mt3 p21-101mt4 p21-Pstmt3
p21-wt p21-BstX p21-Pst p21-101
p21- p21- p21-
p21-wt p21-BstX p21-Pst p21-101 p21-101 101mt3 101mt4 Pstmt3
EAPP→ EAPP →
GAPDH→ GAPDH →
R P R P R P R P R P R P R P R P
shRNA-SC shRNA-EAP shRNA-SC shRNA-EAP shRNA-SC shRNA-EAP shRNA-SC shRNA-EAP shRNA-SC shRNA-EAP shRNA-SC shRNA-EAP shRNA-SC shRNA-EAP shRNA-SC shRNA-EAP
Figure3 EAPPregulatesp21attheleveloftranscriptionviaSp1bindingsites.(a)Westernblotshowingap21decreaseafterknock
downofEAPPandanincreaseuponEAPPoverexpression.b-Actinservedasaloadingcontrol(b)RT–PCRofp21mRNAafter
EAPP knockdown and overexpression. (c) Reporter assay with the full-length p21 promoter. Knockdown of EAPP decreases the
Luciferase activity, whereas overexpression results in an increase. (d) ChIP showing exon 1 associated RNA pol II in EAPP
knockdownandcontrolcells.Thelowerpaneldepictstheactualdata,theupperpanelthequantification.pSuper-Scrambledisthe
RNAi control vector for the expression of an unspecific small hairpin RNA. (e) Schematic illustration of the used p21 luciferase
constructs.(f,g)Luciferaseassayswithp21promoterconstructsundernormalandEAPPknockdownconditions.Aluciferaseactivity
of 1 was assignedto the p21-wt promoter in f and to the p21-101promoter in g. The lower panels are western blotsshowingthe
respectiveEAPPlevelswithGAPDHasaloadingcontrol.Alldataarepresentedasthemean±s.d.fromatleastthreeindependent
experiments.*Po0.05;**Po0.01.
p53-positive U2OS cells to find out whether EAPP is EAPP knockdown cells indicates that besides p53,
not only required for basal expression, but also takes DNA damage-induced EAPP might also contribute to
part in the stress-induced activation of p21. DNA the upregulation of the p21 promoter. Protein levels of
damage was induced with etoposide and p21 mRNA p21 also increased and decreased with EAPP in normal
expression was examined with lowered, normal and and etoposide-treated cells. DNA damage-induced p53
elevated EAPP levels (Figure 5). Overexpression of was not affected by changes in EAPP levels. Inhibition
EAPP not only increased p21 mRNA in control cells, of histone deacetylase (HDAC) activity by TSA also
but also in etoposide-treated cells with already elevated resulted in elevated p21 and EAPP levels. The knock-
p21 mRNA (Figure 5a). In p53 negative T98G cells, downofEAPPloweredp21alsointhepresenceofTSA,
the overall level of p21 mRNA was much lower but confirming that this effect is not acetylation dependent
still stimulated by EAPP, indicating that this effect is (Supplementary Figure 4).
p53-independent(Figure5b).Tofollowtheinductionof
EAPP by etoposide more closely, time-course experi-
ments were performed. Figure 5c shows the level of p21 EAPP has antiapoptotic properties that depend on p21
mRNA and EAPP between 0 and 9h after etoposide In EAPP overexpressing cells, the fraction of apoptotic
addition. Their parallel increase in both, normal and cells following etoposide addition was clearly reduced
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Control p53 5‘ binding p53 3‘ binding Upstream TATA box
(- 5kb) site (- 2.3 kb) site (- 1.4 kb) (- 0.9 kb) (- 0.05 kb) Exon 1 Primer
p21 promoter
2.5 TATA 4 5' BS
% of Input 1.152 ** 53UEC''xpo BBosnSSnttrr eo1alm % of Input 123...152535 3TC'Ao B*Tn*StArol
0.5 0.5
0
0 shRNA- shRNA- shRNA- shRNA-
shRNA-Scrambled shRNA-EAPP Scrambled EAPP Scrambled EAPP
EAPP p53 Pol-II
shRNA-Scrambled
TATA shRNA-EAPP
TATA
5‘ p53 BS shRNA-Scrambled
3‘ p53 BS 3‘ p53 BS shRNA-EAPP
Upstream shRNA-Scrambled
5‘ p53 BS shRNA-EAPP
Exon 1
Control Control shRNA-Scrambled
shRNA-EAPP
shRNA-Scrambled shRNA-EAPP
4.5 4.5
% of Input 312...3541525 * ** TPTEBAOAPFPL1 PII % of Input 231...3554152 * *** CTSEAP2DFF1K149
0.5 * 0.5
0 0
shRNA- shRNA- shRNA- shRNA- shRNA- shRNA- shRNA- shRNA-
Scrambled EAPP Scrambled EAPP Scrambled EAPP Scrambled EAPP
TATA Control TATA Control
TATA shRNA-Scrambled TATA shRNA-Scrambled
Control Control
TATA TATA
shRNA-EAPP shRNA-EAPP
Control Control
Figure4 EAPPbindsaroundtheTATAboxofthep21promoterandinfluencesthebindingorassemblyofthebasaltranscription
machinery and Sp1. (a) Schematic drawing of the p21 promoter region depicting the regions that were amplified after ChIP to
characterizeEAPPbinding.(b)ChIPexperimentsinU2OScellsshowedthatEAPPcouldonlybefoundintheregionamplifiedbythe
TATAboxprimerpair.Thelowerpanelshowsarepresentativeexperiment,theupperpanelthequantification.(c)ChIPassayswith
p53-andPolII-specificantibodiesprobingthep53-bindingsitesandtheTATAboxregion.(d,e)ChIPassayswithTAF1-,TAF4-,
PolII-,CDK9-,E2F1-andSp1-specificantibodiesoftheTATAandcontrolregions.Alldataarepresentedasthemean±s.d.from
atleastthreeindependentexperiments.*Po0.05;**Po0.01.
(Figure 6a). Quantification showed a significant smaller facilitateapoptosis.Totestthishypothesis,EAPPlevels
fraction of apoptotic cells in EAPP overexpressing cells in U2OS and T98G cells were lowered by RNAi. To
even after prolonged exposure to etoposide (Figure 6b). make sure that the observed cell death results from
The same result was obtained by analyzing the apop- apoptosis, we stained the cells with YoPro-1 (Idziorek
totic fraction in the forward/side scatter plot in flow- et al., 1995), which allowed the selection of apoptotic
cytometric analysis. Apoptotic cells have lower forward and dead cells and with propidium iodide (PI) that
scatterandslightlyhigherside-scattervaluesandappear allowed the discrimination between early apoptotic and
as a distinguishable population (not shown). Apoptosis lateapoptoticplusdeadcells.Inaddition,weperformed
can also be induced by overexpression of E2F1 western analysis and looked for cleaved caspase 3, a
(Qin et al., 1994; Shan and Lee, 1994; Wu and Levine, hallmark of apoptosis. The number of cells with a sub-
1994) in p53-dependent and -independent manners G1 DNA content and the amount of cleaved caspase 3
(Phillips and Vousden, 2001), which again was pre- increased dramatically in both, p53-positive U2OS
ventedbythesimultaneousexpressionofEAPP(Figures (Figure 7a) and p53-negative T98G cells (Figure 7b)
6c and d). If apoptosis can be inhibited by EAPP with lowered EAPP, indicating that this effect is p53
overexpression, conversely its knockdown should independent.
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20
el 18 U2OS
RNA Lev 11110246 GAPpD2H1 mmRRNNAA →→ mRNA
el. p21 m 2468 enGdoFgP.- EEAAPPPP →→ otein
R 0 GAPDH → Pr
GFP GFP-EAPP GFP+ GFP-EAPP+
Etoposide Etoposide
7
vel 6 T98G
e
1 mRNA L 345 GAPpD2H1 mmRRNNAA→→ mRNA
Rel. p2 012 enGdoFgPG.- AEEPAADPPHPP→→→ Protein
GFP GFP-EAPP GFP+ GFP-EAPP+
Etoposide Etoposide
EAPP
oposide oposide oposide Etoposide oposide oposide n Amount 12..525 sshh-RRNNAA--ESAcPraPmbled
edLog ed+ 2h Et ed+ 3h Et ed+ 4h Et ed+ 5,5h ed+ 7h Et ed+ 9h Et Rel. Protei 0.105 Log + 2h + 3h + 4h + 5,5h+ 7h + 9h
GAEPADPHP→→ shRNA-Scrambl shRNA-EAPP shRNA-Scrambl shRNA-EAPP shRNA-Scrambl shRNA-EAPP shRNA-Scrambl shRNA-EAPP shRNA-Scrambl shRNA.EAPP shRNA-Scrambl Sh-RNA-EAPP shRNA-Scrambl shRNA-EAPP NA Amount 234...5354552 sshhRRNNAA--SEcAr+Pa pPmE2tb1ole pmdoRsiNdAe
R 1.5
p21 mRNA →→ m 1
GAPDH mRNA→ Rel. 0.05 Log + 2h + 3h + 4h+ 5,5h+ 7h + 9h
+ Etoposide
Figure5 TheincreaseofEAPPuponetoposidetreatmentmightbenecessaryforthefullinductionofp21expression.(a)U2OScells
were transfected with either a GFP- or a GFP-EAPP expression vector in duplicate and one pair was treated with etoposide and
analyzed.TherightpanelshowsthemRNAlevelsofp21andGAPDHandtheproteinlevelsofGFP-EAPP,endogenousEAPPand
GAPDHasdeterminedbyRT–PCRandwesternanalysis,respectively.Theleftpanelshowsthequantificationofp21mRNAlevels.
(b)ThesameexperimentasshowninbbutcarriedoutinT98Gcells.(c)U2OScellsweretransfectedwithanEAPPknockdownvector
orthecorrespondingcontrolvector,treatedwithetoposideandharvestedattheindicatedtimepoints.EAPPlevelsweredeterminedby
westernanalysisandp21mRNAlevelsbyRT–PCR.Forbettercomparison,relativeamountswerequantifiedwithImageQuantwith
GAPDHandGAPDHmRNAservingasareference.Forthep21mRNAPCRsweredoneintriplicateandaveraged.Therightpanels
graphically show the increase of EAPP and p21 mRNA with the duration of etoposide treatment. All data are presented as the
mean±s.d.fromatleastthreeindependentexperiments.
To further investigate the requirement for EAPP, we ToexaminewhethertheantiapoptoticactivityofEAPP
examined U2OS cells and U2OS cells with moderately is also mediated by p21, we determined the fraction of
lowered EAPP levels under normal and DNA damage apoptotic cells at 24, 48 and 72h after the induction of
conditions.Equalnumbersofthesecellswereseededeither DNA double-strand breaks in control cells, cells with
in normal growth medium or in growth medium with elevatedEAPP,cellswithloweredp21andcellswithboth,
low concentrations of etoposide and counted for 4 days. elevated EAPP and lowered p21. It turned out that the
Whereas the number of cells in plain growth medium antiapoptoticactivityofEAPPisgreatlydiminishedbythe
steadilyincreasedwithlittledifferencebetweennormaland knockdown of p21 (Figure 9). This indicates that this
EAPP knockdown cells, the number of etoposide-treated activity of EAPP is indeed mainly mediated by p21 but
cellsdecreasedafteraninitialrise.Thisdecreasewasmuch other factors might also be involved.
more pronounced in the EAPP knockdown cells again
emphasizingtheprotectiveroleofEAPP(Figure8a).This
goes along with a reduced induction of p21 (Figure 8b) Discussion
and a much higher degree of apoptosis in EAPP knock-
downcells.StainingwithYoPro-1andPIandsubsequent Following DNA damage, a cell of a multicellular
flow-cytometric analysisallowed thequantification ofthis organism can repair the damage, withdraw from the
difference (Figures 8c and d). cell cycle and enter senescence, or eliminate itself by
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GFP-EAPP negative GFP-EAPP positive Cell 80 GFP
+ EtopSousb1i-dG8e.15: % + EStoup8bo.-7sGi d%1e: poptotic and Dead 12345670000000 GFP-*E*APP ** ** ** FSC-A (x 1,000)22115050500000 102103104105
A 0
Sub-G1 G1 S G2 Sub-G1 G1 S G2 % Log + 24h +48h +72h DAPI-A
Etoposide Etoposide Etoposide
Sub-G1: Sub-G1: Sub-G1:
2.7 % 18.1 % 5.5 %
GFP-EAPP→
EAPP→
GFP GFP- GFP-E2F1+HA-DP1 GFP-E2F1→
E2F1+HA-DP1 + GFP-EAPP E2F1→
HA-DP1→
β-Actin→
Figure 6 EAPP has antiapoptotic properties. (a) U2OS cells were transfected with a GFP-EAPP expression vector, treated with
etoposidefor16handgatedforGFP-EAPPexpression.ThereisamassivedecreaseinS/G2andsub-G1attheexpenseofanincrease
inG1.(b)Long-termtreatmentwithetoposideofU2OScellsexpressingeitherGFPorGFP-EAPP.GFP-positivecellsweregatedand
apoptosiswasmeasuredthroughanincreaseinHoechst33258intensity,whichbrightlystainsthecondensedchromatinofapoptotic
cells(exampleisshown).Alldataarepresentedasthemean±s.d.fromatleastthreeindependentexperiments.**Po0.01.(c)U2OS
cellsweretransfectedeitherwithaGFPexpressionvectorasacontrol,withexpressionvectorsforGFP-E2F1andHA-DP1,orwith
expressionvectorsforGFP-E2F1,HA-DP1andGFP-EAPP.Fortheflow-cytometricanalysisGFP-positivecellsweregatedandthe
percentageofsub-G1cellswasdetermined.(d)Thelevelsofexpressedproteinswereexaminedinawesternblot.
shRNA- shRNA-
Scrambled shRNA-EAPP Scrambled shRNA-EAPP
Sub-G1: Sub-G1: Sub-G1: Sub-G1:
3.5 % 22.5 % 9.5 % 24.9 %
U2OS T98G
shRNA- shRNA-
Scrambled shRNA-EAPP Scrambled shRNA-EAPP
C
C S
S F
F
EAPP→ YoPro EAPP→ YoPro
Cleaved Cleaved
Casp. 3→ Casp. 3→
GAPDH→ PI GAPDH→ PI
YoPro YoPro
Early Apoptosis : 3.33 % 6.57 % Early Apoptosis : 1.58 % 4.43 %
Late Apoptosis+Dead Cells: 1.86 % 16.57 % Late Apoptosis+Dead Cells: 4.24 % 17.61 %
Figure7 KnockdownofEAPPleadstotheinductionofapoptosis.(a)U2OScellsweretransfectedwithanEAPPknockdownanda
corresponding controlvector and analyzed for apoptosis. We examinedthe sub-G1 fraction, cleaved caspase3 proteinlevels by a
westernblot,andYoPro/PIstainingwithaVybrantApoptosisAssayKit.TheYoPro-positivefractionwasgatedandseparatedinto
PI-negativeand-positivecellstodistinguishbetweenearlyapoptosisandlateapoptosisplusdeadcells,respectively,(FSC¼forward
scatter).(b)Thesamewasdonewithp53-negativeT98Gcellsagainresultinginanincreaseofapoptosis.
apoptosis to avoid the proliferation of aberrant descen- 2002). p21 seems to accomplish this either by inhibiting
dants, possibly resulting in cancer. Among the main CDK activity (Chen et al., 1996) or by modulating the
playersinthisprocessarethetumorsuppressorp53and activity of transcription factors or apoptosis regulators
its transcriptional target p21. Treatment of cancer cells (Janicke et al., 2007).
with certain drugs often induces p21 followed by cell- EAPP is an evolutionary conserved phosphoprotein
cycle arrest and/or cellular senescence and this can with a predominant nuclear localization that is freq-
protect cells from drug-induced apoptosis (Kawasaki uently upregulated in transformed cells. We have pre-
et al., 1996; Waldman et al., 1996; Gartel and Tyner, viously identified it as an E2F-binding protein that
Oncogene
EAPP:Atthecrossroadofapoptosisandarrest
PAndorferandHRotheneder
2686
2500000
shRNA-Scrambled 500000
2000000 shRNA-EAPP
mber 1500000 ber 400000
u m
N u 300000
Cell 1000000 ell N sEhtoRpNoAsi-dSecrambled +
500000 C 200000 shRNA-EAPP +
Etoposide
0 100000
Day 1 Day 2 Day 3 Day 4 Day 1 Day 2 Day 3 Day 4
shRNA-Scrambled shRNA-EAPP
Log +Etoposide Log +Etoposide
Sub-G1: Sub-G1:
EAPP→ 1.7 % 1.6 %
p21→ - Etoposide
Day 1
β−Actin →
EAPP→
p21→ + Etoposide Sub-G1: Sub-G1: Sub-G1: Sub-G1:
1.5 % 1.3 % 2.8 % 2.2 %
β−Actin →
Day 2
Day 2 Day 3 Day 4
50 shRNA-Scrambled Early Apoptosis ** Sub-G1: Sub-G1: Sub-G1: Sub-G1:
s 45 shRNA-Scrambled Late Apoptosis and Dead Cells 3.4 % 5.1 % 5.6 % 20.3 %
d Cell 3450 sshhRRNNAA--EEAAPPPP LEaatrely A Appoopptotossisis and Dead Cells Day 3
ea 30 *
D
d 25
an 20 Sub-G1: Sub-G1: Sub-G1: Sub-G1:
optosis 11055 ** * ** Day 4 2.5 % 39.3 % 5.2 % 59.2 %
Ap 0
% Day 2 Day 3 Day 4 Day 2 Day 3 Day 4
- Etoposide + Etoposide
Figure8 AmoderateEAPPknockdownhasslighteffectsonlogarithmicU2OScellsbutgreatlyreducestheirviabilityuponetoposide
treatment.(a,b)CellsweretransfectedtoaccomplishaknockdownofEAPPofabout50%andtreatedwithorwithout5mMetoposide
for 3 days. Cells were counted with a Casy Counter every day and corresponding protein levels were checked on a western blot.
Thegrowthcurveshowstheaverageofthreeindependentexperiments.(c)Thecell-cycledistributionofthesecellswasexaminedand
thefractionofsub-G1cellsdetermined.(d)YoPro/PIstainingandgatingwascarriedoutasshowninFigure7.Alldataarepresented
asthemean±s.d.fromatleastthreeindependentexperiments.*Po0.05;**Po0.01.
modulates the activity of E2F-dependent promoters could thus not explain our observations (not shown).
(Novy et al., 2005). TransfectionwitheitheraGFPoraGFP-EAPPexpres-
In our current study, we analyzed the role of EAPP sion vector and the separate assessment of transfection-
under DNA damage conditions and examined the positive and transfection-negative cells confirmed that
impact of its overexpression and knockdown. The EAPP overexpression results in G1 arrest. Earlier find-
treatment with drugs that interfered with cell-cycle ings that EAPP stimulates cell-cycle progression (Novy
progression by activating cellular checkpoints revealed etal.,2005)mightbe explainedby adifferingmethodo-
that the fraction of G1 cells was much higher in EAPP- logy.Atthattimewewerenotabletoseparatelyanalyze
overexpressing than normal U2OS cells (Figure 1). transfected and untransfected cells.
This indicates that more EAPP either slowed down the Thegrowth-inhibitoryactivityofEAPPturnedoutto
cell cycle or even arrested these cells in G1, or that bep21-dependentasitwasabrogatedbytheknockdown
EAPP somehow abolished the effects of the drugs. The ofp21.Inlinewiththisfinding,p21proteinandmRNA
onlyplausiblemechanismforblockingthedrugsseemed levels, as well as its promoter activity, went up with
to be the activation of multidrug resistance (MDR) increasing,anddownwithloweredEAPPlevelsshowing
genes, encoding large transmembrane transporters that EAPP influences the expression of p21. This, and
able to remove toxic substances from a cell (Gutmann theobservationthatU2OScellsapproachingconfluency
et al., 2010). To examine this possibility we compared increase both, their EAPP and p21, but not p53 levels
the levels of several MDR proteins in normal EAPP suggestedthatEAPPisamongthedrivingforcesbehind
overexpressing cells. We indeed detected a stimulating the increase of p21 in confluent cells (Figure 2).
effect on the expression of P-glycoprotein (Gros et al., Furthermore, a knockdown of EAPP inhibits this cell
1986; Goda et al., 2009), but the measurement of the density-dependent increase of p21 (Figure 8b).
efflux of a dye revealed that the drug-transport activity The p21 promoter shows a complex pattern of
of EAPP overexpressing cells was not increased and regulation and a variety of DNA-binding factors, as
Oncogene
EAPP:Atthecrossroadofapoptosisandarrest
PAndorferandHRotheneder
2687
12
70 GFP
s GFP GFP-EAPP
Cell 60 GGFFPP-+EsAhRPPNA-p21 * 10 GFP+shRNA-p21
nd Dead 4500 GFP-EAPP+shRNA-p21 ** 1 mRNA 68 GFP-EAPP+shRNA-p21
ptotic a 2300 ** Rel. p2 4
o
Ap 10 ** 2
% *
0 0
Log +24h Etoposide +48h Etoposide +72h Etoposide Log +24h Etoposide +48h Etoposide +72h Etoposide
EAPP p21 G1 arrest
EAPP overexpression
Stress Normal situation G1 arrest /
eg DNA Damage in the cell EAPP p21 Apoptosis
Knockdown
or no induction of EAPP p21 and
EAPP several Apoptosis
other genes
Figure9 TheantiapoptoticactivityofEAPPdependsonp21.(a)U2OScellsweretransfectedeitherwithaGFPvector,aGFP-EAPP
vector,aGFPplusap21knockdownvectororaGFP-EAPPplusap21knockdownvectorwitharatioof3:1(knockdownversusGFP
vector).Subsequently,thepercentageofapoptoticcellswasdeterminedinthecontrolgroup(Log)andafter24,48,and72htreatmentwith
etoposide.Alldataarepresentedasthemean±s.d.fromatleastthreeindependentexperiments.*Po0.05;**Po0.01.(b)Relativeamounts
ofp21mRNAinthesecellsasdeterminedbyRT–PCR.Onlyabout25%ofthecellswereGFPpositive,whichreducedtheoveralleffect.
(c)Amodelshowingtheoutcomeofthreedifferentscenarioswithelevated,normalandreducedlevelsofEAPP.
well as chromatin remodeling and histone-modifying conditions suggested that it contributes to the p21
complexes determine its activity (reviewed in (Gartel induction.Thisisalsosupportedbytheobservationthat
and Radhakrishnan, 2005; Abbas and Dutta, 2009)). the knockdown of EAPP severely hampers the p21
The observed modulation of p21 promoter activity by induction after DNA damage or HDAC inhibition
EAPP could occur either by direct binding to DNA, by (Figure 5; Supplementary Figure 4).
affecting other promoter-binding factors, or chromatin- The observed increase of EAPP in many transformed
modifying processes. ChIP assays demonstrated that cell lines (Novy et al., 2005) suggested that these cells
EAPPassociateswiththeregionaroundtheTATAbox benefitfromhigherEAPPlevelsdespitetheconcomitant
(Figure 4). The histone modification pattern in the stimulation of growth-retarding p21. Inhibition of
vicinity of the TATA box was not affected by differing apoptosisseemedtobealikelymechanism.Andindeed,
EAPP concentrations and thus does not seem to be overexpression of EAPP impeded DNA damage but
involved (Supplementary Figure 3). also E2F1-induced apoptosis (Figure 6), whereas its
Whereas elevating or lowering EAPP had no impact knockdown resulted in apoptosis even without damage.
on the binding of E2F1 and TBP, there was a clear As this apoptosis also occurred in p53-negative T98G
reductionofpromoter-boundTAF1,TAF4,PolIIand cells, it is at least not absolutely dependent on p53
cdk9 (Figure 5b,c and d). EAPP might thus be required (Figure 7). The antiapoptotic activity of EAPP is
fortheefficientassemblyofthepre-initiationcomplexat also mediated mainly by p21 as it almost completely
the p21 promoter. Reduced binding of cdk9, which is abolishedbyaconcurrentknockdownofp21(Figure9).
requiredforelongation(Pengetal.,1998),mightjustbe To find out whether a modest reduction of EAPP levels
a consequence of inefficient initiation or it might itself can also make a difference under stress conditions, we
require EAPP for promoter association. Binding of Sp1 treated cells with low amounts of etoposide, which
ontheotherhandwaselevateduponEAPPknockdown perhapscomesclosertonaturallyoccurringeventsacell
(Figure 4e). This is in line with a very recent report canencounter.Thenwecomparedcellswithnormaland
(Chenetal.,2010)showingthatEAPPcandirectlybind moderately loweredEAPPcontent.Thisrevealed that a
to DNA and compete with Sp1 for its binding site. modest reduction, which only slightly impairs cell
Moreover, two of the six Sp1 binding sites seem to proliferation under normal growth conditions, drasti-
mediate the activity of EAPP on the p21 promoter as cally lowered their viability following DNA damage
their mutation abolished any effect of an EAPP knock- (Figure 8).
down (Figures 3e and g). This could mean that the Taken together our findings suggest that EAPP is
putativeroleofEAPPintranscriptioninitiationismore indispensable for the survival of a cell. The required
importantforp21promoteractivitythanbindingofSp1 amountofEAPPseemstodependontheenvironmental
to these sites. conditions. Under optimal conditions for cell growth
p21 expression is strongly stimulated following DNA less is needed, whereas stress seems to favor cells
damage. This is primarily caused by damage-induced expressing more EAPP (Figure 9c). This might also be
p53 but can also be seen to a lesser degree in p53 an explanation for the high EAPP levels observed in
negative cells. The increase of EAPP under damage many transformed cells. The CDK inhibitor p21 seems
Oncogene
EAPP:Atthecrossroadofapoptosisandarrest
PAndorferandHRotheneder
2688
to have an important role as a mediator of the anti- antibodies, followed by enhanced chemiluminescence detec-
apoptotic activity of EAPP. In the absence of sufficient tion. RNA isolation was done with the TRIzol Reagentfrom
EAPP, the assembly of the transcription initiation Invitrogen(Carlsbad,CA,USA),followingtherecommended
complex at the p21 promoter seems to be perturbed. protocol. The cDNA was prepared with the Revert Aid First
Strand cDNA Synthesis Kit from Fermentas (Burlington,
DependingontheextentofthereductionofEAPP,p21
Canada) and PCR was performed with a Maxima Hot Start
is downregulated or just not upregulated, and under
GreenPCS Master Mixfrom Fermentas.
stress conditions cannot carry out its cell-protecting
functions.Mostlikelyotherproteinsbesidep21arealso
involved in apoptosis caused by the loss of EAPP, as in Luciferase reporter assay
Luciferase reporter assays were carried out as previously
unperturbedgrowingcellsap21knockouthasnosevere
described (Novy et al., 2005) with full-length or truncated
effect (Deng et al., 1995). Preliminary evidence suggests
p21 promoter-luciferase reporter constructs (Sowa et al.,
that the role of EAPP in transcription is not limited to
1997). b-Galactosidase activity was assayed in parallel as a
the p21 promoter. Active promoters are occupied by
controlfor the transfection efficiency(Novyet al.,2005).
multiple types of complexes (Sikorski and Buratowski,
2009) and EAPP seems to be an important compo-
Chromatin immunoprecipitation assays
nent of at least some of them. Lowering EAPP levels
ChIP’s were carried out as previously described (Hauser et al.,
influencedtheexpressionofsomeofthegenesexamined
2002). Following Antibodies were used: Pol II (N-20), TAF1
(unpublished results). Among them are genes enco-
(6B3), TAF4 (4A6), TBP (SI-1), CDK9 (H-169), SP1 (PEP-2)
ding important cell-cycle regulators like proliferating andE2F1(KH95)fromSantaCruzBiotechnology(SantaCruz,
cell nuclear antigen, cyclin B, cdk1 and p27 the level of CA,USA),H3(1791)fromAbcam(Cambridge,UK),H3ac(06-
which went down with EAPP. To examine which genes 599), H4ac (06-866), S10Ph (05-598) and K9ac (06-942) from
are influenced by EAPP and to scrutinize the bio- Upstate (Millipore, Billerica, MA, USA) and the EAPP anti-
chemical details of its activity will be focus of future body. Input was used in a 1/30 dilution and PCRs were
experiments. performed with a Maxima Hot Start Green PCS Master Mix
(Fermentas,St.Leon-Rot,Germany)andproductswereresolved
on a 2% agarose gel. ImageQuant 5.2 (Molecular Dynamics,
Krefeld, Germany) was used to quantify three independent
Materials andmethods experimentsincomparisonwiththeinput.
Plasmids, reporterconstructs andreagents.
Cell-cycle analysis
pCIneo-HA-EAPP, pEGFP-C, pEGFP-EAPP, pGAL-Luc,
The cell-cycle distribution was measured with DAPI (Merck,
pSuper-EAPP-#2 (Novy et al., 2005), p21-Luc and mutations
Darmstadt,Germany,Figure1a),PI(Sigma,Figures2a,7a,b
thereof (Sowa et al., 1997), pSuper-p21 (Karakas et al., 2007),
and8c),Hoechst33258(Invitrogen,Figures1b,2c,and6a),or
pSuper-SCR (Rayter et al., 2008) and pCIneo-HA-DP1(Helin
Draq5 (Biostatus Limited, Leicestershire, UK; Figure 6c).
etal.,1993)havebeendescribed.ForthepGFP-E2F1theE2F1
Cells were trypsinized, washed once with phosphate-buffered
complementary DNA (cDNA) was recloned from the pGex2T-
saline and around 5(cid:3)105 cells were taken for analysis. For
E2F1 (Rotheneder et al., 1999) into the pEGFP-C vector.
Hoechst 33258 and Draq5, which stain viable cells, appro-
ThepSuper-EAPP-#1and#3weregeneratedbycloningadouble
priateconcentrationsofthedyeswereusedandincubatedfor
stranded oligonucleotide with the sequences 50-AAGTTGCAA
CAGCTCCGAC-30 for#1and50-GATTCCAACAAATGACG 20minon41Cinthedark(Draq510mM,Hoechst332585mg/
ml). For DAPI and PI cells were fixed in 85% ethanol and
AA-30 for#3oftheEAPPcDNAasaninvertedrepeatintothe
incubated at least 30min on ice. PI was used in a final
pSupervector.Theimpactofindividualandcombinedtransfec-
concentrationof50mg/mland100mg/mlRNAseTyp1-Awas
tionofpSuper-EAPP-#1,#2and#3isshowninSupplementary
added and incubated for 30min at room temperature in the
Figure 1. Etoposide, nocodazole, methyl-methanesulfonate and
dark. DAPI concentration was 2ng/ml and fixed cells were
colchicinewerepurchasedfromSigma(Taufkirchen,Germany).
incubated for 20min in the dark at 41C. To analyze the cell-
cycle distribution different devices were used. DAPI staining
Cell culture andtransfection was measured with a Pas-III from Partec (Muenster, Ger-
U2OS and T98G cells were cultured in DMEM containing many), PI and Draq5 with a FACS-Calibur (BD Bioscience,
10%(vol/vol) fetalcalf serum. Cells were transfected for 16h San Jose, CA, USA), and Hoechst 33258 with a FACS-Aria
usingPerFectin(Genlantis,SanDiego,CA,USA).Following (BD Bioscience). Hoechst 33258 and Draq5 allowed an
a change of the medium, the cells were grown for addi- additional staining with and gating for GFP. With the
tional 24–48h. As control vector for mock transfec- FACS-Calibur and FACS-Aria cell debris and doublets were
tions we used either pEGFP-C (Clontech, Mountain View, excluded.
CA, USA) for the expression of GFP, pSuper-Scrambled for
the expression of an unspecific small interfering RNA, or an
Apoptosis andcell count
emptypCI-neovector(Clontech).
Cells were counted with a CASY Cell Counter TTC (Scha¨rfe
System, Reutlingen, Germany). To detect apoptosis different
Westernblot analysis,antibodies,RNA isolation assays were carried out. For all procedures cells were
andRT–PCR trypsinized, washed once with phosphate-buffered saline and
Whole-cell extracts were prepared by lysing the cells using a around 5(cid:3)105 cells were taken for measurement. The same
lysis buffer (20mM Tris pH8, 100mM NaCl, 1mM EDTA, procedure as described in the cell cycle analysis part was
0.5% NP-40). The separation was performed by 8–14% appliedforsub-G1analysis.Beforehandathresholdwassetto
sodiumdodecylsulfate–PAGE.Proteinswerethentransferred discriminateagainstthecelldebris.FortheVybrantApoptosis
onto nitrocellulose membranes and probed with indicated AssayKit(Invitrogen)cellswerestainedwithPIandYO-PRO
Oncogene
