Cellular Signalling 23 (2011) 171–179

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Cellular Signalling

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Cyclin D1 regulates p27Kip1 stability in B cells

Sophie Bustany, Guergana Tchakarska, Brigitte Sola ⁎Biologie Moléculaire et Cellulaire de la Signalisation, EA 3919, IFR ICORE, Université de Caen Basse-Normandie, 14032 Caen Cedex, France

Abbreviations: Ab, antibody; CDK, cyclin-dependenCKI, cyclin-dependent kinase inhibitor; CRM1, chromosoexponential growth; FACS, fluorescence-activated cell sinterleukin; IP, immunoprecipitation; KPC, Kip1 ubiquMCL, mantle cell lymphoma; ponA, ponasterone A; rILSCF, SKP1-Cullin 1 F-box protein; SD, standard deviatassociated kinase protein; Thr, threonine.⁎ Corresponding author. EA 3919, UFR de Médecine, C

Cedex, France. Tel./fax: +33 2 31068210.E-mail addresses: sophie.bustany@aliceadsl.fr (S. Bu

tchakarska_guergana@yahoo.fr (G. Tchakarska), brigitte

0898-6568/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.cellsig.2010.09.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 July 2010Accepted 5 September 2010Available online 15 September 2010

Keywords:Cell cycleProliferationDegradationCompartmentalisationMantle cell lymphoma

p27Kip1 is a cyclin-dependent kinase inhibitor that plays a critical role in regulating G1/S transition, and whoseactivity is, in part, regulated through interactions with D-type cyclins. We have generated the BD1-9 cell line, aBaF3 pro-B cells derivative in which cyclin D1 can be induced rapidly and reversibly by ponasterone A. Theinduction of cyclin D1 expression leads to a targeted p27Kip1 accumulation in both cytoplasmic and nuclearcompartments. But, only the p27Kip1 form phosphorylated on serine 10 (pSer10-p27Kip1) accumulates in BD1-9cells.We found that the binding of cyclinD1 and pSer10-p27Kip1 prevents p27Kip1 degradation by the cytoplasmicKip1 ubiquitylation-promoting complex (KPC) proteosomic pathway. Importantly, the nuclear CDK2 activitywhich is crucial for G1/S transition is not altered by p27Kip1 increase. Using siRNA techniques, we revealed thatp27Kip1 inhibition does not affect the distribution of BD1-9 cells in the different phases of the cell cycle. Our studydemonstrates that aberrant cyclin D1 expression acts as a p27Kip1 trap in B lymphocytes but does not inducep27Kip1 relocation from thenucleus to the cytoplasm anddoes notmodulate theG1/S transition. Since our cellularmodel mimics what observed in aggressive lymphomas, our data bring new insights into the understanding oftheir physiopathology.

t kinase; CHX, cycloheximide;me region maintenance 1; EG,orter; IB, immunoblotting; IL,itylation-promoting complex;3, recombinant interleukin 3;ion; Ser, serine; SKP, S phase

HU Côte de Nacre, 14032 Caen

stany),.sola@unicaen.fr (B. Sola).

l rights reserved.

© 2010 Elsevier Inc. All rights reserved.

1. Introduction

Progression through the cell cycle is regulated by sequential events inresponse to extra- and intracellular signals. Two families of protein playan important role in this process: the cyclin-dependent kinases (CDKs)and their regulatory subunits, the cyclins. Activities of cyclin/CDKcomplexes are tightly regulated in particular by specific CDK inhibitors(CKIs). Progression through the G1 phase and transition from G1 to Sphase are promoted by complexes of G1 cyclins (cyclins D1, D2, D3 andE) and CDK4/6 (for cyclins D) or CDK2 (for cyclin E), their kinasepartners. p27Kip1, a CDK inhibitor (CKI) which belongs to the Cip/Kipfamily, also known as a tumour suppressor, prevents specifically theactivity of cyclin D/CDK4/6 and cyclin E/CDK2 complexes and therebyregulates G1 to S phase transition [1–3]. During G0 and early G1 phases,

p27Kip1 is mainly localized in the cell nucleus and inhibits cyclin E/CDK2activity [4]. When cells are stimulated by appropriate signals, p27Kip1

becomes phosphorylated on serine 10 (Ser10) and translocates from thenucleus to the cytoplasm [5,6]. In the cytoplasm, p27Kip1 is thenphosphorylated on two threonine residues (Thr157, Thr198) and helpsthe formation of cyclin D1/CDK4 complexes [7]. Cyclin D1/CDK4 dimersbecome active only after p27Kip1 release and degradation [8–10].

The major pathway of p27Kip1 degradation takes place in the nucleusand is controlled by cyclin E/CDK2 complexes [11]. At the end of G1

phase, cyclin E/CDK2 complexes phosphorylate p27Kip1 on Thr187. Thismodification is necessary for the binding of p27Kip1 to SKP2 (S phasekinase associated protein 2), an F-box protein, component of the SCF(SKP1/cullin-1/SKP2/CKS1B)-type ubiquitin ligase complex. Ubiquityla-tion of p27Kip1 leads ultimately to its degradation by the proteasome[12]. In the cytoplasm, a second ubiquitin/proteasome degradationpathway has been identified. During the G1 phase of cell cycle, freep27Kip1 molecules phosphorylated on Ser10 are the substrate of Kip1ubiquitylation-promoting complex (KPC) which favours their ubiquity-lation and degradation [13]. As a whole, the progression of the cell cyclefrom G1 to S phase is controlled by the sub-cellular localization of p27Kip1

and its phosphorylation status.According to their roles in the early phases of cell cycle, p27Kip1 and

cyclin D1 proteins are often altered in human cancer cell lines andprimary tumours. Cyclin D1 is over-expressed through several mechan-isms in breast, bladder and oesophagus carcinomas, head and necksquamous cell carcinomas, non-small cell lung cancers and haematolo-gical malignancies [3]. The Cdkn1b gene encoding p27Kip1 is rarely

172 S. Bustany et al. / Cellular Signalling 23 (2011) 171–179

mutated or deleted in human cancers but p27Kip1 protein level is oftenreduced or the protein is mislocated. Usually, cyclin D1 and p27Kip1 levelsshow an inverse relationship which has an adverse prognosticsignificance for patients [14]. But in the case of aggressive variants ofmantle cell lymphomas (MCL), a haematological malignancy character-ized by the t(11;14)(q13;q32) and the constitutive activation of theCCND1 gene, a high level of cyclin D1 is associated with a high level ofp27Kip1 [15]. The binding of p27Kip1 by cyclin D1/CDK complexes hasbeen demonstrated in MCL leading to the hypothesis that p27Kip1 isrendered ineffective as an inhibitor of cell proliferation. However, thishypothesis has not been demonstrated yet.

To explore the role(s) of p27Kip1 inB cells,wehave generated amurinepro-B lymphoid cell expressing the human cyclin D1 protein in aninducible and transientmanner [16].Weprovide evidences that cyclin D1expression induces an increase of p27Kip1 level in the nucleus and thecytoplasm but not the relocation of the protein. The increase of p27Kip1

level is due to the formation of cyclin D1/CDK4/p27Kip1 complexes. Thesequestration of p27Kip1 has no impact on cyclin E/CDK2 activity or cellcycle distribution ruling out a direct role of this excess of p27Kip1 in theregulation of cell proliferation. We postulate from our model that theregulationofG1/S phase of the cell cycle inMCL cells escapes the control ofp27Kip1.

2. Materials and methods

2.1. Materials

RPMI 1640 medium and additives for cell culture were purchasedfrom Lonza (Walkersville, MD, USA), foetal calf serum (FCS) from PAALab. GmbH (Pashing, Austria), recombinant (r) interleukin (IL)3 fromR&D Systems (Minneapolis, MN, USA), ponasterone A (ponA) fromStratagene Europe (Edinburgh, Scotland, UK), and cycloheximide(CHX), propidium iodide (PI) and bovine serum albumin (BSA) fromSigma-Aldrich (Saint-Quentin Fallavier, France). All reagents for RT-PCRwere from Invitrogen Life Tech. (Cergy-Pontoise, France). Anti-p27Kip1

(sc-528), anti-pThr187–p27Kip1 (sc-16324), anti-pSer10–p27Kip1

(sc-12939-R), anti-β-actin (sc-47778), anti-SKP2 (sc-7164), anti-cyclinD1 (sc-718), anti-cyclin E (sc-481 and sc-25303), anti-CDK2 (sc-6248),anti-CDK4 (sc-260), anti-cyclin A (sc-751), anti-cyclin D2 (sc-593),anti-cyclin D3 (sc-182), anti-β-tubulin (sc-9104) and anti-pRb (sc-50)antibodies (Abs), p27Kip1 (sc-29430) and scrambled control siRNAs(sc-37007) were purchased from Santa Cruz Biotechnologies (SantaCruz, CA, USA); anti-p27Kip1 (G173-524) from BD Biosciences (Le Pontde Claix, France); anti-pThr198–p27Kip1 (AF3994) from R&D Systems,anti-pThr821–pRb (44-582) from Biosource (Invitrogen); anti-GAPDH(#4300) from Applied Biosystems/Ambion (Austin, TX, USA). Assecondary Abs, we used donkey peroxidase-linked anti-rabbit oranti-mouse immunoglobulins (Ig, Thermo Fisher Scientific, Rockford,IL, USA).

2.2. Cell cultures and cell cycle analysis

The murine IL3-dependent pro-B cell line BD1-9 was generated fromBaF3 cell line as reported elsewhere [16]. Cells were cultured in RPMI1640 medium supplemented with 10% FCS, 2 mM L-glutamine and 5%conditioned medium from WEHI-3B cells as source of IL3. BD1-9 cellstreated with ponA (a structural analogue of muristerone A) producecyclin D1 in a dose- and time-dependent manner. For IL3 deprivation,cells were washed twice and then cultured in RPMI 1640 plus 5% FCS; forIL3 stimulation, cells cultured in complete medium containing rIL3at 1 ng/ml final concentration. For half-life determination, BD1-9 cellswere deprived of IL3 for 18 h and then stimulated with rIL3 with orwithout ponA (10 μM). Six hours later, cells were treated with 50 ng/mlCHX for 0 to 120 min and then harvested. IL3 deprivation for 18 h leadsto apoptosis activation in BD1-9 [16]. However, to assess accurately theeffects of the different culture conditions on cellular viability after PI

staining, both apoptotic and non apoptotic cells were analyzed togetherby FACS as detailed elsewhere [16]. For siRNA transfection, BD1-9 cells(106 cells/ml) were washed twice and cultured for 14 h in RPMImediumplus 5% FCS. Cells were electroporated (250 V, 250 μF, Gene Pulser II,Bio-Rad) with 100 nM p27Kip1 or control siRNA, pooled, cultured incomplete medium and harvested thereafter.

2.3. Immunoblotting and immunoprecipitation

Whole cell lysates were obtained from cultured BD1-9 cells aspreviously described [16]. For the purification of cytoplasmic extracts,cells were lysed in buffer A containing 10 mM KCl, 10 mM HEPES,1.5 mM MgCl2, 50 μM dithiothreitol, 0.1% nonidet P40 (NP40), 1 mM Naorthovanadate, 10 mM NaF, 10 μg/ml leupeptin, 10 μg/ml aprotinin,100 μg/ml phenylmethylsulfonyl fluoride for 6 min at 4 °C. Lysates werethen centrifuged at 16,000×g for 5 min at 4 °C and supernatantscollected. To purify nuclear protein fractions, pellets were washed twicewith buffer A without NP40 and suspended in buffer B containing20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 1 mM EDTAand protease inhibitors for 30 min at 4 °C. Lysates were cleared bycentrifugation at 16,000×g for 10 min at 4 °C and supernatants collected.The purity of cytoplasmic and nuclear extracts was controlled byimmunoblotting (IB) with compartment-specific probes (data notshown). IB and immunoprecipitation (IP) were done as describedpreviously [17]. Densitometry analyses were performed with a FluorSI-mager and QuantityOne software (Bio-Rad, Hercules, CA USA).

2.4. RT-PCR analysis

Total RNA was purified from cultured BD1-9 cells treated or notwith ponA for different time points. RNA was reverse-transcribedwith M-MuLV RT and cDNA submitted to PCR amplification with thefollowing primers: for Cdkn1b, forward primer, 5′-AAC GTG AGA GTGTCT AAC GGG AGC-3′, reverse primer, 5′-TAA CCC AGC CTG ATT GTCTGA-3′; for β-actin, forward primer, 5′-AGG ATG CAG AAG GAG ATTACT-3′, reverse primer, 5′-GTA AAA CGC AGC TCA GTA ACA GTC C-3′.After 30 cycles of denaturation (94 °C, 45 s), annealing (65 °C, 45 s)and elongation (72 °C, 45 s) steps, PCR products were analyzed.

2.5. Statistical analysis

Statistical analyses were performed using Student's t test formatched pairs (*pb0.05; **pb0.01; ns, non significant).

3. Results

3.1. Cyclin D1 induces accumulation of p27Kip1 in both nuclear andcytoplasmic compartments

We have previously described the generation of BD1-9 cells whichsynthesize cyclin D1 upon hormonal treatment [16]. BD1-9 cells weredeprived of IL3 for 18 h to render them quiescent, then stimulated withrIL3 and treated simultaneously with ponA to induce expression of cyclinD1. Cytoplasmic and nuclear extracts were prepared from cultured cellsand analyzed by IB. As presented Fig. 1A, before ponA stimulation, nocyclin D1 was detected in the cytoplasm of BD1-9 cells. Upon ponAtreatment, cyclin D1 was expressed, reached a maximal level at 6 h thendeclined thereafter. In MCL cells in which p27Kip1 is expressed, its level isnot inversely correlated with the proliferation index as in most otherlymphomas [18]. This observation suggests potential interactionsbetween p27Kip1 and cyclin D1 in MCL cells. We analyzed p27Kip1

expression in BD1-9 cells. As expected, p27Kip1 was weakly expressed inthe cytoplasm of exponentially growing (EG) BD1-9 cells and accumu-lated after IL3 withdrawal. The basal level of p27Kip1 was reached 6 hafter rIL3 re-addition and remained low up to 24 h. When cyclin D1synthesis was induced by ponA, we observed a significant accumulation

A

cyclin D1GAPDH

+ rIL3+ ponA

+ rIL3+ ponA+ ponA

cytoplasm nucleus

p27Kip1

-actin

-actinp27Kip1

cyclin D1GAPDH

B

***

nucleuscytoplasm

+ ponA+ IL3

C

ns

ns

cyclin D2

cyclin D3

Nor

mal

ized

p27

Kip

1 ra

tio

Cdk2

-tubulin

EG -IL3 6 h ponA 6 h 24 h ponA 24 h

4

1

2

3

0

p27Kip1

actin

EG -IL3 6h 6 h24 h 24 hEG -IL3 6h 24 h6h 24 h

6 12 24 48 6 12 24 48 h

-IL3 6 h 6 h12 h 12 h24 h 24 h

+ ponAcyclin E

cyclin A

-actin Cdk4

-tubulin

+rIL3D

Fig. 1. Cyclin D1 expression induces p27Kip1 accumulation in both nuclear and cytoplasmic compartments. Exponentially growing (EG) BD1-9 cells were deprived of IL3 for 18 h(−IL3) and then simultaneously treated with rIL3 (+rIL3) and ponA (10 μM) or only rIL3 for 6 or 24 h. A. Cytoplasmic extracts or nuclear extracts were prepared and analyzed bySDS-PAGE. Blots were revealedwith anti-p27Kip1, -cyclin D1, -β-actin and -GAPDH Abs. Experiments have been repeated three times, a representative one is presented. B. The level ofeach protein revealed by IB was quantified by densitometry. For each culture condition, data are expressed as the ratio of p27Kip1/GAPDH (cytoplasm) or β-actin (nucleus) comparedto control assigned to 1 (EG cells). For each histogram, the mean of three independent experiments is presented as well as standard deviation (SD). *pb0.05; **pb0.01; ns, nonsignificant. C. Total RNA was purified from cells, reverse-transcribed and PCR amplified. PCR products were analyzed after electrophoresis. β-actin mRNA amplification served ascontrol. D. BD1-9 cells were treated as in A.Whole cell extracts were prepared and analyzed by IB with the indicated Abs. Anti-β-tubulin Ab served to control gel loading and transfer.

173S. Bustany et al. / Cellular Signalling 23 (2011) 171–179

of p27Kip1 at 6 h and a subsequent decrease to the basal level at 24 h(Fig. 1A, left panel). The kinetics of p27Kip1 and cyclin D1 accumulationand disappearance in the cytoplasm of BD1-9 cells stimulated by IL3 areperfectly superimposed. Within the nucleus, in ponA-treated cells, cyclinD1 reached a maximum level at 6 h and disappeared almost completely24 h later (Fig. 1A, right panel). In quiescent IL3-deprived BD1-9 cellscompared to exponentially growing cells, p27Kip1 level increased. rIL3addition to quiescent cells is accompanied by the down-regulation ofp27Kip1 whereas, in ponA-treated BD1-9 cells, p27Kip1 level wassustained (Fig. 1A). In the nuclear compartment, as observed forcytoplasmic compartment, p27Kip1 and cyclin D1 kinetics were similarwith the highest level at 6 h and then a return to basal level by 24 h. Thiswas confirmed by a statistical analysis (Fig. 1B). We concluded that theincrease of p27Kip1 level was induced by cyclin D1 expression in BD1-9cells. Importantly, the amount of cyclins D2, D3, E, A and CDK4, CDK2 inBD1-9 cells was not modified by cyclin D1 induction (Fig. 1C). p27Kip1

expression is tightly regulated by transcriptional and post-transcriptionalmechanisms [6,19]. Enhanced level of p27Kip1 protein could result froman enhanced transcriptional activity of Cdkn1b gene. We investigatedCdkn1b gene transcription by RT-PCR (Fig. 1D). Comparison with β-actinmRNA expression and densitometry analysis indicated that Cdkn1btranscription was unaltered whatever the culture conditions. Thesustained level of p27Kip1 after cyclin D1 expression was regulated at apost-transcriptional level.

3.2. Cyclin D1 expression leads to an increase of pSer10-p27Kip1 level inthe cytoplasmic and nuclear compartments with no modification ofpSer10-p27Kip1/total p27Kip1 ratio

p27Kip1 localization and degradation are regulated through thephosphorylation of various amino acids but the consequences of thesepost-transcriptional modifications are not completely understood. Inquiescent cells, phosphorylation of p27Kip1 on Ser10 stabilizes theprotein and participates into the inhibition of cyclin E/CDK2complexes [20,21]. In embryonic fibroblast cycling cells, Ser10phosphorylation facilitates p27Kip1 export from the nucleus to thecytoplasm during G1 phase and consequently alleviates the inhibitionof nuclear cyclin E/CDK2 dimers [5]. In BD1-9 cells, we observed anincrease of pSer10-p27Kip1 level in the cytoplasm after cells have beenstarved (Fig. 2A). After stimulation of BD1-9 cells with rIL3, pSer10-p27Kip1 level decreased rapidly from 6 h to 24 h. When cyclin D1 wasinduced, pSer10-p27Kip1 remained high up to 6 h and decreased to thebasal level at 24 h. However, the pSer10-p27Kip1/total p27Kip1 ratiowas not significantly modified whatever the culture conditions. Weconcluded that pSer10-p27Kip1 and total p27Kip1 levels kinetics weresimilar in the cytoplasmic compartment. In the nuclear compartment,the level of pSer10-p27Kip1 was low in BD1-9 cells stimulated with rIL3for 6 h and increased insignificantly by 24 h (Fig. 2B). After ponAtreatment, the level of pSer10-p27Kip1 was sustained after 6 h of rIL3

Ans4

ns

pSer10-p27Kip1p27Kip1 total

pThr198-p27Kip1

ns nsns

2

3

Nor

mal

ized

rat

iocytoplasm

0

1

B ns

EG -IL3 6 h ponA 6 h 24 h ponA 24 h

pSer10-p27Kip1p27Kip1 total

nsns2

nucleus

Nor

mal

ized

rat

io

0

1

EG -IL3 6 h ponA 6 h 24 h ponA 24 h

Fig. 2. Cyclin D1 induction leads to an increase of pSer10-p27Kip1 level in the cytoplasmic and nuclear compartments. Quiescent BD1-9 cells were stimulated with rIL3 and treated or not withponA as in the legend of Fig. 1. A. Cytoplasmic extracts were prepared and analyzed by IB. Blots were revealed with anti-p27Kip1, -pSer10-p27Kip1, -pThr198–p27Kip1 and -β-actin Abs and therespective level of each protein quantified by densitometry (n=3). Data are expressed as in the legend of Fig. 1. B. Nuclear proteins were extracted and blotted as before. Membranes wererevealed with anti-β-actin, -p27Kip1, -pSer10-p27Kip1 Abs and the protein levels quantified as in A. *pb0.05; **pb0.01; ns, non significant.

174 S. Bustany et al. / Cellular Signalling 23 (2011) 171–179

stimulationbefore the return to thebasal level by 24 h (Fig. 2B). As in thecytoplasm, the pSer10-p27Kip1/total p27Kip1 ratio was not modified bycyclin D1 induction. We demonstrated that cyclin D1 expressioninduced an increase of pSer10-p27Kip1 level which is correlated withtotal p27Kip1 level in both cytoplasmic and nuclear compartments.Moreover, our data demonstrated that phosphorylation on Ser10 didnot favour the export of p27Kip1 from the nucleus. An alternativemechanism could explain this delay of pSer10-p27Kip1 export.

Two other sites of phosphorylation of the cytoplasmic form ofp27Kip1 have been identified: Thr157 and Thr198 [7]. These twophosphorylated forms that result from AKT kinase activity, have a highaffinity for cyclin D1/CDK4 dimers and participate into the formation ofcyclin D1/CDK4/p27Kip1 complexes thereby reducing the inhibitoryfunctions of p27Kip1 on cyclin E/CDK2 [8,10]. Moreover, pThr157–p27Kip1 and pThr198–p27Kip1 can interact with 14-3-3 proteininhibiting their binding to α5-importin [7,22]. Then, p27Kip1 phos-phorylated on these two residues, is localized in the cytoplasm andcannot participate into the inhibition of cell cycle progression. OnlypThr198–p27Kip1 was investigated here because Thr157 is not encodedby the murine Cdkn1b gene. We first verified that this phosphorylatedform of p27Kip1 was not detected in the nucleus (data not shown). Inthe cytoplasm, no significant modifications at the protein level wereobserved in quiescent or exponentially growing BD1-9 cells or aftercyclin D1 expression (Fig. 2A). To confirm this result, the activation ofthe AKT signalling pathway was examined. The ponA treatment had noeffects on kinetics or level of Thr308 phosphorylation, the active formof AKT (data not shown). Our data indicated that cyclin D1 productiondid not induce an increase of pThr198–p27Kip1. Importantly, weconfirmed that cyclin D1 did not control the export of p27Kip1 from thenucleus to the cytoplasm.

3.3. p27Kip1 is sequestered by cyclin D1/CDK4 complexes

Sincewe observed a coordinated regulation of cyclin D1 and p27Kip1

proteins, we investigated if these two proteins could associate and if so,

what are the effects of p27Kip1 titration on the activity of cyclin E/CDK2complexes. In BD1-9 cells, only a faint amount of p27Kip1 was detectedafter CDK4 IP (Fig. 3A). When cyclin D1 was induced by ponA, thep27Kip1 amount bound toCDK4was increased. The increase of p27Kip1 inBD1-9 cells expressing cyclin D1 is likely due to the sequestration ofp27Kip1 in cyclinD1/CDK4complexes. By contrast, the amountof p27Kip1

bound to cyclin E and CDK2was notmodified after cyclin D1 expression(Fig. 3B). During the G1/S transition, cyclin E/CDK2 complexesphosphorylate pRb on Thr821 and accelerate the release of E2Ftranscription factor [23]. In BD1-9 cells cultured without IL3, the levelof pThr821–pRbwas low. This level increased after rIL3 stimulation and,as expected from the IP results, remained the same after cyclin D1induction (Fig. 3C). The densitometry analysis confirmed that cyclin D1induction did not impact the phosphorylation level of pRb (Fig. 3C).Cyclin D1 induction did not modify p27Kip1 distribution between thevarious cyclin/CDK complexes.

3.4. Cyclin D1 regulates the degradation of p27Kip1

Since cyclin D1 induction did not influence the localization ofp27Kip1 in BD1-9 cell lines, we looked for a role in p27Kip1 degradation.In the late G1 phase, active cyclin E/CDK2 complexes phosphorylatep27Kip1 on Thr187 [11,24]. This phosphorylation leads to theubiquitylation of p27Kip1 which is catalyzed by the F-box proteinSKP2 [12,25,26]. We analyzed the presence of Thr187–p27Kip1 afterstimulation of BD1-9 cells with rIL3 alone or plus ponA. No majormodifications were observed 6 h and 24 h post-treatment (Fig. 4A).This result confirmed that nuclear CDK2 activity was not altered bycyclin D1 induction. When quiescent BD1-9 cells were stimulatedwith rIL3, the level of SKP2 increased between 6 h and 24 h (Fig. 4B).The same result was observed after cyclin D1 induction by ponA andsuggests a progressive activation of the nuclear degradation pathwayafter IL3 deprivation. We concluded that p27Kip1 accumulation is notdue to a decreased activity of the nuclear SCF-ubiquitin/proteasomecomplex. We next analyzed the second p27Kip1 degradation

A control -

+ ++ponA

control -

+ ++ponA

B

- -+ ++

CDK 4

- -+ ++

cyclin E

IB CDK 2

p27Kip1

IB cyclin D1

p27Kip1

C

input IP CDK4 input IP cyclin E

EG -IL3 rIL3 rIL3

ns1.5

+ ponA

-actin

pThr821-pRb 0.5

1.0

pRb total

pThr

21-p

Rb/

pRb8

tota

lno

rmal

ized

rat

io

0EG -IL3 rIL3 rIL3 +

ponA

Fig. 3. p27Kip1 is sequestered by cyclin D1 and does not inhibit CDK2 kinase activity. Quiescent BD1-9 cells were stimulated with rIL3 and treated or not with ponA as in the legend ofFig. 1. A. Whole cell protein extracts were prepared and immunoprecipitated (IP) with anti-CDK4 Ab. Collected proteins were analyzed by SDS-PAGE. Blots were revealed withanti-CDK4, -cyclin D1 and -p27Kip1 Abs (IB). Negative control was done by using a rabbit non relevant serum. B. Proteins were immunoprecipitated with anti-cyclin E Ab. Blots wererevealed with anti-cyclin E, -CDK2 and -p27Kip1 Abs. C. Whole cell proteins were extracted and analyzed by SDS-PAGE (10%). Blots were revealed with anti-pThr821–pRb, -pRb and-β-actin Abs. One representative experiment out of three independent is presented. In the histograms are presented the data obtained from densitometric analysis of threeindependent experiments (mean±SD); ns, non significant.

175S. Bustany et al. / Cellular Signalling 23 (2011) 171–179

mechanism identified in the cytoplasmic compartment [13,27]. In theearly G1, pSer10-p27Kip1 switches from the nucleus to the cytoplasm.KPC complexes, exclusively present in the cytoplasm, were identifiedas partners of free pSer10-p27Kip1 [13]. Since cyclin D1 induced anincrease of pSer10-p27Kip1 amount in the cytoplasm, we postulatedthat the sequestration of p27Kip1 in cyclin D1/CDK4 complexes couldinhibit the cytoplasmic degradation pathway. We analyzed p27Kip1

A

+ ponA

+ rIL3

-actinEG -IL3 6 h 24 h 6 h 24 h

+ IL3

B

actin

6 h 24 h 6 h 24 h

+ ponA

+ rIL3

-

SKP2

pThr187-p27Kip1

Fig. 4. Cyclin D1 is not involved in the p27Kip nuclear degradation pathway. Quiescent BD1-9Fig. 1. A. Whole cell extracts were analyzed by immunoblotting with anti-pThr187–p27Kip1 awere revealed with anti-SKP2 and -β-actin Abs. For each panel, one representative expexperiments was done and the results presented in the histograms as before (mean±SD);

half-life in the presence (or not) of cyclin D1 (Fig. 5). p27Kip1 half-lifewas higher than 150 min in rIL3-stimulated BD1-9 cells (Fig. 5A).p27Kip1 half-life shortened dramatically to less than 35 min in cyclinD1-expressing cells (Fig. 5B). To explain this unexpected result, weenvisaged that KPC complexes could not bind p27Kip1 as long as it wassequestered in cyclin D1/CDK4 complexes. This implies that free p27Kip1

could be ubiquitylated and degraded only after cyclin D1 degradation.

nsns

1.5

2

β-ac

tin r

atio

0.5

1

Nor

mal

ized

pT

hr18

7-p2

7Kip

1 /

0

ns ns4

β-ac

tin r

atio

2

3

Nor

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ized

SK

P2/

EG -IL3 6 h 24 hponA 6 h ponA 24 h

6 h 24 hponA 6 h ponA 24 h0

1

cells were stimulated with rIL3 and treated or not with ponA (10 μM) as in the legend ofnd -β-actin Abs. The specific SKP2 protein is indicated by an arrowhead. B. Immunoblotseriment out of three is presented. The densitometric analysis of three independentns, non significant.

Fig. 5. Cyclin D1 controls p27Kip1 cytoplasmic degradation. BD1-9 were deprived of IL3 for 18 h (−IL3), simultaneously treated with rIL3 or rIL3 plus ponA for 6 h and then with CHXand harvested different time points later (0–120 min). Cytoplasmic extracts were prepared, separated with SDS-PAGE and immunoblotted with anti-p27Kip1 (A and B) or anti-cyclinD1 (C) and anti-β-actin (for control) Abs. One representative experiment out of three is presented (left part of the figure). The density of each band was measured and p27Kip1

half-life was deduced from the semi-log curve: time in min=f (p27Kip1/β-actin ratio). Data of three independent experiments are presented; the ratio at t0 was the reference (1), foreach time point the mean±SD is indicated.

176 S. Bustany et al. / Cellular Signalling 23 (2011) 171–179

With the same experimental protocol, the cyclin D1 half-life wasestimated to be around 30 min (Fig. 5C).

We concluded that cyclin D1/CDK4 complexes formed in thecytoplasmic compartment of stimulated BD1-9 cells sequesteredp27Kip1 proteins which were free and rapidly degraded by KPCcomplexes in the absence of cyclin D1. In the nuclear compartment,cyclin D1 binding of p27Kip1 induced a delay for pSer10-p27Kip1

relocation without modification of its degradation by SCF-complexes.

3.5. p27Kip1 accumulation is not responsible for the G0/G1 arrest inducedby cyclin D1

Since cyclin D1 and p27Kip1 are major regulators of the cell cycle [1,2],we asked if their increase could modify the distribution of BD1-9 cells inthe different phases of cell cycle (Fig. 6). IL3 deprivation induced anincrease of cells within the sub-G1 and G0/G1 phases and a decrease in Sand G2/M phases (Fig. 6A). We observed the same profile betweenBD1-9 cells expressing or not cyclin D1, 6 h after rIL3-stimulation.However, 24 h later, we observed an increase of the number of BD1-9cells in the sub-G1 and G0/G1 phases (69% vs. 49%) and a concomitantdecrease for the S phase (23% vs. 43%) when cyclin D1 was induced.Since cyclin D1 inhibits cell cycle progression, we investigated if p27Kip1

accumulation could explain this inhibition. We down-regulated p27Kip1

with a specific siRNA in BD1-9 cell lines for at least 24 h with a highefficacy (Fig. 6B). Analyzing the cell cycle distribution of BD1-9 cellstransfected with control or p27Kip1 siRNAs in the various cultureconditions, we observed no major effects of p27Kip1 inhibition (Fig. 6C).The percentage of cells in the sub-G1 phase was higher after treatmentwith siRNA p27Kip1. But, the percentage of cells in sub-G1 plus G0/G1

phases was similar whatever the siRNA transfected (84% vs. 80%). Inparticular, the absence of p27Kip1 did not allow cells to re-enter S phase(14% vs. 15% at 6 h; 16% vs. 13% at 24 h). The same results were obtainedexcluding apoptotic cells present in the sub-G1 fraction from the analysis(data not shown). We concluded that the excess of p27Kip1 is notresponsible for cyclin D1-induced G0/G1 arrest of BD1-9 cells.

4. Discussion

Mantle cell lymphoma is an incurable B-cell neoplasm generallycomposed of monomorphic small to medium-sized lymphoid cellsand characterized by the chromosomal translocation t(11;14)(q13;32) which leads to an abnormal and uncontrolled expressionof cyclin D1, physiologically absent from the B-cell lineage [28]. Tostudy the consequences of cyclin D1 expression in B cells and becausethe translocation is recognised as an initiating event within thetumorigenic process, we established a murine pro-B cell line in which

Fig. 6. The excess of p27Kip1 induced by cyclin D1 has no impact on G1/S phase transition. A. BD1-9 cells were cultured and treated as before, stained with PI and sorted by FACS. Atleast 10,000 events were gated for each culture condition. The percentage of cells in the various phases of cell cycle is indicated in the graph. One representative experiment out offive is shown. B. BD1-9 cells were transfected by electroporation with p27Kip1 or control siRNAs, starved for 15 h (−IL3) and restimulated with rIL3 and/or ponA. Whole cell extractswere analyzed by SDS-PAGE and blotted with anti-β-actin, -cyclin D1 and -p27Kip1 (to control transfection efficiency) Abs. C. BD1-9 cells were treated as before, stained with PI andanalyzed by FACS. At least, 10,000 events were gated. One representative experiment out of three is shown.

177S. Bustany et al. / Cellular Signalling 23 (2011) 171–179

cyclin D1 is expressed in an inducible manner [16]. The BD1-9 cell lineconstitutes an interesting paradigm for understanding the physiopa-thology of MCL.

As they play a central role in the cell cycle regulation, cyclin D1 andp27Kip1 have been studied in various cancerous pathologies [14,29]. Inmost cases, in good agreement with their respective functions, an inverse

178 S. Bustany et al. / Cellular Signalling 23 (2011) 171–179

correlation between the two proteins is observed [14,29,30]. Studies ofvarious lymphomas indicated that p27Kip1 has a prognostic potential; itsweak expression being associated with a high proliferation index, a badoutcome and a shorter global survival [31–33]. But, Quintanilla-Martinezand colleagues described that p27Kip1 and cyclin D1 are both increased inMCL cell lines and primary samples [15]. The same observation waspublished for lymphomas in which cyclin D3 is over-expressed [34]. It isalso well-established that the sub-cellular localization of p27Kip1 is anaccurate prognostic factor for tumours including lymphomas [35,36]. Instimulated BD1-9 cells, the accumulation of p27Kip1 is perfectly super-imposed with the one of cyclin D1. This is the result of the formation ofcomplexes between cyclin D1 and p27Kip1 allowing them to escapedegradation by the ubiquitin/proteasome pathways. We observed inBD1-9 cells what occurs in MCL cells. This validates the use of BD1-9 cellsas a MCL model. We report here for the first time that the induction ofcyclin D1 is accompanied by an increase of p27Kip1 protein both in thecytoplasmic and nuclear compartments. Our data rule out the hypothesisestablishing that cyclin D1 induces a p27Kip1 sequestration and relocationfrom the nucleus to the cytoplasm.

p27Kip1 degradation rate is regulated by the phosphorylation ofseveral Ser, Thr, and Tyr residues [19]. The influence of p27Kip1

phosphorylation on Ser10 in the stability of the protein is not completelyunderstood. In fibroblasts and lymphocytes of transgenic mice, phos-phorylation on Ser10 stabilizes p27Kip1 during G0-phase and early G1 andintensifies cyclin E/CDK2 inhibition [20,21]. After IL3 deprivation, thelevel of pSer10-p27Kip1 increased in BD1-9 cells both in the nucleus andin the cytoplasm confirming Besson' and Kotake' results. In proliferatingcells, pSer10-p27Kip1 could play another role in cell cycle regulation. Inembryonic fibroblasts, pSer10-p27Kip1 has a higher affinity for the CRM1exportin than unphosphorylated form suggesting that pSer10-p27Kip1 isexported from the nucleus [5]. In a contradictory study, p27Kip1 mutantsare exported from the nucleus indicating that phosphorylation on Ser10is not necessary for p27Kip1 to bind exportin [20]. For technical reasons,we were unable to study whether pSer10-p27Kip1 was sequestered bycyclin D1. However, we observed a correlation between p27Kip1 andpSer10-p27Kip1 levels both in nuclear and cytoplasmic compartmentsallowing us to speculate that cyclin D1 binds pSer10-p27Kip1. Conse-quently, in the absence of cyclin D1, p27Kip1 phosphorylation on Ser10displaces it from cyclin E/CDK2 complexes. Free pSer10-p27Kip1 can beexported from nucleus after CRM1 binding. When cyclin D1 is expressedand present in the nucleus, the affinity of pSer10-p27Kip1 for cyclin D1/CDK4 is higher than CRM1 exportin. This explains why the nuclearpSer10-p27Kip1 level is maintained in the presence of cyclin D1. Thus wedemonstrated that p27Kip1 location is regulated by its phosphorylationand by its ability to interact with various partners.

Two p27Kip1degradation pathways have been identified. Severalstudies have demonstrated that the level of SKP2which belongs to theSCFSKP2 nuclear complex, whose activity is maximal during late G1

phase, is inversely correlatedwith the p27Kip1 level [31,37,38]. SCFSKP2

complex deregulation is also implicated in various cancers [33,37,38].However, data obtained using null SKP2−/− transgenic mice revealedthat SKP2 expression is low before S phase and does not participateinto p27Kip1 degradation during G0/G1 transition [20]. In BD1-9 cellline, cyclin D1 expression causes no major modifications of thenuclear degradationway. This observation confirms that SCFSKP2 is notthe main degradation pathway of p27Kip1 in G1 phase as reportedpreviously [14]. The second pathway for p27Kip1 degradation takesplace in the cytoplasm during early G1 phase [26]. The KPC complexinteracts with p27Kip1, ubiquitylates it and directs it to the protea-some. KPC can bind free p27Kip1 phosphorylated or not on Ser10 [13].In the cytoplasm of BD1-9 cells, p27Kip1 and pSer10-p27Kip1 aresequestered by cyclin D1/CDK4 complexes. Thus, when cyclin D1 ispresent, pSer10-p27Kip1 becomes inaccessible to KPC complexes andits level remains high. This conclusion is reinforced by the determi-nation of p27Kip1 and cycline D1 half-lives which were found identical(30 and 35 min respectively). Moreover, we found that p27Kip1 is

rapidly degraded after its release from cyclin D1/CDK4 complexes. Inconclusion, in our model, the excess of p27Kip1 induced by an aberrantcyclin D1 expression is equivalent to a pool of free p27Kip1. In theabsence of cyclin D1, these free proteins are rapidly exported from thenucleus and degraded by cytoplasmic KPC complexes. Moreover,p27Kip1 increase induced by cyclin D1 does not influence theredistribution of cell cycle regulatory proteins between the differentcellular compartments or the composition of cyclin/CDK complexes.

In solid cancers, a low p27Kip1 level is often associated with a badoutcome and a shorter global survival [14]. Studies of lymphomas aremore controversial [32,33,36]. Quintanilla-Martinez and colleaguespublished a cohort of 116 patients suffering of lymphomas and amongthem 40 cases of MCL and 10 cases of blastic variant of MCL [18]. Bycontrast with other lymphomas, p27Kip1 was not expressed in 90% ofMCL and there is no correlation between p27Kip1 level andproliferation index suggesting that p27Kip1 is not crucial for MCLpathogenesis. In the blastic variant, the proliferation index wasparticularly high and associated with a high p27Kip1 expression. Inthese cases, p27Kip1 does not function as a negative regulator of cellproliferation. In another publication, the same authors related that, inMCL caseswhich express p27Kip1, cyclin D1 sequesters p27Kip1 causingthe relocation of the protein from the nucleus to the cytoplasm andthat a low proportion of p27Kip1 is bound to cyclins A and E [15]. Thiswould facilitate MCL cell proliferation. In BD1-9 cells, p27Kip1

sequestration by cyclin D1 does not modify the intracellulardistribution of the protein and its binding to other cyclins.Furthermore, we observed the same distribution of BD1-9 cells inthe different phases of the cell cycle after p27Kip1 down-regulation bysiRNA. This feature questions the importance of this excess of p27Kip1

in the cell cycle regulation of MCL.Recently p27Kip1 was identified as a regulator of cellular motility and

adhesion [39]. Nagahara and colleagues were the first to show that thetransduction of a fusion TAT–p27Kip1 protein in hepatocellular carcinomacells leads to high cytoplasmic p27Kip1 level and promotes cell migration[40]. The same conclusion was reached with the use of p27Kip1 nullmouse embryonic fibroblasts [41]. Mechanisms regulating the role ofp27Kip1 in cell motility and metastasis potential are not fully understood.The cytoplasmic forms of p27Kip1 interact both in vitro and in cells withRhoA, prevent RhoA interaction with guanine nucleotide exchangefactors and decrease actin stress fibber polymerization and stability [42].These observations represent an important link between sub-cellularp27Kip1 location and metastatic potential of tumours. In MCL, such a rolefor p27Kip1 has not been reported yet. Our results indicate that aberrantexpression of cyclin D1 leads to p27Kip1 accumulation in the cytoplasm ofBD1-9 cells. We can speculate that this p27Kip1 pool sequestered bycyclin D1 could influence RhoA-pathway and increase B cells motility.This hypothesis could explain why blastoid variant of MCL, in whichp27Kip1 level is particularly important, presents an aggressive phenotypewith an adverse prognostic feature.

Acknowledgements

This work was supported by the Ligue contre le Cancer–Comité duCalvados (grant to BS). GT had scholarships from Ligue contre leCancer–Comité du Calvados and Société Française d'Hématologie.

Authorship: BS designed the study, SB and GT performed theexperiments, SB, GT and BS analyzed and interpreted the data, SBwrote the first draft, BS revised the manuscript.

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