ORIGINAL ARTICLE

High frequency of development of B cell lymphoproliferationand diffuse large B cell lymphoma in Dbl knock-in mice

Marzia Ognibene & Ottavia Barbieri & Cristina Vanni & Luca Mastracci &Simonetta Astigiano & Laura Emionite & Barbara Salani & Manuela Fedele &

Roberta Resaz & Claudya Tenca & Franco Fais & Federica Sabatini &Amleto De Santanna & Fiorella Altruda & Luigi Varesio & Emilio Hirsch &

Alessandra Eva

Received: 22 June 2010 /Revised: 7 December 2010 /Accepted: 15 December 2010 /Published online: 11 January 2011# Springer-Verlag 2011

Abstract Dbl is the prototype of a large family of GDP–GTP exchange factors for small GTPases of the Rho family.In vitro, Dbl is known to activate Rho, Rac, and Cdc42 andto induce a transformed phenotype in murine fibroblasts.We previously reported that Dbl-null mice are viable andfertile but display defective dendrite elongation of distinctsubpopulations of cortical neurons, suggesting a role of Dblin controlling dendritic growth. To gain deeper insights into

the role of Dbl in development and disease, we attempted aknock-in approach to create an endogenous allele thatencodes a missense-mutation-mediated loss of function inthe DH domain. We generated, by gene targeting technol-ogy, a mutant mouse strain by inserting a mutagenizedhuman proto-Dbl cDNA clone expressing only the Dbl Nterminus regulatory sequence at the starting codon ofmurine exon 1. Animals were monitored over a 21-month

Marzia Ognibene, Ottavia Barbieri, and Cristina Vanni contributedequally to this work.

Electronic supplementary material The online version of this article(doi:10.1007/s00109-010-0712-4) contains supplementary material,which is available to authorized users.

M. Ognibene : C. Vanni : B. Salani :M. Fedele : R. Resaz :L. Varesio :A. Eva (*)Laboratory of Molecular Biology, G. Gaslini Institute,Largo Gaslini 5,16147 Genoa, Italye-mail: alessandraeva@ospedale-gaslini.ge.it

O. Barbieri :C. Tenca : F. Fais :A. De SantannaDepartment of Experimental Medicine (DIMES),University of Genova,Genoa, Italy

O. Barbieri : S. Astigiano : L. EmioniteDepartment of Translational Oncology,National Institute for Research on Cancer,Genoa, Italy

L. MastracciAnatomic Pathology Section, Department of Surgical,Morphological Disciplines and Integrated Methodologies(DICMI), University of Genova,Genoa, Italy

F. SabatiniPulmonary Disease Unit, G. Gaslini Institute,Largo Gaslini, 5,16147 Genoa, Italy

F. Altruda : E. HirschDepartment of Genetics, Biology and Biochemistry,Molecular Biotechnology Center, University of Torino,Via Nizza 52,10126 Turin, Italy

Present Address:B. SalaniDepartment of Endocrinology and Medicine,University of Genova,Viale Benedetto XV 6,16132 Genoa, Italy

Present Address:M. FedeleDepartment of Clinical Pathology, ASL3 Genovese, S. CarloHospital of Genova-Voltri—U.O. Ponente 1,Piazzale Gianasso 2,16158 Genoa, Italy

J Mol Med (2011) 89:493–504DOI 10.1007/s00109-010-0712-4

period, and necropsy specimens were collected for histo-logical examination and immunohistochemistry analysis.Dbl knock-in mice are viable and did not manifest eitherdecreased reproductive performances or gross developmen-tal phenotype but revealed a reduced lifespan compared towild-type (w.t.) mice and showed, with aging, a B celllymphoproliferation that often has features of a frankdiffuse large B cell lymphoma. Moreover, Dbl knock-inmale mice displayed an increased incidence of lungadenoma compared to w.t. mice. These data indicate thatDbl is a tumor susceptibility gene in mice and that lossof function of Dbl DH domain by genetic missensemutations is responsible for induction of diffuse large B celllymphoma.

Keywords Dbl . Knock-in mice . Diffuse large B celllymphoma . Lung tumors . Small lymphocytic lymphoma

Introduction

The Rho-like proteins are small GTPases that function asswitches in a wide variety of signaling pathways regulatingimportant cellular processes ranging from cytoskeletalremodeling, gene expression, cell proliferation, and mem-brane trafficking. The activity of Rho-like GTPases isstrictly regulated, locally and temporally, by three classes ofproteins: the guanine nucleotide exchange factors (GEFs),the GTPase-activating proteins, and the guanine nucleotidedissociation inhibitors. The classical GEFs for RhoGTPases share a motif, designated the Dbl homology(DH) domain, that mediates nucleotide exchange [1] and aPleckstrin homology (PH) domain, responsible for propercellular localization of the protein [2].

In mammals, over 60 DH domain-containing proteinshave been identified, indicating the need for selectiveactivation of Rho proteins by different signaling pathwaysand under diverse conditions. Several lines of evidencesuggest that the biological functions of DH-containingGEFs are intimately dependent upon their ability to interactwith and activate Rho GTPases. Disturbance of RhoGTPase signaling in diseases is caused by alterations atthe level of their regulators, rather than by mutations at thelevel of the small Rho GTPases. In fact, deregulatedexpression of Rho GEFs has been implicated in oncogen-esis, metastasis, and developmental alterations [3].

The founding member of this family of Rho GEFs is theDbl protein, a cytoplasmic phosphoprotein of 115 kDacontaining a long stretch of amino acids within its NH2-terminal half with the potential to form an α-helical coiledcoil [4]. This region negatively regulates the transformingactivity of Dbl through direct interaction with the PHdomain. This interaction limits the access of Rho GTPases

to the DH domain and masks the intracellular targetingfunction of the PH domain [5]. Consistent with thismechanism of inhibition, oncogenic activation of Dbloccurs by truncation of the amino-terminal 497 residues[4, 6].

Dbl was first identified as a transforming agent bytransfection of mouse fibroblasts with DNA derived from ahuman diffuse B cell lymphoma [4, 7]. In naturallyoccurring tumors, Dbl is expressed in a restricted patternof histotypes, including tumors of neuroectodermal andneuroendocrine origin [8, 9]. The role of Dbl in diseaseshas been evaluated utilizing animal models. Dbl transgenicmice failed to develop tumors, unless mice with a p53 (−/−)genetic background were used [10, 11], and Dbl-null miceshow a significant reduction of the length of dendrites indistinct populations of cortical pyramidal neurons, suggest-ing a role for Dbl in dendrite elongation [12].

In order to gain deeper insight into the role of Dbl indisease and development, we generated, by gene targetingtechnology, a mutant mouse strain (proto-Dbl knock-in,pDbl-ki) expressing only the Dbl N terminus regulatorysequence. Our idea was to mimic the mutations of FGD1gene, a GEF for Rho GTPases, whose truncation by pointmutations is responsible for the induction of the Aarskog–Scott syndrome [13]. Because of the alterations made, weexpected developmental alterations, similarly to whathappens with mutant FGD1. Dbl knock-in mice wereviable and did not manifest either decreased reproductiveperformances or gross developmental phenotype but,instead, revealed a reduced lifespan compared to wild-type (w.t.) mice and progressed, with aging, to thedevelopment of diffuse large B cell lymphoma (DLBCL),a type of tumor never found in the w.t. control mice.Moreover, pDbl-ki male mice displayed an increasedincidence of lung adenoma and of small lymphocyticlymphoma compared to w.t. mice. These data indicate thatDbl is a tumor susceptibility gene in mice and thatalterations of Dbl induce tumorigenesis primarily in thelymphocyte lineage.

Materials and methods

Proto-Dbl knock-in mice and animal care

A complete and detailed description of the generation ofpDbl-ki mice is provided in Electronic supplementary File1 and Fig. 1. All animals were maintained in a conventionalanimal facility in 12-h light/dark cycles, fed at libitum, andmonitored for their lifespan. Anymice showing standard signsof suffering (e.g., percentage of weight loss, abdominaldistension, anorexia, decreased activity, ruffled fur, urinaryretention) were sacrificed. Otherwise, all mice were sacrificed

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when they reached the age of 21 months. All animal studieswere approved by the Internal Ethical Animal Experimenta-tion Committee and carried out at the Animal Facility of theNational Institute for Cancer Research (Genoa, Italy).

Cell culture and transfection

The wild-type pDbl cDNA and the mutagenized humanproto-Dbl (pDbl-ki) cDNA were inserted into the BamHIsite of the mammalian pZipneo vector (pZipneo-pDbl andpZipneo-pDbl-ki, respectively) and utilized to transfectmouse fibroblasts. NIH3T3 fibroblasts were cultured inDulbecco’s modified Eagle medium (DMEM) supple-mented with 10% calf serum.

Mass cultures of stable transfected cell lines were generatedby transfecting NIH3T3 cells with 300 ng of each plasmidDNA by the calcium phosphate coprecipitation method, asdescribed by Graham and van der Eb [14] and culturing themin DMEM supplemented with 5% calf serum. Transfectionassays were done on duplicate cultures. One set of eachtransfectant was used to score foci at 10–21 days post-transfection. The second set was grown in the presence of375 μg/ml G418 for colony formation. Colonies of G418-resistant cells were scored 14 days after transfection.

RNA analysis

Total RNA was extracted using RNeasy Minikit (Quiagen)or Nucleo Spin-RNA/protein (Macherey-Nagel). RNA was

retrotranscribed with Advantage RT for PCR kit (Clontech).Amplification by PCR was with the following primers: 5′-TGGGAACTTGCACTTGGTTT-3′ and 5′-CAGGCACTTC-CAGATTTGTT-3′ for Dbl-ki 5′ (amplifying nucleotides 357–793 of the human proto-Dbl cDNA) [4], 5′-TGATCTTATGC-CACCTCTCC-3′ and 5′-TTTGTAGCACCTTTCCGGTG-3′for Dbl 3′ (amplifying nucleotides 1709 to 2251 of the murineDbl cDNA) [12], and 5′-TCCTGCGTCTGGACCTGG-3′ and5′-CCATCTCTTGCTCGAAGT-3′ for actin.

Western blot analysis

Tissue lysates were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and trans-ferred to polyvinylidene fluoride (PVDF) membrane. Anantibody raised against a peptide mapping within Dbl aminoacids 3 to 16 [15] and an antibody raised against a peptidemapping within Dbl amino acids 818–831 [15] were used todetect the mutant and the normal Dbl proteins, respectively.Immunocomplexes were visualized by West Dura extendedchemioluminescent detection (Thermo Scientific) using agoat anti-rabbit horseradish peroxidase (HRP)-conjugatedsecondary antibody (Pierce). Blots were reprobed with ananti-ezrin antibody (Santa Cruz Biotechnology) to evaluateamount of proteins loaded.

NIH3T3 cells were stably transfected with pZipneo,pZipneo-pDbl, or pZipneo-pDbl-ki. Alternatively, spleenscollected from w.t. and diseased mice were minced toobtain a single cell suspension. The lymphocyte population

Fig. 1 Characterization of pDbl-ki construct. NIH3T3 cells, stablytransfected with pZipneo, pZipneo-pDbl, or pZipneo-pDbl-ki, werelysed. Cell lysates were subjected to GST-PAK pull-down assay andanti-Cdc42 a or anti-Rac b Western blot analysis. c Whole cell lysateswere subjected to SDS-PAGE, transferred to PVDF membrane, andprobed with phosphospecific antibody to detect P-JNK and anti-JNKto detect kinase expression levels. d pDbl expression level wasdetermined by Western blot analysis with specific antibodies using

total cell lysates. e Comparison of the focus-forming activity of pDbl-ki. Transfection assays were performed on duplicate cultures bytitration of each DNA (0.3, 0.1, 0.03 mg) on recipient NIH3T3 cells.One set of each transfectant was used to score foci at 10–21 days post-transfection. The second set of transfectants was grown in thepresence of 375 μg/ml G418. Colonies were scored at 14 days aftertransfection. Results shown are the average of three independentexperiments. FFU focus-forming units

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was isolated by Ficoll-Hystopaque (Sigma) densitycentrifugation and selected on mouse CD19 MicroBeads(Miltenyi Biotec). NIH3T3 and B cells were lysed in abuffer containing 20 mM Tris–HCl, 150 mM NaCl,1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mMβ-glycerolphosphate, 1 mM Na3VO4, 2.5 mM sodiumpyrophosphate, 0.5 mM NaF, 1 mM AEBSF, and 10 mg/mleach of aprotinin and leupeptin. Alternatively, purified B cellswere lysed with the Qproteome Cell Compartment Kit(Qiagen) to obtain the nuclear and the cytosolic fractions.Lysates were subjected to SDS-PAGE, transferred to PVDFmembrane (Millipore), and probed with phosphospecificantibodies to detect P-JNK and P-p38 (Cell SignalingTechnology, Beverly, MA, USA), anti-JNK and anti-p38(Santa Cruz Biotechnology, Santa Cruz, CA, USA) to detectkinase expression levels, or anti c-Rel (Santa Cruz Biotech-nology, Santa Cruz, CA, USA) to detect c-Rel expression. Theblot was reprobed with anti-β-actin (Santa Cruz) and anti-lamin (BD Biosciences) antibodies to evaluate proteinloading.

In vivo Rho GTPase activation assay

The GST-PAK-CRIB domain fusion protein, containing theCdc42 and Rac binding region of human PAK1, wasexpressed and purified as described [16]. Aliquots of celllysates were subjected to SDS-PAGE and immunoblottingwith specific polyclonal antibody against the wild type andthe mutant Dbl [4]. To evaluate Cdc42 and Rac activation,the stably transfected cell lines were lysed on the dish in abuffer containing 50 mM Tris–HCl (pH 7.4), 100 mM NaCl,2 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 10 μg/mleach of aprotinin and leupeptin, 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 40 μg of GST-PAK. Lysateswere incubated with 60 μl of glutathione coupled Sepharosebeads (Amersham Biosciences) for 1 h at 4°C under constantrotation. Beads were washed three times with lysis buffer,eluted in Laemmli sample buffer, and subjected to SDS-PAGE. Bound Cdc42 and Rac were detected in Western blotby using monoclonal antibody against Cdc42 (BDBiosciences, Franklin Lakes, NJ, USA) and Rac (UpstateBiotechnology–Millipore, Billerica, MA, USA).

Histology and immunohistochemistry

Necropsy specimens were collected, fixed for 24–48 h in10% neutral buffered formalin, embedded in paraffin, andstained with hematoxylin–eosin. For immunohistochemistry,heat-induced antigen retrieval was performed in citrate bufferpH 6.0 in a microwave oven, and endogenous peroxidaseswere blocked with 3% H2O2 in water. Non-specific bindingwas inhibited by incubating the samples in 10–20% normalgoat serum (Sigma Italia, Milan, Italy). Sections were

incubated overnight at 4°C with the following primaryantibodies: rabbit monoclonal anti-CD3 (1:150, NeoMarker,Fremont, CA, USA), rabbit monoclonal anti-Ki67 (1:250,NeoMarker, Fremont, CA, USA), rat monoclonal anti-B220(1:200, R&D Systems, Inc., Minneapolis, MN, USA), ratmonoclonal anti-C1qRp (1:30, eBioscience), rabbit poly-clonal anti-Fas (1:400, Santa Cruz Biotechnology), mousemonoclonal anti c-Rel (1: 50, Santa Cruz Biotechnology,Santa Cruz, CA, USA), and biotin-conjugated PNA (lectinfrom Arachis hypogaea; 1:200, Sigma). Slides were washedwith Tris-buffered saline (TBS; 0.05 M Tris pH 7.6; 0.15 MNaCl; 0.05% Tween 20) and incubated with biotin-conjugated anti-rabbit, anti-rat, or anti-mouse (Dako Cyto-mation, Glostrup, Denmark). After washing in TBS, HRP-conjugated streptavidin (1:200, BioSPA, Milan, Italy) wasadded for 30 min at room temperature. Slides weredeveloped with DAB (DAKO, LSAB2 system HRP) orAEC (LabVision, Newmarket, UK) and counterstained withhematoxylin. Images were taken with an Olympus BX51microscope. Micrographs were taken on an Olympus(Nagano, Japan) C3030 zoom digital camera and visualizedwith iPhoto 4 (Apple Computer) and Adobe Photoshop 7(Adobe Systems Incorporated) softwares.

TUNEL assay

Terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling (TUNEL) staining was done using a commer-cially available apoptosis detection kit (In Situ Cell DeathDetection, POD, Roche Molecular Biochemicals) accordingto the manufacturer’s instructions.

Ig gene rearrangement studies by Ig complementaritydetermining region 3 spectratyping

Total RNA was extracted using RNeasy Minikit (Quiagen)or Nucleo Spin-RNA/protein (Macherey-Nagel) columnsand retrotranscribed with Advantage RT for PCR kit(Clontech). DNA was amplified with the following fastperformance liquid chromatography purified primers (Invi-trogen): mu1 forward (FW), CAG CTC AGC AGC CTGACA TCT; mu2 FW, GAC CTC CAC AGC CTG ACATCT; mu3 FW, GAC CTT AGT AGATTG ACA TCT; andmuJH reverse (RV), CTYACC TGA GGA GAC KGT GA.MJH RV primer was labeled at 5′ with 6-carboxy-fluoresceinfluorophore. Forward primer design was based on immuno-globulin heavy chain (IgVH) sequence available on theImMunoGeneTics (IMGT) database (http://www.imgt.org/)and took advantage of common sequences existing in theframework 3 region of about 55% of IgVH regions. Forwardprimers were admixed and used in conjunction with the MJHRV primer at a final concentration of 400 nM each. Cyclingreaction was as follows: 30 s at 94°C, 10 s at 54°C, and 5 s

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at 72°C for 35 cycles. PCR products were incubated with0.5 U T4 DNA polymerase (Invitrogen) for 30 min at 37°Cto remove nontemplate nucleotide additions and purifiedusing Montage PCR columns (Millipore, Billerica, MA,USA). PCR products were denatured in 15 ml formamide(Applied Biosystems, Monza, Italy) at 94°C for 5 min andtransferred on ice. Products were run on an automatedsequenator (3100 Genetic analyzer; Applied Biosystems)and analyzed with Peak Scanner 1.0 software (AppliedBiosystems).

Data analysis and statistics

The survival and the tumoral incidence in a matched cohortof 37 female and 35 male pDbl-ki mice and 26 female and28 male w.t. littermates was monitored up to 21 months ofage. The probability of overall survival was calculatedusing Kaplan–Meier method, and the significance of thedifference between Kaplan–Meier curves was calculated bythe log-rank test using Prism 4.03 (GraphPad Software,Inc.). Statistical significance was calculated to comparedisease occurrence between pDbl-ki and w.t. littermatemice by Pearson chi-square analysis on a weightedcontingency table, using SPSS software.

To evaluate the number of apoptotic cells, six mice pergroup were analyzed. Four randomly collected sectionsfrom the spleens of w.t. and diseased mice and five fieldsper section were examined with a Nikon Eclipse E1000fluorescent microscope (Nikon Corp., Tokyo, Japan), andthe number of apoptotic cells was determined. Theaveraged values obtained from these counts were used forgroup comparisons. The statistical significance of thedifferences between the mean values of the two groups ofmice was determined by the two-tailed Student t test(GraphPad software). Findings were considered significantat P<0.01.

Results

Characterization of Dbl expression in pDbl-ki mice

To generate a mutant proto-Dbl allele by gene targeting, ahuman proto-Dbl cDNA clone, containing two stop codonsat nucleotide 1929, was constructed. We expected that thepresence of the two stop codons would allow the expressionof only the Dbl N terminus regulatory sequence, preventingDbl exchange activity. Therefore, we evaluated the trans-forming and biochemical characteristics of the mutanthuman proto-Dbl cDNA. The wild-type proto-Dbl (pZip-neo-pDbl) and pZipneo-pDbl-ki constructs were transfectedinto NIH3T3 cells. As shown in Fig. 1d, under assayconditions in which pDbl displayed 7.5×102 foci/μg of

DNA, the mutant pDbl-ki consistently yielded null foci. Todetermine the exchange activity of pDbl-ki, pZipneo-pDbl-kiwas transfected into NIH3T3 cells, and the relative amounts ofactivated GTPases in cell lysates were measured byGST-PAK1 pull-down, which specifically recognizes theGTP-bound form of Cdc42 and Rac [17]. As shown inFig. 1a, b, pDbl-ki mutant demonstrated no Cdc42 or Racactivating potential. In contrast, pDbl induced the expectedactivation of endogenous Cdc42 and Rac. Moreover, whilepDbl induced a good activation of JNK in NIH3T3 cellsmutant pDbl-ki did not (Fig. 1c). Therefore, as expected,these results indicate that mutant pDbl-ki has no GEFactivity. pDbl-ki construct was then inserted at the startingcodon of murine exon 1, and pDbl-ki mice were generated(Supplementary information File 1 and Fig. 1a).

We analyzed the expression of the Dbl-mutated allele inpDbl-ki mice by RT-PCR with oligonucleotides amplifyingexons 14 to 19 of the murine w.t. and with oligonucleotidesamplifying a region corresponding to the human Dbl Nterminus cDNA. The mouse Dbl transcript was identified asthe expected 542-bp band in total mRNA extracted fromvarious tissues of w.t. mice (Fig. 2a) while correspondingamplification bands were absent in all tissues homozygousfor the Dbl-mutant allele (Fig. 2a). A 400-bp product wasalso identified in testis, in agreement with previouslypublished data [18]. The upstream and downstream primersamplify the C terminus of mouse Dbl cDNA; therefore, theresults of the amplification demonstrate that these sequen-ces are no longer transcribed in the organs of pDbl-ki mice.Conversely, when oligonucleotides amplifying a regioncorresponding to the human Dbl N terminus cDNA wereused, the Dbl transcript was detected in total RNA extractedfrom tissues of pDbl-ki mice (Fig. 2b) while the same RT-PCR product was absent in all tissues from w.t. mice(Fig. 2b). The expression of the mutant protein in pDbl-kimice was proven by Western blot analysis (Fig. 2c). A 115-kDa protein was detected in brain of w.t. mice with anantibody raised against an epitope common to both mouseand human protein and mapping just downstream of the DHdomain. As expected, this antibody did not detect a proteinin brain lysate from pDbl-ki mice (Fig. 2c, lane 2).Conversely, when we used an antibody specific for anepitope mapping at the N-terminal tail of human Dbl,only the mutated protein of 67 kDa was detected,compatible with the expected size of the truncatedprotein (Fig. 2c, lane 4).

Dbl-mutant mice develop B cell lymphoma

The initial observation on the mouse colony evidenced areduced lifespan of pDbl-ki mice compared to w.t.littermate. To define the cause of this mortality, a matchedcohort of 72 pDbl-ki and 54 w.t. littermate mice was

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monitored over a 21-month period. Analysis of survivaldemonstrated that pDbl-ki mice displayed an increaseddeath rate with females being worst respect to males(Fig. 3). Necropsy and successive histological analysisrevealed that 42 out of 66 pDbl-ki mice (50% of the malesand 78% of the females for a total of 64% of the animals)developed malignancy vs a total of four out of 45 (9%) w.t.mice (P<0.0001). The most prominent pathology observedin females (22 out of 25 tumor-bearing animals, 88%) wasconsistent with a lymphoma (Table 1). Indeed, at necropsy,mice presented enlarged spleen and lymph nodes, especiallyin the mesenteric area. Five out of 17 (29%) tumor-bearing

male mice developed the same disease (Table 1). Histologicalanalysis confirmed the diagnosis of lymphoma of DLBCL.Expression of the Dbl-mutant protein in the spleen of theaffected animals was confirmed by Western blot analysiswith an antibody specific for the epitope mapping at theN-terminal tail of human Dbl (Fig. 2c, lane 6). Moreover, wedetermined the GEF activity of mutant Dbl in DLBCL ofpDbl-ki mice. B cells were purified from spleens of w.t. anddiseased pDbl-ki mice by positive selection with CD19MicroBeads, lysed and subjected to Western blot analysis.JNK and p38 activation was determined with phosphospe-cific antibodies. As shown in Fig. 2d, similarly to what was

Fig. 2 Analysis of pDbl-ki expression and activity in pDbl-ki mice. aTotal RNA extracted from brain (B), testis (T), ovaries (O), lung (L),and spleen (S) of w.t. and pDbl-ki mice was analyzed by RT-PCR witholigonucleotides designed to amplify murine Dbl sequences (Dbl 3′;542 bp). b Total RNA extracted from brain (B), testis (T), ovaries (O),lung (L), spleen (S), and lymph nodes (Ly) of pDbl-ki and w.t. micewas analyzed by RT-PCR with oligonucleotides designed to amplifyhuman proto-Dbl cDNA sequence (Dbl-ki 5′; 436 bp). c Western blotanalysis of extracts from w.t. (1, 3) and pDbl-ki (2, 4) mouse brain andfrom w.t. (5) and pDbl-ki (6) spleen. An antibody raised against anepitope (amino acids 818 to 831) mapping just downstream of the DHdomain was used to detect w.t. protein (lanes 1 and 2) while an

antibody raised against an epitope mapping at the N-terminal tail ofhuman Dbl (amino acids 3 to 16) was used to detect the human mutantprotein (lanes 3, 4, 5, and 6). The blot was reprobed with an anti-ezrinantibody to evaluate amount of proteins loaded (bottom). d Lysates ofB cells purified from spleen of w.t. and diseased pDbl-ki mice weresubjected to SDS-PAGE, transferred to PVDF membrane, and probedwith phosphospecific antibody to detect P-JNK and P-p38 and anti-JNK and anti-p38 to detect kinase expression levels. Bars representthe fold of activation over control of the phosphorylated substrates,quantified by densitometry, and normalized to the content of JNK orp38 in cell extracts

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observed in transfected NIH3T3 cells, the mutant proteindoes not have exchange activity as demonstrated by lack ofactivation of JNK and p38 kinases in pDbl-ki B cells.

The spleen of one affected animal is shown in Fig. 4a. Acomplete replacement of red and white pulp by diffuse largeB cell lymphoma can be observed. DLBCL is composed oflarge neoplastic cells, with prominent central nucleoli andfrequent mitosis (Fig. 4b, C′, arrows). Lymphoid infiltrateswere occasionally observed in other tissues, including lung,kidney, and liver. Figure 4c, d shows how the liver and thekidney of the same mouse shown in Fig. 4a, b are diffuselyinfiltrated by DLBCL (arrows in Fig. 4d, D′ indicate residualglomerulous and tubulus, respectively).

pDbl-ki mice also developed small lymphocytic lym-phoma with an incidence, eight out of 66 (12%) vs four outof 45 (9%), that was slightly higher than that observed incontrols (Fig. 4e–g; Table 1). These lymphomas weremainly localized to mesenteric lymph nodes withoutspreading to other tissues and represent a type of lessaggressive lymphoma that is known to develop spontane-ously in aging C57BL mice [19]. However, five out of eightaffected pDbl-ki animals were males, while only one w.t.male mouse developed the disease. Although the differenceis not significant (P=0.39), due to the small number ofevents, these data suggest a role for Dbl in increasinggeneral susceptibility to tumor development. In fact, malepDbl-ki mice also displayed a higher incidence of lungtumors compared to controls (ten out of 34, 29% vs one outof 23, 4%; P=0.01). The tumors were histologicallyclassified as alveolar adenomas (eight cases) or alveolaradenocarcinomas (two cases; Table 1; Fig. 4h).

Immunohistochemical analysis supported the diagnosisof DLBCL with a mature B cell phenotype. In fact, themedium–large-size neoplastic cells expressed the B220marker (Fig. 5a) but were negative for staining of C1qRp(Fig. 5e), a C-type lectin-like type I transmembrane proteinexpressed on early stages of B cell development, as onlyscattered cells were positive, similarly to what can be

Fig. 3 pDbl-ki mice display reduced longevity. Kaplan–Meier curvesof w.t. mice (n=54) and pDbl-ki mice (n=72) for males (top) andfemales (bottom). χ2 test confirmed pDbl-ki mice exhibit a signifi-cantly reduced average survival compared with w.t. cohort. P<0.0004,χ2=12.6 for female mice and P=0.001, χ2=10.9 for male mice.Number of w.t. and pDbl-ki mice is indicated in each survival curveand numbers in parentheses refer to the total number of events thatoccurred during the 21 months of observation

Table 1 Malignancies in Dbl knock-in mice

Disease No. of cases (%) Description

pDbl-ki w.t. P value

M F M F

Large B cell lymphoma 5 (15) 22 (69) 0 0 <0.0001 CD3−, K67+, CD45R/B220+Small lymphocytic lymphoma 5 (15) 3 (9) 1 (4) 3 (14) 0.76 CD3−, K67+, CD45R/B220+Atypical/lymphoid hyperplasia 2 (6) 1 (3) 0 0 0.27 B cell

Lung alveolar adenoma 10 (29) 1 (3) 1 (4) 0 0.01

Hepatic/splenic hemangioma 0 1 (3) 0 0 0.41

Hepatoma 1 (3) 0 0 0 0.41

Tumor-bearing animals/no. evaluated 17/34 (50) 25/32 (78) 1/23 (4) 3/22 (14) <0.0001

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observed in spleen sections from w.t. mice (data notshown). Further phenotypic characterization for the lym-phoma cells indicated that they were CD3 negative(Fig. 5b) and revealed a high proliferation rate using ananti-Ki-67 antibody (Fig. 5c; Table 1). The lymphomaswere of germinal center origin as shown by a diffusepositivity with anti-Fas antibody (Fig. 5f). Moreover, adiffuse positivity (Fig. 5g) was found in the cytoplasm ofneoplastic cells stained with PNA, which is usually used forthe visualization of germinal center B (GCB) cells [20].

We finally evaluated whether lymphoma cells expressc-Rel, a subunit of the REL/NF-κB family. Rel geneamplification or persistent activation has been detected inmany human B cell tumors, including DLBCL [21] and, in

particular, in GCB-type DLBCL [22]. Moreover, nuclearexpression of c-Rel correlates with poorer outcome of thedisease [23]. As shown in Fig. 5, c-Rel was stronglyexpressed in the cytoplasm of the lymphoma cells (Fig. 5j,k) with some nuclear staining, especially evident in affectedlymph nodes (Fig. 5k). These results were confirmed byWestern blot analysis of cytosolic and nuclear cell lysatesobtained from B cells purified from spleen of normal andpDbl-ki mice. As shown in Fig. 6a, cytoplasmic c-Relexpression was stronger in malignant B cells in comparisonwith normal B cells. Moreover, nuclear c-Rel expressionwas significantly augmented in malignant versus normal Bcells (Fig. 6a). The NF-κB/Rel transcription factor familyprotects many cell types from apoptotic signals. In

Fig. 4 Histopathologicalanalysis of tissue sections fromdiseased pDbl-ki mice withDLBCL. A, B Hematoxylin andeosin stained sections of aspleen of a representativepDbl-ki diffuse large B celllymphoma. C Lymphoidinfiltrate to the liver and Dlymphoid infiltrate to the kidneyof the same animal. E, FHematoxylin and eosin stainedsections of A spleen and G alymph node of representativesmall lymphocytic lymphomas.H Hematoxylin and eosinstained section of a representa-tive pDbl-ki lung adenocarcino-ma. Arrowheads in B, C’, F,H indicate mitotic cells. A, C,E ×20; B, C’, D’, F, G ×60; D,H ×40

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particular, it has been demonstrated that c-Rel null micehave greatly reduced B cell proliferation and reducedsurvival in response to mitogenic activation [23]. Therefore,apoptosis was assessed by end-labeling of DNA strandbreaks on paraffin-embedded sections of w.t. and pDbl-kimice (TUNEL assay) as described in “Materials andmethods”. The percentage of TUNEL positive cells wascalculated as a function of the total number of cells in themicroscopic field. Statistically reduced TUNEL staining(P<0.0001) was observed in the lymphoma cells incomparison with normal cells (Fig. 6b). In pDbl-ki mice,the mean value of apoptotic cells was low compared tonormal controls 1.9% (SEM±0.07%, n=6) versus 4.4%(SEM±0.2%, n=6). These results suggest that overexpressionof c-Rel protects lymphoma cells from apoptosis.

In order to analyze the clonality of B cells, Ig generearrangement studies were performed in B cells derivingfrom lymph nodes (L) and/or spleen (S). In all cases ofdiagnosed DLBCL, clonal or oligoclonal B cell expansionswere observed (Fig. 7). In some instances, a clonal expansion

sharing the same VDJ rearrangement length was identified inlymph nodes and spleen from the same animals (Fig. 7,samples Z863L and Z863S and samples Z873L and Z873S),indicating the malignant nature of the B cell clone. Oligoclonalexpansions were also observed in apparently healthy knock-inmice (Fig. 7, samples Z78S and Z80S) signifying that B cellclonal evolution may occur before clinical evidence ofdisease. In w.t. animals, a normal distribution of B cell cloneswas always observed with no evidence of mono- oroligoclonal expansions (Fig. 7, sample wtCTR).

Finally, we performed a study of B cell subsets inspleens over the lifetime of pDbl-ki mice. Spleen popula-tions of B cells were normal based on surface levels ofB220, IgM, IgD, CD25, and CD43 (Supplementaryinformation File 2 and Fig. 2a), as compared to age-matched w.t. mice. Therefore, there were no significantdifferences between wild-type and mutant mice in the cellsubsets analyzed. On the other hand, premalignant lym-phocyte nodular hyperplasia occurring with aging could beobserved (Supplementary information File 2 and Fig. 2b).

Fig. 5 Immunohistochemical analysis of tissue sections from diseasedpDbl-ki mice. Tissue sections of lymphomas of pDbl-ki mice stainedwith antibodies against B220, CD3, Ki-67, C1qRp, Fas, PNA, and c-Rel. Mature B cell phenotype is demonstrated by positive staining oftumor sections with A anti-B-220 antibody and negative staining withB anti-CD3 and E anti-C1qRp antibody. Neoplastic cells also displaya high proliferation rate as revealed by staining with C anti-Ki67antibody. Diffuse positive staining is detected with F anti-Fas antibody

and G PNA demonstrating a germinal center origin of the proliferatingB cells. Strong cytoplasmic and some nuclear staining with anti-c-Relantibody can be detected in tumor sections from the spleen J andlymph node K of a pDbl-ki mice with DLBCL in comparison withstaining of a section from a normal spleen I. D, H, L Negativecontrols with anti-rat, anti-rabbit, and anti-mouse antibody are shown.A–C and F to G ×60; D, H, and I to L ×40. Scale bars represent100 μm

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Discussion

Although a rare tumor in mice, DLBCL is the mostcommon lymphoma worldwide, accounting for approxi-mately 30% of all human lymphoid malignancies [24]. Inour animal model, DLBCL develops at high incidence inadult mice engineered to express a mutant form of Dbl.Alteration of the Dbl protein reduced animal longevity, andthis is associated with lesions that share histologicalfeatures with the most common type of DLBCL. Femalemice displayed a higher incidence of tumor formationcompared to male mice, reaching an overall 78% incidencefor malignancy, 88% of which were DLBCL. These resultssuggest, for the first time, a role for Dbl in DLBCL.

The role of Rho family of GTP binding proteins intumorigenesis is becoming appreciated with the identifica-tion of the overexpression and hyperactivation of some Rhoproteins in human cancers [25]. It has been speculated thatdisturbances of Rho GTPase signaling are caused byalteration at the level of their regulators rather than bymutations at the levels of Rho GTPases themselves [26,27]. Consistent with this idea, many GEFs were originallyisolated as oncogenes, and a few GEFs have been foundmutated or overexpressed in human cancers [3, 28]. Thepoint mutations we have inserted in the human cDNA causethe production of a truncated protein that lacks GEFactivity. In fact, we show here that this mutant does nottransform mouse fibroblasts and does not activate GTPases.

Moreover, no activation of Dbl downstream effectors JNKand p38 kinases could be detected in normal and malignantB cells. Thus, the mechanism of B cell transformation byDbl must be independent of GTPase activation. We canpresume that it is not the lack of Dbl exchange activity thatcauses DLBCL since Dbl-null mice do not developmalignancies but rather defective dendrite elongation [12].Recently published studies indicate that four alternative splicevariants of Dbl exist with tissue specificity of expression,including spleen and peripheral blood [29, 30]. The expres-sion of a specific splice variant of Dbl may be required for Bcell differentiation and function, and thus, altering itsstructure may specifically affect B cell development.

Outside the DH and PH domains, DH-containing GEFsshow significant divergence and typically contain otherprotein domains indicative of functions that are independentof nucleotide exchange. The presence of only the regulatorydomain of Dbl in pDbl-ki mice may alter the function ofmolecules that are normally associated with Dbl to eitherregulate its temporal and tissue-associated activity, or evento mediate signaling events associated with Dbl activity.Dbl N-terminal region contains a Sec14 domain that directsthe subcellular localization of Dbl and its substrate Cdc42[31]. The N-terminal peptide may thus accumulate in Bcells preventing proper cellular functions because ofmisplaced localization. Dbl has been involved in uropodformation, a cellular structure of migrating lymphocytes,and expression of the N-terminal region of proto-Dbl in

Fig. 6 c-Rel expression and apoptosis in pDbl-ki lymphoma cells. a Theamount of c-Rel protein was determined by Western blot both incytoplasmic (cytosol) and nuclear (nuclei) cell lysates of B cells purifiedfrom spleen of w.t. and diseased pDbl-ki mice (pDbl-ki). Loadingcontrols, β-actin for cytosol and lamin for nuclei, are shown. b Paraffin-embedded sections from spleens of w.t. and pDbl-ki mice were stainedwith TUNEL (green) to reveal apoptotic cells. Nuclei were stained with

DAPI (blue). Negative control (ctr) is shown. Representative images areshown. Box–whisker plots display the amount of apoptotic cellsexpressed as percentage of the total number of cells in the microscopicfield. Central horizontal bars indicate median, upper, and lower ends ofboxes indicate 75th and 25th percentiles, and error bars indicatemaximum and minimum values. P value was determined by two-tailedStudent t test. P<0.0001 versus w.t.

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lymphocytes was shown to inhibit uropod formation [32].Therefore, accumulation of the N-terminal region of proto-Dbl may affect proper B cells migration. Verification ofthese points awaits further experimental examination.

We do not know why females are more prone to developDLBCL than male mice. Since Dbl maps to X chromo-some, it is possible that Dbl is one of the genes that escapeX-inactivation [33]. Therefore, if both genes are active, thenegative effect of the truncated Dbl protein may bepotentiated. Alternatively, as male mice are normally moresusceptible than females to lung adenomas, it is possiblethat female mice are more susceptible than males to geneticalterations/mutations that may lead to DLBCL developmentin the presence of a mutated Dbl.

The development of DLBCL in pDbl-ki mice indicates thatthe tumor-prone phenotype of the Dbl-mutant mice is robustdespite the relatively long latency of tumor development.The observation of an abnormal distribution of B cell clones(oligoclonal expansions) in Dbl-mutant mice lymphoidorgans, even before the manifestation of disease, further

reinforces the notion of multiple and likely independentoncogenic events occurring in B cell population.

Very few animal models exist that recapitulate the geneticsand the biology of DLBCL. One of these has been developedby targeted disruption of the S1P2 gene, frequently mutated inhuman DLBCL [34]. In both their and our models, B celllymphomas develop with age and display a uniform pheno-type with histological and molecular characteristics of GC-derived DLBCL, such as expression of the GC markers PNAand c-Rel, strongly resembling the features of humanDLBCL.Therefore, like the model of Cattoretti and coworkers, pDbl-kimice may represent a valuable model of DLBCL derived fromthe GC B cells, the normal counterpart of most human B celllymphomas [35]. Similarly to what occurs in Sip2

−/− mice, theonset of DLBCL in pDbl-ki mice at old age and the clonalityof the developing tumors indicate that a single oncogenic hitis not sufficient for lymphoma genesis. On the other hand,differently from the Sip2

−/− mouse model, pDbl-ki male miceshowed a high incidence of lung tumors, suggesting a role forDbl in increasing general susceptibility to tumor development.

Fig. 7 Ig gene rearrangement studies by Ig complementaritydetermining region 3 spectratyping. RNA was extracted from thelymph nodes (L) and/or the spleen (S) of 18–20-month-old pDbl-kimice (Z78, Z80, Z860, Z863, Z872, Z873, and Z874) and arepresentative matched w.t. mouse (w.t. CTR) and retrotranscribed.PCR amplification was performed by using a set of forward primers,

designed on the IgVH chain sequences available on the IMGTdatabase (http://imgt.cines.fr), that detects approximately 55% ofIgVH regions (see “Materials and methods”). Products were run onan automated sequenator and analyzed with Peak Scanner 1.0 software(Applied Biosystems). Pink-filled peaks represent off-scale peaks

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In conclusion, our data demonstrate that a mutated formof Dbl is a tumor susceptibility gene in mice and indicatethat loss of function of Dbl DH domain by genetic missensemutations is responsible for induction of DLBCL. Whilethe mechanistic details remain to be deciphered, this studyprovides a novel insight into the oncogenic activity of Dbl.

Acknowledgments The authors are indebted to Dr. V. Pistoia,Laboratory of Oncology, and Dr. I. Airoldi A.I.R.C. Laboratory ofImmunology and Tumors, G. Gaslini Institute, for helpful discussions.The authors would like to thankMaria Grazia Calevo, Epidemiology andBiostatistics Section, G. Gaslini Institute, and Fabiola Blengio and PaoloFardin, Laboratory of Molecular Biology, G. Gaslini Institute, for helpwith statistical analysis and Sara Barzaghi, Laboratory of MolecularBiology, G. Gaslini Institute, for secretarial assistance. This work wassupported by grants from the Italian Association for Cancer Research,from the Compagnia di S. Paolo, and from the Ministero della Salute. CTwas supported by Compagnia di S. Paolo (4824 SD/CV-2007.2880).

Conflicts of interest The authors reported no potential conflicts ofinterest.

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