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Leukemia Research 32 (2008) 309–314

Bone marrow pre-B expansion by SL/Kh-Bomb1 locus:Not sufficient for lymphomagenesis

Takuya Hiratsuka a, Tatsuaki Tsuruyama a,b, Richard Kaszynski b, Kohei Kometani c,Nagahiro Minato c, Takuro Nakamura d, Keiji Tamaki b, Hiroshi Hiai a,e,∗

a Department of Pathology and Biology of Diseases, Kyoto University Graduate School of Medicine,Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

b Department of Legal Medicine, Kyoto University Graduate School of Medicine,Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

c Department of Immunology and Cell Biology, Kyoto University Graduate School of Medicine,Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

d Department of Carcinogenesis, The Cancer Institute, Japanese Foundation for Cancer Research,3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan

e Shiga Medical Center Research Institute, 5-4-30 Moriyama, City of Moriyama, Shiga 524-8524, Japan

Received 15 March 2007; received in revised form 15 May 2007; accepted 18 May 2007Available online 6 July 2007

bstract

The pre-B lymphoma in SL/Kh mice is a polygenic trait involving a number of host genes. In prelymphoma-stage bone marrow, transientre-B cell expansion is induced by a host locus, Bomb1, and later followed by the emergence of a monoclonal population with a similarhenotype. To determine whether these pre-B cells represent precursors of lymphomas, we generated a congenic strain, NFS.SL/Kh-Bomb1

ice, by marker-assisted backcrossing to NFS. The congenic mice showed pre-B cell expansion, but pre-B lymphomas were not observed,

ven after 1 year of observation, irrespective of murine leukemia virus inoculation. Disturbed early B cell differentiation per se is not sufficientor SL/Kh lymphomagenesis.

2007 Elsevier Ltd. All rights reserved.

eywords: Mouse lymphoma; Pre-B cells; Bomb1; Prelymphoma; Congenic

Bomto

. Introduction

SL/Kh mice develop pre-B lymphomas by endogenousurine leukemia virus (MuLV) at an incidence >90% withinmonths of age [1]. The SL/Kh lymphoma is a multifactorial

isease involving numerous genetic and epigenetic factorsor its development [2]. Their lymphoma cells commonlyxpress B220, a pan-B cell antigen, and BP1, an immature

Abbreviations: MuLV, murine leukemia virus; BM, bone marrow;omb1, bone marrow pre-B-1; Ig, immunoglobulin; IgH, immunoglobulineavy chain; MMU, mouse chromosome∗ Corresponding author at: Shiga Medical Center Research Institute, 5-4-0 Moriyama, City of Moriyama, Shiga 524-8524, Japan.el.: +81 77 582 6029; fax: +81 77 582 6041.

E-mail address: hiai@shigamed.jp (H. Hiai).

iNthppuete

145-2126/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2007.05.013

cell antigen [3]. In the crosses with NFS mice, expressionf Emv11, an endogenous ecotropic MuLV genome on chro-osome 7 (MMU7), is required for any type of hemopoietic

umors [2]. Types of tumors are dependent on the genotypef host loci (2): a dominant SL/Kh allele at Esl1 on MMU17s essential for pre-B lymphomas, and homozygosity of theFS allele at foc1 on MMU4 is required for follicular cen-

er cell lymphomas. Another unique feature of the SL/Khematopoietic system is a transient expansion of BP1+ B220+

re-B cells in the bone marrow (BM) of 4–6-week-old micerior to the development of overt lymphomas [4,5]. This pop-

lation is not dependent on MuLV, since inhibition of MuLVxpression either by crossing with Fv4R mice or by injec-ion of a maternal resistance factor [6] does not affect thexpansion [4]. Instead, it is a genetic property of SL/Kh BM

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tem cells because lethally irradiated SL/Kh or BALB/c micehowed pre-B cell expansion only when restored with SL/KhM cells but not with BALB/c BM [4]. By analyzing (SL/KhNFS)F2, we have mapped a highly significant quantitative

rait locus (QTL), named bone marrow pre-B-1 (Bomb1),n the distal segment of MMU3 [5] that is responsible forre-B expansion. Considering the close resemblance in phe-otype between the expanded BM pre-B cells and SL/Khymphomas, we asked whether or not the former is a precur-or lesion of the lymphomas. To answer this question, a speedongenic strain NFS.SL/Kh-Bomb1 was constructed in whichhe Bomb1 segment of the SL/Kh was inserted into the geneticackground of NFS/N mice. We studied BM early B cells andheir fate in SL/Kh, NFS.SL/Kh-Bomb1, and NFS. It was con-luded that polyclonal pre-B expansion is not sufficient forymphoma development.

. Materials and methods

.1. Mice

The origin and genetic properties of SL/Kh mice have beenescribed elsewhere [7]. NFS/N is an inbred mouse strainithout an endogenous ecotropic MuLV genome and with

ow spontaneous lymphoma incidence. The mice were main-ained by sister–brother mating for over 100 generations innstitute of Laboratory Animal, Kyoto University. All animalxperiments were carried out under approval of the Ethi-al Committee for Animal Experiments, Kyoto Universityraduate School of Medicine.Congenic strains were developed by a marker-assisted

election method. An (NFS × SL/Kh)F1 male was matedith an NFS female mouse to produce the progenies that were

elected for the SL/Kh-derived Bomb1 segment of MMU3,hich is described later. Males heterozygous at the Bomb1

egment were successively backcrossed to NFS until the N12eneration, at which heterozygous brother and sister sib-ings were intercrossed; among their progeny, homozygousairs were selected for subsequent inbreeding. The congenictrain was designated as NFS.SL/Kh-Bomb1 according tohe nomenclature suggested by Mouse Genome Informatics2004).

SL/Kh and NFS.SL/Kh-Bomb1 mice have been depositedn the National Bio-Resource Project (http://www.anim.

ed.kyoto-u.ac.jp/nbr/home.htm) in RIKEN (Tsukuba,apan).

.2. Genotyping

A 1-cm-long tail segment was sampled from each mouse atweeks of age, and genomic DNA was extracted. All primers

or microsatellite analysis were purchased from Researchenetics, Inc. (Huntsville, AL). Polymorphic microsatel-

ite markers, D3Mit19, D3Mit351, D3Mit319, D3Mit346,nd D3Mit11, were used to define the Bomb1 segment. The

(awU

earch 32 (2008) 309–314

ethod for PCR and agarose electrophoresis of PCR productsas described previously [4]. After establishment of the con-enic mice, 17 marker loci on the distal segment of MMU3nd 58 other microsatellite loci randomly scattered through-ut the genome were examined to characterize the genomeomposition.

.3. Flow cytometry

The single-cell suspension was prepared from both femurM plugs and adjusted to 106 cells/ml. The BM cells were

ncubated with normal rat serum for 15 min and stainedith 1:10,000 diluted monoclonal antibodies for 15 min.fter washing, the cells were analyzed in FACScan (Bectonickinson, Mountain View, CA). The following antibod-

es were purchased commercially: FITC-labeled anti-BP1clone 6C3), PE-labeled anti-B220 (clone RA3-6B2), anti-L7R (clone A7R34), anti-CD24 (clone 30-F1) (Bioscience,an Diego, CA), and anti-CD43 (clone S7) (Pharmingen, Saniego, CA).The BP1+B220+ BM cells were purified from double-

tained BM cells by cell sorting using a FACS VantageBecton Dickinson). The average yield from the BMells of 4-week-old NFS, NFS.SL/Kh-Bomb1, and SL/Khice was 0.52, 6.0, and 2.7%, respectively, and >98%

urified cells were BP1+B220+. The BM cells from 15-eek-old SL/Kh mice, pre-B cells were fractioned into

wo populations depending on intensity of BP1 stain.he gates for the BP-1 low was between 101 and 102,nd those for BP1 high >102 in forward scatter scale.enomic DNA was extracted from the purified cells withIAamp DNA Blood Mini Kit (QIAGEN GmbH, Hilden,ermany).

.4. Analysis of immunoglobulin gene rearrangement

Genomic DNAs were subjected to PCR with primersesigned to amplify four possible junctions between the D-52 and JH regions [9]. The primers for the first round wereAC AGA GAA TTC TCC ATA GTT GAT AGC TC AG

DHQ52-1; sense) and AGG CTC TGA GAT CCC TAGCA G (JH4-1; antisense). PCR conditions were as fol-

ows: denaturation for 1 min at 95 ◦C, annealing for 1 mint 60 ◦C, and extension for 2.5 min at 72 ◦C (28 cycles).wo microliters from each reaction was subjected to a sec-nd round of PCR using a pair of internal primers: GCCCA GAA TTC CTG TGG TCT CTG ACT GGT (DHQ52-; sense) and GGG TCT AGA CTC TCA GCC GGCCC CTC AGG G (JH4-2; antisense). The PCR conditionsere as follows: denaturation for 20 s at 95 ◦C, anneal-

ng for 1 min at 60 ◦C, and extension for 2 min at 72 ◦C

35 cycles). The PCR products were electrophoresed inn agarose gel, blotted on a nylon membrane, and probedith a JH4 probe (Amersham Biosciences, Buckinghamshire,K).

ia Research 32 (2008) 309–314 311

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Fig. 1. The Bomb1 segment of mouse chromosome 3 in congenic mice.White column, definitively SL/Kh-derived segment; shaded column, seg-ment either from SL/Kh or NFS; black column, definitively NFS-derivedsegment. Closed bars, position of microsatellite marker loci with SL/Khallele; open bars, those with NFS allele; broken bar, the position of D3Mit97(not polymorphic). Right on side, microsatellite marker loci used for typing.TnA

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.5. Virus

The ecotropic MuLV was originally isolated from extractsf an SL/Kh pre-B lymphoma and passaged on NIH-3T3broblasts for 3–5 generations. A high-titer stock was keptrozen in liquid nitrogen until use. Newborn mice werenoculated i.p. with 105 XC plaque-forming units of theirus solution. Successful infection was confirmed by an XClaque assay of tail extracts at 2 months of age [6].

.6. Statistic analysis

An unpaired t-test was used. A P value <0.01 was consid-red to be a significant difference.

. Results

.1. Construction of a congenic strain

To examine the biological behavior of the expanded BMre-B cells, a speed congenic strain NFS.SL/Kh-Bomb1 wasenerated by selective transfer of the SL/Kh MMU3 segmentearing Bomb1 to NFS mice by marker-assisted backcross-ng. As shown in Fig. 1, the congenic mice had a minimum of7.2 cM (between D3Mit291 and D3Mit11) and a maximumf 53.9 cM (between D3Mit351 and D3Mit22) introgressedL/Kh MMU3 segment. The QTL peak for Bomb1 mapped

n our previous study was between D3Mit19 and D3Mit2525), as shown by an arrowhead in Fig. 1. Possible recombina-ion break points were between D3Mit351 and D3Mit291 asell as between D3Mit11 and D3Mit22. As shown by shaded

olumns in Fig. 1, a polymorphic marker locus was not foundn these segments. On the other hand, 58 other microsatel-ite loci randomly distributed throughout the mouse genomeere homozygous for the NFS/N allele.

.2. BM pre-B cell expansion

As seen in Fig. 2A, a unique expansion of BP1+B220+

ells was observed among BM cells of SL/Kh andFS.SL/Kh-Bomb1 mice at 4 weeks of age. FACS analy-

is showed that these BP1+B220+ cells were CD43+ CD24+

Fig. 2B and C); therefore, they belonged to early pre-cells of Fractions B to C by Hardy’s classification (8).

n contrast, such a population was much smaller in NFSM of a comparable age (P < 0.001, NFS/N (n = 12) versusFS.SL/Kh-Bomb1 (n = 12), and P < 0.001, NFS/N (n = 12)ersus SL/Kh (n = 12)). The data support the fact that thentrogressed segment contains the genetic locus generatinghe phenotype of SL/Kh-Bomb1.

As reported previously [4], the pre-B cells in SL/Kh mice

ecreased transiently as mice aged, but, when the mice were5 weeks of age or later, a population with very high BP1xpression appeared and rapidly took over BM. In additiono higher BP1 expression, they also expressed higher IL7R

Btfi

he loci polymorphic between SL/Kh and NFS are shown in bold and thoseot polymorphic, in thin. Left on side, map position in cM from centromere.rrow indicates the peak position of Bomb1 in QTL analysis (5).

han that in the pre-B population observed at 4 weeks of ageFig. 2D). In NFS.SL/Kh-Bomb1 mice, such BP1high/IL7Righ population or pre-B lymphoma did not appear even at2 months of age (data not shown), while all SL/Kh miceuccumbed to lymphomas by 6 months of age.

.3. Clonality of expanded pre-B cells

To examine the possible clonal nature of the expandedre-B cells in SL/Kh and NFS.SL/Kh-Bomb1, we exam-ned the PCR-amplified immunoglobulin heavy-chain genesing primers set in the Q52 subset in the immunoglob-lin D-segment and JH4-segment. The DNAs used in thisssay were extracted from FACS-sorted BP1+B220+ cellsrom individual mice of either strain at variable ages. Theata from representative individual mice at each stage arehown in Fig. 3. At 4 weeks of age, both NFS.SL/Kh-

omb1 (lane 1) and SL/Kh pre-B cells (lane 3) showed

hree distinct fragments, DQ52-JH2, JH3, and/or JH4. There-ore, the expanded pre-B cells at this stage were polyclonaln nature and belonged to the early pre-B cells with an

312 T. Hiratsuka et al. / Leukemia Research 32 (2008) 309–314

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ig. 2. Surface phenotype of BP1 positive BM cells in SL/Kh, NFS.SL/KhD) IL7R. Numbers in each upper right quadrants represent percentage of d

gH gene that had completed DJH rearrangement. In 15-eek-old NFS.SL/Kh-Bomb1 mice, polyclonal rearrangedands were detected in the FACS-purified pre-B cells (lane). In contrast, among BM pre-B cells in SL/Kh, popu-ations with a clonally rearranged IgH gene appeared as

ice aged. At 15 weeks of age, the BP1high BM cells pre-ailed in about 60% of the mice, and they represented alonal population with a single band of DQ52-JH2, -JH3, orJH4 (lane 6). Other individuals still had a predominantlyP1low pre-B population, and their IgH remained oligo- or

olyclonal (lane 5). Ultimately, virtually 100% of the miceeveloped pre-B lymphomas by 6–7 months of age. IgHas invariably clonally rearranged in pre-B lymphoma cells

lane 7).

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and NFS at 4 and 15 weeks of age. (A) B220, (B) CD43, (C) CD24, andositive cells.

.4. Fate of Bomb1-driven pre-B cell expansion

NFS mice bearing introgressed SL/Kh-Bomb1 faithfullyhowed pre-B expansion. To examine whether this popula-ion represents a lymphoma precursor lesion, we observedFS.SL/Kh-Bomb1 and NFS mice for 12 months. Some of

hem were inoculated with ecotropic MuLV from SL/Khymphoma as neonates. As shown in Table 1, none ofFS.SL/Kh-Bomb1 and NFS mice developed lymphomas

rrespectively of whether they had received an MuLV injec-

ion, although all virus-injected mice became viremic. Thisbservation indicates that Bomb1-driven pre-B cell expan-ion is not sufficient per se for pre-B lymphoma in the NFSackground.

T. Hiratsuka et al. / Leukemia Res

Fig. 3. Immnoglobulin heavy-chain gene rearrangement in FACS-purifiedbone marrow pre-B cells. Lane 1, NFS.SL/Kh-Bomb1, 4 weeks of age; lane2, NFS.SL/Kh-Bomb1, 15 weeks of age; lane 3, SL/Kh, 4 weeks-old; lane4, SL/Kh, 10 weeks-old; lane 5, SL/Kh, 15 weeks-old, BP1low cells pre-dominant; lane 6, SL/Kh, 15 weeks-old, BP1high cells predominant; lane7, overt lymphoma. Molecular weight shown in base pairs on the left sidewas determined by λ HindIII markers. In lanes 1–5, polyconally rearrangedbIr

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. Discussion

.1. Properties of the congenic strain

Genetically determined pre-B cell expansion is an out-tanding feature in the prelymphoma-stage BM in SL/Khtrain mice, which are highly predisposed to spontaneous pre-

lymphomas [4,5]. A similar expansion is also described inbelson virus-injected BALB/c mice [9] or Eμ-myc trans-enic mice, which are prone to B-lineage lymphoma [10],ut its significance has been unclear. In order to further char-cterize the pre-B expansion in SL/Kh BM, we introgressedn SL/Kh chromosomal segment bearing Bomb1 into NFSice that had no spontaneous lymphoma and no endogenous

cotropic MuLV genome using a speed congenic procedure.he introgressed segment was rather broad. It is possible

hat more than a single gene responsible for B cell pheno-ype. However, it definitively contained a Bomb1 QTL peak

5] faithfully reproducing pre-B cell expansion in BM, aseen in SL/Kh. Thus far in our examination, SL/Kh genomeomponents have not been detected outside this region.

able 1ymphoma incidence

train Virus injection PFUa Lymphomaincidenceb

FS No 0 0/12FS Yes 5.8 × 102 0/12FS.SL/Kh-Bomb1 No 0 0/14FS.SL/Kh-Bomb1 Yes 7.9 × 102 0/15L/Kh No 3.5 × 103 42/42a XC plaque-forming unit of ecotropic MuLV per 0.5 cm tail extract at 2onths of age.b Number of lymphoma-bearing mice/total mice at 12 months of age.

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The expanded pre-B cells in NFS.SL/Kh-Bomb1 micehowed the surface phenotype BP1+B220+CD43+CD24+,ike those in SL/Kh. Hardy [8] classified B220+ CD43+

-lineage cells in mouse bone marrow into three fractionsased on differential cell surface expression of BP1 andD24. The populations with CD24−BP1−, CD24+BP1−,nd CD24+BP1+ are termed Fractions A, B, and C, respec-ively. By PCR amplification with primers for region 5′f JH1, DFL16.1, and Jk1, he also found that the IgHearrangement of the D and J regions occurs between Frac-ions B and C. Both BP1lowIL7Rlow and BP1highIL7Rhigh

re-B cells belong to Fraction C, although it is inconclu-ive that the former is a direct progenitor of the latter.o determine their possible clonal nature, we examined

he pattern of rearrangements of the D regions of thegH using DNAs from FACS-purified BP1+B220+ cells ofach strain of mice using the PCR-Southern method. Itas determined that the IgH gene of 4- and 15-week-oldFS.SL/Kh- Bomb1 mice and 4–10-week-old SL/Kh miceas polyclonal in nature. In contrast, the heavy-chain genef 15-week-old SL/Kh mice was monoclonal or oligoclonal.n particular, when pre-B cells contained a higher fraction ofP1highIL7Rhigh cells, a monoclonal band was consistentlybserved. This indicated that the BM pre-B cells in SL/Kh,ut not those in NFS and NFS.SL/Kh- Bomb1, shifted tolonal growth when the mice were around 15 weeks of age.hese cells shared phenotypes with SL/Kh pre-B lymphomaells in many aspects. We postulate that they progress tolinical lymphoma cells after several genetic changes andelection.

Recently Rumfelt et al. [11] reported that IgH rear-angement in B lymphomas is mostly bi-allelic. In SL/Khymphomas, we have noticed that some lymphomas showedi-allelic whereas others did have mono-allelic rearrange-ent [3]. It is conceivable that the lymphoma withono-allelic rearranged IgH as seen in Fig. 3 may repre-

ent the clone that has an abnormal rearrangement machineryesulting in rearrangement only in one allele, or that has alle-es with different timing of rearrangement and one of whichs arrested in germline configuration during the process ofransformation.

.2. Requirements for lymphomagenesis

Absence of clonal growth of pre-B cells in NFS.SL/Kh-omb1 may indicate that Bomb1-driven pre-B cell expansion

s insufficient for pre-B lymphomagenesis. We have shownhat expression and reinfection of endogenous ecotropic

uLV and genetic factors of SL/Kh mice are critically impor-ant for SL/Kh lymphomagenesis [2,12]. The endogenousirus genome Emv11 mapped on MMU7 induces persistentiremia. In the genomic DNAs of lymphomas, a number

f somatically acquired proviruses are found, and some ofhem are shown to activate cancer-related genes, such astat5a [13] and c-myc [14], by integration to the targetenes. The ecotropic provirus Emv11 is reported to be suf-

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14 T. Hiratsuka et al. / Leuke

cient for the development of a broad range of B-lineagen NFS mice lymphomas [15,16], but insufficient for pre-

lymphomagenesis [2]. For the latter, a dominant SL/Khllele at Esl1 on MMU17 has been shown to be essential.ongenic NFS.SL/Kh-Bomb1 mice lacked both the Emv11nd SL/Kh alleles of Esl1. Consequently, theoretically, theongenic NFS.SL/Kh-Bomb1 mice will not develop pre-Bymphomas. We injected ecotropic MuLV from an SL/Khymphoma into neonates of NFS.SL/Kh-Bomb1 and NFS. Allirus-injected mice became viremic, but none of them devel-ped lymphomas in 12 months of observation, although itay be argued that the titer of MuLV was not high enough

or target cells to transform.

.3. Conclusion

In conclusion, NFS.SL/Kh-Bomb1 mice showed poly-lonal proliferation of BM BP1low IL-7Rlow pre-B cellsnduced by introgressed Bomb1 in the genetic backgroundf NFS/N. Unlike SL/Kh mice, the congenic mice did notevelop monoclonal proliferation of BM BP1high IL-7Rhigh

re-B cells even later in life. The congenic neonates injectedith the virus did not acquire this population. This congenicodel is an important tool for the analysis of host genetic fac-

ors and virologic factors. Although the gene for the Bomb1ocus has not identified, it is a useful target for the investiga-ion of the growth requirement of the early B-lineage as wells their lymphomagenesis.

cknowledgements

We are grateful for constructive discussion by Dr. Akirahimizu. This study was supported by Grant-in-Aid for Sci-ntific Research (no. 09307004) by Ministry of Education,ulture, Sports and Science, Japan to HH.

Contributions. Hiroshi Hiai managed the whole project,unding, virological analysis, and edited the manuscript.akuya Hiratsuka conducted breeding of congenic mice,enetic analysis, characterization of bone marrow pre-Bells and writing the manuscript. Kohei Kometani andagahiro Minato purified BP1+B220+ cells from BM cells

y cell sorting. Tatsuaki Tsuruyama and Takuro Nakamurauided molecular biology of SL/Kh MuLV lymphomagene-is. Richard Kaszynski and Keiji Tamaki performed geneticnalysis.

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earch 32 (2008) 309–314

eferences

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16] Hartley JW, Chattopadhyay SK, Lander MR, Taddesse-Heath L,Naghashfar Z, Morse III HC, et al. Accelerated appearance of mul-tiple B cell lymphoma types in NFS/N mice congenic for ecotropicmurine leukemia viruses. Lab Invest 2000;80:159–69.