T

Cp

MYD1

a

ARRA

KDBG

semtaom(c2aGiipc

0d

Veterinary Immunology and Immunopathology 140 (2011) 166–169

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology

journa l homepage: www.e lsev ier .com/ locate /vet imm

echnical report

haracterization of canine BCL6 cDNA and detection systems for itsrotein expression

asahiko Sato, Yuko Goto-Koshino, Hideyuki Kanemoto, Hiroyuki Mochizuki,asuhito Fujino, Koichi Ohno, Hajime Tsujimoto ∗

epartment of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo13-8657, Japan

r t i c l e i n f o

rticle history:eceived 21 May 2010eceived in revised form 18 August 2010ccepted 22 August 2010

a b s t r a c t

BCL6 is known to be a key molecule in germinal center (GC) formation of lymph nodes,and its expression profiles have been implicated in the prognosis of diffuse large B-celllymphoma in humans. The present study was carried out to characterize canine BCL6 cDNAand to indicate the technical methods for detection of the BCL6 protein in dog tissues. Thededuced amino acid sequence of canine BCL6 showed close homology to that of human BCL6

eywords:ogCL6erminal center

(96.3%), especially in the zinc-finger motifs and POZ (poxvirus and zinc finger) domain withcomplete identity. Immunoblot analysis of a canine lymph node with an anti-human BCL6monoclonal antibody revealed a band of 80 kDa. Immunohistochemical staining using thesame antibody produced positive reactions in the cells exclusively localized in the GC of acanine lymph node. This study will be useful for the molecular classification of canine B-cell

ifferen

lymphomas with d

The human BCL6 gene encodes a 95-kD nuclearequence-specific transcriptional repressor (Deweindtt al., 1995; Chang et al., 1996; Seyfert et al., 1996). It isainly expressed in the nucleus of B-cells in germinal cen-

ers (GCs) (Cattoretti et al., 1995). BCL6 has received muchttention with regard to the classification and prognosisf human non-Hodgkin’s lymphoma (NHL). The most com-on type of human NHL are diffuse large B-cell lymphomas

DLBCL), accounting for 30–40% of adult NHL (Jaffe, 1998). Aomprehensive gene expression profiling study identifiedmolecularly distinct forms of DLBCL: GC-like DLBCL char-cterized by the expression of genes normally expressed inC B-cells and activated B-cell (ABC)-like DLBCL character-

zed by the expression of genes normally induced duringn vitro activation of B-cells. The overall survival time inatients with GC-like DLBCL was reported to be signifi-antly longer than that in patients with ABC-like DLBCL

∗ Corresponding author. Tel.: +81 3 5841 5402; fax: +81 3 5841 5640.E-mail address: atsuji@mail.ecc.u-tokyo.ac.jp (H. Tsujimoto).

165-2427/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.vetimm.2010.08.009

t prognoses.© 2010 Elsevier B.V. All rights reserved.

(Alizadeh et al., 2000). The BCL6 gene is considered to beone of the hallmarks of GC-like DLBCL, and its expression isknown to be strongly indicative of the survival in patientswith DLBCL (Lossos et al., 2001).

Lymphoma is one of the most common malignanttumors in dogs, accounting for 83% of all hematopoi-etic tumors and 7–24% of all neoplasms (Jacobs et al.,2002; Kaiser, 1981). Since the representative histologictype of canine lymphoma is DLBCL (Vezzali et al., 2010),it has been utilized as an attractive model for humanDLBCL (Jamadar-Shroff et al., 2009). Although humanand canine DLBCL share many similarities, molecularapproaches to predict the prognosis of canine lymphomaare still lacking. It can be speculated that measurement ofBCL6 gene expression would be useful for predicting theprognosis of canine patients with DLBCL as indicated in

humans.

To our knowledge, cloning of canine BCL6 cDNA and itsprotein detection system have not been reported. As thefirst step to examine BCL6 gene expression in dogs with B-cell lymphoma, we molecularly cloned and characterized

y and Im

M. Sato et al. / Veterinary Immunolog

canine BCL6 cDNA and assessed the technical methods todetect the BCL6 protein in dog tissues.

A popliteal lymph node was surgically resected from ahealthy adult beagle dog. This procedure was conducted inaccordance with the guidelines of the Animal Care Commit-tee of the Graduate School of Agricultural and Life Sciences,the University of Tokyo.

Total RNA was extracted from the normal lymph nodewith a commercial available kit (RNAqueous, Ambion, TX,USA) and treated with a DNA-free kit (DNase I, Invit-rogen, CA, USA) to prevent contamination by genomicDNA. Reverse transcription of RNA was performed witha cDNA synthesis kit (PrimeScript RT-PCR Kit, Takara,Shiga, Japan). Template cDNA was amplified by PCR withDNA polymerase (Phusion High-Fidelity DNA Polymerase,Finnzymes, Espoo, Finland) using the following primerpairs: 5′-TGA ATC AAG GCA TTG GGT GA-3′ (forward)and 5′-TTT GGG TAG ATT CTG AGA AGG GA-3′ (reverse).These primers were designed on the basis of the predictedsequence of canine BCL6 (GenBank accession number;XM 545248). The PCR product was analyzed by 1% agarosegel electrophoresis and purified from the agarose gel witha commercially available kit (Wizard SV Gel and PCRClean-Up System, Promega Corporation, WI, USA). Follow-

ing A-tailing, the purified PCR product was cloned into aT/A cloning vector (pGEM-T Easy) (Promega Corporation).Plasmid DNA was extracted with a plasmid purificationkit (NucleoSpin Plasmid QuickPure, MACHEREY-NAGEL,Duren, Germany) and sequenced by use of the dideoxy

Fig. 1. Comparison of the deduced amino acid sequence of canine BCL6 cDNA (Ge(GenBank accession number; NP 001124317.1). The amino-terminal POZ domainlines. Dots indicate identical residues.

munopathology 140 (2011) 166–169 167

chain termination method with 4 primers; the 5′- and3′-end were identified with 2 primers complimentary toT7 and SP6 promoter sites of the vector and the innersite was identified with 2 primers complimentary to thepredicted sequences of canine BCL6 (nucleotides 500-519: 5′-CAA GAC ATC ATG GCC TAT CG-3′, nucleotides1026-1044: 5′-GTC TGG TTA GTC CAC AGA G-3′, GenBankaccession number; XM 545248), which were designed tooverlap the each fragment. Eight independent clones of thePCR product were sequenced to avoid errors in sequenceanalysis.

A protein sample was extracted from the normal caninelymph node using lysis buffer (50 mM Tris pH 7.5, 100 mMNaCl, 1.0% NP-40, 10% Glycerol, 1 mM EDTA), analyzedby SDS-PAGE using 12.5% gels, and blotted onto a PVDFmembrane (Amersham Hybond-P PVDF membrane, GEHealthcare, NJ, USA). The membrane was blocked witha commercially available blocking buffer (ECL Advanceblocking agent, GE Healthcare). Immunoblotting was car-ried out with a rabbit monoclonal anti-human BCL6antibody (clone: EP529Y; Abcam, MA, USA) diluted at1:3000 in Tris-HCL buffer saline with 0.1% Tween 20 (TBS-T)by incubation for 1 h at room temperature. After washingin TBS-T, the membrane was incubated with horseradish

peroxidase-labeled goat anti-rabbit IgG (Abcam) diluted at1:12000 in TBS-T for 1 h at room temperature. Immun-odetection was performed by chemiluminescence (ECLAdvance, GE Healthcare) according to the manufacturer’sprotocol.

nBank accession number; HM008708) with that of its human counterpartis boxed with a solid line. Six zinc-finger motifs are boxed with dashed

168 M. Sato et al. / Veterinary Immunology and Immunopathology 140 (2011) 166–169

F immuna toxylin am inal zon

teib5bpaB1wwlfwUh

fTBamcct

ig. 2. Immunoblot analysis of a canine lymph node (A), and histology andnti-human BCL6 antibody (B–E). Serial sections were subjected to hemaonoclonal antibody (C and E). *Germinal center, **Mantle zone, ***Marg

Immunohistochemistry was performed on 5-�m-hick sections obtained from a formalin-fixed, paraffin-mbedded normal canine lymph node sample. Heat-nduced antigen retrieval was performed in EDTAuffer (1 mM EDTA, pH 8.0) using an autoclave formin at 121 ◦C. Endogenous peroxidase was blockedy 3% hydrogen peroxidase in methanol at room tem-erature for 5 min. The slides were incubated with

primary antibody, rabbit monoclonal anti-humanCL6 antibody (clone: EP529Y, Abcam), diluted at:50 for 60 min at room temperature. After reactionith the primary antibody, the slides were incubatedith Envison polymer reagent (Envison + System-HRP

abeled polymer anti-Rabbit, Dako-Japan, Kyoto, Japan)or 60 min at room temperature. All reaction productsere visualized with 3,3′-diaminobenzidine (Sigma, MO,SA). The sections were counterstained with Mayer’sematoxylin.

The PCR product contained the entire open readingrame of canine BCL6 cDNA consisting of 2121 nucleotides.he sequence showed high homology to that of humanCL6 cDNA with 96.3% identity with the deduced amino

cid sequence. The amino acid sequences of the zinc-fingerotifs and POZ (poxvirus and zinc finger) domain were

ompletely identical between the canine and human BCL6DNAs (Fig. 1), indicating the analogous DNA binding func-ion of canine BCL6.

ohistochemistry of a canine lymph node using serial tissue sections withnd eosin stain (B, D) and immunohistochemistry with anti-human BCL6e.

Immunoblot analysis using the anti-human BCL6 anti-body gave a single band with a size of approximately80 kDa, which was identical to the molecular weightexpected from the amino acid sequence of canine BCL6(Fig. 2A). In the immunohistochemical analysis of thenormal canine lymph node, positively stained cells wereexclusively localized in the GC, but not in the mantleand marginal zones in the cortex and in the medulla(Fig. 2B–E). In the positively stained GC cells, a positivereaction was observed in the nucleus but not in the cyto-plasm. This staining pattern, topographically restricted tothe GC but absent from most other lymphocytes, wasconsistent with the BCL6 expression pattern in humans(Cattoretti et al., 1995). The results of the immunoblotand immunohistochemical analyses showed that the anti-human BCL6 antibody used in this study conceivablycross-reacted with canine BCL6. Furthermore, the analo-gous expression pattern of BCL6 in the canine lymph nodestrongly indicates that it has the same role as its humancounterpart.

On the basis of these findings, the technical proce-dure to detect the BCL6 protein in the canine lymph node

will provide useful information for the classification ofcanine B-cell lymphomas with different prognoses. Fur-ther study is needed to indicate the clinical usefulness ofthe detection of BCL6 for prognostic significance in caninelymphoma.

y and Im

Seyfert, V.L., Allman, D., He, Y., Staudt, L.M., 1996. Transcriptional

M. Sato et al. / Veterinary Immunolog

Conflict interest

None of the authors have any conflicts of interest asso-ciated with this study.

References

Alizadeh, A.A., Eisen, M.B., Davis, R.E., Ma, C., Lossos, I.S., Rosenwald, A.,Boldrick, J.C., Sabet, H., Tran, T., Yu, X., Powell, J.I., Yang, L., Marti, G.E.,Moore, T., Hudson Jr., J., Lu, L., Lewis, D.B., Tibshirani, R., Sherlock, G.,Chan, W.C., Greiner, T.C., Weisenburger, D.D., Armitage, J.O., Warnke,R., Levy, R., Wilson, W., Grever, M.R., Byrd, J.C., Botstein, D., Brown,P.O., Staudt, L.M., 2000. Distinct types of diffuse large B-cell lymphomaidentified by gene expression profiling. Nature 403, 503–511.

Cattoretti, G., Chang, C.C., Cechova, K., Zhang, J., Ye, B.H., Falini, B., Louie,D.C., Offit, K., Chaganti, R.S.K., Dalla-Favera, R., 1995. BCL-6 protein isexpressed in germinal-center B cells. Blood 86, 45–53.

Chang, C.C., Ye, B.H., Chaganti, R.S., Dalla-Favera, R., 1996. BCL-6, aPOZ/zinc-finger protein, is a sequence-specific transcriptional repres-sor. Proc. Natl. Acad. Sci. U.S.A. 93, 6947–6952.

Deweindt, C., Albagli, O., Bernardin, F., Dhordain, P., Quief, S., Lantoine, D.,Kerckaert, J.P., Leprince, D., 1995. The LAZ3/BCL6 oncogene encodesa sequence-specific transcriptional inhibitor: a novel function for the

munopathology 140 (2011) 166–169 169

BTB/POZ domain as an autonomous repressing domain. Cell GrowthDiffer. 6, 1495–1503.

Jacobs, R.M., Messick, J.B., Valli, V.E., 2002. Tumors of the hemolymphaticsystem. In: Meuten, D.J. (Ed.), Tumors of Domestic Animals. , 4th ed.State Press, Iowa, p. 138.

Jaffe, E.S., 1998. Histopathology of the non-Hodgkin’s lymphomas andHodgkin’s disease. In: Canellos, G.P., Listrer, T.A., Sklar, J.L. (Eds.), TheLymphomas. WB Saunders, Philadelphia, PA, pp. 77–106.

Jamadar-Shroff, V., Papich, M.G., Suter, S.E., 2009. Soy-derived isoflavonesinhibit the growth of canine lymphoid cell lines. Clin. Cancer Res. 15,1269–1276.

Kaiser, H.E., 1981. Animal neoplasms: a systemic review. In: Kaiser, H.E.(Ed.), Neoplasms – Comparative Pathology in Animals, Plants and Man.Williams and Wilkins, Baltimore, pp. 747–812.

Lossos, I.S., Jones, C.D., Warnke, R., Natkunam, Y., Kaizer, H., Zehnder,J.L., Tibshirani, R., Levy, R., 2001. Expression of a single gene, BCL-6,strongly predicts survival in patients with diffuse large B-cell lym-phoma. Blood 98, 945–951.

repression by the proto-oncogene BCL-6. Oncogene 12, 2331–2342.

Vezzali, E., Parodi, A.L., Marcato, P.S., Bettini, G., 2010. Histopathologicclassification of 171 cases of canine and feline non-Hodgkin lym-phoma according to the WHO. Vet. Comp. Oncol. 8, 38–49.