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Am J Pathol. Mar 2004; 164(3): 967–974.
PMCID: PMC1614712

Integration of Epstein-Barr Virus into Chromosome 6q15 of Burkitt Lymphoma Cell Line (Raji) Induces Loss of BACH2 Expression

Abstract

Epstein-Barr virus (EBV) initially isolated from cultured Burkitt lymphoma (BL) cells, is a well-known oncogenic virus. The Raji cell line was established from BL tissue and used for research worldwide. Previous study showed that each Raji cell contains an average of 50–60 EBV genome equivalents, and a significant proportion of the EBV genome is linearly integrated into host genome through BamHI-W close to the BamHI-Y fragment. However, a definitive EBV integration site in the chromosome has not been identified as yet. In this study, direct evidence that EBV DNA is integrated into the host genome was provided through cloning of the fragments containing nucleotide sequence of Raji integration sites. Integrated EBV DNA consisted of the BamHI-W fragment at one end and BamHI-D fragment at another end. Both junction sites were highly guanine/cytosine-rich. The BamHI-W fragment and the adjacent part of chromosome 6 showed 70% homology, while no homology was found between the BamHI-D and adjacent host sequences. EBV was present at intron 1 of the BACH2 gene located on chromosome 6q15. BACH2 was not expressed in the Raji cell line. Because BACH2 is a putative tumor suppressor gene, loss of its expression through EBV integration might contribute to lymphomagenesis.

Epstein-Barr virus (EBV) is a human herpesvirus with a double-stranded DNA genome, and is reported to be associated with development of malignancies including Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC).1–3 EBV DNA is detected as a linear form in the cells at the lytic phase of EBV infection. During the latent phase of infection, the viral genome is maintained as an episomal form through the fusion of genomic termini.4–6 Most BL and NPC tumors harbor episomal EBV genomes, representing the persistently latent infection.

Chromosomal integration of EBV DNA provides an alternative way of persistent infection, which may represent another state of virus-cell interaction.7–12 Integration of EBV into the host genome might be a more common event in lymphoma cells, but analysis of integrated EBV DNA is complicated because highly methylated DNA hinders mapping of EBV genomes and multiple copies of the viral episomes give interfering noise at EBV integration sites.10,13 Large genome size of EBV compared with other virus such as human papilloma virus (HPV), Hepatitis B virus, Polyoma virus, and retrovirus also make identification of the integration site into host genome and analysis of its effect difficult. Therefore the biological influence of EBV integration on the host cells is not assessed as yet.

A majority of previous studies on the intracellular state of EBV DNA have been performed using the Raji cell line, which was established by Pulvertaft14 from BL tissue biopsied from the left maxilla of an 11-year-old black male. Each Raji cell contains an average of 50 to 60 EBV genome equivalents. Anvret et al13 reported that a significant proportion (10 to 12 copies) of the EBV genome might be integrated as a linear form into cellular DNA of the Raji cells through BamHI-W close to the BamHI-Y fragment.

Inverse polymerase chain reaction (PCR) is a useful method for amplification of unknown DNA that flanks one end of a known DNA sequence for which no primers are available.15–17 Using this method, we cloned the fragments containing the nucleotide sequence of Raji integration sites. The present study provides an evidence that the integration of EBV into 6q15 cause in loss of BACH2 expression. Sasaki et al18 previously reported loss of BACH2 expression in the Raji cell line, and showed that enforced BACH2 expression in the Raji cell line resulted in marked reduction of clonogenic activity. They suggested that the loss of BACH2 expression might contribute to lymphomagenesis in B-cell lymphoma including BL. EBV could be an etiological factor for lymphomas through not only expression of its latent genes, such as latent membrane proteins (LMP) and EB nuclear antigens (EBNA), but also integration of virus genome into host chromosome.

Materials and Methods

Cell Lines

EBV-positive cell line Raji was obtained from the Japanese Cancer Research Resources Bank, and was grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum at 37°C in 5% CO2 in air. Other BL-derived cell lines, Daudi, Ramos, Namalwa, BJAB, BL-36, BL-60, BL-137, and NAB-2 were also included in the study. Daudi, Ramos, Namalwa, BJAB, and K562 were obtained from the Japanese Cancer Research Resources Bank. BL-36, -60, and -137 were kindly donated from Dr. G. W. Bornkamm (GSF, Forschungszentrum, Germany). NAB-2 was kindly donated from Dr. P. W. Turker (University of Texas). Lymphoblastoid cell line (LCL) was established in our laboratory by infection of B lymphocytes from healthy volunteer with EBV. OPL-1 was established from the clinical samples of pyothorax-associated lymphoma which developed in patients with chronic pyothorax, shows B-cell immunophenotype, and contains EBV genome.19

Clinical Samples

Clinical samples from 174 cases with B-cell lymphomas were collected and used for immunohistochemical analysis of BACH2 expression. The patients were admitted to hospitals in Osaka, Japan during the period from 2000 to 2003. Age of the patients ranged from 24 to 90 (median, 64.0) years with a male to female ratio of 98:76. Histological specimens were fixed in 10% formalin and routinely processed for paraffin embedding. Histological sections, cut at 4 μm, were stained with hematoxylin and eosin. Immunoperoxidase procedures (avidin-biotin-peroxidase complex method) were performed to determine the immunophenotype of lymphoma: all cases showed a B-cell immunophenotype, ie, CD20+ and/or MB-1+, CD3, CD45RO. Histological slides were reviewed by two of the authors (E. I. and K. A.) to classify the cases according to the World Health Organization classification; diffuse large B-cell lymphoma (DLBCL) in 99 cases, mantle cell (MC) lymphoma in 15, follicular cell lymphoma (FC) in 45, and marginal zone B-cell lymphoma of extranodal type (MALT) in 15. Tonsils from three patients with chronic tonsillitis were used as control to characterize BACH2 expression in lymphoid tissues.

Inverse PCR for Recovery of Junction Site Adjacent to BamHI-W Fragment

Inverse PCRs were designed according to the restriction map indicated by Anvret et al13 (Figure 1A). They demonstrated that HindIII fragments containing BamHI-W sequences joined to host chromosome fragments. DNA isolated from the Raji cell line was digested with HindIII. Ten to 100 ng/μl of DNA were circularized by ligation using T4 DNA ligase, and were subjected to inverse PCR as a template. The primers EbYF, EbWR1, and EbWR2 were designed to extend outward from the BamHI-W and -Y fragments of EBV (Figure 1B; Table 1). Thermocycling conditions for each reaction were 35 cycles of denaturation at 98°C for 5 seconds, annealing and extension at 68°C for 5 minutes, followed by a final 7-minute extension at 72°C. Under the present experimental condition, episomal EBV sequence may not be amplified because of the too large size of the expected products through PCR reaction, ie, more than 40 kbp in size. PCR products were purified using a QIAquick PCR Gel Extraction Kit (Qiagen, Santa Clarita, CA) and cloned into pCR 2.1-TOPO (Invitrogen, Carlsbad, CA). Sequencing was performed by the dideoxy chain termination method using the DNA sequencing kit (Applied Biosystems, Foster City, CA). The samples were analyzed using the Genetic Analyzer (ABI PRISM 310′; Applied Biosystems).

Figure 1
A: A portion of the restriction endonuclease cleavage map of circular EBV DNA from Raji cells and schematic representation of Eco exA fragment reported by Anvret et al13 Restriction site of the BamHI, EcoRI, and HindIII are indicated. The striped area ...
Table 1
Oligonucleotide Primers Used for PCR Reactions

Inverse PCR for Discovery of the Junction at the Other End of EBV

Because integration event could affect the adjacent structure of the host genome, junction at the other end of the EBV insert was investigated. The primer pairs of Ch6F2 and Ch6R were designed to extend outward from the nucleotides that were approximately 850-bp apart from the junction site discovered by the method described above. DNA isolated from the Raji cell line was digested with BglII, BamHI+BglII, and EcoRI. Ten to 100 ng/μl of DNA were circularized by ligation using T4 DNA ligase and were subjected to inverse PCR as a template. Conditions of thermocycling, cloning, and sequencing were the same as mentioned above.

Analysis of Polymorphic Sites Near the BACH2 Locus

Three microsatellite loci, D6S458, D6S1644, and BACH/CA-1, which locate closely to the BACH2 locus, were analyzed by PCR. Primer pairs used were shown in Table 1. After amplification, reaction products (3 μl) were denatured and separated on 6% polyacrylamide gels containing 7 mol/L urea.

PCR Flanking the EBV Integration Site

According to the nucleotide sequences obtained by each inverse PCR, three pairs of primers, Ch6F and EbYR, EbDF and Ch6R, and Ch6F and Ch6R, flanking the EBV integration sites, were designed (Table 1). DNA from the Raji cell lines was used as template. DNA from the peripheral leukocytes of a healthy volunteer was used as control. Thermocycling conditions used were the same as described above.

Southern Blot Analysis and DNA Probes

Ten micrograms of DNA from the cell line were digested with HindIII, BamHI, or EcoRI, electrophoresed on 0.7% to 0.9% agarose gels, and transferred to a Hybond N+ (Amersham Biosciences, Piscataway, NJ) nylon membrane. The filters were hybridized with BamHI-Y and EcoRI-E fragments of EBV, PstI fragments on chromosome 6 and exon 1 of BACH2. BamHI-Y and EcoRI-E fragments in the vector pUC119 were kind gifts from Dr. Kenzo Takada (Hokkaido University). PstI fragments on chromosome 6, and exon 1 of BACH2 were made with use the combination of PCR reaction and restriction enzyme cutting. Probes were radiolabeled by the random prime method according to the indications of the manufacturer (Amersham).

RT-PCR for Detection of BACH2 Expression

RNA from 105 to 2 × 107 cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA), and reverse-transcribed according to the manufacturer’s instructions. (Invitrogen). BACH2 was amplified with primer pair of BaF on exon 7 and BaR on exon 9, which enable to distinguish cDNA from genomic DNA by amplicon sizing. β-actin was amplified with primer pair of AcF and AcR. Thermocycling conditions were 25 cycles of denaturation at 94°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 45 seconds, followed by a final 7-minute extension at 72°C.

Immunohistochemistry

Immunohistochemical study on the paraffin sections was carried out using envision+ system (DAKO, Carpinteria, CA). Rabbit polyclonal anti-human BACH2 antibody (F69-220; kind gift from K. Igarashi, Hiroshima University) was used as primary antibody.

1 × 105 to 107 cells from nine BL cell lines (Raji, Daudi, Ramos, Namalwa, BJAB, BL-36, BL-60, BL-137, and NAB-2), pyothorax-associated lymphoma cell line OPL-1 which contains EBV genome,19 and lymphoblastoid cell lines were collected by centrifuge, fixed in 10% formalin, and routinely processed for paraffin embedding. Clinical samples from 174 cases with B-cell lymphomas including 99 of DLBCL, 15 of MC lymphoma, 45 of FC lymphoma, and 15 of low-grade marginal zone B-cell lymphoma of MALT type were also included in this study.

Results

Inverse-PCR for Recovery of the Junction Site Adjacent to BamHI-W Fragment

Inverse-PCRs were designed according to the restriction map reported by Anvret et al (Figure 1). The primers EbYF, EbWR1 and EbWR2 were designed to extend outward from the BamHI-W and -Y fragments of EBV.

Through two inverse PCR reactions with use of primer pairs of EbYF and EbWR1, and EbYF and EbWR2, two single bands with sizes of 1.75 and 0.65 kbp were obtained respectively (Figure 2A). Each PCR product was cloned into pCR 2.1-TOPO, and named as pIVWYL and pIVWYS, respectively. Sequencing analysis revealed that pIVWYL contained the whole pIVWYS sequence. Both pIVWYL and pIVWYS contained approximately 400 bp of unknown sequences that were sandwiched between BamHI-Y and BamHI-W fragments (Figure 2B). The orientation of BamHI-W and -Y sequences were opposite. Only one HindIII cleavage site present between the unknown sequence and BamHI-Y fragment was found, indicating that this cleavage site was ligated artificially. Standard nucleotide-nucleotide BLAST analysis revealed that the unknown sequences corresponded to nucleotides 59690 to 60124 of clone RP1-104D1 on chromosome 6 (ACC AL159164), which imply that nucleotide 59690 of the clone RP1-104D1 was a candidate for the junction.21

Figure 2
A: Products of inverse PCR reactions with use of primer pairs of EbYF and EbWR1, and EbYF and EbWR2 were separated by electrophoresis on a 1.0% agarose gel. Primers used are shown on the lane of electrophoresis. B: Schematic representation of pIVWYL and ...

Inverse PCR for Discovery of another Junction Site of EBV

According to the information of GenBank of National Center for Biotechnology Information (NCBI), exon 1 of BACH2 was mapped close to the junction site. Fragments containing exon 2 and exon 7 of BACH2 and also fragments between nucleotide 58095 and 58876 of the clone RP1-104D1 could be amplified as expected sizes with use of the genomic DNA from Raji cells as a template (data not shown). From these findings, it was expected that another junction site of EBV might be located in the restricted regions between nucleotide 58876 and 59690 of the clone RP1-104D1. To define another junction site of EBV, the primers Ch6F2 and Ch6R were designed to extend outward around nucleotide 58800 of the clone RP1-104D1 (Figure 3A). DNA isolated from the Raji cell line was digested with BglII, BamHI+BglII, and EcoRI. One hundred to ten hundred ng/μl of the DNA were circularized by ligation and were subjected to inverse PCR as a template. Three independent inverse PCR reactions revealed three single bands with sizes of 3.9 kbp, 2.4 kbp, and 6.1 kbp, respectively. Each PCR product was cloned into pCR 2.1-TOPO, and named as pIV6DM, pIV6DS, and pIV6DL, respectively. Sequencing analysis revealed that the pIV6DL began at nucleotide 58845 and joined at nucleotide 59672 of the clone RP1-104D1 and continued to the BamHI-D fragment of EBV at nucleotide 129728 (Figure 3B). The other plasmids, pIV6DM and pIV6DS, also contained the same junction site.

Figure 3
A: Schematic representation indicating the sites and direction of primers Ch6F2 and Ch6R used for inverse PCR for recovery of another junction site. B: Schematic representation of pIVD6 mol/L, pIVD6S, and pIVD6L, which were obtained by inverse PCR. All ...

Analysis of Polymorphic Sites Near the BACH2 Locus

All three microsatellite markers near the junction (D6S458, D6S1644, and BACH/CA-1) revealed the heterogeneous results, indicating the presence of both paternal and maternal chromosomes. Large deletion events did not occur around the junction (data not shown).

PCR Flanking the EBV Integration Sites

Both of the predicted integration sites were located on clone RP1-104D1 of chromosome 6q15. Each integration site was separated by only 17 bp. To examine whether the nucleotide sequences obtained by the inverse PCRs were identical to the EBV integration sites of the Raji cell line, two pairs of oligonucleotide primers flanking the junction site between chromosome 6 and the BamHI-Y fragment, and the BamHI-D fragment and chromosome 6, and one pair of primers flanking the two junction sites were designed as shown in Figure 4. PCR reactions using DNA from Raji with the primer pairs Ch6F and EbYR, and EbDF and Ch6R revealed two single bands with the expected sizes, 2.4 kb and 1.0 kb, respectively. The sequencing analysis revealed that both PCR products consisted of the junction sites identical to those indicated by inverse PCR (data not shown). An expected single band of 1.1 kbp in size was obtained with the primer pairs, Ch6F and Ch6R, using the control DNA as template. However, no bands were observed when DNA from Raji was used as template. These findings indicate that both paternal and maternal chromosomes 6 were interrupted at the same regions where integration of EBV was demonstrated.

Figure 4
A: Schematic representation of EBV integration on clone RP1-104D1 on chromosome 6. B: Restriction enzyme map around the junction sites (I) and (II) in A based on the results of inverse PCR and genome database from NCBI. Site and orientation of the primers ...

Southern Blot Analysis

According to the nucleotide sequence of the junction sites and information from GenBank of NCBI, the junction site at the BamHI-W consisted of 3.3 kbp of BamHI fragments together with exon 1 of BACH2. The junction site at the BamHI-D consisted of 6.1 kbp of EcoRI fragments. To further confirm these findings, genomic Southern blotting analysis was performed using the probes shown in Figure 4B. The BamHI-Y fragment can detect both BamHI-W and -Y fragment because of its homology. Probes BamHI-Y and exon 1 of BACH2 hybridized to the restriction fragments with an identical size of 3.3 kbp, which corresponded to the fragments containing the junction site at BamHI-W. EcoRI-E and Ch6 PstI fragments hybridized to 6.1 kbp of EcoRI restriction fragments, which corresponded to the fragments containing the junction site at BamHI-D (Figure 5).

Figure 5
Southern blot autoradiographs of BamHI-, and EcoRI-digested DNA probed with BamHI-Y fragment of EBV (Y), exon 1 of BACH2 (6Ba), EcoRI-E fragment of EBV (E), and PstI fragment of the chromosome 6 (6P). E, W, and Y indicate the bands corresponding to Eco ...

Sequence Analysis of the Raji-EBV Junction Sites

Sequences of the Raji-EBV junction sites were compared to those of chromosome 6 and BamHI-W and -D fragments (Figure 6). The flanking region of chromosome 6 at the junction site showed 70% homology to the junction site of BamHI-W and was highly guanine/cytosine (G/C)-rich (more than 70%), while no homology was observed around the junction at BamHI-D. G/C content in the regions flanking the junction site of chromosome 6 and EBV were 72% and 62%, respectively.

Figure 6
Sequences of the Raji-EBV junction sites. Sequence from the Raji cell line are shown in italic letters. The unrecombined site obtained from clone RP1-104D1 are shown in the upper line and BamHI-W and -D fragments of EBV genome from strain B95-8 in the ...

Expression of BACH2 in Burkitt Cell Lines and B-Cell Lymphomas

According to the GenBank of NCBI, the integration site of EBV is located between exon 1 and 2 of BACH2 (Figure 4). To investigate whether the Raji cell line expresses BACH2 or not, RT-PCR analysis was performed (Figure 7A). Raji cells did not expressed BACH2 mRNA, which was consistent with the previous report.18

Figure 7
A: Expression of BACH2 in Raji, other cell lines, and tissue sample from tonsil determined by RT-PCR. β-actin was used as a control. Raji cells did not express the BACH2 mRNA, which was consistent with the previous report. Cell lines used are ...

Immunohistochemistry revealed that BACH2 protein was detected in the cytoplasm in the control cell lines, LCL, and OPL-1 as well as germinal center of the tonsil, but never in Raji cell line (Figure 7B).

To delineate the role of BACH2 expression in lymphomagenesis, clinical samples from 174 cases with B-cell lymphomas together with 9 BL cell lines were examined immunohistochemically. Germinal center cells and occasionally mantle zone lymphocytes in the tonsil showed positive immunoreactivity for BACH2. Among B-cell lymphomas, positive reactivity was found in 1 of 15 cases (6.7%) of marginal zone-B cell lymphoma of MALT type, 39 of 45 (86.7%) of FC lymphoma, 1 of 15 cases (6.7%) of MC lymphoma, 33 of 99 cases (33.3%) of DL, respectively. Among BL cell lines, all but Raji cell lines expressed BACH2 protein.

Discussion

Previous studies demonstrated EBV integration in the chromosomes of tumor cells in biopsy specimens or cell lines of BL,7,12,22 other kinds of B-cell lymphomas,8,23 and NPC.10,24 In these studies, integration sites of EBV were demonstrated indirectly by Gardella gels25 and/or by Southern blot analysis. Localizations of EBV on the chromosomes were shown by in situ hybridization method. Although integration might be an important mechanism for interaction of EBV with cellular genes, particularly those genes involved in cell-growth regulation and tumorigenesis, neither direct evidence of integration nor inactivation of suppressor genes through integration were provided until present. This might be due to the presence of highly methylated DNA, which hinders mapping of EBV genomes and multiple copies of the viral episomes which give interfering noise at investigation of EBV integration sites.10,13 Large genome size of EBV compared with other viruses such as HPV, Hepatitis B virus, Polyoma virus, and retrovirus also make identification of the integration site and analysis of its influence difficult. Here we provided the direct evidence that EBV is integrated into host genomes, which encode the putative suppressor gene BACH2 in BL cell line (Raji). Loss of BACH2 expression was demonstrated at mRNA and protein level.

The present results showed that both paternal and maternal chromosome 6 were interrupted in the same region. Possible mechanisms for this phenomenon are as follows: two junctions come from the same allele, resulting in deletion of the intervening 17 nucleotides of chromosomal DNA. In this case, the same region in the paired allele might be interrupted by some chromosomal abnormalities such as deletion, inversion, or EBV integration. Another possibility is that two junctions come from the different alleles. In this case, two additional junction sites must be present. Each Raji cell contains an average of 50 to 60 EBV genome equivalents; thus, Southern blot analysis to examine state of EBV genome in Raji cell line is complicated.6 There are several aberrant bands in the present analysis (Figure 6), possibly due to the heterogeneity of EBV. Therefore, additional integration sites could be cloned by analyzing each aberrant band.

BamHI-W and the adjacent part of chromosome 6 showed a strong homology and G/C-rich sequences, suggesting that homologous recombination is the mechanism of integration. No obvious homology was found between the BamHI-D and adjacent host sequences. These findings at both EBV junction sites in Raji were different from those in Namalwa, in which no homology was present between G/C-rich EBV terminal repeat and adenine thymine-rich adjacent host sequences.11

In the Raji cell line, the integration site of EBV was mapped to chromosome 6q15, which is intron 1 of BACH2. The present study further showed that Raji cells do not express BACH2 mRNA, which is consistent with the finding of a previous report.18 Immunohistochemistry revealed the loss of BACH2 protein expression. BACH2 is a transcription factor belonging to the Bric-a-bric, Tramtrack, and Broad complex (BTB)-basic region leucine zipper (bZip) factor family.19,26 BACH2 forms homodimers or heterodimers with Maf-related transcription factors, which bind to a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element and a related Maf-recognition element (MARE), and regulate transcription of these elements.18 BACH2 is abundantly expressed in lymphocytes of B-cell lineage as well as lymphoma cell lines of B-cell origin including other BL cell lines than Raji cell lines.18 Sasaki et al18 demonstrated that enforced expression of BACH2 through infection with the retrovirus harboring the human BACH2 cDNA in the Raji cell line resulted in marked reduction of clonogenic activity, indicating that BACH2 possesses an inhibitory effect on cell proliferation and thus might be involved in tumorigenesis of Raji cells. Vieira et al27 suggest that repression of BACH2 by products of a BCR/ABL chimera gene generated in chronic myeloid leukemia and Ph1-positive acute lymphoblastic leukemia cell lines might be involved in lymphoid transformation. LOH of BACH2 gene was found in B-cell non-Hodgkin’s lymphoma.18 All of these findings might suggest a role of BACH2 in lymphomagenesis.

Among BL cell lines, BACH2 was commonly expressed except for Raji, suggesting that loss of BACH2 expression might not be common events in BL. In the clinical samples from B-cell lymphomas, the frequency of BACH2 positivity, ie, high in FC lymphoma and low in MC lymphoma and marginal zone B-cell lymphoma of MALT type, might reflect the origin of the tumors, because BACH2 were strongly positive in the germinal center cells and weakly positive in mantle and marginal zone cells of tonsil. EBV is known to be exclusively detected in large cell lymphoma among B-cell lymphomas. Two third of cases with DL examined here did not express BACH2. Further study to examine correlation between integration of EBV genome and BACH2 protein expression is necessary.

In HPV, the significance of the integration on the tumorigenesis is well documented. Premalignant cervical lesions usually harbor HPV as episomal form. Whereas HPV is integrated into a single locus within the host cell genome of cervical carcinoma cells.28 Thus, HPV integration seems to be associated with the acquisition of the malignant phenotype. Integrations, deletions, and/or rearrangements of HPV genome, which express E6 and E7 genes, underscore the importance of the continued expression of these genes in the maintenance of the malignant phenotype. Integration events preferentially occur within the common fragile sites containing genes that are important in the development of cervical tumors.29 HPV integration causes deletions and complex rearrangements of the cellular DNA that may affect the expression of cellular genes. These findings suggest that HPV integration might play important role in cervical tumorigenesis.

When compared to role of HPV in cervical carcinogenesis, the effect of EBV integration into the host genome remain to be clarified. Technique for efficient amplification of the junctions between the EBV genome and flanking cellular sequences at viral integration sites has not been established as yet. The present study demonstrated that EBV could be a causative factor for lymphomagenesis through not only its latent gene expression but also through reduced expression of tumor suppressor gene by integration of the virus into the specific sites of host genome. The Raji cell line, which is commonly used worldwide, could be a suitable tool for analysis of the integration effect. Further study is necessary to clarify the sequel of EBV integration. In any way, present findings give an insight for paradigm in EBV oncogenesis.

Acknowledgments

We thank Dr. Kenzo Takada for critically reviewing the manuscript, Dr. Yoichi Tani for technical advice about immunohistochemistry, Dr. G.W. Bornkamm and Dr. P.W. Turker for providing us the BL-36, -60, -137, and NAB-2 cell lines, respectively, and Dr. Kazuhiko Igarashi for providing the polyclonal antibody (B79-2).

Footnotes

Address reprint requests to Tetsuya Takakuwa, Department of Pathology (C3), Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. .pj.ca.u-akaso.dem.htaplom@awukakat :liam-E

Supported by grants from the Ministry of Education, Science, Culture, and Sports, Japan (14031213, 15026209, 15406013, 15590340).

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