Unité de Physiopathologie Cellulaire et Moléculaire, UPR 2163-CNRS, CHU-Purpan, Avenue de Grande-Bretagne, 31059 Toulouse Cédex, France1
Author for correspondence: Fabienne Meggetto. Fax +33 5 61 49 90 36. e-mail fabienne.meggetto{at}immgen.cnrs.fr
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Abstract |
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Introduction |
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In the present study, we studied 13 patients with EBV-associated HD in which EBV-positive RS cells co-existed with rare, EBV-positive bystander small lymphocytes. Of these cases, frozen specimens were available for three cases and in two of them we also had the corresponding lymphoblastoid cell lines. These cases are different from those reported previously, which were not included in the present study because frozen lymph node biopsy specimens were exhausted (Meggetto et al., 1997 ). In each case, we performed a PCR assay to determine the LMP-1 gene sequence, using DNA extracted from (i) whole lymph node cells and (ii) single RS cells isolated from tissue sections by micromanipulation. The analysis of LMP-1 sequence polymorphism using single-cell PCR demonstrates that, in each case, all RS cells were infected by the same EBV genome, which may be different from that infecting bystander B lymphocytes.
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Methods |
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PCR analysis of the LMP-1 gene.
Total DNA extracted from lymph node biopsies affected by HD was amplified by the PCR strategy described by Mehl et al. (1998) . Briefly, LMP-1 PCR products were amplified by nested PCR with two sets of primers. For the 5'-fragment, the primers were as follows: 8617 (5' GGTCCGTCGCCGGCTCCACTCACGAGCAGG 3'; B95.8 coordinates 168617168646) and 9580 (5' CCAAGAAACACGCGTTACTCTGACGTAGCC 3'; 169551169580); for nested PCR, 8667 (5' GTTAGAGTCAGATTCATGGCCAGAATCATCG 3'; 168667168697) and 9494 (5' CCTGACACACTGCCCTCGAGG 3'; 169471169494). For the 3'-fragment, the primers were as follows: 7832 (5' GCCTGGTAGTTGTGTTGTGCAGAGGTC 3'; 167832167858) and 8785 (5' CGATTTTAATCTGGATGTATTACCATGG 3'; 168758168785); for nested PCR, 7901 (5' GGCGGAGTCTGGCAACGCCCGGGTCCTTG 3'; 167901167929) and 8702 (5' GCTACCGATGATTCTGGCCATGAATCTGAC 3'; 168673168702).
PCR amplification was performed in a final volume of 50 µl containing 1 µl of each primer (50 µM), 5 µl 10x polymerase buffer, 8 µl each dNTP (1·25 mM), 5 µl DMSO, 500 ng genomic DNA and 1·25 U AmpliTaq Gold polymerase (Perkin Elmer). Reaction mixtures were covered with mineral oil, placed in a 480 thermal cycler (Perkin Elmer) and subjected to the following program: 35 cycles at 94 °C for 1 min, 65 °C (or 55 °C for the nested PCR) for 1 min and 72 °C for 1 min. For all PCR amplifications, a pre-PCR heating step at 94 °C for 10 min to activate the AmpliTaq Gold enzyme and a final period of 10 min at 72 °C to complete the reaction were included. PCR products were analysed by agarose gel electrophoresis.
DNA sequencing.
Fifty ng of each PCR product was sequenced with an ABI PRISM dye terminator kit (Perkin Elmer) supplemented with 7·5 pmol of each primer. Reaction mixtures were placed in a 2400 thermal cycler (Perkin Elmer) and subjected to a program that followed the manufacturers recommendations. Sequence analysis was performed with the OMIGA software. In addition to the primers used for amplification, internal primers used for sequencing were: LMP-2 (5' GACTGGACTGGAGGAGCCCTC 3'; B95.8 coordinates 169319169339), LMP-4 bis (5' CTCTCTGGAATTTGCACGGA 3'; 169091169110), LMP-9S (5' ATCATTTCCAGCAGAGTCGC 3'; 168370168389), LMP-13AS (5' AACGAGGGCAGACACCACCT 3'; 168644168663) and LMP-11AS (5' TGATTAGCTAAGGCATTCCCT 3'; 168075168095).
Extraction of DNA from single cells.
Each isolated cell was placed in a tube containing 12 µl extraction solution (50 mM TrisHCl, pH 8, 10 mM EDTA, 100 mM NaCl, 200 µg/ml proteinase K). After an overnight proteinase K digestion at 37 °C followed by 10 min inactivation of the proteolytic enzyme at 95 °C, the extracted DNA was used for PCR amplification.
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Results and Discussion |
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PCR amplifications of the 5'-segment of the LMP-1 gene of total DNA extracted from the lymph node of the three cases yielded single fragments. PCR amplification of the 3' region of cases 2 and 3 yielded only single fragments, whereas two distinct fragments were obtained for case 1 (designated Ta and Tb), which were cloned into pGEM-T. Fragments Ta and Tb were of different sizes compared with the 3'-fragment amplified from the standard B95.8 used as a control. These results suggested that the lymph node of case 1 contained two EBV strains (Table 1).
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Single-cell PCR amplification of the LMP-1 gene in RS cells
For this purpose, 8 µm frozen sections were immunostained with monoclonal antibodies directed against the LMP-1 protein of EBV [anti-LMP-1 antibody (CS1-4); Dako] to visualize EBV-positive RS cells by the alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP) technique (Fig. 2a) (Cordell et al., 1984
). After overlaying the frozen section with PBS, eight single cells were picked under the microscope with a closed glass capillary using a hydraulic micromanipulator and then transferred by aspiration to an open glass capillary with another micromanipulator (Fig. 2b
, c
) (Gravel et al., 1998
). DNA was extracted from each isolated cell as described in Methods and used for PCR amplification.
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In most cases of EBV-associated HD, EBV-positive RS cells and rare, EBV-positive bystander B lymphocytes co-exist in the same lymph node (Hummel et al., 1992 ; Brousset at al, 1993
). In our previous study, based on two cases of EBV-associated HD, we amplified two fragments with a different LMP-1 polymorphism (Meggetto et al., 1997
). We showed a dual EBV infection in lymph nodes affected by HD, but we could not completely exclude the possibility that subsets of tumour cells and small lymphocytes shared the same virus strain. Using single-cell PCR and sequence analysis of the LMP-1 gene, we now strengthen the argument that RS cells and bystander B lymphocytes may be infected by different EBV isolates.
In previous reports, EBV clonality in HD was based on RFLP analysis of EBV terminal repeats (Raab-Traub, 1989 ). However, we now know that two different EBV strains may have the same number of terminal repeats (Sato et al., 1990
). On the basis of the LMP-1 sequence, the results of the present study further confirm that, in RS cells, the EBV genome is clonal. Our findings are in keeping with those reported by other groups, which demonstrated by single-cell PCR that RS cells showed clonal Ig gene rearrangement (Kuppers et al., 1998
; Stein & Hummel, 1999
).
The question arises as to the origin of these two strains and whether they could be derived from a unique strain by accumulation of mutations and recombination events. Co-infection with multiple EBV genotypes is commonly found in human immunodeficiency virus-positive patients (Berger et al., 1999 ; Sixbey et al., 1989
), but the identification of multiple virus isolates has not been documented in lymphocytes from non-immunocompromised patients. Our preliminary results suggest that EBV strains infecting RS and bystander lymphocytes are phylogenetically related but different from those found in reactive lymph nodes from patients with non-neoplastic lymphoproliferative disorders (unpublished data).
Whatever the origin of the EBV strains infecting RS cells, it is possible to speculate that LMP-1 sequence variations may alter the oncogenic properties and/or the immunogenicity of the LMP-1 protein.
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Acknowledgments |
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References |
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Baer, R., Bankier, A. T., Biggin, M. D., Deininger, P. L., Farrell, P. J., Gibson, T. J., Hatfull, G., Hudson, G. S., Satchwell, S. C., Seguin, C. and others (1984). DNA sequence and expression of the B95-8 EpsteinBarr virus genome. Nature 310, 207211.[Medline]
Baichwal, V. R. & Sugden, B. (1988). Transformation of Balb 3T3 cells by the BNLF-1 gene of EpsteinBarr virus. Oncogene 2, 461-467.[Medline]
Berger, C., van Baarle, D., Kersten, M. J., Klein, M. R., Al-Homsi, A. S., Dunn, B., McQuain, C., van Oers, R. & Knecht, H. (1999). Carboxy terminal variants of EpsteinBarr virus-encoded latent membrane protein 1 during long-term human immunodeficiency virus infection: reliable markers for individual strain identification. Journal of Infectious Diseases 179, 240-244.[Medline]
Brousset, P., Meggetto, F., Chittal, S., Bibeau, F., Arnaud, J., Rubin, B. & Delsol, G. (1993). Assessment of the methods for the detection of EpsteinBarr virus nucleic acids and related gene products in Hodgkins disease. Laboratory Investigation 69, 483-490.[Medline]
Cordell, J. L., Falini, B., Erber, W. N., Ghosh, A. K., Abdulaziz, Z., MacDonald, S., Pulford, K. A., Stein, H. & Mason, D. Y. (1984). Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). Journal of Histochemistry and Cytochemistry 32, 219-229.[Abstract]
Delsol, G., Brousset, P., Chittal, S. & Rigal-Huguet, F. (1992). Correlation of the expression of EpsteinBarr virus latent membrane protein and in situ hybridization with biotinylated BamHI-W probes in Hodgkins disease. American Journal of Pathology 140, 247-253.[Abstract]
Gravel, S., Delsol, G. & Al Saati, T. (1998). Single-cell analysis of the t(14;18)(q32;q21) chromosomal translocation in Hodgkins disease demonstrates the absence of this translocation in neoplastic Hodgkin and ReedSternberg cells. Blood 91, 2866-2874.
Herbst, H., Niedobitek, G., Kneba, M., Hummel, M., Finn, T., Anagnostopoulos, I., Bergholz, M., Krieger, G. & Stein, H. (1990). High incidence of EpsteinBarr virus genomes in Hodgkins disease. American Journal of Pathology 137, 13-18.[Abstract]
Herbst, H., Dallenbach, F., Hummel, M., Niedobitek, G., Pileri, S., Muller-Lantzsch, N. & Stein, H. (1991). EpsteinBarr virus latent membrane protein expression in Hodgkin and ReedSternberg cells. Proceedings of the National Academy of Sciences, USA 88, 4766-4770.
Hummel, M., Anagnostopoulos, I., Dallenbach, F., Korbjuhn, P., Dimmler, C. & Stein, H. (1992). EBV infection patterns in Hodgkins disease and normal lymphoid tissue: expression and cellular localization of EBV gene products. British Journal of Haematology 82, 689-694.[Medline]
Izumi, K. M. & Kieff, E. D. (1997). The EpsteinBarr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-B. Proceedings of the National Academy of Sciences, USA 94, 12592-12597.
Kaye, K. M., Devergne, O., Harada, J. N., Izumi, K. M., Yalamanchili, R., Kieff, E. & Mosialos, G. (1996). Tumor necrosis factor receptor associated factor 2 is a mediator of NF-B activation by latent infection membrane protein 1, the EpsteinBarr virus transforming protein. Proceedings of the National Academy of Sciences, USA 93, 11085-11090.
Knecht, H., Bachmann, E., Brousset, P., Sandvej, K., Nadal, D., Bachmann, F., Odermatt, B. F., Delsol, G. & Pallesen, G. (1993). Deletions within the LMP1 oncogene of EpsteinBarr virus are clustered in Hodgkins disease and identical to those observed in nasopharyngeal carcinoma. Blood 82, 2937-2942.[Abstract]
Kuppers, R., Hansmann, M. L. & Rajewsky, K. (1998). Clonality and germinal centre B-cell derivation of Hodgkin/ReedSternberg cells in Hodgkins disease. Annals of Oncology 9, S17-S20.[Medline]
Li, S. N., Chang, Y. S. & Liu, S. T. (1996). Effect of a 10-amino acid deletion on the oncogenic activity of latent membrane protein 1 of EpsteinBarr virus. Oncogene 12, 2129-2135.[Medline]
Meggetto, F., Muller, C., Henry, S., Selves, J., Mariame, B., Brousset, P., Al Saati, T. & Delsol, G. (1996). EpsteinBarr virus (EBV)-associated lymphoproliferations in severe combined immunodeficient mice transplanted with Hodgkins disease lymph nodes: implications of EBV-positive bystander B lymphocytes rather than EBV-infected ReedSternberg cells. Blood 87, 2435-2442.
Meggetto, F., Brousset, P., Selves, J., Delsol, G. & Mariame, B. (1997). ReedSternberg cells and bystander lymphocytes in lymph nodes affected by Hodgkins disease are infected with different strains of EpsteinBarr virus. Journal of Virology 71, 2547-2549.[Abstract]
Mehl, A. M., Fischer, N., Rowe, M., Hartmann, F., Daus, H., Trumper, L., Pfreundschuh, M., Muller-Lantzsch, N. & Grasser, F. A. (1998). Isolation and analysis of two strongly transforming isoforms of the EpsteinBarr-virus (EBV)-encoded latent membrane protein-1 (LMP1) from a single Hodgkins lymphoma. International Journal of Cancer 76, 194-200.
Raab-Traub, N. (1989). The human DNA tumor viruses: human papilloma virus and EpsteinBarr virus. Cancer Treatment and Research 47, 285-302.[Medline]
Sato, H., Takimoto, T., Tanaka, S., Tanaka, J. & Raab-Traub, N. (1990). Concatameric replication of EpsteinBarr virus: structure of the termini in virus-producer and newly transformed cell lines. Journal of Virology 64, 5295-5300.[Medline]
Sixbey, J. W., Shirley, P., Chesney, P. J., Buntin, D. M. & Resnick, L. (1989). Detection of a second widespread strain of EpsteinBarr virus. Lancet ii, 761-765.
Stein, H. & Hummel, M. (1999). Cellular origin and clonality of classic Hodgkins lymphoma: immunophenotypic and molecular studies. Seminars in Hematology 36, 233-241.[Medline]
Wang, D., Liebowitz, D. & Kieff, E. (1985). An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell 43, 831-840.[Medline]
Weiss, L. M., Strickler, J. G., Warnke, R. A., Purtilo, D. T. & Sklar, J. (1987). EpsteinBarr viral DNA in tissues of Hodgkins disease. American Journal of Pathology 129, 86-91.[Abstract]
Weiss, L. M., Movahed, L. A., Warnke, R. A. & Sklar, J. (1989). Detection of EpsteinBarr viral genomes in ReedSternberg cells of Hodgkins disease. New England Journal of Medicine 320, 502-506.[Abstract]
Received 7 September 2000;
accepted 22 December 2000.