Integration of Epstein-Barr virus in Burkitt's lymphoma cells leads to a region of enhanced chromosome instability

Annals of Oncology 8 (Suppl. 2): S131-S135, 1997. © 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Original article Integration of Epstein-Barr virus in Burkitt's lymphoma cells leads to a region of enhanced chromosome instability A. Jox,1 C. Rohen,2 G. Beige,2 S. Bartnitzke,2 M. Pawlita,3 V. Diehl,1 J. Bullerdiek2 & I Wolfl 'Department ofInternal Medicine I. University of Cologne, Cologne; 'Centerfor Human Genetics and Genetic Counseling, University of Bremen, Bremen; 3Research Program Applied Tumor Virology, Deutsches Krebsforschungszenlrum, Heidelberg, Germany

Summary

Introduction

The Epstein-Barr virus (EBV) is a human herpesvirus with the capacity to immortalize human B lymphocytes in vitro [1]. In EBV-infected B cells, the virus usually persists in an episomal state forming multiple copies of covalently closed circles. So far, integration of the virus in the host cell genome has been described only in a few Burkitt's lymphoma (BL) cell lines [2-5] and one lymphoblastoid cell line (LCL) [6]. Several malignant lymphomas are associated with EBV infection. In individuals lacking efficient T-cell function, e.g., AIDS patients or transplant recipients, EBVimmortalized B lymphocytes can grow into immunoblastic lymphomas [7]. In addition, Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HD) have been linked to EBV infection. In endemic Burkitt's lymphoma, which represents the most common malignancy of children in certain areas of central Africa, the viral DNA was detected in 96% of cases in the tumor cells [8]. However, in 4% of endemic BL as well as in 85% of sporadic BL in the United States and Western Europe no viral DNA could be found [9]. Similarly, in HD in developing countries Hodgkin/Reed-Sternberg (H-RS) cells are EBV-infected in about 90%~100% of cases; by comparison in industrial

Key words: EBV-associated lymphomas, Epstein-Barr virus, FISH, hit-and-run mechanism, viral integration

countries the virus is found only in 40%—50% of cases in the malignant cells [10]. Possibly, transformation in the EBV-negative cases of BL and HD is EBV-independent, e.g., due to another tumor virus or a still undefined oncogene activation or suppressor-gene inactivation, respectively. If, however, EBV plays a causal role in the transformation process of these B-cell lymphomas, two mechanisms may underlie the observation of 'EBV-negative' cases. EBV DNA may be present in the malignant cells but be undetected by standard screening procedures because of deletions in the viral genome. Alternatively, the virus may have been lost after initiating infection. Here we present an experimental model where both of these possibilities hold true. EBV integration into the host cell genome of a BL cell line is associated with a large deletion in the viral genome and generates a region of enhanced chromatin instability in the host cell genome.

Materials and methods Cell lines The Burkitt's lymphoma (BL) cell line BL60 and the autologous lymphoblastoid cell line (LCL) IARC 277 were established and kindly

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Background: Several lymphomas are associated with EpsteinBarr virus (EBV) infection. However EBV is not detectable in 100% of cases using standard staining techniques. It still remains an open question whether in these EBV-negative cases EBV has never infected the cell, whether it has infected the cell and escapes conventional screening methods, or whether it has been lost again after initial infection. Materials and methods: The physical status of EBV in the Burkitt's lymphoma cell line BL60-P7 as well as in three somatic cell hybrids between BL60-P7 and its autologous EBVimmortalized lymphoblastoid cell line IARC 277 was analyzed using conventional cytogenetics, Southern blotting, and fluorescence in situ hybridization (FISH). Results: Integration of EBV into the host genome of the lymphoma cell line BL60-P7 leads to an achromatic gap which

causes a 'vulnerable site'. In hybrid cells, loss of integrated EBV, together with an adjacent chromosomal fragment, occurs during long-term cultivation. The integrated EBV genome, including genes encoding for LMP and EBER, is partly deleted. Conclusion: We assume that integration of EBV into the host cell genome could be a more common event in lymphoma cells. Partially deleted EBV might escape standard detection assays. The integration might constitute a chromosomal region prone to break events akin to the phenomenon of fragile sites, leading to the loss of viral DNA as well as chromosomal DNA. This observation makes it tempting to speculate that under certain conditions EBV can act in lymphomagenesis by a so-called hitand-run mechanism.

132 provided by G. Lenoir [11]. BL60-P7 is a neomycin-resistant and hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) deficient subclone of BL60 [12]. Establishment and growth characteristics of the three BL60-P7 x IARC 277 somatic cell hybrid sublines 1, 2, and 4 have been described [12]. The phenotype of these hybrid cells is comparable with the phenotype of the parental LCL with regard to expression of B-cell-associated differentiation antigens as well as growth characteristics in vitro and in vivo [12, 13]. Parental cell lines and hybrid cell lines with the additional letter e are defined as short-term cultured for up to six months after fusion. Long-term cultured hybrid cell lines and parental cell lines are marked with / and have been continuously kept in culture for at least two years after fusion. All cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 raM glutamine, 100 IU of penicillin, and 100 ug of streptomycin/ml.

Cytogenetic analysis

der(19) Fluorescence in situ hybridization For FISH experiments, the EBV cosmid clone cM Sal-A [15], kindly provided by A. Polack, was used. The cosmid was labeled with biotinlldUTP (Sigma, St.Louis, MO) using a nick translation kit (Gibco BRL, Gaithersburg, MD) Unincorporated nucleotides were removed by chromatography (Sephadex G50, Pharmacia, Uppsala, Sweden); 125 ng cosmid DNA were ethanol-precipitated and vacuum dried; 5 ug salmon sperm DNA were added; and the DNA was resuspended in 25 ml hybridization mixture, containing 50% formamide/2 x SSC/2 x SSPE/20% dextran sulfate and 1% Tween 20. Probe denaturation, prehybridization, hybridization, and posthybridization washes were done as previously described [16]. Hybridized probes were detected by fluorescein isothiocyanate conjugated to avidin (Vector Laboratories, Burlingame, CA). Chromosomes were counterstained with propidium iodide.

Southern blotting Cellular DNA (10 ug) was cleaved with the restriction enzyme Hindlll, separated by agarose gelelectropheresis and transferred onto a nylon filter (Gene Screen Plus, NEN, Boston, MA). The HindUl II fragment which is part of the EBNA-1 gene [17] was 32P labeled and hybridized using standard methods [18].

Results The BL subline BL60-P7 exclusively harbors 6-12 copies of the EBV genome integrated into the host cell genome, whereas its autologous LCL carries only episomal EBV DNA [3]. The integrated EBV in BL60-P7 carries a large deletion, including genes encoding for LMP and EBER [19]. By G-banding and subsequent fluorescence in situ hybridization, it has been confirmed that viral integration takes place near the breakpoint of chromosomal translocation t(ll;19), resulting in a der(19) carrying the integrated EBV copies (3, for scheme see Figure 1). To investigate the physical status of the integrated EBV metaphases of cultured BL60-P7, cells were prepared and Giemsa stained. Twenty-five metaphases with well-spread chromosomes were documented and the slides were then subjected to fluorescence in situ hybridization to detect

Figure I. Putative cytogenetic structure of the derivate (der) chromosome 19 carrying the EBV integration. The scheme is based on the assumption that the EBV is integrated directly between the parts derived from chromosome 11 and 19, respectively.

EBV. Comparison of the metaphases before and after FISH allowed the integration site to be correlated with the presence of an achromatic gap in the Giemsa-stained metaphases (Figure 2). This achromatic gap was only detectable in less condensed metaphases, but not in metaphases with a higher degree of condensation (Figure 3). In some metaphases, the integration site was at the end of the chromosome, most probably because the part of the chromosome distal to the integration site was lost. In about 3% of evaluated metaphases, no integrated EBV has been found. Three somatic hybrid cells between the BL60-P7 cell line and the autologous LCL IARC 277 which were known to contain the integrated EBV from the parental BL cell line as well as the episomal EBV copies from the parental LCL [20] were analyzed by the same method. In short-term cultured hybrid cells, integrated as well as episomal EBV DNA could be demonstrated by FISH. In long-term cultured hybrid cells, FISH failed to demonstrate the integrated EBV in any of the metaphases, indicating the loss of integrated EBV in all of these cells. However, episomal EBV DNA remained always detectable. In contrast, after long-term cultivation in both parental cell lines, EBV remains integrated in the BL60-P7 cell line and episomal in the LCL IARC 277, respectively (data not shown). These results were confirmed by Southern blotting. Because of polymorphism in the Hindlll II fragment of EBV that represents a part from the EBNA-1 gene, the EBV derived from BL60-P7 and the EBV derived from IARC 277 can be differentiated after hybridizing the ifrndlll-digested cellular DNA with the EBV Hindlll II fragment. After long-term cultivation, hybrid cells selectively lose the 3.7 kb fragment representing the BL60-derived integrated EBV while the 4.2 kb fragment representing the episomal LCL-EBV remains detectable.

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Cytogenetic preparations were performed according to routine methods. For G-banding, a modification of the trypsin-Giemsa-banding technique was used [14].

133

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4.2 kb

'3.7 kb

long-term cultured hybrid cells were karyotyped to reidentify the aberrrant BL60-derived chromosome containing the integrated EBV copies. The G-banding patterns of the analyzed metaphases revealed that loss of integrated EBV usually resulted from a chromosomal break proximal to the integration site (Figure 1).

Discussion Figure 2. Giemsa staining of three moderately condensed metaphases (a, b, c) and subsequent fluorescence in situ hybridization (A, B, C) to detect the integrated EBV copies in BL60-P7 cells. The integration site can be detected as an achromatic gap in Giemsa staining (arrows) and as a fluorescence signal after probing with the EBV cosmid clone cM Sal-A (arrows).

By comparison, both parental cell lines retain their EBV even after long-term culture (Figure 4). In short-term cultured hybrid cells, the integration site of EBV detected by FISH did coincide with the presence of an achromatic gap in the Giemsa staining similar to the achromatic region seen in BL60-P7 metaphases. In addition, in some metaphases the integration site represented a secondary constriction site [20]. EBV short-term and

i

A Figure 3. Giemsa staining of a highly condensed BL60-P7 metaphase (a) and subsequent fluorescence in situ hybridization (A). An achromatic gap is not visible.

Integration of EBV in the BL cell line BL60-P7 has been shown to correlate with alterations in the viral genome as well as in the host cell genome. In addition, the integration site represents an achromatic gap like lesion leading to a 'vulnerable site'. Owing to chromosomal breakage at this achromatic gap, there is loss of cellular and integrated viral DNA after fusion of the BL-cell line with its autologous LCL. The integrated EBV in BL60-P7 carries a large deletion consisting of about 10% of the viral genome including, among others, the genes encoding for the EBER transcripts and the LMP protein. The biological relevance of this deletion is not known. The deletion of parts of the viral genome, however, can prevent their detection by standard screening procedures. In situ hybridization of the viral EBER transcripts (EBER-ISH) [21] represents the most sensitive and most frequently used technique to detect EBV in Hodgkin/Reed-Sternberg cells. Another commonly used method is immunostaining of H-RS cells for LMP-expression [22]. With both of these methods, an EBV carrying the deletion of the integrated BL60-P7 EBV cannot be detected. Loss of the integrated EBV genomes in long-term cultured BL60 x IARC 277 hybrids was not simply a result of loss of the whole t(l 1; 19) translocation chromosome carrying the EBV integration, but of a chromosomal breakage leading to loss of integrated EBV molecules along with a fragment of chromosome 11 distal to the integration site. The integration site itself represents a

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Figure 4. Southern blot analysis to detect the presence of integrated as well as episomal EBV genomes in lymphoma cells. During long-term cultivation, hybrid cells selectively lose their integrated EBV copies deriving from the parental lymphoma cell line BL60 (3.7 kb fragment), wheras the episomal LCL-EBV DNA remains detectable (4.2 fragment). Both parental cell lines retain their EBV DNA despite long-term culture. (B) BL60-P7; (L) LCL IARC 277; (1, 2, 4) hybrid cells; (e) short-term cultured, (1) long-term cultured.

134

Hit and run mechanism

EBV infection of host cell carrying two alleles of a tnmor suppressor gone

local ctiromaltn destabilization il GBV integration site leading lo a loss olisitegiated EBV-DtIA along with pait ol Ihe IKJSI chromosome

loss ol one allele of the putative mor suppressor gene and loss ol (unction of its other allele either preceding or followed by loss of episomal EBV-DNA

Figure 5. Hypothesis of EBV-associated 'hit-and-run' mechanism in lymphoma cells.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG, grant Di 184/9-5) and by a grant from the Tonjes-Vagt Stiftung.

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region of enhanced chromosome instability (secondary constriction, achromatic gap). Loss of the integrated EBV DNA in the lymphoma hybrid cells thus most probably results from two phenomena: an enhanced chromosomal instability and a selection pressure against products of the integrated EBV or the fragment of chromosome 11 distal to the integration site. The same achromatic gap site occurs also in BL60-P7 cells, although BL60-P7 cells loosing EBV did not gain a growth advantage. Popescu et al. [5] demonstrated integration of EBV in the BL cell line NAB-2 with the integration site also representing an achromatic gap site. Our studies show that such an altered chromosomal region can break, resulting in loss of the virus and of cellular DNA. The frequency and biological significance of EBV integration is still not known. The BL60 x IARC 277 hybrid cells represent an artificial experimental model. Nevertheless our observations in this model provide a first suggestion that integrated EBV DNA can be lost. If the loss of the viral as well as adjacent cellular DNA also occurs in vivo, one might postulate on a so-called hit-andrun mechanism. Loss of one allele of a tumor-suppressor gene may thus predispose a cell to malignant transformation after loss of function of the second allele, e.g., through the chromosomal instability described in Hodgkin's disease (Figure 5). This hypothesis may be confirmed by recent results of Razzouk et al. [23] in analyzing sporadic North American BL cases classified as EBV-negative by EBNA-1 immunostaining. Southern blot analysis with probes from different regions of the viral genome revealed the presence of integrated, defective EBV genomes in three of nine cases. These authors also postulated a hitand-run mechanism of EBV. EBV integration and subsequent loss of viral and cellular DNA might play a role as an initiating event in the transformation of 'EBV-negative' lymphomas, although it might not be simple to prove this hypothesis in primary lymphoma tissue.

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