Mutagenesis, Vol. 16, No. 3, 203-208,
May 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Enhancement of genetic instability in human B cells by Epstein–Barr virus latent infection
DABAC, Università degli Studi della Tuscia, via SC deLellis snc, 01100 Viterbo, Italy
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Abstract |
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The level of genetic instability, as assessed by micronucleus (MN) formation, was higher in Epstein–Barr virus (EBV)-converted B-cell lines with one copy of the EBV genome integrated in each cell than in the parental, EBV-negative, B lymphoma cells. MN induced by EBV latency, as analysed by in situ hybridization, contained mainly centromeric regions, indicating that the presence of EBV affects the segregation of entire chromosomes. The instability was inhibited by treatment with antioxidants. Flow cytometric analysis indicated that there was a higher basal level of peroxides in EBV+ cells. Direct oxidative stress caused by hydrogen peroxide (which is known to be both apoptogenic and mutagenic) enhanced the number of MN only in an EBV-converted clone. These cells were also resistant to apoptosis, as expected, suggesting that in the parental EBV cells apoptosis may efficiently eliminate cells with genetic damage. These results show for the first time a direct involvement of EBV in the induction of genetic instability, suggesting that it could contribute to tumour progression.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Epidemiological studies have shown that Epstein–Barr virus (EBV), a member of the herpesvirus group, is associated with lymphomagenesis and nasopharyngeal carcinoma (Epstein, 1986
). Although the role of EBV in cell immortalization is well established, its role in the full development of tumoral phenotypes in B cells is still unclear (Magrath, 1991).
Some authors have suggested that EBV, by allowing the clonal expansion of infected cells, as a result of its immortalization ability and its inhibition of physiological cell death (Henderson et al., 1991), can indirectly facilitate tumour progression, allowing the cell to accumulate additional spontaneous genetic changes. Inhibition of apoptotic cell death is a feature of EBV-converted cell lines (Gregory et al., 1991) and apoptosis is a fundamental factor in carcinogenesis (Wyllie, 1997). Impairment by mutation of some pro-apoptogenic gene products (e.g. p53) reduces the extent of apoptosis and allows the propagation of mutant cells following genotoxic damage, eventually promoting tumorigenesis (Griffiths et al., 1996). Over-expression of the more general anti-apoptosis genes, bcl-2 and bcl-xl, which are not specific towards DNA damage, also enhances mutagenesis via other mechanisms, for example by inhibiting the selective elimination of heavily mutated cells from the population (Cherbonnel-Lasserre et al., 1996).
Other authors have suggested that EBV may directly prime genetic instability, for instance via activation of the B-cell-specific recombinases RAG-1 and -2 (Kuhn-Hallek et al., 1995), or via cycles of integration–excision of its DNA into the host genome (Wolf et al., 1993; Gargano et al., 1995; Gualandi et al., 1995), while the expression of its latent genes could promote the excision of EBV DNA from the host genome (Wolf et al., 1995).
Despite intensive studies, direct demonstration of the `mutagenic' potential of EBV has not been provided.
Genetic instability is a known characteristic of transformed cells and cells carrying viral genomes, as is the case of cells infected with SV40 (Walen, 1987), transformed by adenovirus (Schamayr et al., 1990) or HTLV-I (Majone et al, 1993) or carrying papillomavirus DNA (Stich et al., 1990). In the latter case, the increased genetic instability in papillomavirus-transformed cells was counteracted by antioxidants, suggesting that chronic oxidative stress may have a role in some cases of viral-induced genotoxicity.
The purpose of this study was to verify if the presence of EBV genome in converted human B lymphoma lines might enhance their genetic instability, as measured by the formation of spontaneous or stress-induced micronuclei (MN) in binucleated cells by the cytokinesis-block assay (Fenech and Morley, 1985; Kirsch-Volders et al., 1997). The MN test allows estimation of the genotoxic effect of agents acting at each point of the cell cycle; this is an advantage in testing the action of agents whose mechanism has not been characterized.
We also investigated the possibility that endogenous, constitutive oxidative stress, elicited by the presence of EBV, may contribute to the formation of cells containing MN. Finally, we wanted to test, in our system, the role of apoptosis inhibition in causing genetic damage cells after exogenous oxidative stress. We therefore compared the genotoxic/cytotoxic effects in EBV–, apoptosis-prone cells, with that in EBV+, apoptosis-resistant cells after exposure to hydrogen peroxide.
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Materials and methods |
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Cell lines
BL41 is an EBV– B-cell line obtained from a Burkitt lymphoma (Calender et al., 1987
) carrying a mutant form of the p53 gene (Farrel et al., 1991); it is very prone to apoptosis (unpublished). The ClA, HS1 and E2 clones were obtained after infection of the parental BL41 line with a non-defective B85-8 EBV strain. Virus filtrate from aged B95-8 marmoset cells was concentrated by centrifugation at 14 000 x g and 2 x 105 c.f.u. were added to dishes containing 106 cells. Cloning was achieved by limiting dilutions or by directly picking cells with a micro-needle under an inverted microscope. Clones showing the characteristic EBV+ phenotype (growth in clumps) were isolated. The presence of EBV and monoclonality were checked by in situ hybridization on metaphase spreads and by Southern analysis using the BamHI `W' probe, which represents the repeat unit of the IR1 region (this is commonly involved in recombination with the human genome) (Hurley et al., 1991; Gargano et al., 1992).
Chemicals and media
The Ca2+ ionophore, ionomycin (Sigma), the topoisomerase II inhibitor, 4'-(9-acridinylamino) methanesulfon-m-anisidine (m-AMSA) (Sigma) and the alkylating agent, trimethyil tin-chloride (TMT) (Aldrich) were dissolved in dimethylsulfoxide (DMSO; Sigma). Hydrogen peroxide, 
-thioglycerol, sodium pyruvate, bathocuproindisulfonate salt, N-acetyl-cysteine (NAC) and buthionine sulfoximine (BSO; Sigma) were dissolved in water.
The cell lines were cultured in RPMI 1640 medium (Gibco BRL) supplemented with 10% fetal calf serum (FCS; Bio-Whittaker) and 2 mM L-glutamine.
Analysis and quantification of apoptotic cells
For morphological analysis of apoptosis, 2–6 x 105 cells were fixed in 4% (v/v) paraformaldehyde, loaded on to a gelatinized slide, stained with 0.1% haematoxylin and checked by direct optical microscopy. Apoptosis was quantified by scoring cells with condensed and fragmented nuclei, according to Ghibelli et al. (1995); 
500 cells in random fields were scored. Evaluation of apoptosis by morphological analysis is rapid, quantitative and unambiguous; the scoring of cells showing extensive nuclear condensation or fragmentation rules out any possible mis-interpretation.
Micronucleus assay
Analysis of MN was performed in binucleated cells using the cytokinesis inhibitor cytochalasin B (CB; Sigma) at 1 µg/ml for 18–22 h. Cells from all the EBV-converted clones replicate more slowly than cells from the parental line, BL41; the cell cycle times are ~12 h for BL 41, 16 h for HS1 and E2, and 18 h for ClA. The cells were resuspended in 4 ml of hypotonic solution (0.1 M KCl) added dropwise for 7 min. Cells collected by centrifugation were fixed in acetic acid:methanol (1:5 v/v) at 4°C for 30 min, spread on slides and stained in 5% Giemsa for 7 min. MN were scored by optical microscopy at 1000x and expressed as the number of MN divided by the number of binucleated cells (1000 cells scored). Standard error (S.E.) was calculated for results from three or four experiments. Nuclear fragmentation due to apoptotic events can be easily recognized in both mononucleated and binucleated cells (Kirsch-Volders et al., 1997; Fenech et al., 1999).
Modulation of glutathione concentration
Cells were treated continuously for 20 h in the presence of CB with one of the following glutathione (GSH)-modulating agents: (i) `TBP mix' (50 µM -thioglycerol, 1 mM sodium pyruvate, 20 nM bathocuproindisulfonate salt); (ii) 5 mM NAC; or (iii) 0.6 mM BSO.
In situ hybridization
In order to discriminate centromere-positive and centromere-negative MN, slides were hybridized with a biotin-labelled pancentromeric probe (Oncor) consisting of a selection of alphoid sequences that hybridize to the centromeres in all human chromosomes. Slides bearing MN were denatured in 70% formamide for 3 min at 65°C. The probe (30 µl/slide) was denatured at 70°C for 10 min, placed on ice, dropped on to each slide and covered with a coverslip. Slides were incubated overnight at 37°C in a humid chamber. After incubation, preparations were washed twice at 42°C in 50% formamide in 2x SSC followed by two washes in 0.1x SSC for 3–5 min at 62°C.
Slides were then incubated with 5% non-fat dried milk for 15 min at room temperature. Biotinylated probe was detected by incubating 5 µg/ml fluorescein isothiocyanate (FITC) conjugated with avidin D (Vector Labs) for 30 min at room temperature followed by three washes 2 x SSC. Signals were amplified by reincubation with 5 µg/ml biotin-conjugated anti-avidin D antibodies (Vector Labs) for 30 min at room temperature followed by another incubation with avidin D and FITC for 30 min at room temperature. Slides were dehydrated and then mounted in 20 mM Tris–HCl (pH 7.5), 90% glycerol containing 2% antifade. Preparations were examined under a Zeiss microscope equipped with DAPI 4,6 Diamidino-2-phenylindole and FITC epifluorescence optics.
Flow cytometric analysis of endogenous peroxides
Intracellular peroxide levels were assessed using an oxidation-sensitive fluorescent probe, 2',7'-dichloroflurorescein diacetate (DCFH-DA; Sigma– Aldrich). In the presence of a variety of intracellular peroxides, DCFH-DA is oxidized to 2', 7'-dichloroflurorescein. After treatment, cells were incubated with 5 mM DCFH-DA for 30 min at 37°C in a CO2 incubator (Kayanoki et al., 1994) and then analysed using a FACScalibur cytometer (Becton Dickinson) with excitation at 488 nm and emission at 533 nm recorded in FL-1; 10 000 events were recorded for each sample.
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Results |
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EBV in the latent phase enhances genetic instability
The EBV-converted clones, E2, HS1, ClA, were obtained independently upon infection with the EBV B95-8 strain. The E2, HS1 and ClA2 clones have only one copy of the EBV genome integrated on a single chromosome; the site of integration differs among the various clones.
Southern analysis confirmed the presence, in each cell clone, of a single copy of the EBV genome in each cell as a consequence of independent integrative events (not shown).
The cell lines showed a spontaneous rate of apoptosis ranging between 2% and 6%, not very dissimilar from the spontaneous level observed in the parental line. All EBV+ clones show a generalized resistance to apoptosis induced by various compounds with unrelated mechanism of action when compared with the parental uninfected cell line; the degree of resistance was a characteristic feature of each clone (Figure 1a), with E2 and ClA clones showing the highest level of resistance to apoptosis (Figure 1a).
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Next, we analysed the level of spontaneous MN formation in the cell clones, using standard protocols based on the promotion of binucleated cells with CB (see Materials and methods). Doses of CB and duration of treatment required for induction of binucleated cells in the MN test were adjusted so that they were non-toxic and did not induce apoptosis; at all tested concentrations, CB did not affect the level of micronucleated cells in our system. The clones with the highest level of resistance to apoptosis displayed significantly more genomic instability, as judged by the presence of MN, than the parental cell line, BL41 (Figure 1b,
top).
Slides for MN analysis from BL41 and the converted clone E2 (showing a higher incidence of spontaneous MN formation) were hybridized to the centromeric probe (Figure 2); in this experiment the MN induced by colcemid represent the positive control. The results indicate that the EBV+ cells mainly show an increase in mis-segregation events, being positive for the centromeric probe. Half of the centromere-positive MN displayed more than one dot, indicative of a multiple kinetochore; a slight increase in the number of centromere-negative MN was also found in EBV+ cells.
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Genetic instability due to EBV latent infection is associated with production of endogenous oxidants
Genetic instability related to virus presence is, in some instances, associated with oxidative stress (Stich et al., 1990
), so we investigated whether this might be happening in our system by two techniques: (i) measuring endogenous peroxides via flow cytometry with the oxidation of DCFH-DA; and (ii) checking if antioxidants can reduce MN formation and/or apoptosis.
The results, shown in Figure 3b, indicate that the E2 line consistently shows more DCFH-DA oxidation than the parental BL41 line: the mean obtained from six experiments gave an average 3-fold increase in fluorescence in the E2 cells. The fluorescence in the HS1 line was in an intermediate position, in accordance with the intermediate level of spontaneous MN formation in these cells. The increased oxidation appears to be specifically due to peroxide production rather than depending on different uptake or retention of the fluorochrome, since CMF-DA or calcein-AM uptake (measures of viability) was the same in both these cell lines (not shown).
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The TBP mix, a mixture of antioxidants known to reduce the level of apoptosis induced by cell density stress (Falk et al., 1998
), was tested in our cells. In human B-cell lymphoma lines, this mixture increases the intracellular content of GSH, the main and general cellular antioxidant. The thioglycerol in the TBP mix increases the uptake of cysteine (necessary for GSH synthesis) from the medium, while the sodium pyruvate destroys hydrogen peroxide generated by thioglycerol (Falk et al., 1998). The TBP mix worked well in reducing apoptosis in E2 cell line (Figure 3a). The level of cells with spontaneous MN formation (Figure 3a) was also significantly reduced. In cells treated with sodium pyruvate alone, there was a significant decrease in the numbers of micronucleated and apoptotic cells (Figure 3a).
To analyse further the possible role of oxidative stress, we treated the cells with NAC, which after deacylation is converted to cysteine, a precursor of GSH. A significant decrease in the spontaneous level of MN formation was obtained. To confirm the role of GSH, we depleted E2 cells of GSH using BSO. Depletion of GSH content to quarter of the control value after BSO treatment led to a significant increase in the number of micronucleated E2 cells (Figure 3a).
Hydrogen peroxide increases genetic instability in EBV+ but not in EBV – cells
We next investigated the effect of a direct oxidative insult in inducing genetic changes in apoptosis-sensitive (EBV–) BL41 cells compared with apoptosis-resistant (EBV+) E2 cells. Hydrogen peroxide was used at doses (30–50 µM) that were mildly apoptogenic in our system and still allowed analysis of an appreciable percentage of binucleated cells (25%). Hydrogen peroxide was apoptogenic in all cells but was more effective in the sensitive BL41 cell line (Figure 4a, bottom). MN were efficiently induced only in the E2 line (Figure 4a, top). In the DCFH-DA assay, the increase in fluorescence (after 1 h of incubation with hydrogen peroxide at the same concentration used in the toxicity experiments (50 µM)) was similar in the two cell lines (Figure 4b); measurements made after 1 h of recovery indicated that the values returned to the control level in both cell lines (not shown). These results suggest that the differences in MN induction cannot be ascribed to a different efficiency of the detoxifying apparatus between the EBV– and EBV+ cells.
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Discussion |
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The findings that EBV confers genetic instability upon converted human B-cell lines is clearly of interest. A previous study on papillomavirus-transformed cells indicated that the enhancing effect of virus on MN formation can be counteracted by antioxidants (Stich et al., 1990
), suggesting that peroxide intermediates may be responsible for such an effect. In this and another paper (Walen, 1987) it was not clear if the genome status (episomal versus integrated) of the viruses played a role in the genetic instability, although it is known that EBV appears to produce fragile sites at the point of integration (Popescu et al., 1993).
Our results with the E2 clone, which carries one integrated copy of the EBV genome, indicate that the different antioxidants (TBP mix, sodium pyruvate and NAC; see Figure 3) significantly reduced MN formation, suggesting that the level of endogenous oxidants may modulate MN frequency in EBV+ cells. Apoptosis in control cells was also reduced by antioxidants acting directly (sodium pyruvate) or indirectly (by increasing GSH synthesis), raising the possibility that, in this system, these two processes are induced by a common upstream signal. We are presently investigating the nature of the oxidant intermediate(s) responsible for these effects. Either hydrogen peroxide (via peroxidase) or peroxinitrite may be responsible for DCFH-DA oxidation (Ischiropoulos et al., 1999).
The MN induced by the presence of EBV were mainly positive for the centromeric probe, indicating chromosome mis-segregation, but other events leading to chromosome fusion, formation of dicentric, etc. cannot be exluded at present (indeed, a large proportion of MN analysed by in situ hybridization contained more than one positive signal). Similar results were obtained in T-lymphocytes after expression of the transforming protein Tax of HTLV-I (Majone et al., 1993); also in that case, most MN contained kinetochore material.
Why should the endogenous oxidative stress preferentially induce segregation of whole chromosomes rather than of acentric fragments? Some data in the literature partially support this finding. It is known that some DNA-damaging agents that also have oxidative power (such as X-rays and nitro-quinoline-1-oxide) can induce both centromere-positive and -negative MN (Schuler et al., 1997). Moreover, the ageing process is associated with oxidative problems (Ames and Shigenaga, 1992) and there is increased chromosome loss in the lymphocytes from elderly donors (Carere et al., 1999). Another possibility is that damaged DNA, which may be more likely to pass checkpoints that are weakened as a consequence of the ageing process or because of the interference of viral genes, may evolve, producing abnormal mitotic segregation (Lane, 1992). As an alternative, EBV-specific, explanation for the kinetochore-containing MN, we speculate that the EBV genome may act as a cis- (in its integrated status) or trans-acting agent on chromosomal instability, peroxides being the triggers and/or mediators of the process.
The instability in EBV+ (E2) cells can be further increased (
15% of micronucleated cells) following exogenous oxidative stress, which was only weakly mutagenic in EBV– cells. In this respect, apoptosis may be a powerful means of eliminating damaged cells carrying genetic changes (Schwartz et al., 1995). In fact, in the BL41 (EBV–) cell line, which is particularly prone to cell death, MN induction via direct pro-oxidant treatment with hydrogen peroxide (a strongly cytotoxic compound) was rare, while the E2 apoptosis-resistant line was highly inducible. BL41, on the other hand, can be readily induced to form MN using sublethal X-rays doses (unpublished), which, with some treatments, can cause genotoxicity in the absence of cytotoxicity. A p53-independent pathway has to be evoked in interpreting the above results in that both BL41 and its converted line carry a mutated form of these proteins (Farrel et al., 1991).
The abovementioned findings may help in interpreting the results obtained in other systems with hydrogen peroxide and in general with oxidants, where the genotoxic/cytotoxic ratio may greatly vary in relation to the systems employed and allow discrimination between agents that primarily induce cytotoxic effects as opposed to genotoxic effects (Fenech et al., 1999).
The links between cellular and genetic damage could be rather complex. The development of human B-cell lymphoma is very complex, and the precise role played by EBV is still controversial (Lenoir and Bornkamm, 1997). The observation that the presence of EBV enhances the frequency of spontaneous MN formation suggests a novel mechanism through which EBV could directly contibute to the malignant progression of cells carrying EBV in the latent state, by enhancing the `background' level of genetic instability. In addition, the EBV-dependent increase in damage-induced MN, together with the enhanced resistance to cell death, indicate that genetically damaged cells may be rescued and survive due to the presence of EBV. This aspect may be particularly relevant in the case of treatments with antineoplastic, genotoxic agents, where EBV-carrying cells may be particularly prone to accumulating treatment-induced genetic alterations. We are planning to verify the differing MN inducibility in EBV– and EBV+ cells by some compounds such as bleomycin or cis-platinum whose apoptogenic power is reduced in response to EBV infection of BL41 cells (Wade and Allday, 2000).
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Acknowledgments |
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We are very grateful to Dr L.Ghibelli for helpful discussions. This work was supported by an EEC grant (contract ENV4-CT96-0169) and by fondi ricerca Ateneo (ex 60%).
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Notes |
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1 To whom correspondence should be addressed. Email: gualandi{at}unitus.it
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References |
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Received on August 14, 2000; accepted on November 20, 2000.
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