Oncogene (2003) 22, 4459–4468 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc ORIGINAL PAPERS BCL6 overexpression prevents increase in reactive oxygen species and inhibits apoptosis induced by chemotherapeutic reagents in B-cell lymphoma cells Tetsuya Kurosu1, Tetsuya Fukuda1, Tohru Miki1 and Osamu Miura*,1 1Department of Hematology and Oncology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo 113-8519, Japan Chromosomal translocations and somatic mutations diffuse large B-cell lymphoma (DLBCL) (Kerckaert occurring in the 50 noncoding region of the BCL6 gene, et al., 1993; Ye et al., 1993; Miki et al., 1994). The BCL6 encoding a transcriptional repressor, are most frequent gene encodes a transcriptional repressor containing a genetic abnormalities associated with non-Hodgkin B-cell carboxy-terminal DNA-binding zinc-finger domain and lymphoma and result in deregulated expression of BCL6. an amino-terminal BTB/POZ domain, which can bind However, the significance of deregulated expression of to transcriptional repressor cofactors, including N-CoR BCL6 in lymphomagenesis and its effect on clinical and SMRT, and, thereby, recruit the histone deacetylase outcomes of lymphoma patients have remained elusive. In protein complex (reviewed in Dent et al., 2002; Niu, the present study, we established Daudi and Raji B-cell 2002). BCL6is preferentially expressed in germinal lymphoma cell lines that overexpress BCL6 or its mutant, center B cells and has been demonstrated to be required BCL6-Ala333/343, in which serine residues required for for the formation of germinal centers (Cattoretti et al., degradation through the proteasome pathway in B-cell 1995; Onizuka et al., 1995; Dent et al., 1997; Fukuda receptor-stimulated cells are mutated. BCL6 overexpres- et al., 1997; Ye et al., 1997). A recent study with DNA sion did not have any significant effect on cell prolifera- microarray analysis revealed that BCL6represses a tion, but significantly inhibited apoptosis caused by discrete set of genes involved in B-cell activation, etoposide, which induced a proteasome-dependent degra- terminal differentiation, and inflammation (Shaffer dation of BCL6. BCL6-Ala333/343 was not degraded et al., 2000). It has thus been hypothesized that after etoposide treatment and strongly inhibited apoptosis. downregulation of BCL6is required for germinal center In these lymphoma cell lines, etoposide increased the B cells to differentiate into plasma cells. Consistent with generation of reactive oxygen species (ROS) and reduced this hypothesis, BCL6expression is regulated by signals mitochondria membrane potential, both of which were important for the differentiation of germinal center B inhibited by the antioxidant N-acetyl-l-cysteine (NAC). cells. For instance, the expression of BCL6is down- NAC also inhibited apoptosis. Furthermore, BCL6 over- regulated at the transcriptional level by the CD40 expression was found to inhibit the increase in ROS levels receptor signaling (Allman et al., 1996) and at the and apoptosis in response to etoposide and other protein level by the B-cell receptor signaling, which chemotherapeutic reagents. These results raise the possi- induces phosphorylation of Ser333 and Ser343 of BCL6 bility that deregulated expression of BCL6 may endow through activation of Erks and, thereby, targets BCL6 lymphoma cells with resistance to chemotherapeutic for degradation by the ubiquitin proteasome pathway reagents, most likely by enhancing the antioxidant defense (Niu et al., 1998). systems. Chromosomal translocations involving the BCL6 Oncogene (2003) 22, 4459–4468. doi:10.1038/sj.onc.1206755 gene represent the most common genetic abnormality in non-Hodgkin B-cell lymphomas and have been Keywords: BCL6; apoptosis; chemotherapeutic re- demonstrated in 27–45% of DLBCL and 5–14% of agents; reactive oxygen species; etoposide follicular lymphomas (Bastard et al., 1994; Lo Coco et al., 1994; Otsuki et al., 1995; Ohno and Fukuhara, 1997). These translocations typically juxtapose the intact BCL6-coding sequences with heterologous pro- moters from genes expressed in B cells, including the Introduction immunoglobulin genes, thus causing deregulated expres- sion of BCL6(Ye et al., 1995; Chen et al., 1998). Besides The BCL6gene was originally identified on the break this promoter substitution mechanism, occasional mi- point of a chromosomal translocation involving 3q27 in crodeletions and frequent multiple somatic mutations in the 50 noncoding negative autoregulatory region of the *Correspondence: O Miura; E-mail: [email protected] BCL6gene have been shown to deregulate the expres- Received 11 February 2003; revised 1 May 2003; accepted 1 May 2003 sion of BCL6in lymphoma cells (Kikuchi et al., 2000; Antiapoptotic effect of BCL6 in lymphoma cells T Kurosu et al 4460 Nakamura, 2000; Wang et al., 2002). Since the BCL6 a gene is most frequently affected in non-Hodgkin B-cell Daudi Daudi/Ton-BCL6 lymphomas, it has been suggested that deregulated DOX - - + expression of BCL6should play an important role in Blot: lymphomagenesis. However, the oncogenic ability of anti-BCL6 BCL6in B cells has remained elusive. It has also remained very controversial as to whether the BCL6 anti-α -tubulin gene alterations affect the clinical outcome of patients with Non-Hodgkin lymphomas (reviewed in Lossos et al., 2001; Barrans et al., 2002; Dent et al., 2002; Niu, b 2002). 120% To gain insight into the significance of deregulated BCL6expression in the pathogenesis and prognosis of 100% non-Hodgkin lymphomas, we overexpressed BCL6in germinal center B-cell-derived lymphoma cell lines, 80% Daudi and Raji, and examined the effects on cell proliferation and apoptosis. BCL6overexpression sig- DOX(-) 60% nificantly inhibited apoptosis of lymphoma cells in DOX(+) response to etoposide and other chemotherapeutic Viable cells reagents. BCL6overexpression also inhibited the 40% increase in reactive oxygen species (ROS) levels and the reduction of mitochondria transmembrane potential 20% (Dcm) in response to etoposide. The ROS scavenger N- l acetyl- -cysteine (NAC) also inhibited the reduction of 0% Dcm and apoptosis as well as the increase in ROS levels 0 0.1 0.25 0.5 1 2 5 10 20 in etoposide-treated lymphoma cells. These observations Etoposide (µM) indicate that BCL6overexpression inhibits apoptosis of lymphoma cells treated with chemotherapeutic reagents, Figure 1 Inducible overexpression of BCL6enhances the survival most likely through enhancement of the antioxidant of Daudi cells treated with etoposide. (a) Parental Daudi cells and Daudi/Ton-BCL6cells were cultured overnight in the absence ( À) defense systems. or presence ( þ ) of DOX (1 mg/ml), as indicated, and harvested. Total cell lysates were subjected to Western blot analysis with anti- BCL6antibody followed by reprobing with anti- a-tubulin. (b) Daudi/Ton-BCL6cells, precultured overnight in the absence ( À)or Results presence ( þ ) of DOX as indicated, were treated with indicated concentrations of etoposide for 24 h in the absence (À) or presence BCL6 overexpression inhibits apoptosis of B-cell ( þ ) of DOX. Numbers of viable cells were measured by the XTT assay. Each data point represents the mean of triplicate determina- lymphoma cells treated with etoposide tions, with error bars indicating standard errors, and is expressed as To explore the possibility that overexpression of BCL6 a percentage of cell numbers without etoposide may have an effect on the proliferation of lymphoma cells or on their responses to chemotherapeutic reagents, we established a clone of the Burkitt’s lymphoma Daudi cell line, Daudi/Ton-BCL6, as described under Materi- absence of DOX, and the numbers of viable cells were als and methods. As shown in Figure 1a, Daudi/Ton- measured by the XTT assay. As shown in Figure 1b, BCL6cells express a low level of BCL6,comparable etoposide decreased the number of viable cells in a dose- with that in parental Daudi cells, in the absence of dependent manner in the presence or absence of DOX. doxycycline (DOX) and inducibly overexpress BCL6 However, a significantly higher number of cells survived when cultured in the presence of DOX. We first the treatment of etoposide at concentrations as high as examined the effect of BCL6overexpression on cell 2 mm when cultured with DOX, which indicates that growth by culturing Daudi/Ton-BCL6cells in the Daudi cells overexpressing BCL6are more resistant to presence or absence of DOX and by measuring the etoposide treatment than cells expressing the endogen- number of viable cells for up to 4 days by the XTT ous level of BCL6. colorimetric assay, as described under Materials and Chemotherapeutic reagents, including etoposide, have methods. However, we did not observe any significant been known to induce apoptosis of cancer cells. There- effect of BCL6overexpression on cell proliferation in fore, we next examined the effect of BCL6overexpres- repeated experiments (negative data not shown). sion on etoposide-induced apoptosis by the annexin V We next examined the effect of BCL6overexpression staining method, as described under Materials and on the response of Daudi cells to etoposide, a methods. Etoposide induced apoptosis of Daudi/Ton- topoisomerase II inhibitor commonly used as a che- BCL6cells in time- and dose-dependent manners motherapeutic reagent in the treatment of lymphoma. (Figure 2 and data not shown). Notably, the fraction Daudi/Ton-BCL6cells were cultured for 24 h with of annexin V-positive, apoptotic cells was consistently various concentrations of etoposide in the presence or smaller in the presence as compared with the absence of Oncogene Antiapoptotic effect of BCL6 in lymphoma cells T Kurosu et al 4461 a DOX (-) DOX (+) DOX (-) DOX (+) Etoposide (h) 0 2 4 6 0 2 4 6 Blot: 6.4 % 5.5 % Control anti-PARP * 0.7 % 1.3 % anti-α-tubulin Etoposide 20.7 % 17.3 % anti-BCL6 16.3 % 12.7 % PI Figure 3 Reduction in etoposide-induced cleavage of PARP by BCL6overexpression and etoposide-induced downregulation of Annexin V BCL6. Daudi/Ton-BCL6 cells, precultured in the absence (À)or presence ( þ ) of DOX as indicated, were further cultured with 50 mm etoposide for indicated times.