Original Manuscript

Leukemia (2003) 17, 1390–1397. doi:10.1038/sj.leu.2402997

The BCL6 gene in B-cell lymphomas with 3q27 translocations is expressed mainly from the rearranged allele irrespective of the partner gene

I S Lossos1, T Akasaka1, J A Martinez-Climent2, R Siebert3 and R Levy1

  1. 1Division of Oncology, Department of Medicine, Stanford University Medical Center, Stanford, CA, USA
  2. 2Department of Hematology and Medical Oncology, Hospital Clinico, University of Valencia, Spain
  3. 3Institute of Human Genetics, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany

Correspondence: Dr R Levy, Stanford University School of Medicine, Division of Oncology, Room 1105, Stanford, CA 94305-5151, USA. Fax: +1 650 725 1420

Received 28 January 2003; Accepted 17 March 2003.

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Abstract

The BCL6 gene, which functions as a transcription repressor, is the target of multiple chromosomal translocations in non-Hodgkin's lymphomas (NHL). These translocations occur in the nontranslated region of the BCL6 gene, juxtaposing regulatory sequences of the diverse partner genes to the open reading frame of the BCL6 gene and thus are thought to deregulate BCL6 gene expression. The levels of expression of the BCL6 gene and protein have been demonstrated to predict the clinical outcome of diffuse large B-cell lymphomas. By contrast, the prognostic significance of BCL6 gene translocations is unclear. In this study we have sought an explanation for this apparent discrepancy. We examined tumors with a variety of different BCL6 translocations and therefore with a variety of potentially substituted promoters. We found no increase in total BCL6 mRNA levels in the NHL specimens harboring BCL6 gene translocation. Indeed, some of these tumors expressed relatively low quantities of the BCL6 mRNA. We also sought to determine whether BCL6 transcription occurs from the rearranged or from the normal untranslocated allele in these tumors. We demonstrate that lymphoma cell lines and majority of NHL tumor specimens expressed BCL6 mRNA predominantly from the rearranged allele that may come under the control of various partner gene promoters. However, few NHL tumors with BCL6 gene translocations expressed BCL6 mRNA equally from the rearranged and the nonrearranged alleles. Neither the nature of the substituted promoters nor the presence of activating mutations in the BCL6 regulatory sequences correlated with the allelic expression of the BCL6 gene in these tumors.

Keywords:

BCL6, lymphoma, translocation, gene expression, allele suppression

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Introduction

The BCL6 gene on chromosome 3q27 encodes a 96 kDa sequence-specific transcriptional repressor protein with 6 C-terminal zinc-finger motifs and an N-terminal POZ/ZIN domain homologous to a family of zinc-finger proteins.1,2,3 BCL6 gene expression is tightly regulated during B-cell differentiation, being restricted to B cells in the germinal centers (GC).4,5,6 The requirement for BCL6 gene expression for GC formation,7,8 an immune reaction characterized by high proliferation rate, oligoclonal expansion of B cells and the presence of active somatic mutations, and downregulation of its expression in cells exiting GC microenvironment suggest that a block in the normal downregulation of this gene might favor genetic instability, which could ultimately lead to neoplastic transformation. Indeed, chromosomal translocations involving the chromosome 3q27 at the site of the BCL6 gene are observed in 30–40% of diffuse large B-cell lymphomas (DLBCL) and in 6 to 15% of follicle center lymphomas (FCL).1,9,10,11 These translocations occur in a highly conserved 4.0 kb regulatory region of the BCL6 gene (the major translocations cluster – MTC) spanning the promoter, the first noncoding exon and the 5' region of the first intron.1,9,12 This distribution overlaps with the region affected by somatic mutations in the BCL6 gene suggesting a common mechanism for both aberrations.12,13

The partners of the BCL6 chromosomal translocations most often involve the immunoglobulin (IG) genes on chromosome bands 14q32, 2p12 and 22q11, but also a variety of other loci on other chromosomes, a phenomenon termed promiscuous translocation.12,14,15 As a result of the translocation, the 5' regulatory region containing the BCL6 promoter sequence is either removed or truncated leading to the juxtaposition of BCL6 exons 2–10 downstream from the partner gene with its own promoter or other regulatory elements.16,17 In the OCI-Ly8 DLBCL cell line which harbors an IGHG3-BCL6 recombination, BCL6 gene expression is initiated from the IGHG3 promoter (Iitalic gamma3), a phenomenon termed promoter substitution.16 This observation together with a non-GC restricted expression of the partner genes17 suggested that the heterologous promoters might deregulate BCL6 expression. Recent studies demonstrated that BCL6 may have an antiapoptotic effect18 thus suggesting that deregulation of BCL6 expression would have an important role in lymphomagenesis. BCL6 gene and protein expression have been demonstrated to predict clinical outcome in DLBCL.19,20 However, there is no consensus on the effect of BCL6 gene rearrangements on patient outcome. Previous studies have reported a favorable prognosis21 or no effect on DLBCL outcome.9,22 Recently it was demonstrated that BCL6 gene translocations do not necessarily increase BCL6 mRNA expression, since its mRNA expression is similar in DLBCL tumors with and without BCL6 gene translocation.23 It is presently unknown whether BCL6 promoter substitution mechanism is active in actual lymphoma tumors, similar to previous reports in lymphoma cell lines16 and how its activity correlates with BCL6 mRNA levels. This information is critical for understanding the reason for the discrepancy in prognostic significances of BCL6 mRNA expression and BCL6 gene translocations in DLBCL.

We have designed a real-time PCR method to address these questions. Our study demonstrates that the BCL6 is expressed from the translocated allele; however, the amount of BCL6 gene expression may be high, normal or even low in these tumors.

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Materials and methods

Patient material and cell lines

Nine B-cell NHL cell lines (Raji, SU-DHL6, HF1, OCI-Ly8, OCI-Ly10, RC-K8, VAL, KHM10 and SSK41) were selected for this study. OCI-Ly8 and VAL cell lines have been previously reported to harbor BCL6 translocations (t(3;14)(q27; q32) and t(3;4)(q27; p11), respectively).16,24 The SSK41 cell line, originally derived from a patient with FCL without BCL2 gene rearrangement,25 harbors gene amplification, encompassing chromosomal bands 3q27–3q29. This amplicon includes four copies of the BCL6 gene locus, as demonstrated by comparative genomic hybridization and fluorescence in situ hybridization (FISH) studies (data not shown). All cell lines, except OCI-Ly10, were grown in RPMI 1640 medium (Fisher Scientific CO, LLC, Santa Clara, CA, USA), supplemented with 10% fetal calf serum, 2 mM/l glutamine (GIBCO BRL, Grand Island, NY, USA), and penicillin/streptomycin (GIBCO BRL, Grand Island, NY, USA). The OCI-Ly10 cell line was grown in IMDM essential medium (Fisher Scientific CO, LLC, Santa Clara, CA, USA), supplemented with 20% fresh human plasma and 50 muM 2-beta mercaptoethanol.

Biopsy specimens from a total of 110 patients either with FCL and six patients with DLBCL, classified according to the Revised European-American Lymphoma Classification26 were analyzed for the presence of BCL6 rearrangement. For the analysis of BCL6 mRNA expression in this study, we have chosen 20 FCL specimens enriched for the BCL6 rearrangement and the 6 DLBCL specimens. All specimens had been stored in liquid nitrogen as viable cell suspensions of the biopsies. Tumor samples were immunophenotyped by flow cytometry for expression of immunoglobulin (Ig) heavy and light chains and B- and T-cell markers. The percentage of the tumor cells in the specimens was estimated from the light-chain restriction as determined by flow cytometry [goat F(ab')2 FITC anti-human kappa and lambda antibodies, Biosource International INC, Camarillo, CA, USA] in 20 specimens, by immunohistochemistry in four specimens and by FISH analysis of the BCL6 gene translocation in three specimens. In addition, we used total GC-B cells, purified from three human tonsils as described.27

RNA and DNA isolation

Total cellular RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. High-molecular-weight DNA was extracted from 5.0 times 106 cells using a commercially available kit as described by the manufacturer (QIAamp Tissue Kit; Qiagen, Valencia, CA, USA).

Detection of the BCL6 gene translocation

Long-distance inverse (LDI)-PCR was used to detect BCL6 gene translocations and to identify the BCL6 translocation partner genes in the cell lines and in all the tumor samples. The LDI-PCR was performed as previously reported.12 Briefly, 100–500 ng of genomic DNA was digested with BamHI or XbaI, and purified by standard methods. The DNA was diluted to a concentration of 1 mug/ml and incubated at 4°C overnight in the presence of T4 DNA ligase to facilitate intramolecular ligation. The self-ligated circular DNA was used as a template for a nested PCR,12 with additional primers (Akasaka et al, submitted).

In the RC-K8 cell line and IL696 specimen, we also performed 5' SMART rapid amplification of cDNA ends (RACE) PCR using primer, 5'-GTTAGTCCACAGAGCCCCCAGAA-3' and Advantage 2 polymerase mix according to the manufacturer's protocol (Clontech). PCR amplicons were cloned into a TA-PCR cloning vector (Invitrogen, Carlsbad, CA, USA). After the transformation of competent Escherichia coli (1 Shot INV F; Invitrogen) and plating on selective agar (50 mug/ml kanamycin, 40 mul of 40 mug/ml X-gal), several white colonies were picked for plasmid purification using QIAprep kit (Qiagen, Valencia, CA, USA). DNA sequencing was performed on a 373 automatic DNA sequencer (Applied Biosystems) using ABI Prism Big Dye Terminator Kit (Perkin Elmer, Foster City, CA, USA) as recommended by the manufacturer.

FISH studies

Interphase FISH as well as combined fluorescence R banding and FISH analysis were performed using the LSI BCL6 probe (Vysis, Downers Grove, IL, USA), as recently described.28

Measurement of total BCL6 mRNA expression and of BCL6 mRNA expression by specific alleles

Total BCL6 mRNA expression was measured by real-time quantitative reverse transcription (RT)-PCR using the TaqMan technology on an ABI Prism 7900HT Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA) as was previously reported with minor modifications.19 The RNA was reverse transcribed by High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol with minor modification that consisted of an addition of RNase inhibitor (Applied Biosystems, Foster City, CA, USA) at final concentration of 1 U/mul. Samples were incubated at 25°C for 10 min and 37°C for 120 min. PCR reactions were prepared in a final volume of 20 mul, with final concentrations of 1 times TaqMan Universal PCR Master Mix (PE Applied Biosystems, Foster City, CA, USA) and cDNA derived from 20 ng input RNA. Since the precise amount of total RNA added to each reaction mix and its quality (ie, extent of RNA degradation) are both difficult to assess and may vary between the specimens, total BCL6 mRNA expression was normalized on the basis of an endogenous control – glyceraldehydes-3-phosphate dehydrogenase (GAPDH), as was reported previously.19 Total BCL6 mRNA expression was measured by previously reported set of primers19 located in exons 2 and 3 and probe located across exons 2–3 boundary and labeled with 6-carboxy-fluorescein phosphoramidite (FAM) at the 5' end and with 6-carboxy-tetramethyl-rhodamine (TAMRA) as quencher at the 3' end (Table 1). GAPDH mRNA expression was measured by Pre-Developed TaqMan Assay Reagents Endogenous Control kit (Applied Biosystems, Foster City, CA, USA). To allow the total BCL6 mRNA expression to be compared across all the tested samples, the BCL6/GAPDH expression ratio for each sample was also normalized to the BCL6/GAPDH ratio concomitantly measured in Raji cells (calibrator) as suggested in ABI 7700 user Bulletin #2 (PE Applied Biosystems, Foster City, CA, USA) and previously reported.19


To determine the relative contribution of the two alleles to the BCL6 mRNA expression, BCL6 mRNA expression was simultaneously assessed by an additional set of primers located in exons 1 and 2, respectively, and a probe located at the exons 1–2 boundary and labeled with FAM at the 5' end and with TAMRA as quencher at the 3' end (Table 1).

Each RT-PCR run included the five points of the calibration curve for each set of BCL6 primers and probes and for GAPDH (five-fold serially diluted human RNA), a no-template control, the calibrator Raji RNA and patient's samples, all in triplicate.

Analysis of the BCL-6 gene mutations

The first exon region of the BCL-6 gene, which has previously been shown to contain BCL6 binding regulatory sequences,29,30 was amplified by PCR using 5'-ACGCTCTGCTTATGAGGA and 5'-CGGCAGCAACAGCAATAA primers. The PCR amplicons were sequenced on a 373 automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA) using ABI Prism Big Dye Terminator Kit (Perkin Elmer, USA) as recommended by the manufacturer. The same primers used for the PCR were used for forward and backward sequencing.

Statistical analysis

Comparisons of total BCL6 mRNA expression and BCL6 exon 2–3/BCL6 exon 1–2 ratios between specimens were performed by two-tailed Mann–Whitney test, with P-values <0.05 considered to be significant.

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Results

Determination of allele-specific BCL6 expression

To establish a method for determination of allele-specific expression of the BCL6 gene, we designed a real-time quantitative RT-PCR assay based on previously accumulated data on the molecular anatomy of the BCL6 rearrangements1,12 (Figure 1). The vast majority of the BCL6 gene rearrangements result from breakpoints clustering in intron 1 of the BCL6 gene leading to substitution of its nontranslated exon 1 with a sequence from a partner gene. Consequently, while the BCL6 mRNA transcribed from the nonrearranged allele should contain BCL6 gene sequences from exons 1, 2 and 3, the BCL6 mRNA transcribed from the BCL6 allele rearranged at intron 1 should contain exons 2 and 3, but not exon 1. Therefore, in cells without BCL6 gene rearrangement similar amounts of the BCL6 exons 2–3 and exons 1–2 mRNA should be detected. In contrast, in cells harboring both rearranged and un-rearranged BCL6 alleles, a disproportional quantity of the BCL6 exons 2–3 and exons 1–2 mRNAs should be detected unless the rearranged allele is silent. We designed TaqMan primers and probes that would specifically amplify BCL6 exons 1–2 and exons 2–3 mRNA. As expected, genomic DNA was not amplified by either assay (data not shown). The two assays had similar efficiencies of PCR amplification (1.97-fold increase/PCR cycle) resulting in a perfect correlation (R2=0.9978) between the amounts of BCL6 mRNA measured by both assays over a range of 0.08–50 ng total RNA input (Figure 2).

Figure 1.
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Schematic representation of the BCL6 germline exons 1–3 and rearranged BCL6 gene and of the real-time RT-PCR assays. In cells with germline BCL6 gene, both the exons 1–2 and the exons 2–3 set of primers and probes (Forward-1, Reverse-1, P-1 and Forward-3, Reverse-3 and P-3, respectively) will amplify the transcript, resulting in approximately similar amounts of these two amplicons. In cells with BCL6 gene rearrangement, exon 1 is replaced by a partner gene, thus allowing amplification of exons 2–3, but not of exons 1–2 from the rearranged allele transcript, leading to disproportional quantities of the two amplicons.

Full figure and legend (65K)

Figure 2.
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Correlation between the threshold cycles (Ct) of the BCL6 mRNA exons 2–3 and exons 1–2 assays. BCL6 mRNA exons 2–3 and exons 1–2 quantities, as reflected by the Ct, were measured in 50, 10, 2, 0.4 and 0.08 ng input human RNA and the correlation between the quantities (Cts), measured by the two assays, was performed.

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To test the usefulness of this assay and to measure allele-specific BCL6 mRNA expression, we evaluated its performance in nine cell lines with and without BCL6 rearrangement and calculated the BCL6 mRNA exons 2–3/exons 1–2 ratios. In the OCI-Ly8 and the VAL cell lines which harbor BCL6 gene rearrangements,31 BCL6 mRNA exons 2–3/exons 1–2 ratios were 51.19 and 52.92, respectively, with minute amounts of BCL6 mRNA exons 1–2 detected, thus suggesting that almost all the transcribed BCL6 mRNA originated from the rearranged allele. This finding confirmed the previous observation in the OCI-Ly8 cell line, which also showed by an RNA protection assay that almost all the BCL6 mRNA was transcribed from the rearranged allele.16 By contrast, in all the cells lines previously known not to harbor BCL6 gene rearrangements32,33 (and data not shown), the BCL6 mRNA exons 2–3/exons 1–2 ratios were in the range of 0.42–1.02 (meanplusminuss.d.=0.81plusminus0.21), demonstrating equal or excess of BCL6 mRNA originating from exons 1 to 2. The single exception was the RC-K8 cell line,34 in which the exons 2–3/exons 1–2 ratio was 55.0, a result similar to that observed in cell lines with known BCL6 gene rearrangements and suggesting the presence of a rearrangement in this cell line. The original cytogenetic studies of the RC-K8 cell line revealed a complex karyotype that contained t(3;4) (q29;q31).34 Nevertheless, combined banding and FISH analyses using differently labeled probes flanking the BCL6 locus detected a translocation t(3;7) affecting the BCL6 locus which was confirmed by LDI-PCR (Figure 3). Moreover, performance of the 5'RACE PCR using a primer originating in the BCL6 gene 3' to exon 2 disclosed a single amplicon corresponding to a fusion of the exon 2 of the BCL6 gene to a sequence from chromosome 7 that skipped exon 1 of the BCL6 gene. Therefore, the BCL6 mRNA exons 2–3/exons 1–2 ratio in cell lines harboring a BCL6 gene rearrangement was significantly higher than the ratio in cell lines not harboring a BCL6 gene rearrangement (Figure 4 and Table 2). This observation demonstrates that in the analyzed cell lines with BCL6 gene rearrangement BCL6 mRNA is transcribed almost exclusively from the rearranged allele.

Figure 3.
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Fluorescence R-banding analysis and subsequent FISH evaluation of the RC-K8 cell line. BCL6 gene translocation was assessed using the LSI BCL6 Dual Color Break Apart Rearrangement probes (Vysis Inc, IL, USA). Nonrearranged BCL6 gene is represented by orange/green fusion signal. Rearrangement results in separation of the two colors and their appearance on two separate chromosomes.

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Figure 4.
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BCL6 mRNA exons 2–3/exons 1–2 ratios in NHL cell lines with and without BCL6 gene rearrangement.

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In contrast to cell lines, NHL tumor samples contain nonmalignant cells, some of which express BCL6 mRNA (GC B cells and some CD4+ T cells). We therefore determined the sensitivity of this method by detecting imbalanced expression contributed by the tumor. We have measured BCL6 mRNA exons 2–3/exons 1–2 ratios in different proportional mixtures of the Raji and the OCI-Ly8 cell lines (Figure 5). The Raji cell line harbors only the germline BCL6 gene and expresses high levels of normal BCL6 mRNA. The OCI-Ly8 cell line harbors a rearranged BCL6 gene on one allele.16 By choosing a threshold for the mRNA exons 2–3/exons 1–2 ratio of 1.2 (the mean ratio +2 s.d. in the cell lines without BCL6 gene rearrangement), this method could detect a disproportional increase in the quantity of the BCL6 exons 2–3 mRNA in cell mixtures containing at least 70% of OCI-Ly8 cells, even though the gene expression by the OCI-Ly8 cell line is lower than that of Raji cell line.

Figure 5.
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BCL6 mRNA exons 2–3/exons 1–2 ratios in various mixtures of cells with and without BCL6 gene rearrangement. BCL6 mRNA exons 2–3/exons 1–2 ratios were calculated in Raji cells (assumed to represent normal cells without BCL6 gene rearrangement), OCI-Ly8 cells (assumed to represent tumor cells with BCL6 gene rearrangement) and 1:1, 1:2, 1:4, 1:8 and 1:16 mixtures of these cells.

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Allele-specific BCL6 mRNA expression in NHL specimens

Having confirmed in cell lines the predominant expression of the BCL6 gene from the translocated allele, we wished to extend these observations to actual NHL tumor specimens. In all, 20 specimens of FCL and six specimens of DLBCL, analyzed by LDI-PCR to detect the presence of BCL6 gene rearrangement, were included in the study. BCL6 gene rearrangement was detected in 10 specimens and the translocation partner genes are summarized in Table 3. All these specimens were assessed for the BCL6 mRNA exons 2–3/exons 1–2 ratios to determine whether the rearranged BCL6 allele is disproportionally transcriptionally active. The BCL6 mRNA exons 2–3/exons 1–2 ratios were significantly higher in specimens harboring BCL6 gene rearrangement than in specimens without BCL6 gene rearrangement (P=0.0002; meanplusminuss.d. of 4.23plusminus4.18 and 1.10plusminus0.16, respectively, Figure 6). The BCL6 mRNA exons 2–3/exons 1–2 ratios were significantly higher in cell lines harboring BCL6 gene rearrangement than in real tumors with the rearrangement (mean of 53.04 vs 4.23, respectively). This could be attributed to the presence of normal cells in the specimens or presence of the translocation in a fraction of the tumor cells. Moreover, in four of the 10 NHL specimens with BCL6 gene translocation, the observed BCL6 mRNA exons 2–3/exons 1–2 ratios were in the same range or only slightly higher than the ratios detected in the specimens not harboring BCL6 gene rearrangement. In one of these specimens (IL122), the percentage of the tumor cell in the specimens (55%) was below the sensitivity threshold of 70% established above, thus providing an explanation for the observed low ratio. In the remaining three specimens (Nos. 44, IL351 and IL696), the percentage of the tumor cells in the biopsy was above 78%, which should have been high enough to detect a high exons 2–3/exons 1–2 ratio if it existed. However, in specimen No. 44 only 5% of the cells harbored BCL6 gene translocation as determined by FISH, thus possibly explaining the low BCL6 mRNA exons 2–3/exons 1–2 ratio. In contrast, 78 and 85% of cells harbored BCL6 gene translocation as determined by FISH in specimens IL696 and IL351, respectively (Table 3). The breakpoints of the BCL6 gene rearrangements were located in the intron 1 of the BCL6 gene in three of these cases (Nos. 44, IL351 and IL122). In specimen IL696, the BCL6 rearrangement breakpoint was located in 5' UTR (5' to exon 1) of the BCL6 gene – a possible explanation for the relatively low BCL6 mRNA exons 2–3/exons 1–2 ratio observed by the applied method. However, in the RC-K8 cell line, which harbors a similar location of the break point, the applied method predicted the presence of the BCL6 gene rearrangement due to exon 1 skipping (data not shown), as was recently reported in the YM cell line harboring t(3;16)(q27;p11) translocation.35 Consequently, we have performed a 5' RACE in this IL696 specimen using BCL6 primer located 3' to exon 2. Sequencing of the amplicons disclosed presence of two amplification products, one corresponding to a nonrearranged BCL6 mRNA and the second originating from the rearranged allele and indeed demonstrating exon 1 skipping. Consequently, we do not think that breakpoint location explains the low BCL6 mRNA exons 2–3/exons 1–2 ratio. Moreover, 5'RACE analysis collaborated our data on relative contribution of individual alleles to BCL6 mRNA expression: while in IL696 specimen, in which the exons 2–3/exons 1–2 ratio was low (1.45) we detected 5'RACE amplicons from both the rearranged and the nonrearranged alleles, in RC-K8 cell line in which the exons 2–3/exons 1–2 ratio was high (55.0), we detected 5'RACE amplicon only from the rearranged allele. The partner gene of the translocation in specimens No. 44 and IL351, both with low BCL6 mRNA exons 2–3/exons 1–2 ratios, was CIITA. However, this partner gene (CIITA) significantly deregulated BCL6 expression in another specimen (IL124), thus suggesting that the low BCL6 mRNA exons 2–3/exons 1–2 ratios observed in these two specimens could not be attributed to the nature of the partner gene. Consequently, our findings demonstrate that BCL6 mRNA is transcribed from the rearranged allele in majority of the NHL specimens harboring BCL6 gene rearrangement, irrespective of the partner gene. The reason why BCL6 gene was equally expressed from translocated and nontranslocated alleles in tumor specimens IL351 and IL606 remains unclear.

Figure 6.
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BCL6 mRNA exons 2–3/exons 1–2 ratios in NHL tumor specimens with and without BCL6 gene rearrangement.

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When it does occur, the reduced expression of the normal BCL6 allele has been recently shown to be caused by negative feedback effects of BCL6 protein which binds to regulatory sequences in the first exon.30 These sequences are removed by the translocation, thus allowing unregulated BCL6 mRNA expression from the rearranged allele. Alternatively, mutations in these sequences might disturb the auto-repressive effects of the BCL6.30 Consequently, we have searched for mutations in the BCL6 gene exon 1 regulatory sequences in the four tumors in which the observed BCL6 mRNA exons 2–3/exons 1–2 ratios were in the same range as the ratios detected in the specimens not harboring BCL6 gene rearrangement. Mutations were not found in any of these tumors. Deletions of the exon 1 in the nontranslocated allele,36 might also explain the normal BCL6 mRNA exons 2–3/exons 1–2 ratios in these cases. However, no deletions were detected in these cases by LDI-PCR. Finally, no consensus BCL6 binding motif30 was detected in the regulatory sequences of CIITA translocated next to the BCL6 gene.

Total BCL6 mRNA expression in NHL with and without BCL6 gene rearrangement

Next we examined the total BCL6 mRNA expression in NHL specimens and cell lines with and without BCL6 gene rearrangement (Figure 7). Since BCL6 mRNA expression levels were previously reported to differ between FCL and DLBCL,19 each NHL subtype was analyzed separately. Indeed, normalized total BCL6 mRNA expression levels in FCL overlapped with its expression in GC and were higher than in DLBCL specimens and cell lines. In both FCL and DLBCL samples, there was no difference in the BCL6 mRNA expression between specimens with and without BCL6 gene rearrangement. Interestingly, total BCL6 mRNA expression levels were very low in some DLBCL specimens and cell lines that had a BCL6 gene rearrangement. Normalization of the total BCL6 mRNA expression to the tumor cell content in each specimen did not change these conclusions (data not shown). In cell lines and tumors with translocation and with BCL6 expression predominantly from the translocated allele, both high (eg IL351, IL126) and low (eg OCI-Ly8, RC-K8) BCL6 mRNA expression levels were observed. Interestingly, relatively low BCL6 mRNA expression was observed in one of the two NHL tumors (IL696) with BCL6 gene rearrangements, but without biased allelic expression.

Figure 7.
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Total normalized BCL6 mRNA in FCL and DLBCL specimens and cell lines with and without BCL6 gene rearrangement. BCL6 mRNA expression normalized to GAPDH and Raji cell line expression (see Materials and methods) was evaluated in 20 FCL specimens, 13 DLBCL specimens and cell lines and in GC cells obtained from three tonsils from normal individuals. There is no statistical difference in BCL6 mRNA expression between FCL specimens with and without rearrangement (P=0.33) and between DLBCL specimens with and without rearrangement (P=0.52).

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Discussion

The BCL6 gene, commonly affected by chromosomal translocations, deletions and mutations in NHL specimens, is implicated in the pathogenesis of lymphoma, but its role in this process is still not clear. When highly expressed in GC lymphocytes, the BCL6 protein represses transcription of several genes, including the BLIMP1 and programmed cell death-2 (PDCD2) genes, which are involved in plasma cell differentiation and apoptosis, respectively.37,38 In addition, BCL6 may act as an immortalizing oncogene by rendering cells resistant to the antiproliferative signals emanating from the p19ARF-p53 pathway, as has been demonstrated in the senescence response of fibroblasts.39 Therefore, it is conceivable that inappropriate expression of BCL6 may result in a block of terminal differentiation, prevention of apoptosis and continued proliferation, predisposing the B cells to the neoplastic transformation.

In this study, we sought to evaluate the effects of chromosomal translocation involving the BCL6 gene in NHL tumors. We examined BCL6 mRNA expression to determine whether the BCL6 gene is preferentially expressed from the rearranged allele and whether BCL6 gene expression levels are elevated as a result of substitution of the promoter of a different gene.

In NHL, the effects of mutations located in exon 1 and in the intron 1-silencer sequences on the BCL6 mRNA expression were previously reported.30,40,41 Such mutations may deregulate BCL6 mRNA expression by alleviating the inhibitory effects of the silencers. Rearrangements of the BCL6 gene, which replace its transcription regulatory sequences by those of the partner genes (promoter substitution), have been shown to deregulate BCL6 gene expression.17 Indeed, preferential expression of the BCL6 gene from the translocated allele has been demonstrated in the OCI-Ly8 cell line by the RNA protection assay.16 General proof for this mechanism, in NHL tumor specimens with BCL6 gene rearrangement to variable promoters, has not been reported, most probably due to the technical difficulties and the labor-intensive nature of the RNA protection assay and the requirements for this assay of large amounts of high-quality RNA. Owing to this, we designed a quantitative assay to address this question based on the TaqMan technology, which requires only 20 ng of RNA. Using this method we detected the presence of a previously unknown BCL6 gene rearrangement in the RC-K8 cell line. Applying this method, we demonstrated that preferential expression of BCL6 mRNA from the translocated allele occurs in multiple cell lines and in most of the evaluated tumor specimens, thus confirming the initial observation by Ye et al16 in the OCI-Ly8 cell line. However, examination of actual NHL tumors with BCL6 translocation also disclosed that both the translocated and the nontranslocated alleles contributed equally to the BCL6 mRNA expression in two tumors. This might indicate that heterologous promoters do not always dominate. The phenomenon of silencing of the nontranslocated allele has recently been shown to be caused by an auto-regulatory repression of the nontranslocated allele by the BCL6 protein.30 Our data demonstrate that this phenomenon occurs in majority but not all actual NHL specimens with BCL6 rearrangement. Neither the nature of the substituted promoters nor the presence of activating mutations in the BCL6 regulatory sequences could explain the equal allelic expression of the BCL6 gene in the two cases that expressed BCL6 from both the rearranged and the nonrearranged alleles. Another recent evaluation of actual NHL tumors also found that BCL6 activating mutations do not always deregulate its expression.30 In that study BCL6 gene mutations ameliorated the BCL6 negative autoregulatory effect, but this occurred in only three out of four lymphoma specimens.

We also addressed the issue of the effects of the BCL6 gene rearrangements on total BCL6 mRNA expression levels. Numerous previous studies have demonstrated that the presence of BCL6 gene rearrangements is not a sine qua non for increased BCL6 protein expression. However, in these studies BCL6 mRNA expression was not evaluated.42,43 Only 50% of NHL harboring a BCL6 gene rearrangement demonstrated stainable BCL6 protein. It was assumed that BCL6 gene rearrangements increase BCL6 mRNA expression and that the observed lack BCL6 protein expression was due to post-translational regulation of the BCL6 protein expression.18,44 However, low BCL6 mRNA levels, despite the presence of the BCL6 gene rearrangement, could also account for the observed absence of the BCL6 protein expression in some of these tumors. Indeed, recent evaluation of BCL6 mRNA expression in 23 DLBCL cases demonstrated similar BCL6 mRNA expression in DLBCL specimens without BCL6 translocation and with BCL6 translocation to Ig genes; however, lower BCL6 mRNA expression was observed in cases with non-Ig partners.23 Our results, demonstrating that high BCL6 mRNA levels need not be the consequence of BCL6 translocation, provide an explanation for why the presence of BCL6 rearrangements is not associated with improved clinical outcome of DLBCL.

In conclusion, our study demonstrates that BCL6 is usually expressed predominantly from the rearranged allele in NHL tumors harboring BCL6 gene rearrangement. The presence of BCL6 gene rearrangement in NHL is not necessarily associated with elevated BCL6 mRNA levels. Therefore, if the BCL6 rearrangements play a role in lymphomagenesis, they do so by deregulating and not necessarily by increasing BCL6 gene expression. Further studies, such as stable transfections of rearranged BCL6 gene constructs, are needed to clarify the mechanism by which deregulated BCL6 gene expression contributes to lymphoma pathogenesis.

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References

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Acknowledgements

We thank Dr Stephan Gesk for assistance with FISH analysis. This work was supported by grants CA33399 and CA34233 from the USPHS-NIH and grant FIS 01/0015 from Spanish Ministry of Health. RL is an American Cancer Society Clinical Research Professor.

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