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Prepublished online as a Blood First Edition Paper on November 27, 2002; DOI 10.1182/blood-2002-08-2464.
NEOPLASIA
From the Department of Hematology and Oncology,
Graduate School of Medicine, Kyoto University, Japan.
Anaplastic large cell lymphoma (ALCL) with t(2;5)(p23;q35) and
Hodgkin disease (HD) share many cellular features, including expression
of CD30. We compared gene expression profiles of 4 ALCL (Karpas 299, SU-DHL-1, DEL, SR-786) and 3 HD cell lines and found that
BCL3, which encodes a nuclear protein belonging to the
I Anaplastic large cell lymphoma (ALCL) was initially
defined on the basis of the anaplastic appearance of tumor cells,
characteristic intrasinusoidal spread, and consistent expression of the
cytokine receptor CD30.1 However, heterogeneity in the
cytology as well as in the clinical features has been
described.2,3 Anaplastic large cell lymphoma
kinase-positive (ALK+) ALCL was subsequently identified as
a distinctive disease entity within the heterogeneous group of
ALCL.4,5 Most cases with ALK+ ALCL carry
t(2;5)(p23;q35), which leads to the generation of nucleophosmin
(NPM)/ALK fusion protein.2,3,6 Introduction of the
NPM/ALK fusion gene in mouse cell lines
leads to a transformed phenotype, and several downstream signaling
pathways through which this particular fusion protein exerts its
oncogenic action have been identified.7-9
Hodgkin disease (HD) is another lymphoid neoplasm, in which neoplastic
Reed-Sternberg (RS) cells express the CD30 molecule on their cell
surface.10 Studies of HD-derived cell lines and micromanipulated RS cells have revealed recurrent molecular
abnormalities of HD.11 An initial study demonstrated
constitutive nuclear factor- Although both ALCL and HD express CD30 at high levels, stimulation of
this molecule exerts contradictory effects on these 2 cell
types.17-20 ALCL cells upon stimulation with a CD30
agonistic antibody show apoptotic cell death along with the impairment
of activation of prosurvival NF- Cells
Complementary DNA array hybridization
Southern and Northern blot analyses and long-distance polymerase chain reaction Southern and Northern blotting were carried out as described previously.32 The cDNA probe for BCL3 was clone cLK2 containing a 1.8-kb cDNA fragment.31 Probe A representing the 5' cytosine-guanine dinucleotide (CpG) island of BCL3 was a 1.4-kb PstI fragment containing exon 1 and part of intron 1, whereas probe B for the 3' CpG was a 0.92-kb PstI/BamHI containing exon 7 and neighboring introns.30 The BCL6 probe represented the major translocation cluster.38 Long-distance polymerase chain reaction (LD-PCR) amplifying a DNA fragment encompassing the NPM/ALK fusion point was as described previously.37 The PCR was carried out using an automated thermal cycler (GeneAmp PCR System 2400, Applied Biosystems, Foster City, CA).Real-time reverse transcriptase-mediated polymerase chain reaction Real-time PCR analysis based on the TaqMan methodology was performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems). The sequences of oligonucleotide primers and a fluorogenic probe for BCL3 mRNA were 5'-TCGACGCAGTGGACATTAAGAG-3' (forward); 5'-ACATTTGCGCGTTCACGTT-3' (reverse); and 5'-TCATCCACGCCGTGGAAAACAACAG-3' (probe). Complementary DNA was synthesized from 1 µg of total cellular RNA using random primers. The DNA standard template containing the BCL3 cDNA sequence was generated by cloning into pGEM-T EASY vector (Promega, Madison, WI). An aliquot of cDNA or standard template DNA (1 µL) was added to the PCR reaction mixture containing 1 × TaqMan Universal PCR Master Mix (Applied Biosystems), 400 nM of each primer, and 100 nM of probe in a total reaction volume of 50 µL. After an initial incubation for 2 minutes at 50°C and 10 minutes at 95°C to activate the Taq polymerase, 50 cycles of 15 seconds at 95°C and 1 minute at 60°C were carried out. The CT (threshold cycle) parameter was defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe passed a preset threshold. The standard curve, in which CT decreased in linear proportion to the log of the template copy number, was established by serially diluted pGEM-T-BCL3 plasmid DNA. The CT values of test materials were plotted on standard curve, and the corresponding copy number was calculated by Sequence Detector version 1.6 software (Applied Biosystems). The amount of BCL3 cDNA copy number of the test materials was divided by the endogenous reference, 18S ribosomal RNA (rRNA) (TaqMan Ribosomal RNA Control Reagents; Applied Biosystems), and the BCL3/18S rRNA ratio was further normalized with that of adult T-cell leukemia HUT 102 cells. All assays were performed in triplicate.Western blot analysis Cells were lysed in 1 × loading buffer containing protease inhibitor cocktail.39 The lysate was loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels and electrotransferred onto Immobilon polyvinylidene difluoride (PVDF) transfer membranes (Millipore, Bedford, MA). The membranes were blocked in phosphate-buffered saline-Tween (PBS-T) buffer containing dried milk and then incubated with the following antibodies purchased from Santa Cruz Biotechnology (Santa Cruz, CA): polyclonal rabbit anti-Bcl-3 antiserum raised against a peptide at the carboxy-terminus of human Bcl-3 (sc-185); monoclonal immunoglobulin G1 (IgG1) mouse anti-p65 (sc-8008); monoclonal IgG1 mouse anti-p50 (sc-8414); monoclonal IgG2a mouse anti-p52 (sc-7386); and polyclonal goat anti-ALK antiserum (sc-6344). After extensive washing in PBS-T buffer, the blots were incubated for 1 hour with horseradish peroxidase-conjugated secondary antiserum followed by enhanced chemiluminescence reaction (Amersham Pharmacia Biotech, Piscataway, NJ).Electrophoretic mobility shift assay for NF-  B site of the
immunoglobulin light chain gene was 5'-AGTTGAGGGGACTTTCCCAGGC-3'.
Binding reactions were performed in a total volume of 10 µL
containing 5 µg extract, 2 µL of 5 × binding buffer (Promega),
and 1 µL of 32P-end-labeled probes.38
Incubations were carried out for 30 minutes at 25°C, and the
resulting complexes were resolved on 5% nondenaturing polyacrylamide
gels in 0.5 × TBE. For competition experiments, incubations were
performed with a 100-fold excess of unlabeled probe. For antibody
supershift assays, the lysates were preincubated for 10 minutes at
25°C with 4 µg antibody before the addition of the radiolabeled gel
shift probe. Antibodies used for the supershift assay were from Santa
Cruz Biotechnology: monoclonal IgG1 mouse anti-p65 (sc-8008X);
polyclonal goat anti-p50 (sc-1190X); and monoclonal IgG2a mouse
anti-p52 (sc-7386X).
Fluorescence in situ chromosomal hybridization Metaphase spreads were prepared as described previously.39 A bacterial artificial chromosome (BAC) clone containing the BCL3 locus (clone no. 84C16 of the RPCI-11 Human BAC Library; Invitrogen, Carlsbad, CA) was labeled with digoxigenin-11-deoxyuridine triphosphate (digoxigenin-11-dUTP) (Roche Diagnostics, Mannheim, Germany) by nick translation. Hybridization and washing were performed according to the manufacturer's instructions, and hybridization signals were detected by a fluorescein isothiocyanate (FITC)-conjugated antidigoxigenin antibody (Roche Diagnostics). The chromosomes and nuclei were counterstained with propidium iodide. Fluorescence in situ chromosomal hybridization (FISH) results were analyzed with a fluorescence microscope (Olympus, Tokyo, Japan).39 A charge-coupled device camera (CoolSNAP/OL; Olympus) attached to the fluorescence microscope and LuminaVision software (Mitani, Fukui, Japan) were used to capture and process images.Bisulfite DNA sequencing Bisulfite conversion of DNA was performed using a CpGenome DNA Modification Kit (Intergen, Purchase, NY). Bisulfite-modified DNA (50 ng) was subjected to PCR amplification of the CpG island of BCL3 using a reverse primer 5'-CAAAATCAACCAAACCATC-3' and a forward primer 5'-GAGTAGAGTTTGGAGAAAT-3'. The PCR products were ligated into pGEM-T EASY vector, and transformation and extraction of DNA were performed by established methods. Nucleotide sequencing to determine the methylation status of the insert was performed with a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems), and the sequencing reactions were resolved on an automated sequencer (ABI Prism 310 Genetic Analyzer, Applied Biosystems).Indirect immunofluorescence Cells were pelleted onto poly-L-lysine-coated coverslips (Nacalai Tesque, Kyoto, Japan) by a cytocentrifuge (600 rpm, 3 minutes). The cells were washed with PBS and fixed for 15 minutes with 3.0% paraformaldehyde in PBS at room temperature. The fixed cells were treated with 0.1% Triton X-100 for 4 minutes and cold methanol for 5 minutes. The cells were then permeabilized with 0.05% PBT solution (0.05% Tween 20 in PBS containing 0.1% fetal calf serum [FCS]) for 5 minutes and blocked with 2 Blocking (DAKO, Carpinteria, CA). After 3 washes with 0.05% PBT, the specimens were incubated with primary antibodies (polyclonal rabbit anti-Bcl-3; polyclonal rabbit anti-p65 (sc-372), Santa Cruz Biotechnology; polyclonal rabbit anti-p50 (no. 06-414), Upstate Biotechnology, Lake Placid, NY), which were diluted 1:100 in 0.05% PBT for 1 hour at room temperature. They were washed 3 times with 0.05% PBT and incubated with a secondary antibody conjugated to Alexa 488 (Molecular Probes, Eugene, OR) for 20 minutes at room temperature. After rinsing in PBS, the DNA was stained with DAPI (4,6 diamidino-2-phenylindole) (Vysis, Downers Grove, IL). Preparations were examined and photographed using a camera-equipped fluorescence microscope (Olympus).
Hierarchic clustering that differentiates ALCL and HD Table 1 summarizes the characteristics of ALCL and HD cell lines used in this study. Three of the 4 ALCL cell lines carried cytogenetically identified t(2;5)(p23;q35), whereas the translocation of DEL was t(5;6)(q35;p21). Genomic DNA prepared from the 4 ALCL lines was subjected to LD-PCR amplifying a DNA fragment encompassing the NPM/ALK fusion point. The results showed that the 4 cell lines were positive for amplification and the sizes of the PCR products were unique to each cell line. Western blot analysis confirmed restrictive expression of ALK protein in ALCL cells. Deletion and mutation within the IKBA gene of KM-H2 and L428 were detected by appropriate reverse transcriptase (RT)-PCR and sequencing analysis. The genotype was determined by receptor gene-rearrangement studies.
We profiled 4 ALCL and 3 HD cell lines using Atlas Human 1.2 Array
containing 1176 cDNA fragments of known human genes, including several
housekeeping genes. After global normalization, we selected a total of
110 genes that were differentially expressed between the 2 disease
groups at a P value of less than 0.1 by the
Student t test. Hierarchic clustering analysis based upon
the levels of these genes was applied to both axes using the GeneSpring
software. As shown in Figure 1A, the
analysis effectively separated ALCL and HD and highlighted the
gene expression pattern characterizing the 2 groups. The ALCL branch
diverged to Karpas 299 and to the remaining 3 cell lines, generating a
tight cluster, whereas the T-cell type HDLM-2 was separated
from the other 2 B-cell type HD cell lines. Examination of the gene
axis revealed that 69 genes were expressed to a higher degree in ALCL
than HD, whereas 41 genes were expressed at a higher level in HD than
ALCL. The ALCL-associated genes included BCL3, which lay
close to clusterin and whose preferential expression in ALCL was
described previously40 (Figure 1B). In contrast, the gene
for NF-
High-level expression of BCL3 in ALCL cells To confirm the higher levels of BCL3 expression in ALCL than HD, we performed Northern blotting using the BCL3 cDNA clone as a probe and Western blotting using an anti-Bcl-3 polyclonal antibody. As shown in Figure 2, the levels of BCL3 expression at both the mRNA and protein levels were in good accordance with those of Atlas array analysis. The level of T-cell type HDLM-2 was higher than the other 2 B-cell type HD cell lines. A comparison with other types of cell lines revealed a predominant level of BCL3 expression in ALCL.
We next developed real-time RT-PCR to quantify the BCL3 mRNA
levels. The forward primer was designed for exons 4 through 5 and the
reverse primer for exon 6 to prevent amplification of genomic DNA. The
PCR product was sequenced to verify that the expected fragment
was amplified. The calculated BCL3 mRNA copy number was
normalized by the level of 18S rRNA of each sample, and the ratio of
BCL3/18S rRNA of HUT 102 cell line was arbitrarily defined
as 1. Figure 3 summarizes the
BCL3 mRNA levels in a variety of hematologic tumor cell
lines and clinical materials. The levels of the 4 ALCL cell lines and 3 clinical materials with t(2;5)+ ALCL were comparable to or
higher than that of a B-CLL carrying t(14;19)(q32;q13), in which high
BCL3 mRNA expression resulting from the translocation was
previously determined.32 Among t(2;5)+ ALCL
cells, SR-786 showed the highest level of BCL3 mRNA. Other types of lymphoid and myeloid disease expressed BCL3 mRNA at
low levels. These data clearly confirmed that t(2;5)+ ALCL
cells express a predominant level of BCL3 among hematologic tumors.
BCL3 gene copy number in ALCL cells To investigate the molecular mechanism underlying the high BCL3 expression in ALCL, we first performed Southern blotting analysis of DNA extracted from ALCL cell lines using the BCL3 cDNA probe (Figure 4A). Although no rearrangement was detected within the region covered by the restriction enzymes used, hybridizing bands of SU-DHL-1 cells were intensified compared with those of other cell lines.
We next performed FISH analysis of metaphase spreads of ALCL cells using a fluorescence-labeled BAC clone containing the BCL3 locus. The chromosome numbers of the 4 ALCL cell lines were in the near-triploid range. As shown in Figure 4B, SU-DHL-1 cells carried 3 normal-appearing chromosome 19's and 2 marker chromosomes containing multiple BCL3 gene copies. The amplification of BCL3 gene copies was also observed in interphase nuclei (Figure 4C). Karpas 299 cells showed 4 hybridizing signals on interphase nuclei (Figure 4C), and the BCL3 gene was duplicated on one chromosome 19 (Figure 4B). In contrast, interphase cells of DEL and SR-786 carried 3 fluorescence signals (Figure 4C); on metaphase cell analysis, the latter had 3 copies of normal chromosome 19, whereas 2 of the 3 chromosome 19's of the former were affected by undetermined structural aberrations (data not shown). Thus, a gene dosage effect was potentially responsible for the high-level BCL3 expression in SU-DHL-1 and Karpas 299. The 3' CpG island of BCL3 was demethylated in SU-DHL-1 and DEL cells A characteristic structural feature of BCL3 is that it contains 2 independent CpG islands30 (Figure 5A). The 5' CpG island containing exon 1 was unmethylated in all the tissues tested,30 and this previous observation was confirmed in DNA from a variety of hematologic tumor cell lines (data not shown). In contrast, the methylation status of the 3' CpG island, which encompasses exons 4 through 7 covering approximately 1.6 kb of DNA, varied among cell types and tissues.30 We therefore studied whether ALCL and HD cells exhibit different methylation patterns of the 3' CpG and whether the degree of methylation is related to the level of expression of BCL3. Because 6 methylation-sensitive restriction enzyme sites that is, 2 NotI, 2 SacII, and 2 BssHII siteswere identified within the 3' CpG, we
performed a double digest of DNA with BglII and either one
of the 3 enzymes and hybridized the DNA with probe B representing the
3' CpG. The results showed no digestion of DNA from the hematologic
tumor cell lines tested, with the exceptions of 2 ALCL cell lines,
SU-DHL-1 and DEL (Figure 5B; the data of the other cell lines are not
shown). Because the restriction sites were distributed within a 1.2-kb
region, the 3' CpG of these 2 cell lines was determined to be entirely
unmethylated.
We next prepared bisulfite-treated DNA of the ALCL and HD cell lines,
in which unmethylated cytosine was selectively converted to uracil. A
360-bp fragment within the 3' CpG island, which contained 22 CpG sites, was amplified by PCR using appropriately designed primers.
Sequencing analysis revealed that 3 of 3 and 2 of 3 clones of SU-DHL-1
and DEL, respectively, showed conversion from C to T at almost all the
CpG sites, whereas the sequenced areas of Karpas 299, SR-786,
and the 3 HD cell lines were more than 90% methylated (Figure
6). We applied the bisulfite DNA
sequencing to 3 clinical materials of t(2;5)+ ALCL. The
frequencies of demethylation calculated from 10 independent clones
for each sample were 0.9% (no. 646), 8.6% (no. 1029), and 7.3%
(no. 1078).
Comparison of NF- B family of inhibitors of NF-B transcriptional
factors,21-29 high-level expression of BCL3 is
likely to affect the NF-B activity of ALCL cells. Whole-cell lysates
of ALCL and HD cell lines were incubated with the
32P-labeled oligonucleotide probe containing a B binding
site and analyzed by EMSA (Figure 7A).
High levels of NF-B binding activity in the HD cell lines were
clearly demonstrated as reported previously.12 On the
other hand, the ALCL cells exhibited considerable levels of
(p50)2 homodimer activity determined by supershift
analysis. However, the shifted bands representing the p65/p50
heterodimer were much less intense than those of HD; SU-DHL-1
cells lacked a measurable level of p65/p50 activity. The activities of
both (p50)2 and p65/p50 of Karpas 299 cells were higher
than those in the remaining 3 ALCL cells, potentially accounting for
the divergence of ALCL cell lines on array analysis.
To clarify the difference in NF- We finally stained Karpas 299 and KM-H2 cells for Bcl-3, p65, and p50
by indirect immunofluorescence to study whether these proteins exhibit
differential subcellular localization between ALCL and HD.
Cytocentrifuged preparations of the cells were incubated with primary
antibodies against these proteins and labeled with the Alexa
488-conjugated secondary antibody. As shown in Figure 8A, Karpas 299 cells displayed bright
nuclear staining against Bcl-3, confirming the nuclear localization of
this particular protein belonging to the I
Immunofluorescence against p50 was detected in both the nucleus
and cytoplasm of these 2 cell types; the nuclei of Karpas 299 were more
intensely labeled than those of KM-H2 (Figure 8C). These results
suggest that high-level nuclear Bcl-3 in ALCL cells may significantly
affect the localization of NF-
The cDNA array technology has identified global gene expression differences among subtypes of hematologic tumors and provided new diagnostic and prognostic markers.40-44 However, the studies have not been able to predict whether these markers are implicated in the oncogenic process of particular tumors or whether the expression of each gene reflects only the characteristics of normal counterpart cells from which the tumor developed. Here, we showed the gene expression profiles differentiating t(2;5)+ ALCL and HD, both of which share many clinical and pathologic features. The array analysis and additional studies at both the mRNA and protein levels clearly demonstrated predominant expression levels of BCL3 in t(2;5)+ ALCL. We next found that the BCL3 gene of ALCL cells was altered by gene amplification and/or demethylation of the intragenic CpG island, potentially accounting for the high-level expression of the gene. These 2 alterations, however, were not common to all t(2;5)+ ALCL cells tested; SR-786, showing the highest level of BCL3 mRNA, lacked amplification or demethylation. Nevertheless, it is most likely that high-level expression of BCL3 plays an oncogenetic role in the development of t(2;5)+ ALCL. The Bcl-3 protein contains 7 ankyrin repeats and shares structural
features with the members of the I Evidences for the involvement of NF-
BCL3 was originally identified as a putative oncogene by
cloning the breakpoint of t(14;19)(q32;q13), which is a recurrent chromosomal translocation in B-CLL.31-33 As a result of
translocation, BCL3 on 19q13 is juxtaposed to the The BCL3 gene contains 2 CpG islands.30 The 5' CpG, covering the promoter and the transcription initiation site, was unmethylated in all the tissues and hematologic tumor cell lines tested. Sequencing analysis of the promoter of BCL3 of ALCL disclosed a germ-line configuration in this region (data not shown), indicating that genetic alteration of the promoter at the nucleotide level does not account for the high-level expression of BCL3 in ALCL. In contrast, the 3' CpG was unmethylated in sperm DNA and methylated to varying degrees in several other tissues,30 whereas essentially methylated in DNA from peripheral blood leukocytes30 and most hematopoietic tumor cells. The function of 3' CpG is currently unknown. A study of the apolipoprotein E gene showed that transcription of the gene was not related to methylation status of the 3' CpG.48 On the other hand, methylation of the intronic CpG of mouse insulinlike growth factor type 2 receptor gene (Igf2r) acted as an imprinting signal and was positively correlated to the transcriptional activity of Igf2r.49 We showed here that the 2 particular ALCL cells expressing a high level of BCL3 mRNA carried almost entirely unmethylated 3' CpG islands. Thus, it is possible that transcription of BCL3 is negatively controlled, at least in part, by methylation of the 3' CpG. The previous studies and our present work suggest that t(2;5)+ ALCL cells are characterized by the presence of NPM/ALK fusion protein predominantly in the cytoplasm, expression of the CD30 molecules on the cell surface, and high-level expression of Bcl-3 in the nucleus. Because there is no link between the signaling cascades activated by NPM/ALK and those by CD30 stimulation, t(2;5) leading to the generation of NPM/ALK may occur at a stage of T-cell differentiation that expresses CD30, and therefore CD30 expression itself may not be involved in the development of ALCL9; indeed, ALCL as well as HD cells carry the CD30 gene with a germ-line configuration.50 In contrast, the BCL3 gene of ALCL cells showed genetic and epigenetic modifications, both of which are likely associated with oncogenesis. We suggest that the high-level expression of BCL3 is an important process in the development of ALCL and contributes to the cellular features of this particular lymphoid tumor.
The authors thank Mitsubishi Kagaku Bio-clinical Laboratories (Tokyo, Japan) for its technical assistance with the FISH analysis.
Submitted August 12, 2002; accepted November 15, 2002.
Prepublished online as Blood First Edition Paper, November 27, 2002; DOI 10.1182/blood-2002-08-2464.
Supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (no. 13470204) of Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hitoshi Ohno, Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, 54-Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606-8507, Japan; e-mail: hohno{at}kuhp.kyoto-u.ac.jp.
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Overexpression of I | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
activity. Although the genetic lesions of the IKBA
gene are not common to all RS cells, as yet unidentified defects in the
I
ALCL, and HD are highlighted with
appropriate symbols. B-CLL indicates B-cell chronic
lymphocytic leukemia; FL, follicular lymphoma; DLBCL, diffuse large
B-cell lymphoma; MCL, mantle cell lymphoma; and LPL, lymphoplasmacytoid
lymphoma. Molecular analyses of t(14;19)+ B-CLL and
t(2;5)+ ALCL (cases no. 646, 1029, 1078) were
described previously.
phage DNA was a molecular weight
marker. (B) Chromosomal FISH analysis of Karpas 299 (i) and SU-DHL-1
(ii). Metaphase preparations were hybridized with a BAC clone
containing the BCL3 locus, which was labeled with
digoxigenin/antidigoxigenin-FITC. The chromosomes and nuclei were
counterstained with propidium iodide (PI). The arrowheads
indicate duplicated (i) and amplified (ii) BCL3 gene copies.
(C) Interphase FISH analysis of Karpas 299 (i), SU-DHL-1 (ii), DEL
(iii), and SR-786 (iv). Original magnification Bi-ii,
× 1000; Ci-iv, × 1000.
to mediate its mitogenicity.
Mol Cell Biol.
1998;18:6951-6961