High-level expression of BCL3 differentiates t(2;5)(p23;q35)-positive anaplastic large cell lymphoma from Hodgkin disease by Momoko Nishikori, Yoshitomo Maesako, Chiyoko Ueda, Masayuki Kurata, Takashi Uchiyama, and Hitoshi Ohno Blood Volume 101(7):2789-2796 April 1, 2003 ©2003 by American Society of Hematology

Hierarchic clustering analysis of gene expression of 4 ALCL and 3 HD cell lines.(A) Expression data of 110 genes were selected to optimally separate ALCL and HD. Analysis was performed with GeneSpring software. Hierarchic clustering analysis of gene expression of 4 ALCL and 3 HD cell lines.(A) Expression data of 110 genes were selected to optimally separate ALCL and HD. Analysis was performed with GeneSpring software. The unrooted tree of each axis, where the length of the branches represents a similarity in the distance of expression profiles, shows the relationship of the samples (left) and the genes (top). The gene expression values and trust are color-coded as indicated by the scale on the bottom: red indicates the expression level above a median level across all samples, green indicates below the median, and the brightness represents the trust of each value. (B) Enlargement of proportions of the expression profile indicated by stars in panel A. In this matrix, each column represents a sample and each row a gene. Momoko Nishikori et al. Blood 2003;101:2789-2796 ©2003 by American Society of Hematology

Expression of BCL3 mRNA and Bcl-3 protein in ALCL, HD, and other hematologic tumor cell lines.(A) Northern blotting analysis of RNA extracted from ALCL, HD, and other hematologic tumor cell lines. Expression of BCL3 mRNA and Bcl-3 protein in ALCL, HD, and other hematologic tumor cell lines.(A) Northern blotting analysis of RNA extracted from ALCL, HD, and other hematologic tumor cell lines. Total RNA samples (10 μg) were electrophoresed on a MOPS (3-[N-Morpholino]propanesulphonic acid)–formaldehyde gel, and the patterns of the 28S and 18S ribosomal RNA bands were observed to verify the comparable amounts of RNA in each lane (bottom). (B) Western blotting analysis of ALCL, HD, and other hematologic tumor cell lines for Bcl-3 protein expression. Cell lysates were prepared from 1 × 106 cells, and an aliquot was loaded. Bcl-3 migrates around 58 kDa in sodium dodecyl sulfate (SDS)–polyacrylamide gels. Momoko Nishikori et al. Blood 2003;101:2789-2796 ©2003 by American Society of Hematology

Real-time quantitative RT-PCR to measure theBCL3 mRNA levels in hematologic tumor cells.The BCL3/18S rRNA ratio of each test material was normalized to that of HUT 102 (= 1). Real-time quantitative RT-PCR to measure theBCL3 mRNA levels in hematologic tumor cells.The BCL3/18S rRNA ratio of each test material was normalized to that of HUT 102 (= 1). The t(2;5)+ ALCL, t(2;5)− 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.32 37 Momoko Nishikori et al. Blood 2003;101:2789-2796 ©2003 by American Society of Hematology

BCL3 gene copies in ALCL cell lines BCL3 gene copies in ALCL cell lines.(A) Southern blot analysis of DNA extracted from ALCL and HD cell lines. BCL3 gene copies in ALCL cell lines.(A) Southern blot analysis of DNA extracted from ALCL and HD cell lines. The membranes were sequentially hybridized with BCL3cDNA and BCL6 genomic DNA probes.HindIII-digested λ 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. Momoko Nishikori et al. Blood 2003;101:2789-2796 ©2003 by American Society of Hematology

Methylation status of the 3′ CpG island ofBCL3. Methylation status of the 3′ CpG island ofBCL3. (A) A map of BCL3 focusing upon the 5′ and 3′ CpG islands according to the draft sequence registered in the database (accession no. NT011109). Restriction sites for BglII, NotI,SacII, and BssHII are indicated. (B) Southern blotting analysis of DNA from ALCL and HD cell lines digested byBglII and either one of the 3 methylation-sensitive restriction enzymes. The blots were hybridized with probe B representing the 3′ CpG island. Momoko Nishikori et al. Blood 2003;101:2789-2796 ©2003 by American Society of Hematology

Sequencing analysis of bisulfite-treated DNA from ALCL and HD cell lines for the 3′ CpG island of BCL3. Sequencing analysis of bisulfite-treated DNA from ALCL and HD cell lines for the 3′ CpG island of BCL3. Numbers indicating the nucleotide positions refer to the 5′ boundary ofBCL3 exon 6. The unmethylated CpG sites are indicated by open cells, whereas the methylated CpG sites are indicated by gray cells. Momoko Nishikori et al. Blood 2003;101:2789-2796 ©2003 by American Society of Hematology

Comparison of NF-κB activity between ALCL and HD Comparison of NF-κB activity between ALCL and HD.(A) NF-κB DNA binding activity of ALCL (lanes 1 to 4) and HD (lanes 5 to 7) cells was analyzed by EMSA. The whole-cell lysates were incubated with 32P-labeled oligonucleotide probe containing a κB site and re... Comparison of NF-κB activity between ALCL and HD.(A) NF-κB DNA binding activity of ALCL (lanes 1 to 4) and HD (lanes 5 to 7) cells was analyzed by EMSA. The whole-cell lysates were incubated with 32P-labeled oligonucleotide probe containing a κB site and resolved on a 5% nondenaturing polyacrylamide gel. Supershift assay of Karpas 299 cells using antibodies against p65, p50, and p52 determined the components of NF-κB binding complexes (lanes 11 to 13). Lane 9, addition of an excess amount of unlabeled probe; lane 10, addition of the unlabeled AP2 probe (5′-GATCGAACTGACCGCCCGCGGCCCGT-3′) to eliminate nonspecific binding. (B) Western blotting analysis of ALCL and HD cell lines for the expression of p65, p50, and p52. Cell lysates were prepared from 1 × 106 cells, and an aliquot was loaded. Momoko Nishikori et al. Blood 2003;101:2789-2796 ©2003 by American Society of Hematology

Indirect immunofluorescence of Karpas 299 and KM-H2 cells against Bcl-3, p65, and p50.Cytocentrifuged cells were incubated with anti–Bcl-3 (A), p65 (B), and p50 (C) antibodies and labeled with the Alexa 488–conjugated secondary antibody. Indirect immunofluorescence of Karpas 299 and KM-H2 cells against Bcl-3, p65, and p50.Cytocentrifuged cells were incubated with anti–Bcl-3 (A), p65 (B), and p50 (C) antibodies and labeled with the Alexa 488–conjugated secondary antibody. The positions of nuclei, as revealed by DAPI staining in the lower panels. Original magnification × 1000. Momoko Nishikori et al. Blood 2003;101:2789-2796 ©2003 by American Society of Hematology

A model for NF-κB/IκB/Bcl-3 interaction in HD and ALCL cells A model for NF-κB/IκB/Bcl-3 interaction in HD and ALCL cells.A hypothetical model for differential NF-κB binding activity between HD and ALCL focusing upon the inhibitory molecules IκBα and Bcl-3. A model for NF-κB/IκB/Bcl-3 interaction in HD and ALCL cells.A hypothetical model for differential NF-κB binding activity between HD and ALCL focusing upon the inhibitory molecules IκBα and Bcl-3. Momoko Nishikori et al. Blood 2003;101:2789-2796 ©2003 by American Society of Hematology