Activation of EVI1 Transcripts with Chromosomal Translocation Joining

Leukemia (1999) 13, 1359–1366  1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu Activation of EVI1 transcripts with chromosomal translocation joining the TCRV␤ locus and the EVI1 gene in human acute undifferentiated leukemia cell line (Kasumi- 3) with a complex translocation of der(3)t(3;7;8) K Suzukawa1,2, T Kodera1, S Shimizu1, T Nagasawa2, H Asou3, N Kamada3, M Taniwaki4, J Yokota1 and K Morishita1

1Biology Division, National Cancer Center Research Institute, Tokyo; 2Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, Ibaraki; 3Department of Cancer Cytogenetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima; and 4Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan

A cell line (Kasumi-3) established from acute myeloid leukemia AML, MDS and CML-BC through chromosomal abnormalities (AML-M0) had unique phenotypes of undifferentiated leukemia involving 3q26. cells with expression of both T cell and myeloid antigens. Kas- umi-3 cells with t(3;7)(q26;q22) highly expressed a 6 kb tran- Translocational breakpoints of t(3;3) or t(3;12) or t(3;21) script of EVI1, which is located on chromosome 3q26. There- spanned around 300 to 400 kb at the 5Ј region of EVI1, and fore, we further characterized the chromosomal breakpoint by the breakpoints of inv(3)(q21q26) spanned from the 3Ј end of pulsed-field gel electrophoresis near EVI1. We identified and the coding region to around 200 kb downstream of EVI1.In isolated the chromosomal breakpoint at approximately 80 kb cases of t(3;21), the 5Ј region of the AML1 gene was translo- upstream from the 5Ј end of EVI1. Sequence analysis of the breakpoint revealed that the whole V␤ region from T cell recep- cated to upstream of the EVI1 gene, and the AML1 gene was 5 tor beta (TCR␤) at 7q35 was translocated to the upstream of fused with multiple genes (EAP, MDS1 and EVI1). In cases of EVI1. A 1.0 kb TCR␤ transcript was expressed in the Kasumi- t(3;12), some cases showed that the 5Ј region of the TEL gene 3 cells, suggesting that TCR␤ rearrangement occurred as D␤– was fused with MDS1 and EVI1,6 but other cases showed that ␤ J joining events. Fluorescence in situ hybridization analysis only the promoter region of the TEL gene translocated revealed that the inverted chromosome 7q22-q35 segment between TCR␤ and the region proximal to the erythropoietin upstream of the EVI1 gene without making any fusion tran- 9 gene at 7q22 was translocated to the region distal to EVI1 in scripts. In 3q21q26 syndrome, the 3q21 region containing der(3). Since the telomeric region of chromosome 8 q was also Ribophorin I (RPN1) translocated to upstream of EVI1 in the translocated to the inverted chromosome 7q22-q35 segment in cases with t(3;3), but the same region was translocated to der(3), the chromosomal abnormalities of der(3) were defined downstream of EVI1 in the cases with inv(3), suggesting that as being der(3)t(3;7;8)(3pter-3q26::7q35-7q22::8q22-8qter). It is suggested that a translocated enhancer element in the TCR␤ the translocated 3q21 region containing RPN1 might activate locus and/or loss of a negative regulatory element near EVI1 expression of EVI1 in the cases with both t(3;3) and inv(3) as might function to enhance the EVI1 expression. Therefore, the the transcripitonal enhancer mechanism. Based on the reports enhanced EVI1 expression may contribute to the development of these leukemia cases with translocations at 3q26, it is sug- of a subset of undifferentiated leukemia. gested that EVI1 expression in these cases with t(3;3), inv(3), Keywords: EVI1; TCR␤; Kasumi-3; chromosomal breakpoint; AML (FAB-M0) t(3;12), or t(3;21) might be activated by the translocated promoter or enhancer region(s). Recently, a unique undifferentiated leukemia cell line 10 Introduction (AML-M0), Kasumi-3, was established by our group. It has a unique chromosomal abnormality of t(3;7)(q27;q22) with The proto-oncogene EVI1 was initially identified as being acti- other additional chromosomal abnormalities. In this study, we vated in murine myeloid leukemia by proviral insertions in showed that the EVI1 gene was transcriptionally activated in the Evi-1 common viral integration site.1 In human leukemia, Kasumi-3 cells to the same level as that in UCSD/AML1 cells the EVI1 gene is activated in myeloid leukemia (M1 to M7) and we identified that the translocation breakpoint was and myelodysplastic syndromes (MDS) by chromosomal located at 80 kb upstream of the EVI1 gene at 3q26 and at rearrangements at either the 5Ј region of the gene in the 70-kb region between the V␤ and D␤ elements of the T t(3;3)(q21;q26) or the 3Ј region of the gene in inv(3)(q21q26) cell receptor (TCR) ␤ gene at chromosome 7q35. A 1.0 kb by juxtaposition of the EVI1 gene to enhancer elements in the TCR␤ transcript was expressed in the Kasumi-3 cells, suggest- 3q21 region with the Ribophorin I gene.2,3 Moreover, the ing that TCR␤ gene rearrangement might have occurred as fusion gene, AML1/MDS1/EVI1, was activated in chronic mye- aD␤–J␤ joining event. The promoter region with whole V␤ logenous leukemia-blastic crisis (CML-BC)4 or MDS5 with elements of TCR␤ gene was translocated to upstream of the t(3;21) translocation, and TEL/MDS1/EVI1 was activated in EVI1 gene and might contribute to activating EVI1 expression. acute myeloid leukemia with t(3;12) translocation.6 Although Moreover, the inverted chromosome 7q22-q35 segment the exact mechanism by which EVI1 exerts its leukemogenic between the TCR␤ gene and the region proximal to the effect is not clear, it may involve inhibition of terminal differ- erythropoietin gene at 7q22 was translocated to the region entiation through the abnormal repression of genes necessary distal to the EVI1 gene with the telomeric region of chromo- for cellular maturation.7,8 Therefore, the EVI1 gene is one of some 8 q in der(3)t(3;7;8). Therefore, activation of the EVI1 the leukemogenic genes mapped to 3q26 and activated in gene by translocation of the TCR␤ gene was suggested to be one of the pathogenetic mechanisms for the development of a part of undifferentiated myeloid leukemia. Correspondence: K Morishita, Biology Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104, Japan; Fax: 81 3 3542 0807 Received 12 November 1998; Accepted 23 April 1999 Chromosomal translocation joining the TCR␤ and EVI1 genes K Suzukawa et al 1360 Materials and methods

Kasumi-3 cells and other cell lines

Kasumi-3 was established and the phenotype previously analyzed in detail.10 Briefly, flow cytometric analysis showed cell surface expression of CD4, CD7, CD13, CD33, CD34, HLA- DR and c-kit. The cytogenetic study indicated that Kasumi- 3 cells carried chromosomal abnormalities of t(3;7)(q27;q22), del(5)(q15), del(9)(q32) and add(12)(p11). Kasumi-3 cells pro- liferated by treatment of IL-2, IL-3, IL-4, granulocyte–macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF), suggesting that the cells have the characteristics of undifferentiated leukemia. Cells were maintained in Iscove’s modified Dulbecco’s Eagle medium (IMDM) with 10% fetal ° bovine serum at 37 C in humid conditions in a 5.0% CO2 atmosphere. The UCSD/AML1 cell has been described as an acute myeloid leukemia with t(3;3)(q21;q26).11 The F36 cell line has been established as factor-dependent myeloid leukemia cells.12 The KP-L-RY cell line was kindly provided by Drs Imashuku and Ikushima, Kyoto Prefectural University of Medi- cine. HEL, Jurkat, SKW3 and MOLT16 cell lines were kindly provided by Drs Matsuo and Minowada at Hayashibara Biochemical Laboratories.

Southern and northern blot analysis Figure 1 Expression of the EVI1 gene and identification of chromo- High molecular weight DNA was extracted from mononuclear somal rearrangements in Kasumi-3 cells. (a) Northern blot hybridiz- cells (MNCs) of a normal donor and cell lines by proteinase ation performed on mRNA from various leukemia cell lines. The name K digestion and phenol/chloroform extraction.13 Ten of each cell line is indicated at the upper side of the figure. A 6 kb micrograms of DNA were digested with appropriate restriction band of EVI1 transcripts was found in mRNA from USCD/AML1 and Kasumi-3 cells and a 2 kb band of ␤-actin was detected in all of the enzymes, subjected to electrophoresis on 0.8 or 1.0% agarose cell lines. (b) The C␤ probe for the TCR␤ gene was hybridized to gel, transferred to charged nylon filters (Pall BioSupport, Port the same Northern blot fiter. (c) Identification of the chromosomal Washington, NY, USA), and hybridized to DNA probes lab- breakpoint by pulsed-field gel electrophoresis. Three probes (Figure eled by the random hexamer method. 2a) were hybridized to SfiIorBssHII-digested DNA from normal per- mRNA was extracted from cells growing in culture using ipheral blood leukocytes (N) and Kasumi-3 cells (K). The sizes of DNA the FastTrack 2.0 mRNA isolation kit according to the manu- markers are shown on the left. Arrowheads represent rearranged bands. (d) Hybridization of the P2 probe in Figure 2 to EcoRI, BamHI facturer’s instructions (Invitrogen, Carlsbad, CA, USA). ␮ and HindIII-digested DNA from normal peripheral blood leukocytes Approximately 5 g of mRNA was denatured with 37% (C) and Kasumi-3 cells (K). The sizes of DNA markers (kb) are shown formamide/15% formaldehyde and was electrophoresed on on the left. 1% agarose gel containing 17% formaldehyde. mRNA was transferred to nylon filters. The filters were hybridized with a [␣-32P] dCTP-labeled human EVI1 and ␤-actin probes and were exposed to Kodak Biomax film (Kodak, Rochester, NY, Screening of P1 clones USA) at −70°C. Pre-hybridization, hybridization and post- hybridization washes were performed as described.3 Human P1 library (series B; Du Pont, Boston, MA, USA) was screened by a PCR-based strategy.14 Amplification was perfor- Isolation of phage clones med by 35 cycles in a thermal cycler (Perkin Elmer, Foster City, CA, USA) under the cycling conditions of 1 min at 94°C, Approximately 5 × 105 recombinant phages of an EMBL3 1 min at 55°C, and 2 min at 72°C. Primer sequences were as human genomic DNA library from normal peripheral blood follows: C-forward: 5Ј-TCACAACCAGAATCCAGACC-3Ј;C- cells were screened with the 32P-labeled probe according to reverse: 5Ј-CATTGAGGGCCAGG- TTACTA-3Ј. P1 DNAs standard procedures.11 Hybridization with a random-primer- were prepared by a standard alkaline lysis method. labeled probe was performed at 42°Cin6× standard saline citrate (SSC)/10% dextran sulfate/1% sodium dodecyl sulfate (SDS)/1 × Denhardt’s solution/50% formamide containing denatured salmon sperm DNA (100 mg/ml). When probes Nucleotide sequencing containing repetitive sequences were used, human placenta DNA was added to the hybridization mixture. The final wash- Nucleotide sequences of genomic phage clones were determ- ing was in 0.1 × SSC/0.1% SDS at 65°C. Autoradiography was ined by the PCR cycling DNA sequence method with the fmol performed using a Bioimage analyzer, Fujix BAS2000 (Fuji DNA Sequencing System (Promega, Madison, WI, USA), Film, Tokyo, Japan). based on the dideoxynucleotide chain termination reaction.13 Chromosomal translocation joining the TCR␤ and EVI1 genes K Suzukawa et al 1361

Figure 1 Continued.

Probes

The C␤ probe for detecting the TCR␤ gene expression was a 720-bp fragment, isolated from the HBVT 96 cDNA clone,16 which was kindly provided by Dr Kimura, the First Depart- ment of Internal Medicine, Fukuoka University School of Medicine. The EVI1 probe for Northern blot hybridization was derived from a human cDNA clone (H1-1) as previously described,3 and a 300-bp fragment of MDS1 was derived from amplified cDNA by reverse transcriptase-PCR (RT-PCR). Amplification was performed by 35 cycles in a thermal cycler (Perkin Elmer) under the cycling conditions of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C. Primer sequences were as follows: MDS1F1: 5Ј-ACTGGGCACAGCATGAGATC-3Ј; MDS1R1: 5Ј-TCCCATCCATAACTGGGGTC-3Ј. Three DNA probes for pulsed-field gel electrophoresis (PFGE) analysis were previously described.2 Briefly, the human FIM3 probe is a 0.5 kb single copy clone, which is derived from the murine fim-3 equivalent region. The B3N probe is a 1.0 kb single copy fragment isolated from the breakpoint of UCSD/AML-1 cells. H1-1 is a cDNA clone of the EVI1 gene.

Results Figure 1 Continued. Expression of EVI1 transcripts in Kasumi-3 cells

Since the Kasumi-3 cell line was found to have translocation Fluorescence in situ hybridization (FISH) t(3;7)(q26;q22), we initially determined expression of the EVI1 gene by Northern hybridization. As shown in Figure 1a, Chromosomal mapping of the genomic clones was performed expression of a major 6 kb EVI1 transcript in Kasumi-3 was on R-banded metaphase. To confirm the origin of cloned detected to the same level as that in the UCSD/AML1 cells DNAs, we also mapped the genomic clones with Kasumi-3 with t(3;3)(q21;q26).2 No other cell lines (three lymphoid and cellsЈ metaphase chromosomes. FISH analysis was carried out two myeloid cell lines) expressed a high level of EVI1 tran- as described previously.15 scripts, except HEL cells with weak expression of EVI1.A Chromosomal translocation joining the TCR␤ and EVI1 genes K Suzukawa et al 1362 MDS1 or MDS1/EVI1 transcript was not detected by Northern it was revealed that the sequence along with the breakpoint blot hybridization (data not shown). Therefore, it is possible completely matched the T cell receptor ␤ locus at chromo- that EVI1 transcripts in leukemia cells were activated by some 7q34-35 (Figure 2f). Based on the physical map of the chromosomal translocation at the 3q26 region. Next, we per- TCR␤ locus reported previously,17 the breakpoint was mapped formed pulsed-field gel electrophoresis (PFGE) to identify the within the 70-kb region between the V␤4S1 and D␤1 chromosomal breakpoint near EVI1 in Kasumi-3 cells. elements (Figure 2d and e). The centromeric region of the TCR␤ locus with the entire V␤ region was translocated to the telomeric side of the EVI1 gene at chromosome 3q26, suggest- Identification of the chromosomal breakpoint ing that the translocated part of the long arm of chromosome 7 with the TCR␤ locus was possibly inverted in chromosome To identify the chromosomal breakpoint, we performed PFGE der(3). Furthermore, we confirmed that the genomic clone using three probes, H1-1 EVI1 cDNA, B3N and FIM3 (Figure containing the chromosomal breakpoint was hybridized to 2a).3 The H1-1 EVI1 cDNA probe detected a rearranged band chromosome 7q35 by FISH analysis (data not shown). in SfiI-digested DNA, but did not detect any rearranged band To determine whether the TCR␤ gene was expressed in the in BssHII-digested DNA (Figure 1c). The B3N probe detected Kasumi-3 cells, Northern blot hybridization was performed the same rearranged band in SfiI-digested DNA and a novel using a C␤ probe from TCR␤ cDNA. As shown in Figure 1b, rearranged band with a germ line band in BssHII-digested a 1.3 kb of full-length TCR␤ transcript was detected in two T DNA, suggesting that the chromosomal breakpoint was cell leukemia cell lines (Jurkat, MOLT16) but around 1.0 kb localized between the CpG island at the 5Ј end of the EVI1 of TCR␤ transcript was detected in the mRNA from SKW3 and gene and the SfiI site of 170 kb upstream from the EVI1 gene Kasumi-3 cell lines. The short 1.0 kb transcript was reported (Figure 1c). However, the FIM3 probe did not detect a to be derived from the D␤–J␤–C␤ joining event without the rearranged band but only detected a germ line band, suggest- V␤ region.18 Based on these results, it was suggested that the ing that the region with the FIM3 fragment was deleted in the whole V␤ region with promoter from the TCR␤ locus at 7q35 rearranged allele. Thus, it is likely that the chromosomal was translocated to the upstream of the EVI1 gene with breakpoint at 3q26 is localized at the region between the B3N expression of the TCR␤ and EVI1 genes. and FIM3 probes, if the translocation at 3q26 was accompanied by deletion of the FIM3 region. To identify the chromosomal breakpoint of t(3;7), we iso- Sequence analysis of chromosomal breakpoints lated 80 kb of a P1 genomic clone (70-1) covering the region between the FIM3 locus and the B3N probe (Figure 2a). Since Translocation preferentially occurs in regions that have DNA the B3N probe was located 15 kb upstream from the 5Ј end sequences with homology to nonamer and heptamer signal of the EVI1 gene, the distance between exonl of EVI1 and the sequences in T cell malignancy.19 The germ line DNA FIM3 locus was estimated as approximately 100 kb. The sequence at chromosome 3 was analyzed for both structural 3.5 kb Bam H1 fragment (designated P2) in the 70-1 clone motifs. However, a definite set of heptamer and nonamer detected rearranged bands in DNA digested with BamHI and sequences was not found in the germ line sequence of chro- HindIII by Southern blot analysis (Figure 1d). The P2 probe mosome 3q26 (Figure 2f). An imperfect heptamer sequence Ј A was localized at approximately 80 kb upstream from the 5 (CACTTAT vs the predicted CAC /TGTG) was present adjacent end of the EVI1. The sizes of rearranged BamHI and HindIII to the breakpoint at 3q26, and a nonamer-like sequence fragments were 14 kb and 8 kb, respectively. We could not (TGTTTTTGA vs predicted GGTTTTTGC) was present at the find a rearranged fragment in the EcoRI-digested DNA, since other side of the breakpoint at 3q26. On the other hand, an the size of a rearranged fragment was almost the same as that imperfect heptamer sequence (CACTAAA) and a nonamer-like of the germ line fragment by EcoRI digestion. Only one sequence (GGTTTAGTG) were also found near the breakpoint rearranged fragment was detected in DNA with BamHI or at chromosome 7q35. Since complete matched heptamer and HindIII digestion by the P2 probe. Since the FIM3 probe did nonamer sequences were not found surrounding the break- not hybridize to both der(3) and der(7) chromosomes by FISH point, the sequence data did not support the idea that analysis (data not shown), it is likely that the other side of abnormality of V–D–J recombinase was important in the the translocated allele surrounding the breakpoint was deleted development of an aberrant rearrangement in this case. with the FIM3 locus.

An inverted and translocated segment of chromosome The TCR␤ locus was translocated to chromosome 7(q22q35) was identiﬁed by FISH analysis in 3q26 with expression of the TCR␤ gene in Kasumi-3 der(3)t(3;7;8) cells Since a part of the long arm of chromosome 7 with the TCR␤ Using the P2 probe, a 14 kb fragment was isolated from a locus was possibly inverted in the der(3) chromosome, we Bam-HI complete-digested genomic DNA library of Kasumi- performed FISH analysis of Kasumi-3 metaphasic chromo- 3 cells. The isolated 14 kb clone was derived from der(3)t(3;7) somes using painting probes for chromosome 3 and chromo- (Figure 2c). The translocational breakpoint was determined by some 7. The painting probe for chromosome 3 hybridized to comparison of the restriction map between the germ line chro- the short arm and a half of the long arm of der(3), and a part mosome 3 (Figure 2b) and the rearranged BamHI fragment of chromosome 7 (red in Figure 3a and e) was hybridized at (Figure 2c). We sequenced a region surrounding the breakpo- the proximal end of der(3) (green in Figure 3a and d). How- int in the phage clone and performed FISH analysis of this ever, the distal end of der(3) did not hybridize both probes, fragment to determine the chromosome location of the trans- suggesting that a part from an unknown chromosome was location counterpart. By comparison of the sequence around translocated to the telomeric end of the der(3). To conﬁrm the breakpoint with that of the DNA database in GenBank, which chromosome was translocated at the end of der(3), we Chromosomal translocation joining the TCR␤ and EVI1 genes K Suzukawa et al 1363

Figure 2 Mapping of the chromosomal breakpoint at 3q26 in Kasumi-3 cells with der(3)t(3;7;8). (a) Mapping of the chromosomal breakpoint at 3q26 in Kasumi-3 cells with t(3;7). Three DNA probes (FIM3, B3N and H1-1) for pulsed-field gel electrophoresis (PFGE) analysis are represented as black boxes. (b) Restriction map of germ line chromosome 3 surrounding the breakpoint. The P2 probe is a 3.5 kb BamHI fragment isolated from a P1 phage clone 70-1, which was used for Southern hybridization as a probe (Figure 1d). (c) Restriction map of der(3)t(3;7;8). A narrow line with arrows represents the isolated phage clone of a 14 kb BamHI fragment. (d) Restriction map of germ line chromosome 7. (e) Physical map of the TCR␤ gene on chromosome 7. An arrow indicates the chromosomal breakpoint of der(3)t(3;7;8) Restriction sites are indicated by capital letters: Bs (BssHII), Sf (SfiI), Nr (NruI), B (BamHI), E (EcoRI) and H (HindIII). (f) Sequence comparison of germ line chromosome 3, der(3) spanning the chromosomal breakpoint, and germ line chromosome 7. Underline represented heptamer-like motifs near the site of breakage and nonamer-like sequences are boxed. used many painting probes, and finally the probe for chromo- next to the EVI1 gene on chromosome 3q26, and the proximal some 8 (pBS8) did hybridize the telomeric part of der(3). A breakpoint was localized centromeric to the EPO gene at part of chromosome 8 (green color in Figure 3f) was 7q22. Finally, the der(3) chromosome was defined as being hybridized at the telomeric end of der(3) (red in Figure 3f by der(3)t(3;7;8)(3pter→3q26::7q35→7q22::8q22→8qter) (Figure the painting probe for chromosome 3) with a gap of another 3j). chromosome, which was hybridized by the probe for chromosome 7 (red in Figure 3g). G-banding pattern of der(3) indicated that segment 8q22-ter was involved in this complex Discussion chromosomal rearrangement. To confirm the direction of translocated chromosome 7 in der(3), we hybridized a gen- In this study, we showed a unique translocation of t(3;7;8), omic phage clone with the erythropoietin gene (EPO) at chro- involving the TCR␤ gene and the EVI1 gene, in the undifferen- mosome 7q22 as a probe. The EPO probe hybridized to the tiated myeloid leukemia cell line, Kasumi-3. The whole V␤ region adjacent to the distal chromosomal breakpoint at the region of the TCR␤ gene at chromosome 7q35 was translo- region between chromosome 7 and chromosome 8 in der(3) cated to upstream of the EVI1 gene and the chromosomal (Figure 3h and i), suggesting that a part of the chromosome aberrancy near EVI1 might have an important role for acti- 7q22-q35 segment with the EPO gene was inverted and trans- vation of the EVI1 expression. Until now, we had examined located to der(3)t(3;7;8) (Figure 3j). Therefore, the distal break- expression of the EVI1 gene in several leukemia cells and point of the inverted chromosome 7q22-q35 segment in found Kasumi-3 cells only in the case of FAB-M0 with der(3)t(3;7;8) was localized within the TCR␤ locus at 7q35 expression of the EVI1 gene. Furthermore, the long arm of Chromosomal translocation joining the TCR␤ and EVI1 genes K Suzukawa et al 1364

Figure 3 Fluorescence in situ hybridization analysis of leukemia metaphase chromosomes. Dual-color FISH analysis revealed that a part of chromosome 7 (red) translocated at the telomeric side of der(3) (green) (a). Each probe hybridization was confirmed by single-color FISH analyses (d and e). Each derivative chromosome was confirmed by Q-banded chromosomes (b) and also a part of chromosome 7 (red) was translocated to der(3) using Q-banded chromosomes (c). The telomeric end of der(3) was derived from chromosome 8 (green), which was detected by the painting probe (pBS8) (f). Arrows with a and b in panel (f) indicate der(3). The painting probe for chromosome 3 was hybridized to der(3) by red in dual color (f). Chromosomes 7 and 8 are indicated as red and green, respectively (g) and each chromosome was confirmed by Q-banded chromosomes. The EPO gene (arrows) was assigned to band 7q22 on normal chromosome 7 (small arrow) and to the distal breakpoint on der(3)t(3;7;8), which is next to the region of chromosome 8 (q22-ter) (large arrow) (h). The chromosome bandings were confirmed using Q-banded chromosomes (i). Schematic representation of chromosomal rearrangements in Kasumi-3 cells with der(3)t(3;7;8) (j).

chromosome 7 between the centromeric side of the EPO gene EVI1 gene in other leukemia cases with t(3;3), inv(3), t(3;12) at 7q22 and the TCR␤ locus was inverted and translocated in or t(3;21), it is suggested that an unknown enhancer element der(3)t(3;7;8). Therefore, the translocated TCR␤ locus and/or in the TCR␤ locus translocated near the EVI1 gene and/or loss deletion of the 5Ј region of the EVI1 gene may enhance the of a negative regulatory element might contribute to transcrip- EVI1 gene expression and may contribute to leukemogenesis tional activation of the EVI1 gene. of a subset of AML-M0. It is a unique finding that the TCR␤ locus was translocated Disregulation of proto-oncogene by translocation of the to the 5Ј region of the EVI1 gene with activation of the EVI1 TCR gene was originated by at least two mechanisms. One is expression in AML-M0. This is the first report that the TCR␤ Ј ␤ that the enhancer element at the 3 end of the TCR gene was locus was rearranged to the locus of a proto-oncogene in Ј Ј translocated to the 5 or 3 region of proto-oncogenes, as in AML-M0. Since patients with AML-M0 have frequently been 20 21 22,23 the case of the LYL1, TAL1, and LCK genes. The other found to have complex karyotypes and unbalanced chromo- ␤ is that the V region of the TCR gene without the usual some changes involving abnormalities of 5 q or 7 q,26–29 Kas- enhancer element was translocated near the Rhom-1 and umi-3 is a typical case of complex abnormalities of Rhom-2 genes.24,25 In these cases, the TCR enhancer element Ј ␤ der(3)t(3;7;8) with insertion of the inverted chromosome at the 3 end of the TCR gene cannot influence the 7q22-q35 segment and/or del(5)(q15). Thus, these findings expression of these proto-oncogenes, but it is suggested that indicate that EVI1 gene activation with complex chromosomal the loss of a negative regulatory element or generation of a abnormalities might contribute to the leukemogenesis of cryptic promoter/enhancer may contribute to the activation of Kasumi-3 cells. these genes. Since the breakpoint was located at approximately 80 kb upstream from the 5Ј end of the EVI1 gene, and enhancer or promoter elements were translocated near the Chromosomal translocation joining the TCR␤ and EVI1 genes K Suzukawa et al 1365

Figure 3 Continued. Chromosomal translocation joining the TCR␤ and EVI1 genes K Suzukawa et al 1366 Acknowledgements tory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. This work was supported in part from the Ministry of Health 14 Ohira M, Ichikawam H, Suzuki E, Iwaki M, Suzuki K, Saito-Ohara F, Ikeuchi T, Chumakov I, Tanahashi H, Tashiro K, Sakaki Y, Ohki and Welfare by a Grant-in-Aid for Research on Human Gen- M. A 1.6-Mb P1-based physical map of the down syndrome region ome and Gene Therapy of Japan. We are grateful to Dr on chromosome 21. Genomics 1996; 33: 65–69. Yasuhide Hayashi of the Department of Pediatrics, University 15 Kimura N, Akiyoshi T, Uchida T, Okamura J, Kozuru M, Nishi- of Tokyo, and Dr Masao Seto, Aichi Cancer Center for helpful mura J, Ohyashiki JH, Kikuchi M, Niho Y, Okumura M. High discussions and a critical review of the manuscript. 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