HUMAN CELL(Hum Cell) Copyright 2005 by The Japan Human Cell Society Vol 18 No Printed in Japan -Original Article Cell Line Establishment And Characterization Of A Novel PhiladelphiaChromosome Positive Chronic Myeloid Leukemia Cell Line, TCC-S, Expressing P210 And P190 BCR/ABL Transcripts But Missing NormalABL Gene Phan Nguyen Thanh Van”3,Phan Thi Xinhle3,Yasuhiko KANO’, Katsushi TOKUNAGA3,Yuko SATO’ &stracb A novel Philadelphia-chromosome positive (ph+) cell line, TCC-S, has been established from a patient with Ph+ chronic myeloid leukemia (CML) in the blastic crisis TCC-S cells were shown to express both P210 and P190 BCR/ABL transcripts by reverse transcriptase-polymerasechain reaction (F’CR), although quantitative-PCRrevealed that TCC-S cells mainly expressed P210 BCWABL transcript Karyotype analysis revealed several triploid clones which constantly harbored two der(9)del(9) (p12)t(9;22) (q34;qll)s and two de1(9)(q21)s The der(9)de1(9)@12)t(9;22)(q34;q11) is rarely found in other CML cell lines Moreover, to the best of our knowledge, del(9) (q21) resulting in missing of a restrict region including normal ABL gene has not been found among CML cell lines previously described Thus, X C - S cells with only BCR/ABL gene and no normal ABL gene may be a useful tool for functional study of ABL in Pht CML Keywords: Philadelphia (ph) chromosome, chronic myeloid leukemia (CML.), P210 BCWABL transcript, P190 BCWABL transcript, ABL [HUMAN CELL 18(1) :25 - 33,20051 Introduction Chronic myeloid leukemia (CML) is a pluripotent stem cell disease resulting from oncogenic transformation The hallmark of CML, Philadelphia translocation, t(9;22) (q34;qll) is found in 90 to 95% 1:Division of Ultrafine Structure, Department of Pathology, Research Institute of International Medical Center of Japan, Tokyo, Japan 2: Division of Hematology and Medical Oncology, Tochigi Cancer Center, Tochigi, Japan 3: Department of Human Genetics, School of International Health, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan patients with CML”.”.As the result, a fusion of the ABL (Abelson) gene3’ at chromosome band 9q34 and the BCR (breakpoint cluster region) gene’),‘) a t chromosome band 22qll occurs forming a chimeric BCR/ABL gene a t 2 q l l T h i s chimeric gene is reported to be transcribed to P190”’5’,P210”“, or P230” kDa BCR/ABL oncoprotein according to t h e breakpoint within BCR These BCYVABL oncoproteins show constitutively active tyrosine kinase activity and are implicated in the pathogenesis of CML with diverse actions on hematopoietic cells, including transformation, protection of apoptosis, cell cycle progression, altered cell migration and altered adhesion to the extracellular matrix Here, we report establishment of a novel Ph 25 chromosome positive (Ph+) CML cell line, designated TCC-S, which was derived from a patient with Ph+ CML in the blastic crisis (BC) Karyotype analysis of TCC-S cells revealed several triploid clones which constantly harbored two der(9)del(9) (p12)t(9;22) (q34;qll)s, two del(9) (q21)s and two der(22)t(9;22) (q34;qll)s More than 40 Ph+ CML cell lines have been established so far, and a missing of whole normal chromosome is occasionally reported among them However, to the best of our knowledge, this del(9) (q21), resulting in the missing of a restricted region of the long arm of chromosome including normal ABL gene at 9q34, has never been found Thus, TCC-S cells, with only BCWABL gene and no normal ABL gene may provide a useful tool for functional study of normal or altered ABL gene in Ph+ CML Materials and Methds Case report A 4Gyear-old Japanese man was found to have a leukocytosis at the health examination in August 1988 In June 1989, he took an examination for hematologic malignancies at Utsunomiya Social Insurance Hospital His bone marrow (BM)was found to be hypercellular with marked increase of myeloid lineage cells without a leukemic hiatus Cytogenesis study of the BM cells showed 46, XY, t(9;22) (q34;qll) [20/201 T h e diagnosis of Ph+ CML in the chronic phase was made Treatment with 1-2 mg/day carboquone was started His hematologic findings in December 1989 were a s follows: white blood cell (WBC) 10.1 X 106/L (1.0% meta-myelocytes, 74.5% neutrophils, 3.1% eosinophils, 6.1%basophils, 6.1%monocytes and 9.2%lymphocytes), red blood cell 5,150 X 109/L,hemoglobin 158 g/L, and platelet 595X 109/L In September 1991, WBC began to increase with 78% myeloid blasts The BM aspirate showed hypercellular BM with 73% myeloid blasts which expressed the positivity of CD13 and CD33 and the negativity of peroxidase staining In October 1991, chromosome study of BM cells showed 46, XY, t(9;22) (q34;qll) as a main clone together with several sub-clones with Ph translocation (Table 1) T h e diagnosis of the myeloid blastic crisis was made He was treated with a combined chemotherapy with behenoyl cytarabine (a long-acting depot form of 26 cytarabine)*),6-mercaptopurine, daunomycine and prednisone However, he died from pneumonia on October 31,1991 Cell culture The leukemic cells were obtained from the patient’s BM during the blastic crisis in October 1991 with informed content The cells were cultured in a flask containing RPMI 1640 medium (Sigma Chemical Co., MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (ICN Biochemicals, Irvine, USA), 100 U/mL penicillin and 100 pg/mL streptomycin (Nacalai Tesque, Inc., Kyoto, Japan) (later called as “culture media”), in a humidified atmosphere of 5% COZ.Half of the culture media was replaced once a week Cell morphology to X 10‘ cells were used to prepare slides by using a Shandon Cytospin (Thermo Electron Corporation, Waltham, USA) Cell morphology was observed under a light microscopy after WrightGiemsa staining Immuno-phenotypeanalysis Cell surface antigens were analyzed by using a FACsCalibur (BD Bioscience, San Jose, USA) The monoclonal antibodies, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD14, CD19, CD20, CD33, CD34, CD56 and HLA-DR, were used with a direct staining technique Cytogenetic study Metaphase slides were prepared with a highresolution method described elsewhereg’.In brief, X lo6 cells were cultured in 10 mL culture media, and were harvested after exposure in 300 p g / m L thymidine (Sigma Chemical Co.) for 16 hours Then, the cells were exposed to 12.5 p g / m L bromodeoxyuridine (Wako Pure Chemical Industries, Osaka, Japan) for 5.5 hours, and 0.05 pg/mL demecolchin (Invitrogen Ltd., Carlsbad, USA) for 30 minutes, followed by treatment with 0.05 M KCL for 20 minutes The cells were fixed with mix of methanol and glacial acetic acid (ratio 3:l) Slides were made with air-dry HUMAN CELL Vol 18 No (2005) method and stained with a dual Q-banding using quinacrine and H o e c h s t 33258 Twenty-eight metaphases were analyzed by using Macktype v 5.4.2 software (Appied Imaging, Newcastle, UK) Fluorescence in situ hybridization (FISH) study T o detect a BCR/ABL fusion gene, t h e abovementioned metaphase slides were used for FISH study, with LSI BCR/ABL ES dual color translocation probe (Vysis Inc Downers Grove, USA) The probe was hybridized to c h r o m o s o m e s according to t h e manufacturer’s protocol The metaphase images were captured under a fluororescence microscope, and more than 15 metaphases were analyzed by using Macktype v 5.4.2 software (Appied Imaging) Detection of P and P BCR/ABL transcripts by reverse transcriptase- polymerase chain reaction (RT-PCR) Total RNA was extracted by using Sepazol-I reagent (Nacalai Tesque Inc.) according to the manufacturer protocol All steps were carried out in a laminar hood RNA pellet was dissolved in 20 p L DEPC water and s t o r e d at - T until u s e cDNA s y n t h e s i s w a s performed in a 30 ,vL volume containing 1.5 ,ug RNA, 1.25 !tg random hexamers (New England Biolabs, Table 1: Cytogenetic findings of the patient’sbone marrow cells I Date I Stagc _~ Jan 1YW April IYYO Dcc 13 1YY(l Jan 16 1991 Dx* I 46 XY t(Y:22)(q34:qlI) 46 XY t(Y:22)(q34:ql I ) 2(I 30 IY 2(I CPt 46 X Y CPt 46 XY t(Y:22)(q34:ql ) CPi 46,XY t(Y:22)(q31:ql1) I 46 XY t(Y:12)(q34:q1 1) 44 XY -Y 20 20 3) 3J t(Y:22}(q34:qll) -17 -18 +mar 45, XY 7q+, -Y t(Y:22)(q34:qlI).-17 Oc.1 lYY1 45 XY 7q+, -9 I(Y:22)(q34:qI I) 14q+ -17 -18, +2mar 17 44 XY -Y, 1(9:22)(q34:qll) + I % -17 -18 I 45 XY 2q- -Y 1(Y:Z2)(q34:qlI) +14 -17 -18 +21 47, XY -9, -9, +22q- +5mar ‘Dx,diagnosis: + CP chronic phase; -16 -17 -18 Xq- Z BC blastic crisis 27 Hertfordshire LK) 200 I1 M-MLV RT 2mM dNTPs, O.lmM IITT (Invitrogen Ltd.) V L RNase inhibitor (Promega Madison, USA) and 'IL first strand RT buffer according to the manufacturer's instructions cDNA product was diluted folds Two V L diluted cDNA was subsequently mixed with 0.1 :fL (0.5 IT) AmpliTaq Gold (Applied Biosystems, Foster City lJSA).and the following primer set of L (10 pmol) forward and reverse in a 20 ;1L reaction mixture To check quality of RNA and eficiency of cDNA synthesis the internal control gene, GAPDH, was used wiith a primer set forward GAPDH-F (5'-GCACCGTCk~GGCTGAGAA-:~* GAPDH exon ) and reverse GAPDH-R (5'CAACGTAGGTCCACCACTGACACG-3' GAPDH exon 8) T h e primers used in first PCR to detect P-310 BCRIABL transcript were forward M-BCR-1 (5'ATCCAAGGCTACGGAGAGGC-3' RCR exon 11) and reverse ABL-R1 (5',4TGGTtlCCAGGAGTGTCTC C3 ' ABL exon 3) To detect P190 BCR/ABL transcript forward m-BCR-1 (S'-CAACAGTCCTTCGACAGC-3' BCR exon 1) and reverse ABL-R1 were used T h e thermal cycling profile was: 95T for 12 minutes 35 cycles at 9ST for 30 second 62°C for 45 second and a final extension at 72°C for 10 minutes The second PCR was perfornied by using ,I L of the first PCR product The primers used for the second PCR to detect Pi10 BCR/,4BL transcript were forward 34-BCR-2 (5'GGAC;Cn;CAGATGCTGACCAAC-3, BCR exon 13), and reverse ABL-R2 (S'-TTCCTTGGAGTTCCAACGAGC3', ABL exon ) To detect P190 BCR/ABL transcript, forward in-BCR-2 (5'-CAGTGCCATAAGCGGCACC-3' BCR exon ) and reverse ABL-R2 were used T h e thermal cycling profile of the second PCR was the same as that of the first PCR PCR was performed by u s i n g a GeneAmp PCR system 9700 (Applied Biosystems) T h e second PCR product w a s electrophoresed in a 2% agarose gel containing 0.5 erg/mL ethidium bromide in 0.5xTRE The bands of P"10 RCR/ABL and P190 BCR/ABL transcripts were visualized under Liv light Detection of P210 or P190 BCWABL transcript amount by quantitative-polymerasechain reaction (QT-PCR) To detect BCR/ABL transcript amount QT-PCR was performed by using an ABI PRISM 7700 sequence detector (Applied Biosystems) The primers and probe used for P210 BCR/ABL transcript were forward BCR-F (S'-GATGCTC;ACCAACTGTGT(;T~*3'.BCR exon 13) reverse ABL-R (5'-TGGCCACAAAATCATACAGTGC3' ABL exon ) and probe ABL-P (5'CCTTCAGCGGCC,4GTAGCd4TCTGACTTT-3', ARL exon -3) T h e p r i m e r s and probe for P l BCR/ABL transcript were forward bcr-f (5'CAGTGCCATAAGCGGCACC-3', BCR exon ) reverse abl-r (5'-1TCCTTGGAGTTCCAACGAGC-3' ABL exon 2) and p r o b e abl-p (5'CGCCCTCGTCATCGTTGGGCCAGATCT-3' ABL exon ) T h e primers and probe for GAPDH were forward GAP-F (S-(;AA(;GT(;AACI(;TCGGA(;TC-~ exon Fig 1: The morphology of TCC-S cells A TCC-S cells lookrd as round-shaped and non-adhrrent cells (magnification X 1O()) B In Wright-Girmsa staining TCC-S cells showcad finr chromatin and round nuclei Cytoplasmic protrusions and small vacuoles wcrc occasionally obsrnvd (magnification S 1.(H)O) 28 2) reverse GAP-R (Y-GAAGATGGTGATGGGATTTC3’ exon 4) and probe GAP-P (5’CAAGCTTCCCGTKTCAGCC-3’ exon 4) The result was calculated a s a ratio between the amount of BCR/ABL and GAPDH transcripts Results Establishment and morphology of TCC-Scells After six weeks’ subculture, cells began to grow In December 1991, a cell line was established and designated TCC-S TCC-S cells grew well with a doubling time of 27.96 + 0.97 hours T h e cell morphology was observed under a light microscopy after Wright-Giemsa staining by using a cytospin preparation TCC-S cells had round nuclei with fine chromatin Cytoplasmic protrusions and small vacuoles were occasionally observed (Figure 1) Kaqyotypes and FISH findings Cytogenetic findings are summarized in Table Table 2: Cytogenetic findings of the TCC-S cells No of melaphases Karyot ype , -x -x.Y,+ I +l -2 +3 -1.-5 -6 +add(K)(p?l) der(Y)del(Y)(pl2)t(Y:22)(q31:q 1 ) der(Y)deI(Y)(pl2)1( Y:22)( q33:q 1 ) dcl(Y)(q21) +del( Y)( $1 ), +add(lO)(pll) +11 +11 add(l2)(plZ),+add(IZ)(plZ), +15 +lh, add( 17)(p12), +20, -22 dcr(’Z)t(Y;22)(q34:qI der(’2)t(Y:ZZ)(q31:qll) 1) +mar 77 XY.-X +l add(2Xp23), -3 -3 +h +7, der(Y)deI(9)(pl2)1(9:22)(q34:q 1), der(Y)drl(Y)(pl2)1(9:22)(q34:q 1 ) del(Y)( qZ ), +de1(9)(qZ ), +add(lO)(pl4) + 11 +add( lZ)(p 12) + 15 + 16 add( 17)(pI 2) -18 + l U +:(I dcr(22)1(C):2~)(q33:ql 1) +dcr(22)t(Y:X!)( q31:q 1 ) h 76 XY -X,+1 add(Z)(p23) -4.+h +7 add(8)(p21) dcr(Y)Jel(Y)(pI2)t(Y:22)(q33:ql I ) der(Y)dcl(Y)(pl2)t(Y:22)(q34:q I 1) deI(Y)(q21) +dcl(Y)(q?l) 16 add(lO)(pll) +11 +add(l’)(pl2) +15 add(17)(p12) -18 + l Y +‘I Jer(27)t(Y:22)(q~l;q11).+Jcr(2’)t(Y:l:!)(q3.I:qI 1) Total No of mctaphascs ‘8 29 The TCC-S cells showed triploid karyotypes with 67 to 82 chromosomes which constantly harbored two der(9)deI(9)(p12)t(9:22)(q34:qll)s and two del(9) (q21)s A clone with 76 chromosomes was a main one One of karyotypes with 73 chromosomes is shown in Figure 2.4 FISH study revealed red signal (ABLgene) on each of two der(9)s, green signal (BCR gene) on one nI(22) and yellow fusion signal (BCR/ABL gene) on each of two der(22)s (Figure 2B) Immuno-phenotypeanalysis Expression of surface markers on TCC-S cells and the patient's BM cells is compared in Table The majority of the primary BM cells showed positive for the myeloid markers (CD13 and CD33) CD4 and HLAD R Stem cell antigen CD34 was not detected After establishment of the cell line, expression of HLA-DR was lost, and that of CD33 and CD13 were increased Expression of P BCR/ABL and P BCWABL transcripts RT-PCR detected both P210 BCR/ABL (b3a2 type) and P190 BCR/ABL transcripts in TCC-S cells (Figure 3) K562 cells which expressed both transcripts were used a s a positive control QT-PCR detected 209,432 copy/,ug of P210 BCR/ABL and 1.553 copy/,ug of P190 BCR/ABL transcript in TCC-S ceIls Fig 2: fiaryotyptt and FISH findings of TCC-Scells A One of k a r y o t p s showed 73 XYY, +l addO)(p23), add(8)@21).dt.r(9)de1(9)(p12)1(Y;P)(q34:qll),der(9)del(9)@12)1(9;r)(q34:qll) del (Y) ( q ) +del(9)(q21) + 11, add (12)( p l l ) , +add ( ) ( p l ) , + 15 add (17) ( ~ ) -.18 -21, d e r ( 2 )t (9;22) (q34:ql l ) , +der(Z)t(l):Z)(q34.qll) 1% The precenct of Ph chromosome was confinncd by FISH FISH revealed red signal ( A H L gene) on drr(9h L T w n signal (RCR g m r ) on a n I ( ) and yellow fusion signal (BCK/.4RL gme) on der(2'2)s Fig 3: BCWABL transc7ipts in TCC-S cells by RT-PCR A P210 BCWABL (Z2Y bp b3a2 type) was detected in TCC-Scells R PIN) BCR/ABL (198 bp el& type)was also detected in TCC-S cells C GAPDH was detec-ted in TCC-S and positive control cells M l(K1 bp ladder (0.5 ttg); P K5K2 cells used as a positive control: N no cells as a negative control 30 HUMAN CELLVol 18 No (2005) Discussion Here, we report establishment and characterization of a novel Ph+ chronic myeloid leukemia cell line, TCCS which was derived from a patient with Ph+ CML in BC It grew well with the doubling time of 27.96 0.97 hours, a similar time to other CML cell lines*o’.”’ More than 40 Ph+ CML cell lines have been established so far It is known that the majority of P210 BCR/ABL-expressing Ph+ CML cell lines simultaneously express P190 BCR/ABL transcript, * although the expression level of P190 BCR/ABL is low’?) The mechanism of this co-expression is considered to be due to the alternative splicing from the same pre-mRNA of BCR/ABL, not due to the existence of two clones, one which has the breakpoint within the major BCR and the other withim the minor BCR in the BCR gene In TCC-S cells, coexpression of P210 and P190 BCR/ABL transcripts was also observed, although the expression level of P190 BCR/ABL was much lower than that of P210 BCR/ABL transcript Since we did not observe the Table 3: Immunc-phenotype of the patient’s bone marrow cells and TCC-S cells Patient’s bone marrow Markers TCC-S cells (%) cells (7%) OCI 2, Nov 29, Feb 27, Feb 13, 1991 1991 1992 2004 CD2 1.9 1.5 8.3 1.7 CD3 1.1 1.1 9.5 2.1 CD4 16.9 44.4 21.2 77.5 CD5 2.6 2.6 7.1 2.0 CD7 7.0 3.8 15.5 1.7 CD8 3.2 3.2 10.4 1.6 CDlO 0.1 0.1 10.4 1.5 CD13 16.7 55.2 35.3 63.8 CD14 6.2 6.2 10.3 2.0 CD19 0.1 0.1 11.2 2.8 CD20 0.2 0.2 10.2 1.5 CD33 51.6 57.4 73.6 99.7 14.4 1.3 0.4 11.1 1.2 1.4 9.3 1.3 CD34 HLA-DR 39.7 31 existence of clones with a different breakpoint with FISH study, coexpression mechanism should be also due to the alternative splicing When the patient’s BM cells were obtained for establishment of a cell line, the majority of the cells expressed myeloid antigens (CD13 and CD33), CD4 and HLA-DR However, TCC-S cells showed a drastic increase of CD13 and CD33 expression and loss of HLA-DR expression, while they still retained CD4 expression (Table 3) CD4 is expressed in T-cells, but also in monocytes, and CD4 expression is usually observed in myeloid BC-derived CML cell lines In the process of sub-culture, a lineage switch to myeloid direction must have occurred in TCC-S cells We defined TCC-S cells as triploid cells according to t h e ISCN (International System for Human Cytogenetic Nomenclature), because the chromosome number was 67 to 82 with 76 chromosomes as a mode number However, the majority of the cells retained two der(9)del(9) (p12)t(9;22) (q34;qll)s, two del(9) (q21)s, two der(22)t(9;22) (q34;qll)s and two normal chromosome 22 Moreover, of 28 cells showed XYY sex chromosome pattern with two Y chromosomes, nevertheless usually triploid karyotype shows XXY Thus, it is likely that TCC-S was derived from a tetraploid cell Missing of normal chromosome is occasionally seen among Ph+ CML cells lined’)’ 14) or patients, which gives rise to missing of a huge amount of genes However, a partial loss of the long arm of normal chromosome h a s been seldom seen among themlo),11) 13) , which results in missing of a restricted region including normal ABL gene at 9q34 TCC-S cells have del(9) (q21) with no existence of ABL genes which is also confirmed by FISH study Recently, submicroscopic deletions on t h e derivative chromosome called “der(9) deletions” are identified in 10-15% of patients with CML’” T h e deletions are usually large, spanning several megabases They are located in the region flanking the BCR/ABL breakpoint on the der(9), involving the loss of sequences from chromosome 9, chromosome 22 or both, although deletions of sequence only from chromosome 22 represent only 510% of all deletions CML patients carrying such deletions are known to 32 have significantly an unfavorable prognosis than those without them if they are treated with interferon-alpha and cytosine arabinoside, o r bone marrow transplantation, probably due to the loss of several tumor suppressor genes (TSGs) involved in the deleted region, although more recently, it has been reported that imatinib mesylate can overcome this disadvantage16’.However, the TSGs responsible for a poorer prognosis in CML patients with der(9) deletions have not yet been determined Thus, if a candidate TSG in these deleted regions is transfected to TCC-S cells to investigate the therapeutic effect, TCC-S cells may provide a good tool to determine such TSGs ABL protein is ubiquitously expressed, and is considered to play a complex and important role as a cellular module that integrates signals from various extra- and intra-cellular sources3’ This protein influences decisions in regard t o cell cycle and apoptosis, although this function still remains not fully understood due to lack of an adequate model system to investigate TCC-S cells will be a useful tool also for studying the biological properties of ABL protein, if ABL gene is transfected and expressed in them In conclusion, we have established a novel triploid CML cell line which harbors only BCR/ABL gene and no normal ABL gene This cell line will provide a useful tool for functional study of ABL in Ph+ CML Acknowledgements T h i s study was supported in part by Japan Foundation for the Promotion of International Medical Research Cooperation UF-PIMRC) References 1) Kantarjian HM, Deisseroth A, Kurzrock R, et al.: Chronic myelogenous leukemia Blood 82: 691703,1993 2) Drexler HG, Macleod RAF and Uphoff CC: Leukemia cell lines: in vitro models for the study of Philadelphia chromosome-positive leukemia Leukemia Res 23: 207-215, 1999 3) 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and the expression of a bcr-ubl fusion gene and apoptotic cell death in a new human chronic myelogenous leukemia cell line, KT-1, by interferon- a Blood 91: 641-648, 1998 15) Kolomietz E, Al-Maghrabi J, Brennan S, et al.: Primary chromosomal rearrangements of leukemia are frequently accompanied by extensive submicroscopic deletions and may lead to altered prognosis Blood 97: 3581-3588,2001 Blood 72: 1261-1268,1988 16) Quintas-Cardama A, Kantajian H, Talpaz M, et al.: Imatinib mesylate therapy may overcome the poor prognostic significance of deletions of derivative chromosome in patients with chronic myelogenous leukemia Blood 105: 2281-2286, 2005 11) Beran M, Pisa P, O’Brien S, e t al.: Biological Received 2005.3.15,Accepted 2005.5.23 Corresponding Author: Yuko Sato, M.D., Ph.D., Division of Ultrafine Structure, Department of Pathology, Research Institute, International Medical Center of Japan, Toyama 1-21-1,Shinjuku-Ku,Tokyo, 162-0052,JAPAN Direct TEL: 81fJapan)-352758602, F m 81Uapan)-352738603 ernail:ysato@ri.imcj.go.jp 33