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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 3088-3095
By
From the Department of Pathology, Hematology Laboratory, and UPCM-ERS
1590 CNRS, CHU Purpan, Toulouse, France; and the LRF Immunodiagnostics
Unit, Department of Cellular Science, John Radcliffe Hospital,
Oxford, UK.
Anaplastic large cell lymphomas (ALCL) are frequently associated
with the t(2;5)(p23;q35). This translocation fuses the nucleophosmin (NPM) gene at 5q35, which encodes a nucleolar protein involved in shuttling ribonucleoproteins from the cytoplasm to the nucleus, to
the anaplastic lymphoma kinase (ALK) gene at 2p23, encoding a
tyrosine kinase receptor. In this report, we describe a typical case of
ALCL whose malignant cells exhibited a novel (1;2)(q25;p23) translocation. These cells expressed ALK protein, but, in contrast to
t(2;5)-positive ALCL (which show cytoplasmic, nuclear, and nucleolar
staining), labeling was restricted to the malignant cell cytoplasm.
Using a polymerase chain reaction (PCR)-based technique to walk on
chromosome 2 from the known ALK gene across the breakpoint, we
showed that the gene involved at 1q25 is TPM3, encoding a
nonmuscular tropomyosin. We subsequently identified, using reverse
transcription-PCR analysis of cases showing similar ALK
cytoplasm-restricted staining, fusion of the ALK and
TPM3 genes in 2 other cases of ALCL. The TPM3 gene has
been previously found in papillary thyroid carcinomas as a fusion
partner with the TRK kinase gene. We showed that TPM3 is
constitutively expressed in lymphoid cell lines, suggesting that, in
these t(1;2)-bearing ALCL cases, the TPM3 gene contributes an
active promoter for ALK expression. Activation of the
ALK catalytic domain probably results from homodimerization of the
hybrid protein TPM3-ALK, through the TPM3 protein-protein interaction
domain. The present cases of ALCL associated with a novel
t(1;2)(q25;p23) demonstrate that at least one fusion partner other than
NPM can activate the intracytoplasmic domain of the ALK kinase.
ANAPLASTIC large cell lymphoma (ALCL) is
associated with a recurrent translocation, the
t(2;5)(p23;q35).1-7 This translocation involves the
ALK (anaplastic lymphoma kinase) gene at 2p23 and the
NPM (nucleophosmin) gene at 5q35. The ALK gene encodes
a tyrosine kinase receptor belonging to the insulin growth factor
receptor superfamily,8 which is normally expressed in nerve
cells but silent in normal lymphoid cells.9,10
The NPM gene is a housekeeping gene encoding a nucleolar
phosphoprotein involved in shuttling ribonucleoproteins from the cytoplasm to the nucleus.11,12 It is postulated that
NPM gene promotes the expression of the ALK catalytic domain
present in the chimeric NPM-ALK protein, and it has recently been
demonstrated that constitutive ALK activity contributes to the
malignant transformation of lymphoid cells.13,14
Since the first report of the t(2;5) in ALCL,15 cytogenetic
or molecular variants implicating the ALK gene have been
described.16-18 The recent report of a complex
t(2;5)(q37;q31), which brings the ALK gene into the vicinity of
the NPM gene, and of an inv(2)(p23q35), both of which are
associated with the expression of the ALK intracytoplasmic domain,
suggests that genes other than NPM can promote ALK gene expression.19
In a previous study,5,20 we described a case of ALCL (case
GEL) with typical morphologic and phenotypic features whose malignant
cells did not show the (2;5) translocation but instead a variant
translocation involving a breakpoint at q25 on chromosome 1 and at p23
on chromosome 2. The malignant cells expressed ALK protein strongly, as
detected with polyclonal (p80)5,21 and monoclonal (ALK1)
antibodies.20 Because both of these antibodies react with
the tyrosine kinase domain of the ALK protein, we postulated that a
gene located at 1q25 contributed an active promoter for the expression
of the ALK catalytic domain in the malignant cells in this case.
To identify the gene on chromosome 1q25 involved in the above-mentioned
case, we used a polymerase chain reaction (PCR)-based technique
described by Siebert et al22 to walk on chromosome 2 from
the ALK gene across the breakpoint to the unknown gene on
chromosome 1. This analysis showed that ALK expression probably results
from a chromosomal rearrangement fusing the last exons of the
ALK gene to sequences of the TPM3 gene, encoding
nonmuscular tropomyosin.23,24 The latter gene has been
reported as fusing to the TRK kinase gene in human thyroid
papillary carcinomas.25 Subsequently, we identified 3 other
cases of ALCL, on which no cytogenetic data were available, but which
also showed ALK staining restricted to the cytoplasm. When investigated
by reverse transcription-PCR (RT-PCR), 2 of these cases
were also positive for TPM3-ALK transcripts.
Material
Immunostaining
Cytogenetics Karyotyping was performed on cells from the diagnostic lymph node from patient GEL after 24 hours of culture. Reverse-Heating-Giemsa (RHG) banding was performed using hot phosphate buffer, and chromosomal abnormalities were described according to the ISCN 95 nomenclature.30aDNA Extraction and Digestion DNA from case GEL was extracted from 10 deparaffinized ModAMeX-processed sections (10-µm thick) by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation.31 A small quantity (70 µg) of genomic DNA could be extracted from this material, but this DNA was of poor quality, with a maximum size of 6.5 kb when analyzed by agarose gel electrophoresis, precluding the construction and the screening of any genomic library for cloning the breakpoint.PCR-Based Technique for Walking on Genomic DNA This technique, described by Siebert et al,22 involves a PCR between a gene-specific primer and a primer annealing to an adaptor sequence previously ligated to the ends of the genomic DNA fragments. The adaptor is designed in such a way that its specific primer cannot anneal to the native adaptor, because the target sequence is only created by the elongation of the gene-specific primer after the first PCR cycle.22 Nonspecific amplifications between adaptors are thus avoided and specific amplification occurs despite the use of only a single gene-specific primer (Fig 1). To increase sensitivity and specificity, the first amplification is followed by a second nested amplification.
Adaptor ligation. The blunt ends of DNA fragments generated by digestion with Dra I, EcoRV, Pvu II, Sca I, and Ssp I enzymes were ligated to an excess of adaptors, using T4 DNA ligase (New England Biolabs), following the manufacturer's recommendations. The adaptor was made of two complementary oligonucleotides, one long and one short (Fig 1). Once the two complementary oligonucleotides annealed, the adaptor presented with one blunt end that was able to ligate to any blunt end fragment of genomic DNA. The 3' extremity of the short oligonucleotide was blocked by an amine group, preventing its elongation by DNA polymerase (Fig 1). DNA amplification.
Long-range PCR was performed with the Expand Long Template PCR system
(Boehringer Mannheim, France SA, Meylan, France) using a unique enzyme
mix containing thermostable Taq and Pwo DNA polymerases. This was
performed using GEL DNA fragments for the three first steps and JAR DNA
fragments for the fourth (because of the limited amount of DNA from
case GEL; Fig 2). Primary PCR reactions
were conducted in 50 µL volumes containing 0.5 µg of ligated DNA,
0.3 µmol/L adaptor primer AP122 and ALK
gene-specific primer, 1.75 mmol/L MgCl2, 0.35 mmol/L dNTP,
and 6.125 U of DNA polymerase. An initial denaturation step at 94°C
for 2 minutes was followed by 30 cycles of denaturation at 94°C for
30 seconds, annealing-extension at 68°C for 5 minutes, and a final
extension time of 7 minutes at 65°C. The second (nested) PCR
reaction was performed under the same conditions on 1 µL of the
primary PCR, using adaptor primer AP222 and a nested
ALK gene-specific primer. The adaptor primers AP1 and AP2 and
the different ALK gene-specific primers are listed in
Table 1.
Identification of the ALK Gene Breakpoint To determine whether a given PCR fragment contained the breakpoint of interest, the fragment was purified, labeled, and used as a probe in Southern blot experiments on an unrelated DNA (CNE2) digested with the same panel of restriction enzymes: Dra I, EcoRV, Pvu II, Sca I, and Ssp I. We considered that a PCR fragment generated from the GEL DNA (following digestion with a single specific enzyme) that annealed with two different fragments of the unrelated CNE2 DNA digested with the same enzyme probably contained the ALK gene breakpoint.
Direct Cycle Sequencing of PCR Products Long-range PCR fragments and RT-PCR fragments were sequenced using an original protocol32 with DeepVent(exo )
DNA polymerase (New England Biolabs) and  -35S-dATP. For
each PCR fragment, 5' sequences (located close to the adaptor)
were first determined to prepare primers for the next PCR step.
Total RNA Extraction Total RNA was extracted from frozen sections of normal thyroid and lymph nodes involved by ALCL (cases no. 1, 2, and 3) and from the Jurkat, CEM, HSB-2, and SU-DHL-1 cryopreserved cell pellets (10 × 106) using the RNeasy Midi Kit (Qiagen, Courtaboeuf, France).Detection of Tropomyosin (TPM3) Transcripts by RT-PCR Two TPM primers pairs, C-TPM1/C-TPM2 (positions 149-169/451-431) and C-TPM3/C-TPM4 (positions 497-515/809-788; see Table 1), were chosen according to the cDNA sequence of TPM3 published by MacLeod et al,33 yielding RT-PCR products of 303 and 313 bp, respectively. Synthesis of the first cDNA strand and PCR amplification were performed in a single tube with the RT-PCR Access Kit (Promega France, Charbonnières, France), following the manufacturer's recommendations. The first step consisted of a gene-specific reverse transcription at 48°C for 45 minutes, followed by 30 cycles comprising a denaturation step at 94°C for 45 seconds, an annealing step at 58°C for 45 seconds, and an elongation step at 72°C for 35 seconds.Detection of Hybrid TPM3-ALK Transcripts by RT-PCR This study was performed in 3 ALCL cases that were negative for NPM-ALK transcripts and that showed ALK staining restricted to the cytoplasm, resembling that observed in the case GEL. The first round of RT-PCR was performed as described above, using C-TPM1 (position 149-169) and ALK-1 (position 4211-4187 on the cDNA ALK sequence) primers. The second round was performed on a 1-µL aliquot from the first amplification, using nested primers C-TPM3 (position 497-515) and ALK-2 (position 4171-4148), yielding a RT-PCR product of 307 bp (see Table 1 for primers sequence).
Immunomorphologic Features The tumor from patient GEL and the 3 other cases of ALCL investigated subsequently all contained large neoplastic cells with horseshoe or kidney-shaped eccentric nuclei (Fig 3). Their phenotype was also typical of systemic t(2;5)-positive ALCL in that they were of null (cases no. 1 and 2) or T phenotype (case no. 3) and coexpressed CD30 (Fig 3) and epithelial membrane antigen (EMA). Malignant cells were also positive with the antibody BNH.9. All cells stained for ALK protein as detected by p80 and ALK1 antibodies. However, in contrast to most ALCL, which show labeling of both the cytoplasm and the nucleus,14,20 ALK staining in these cases was limited to the cytoplasm (Fig 3).
Cytogenetics Cytogenetic analysis of the diagnostic biopsy from patient GEL showed only abnormal metaphases, with the karyotype 46,XY,t(1;2)(q25;p23),der(22)t(1;22)(q12;p11)t(1;2)(q25;p23)[20].Cloning of the t(1;2) Breakpoint Long-range PCR was performed on DNA from the biopsy of patient GEL and from the JAR cell line, after digestion with different restriction enzymes and ligation to adaptors, as described in Materials and Methods. Each PCR step was realized with a couple of nested gene-specific primers and the nested adaptor-specific primers, AP1 and AP2. A PCR walk on chromosome 2 was performed using ALK-1 and ALK-2 gene-specific primers, located in the exon adjacent to the intron where the classical t(2;5) breakpoint occurs (Fig 2).
Detection of Hybrid TPM3-ALK Transcripts and Direct Cycle Sequencing of RT-PCR Products The 3 cases of ALCL that showed a cytoplasm-restricted staining pattern for ALK (from which no cytogenetic data were available) were analyzed for the presence of TPM3-ALK transcripts. A 307-bp band was found in cases no. 1 and 3 (Fig 4A), and sequencing of nested RT-PCR products confirmed the presence of hybrid TPM3-ALK transcripts (Fig 4B).
Detection of Tropomyosin Transcripts The tyrosine kinase domain of ALK is constitutively expressed in ALCL carrying the classical t(2;5), under control of the ubiquitously expressed NPM gene promoter. We therefore wished to know whether the TPM3 gene is also active in lymphoid cells. RT-PCR assays of RNA extracted from the Jurkat, CEM, HSB-2, and SU-DHL-1 T-cell lines (and also from thyroid tissue) demonstrated that the TPM3 gene was constitutionally active,34 because bands of the expected size (303 and 313 bp) were detected with C-TPM1/C-TPM2 and C-TPM3/C-TPM4 primer pairs, respectively (data not shown).
Recurrent chromosomal translocations play an important role in many human lymphoid tumors and are often responsible for the deregulation of genes involved in the control of cell proliferation.35 A number of recent studies have investigated the (2;5)(p23;q35) translocation, an anomaly associated with ALK-positive ALCL.3-7 The t(2;5) fuses the nucleophosmin gene (NPM) with the ALK receptor tyrosine kinase gene (anaplastic lymphoma kinase) to produce a chimeric protein in which the 116 aminoterminal residues of NPM are linked to the intracytoplasmic portion of ALK.8 The NPM oligomerization domain present in NPM-ALK leads to homodimerization, thus mimicking ligand binding and leading to activation of the ALK catalytic domain.13,14 Furthermore, normal NPM contains two potential nuclear-localization signals located between aminoacids 152-157 and 191-197.11,12,36 These sequences are lost from the chimeric NPM-ALK protein, but the oligomerization domain located in the aminoterminal portion of NPM is retained11,13,37 and heterodimers can thus form between NPM-ALK and normal NPM. These translocate to the nucleus, accounting for the characteristic combination of cytoplasmic and nuclear staining observed with the anti-ALK antibodies.14,20
The authors gratefully acknowledge Prof David Y. Mason for helpful suggestions and critical reading of the manuscript.
Submitted August 25, 1998; accepted January 6, 1999.
Supported by the "Projet Hospitalier de Recherche Clinique (PHRC98)," "Ligue Nationale Contre le Cancer," the "Groupe d'Etude des Lymphomes de l'Adulte (GELA)," and the Leukemia Research Fund, UK.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
Address reprint requests to Georges Delsol, MD, Laboratoire d'Anatomie Pathologique, CHU Purpan, Place du Dr Baylac, 31059, Toulouse Cedex, France.
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N. S. Reading, S. D. Jenson, J. K. Smith, M. S. Lim, and K. S. J. Elenitoba-Johnson 5'-(RACE) Identification of Rare ALK Fusion Partner in Anaplastic Large Cell Lymphoma J. Mol. Diagn., May 1, 2003; 5(2): 136 - 140. [Full Text] [PDF]
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G. Piccinini, R. Bacchiocchi, M. Serresi, C. Vivani, S. Rossetti, C. Gennaretti, D. Carbonari, and F. Fazioli A Ligand-inducible Epidermal Growth Factor Receptor/Anaplastic Lymphoma Kinase Chimera Promotes Mitogenesis and Transforming Properties in 3T3 Cells J. Biol. Chem., June 14, 2002; 277(25): 22231 - 22239. [Abstract] [Full Text] [PDF]
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L. Passoni, A. Scardino, C. Bertazzoli, B. Gallo, A. M. L. Coluccia, F. A. Lemonnier, K. Kosmatopoulos, and C. Gambacorti-Passerini ALK as a novel lymphoma-associated tumor antigen: identification of 2 HLA-A2.1-restricted CD8+ T-cell epitopes Blood, March 15, 2002; 99(6): 2100 - 2106. [Abstract] [Full Text] [PDF]
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B. Falini and D. Y. Mason Proteins encoded by genes involved in chromosomal alterations in lymphoma and leukemia: clinical value of their detection by immunocytochemistry Blood, January 15, 2002; 99(2): 409 - 426. [Abstract] [Full Text] [PDF]
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S. J. Meech, L. McGavran, L. F. Odom, X. Liang, L. Meltesen, J. Gump, Q. Wei, S. Carlsen, and S. P. Hunger Unusual childhood extramedullary hematologic malignancy with natural killer cell properties that contains tropomyosin 4-anaplastic lymphoma kinase gene fusion Blood, August 15, 2001; 98(4): 1209 - 1216. [Abstract] [Full Text] [PDF]
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G. Z. Rassidakis, A. H. Sarris, M. Herling, R. J. Ford, F. Cabanillas, T. J. McDonnell, and L. J. Medeiros Differential Expression of BCL-2 Family Proteins in ALK-Positive and ALK-Negative Anaplastic Large Cell Lymphoma of T/Null-Cell Lineage Am. J. Pathol., August 1, 2001; 159(2): 527 - 535. [Abstract] [Full Text]
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B. Maes, V. Vanhentenrijk, I. Wlodarska, J. Cools, B. Peeters, P. Marynen, and C. De Wolf-Peeters The NPM-ALK and the ATIC-ALK Fusion Genes Can Be Detected in Non-Neoplastic Cells Am. J. Pathol., June 1, 2001; 158(6): 2185 - 2193. [Abstract] [Full Text] [PDF]
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C Greenland, N Dastugue, C Touriol, L Lamant, G Delsol, and P Brousset Anaplastic large cell lymphoma with the t(2;5)(p23;q35) NPM/ALK chromosomal translocation and duplication of the short arm of the non-translocated chromosome 2 involving the full length of the ALK gene J. Clin. Pathol., February 1, 2001; 54(2): 152 - 154. [Abstract] [Full Text] [PDF]
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R.-Y. Bai, T. Ouyang, C. Miething, S. W. Morris, C. Peschel, and J. Duyster Nucleophosmin-anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway Blood, December 15, 2000; 96(13): 4319 - 4327. [Abstract] [Full Text] [PDF]
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H. Stein, H.-D. Foss, H. Durkop, T. Marafioti, G. Delsol, K. Pulford, S. Pileri, and B. Falini CD30+ anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features Blood, December 1, 2000; 96(12): 3681 - 3695. [Abstract] [Full Text] [PDF]
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M. Ladanyi Aberrant ALK Tyrosine Kinase Signaling : Different Cellular Lineages, Common Oncogenic Mechanisms? Am. J. Pathol., August 1, 2000; 157(2): 341 - 345. [Full Text] [PDF]
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B. Lawrence, A. Perez-Atayde, M. K. Hibbard, B. P. Rubin, P. Dal Cin, J. L. Pinkus, G. S. Pinkus, S. Xiao, E. S. Yi, C. D. M. Fletcher, et al. TPM3-ALK and TPM4-ALK Oncogenes in Inflammatory Myofibroblastic Tumors Am. J. Pathol., August 1, 2000; 157(2): 377 - 384. [Abstract] [Full Text] [PDF]
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M. W. Bekkenk, F. A. M. J. Geelen, P. C. v. V. Vader, F. Heule, M.-L. Geerts, W. A. van Vloten, C. J. L. M. Meijer, and R. Willemze Primary and secondary cutaneous CD30+ lymphoproliferative disorders: a report from the Dutch Cutaneous Lymphoma Group on the long-term follow-up data of 219 patients and guidelines for diagnosis and treatment Blood, June 15, 2000; 95(12): 3653 - 3661. [Abstract] [Full Text] [PDF]
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C. Touriol, C. Greenland, L. Lamant, K. Pulford, F. Bernard, T. Rousset, D. Y. Mason, and G. Delsol Further demonstration of the diversity of chromosomal changes involving 2p23 in ALK-positive lymphoma: 2 cases expressing ALK kinase fused to CLTCL (clathrin chain polypeptide-like) Blood, May 15, 2000; 95(10): 3204 - 3207. [Abstract] [Full Text] [PDF]
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Z. Ma, J. Cools, P. Marynen, X. Cui, R. Siebert, S. Gesk, B. Schlegelberger, B. Peeters, C. De Wolf-Peeters, I. Wlodarska, et al. Inv(2)(p23q35) in anaplastic large-cell lymphoma induces constitutive anaplastic lymphoma kinase (ALK) tyrosine kinase activation by fusion to ATIC, an enzyme involved in purine nucleotide biosynthesis Blood, March 15, 2000; 95(6): 2144 - 2149. [Abstract] [Full Text] [PDF]
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G. W. B. Colleoni, J. A. Bridge, B. Garicochea, J. Liu, D. A. Filippa, and M. Ladanyi ATIC-ALK: A Novel Variant ALK Gene Fusion in Anaplastic Large Cell Lymphoma Resulting from the Recurrent Cryptic Chromosomal Inversion, inv(2)(p23q35) Am. J. Pathol., March 1, 2000; 156(3): 781 - 789. [Abstract] [Full Text] [PDF]
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M. Trinei, L. Lanfrancone, E. Campo, K. Pulford, D. Y. Mason, P.-G. Pelicci, and B. Falini A New Variant Anaplastic Lymphoma Kinase (ALK)-Fusion Protein (ATIC-ALK) in a Case of ALK-positive Anaplastic Large Cell Lymphoma Cancer Res., February 1, 2000; 60(4): 793 - 798. [Abstract] [Full Text]
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R. Siebert, S. Gesk, L. Harder, D. Steinemann, W. Grote, B. Schlegelberger, M. Tiemann, I. Wlodarska, and V. Schemmel Complex Variant Translocation t(1;2) With TPM3-ALK Fusion Due to Cryptic ALK Gene Rearrangement in Anaplastic Large-Cell Lymphoma Blood, November 15, 1999; 94(10): 3614 - 3617. [Full Text] [PDF]
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L. Hernandez, M. Pinyol, S. Hernandez, S. Bea, K. Pulford, A. Rosenwald, L. Lamant, B. Falini, G. Ott, D. Y. Mason, et al. TRK-Fused Gene (TFG) Is a New Partner of ALK in Anaplastic Large Cell Lymphoma Producing Two Structurally Different TFG-ALK Translocations Blood, November 1, 1999; 94(9): 3265 - 3268. [Abstract] [Full Text] [PDF]
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G. E. Stoica, A. Kuo, A. Aigner, I. Sunitha, B. Souttou, C. Malerczyk, D. J. Caughey, D. Wen, A. Karavanov, A. T. Riegel, et al. Identification of Anaplastic Lymphoma Kinase as a Receptor for the Growth Factor Pleiotrophin J. Biol. Chem., May 11, 2001; 276(20): 16772 - 16779. [Abstract] [Full Text] [PDF]
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TOP
ABSTRACT
INTRODUCTION
CEM, Jurkat, HSB-2 (all acute T
leukemia; ATCC CCL 119, ATCC TIB 153, and ATCC CCL 120.1, respectively) and SU-DHL-1 [established from a t(2;5)-positive anaplastic lymphoma; kindly provided by Dr M.L. Cleary, Stanford University Medical Center,
Stanford, CA]
80°C.
1-2R primers, with the latter primer being chosen from the chromosome 1-specific 80 bp of the GEL
Dra I fragment. This amplification yielded the expected 700-bp fragment.
