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NEOPLASIA
From the Department of Virology, National Children's
Medical Research Center, Tokyo; Department of Cancer Cytogenetics,
Research Institute for Radiation Biology and Medicine, Hiroshima
University, Hiroshima; Division of Pediatrics, Hamanomachi Hospital,
Fukuoka; Department of Pediatrics, Saga University School of Medicine,
Saga; and Department of Pediatrics, Hiroshima University School of
Medicine, Hiroshima, Japan.
TEL-AML1 fusion resulting from the t(12;21)(p13;q22) is
one of the most common genetic abnormalities in childhood acute
lymphoblastic leukemia. Recent findings that site-specific cleavage of
the MLL gene can be induced by chemotherapeutic agents such
as topoisomerase-II inhibitors suggest that apoptogenic agents can
cause chromosomal translocations in hematopoietic cells. This study
demonstrates a possible relationship between exposure to apoptogenic
stimuli, TEL breaks, and the formation of
TEL-AML1 fusion in immature B lymphocytes. Short-term
culture of immature B cell lines in the presence of apoptogenic stimuli
such as serum starvation, etoposide, or salicylic acid induced
double-strand breaks (DSBs) in intron 5 of the TEL gene and
intron 1 of the AML1 gene. TEL-AML1
fusion transcripts were also identified by reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis in cell
lines treated by serum starvation or aminophylline. DSBs within the
TEL gene were also associated with fusion to other unknown
genes, presumably as a result of chromosomal translocation. We also
examined 67 cord blood and 147 normal peripheral blood samples for the
existence of in-frame TEL-AML1 fusion
transcripts. One cord blood sample (1.5%) and 13 normal peripheral
blood samples (8.8%) were positive as detected by nested RT-PCR. These
data suggest that breakage and fusion of TEL and
AML1 may be relatively common events and that sublethal
apoptotic signals could play a role in initiating leukemogenesis via the promotion of DNA damage.
(Blood. 2001;97:737-743) The t(12;21)(p13;q22) is one of the most common
chromosome translocations in childhood acute lymphoblastic leukemia
(ALL).1-3 This translocation is associated with
rearrangement of the TEL (ETV6) gene on chromosome 12 and
the AML1(CBFA2) gene on chromosome 21 resulting in
TEL-AML1 fusion. Although many studies on the TEL-AML1 fusion gene focused on the biologic
significance of leukemogenesis, little is known about the mechanisms
involved in the cleavage of TEL and AML1 and
subsequent fusion of the TEL-AML1 gene.
Chemotherapy-related secondary leukemia is frequently associated with
rearrangement of the MLL(ALL1) gene. Several
clinicoepidemiologic studies have indicated that topoisomerase-II
(topo-II) inhibitors cause rearrangement of the MLL
gene.4 These findings are supported by in vitro studies
that have demonstrated that MLL rearrangement could be induced by topo-II inhibitors in human hematopoietic
cells.5 Site-specific cleavage within the MLL
gene can also be induced by nongenotoxic stimuli involved in apoptotic
cell death, suggesting that this cleavage is not specific to topo-II
inhibitors but is rather part of a chromatin fragmentation similar to
the generalized cellular response to apoptotic stimuli.6
Based on these early findings, we speculated that general apoptotic
stimuli on hematopoietic cells could play a key role in chromosomal
translocation involving not only MLL but also
TEL, another gene frequently altered in hematopoietic
malignancies. To investigate whether apoptotic stimuli could trigger a
cascade of events that ultimately leads to the formation of the
TEL-AML1 fusion gene, we studied the in vitro conditions inducing the formation of DNA double-strand break (DSB) ends
within TEL and AML1 genes. Our study indicates
that DSBs of TEL and AML1 can be induced by
apoptotic stimuli and this DNA cleavage appears to be the first step in
the generation of TEL-AML1 fusion in immature B
lymphocytes, which is considered to be an early or initiating event in
childhood ALL.7,8
Cell lines
Peripheral blood and cord blood samples
Serum starvation and chemicals The ALL cell lines at exponential growth phase were centrifuged and washed twice with phosphate-buffered saline (PBS). Cells were resuspended in RPMI 1640 without FBS at a concentration of 5 × 105 cells/mL. As primary hematopoietic cells, mononuclear cells were separated from fresh cord blood samples and suspended in RPMI 1640 without FBS. VP-16 (10 µM), H2O2 (5 µM), salicylic acid (2 mM, 2-hydroxybenzoic acid, Sigma Co, St Louis, MO), caffeine (2 mM, Sigma), or aminophylline (2 mM, Neophyllin; Eisai Pharmaceuticals, Tokyo, Japan) was added to RPMI 1640 supplemented with 10% FBS. Cells were cultured at 37°C in 5% CO2 in air for indicated times and collected for further analysis.Ligation-mediated PCR for detection of broken-ended DNA To detect DSB ends of DNA, ligation-mediated PCR (LM-PCR) was performed as described previously.13-15 BW linker was produced by annealing the BW-1 and BW-2 oligonucleotides (Table 1A). Two micrograms of high-molecular-weight DNA was ligated to the BW linker with T4 DNA ligase (Takara Shuzo, Otsu, Japan), precipitated with ethanol, and dissolved in 20 µL distilled water. One microliter of the ligated sample was analyzed by PCR with Ex Taq (Takara) using BW-1 linker primer (0.02 µM) and gene-specific primers (0.4 µM) of target genes (Figure 1, Table 1A). Seminested PCR was performed with a specific primer and BW-1 primer for first round PCR, and with internal specific primer and BW-1 for the second round PCR. The conditions for PCR were as follows: 94°C for 2 minutes, followed by 30 cycles of 94°C for 1 minute, 65°C for 1 minute, 72°C for 1 minute, and final extension at 72°C for 10 minutes. PCR products were purified using a PCR purification kit (Qiagen, Santa Clarita, CA) and subcloned to pGEM-T easy vector with a TA-cloning kit (Promega, Madison, WI). Plasmid DNA was extracted using the Qiaprep Spin Plasmid system (Qiagen) and insert DNAs were sequenced with 373A Autosequencer (Applied Biosystems, Urayasu, Japan). Obtained nucleotide sequences were compared with the sequences of intron 5 of TEL and intron 1 of AML1 (GenBank accession number: HSU6137516 and AJ229043, respectively). DSB of BCR, -globin, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes
(GenBank accession number U07000, AF007546, and J04038, respectively)
were also studied with LM-PCR analysis.
Inverse PCR for detection of rearrangement of the TEL gene Inverse PCR strategy was performed as previously reported.17 High-molecular-weight DNA (2 µg) was digested with HindIII and ligated at 15°C for 30 minutes in a total volume of 250 µL with 5 U T4 DNA ligase (Takara). The ligated DNA was then purified using the Geneclean (BIO 101 Inc, La Jolla, CA) and eluted in a final volume of 50 µL distilled water. Ligated DNA (4 µL) was used as the template and amplified by nested PCR using LA Taq (Takara). First-step PCR amplification was performed with E3F as the sense primer and E1R as the antisense primer. Second-step PCR was performed with E4F and E2R (Figure 1, Table 1A). The PCR amplification condition was as follows: 94°C for 2 minutes, followed by 30 cycles of 98°C for 20 seconds and 68°C for 7 minutes and terminal extension at 72°C for 10 minutes. PCR products were electrophoresed onto 0.8% agarose gel and positive bands were eluted from the gel, purified with Geneclean and subcloned into pGEM-T easy vector. The extracted plasmid DNAs were sequenced using 373A Autosequencer. Obtained nucleotide sequences were compared with the sequence of intron 5 of TEL and unknown sequences fused to TEL were analyzed by NCBI BLAST server.Fluorescence in situ hybridization analysis Mononuclear cells separated from peripheral blood of healthy volunteers were cultured with phytohemagglutinin (PHA) for 72 hours and used for the normal metaphase sample for fluorescence in situ hybridization (FISH) analysis. DNA fragments for chromosome mapping were labeled with digoxigenin-11-dUTP (Boehringer Mannheim, Mannheim, Germany) by PCR after sequence-independent amplification method18 and were hybridized to metaphase samples as described previously.19RT-PCR Nested RT-PCR amplification for the TEL-AML1 fusion gene was performed using the specific primers (Table 1B) as described previously.20 GAPDH was used for control expression to confirm RNA integrity. Strict precautions were followed to prevent cross-contamination of samples and negative controls were included in each PCR amplification step. RNA for positive control experiments (extracted from the REH cell line) were handled only in the first experiment for setting of PCR conditions and sensitivity assay of the RT-PCR analysis. Positive RNA was never handled in whole step while analyzing RNA samples from nonleukemic volunteers.
DSBs of TEL and AML1 after serum starvation Serum starvation induced apoptosis in TS-2 and RS4;11 cell lines and the remaining surviving cells showed growth arrest within 48 hours. Cell lines cultured for 12 hours under serum starvation were subjected to detection of DSBs by LM-PCR analysis. About 15% and 30% of TS-2 and RS4;11 cells were identified to have undergone apoptosis, respectively (data not shown). Representative results of LM-PCR are shown in Figure 2A. A clear but weak band representing DSB of intron 5 of TEL was detected with E1F and E2F primers in the RS4;11 cell line. By PCR with E1F and E3F primers both leukemic cell lines showed smearing bands ranging in size from 400 base pairs (bp) to 1 kilobase (kb). As for AML1, the 2 cell lines showed a clear band with A1R and A2R primers and a broad 1- to 2-kb band with A3R and A4R primers, both indicating DNA break in intron 1. Sequence analysis revealed that 5 independent clones obtained from E1F/E2F product in the RS4;11 cell line included the same DSB DNA fragment, which corresponded to intron 5 of TEL (BP1 in Figure 3A). As for AML1, 5 and 6 independent clones from RS4;11 and TS-2, respectively, were obtained from A1R/A2R products and 4 different sites of breakpoints in intron 1 were isolated (BP2-5 in Figure 3A). Interestingly, sequences of the DNA break ends from these immature B lymphocytes showed nontemplated base addition to the break ends. HL60 cell line was also examined with the same primer sets and DSBs of TEL and AML1 were also detected after 12 hours of serum starvation. The sequences of DNA break ends from HL60 cell line showed no addition of nontemplated bases to the break ends (data not shown). We also studied DSB formation of the BCR gene in both ALL cell lines, which is also frequently involved in pathogenesis of leukemia. As a result DSB ends of BCR were detected after 12 hours of serum starvation. DSBs of the-globin and
GAPDH genes were also studied and only GAPDH showed DSB ends in the same condition (Figure 2B). To confirm whether
the formation of DSB ends of TEL and AML1 genes
is a general event that occurs in not only immortal cell lines but also
primary hematopoietic cells, we applied LM-PCR analysis to mononuclear cells of 3 cord blood samples. Although we could not obtain clear bands
of the LM-PCR products, subcloning and sequencing of the LM-PCR
products revealed that DSBs occurred in TEL and
AML1 also in the cord blood after 24 hours of serum
starvation (Figure 3C).
DSBs of TEL and AML1 are induced by chemicals Formation of DSB by topo-II inhibitor (VP-16), H2O2, and salicylic acid was also examined to determine whether these agents could induce DNA breaks in TEL and AML1 genes. After 24 hours' culture with VP-16 or H2O2, DSBs of AML1 were observed by LM-PCR analysis (data not shown). In addition DSBs of the AML1 gene could be induced by 24 hours' culture of RS4;11 with salicylic acid (Figure 2A). Nucleotide sequences of the DSB ends induced by salicylic acid were determined and DNA breaks in intron 1 of AML1 were confirmed in all examined clones.The DSBs of TEL and AML1 were tested in 4 EB-LCLs after apoptogenic stimuli with salicylic acid, caffeine, or H2O2. After 24 hours of culture with the chemical agents, cells were harvested and formation of DSBs was analyzed by LM-PCR (Figure 2C). DSBs of TEL were observed as weak bands in EB-LCL4 after salicylic acid treatment and in EB-LCL1 after H2O2 treatment. DSBs of AML1 were also detected as clear bands in EB-LCL1 and 3 after salicylic acid treatment, in EB-LCL3 and 4 after caffeine treatment, and as smearing band in all 4 EB-LCLs after H2O2 treatment. When cells were cultured without the addition of any chemical agent, all 4 EB-LCLs showed no positive band by LM-PCR analysis (data not shown). Representative nucleotide sequences of DSB ends of AML1 are shown in Figure 3B. As with HL60, the addition of nontemplated bases to the break ends was not observed in DSB ends detected in EB-LCLs. Fusion gene formation of the TEL gene after serum starvation Because DSBs of TEL and AML1 were frequently detected in ALL cell line after serum starvation, we investigated whether formation of broken DNA ends was followed by chromosome translocation after repair of broken DNA ends. For this purpose, we applied inverse PCR strategy to detect rearrangement of the TEL gene. DNA samples extracted from cells before serum starvation were used for negative control samples (0 hour). As shown in Figure 4A, only the 4-kb germline band was detected in these control samples. However, both cell lines cultured for 12 hours under serum starvation showed PCR fragments with smaller size than the germline band. By subcloning the PCR fragments to pGEM-T easy vector, 17 clones were isolated, which exhibited rearrangement within the 4-kb HindIII fragment of intron 5. Twelve of 17 clones had interstitial deletion of about 100 to 200 bp within the 4-kb germline HindIII fragment. The other 5 clones showed fusion of TEL genomic fragment to unknown genes whose length ranged from 30 bp to 2 kb. These 5 fusion partner genes showed no homology to AML1 or to any published sequences based on BLAST homology search. Sequences of the 5 clones are shown in Figure 4B. Clone 1 was the only one that was long enough for chromosome mapping by FISH. This clone was mapped to chromosome 19 (Figure 5), indicating that chromosome translocation had occurred between TEL gene and clone 1 on chromosome 19.
TEL-AML1 fusion transcript is detected after serum starvation AML1 was not included as the fusion partner in isolated 5 TEL-rearranged clones. Therefore, we performed RT-PCR analysis to detect possible TEL-AML1 fusion transcript in the RS4;11 and TS-2 cell lines after serum starvation. Sensitivity analysis of RT-PCR was performed using RNA extracted from the REH cell line mixed with the HL60 cell line at different ratios, which showed that a single cell with TEL-AML1 fusion transcript in 105 cells could be detected (data not shown). Representative results of RT-PCR analysis are shown in Figure 6A. Samples obtained from cells before serum starvation showed no amplified product using combination of TEL exon 5 forward primer and AML1 exon 3 or exon 4 reverse primer. Twelve hours after serum deprivation RS4;11 showed positive TEL-AML1 fusion transcript with combination of TEL exon 5 forward primer and AML1 exon 3 reverse primer. Sampling of cell lines before and after serum starvation, RNA extraction, and RT-PCR analysis was repeated 3 times and only RS4;11 showed positive results. Amplified PCR products were directly sequenced and confirmed to be identical to the sequence of fusion transcript in ALL with TEL-AML1 rearrangement.21
TEL-AML1 fusion transcript is detected after aminophylline treatment It is important to study whether chemicals that can induce apoptosis of immature B lymphocytes could also induce the formation of TEL-AML1 fusion as serum starvation. Aminophylline was added to the culture, which resulted in growth impairment of both ALL cell lines. At a concentration of 3 mM, aminophylline caused apoptosis of almost all the cell populations after 72 hours of culture. However, after the addition of 2 mM aminophylline, cells were still alive and proliferated slowly up to day 7. Total RNA was extracted every 24 hours after the addition of 2 mM aminophylline and TEL-AML1 fusion transcripts were analyzed by RT-PCR. As shown in Figure 6B, TEL-AML1 fusion transcripts were detected in both cell lines at 5 and 6 days after addition of aminophylline. Nucleotide sequences of these products showed that all positive bands corresponded to one of the 4 isoforms of TEL-AML1 fusion transcript observed in ALL.21 Although TS-2 cell line showed only one isoform, RS4;11 cell line showed 2 isoforms depending on the time points analyzed.TEL-AML1 fusion transcripts are present in normal blood We examined the presence of TEL-AML1 fusion transcript in lymphocytes from peripheral blood of nonleukemic individuals to assess whether the fusion gene is frequently generated in vivo (Table 2). Surprisingly, 13 of 147 samples (8.8%) showed at least one type of TEL-AML1 fusion transcript as detected by nested RT-PCR. Specifically, 11 of 99 samples (11.1%) obtained from individuals younger than 20 years of age and 2 of 48 samples (4.2%) from subjects older than 20 years of age were positive for the fusion transcript. Representative results of RT-PCR analysis are shown in Figure 7. All positive products were directly sequenced, which confirmed that fusion of TEL to AML1 was identical to that of leukemic patients (data not shown). All the 4 isoforms of TEL-AML1 fusion transcript were identified by DNA sequence analysis. Different types of isoforms were observed in each of the positive individuals and some of them had more than one type of fusion transcripts (data not shown), thus making contamination of the tested samples unlikely. We also examined 67 cord blood samples for the existence of TEL-AML1 fusion transcripts and one sample was positive.
Chromosome translocation and consequent fusion gene formation with acquired oncogenic function is a frequent event in hematopoietic malignancy. The mechanism of chromosome translocation and fusion with each other is an important issue in leukemogenesis. The general mechanism of gene fusion appears to be error-prone DNA repair of DSBs via nonhomologous recombination.17,22,23 MLL gene is considered to be highly susceptible to topo-II inhibitors, and DSB of MLL gene after chemotherapy may produce translocations and gene fusion resulting in secondary leukemia.4 Previous studies have shown that site-specific cleavage of MLL is induced not only by topo-II inhibitors but also by more general nongenotoxic, apoptogenic stimuli such as serum starvation.5,6 These findings suggest that apoptogenic stimuli in general can cause DSB in leukemogenic genes. In our study, we hypothesized that in immature B lymphocytes, apoptogenic stimuli might trigger DSBs of TEL and AML1 genes and that the TEL-AML1 fusion gene could result from aberrant or error-prone DSB repair. In 12;21 translocation, the breakpoint of the TEL gene is located at intron 5, and more than half of the cases have breakpoints in the 4-kb HindIII fragment of intron 5 (Figure 1).17,24 As shown in this study, this region is indeed susceptible to DNA cleavage and produces DSB ends after exposure to several apoptogenic stimuli. In contrast to TEL, breakpoints in AML1 of leukemic cells are dispersed throughout more than one region of the large intron 1.17 Because some previous reports indicated the relationship between DNA cleavage/chromosome translocation and alternating purine-pyrimidine tract25,26 that is also present in the 4-kb HindIII fragment of TEL gene, we tentatively focused on the purine-pyrimidine tracts in intron 1 of AML1 as the target region for LM-PCR analysis. Our data indicated that this region and, we assume, other regions of AML1 intron 1 included in TEL-AML1 fusion might produce DNA break ends under apoptotic stress. Because efficiency of detecting DSB ends by LM-PCR analysis may depend
on the selection and location of gene-specific primers, we have no
insight into the relative frequency of DSB formation between analyzed
genes with our LM-PCR strategy. The level of DSB formation may depend
on the level and duration of apoptogenic stress. We have no
grounds for suspecting that TEL and AML1
genes are unusually susceptible to breakage and recombination.
Vulnerability to DSB may be associated with some general features of
the gene including open or accessible chromatin and transcriptional
activity. We found that like TEL and AML1, other
genes that are widely expressed, BCR and
GAPDH also underwent DSB in response to apoptotic
signals in contrast to transcriptionally silent (in lymphoid cells)
As reported by Richardson and Jasin,28 the presence of DSB ends of both the TEL and AML1 genes simultaneously in a single cell might be expected to increase substantially the frequency of TEL-AML1 fusion gene formation. Our results showed that DSBs of the TEL gene under serum starvation were repaired and rearrangement and fusion to other genes occurred. Although genomic fusion of TEL to AML1 gene was not detected by inverse PCR, RT-PCR analysis demonstrated TEL-AML1 fusion transcripts were formed after 12 hours of serum starvation or aminophylline treatment. These findings suggest that the DSB end of the TEL gene may fuse to the break end of AML1 gene during the repair process. Like TEL and AML1 genes, apoptogenic stimuli on normal hematopoietic cells could induce DSB ends of some other genes leading to misjoining of the leukemia-associated genes. Several studies have previously shown that fusion genes specific to hematopoietic malignancy, such as BCR-ABL,29 IGH-BCL2,30-33 and tandem duplication of MLL,34 could be detected in peripheral blood of healthy volunteers at a relatively high frequency. Based on the results of our study, we have added TEL-AML1 to the catalog of leukemic fusion genes that can be detected in normal blood samples. A limitation of this finding on normal blood is that the cell type harboring the fusion gene is unidentified. It is therefore unclear if the presence of a fusion protein is associated with a transformation event (or is neutral in an "irrelevant" cell). Even if these fusion genes are functionally active in some samples it is likely that they are insufficient for leukemogenesis. Studies on identical twins with ALL and TEL-AML1 fusion provide a strong argument for the fusion gene being an initiating event (prenatally) but insufficient to generate ALL.8,35 The failure of a TEL-AML1 transgene to induce leukemia in mice (A. M. Ford and M. F. Greaves, personal communication, June 2000) leads to a similar conclusion. Recently, observations on identical twins with ALL and retrospective PCR analysis of neonatal blood spots of patients with ALL have established an in utero origin of leukemic cells with t(12;21) in most cases of childhood ALL.7,8,35,36 Considered together with our findings, it is possible that exposure to apoptogenic stimuli to immature B lymphocytes in utero can trigger the initial molecular change in childhood ALL. Whether this mechanism is dependent on exposure of the pregnant mother and fetus to exogenous agents (eg, pesticides, organic solvents or other chemicals, viruses) or the endogenous "physiologic" apoptotic stress of development36 is unknown but may be clarified by ongoing case/cohort epidemiologic studies. In summary, we have demonstrated that DSB of TEL and AML1 genes occurs after exposure of immature B lymphocytes to apoptotic stimuli and that TEL-AML1 fusion gene can be generated during the apoptotic response in these cells. Based on these results we speculate that apoptotic stimuli triggering DSB with subsequent fusion gene formation might play a role in initiating childhood ALL.
We thank Prof Kiyoshi Miyagawa (Department of Pathology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan) for providing adult peripheral blood samples. We also thank Dr Masataka Imaizumi (Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan) for providing the TS-2 cell line, Dr Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Institute, Okayama, Japan) for providing the REH cell line, and Prof Mel Greaves (Leukaemia Research Fund Centre, Institute of Cancer Research, London) for helpful advice.
Submitted March 29, 2000; accepted October 2, 2000.
Supported by a Grant-in-Aid for Pediatric Research from the Ministry of Health and Welfare, Japan; by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan; by a Grant-in-Aid from the Ministry of Health and Welfare, Japan, as part of a comprehensive 10-year strategy for cancer control; by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan; by Health Science Research Grants; and by Organized Research Combination System.
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: Minenori Eguchi-Ishimae, Leukaemia Research Fund Centre, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Rd, London SW3 6JB, United Kingdom; e-mail: mieguchi{at}icr.ac.uk.
1. Romana SP, Le Coniat M, Berger R. t(12;21): a new recurrent translocation in acute lymphoblastic leukemia. Genes Chromosomes Cancer. 1994;9:186-191[Medline] [Order article via Infotrieve].
2.
Romana SP, Poirel H, Leconiat M, et al.
High frequency of t(12;21) in childhood B-lineage acute lymphoblastic leukemia.
Blood.
1995;86:4263-4269 3. Shurtleff SA, Buijs A, Behm FG, et al. TEL/AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent prognosis. Leukemia. 1995;9:1985-1989[Medline] [Order article via Infotrieve]. 4. Felix CA. Secondary leukemias induced by topoisomerase-targeted drugs. Biochim Biophys Acta. 1998;1400:233-255[Medline] [Order article via Infotrieve].
5.
Aplan PD, Chervinsky DS, Stanulla M, Burhans WC.
Site-specific DNA cleavage within the MLL breakpoint cluster region induced by topoisomerase II inhibitors.
Blood.
1996;87:2649-2658 6. Stanulla M, Wang J, Chervinsky DS, Thandla S, Aplan PD. DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis. Mol Cell Biol. 1997;17:4070-4079[Abstract]. 7. Wiemels JL, Cazzaniga G, Daniotti M, et al. Prenatal origin of acute lymphoblastic leukaemia in children. Lancet. 1999;354:1499-1503[CrossRef][Medline] [Order article via Infotrieve].
8.
Wiemels JL, Ford AM, Van Wering ER, Postma A, Greaves M.
Protracted and variable latency of acute lymphoblastic leukemia after TEL-AML1 gene fusion in utero.
Blood.
1999;94:1057-1062
9.
Stong RC, Korsmeyer SJ, Parkin JL, Arthur DC, Kersey JH.
Human acute leukemia cell line with the t(4;11) chromosomal rearrangement exhibits B lineage and monocytic characteristics.
Blood.
1985;65:21-31 10. Yoshinari M, Imaizumi M, Eguchi M, et al. Establishment of a novel cell line (TS-2) of pre-B acute lymphoblastic leukemia with a t(1;19) not involving the E2A gene. Cancer Genet Cytogenet. 1998;101:95-102[CrossRef][Medline] [Order article via Infotrieve]. 11. Uphoff CC, MacLeod RA, Denkmann SA, et al. Occurrence of TEL-AML1 fusion resulting from (12;21) translocation in human early B-lineage leukemia cell lines. Leukemia. 1997;11:441-447[CrossRef][Medline] [Order article via Infotrieve]. 12. Pelloquin F, Lamelin JP, Lenoir GM. Human B lymphocytes immortalization by Epstein-Barr virus in the presence of cyclosporin A. In Vitro Cell Dev Biol. 1986;22:689-694[Medline] [Order article via Infotrieve].
13.
Schlissel M, Constantinescu A, Morrow T, Baxter M, Peng A.
Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated.
Genes Dev.
1993;7:2520-2532
14.
Schlissel MS.
Structure of nonhairpin coding-end DNA breaks in cells undergoing V(D)J recombination.
Mol Cell Biol.
1998;18:2029-2037 15. Staley K, Blaschke AJ, Chun J. Apoptotic DNA fragmentation is detected by a semiquantitative ligation-mediated PCR of blunt DNA ends. Cell Death Differ. 1997;4:66-75[CrossRef][Medline] [Order article via Infotrieve].
16.
Baens M, Peeters P, Guo C, Aerssens J, Marynen P.
Genomic organization of TEL: the human ETS-variant gene 6.
Genome Res.
1996;6:404-413
17.
Wiemels JL, Greaves M.
Structure and possible mechanisms of TEL-AML1 gene fusions in childhood acute lymphoblastic leukemia.
Cancer Res.
1999;59:4075-4082 18. Bohlander SK, Espinosa Rd, Fernald AA, Rowley JD, Le Beau MM, Diaz MO. Sequence-independent amplification and labeling of yeast artificial chromosomes for fluorescence in situ hybridization. Cytogenet Cell Genet. 1994;65:108-110[Medline] [Order article via Infotrieve].
19.
Eguchi M, Eguchi-Ishimae M, Tojo A, et al.
Fusion of ETV6 to neurotrophin-3 receptor TRKC in acute myeloid leukemia with t(12;15)(p13;q25).
Blood.
1999;93:1355-1363 20. Eguchi-Ishimae M, Eguchi M, Tanaka K, et al. Fluorescence in situ hybridization analysis of 12;21 translocation in Japanese childhood acute lymphoblastic leukemia. Jpn J Cancer Res. 1998;89:783-788[CrossRef][Medline] [Order article via Infotrieve]. 21. Satake N, Kobayashi H, Tsunematsu Y, et al. Minimal residual disease with TEL-AML1 fusion transcript in childhood acute lymphoblastic leukaemia with t(12;21). Br J Haematol. 1997;97:607-611[CrossRef][Medline] [Order article via Infotrieve].
22.
Zucman-Rossi J, Legoix P, Victor JM, Lopez B, Thomas G.
Chromosome translocation based on illegitimate recombination in human tumors.
Proc Natl Acad Sci U S A.
1998;95:11786-11791 23. Gillert E, Leis T, Repp R, et al. A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells. Oncogene. 1999;18:4663-4671[CrossRef][Medline] [Order article via Infotrieve].
24.
Thandla SP, Ploski JE, Raza-Egilmez SZ, et al.
ETV6-AML1 translocation breakpoints cluster near a purine/pyrimidine repeat region in the ETV6 gene.
Blood.
1999;93:293-299
25.
Spitzner JR, Chung IK, Muller MT.
Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats.
Nucleic Acids Res.
1990;18:1-11 26. Boehm T, Mengle-Gaw L, Kees UR, et al. Alternating purine-pyrimidine tracts may promote chromosomal translocations seen in a variety of human lymphoid tumours. EMBO J. 1989;8:2621-2631[Medline] [Order article via Infotrieve]. 27. Dann EJ, Fears S, Arad-Dann H, Nucifora G, Rowley JD. Lineage specificity of CBFA2 fusion transcripts. Leuk Res. 2000;24:11-17[CrossRef][Medline] [Order article via Infotrieve]. 28. Richardson C, Jasin M. Frequent chromosomal translocations induced by DNA double-strand breaks. Nature. 2000;405:697-700[CrossRef][Medline] [Order article via Infotrieve].
29.
Biernaux C, Loos M, Sels A, Huez G, Stryckmans P.
Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals.
Blood.
1995;86:3118-3122
30.
Limpens J, Stad R, Vos C, et al.
Lymphoma-associated translocation t(14;18) in blood B cells of normal individuals.
Blood.
1995;85:2528-2536
31.
Liu Y, Hernandez AM, Shibata D, Cortopassi GA.
BCL2 translocation frequency rises with age in humans.
Proc Natl Acad Sci U S A.
1994;91:8910-8914
32.
Ji W, Qu GZ, Ye P, Zhang XY, Halabi S, Ehrlich M.
Frequent detection of bcl-2/JH translocations in human blood and organ samples by a quantitative polymerase chain reaction assay.
Cancer Res.
1995;55:2876-2882
33.
Dolken G, Illerhaus G, Hirt C, Mertelsmann R.
BCL-2/JH rearrangements in circulating B cells of healthy blood donors and patients with nonmalignant diseases.
J Clin Oncol.
1996;14:1333-1344
34.
Schnittger S, Wormann B, Hiddemann W, Griesinger F.
Partial tandem duplications of the MLL gene are detectable in peripheral blood and bone marrow of nearly all healthy donors.
Blood.
1998;92:1728-1734
35.
Ford AM, Bennett CA, Price CM, Bruin MCA, Van Wering ER, Greaves M.
Fetal origins of the TEL-AML1 fusion gene in identical twins with leukemia.
Proc Natl Acad Sci U S A.
1998;95:4584-4588 36. Greaves M. Molecular genetics, natural history and the demise of childhood leukaemia. Eur J Cancer. 1999;35:1941-1953.
© 2001 by The American Society of Hematology.
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Abstract
Introduction
) indicates cells
treated with serum starvation; salicylic acid, cells treated with
salicylic acid as described in "Materials and methods." (B)
Representative results of LM-PCR for BCR,
