[CANCER RESEARCH 58. 421-425. February I. 1<W8| Selective Induction of Leukemia-associated Fusion Genes by High-Dose Ionizing Radiation1 Michael W. N. Deininger, Shikha Bose, Joanna Gora-Tybor, Xiu-Hua Yan, John M. Goldman, and Junia V. Melo2 Leukaemia Research Fumi Centre for Adult Leukaemia. Department of Haemah>li>/;\: Royal Postgraduale Medical School, Ducane Road, London W12 ONN, United Kingdom ABSTRACT event involves the acquisition of the genetic abnormality whose "success" in the production of a leukemic phenotype will depend on There is strong clinical and epidemiológica! evidence that ionizing its capacity to impart to the target cell a proliferative and/or survival radiation can cause leukemia by inducing DNA damage. This crucial advantage over its normal neighbors. In molecular terms, the gener initiation event is believed to be the result of random DNA breakage and misrepair, whereas the subsequent steps, promotion and progression, ation of a potentially successful reciprocal chromosomal translocation must rely on mechanisms of selective pressure to provide the expanding requires that: (a) at least two independent DNA DSBs occur, one in leukemic population with its proliferative/renewal advantage. To investi each chromosome partner; (b) the two breaks occur simultaneously, gate the susceptibility of human cells to external agents at the genetic i.e., within the same cell cycle, so that the two ends of one broken recombination stage of leukemogenesis, we subjected two hematopoietic chromosome are available to interact and be ligated (misrepaired) to cell lines, KG1 and III.6(1, to high doses of y-irradiation. The irradiation the respective complementary broken ends of the other chromosome; induced the formation of fusion genes characteristic of leukemia in both and (c) the recombination observes the polarity of the DNA molecule. cell lines, but at a much higher frequency in M.I than in 111,6».In KG1 Various physical and chemical mutagens are known to induce DNA cells, the AML1-ETO hybrid gene [associated with the t(8;21) transloca DSBs that may ultimately be misrepaired in the form of reciprocal tion of acute myeloid leukemia] occurred significantly more often than the BCR-ABL [associated with t(9;22) chronic myeloid leukemia) or the chromosomal translocations. IR is the most extensively studied and DEK-CAN [associated with t(6;9) acute myeloid leukemia] fusion genes. has been shown to produce DNA DSBs directly, in contrast to UV These findings support the notion that ionizing radiation can directly radiation and most chemical mutagens that induce DSBs only sec generate leukemia-specific fusion genes but emphasize the differing sus ondarily in the course of DNA replication. ceptibility of different cell populations and the differing frequency with There is no doubt that IR can cause leukemia. Most of the evidence which the various fusion genes are formed. The selectivity observed at the for this association in humans is provided by studies of people who primary level of gene fusion formation may explain at least in part the received irradiation as a consequence of medical (diagnostic X-ray differential risk for development of some but not other forms of leukemia and radiotherapy), occupational (radiology and nuclear industry) or after high-dose radiation exposure. accidental (nuclear explosion) exposure (3). However, little is known of the molecular mechanism(s) by which IR generates leukemia- INTRODUCTION specific fusion genes or of the reasons why some forms of leukemia are associated with exposure to IR, whereas others are not. It is Leukemia is the commonest neoplastic disorder of the hematopoi generally believed that in the multistep process of leukemogenesis, the etic system. Like other human malignancies, leukemia arises as a initiation step represented by DNA damage and misrepair is a random consequence of an acquired genetic change in a cell capable of clonal phenomenon, and that selectivity in the establishment of a leukemic expansion. The commonest and most extensively characterized ge phenotype operates only at the subsequent stages of promotion and netic abnormalities that cause leukemia are the chromosomal translo progression. To test this concept, we have established an experimental cations that give rise to fusion genes encoding oncogenic proteins. The classical example of this type of abnormality is found in CML,3 system in which the formation of different types of fusion genes in hematopoietic cells can be assessed independently of the biological in which a t(9;22)(q34;ql 1) chromosomal translocation ( 1) generates consequence of the individual translocation, thereby eliminating the a BCR-ABL hybrid gene, which is transcribed into a chimeric mRNA and translated into a p210BCR"ABLfusion protein with elevated tyro- proliferative advantage component of the mechanism that gives rise to the actual leukemic process in vivo. This system is similar to that used sine kinase activity and transforming abilities (2). Several other types by Ito et al. (4) in their demonstration of radiation-induced BCR-ABL of chromosomal translocations and their derived fusion genes have translocations. We show here that the generation of leukemia-associ now been identified in acute leukemia. Some types seem to be lineage ated fusion genes by high-dose IR in different cell lines varies in specific, such as the t(l;19) PBX1-E2A gene and the t(l;ll) MLL- frequency according to the specific type of gene fusion, suggesting AF1P gene of acute lymphoblastic leukemia, the t(8;21) AML1-ETO that selectivity at the basic genetic recombination level may also gene, and the t(6;9) DEK-CAN gene of AML. or even restricted to a underlie the preferential induction of some forms of leukemia by IR. defined morphological subtype, such as the t(15;17) PML-RAR-a gene of M3-AML. MATERIALS AND METHODS It is widely accepted that leukemogenesis is a multistep process, consisting of initiation, promotion, and progression. The initiation Cell Cultures. A single batch from each of the HL60 (5) and KG1 (6) cell lines was used for all of the experiments after thorough karyotypic analyses to confirm the absence of t(9;22), 1(8:21), and t(6:9) translocations. The lines Received 10/7/97; accepted 11/25/97. were cultured in RPMI 1640 supplemented wilh 109f- FCS. y-irradiation of The cosls of publication of this article were defrayed in part by the payment of page replicale l()7-cell aliquots was carried out from a ' "Cs source operating al 1.85 charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely lo indicate this fact. Gy/min 24 h after subculturing. In the initial experiments, culture aliquots of ' Supported in pan by grants from the Leukaemia Research Fund (United Kingdom) the cell lines were irradiated in parallel with fast neutrons al upprnximalely and the lit. Mildred Scheel-Stiliung fur Krebsforschung (Germany). 2 To v.hom requests for reprints should be addressed, at LRF Centre for Adult 0.75 Gy/min. produced by a Van de Graat accelerator (7). After irradiation, the Leukaemia. Department of Haematology. Royal Postgraduate Medical School. Hammer cells were reincubated at 37°C,5% CO2, and 95% humidity for 24 or 48 h smith Hospital. Ducane Road, London W12 ONN. United Kingdom. Phone: 44-181-383- before being harvested. Cell viability in cultures before and alter irradiation 2167; Fax: 44-181-742-9335; E-mail: jmelo^rpms.ac.uk. 3 The abbreviations used are: CML. chronic myeloid leukemia; AML, acute myelo- was assessed by Irypan blue exclusion. blastic leukemia; DSB, double-strand break; IR. ioni/.ing radiation; RT-PCR, reverse RT-PCR Assays. RNA extraction and random hexamer-primed cDNA Iranscription-PCR: nt. nucleotide. synthesis from each cell aliquot were done as described previously (8. 9). 421 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1998 American Association for Cancer Research. INDUCTION OF LEUKEMIA FUSION GENES BY IR Quality control of the cDNA samples was assessed by a one-step PCR RESULTS amplification of the normal ABL message from 1 fil of cDNA. The remaining 79 /J.1of cDNA from each cell aliquot were then divided into 4 X KKl-^il Experimental Design. For establishment and optimization of the replicate PCR tests for the first-step amplification of a given fusion transcript, test system, several pilot experiments were carried out with variable followed by a second-step (nested) amplification of 1 p.1 of the first-step doses of low and high linear energy transfer irradiation produced by products. Amplification of BCR-ABL and of AML1-ETO fusion transcripts y-rays and neutron particles, respectively. Other variables in these were done as described previously (10-12), with minor modifications of the experiments included the number of cells/irradiated aliquot (from thermocycling conditions for the AMLI-ETO PCRs to increase the stringency 106-10S) and the time interval between irradiation and analysis. In the and specificity of the amplification: the annealing temperature was increased basic protocol, cells from exponentially growing myeloid cell lines from 62°Cto 66°Cand from 60°Cto 64°Cfor the first- and second-step PCRs. were exposed in liquid suspension to y-rays or to neutron particles, respectively: and the number of cycles in the first step was increased from 40 returned to standard culture for 24-48 h, and harvested for RT-PCR to 45. Positive controls for these PCRs were diluted cDNA preparations from the BV173 (for BCR-ABL) and the KASUMI-1 (for AMLI-ETO) cell lines, analysis for specific fusion gene transcripts. The cell viability in these cultures 24 and 48 h after y-irradiation was, on average, 55-79% and previously standardi/.ed in our single-test diagnostic protocols for reproducible detection of one leukemia cell in IO5 nonhematopoietic cells (murine fibro- did not differ significantly between the two cell lines studied (see blasts).