[CANCER RESEARCH 52, 4534-4539, August 15, 1992] Restricted Oncogenicity of BCR/ABL pl90 in Transgenic Mice1 Jan Willem Voncken, Stephen Griffiths, Melvyn F. Greaves, Paul K. Pattengale, Nora Heisterkamp, and John Groffen2 Section of Molecular Diagnosis ¡J.W. V., N. H., J. G.J and Section of Hematopathology [P. K. P.], Department of Pathology, Childrens Hospital of Los Angeles, Los Angeles, California 90027, and Leukemia Research Fund Centre at the Institute for Cancer Research, Chester Beany Laboratories, 237 Fulham Road, London SW3 6JB, United Kingdom fS. G., M. F. G.] ABSTRACT A chimeric BCR/ABL oncogene encoding the pl90 protein has been introduced into the mouse germline using microinjection of one-cell fertilized eggs. Founder and progeny transgenic animals, when becom ing ill, were found to develop lymphoblastic leukemia/lymphoma which was transplantable to compatible recipients. Lymphoblasts were ar rested at the pre-B stage of development. Expression of BCR/ABL was not detected in peripheral blood during the early stages of leukemia but became evident as the disease progressed. However, the transgene was expressed early in development in bone marrow and was also tran scribed in nonhematopoietic tissues although this did not result in tu- morigenesis. These results strongly suggest that the oncogenicity of BCR/ABL is limited to hematopoietic cells, including pre-B cells or their progenitors. INTRODUCTION The Ph chromosome, the result of a reciprocal translocation between chromosomes 22 and 9 (1-3), is specifically associated with CML3 and is also found in a percentage of adult and pediatrie patients with ALL (4, 5). Due to the translocation, the ABL protooncogene from chro mosome 9 is fused to the BCR gene on chromosome 22 and together they form a chimeric gene. The BCR gene contributes promoter and 5' exons, and the ABL gene contributes its 3' exons. The activity of the ABL gene product, normally a ty- rosine kinase with a low level of autophosphorylation activity, is substantially increased in the BCR/ABL protein. Moreover, the fusion of parts of the BCR gene with the ABL combines two kinase activities within one protein (6, 7). The two major forms of BCR/ABL proteins, p210 and p 190, differ in the amount of BCR amino acid residues included. On a genomic level, p 190 contains only BCR gene exon 1, whereas p210 contains exon 1 as well as several other BCR exons. p 190 appears to be associ ated mainly with Ph-positive ALL, whereas p210 is found both in Ph-positive ALL and in CML (for reviews see Refs. 8-10). To investigate the role of the chimeric gene in leukemia, BCR/ABL p210 retroviral constructs have been introduced into 5-fluorouracil-treated mouse bone marrow and transplanted into lethally irradiated recipients. Different types of hemato poietic disease were found. In one study 13 of 30 recipients (43%) developed either a CML-like myeloproliferative disease, lymphoblastic leukemia, or macrophage cell type tumors within 5 months (11). In a different study recipients eventually died of hematopoietic disease involving macrophage, lymphoid, eryth- Received 4/6/92; accepted 6/9/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accord ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This study was supported by American Cancer Society Grant CD-421 and USPHS Grants CA47073 and CA50248 (N. H.), The Leukaemia Research Fund of Great Britain (M. F. G.), and the United Kingdom Coordinating Committee on Cancer Research (S. G.). 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; RT/PCR, reverse transcriptase/polymerase chain reac tions. roid, and mast cell lineages with the incidence (69-100%) of the exact type of disease being strain dependent (12). Myelomono- cytic leukemias, granulocytic leukemias, and lymphocytic leukemia/lymphomas were found with p210 constructs (13). These types of studies have tested only the oncogenic poten tial of the BCR/ABL gene in cells types limited to the hemato poietic system and even there the outcome could have been determined by selection of hematopoietic cells capable of being infected by these viruses. A different model system is created through the use of transgenic mice because the gene of interest will be present in every cell. In a previous study, we have gen erated mice transgenic for a BCR/ABL p 190 construct. Often founder mice, eight died rapidly, within 58 days of birth, of acute leukemia (the majority lymphoid and possibly some my eloid) (14). The two remaining mice were found to be chimeric and did not express the construct in their bone marrow; the unexpected early onset of the disease precluded further studies or breeding with these mice. To examine pl90-associated malignancies in more detail and over a prolonged period of time, we have now generated new founders and have established a transgenic line from one, in which the progeny reproducible develop cancer. We find that BCR/ABL expression is not restricted to bone marrow cells. Surprisingly, however, the types of malignancies found were limited to pre-B leukemias and lymphomas even in animals with prolonged survival time and over 4 generations. MATERIALS AND METHODS Transgenic Mice. Mice transgenic for the BCR/ABL pl90 construct were generated as previously described (14). Founder animals were the offspring of matings between C57BL x CBA F! animals. Transgenic progeny was the result of matings between transgenics and C57BL x CBA F! mice. FI progeny are identified by six-digit numbers. The first three digits are those of the founder of that specific line. Autopsies on terminally ill mice were performed as described previ ously (IS). Routine histology examinations included brain, heart, lung, liver, kidney, spleen, and thymus. Tissue sections were fixed in 10% formalin, 90% B5 (90% B5 = 66 g HgCl2 and 14 g NaAc/liter). Pe ripheral blood and bone marrow smears were stained with Wright- Giemsa and evaluated histologically. WBC were performed manually. Mice were diagnosed with lymphoblastic lymphoma/leukemia or ALL alone. Of all founder and progeny transgenic mice included in this study (n = 100), 50% had died within 70 days of birth and 72% had died (range, 31-199 days) of leukemia/lymphoma at an arbitrarily chosen experiment end point. Of the line established from founder 623, ani mals died of or were sacrificed with terminal disease on average 68 days after birth (range, 41-147 days). Southern Blot Analysis. DNAs were isolated from spleens and in volved lymph nodes as described (14). DNAs digested with £coRIwere run on 0.7% agarose gels, blotted to nitrocellulose, and hybridized to a 1.2-kilobase Mspl/EcoRl JM probe located immediately 3' to J1-J4. Posthybridization washings were at 0.3x standard saline-citrate, 65°C. Analysis of BCR/ABL Expression ¡nPeripheral Blood. WBC and BCR/ABL expression assays were performed regularly on peripheral blood from transgenic and control mice. Briefly, approximately 10 M'of blood were withdrawn from the tail artery using a 25-gauge needle, and 4534 Research. on September 15, 2015. © 1992 American Association for Cancer cancerres.aacrjournals.org Downloaded from