Biological Properties and Growth in SCID Mice of a New Myelogenous Leukemia Cell Line ( KBM-5 ) Derived from Chronic Myelogenous Leukemia Cells in the Blastic Phase 1

The establishment and the biological properties of a new leukemic cell line (KBM-5) derived from a patient in the blastic phase of chronic inyelogenous leukemia are described. The cells exhibited multiple copies of the Philadelphia chromosome, and a high level of p210Bcr"Ablkinase ac tivity was detected with rabbit anti-Abl and anti-Bcr (exon 3) peptide antisera. Use of specific primers and polymerase chain reaction followed by Southern blotting revealed that KBM-5 cells carried a bcr3-ABLII splice junction. While a normal BCR message was detected, no normal ABL message was found. The cells were phenotypically myeloid with monocytic differentiation. The high cloning efficiency in semisolid media was independent of the presence of exogenous colony-stimulating factors. In vitro exposure to Â¡mincers of differentiation, such as retinoic acid, dimethyl sulfoxide, or hemin, failed to influence the growth rate of the cells and their level of differentiation. KBM-5 cells are highly resistant to the antiproliferative action of recombinant aand y-interferons. Although sensitive to recombinant tumor necrosis factor a, they were completely resistant to natural killer cell action. KBM-5 cells constitutively expressed mRNA for tumor necrosis factor a but not for y-interferon, other interleukins, or hematopoietic growth factors. The KBM-5 cells that were transplanted into SCID mice manifested metastatic potential and tissue invasiveness similar to the way leukemic cells in humans do. This new KBM-5 cell line represents a helpful model for examining in vitro and in vivo modulation of the growth and properties of leukemic cells by using biological and chemotherapeutic agents. INTRODUCTION In CML,3 the long-term prognosis is poor despite recent advances in biological therapy and chemotherapy (1-3). Although a fraction of patients can be cured with allogeneic bone marrow transplantation (4), in the majority of patients, the development of a blast phase heralds a terminal stage of the disease for which there is no effective treatment (5). The cellular processes that cause such a transformation are un known. Once developed, the blastic phase of CML is highly resistant to therapy, including chemotherapy combinations and allogeneic bone marrow transplantation (4-6). To develop new treatments, an understanding of the events of blastic transformation is necessary, which is best gained through the study of in vivo and in vitro preclinical models. Studying leukemic cell Received 11/16/93; accepted 5/16/93. The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by West Fund for Drug Resistance in Leukemia (to M. B.). 2 To whom requests for reprints should be addressed, at Department of Hematology, Box 61, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd. Houston. TX 77030. 'The abbreviations used are: CML. chronic myelogenous leukemia; Ph + . positive Philadelphia chromosome; IMDM, Iscove's modification of Dulbecco's medium; PCS, fetal calf serum; MPO, myeloperoxidase; EBV, Epstein-Barr virus; PCR, polymerase chain reaction; DMSO, dimethyl sulfoxide; RA, retinoic acid; TPA, 12-O-tetradecanoylphorbol-13-acetate; DPBS, Dulbecco's phosphate-buffered saline; NK. natural killer cells; NBT. nitro-blue tetrazolium dye; BCR, breakpoint cluster region; ABL, Abelson oncogene; SCID, severe combined immunodeficiency; TNFa, tumor necrosis factor a; IFN, interferon; cDNA, complementary DNA; G-CSF, granulocyte colony-stimulating factor; AML, acute myelogenous leukemia. lines can help us to obtain this information. By manipulating these cell lines, we can study the mechanisms that regulate the proliferation and differentiation of leukemic cells as well as evaluate the response of the cells to chemotherapeutic and biological agents. Myeloid leukemic cell lines are relatively difficult to establish, and their availability is limited. It is difficult to generalize the validity of observations made from one particular cell line, because it may represent a selected subclone of leukemic cells that are able to continue growth in vitro. Most existing lines are also poorly characterized. Thus, there is a need to establish cell lines that have defined biological properties to (a) study the leukemic process and (b) test the efficacy of various treat ment approaches. Ph+ cell lines derived from patients in the CML blastic phase may be valuable for investigating transformation events, the production of autocrine and paracrine growth factors, the significance of additional chromosomal abnormalities, and the role of cellular oncogenes in leukemic transformation. Only a limited number of Ph+ cell lines have been established (7-17). Here we describe a new leukemic cell line, KBM-5, established from the peripheral blood of a patient in the blastic phase of CML. Because these cells were found to have a metastatic potential in SCID mice, their grafting in such animals creates a new human/animal model of the disease that could be used to compare in vitro with in vivo results and that could provide unique opportunities for preclinical in vivo studies on chemotherapeutic and biological agents potentially active in the blastic phase of CML. MATERIALS AND METHODS Case Report of the Patient from Whom the KBM-5 Cell Line Was Derived. A 67-year-old female with a 2-year history of Ph+ CML and a recent rapid increase in peripheral WBC presented with a hemoglobin of 6.0 g/dl, a platelet count of 188 X 103//j,l, and a WBC of 74 x 103/^il, 50% of which were blasts. The morphological and cytochemical characterization of the blasts was compatible with the myeloid blastic phase of CML. The patient was treated with 9 doses of 1-ÃŸ-o-arabinofuranosylcytosine (3 g/m2 for 2 h every 12 h). Although this treatment reduced circulating blast cells, on day 15 of therapy, the peripheral blast count rose again. At this time WBC were obtained for cultures from which the KBM-5 cell line was derived. Culture of Leukemic Cells and Establishment of the Cell Line. Mononuclear cells were separated by Hypaque-Ficoll density gradient sedimentation (1.077 g/cm3), 10 million cells were suspended in IMDM (GIBCO, New York, NY) with 25% PCS (GIBCO) and placed in 25-cm2 plastic flasks (Corning Glass Works, Corning, NY). The cultures were maintained in closed boxes at 37Â°Cin an atmosphere of 5% CO2, 12% 02, and 83% N2. After a lag period of 3 weeks, the cells resumed proliferation and were split and reseeded, initially at a cell density of 5 x 105/ml. After the tenth passage, the cell line was maintained by biweekly passages in IMDM supplemented with 15% PCS at an initial density of 3 X 105/ml, but without the addition of conditioning factors. For clonogenic assays, the cells were cloned in IMDM supplemented with 15% PCS and 0.3% agar (Bacto Difco Agar) and plated in 35-mm Petri dishes at 1 X IO4 cells/ml/dish. After incubation for 7-8 days in a fully humidified atmosphere of 5% CO2, 12% 02, and 83% N2, clones of more than 50 cells were counted using an inverted microscope.