Continuous Cell Culture From a Patient With Chronic Myelogenous Leukemia. I. Propagation and Presence of Philadelphia Chromosome 1

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1 Continuous Cell Culture From a Patient With Chronic Myelogenous Leukemia. I. Propagation and Presence of Philadelphia Chromosome 1 LINDA S. LUCAS,2 JACQUELINE J. K..WHANG,3 J. H. TJIO,4 ROBERT A. MANAKER,2 anc/ VIGOR H. ZEVE,2. 5 National Cancer Institute, 6 Bethesc/a, Marylana SUMMARY-A continuous culture, designated the M-2 line, was established from white cells from the peripheral blood of a patient with chronic myelogenous leukemia. The cells grew singly and in aggregates unattached to the surfaces of the culture vessel. During logarithmic growth, the cell doubling time was about 24 houn. The maximal viable cell concentration observed was about 1 X 10 6 cells per mi. Cells could be grouped into 3 categories according to size. About 23% of the cells contained the Philadelphia chromosome without other chromosome abnormalities. Some of the remaining cens contained a small metacentric chromosome and others had chromosomes and an abnormal karyotype. Polyploidy and multiple chromosomal aberrations were in 15-20% of the cells.-j Nat Cancer Inst 37: , IN THESE and other laboratories, many attempts are being made to propagate cells derived from patients with leukemia. Continuous cultures of cells that characteristically grow in suspension have been established from human lymphomas (1-3). Similar cultures derived from the buffy coat of peripheral blood from patients with myelogenous leukemia were also successfully established, but the Philadelphia chromosome was not detected in the cells (4, 5). This paper reports the long-term propagation of cells obtained from the buffy coat of a patient with chronic myelogenous leukemia and the retention of a cell type with the Philadelphia chromosome, associated with this disease. The cell line was designated M-2. The ultrastructure of the cells and of virus particles detected in the culture will be described in another report (6). MATERIALS AND METHODS Cellular material.-about 30 cc of partially purified white blood cells was obtained by plasmapheresis of whole blood from a patient at the Clinical Center of the National Institutes of Health. This patient (M.E.S.) was a 57-yearold female with chronic myelogenous leukemia, first diagnosed in June At the time of 1 Received May 11, Viral Biology Branch, National Cancer Institute. 3 Medicine Branch, National Cancer Institute. 4 Laboratory of Experimental Pathology, National Institute of Arthritis and Metabolic Diseases. 5 The assistance of Dr. G. Rabotti is gratefully acknowledged. 6 Special Virus-Leukemia Program, National Cancer Institute, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare. 2,

2 754 LUCAS, WHANG, TJIO, MANAKER, AND ZEVE initiation of the cells into culture, the patient's peripheral white blood count was 67,700, of which 89% was neutrophils, 2 % lymphocytes, 2% monocytes, 5% eosinophils, and 2% abnormal unidentifiable cells. Of the neutrophils, 24% was mature neutrophils, 24% nonfilamented, 29% metamyelocytes, 6 % myelocytes, 5 % promyelocytes, and 1% myeloblasts. Medium.-The cells were propagated in Eagle's Minimal Essential Medium supplemented with 30% fetal calf serum (Hyland Laboratories, Los Angeles, Calif.) previously heated at 56 C for 30 minutes, containing 200 units of penicillin, 200 p.g streptomycin, 50 p.g aureomycin, and 50 J,A.g Kantrex per ml. Glutamine was added to a final concentration of 2 mm. Procedure for initiation of culture.-the white cell fraction obtained by plasmapheresis was diluted with an equal volume of culture medium and centrifuged at 500 X g for 15 minutes at 5 C. The supernatant was discarded and the upper layer of cells, the "buffy coat," was withdrawn with a premoistened capillary pipette. Two or three tenths of an m1 of packed cells was added to each of a number of screw-capped prescription bottles containing 5 m1 of medium. The cultures were incubated in a horizontal position at 37 C. Culture method.-medium changes were made after the cells were sedimented within the culture bottles by centrifugation at 200 X g for 5 minutes. Depending on ph shift, up to 50 % of the fluids was changed every 3-5 days. Cell counts.-the kinetics of cellular growth were studied by daily counts of viable cells in a hemocytometer, with dye-exclusion of trypan blue as an indicator. Morphological studies.-about 10 ml of cell suspension was removed from cultures during logarithmic growth. The cells were separated from the culture fluids by centrifugation at 500 X g for 15 minutes and resuspended in an equal volume of horse serum. Films of one drop of the cell suspension were made on glass slides, air-dried, and colored with Wright's stain. Seven weeks after initiation of the culture, samples were removed for electron microscopy and chromosome studies. Portions of the culture were stored in liquid nitrogen. For this purpose, the cells were suspended in a mixture composed of 10% dimethylsulfoxide, 30% fetal calf serum, and 60% Eagle's Minimal Essential Medium. RESULTS Five weeks after initiation of the culture, cell growth was observed by the increasing cell density and clumping of cells. Scattered fibroblastic cells were attached to the glass. Floating cells were removed and distributed to screw-capped culture tubes, and an equal volume of fresh medium was added. The tubes were placed in the 37 C incubator in stationary racks. Under these conditions, the cells began to proliferate rapidly and the culture was quickly expanded. The cells were then carried routinely in screw-capped, milk-dilution bottles. Morphology Cultures of the M-2 cell line were composed of free-floating cells, occurring both singly and in aggregates. Figure 1 shows a representative field of the cells cultured in glass bottles. Most of the cells were round and refractile; large aggregates settled rapidly to the bottom of the culture bottle. Aggregates were also in fixed and stained preparations; they usually consisted of a large cell surrounded by groups of two other sizes of cells delineated below. On the basis of size, 3 classes of cells (fig. 2) could be distinguished in fixed and stained preparations. The smallest cells, measuring approximately 10 p. in diameter, stained very intensely. The nucleus was regularly round and surrounded by an intensely basophilic thin rim of cytoplasm. One nucleolus was usually present, but was indistinct due to the density of the nucleoplasm. Medium cells approximately 15 p. in diameter contained a round nucleus with coarse chromatin and up to 5 large, irregularly shaped nucleoli. The cytoplasm was less basophilic than that of the smaller cells. A perinuclear halo was usually in these medium and larger cells. Scattered eosinophilic granules similar to those present in early promyelocytes were observed in several of the medium cells. The third type of cell had an average diameter of approximately 30 p.. The cytoplasm was less basophilic than that of the JOURNAL OF THE NATIONAL CANCER INSTITUTE

3 LEUKEMIC CELLS IN VITRO 755 smaller cells. Polykaryocytosis was frequently in the large cells, suggesting the existence of endoreduplication or endomitosis. In cultures examined during the log phase of growth, mitotic figures were noted in cells of all 3 sizes. Kinetics of Cellular ProliFeration Early observations suggested that the M-2 cell line grew rapidly. Under ideal conditions, with partial fluid replacement every 3 days, the doubling time during the log phase of growth was about 24 hours. Cultures initiated with I X 10 5 viable cells per ml multiplied at a higher rate than those initiated with 4 times as many cells. Regardless of the initial cell concentration in a culture, proliferation gradually decreased to yield a final concentration of about 1 million cells per ml. Thereafter, the percentage of viable cells gradually decreased. Observations made on films colored with Wright's stain supplemented by chromosome analysis (see table 1) indicated that the fraction of the cells representing each class remained constant over several months of culture. At present, there is no indication that cells of a specific size or karyotype will become dominant on continued propagation of the M-2 cell line. Chromosome Studies The chromosomes of cells from the patient's bone marrow, short-term blood cultures, and the continuous M-2 culture were studied. The number of metaphases analyzed per sample ranged from 50 to 137. From each sample, 100 metaphases, randomly selected, were analyzed for polyploidy and multiple aberrations, e.g., breaks, fragments, and multicentric chromosomes. The observations are presented in table 1. Figure 3 shows a metaphase representing cells from the M-2 culture with normal chromosomes of the group A metaphase with the Philadelphia chromosome is illustrated in figure 4. This cell is comparable in all respects to the pathologic cell found in patients with chronic myelogenous leukemia. The M-2 culture also contained an abnormal cell not observed in the marrow and short-term blood cultures. This cell had 46 chromosomes with a small metacentric chromosome of the group (fig. 5). The number of the polyploid cells and cells with multiple aberrations was considerably higher in the M-2 culture as compared with bone marrow and short-term blood cultures. REFERENCES TABLE I.-Results of chromosome studies (1) EpSTEIN, M. A., and BARR, Y. M.: Characteristics and mode of growth of a tissue culture strain (EB1) of human lymphoblasts from Burkitt's lymphoma. J Nat Cancer Inst 34: , (2) PuLVERTAFT, R. J. V.: A study of malignant tumours in Nigeria by short-term tissue culture. J Clin Path 18: , Percentage of cells with: 46 chromosomes Sample Days in chromoculture somes Percentage in 100 metaphases PhI with PhI + PhI - small Polyploid Multiple normal normal meta- PhI aberrations centric* abnormalt Bone marrow... None Bone marrow... None Peripheral bloodt Peripheral bloodt Peripheral blood Peripheral blood *Small metacentric chromosome of the size twith variable karyotypes. ~Short-term blood culture. Continuous long-term culture (M-2). VOL. 37, No.6, DECEMBER 1966

4 756 LUCAS, WHANG, TJIO, MANAKER, AND ZEVE (3) O'CONOR, G. T., and RABSON, A. S.: Herpes-like particles in an American lymphoma: Preliminary note. J Nat Cancer lnst 35: , (4) IWAKATA, S., and GRACE, J. T., JR.: Cultivation in vitro of myeloblasts from human leukemia. New York J Med 64: , (5) MOORE, G. E., ITO, E., ULRICH, K., and SANDBERG, A. A.: Culture of human leukemia cells. Cancer 19: , (6) ZEVE, V. H., LUCAS, L. S., and MANAKER, R. A.: Continuous cell culture from a patient with chronic myelogenous leukemia. II. Detection of a herpeslike virus by electron microscopy. J Nat Cancer lnst 37: , 1966.

5 JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 37 PLATE 103,. 2 FIGURE 1.-M-2 cells in continuous suspension culture. Aggregates and single cells are present. Approximately X 110 FIGURE 2.-Air-dried smear of M-2 cells. Note small, medium, and large cells. Wright's stain. X 1,000 LUCAS, WHANG, TJIO, MANAK,ER, AND ZEVE

6 PLATE 104 JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL FIGURE 3.-Metaphase with normal chromosomes (arrows). I \ 758 LUCAS, WHANG, TJIO, MANAKER, AND ZEVE

7 JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 37 PLATE FIGURE 4.-Metaphase with Ph I chromosome. 5.-Metaphase with FIGURE very small metacentric chromosome (SM) in group (arrows). 5 LUCAS, WHANG, TJIO, MANAKER, AND ZEVE 759