Hum Genet (1984) 66 : 220-224 Synchronization of human leukemic cells: Relevance for high-resolution chromosome banding © Springer-Verlag1984 J. H. Gallo, J.V. Ordonez, G. E. Brown, and J. R. Testa Cytogenetics Section, 9-031 BRB, University of Maryland Cancer Center, 655 W. Baltimore Street, Baltimore, MD 21201, USA Summary. The cell-cycle kinetics of synchronized K562 human leukemic cells and bone marrow cells from adults with acute leukemia were studied in order to develop more reliable methods for producing increased numbers of mitoses, particu- larly those with elongated chromosomes suitable for high- resolution banding. Parameters examined included DNA con- tent, mitotic index (MI), and chromosome preparations. K562 cells synchronized with methotrexate (MTX), thymidine (Tdr), or hydroxyurea (HU) showed two-fold increases in peak MI. Optimal harvesting times after release from block were approximately 10.5, 12.5, and 14.5 h for MTX, HU, and Tdr, respectively. MTX was selected for studies with cells from patients. Cells from 7 of the 10 patients studied showed 4.4-fold increases in peak MI. The optimal harvesting time was 9.5 to 11.5 h after release from block, considerably later than the 6 h time previously assumed in studies using stimulated lympho- cytes. Cells from the three remaining patients showed no increase in MI after synchronization; and the lack of response may have been related to the high proportion of cells in G0+G 1 prior to MTX exposure. For both the K562 cell line and most patient specimens, the combination of synchronization with appropriate release times and short Colcemid exposure (10 rain) resulted in substantially improved chromosome preparations. Introduction The application of chromosome banding techniques to the study of bone marrow cells from patients with acute leukemia has resulted in the identification of specific chromosome re- arrangements of diagnostic and prognostic value, such as the t(15;17) in promyelocytic leukemia and the t(8;21) in acute myeloblastic leukemia with maturation (Rowley and Testa 1982). However, the usefulness of banding analysis has some- times been limited by the low mitotic index (MI) of leukemic cells (Brandt et al. 1975) and by the fuzzy, contracted nature of chromosomes obtained from these cells using conventional techniques (Sandberg et al. 1961). Various approaches have been used to overcome these limitations. The number of mitotic cells obtained for study can be increased by using a prolonged (1-2 h) exposure to a mitotic-arresting agent such as colchieine (Tij o and Whang 1962). Improved mitotic yield and chromosome morphology have been obtained by exposing cells to cell cycle blocking agents such as methotrexate (MTX) (Hagemeijer et a1.1979; Yunis 1981), excess thymidine (Tdr) (Viegas-Pequignot and Dutrillaux 1978), fluorodeoxyuridine (FUdr) (Webber and Garson 1983), or low temperature Offprint requests to: Joseph R. Testa (Boucher and Norman 1980) followed by release from the block and harvest at appropriate times. The application of these pro- cedures has led to the detection of chromosome abnormalities in a much higher proportion of patients with acute leukemia than had been reported previously (Yunis et al. 1981; Testa et al. 1983). The increased rate of detection appears to be due, partly, to the ability to recognize more subtle rearrangements, such as the t(9;ll) in acute monoblastic leukemia (Hagemeijer et al. 1982). Although cell synchronization procedures have the poten- tial for improving preparations for cytogenetic analysis, there has been insufficient quantitative data defining the optimal conditions for the use of this procedure. Yunis (1982) reported that the application of the MTX synchronization method to leukemic cells resulted in improvement in MI and chromo- some length in specimens from many patients. Several factors may influence the response to synchronization techniques, including efficiency of blocking by the agent, degree of toxic- ity, reversibility of the block, harvesting time, and cell type. For example, stimulated lymphocytes are usually harvested 5 to 6h after release from MTX block (Yunis et al. 1978; Camargo and Cervenka 1980), whereas leukemic cells may require at least 7 to 8 h (Webber and Garson 1983). In the present study, the K562 leukemic cell line was used as a model system to compare the synchronization produced by MTX, Tdr, and hydroxyurea (HU), which reversibly inhibit DNA synthesis (Rueckert and Mueller 1960; Xeros 1962; Sin- clair 1967). The effects of each drug on the cell-cycle were determined by measuring DNA content. Changes in the MI and chromosome length following synchronization also were quantitated. In the second part of the study, these techniques were applied to bone marrow cells from patients with acute leukemia. Materials and methods Cell culture K562 cells were derived from a patient in the acute phase of chronic granulocytic leukemia (Lozzio and Lozzio 1975). The cells were maintained in suspension culture consisting of RPMI 1640 medium supplemented with 15% fetal calf serum (Gibco Laboratories, Grand Island, NY), 2 mMglutamine, and 1% penicillin-streptomycin solution (Gibco). Cells were incu- bated at 37°C in plastic tissue culture flasks (Coming Glass Works, Corning, NY) in humidified air containing 5% CO2. All experiments were performed using cells in the exponential phase of growth. Patient cells were obtained prior to therapy from six adults with acute nonlymphoblastic leukemia (ANLL) and from four adults with acute lymphoblastic leuk-