Double Minute Chromatin Bodies and Other Chromosome Alterations in Human Myeloid HL-60 Leukemia Cells Susceptible or Resistant to Induction of Differentiation by Phorbol-12-myristate-13-acetate1

An analysis of the chromosomal karyotype of the human promyelocytic HL-60 leukemia cell line and of a number of its sublines that exhibit varying degrees of resistance to induction of differentiation by phorbol-12-myristate-13-acetate was con ducted. The HL-60 cell line and the derived sublines contained two consistent marker chromosomes [9pand t(10;13)], which suggested that they have a common and possibly clonal origin. HL-60 cells that are susceptible to phorbol-12-myristate-13acetate-induced cell differentiation contained double minute chromatin bodies. The sublines with different degrees of resistance showed a corresponding sequential reduction of double minute chromatin bodies in metaphase cells. This loss of double minute chromatin bodies was not associated with an appearance of homogeneously staining chromosomal regions. Resistant and susceptible HL-60 cells differed also in a number of other chro mosomal alterations, including gains or losses involving chro mosomes 5, 8, 11, 13, 16, and 17. Thus, it is suggested that acquisition of resistance to phorbol-12-myristate-13-acetate-induced cell differentiation in the HL-60 cells may involve one or more of the above chromosomal changes. INTRODUCTION Cultured human promyelocytic (HL-60) leukemia cells (6) can be induced to differentiate into mature cells after treatment with a number of chemical agents (5,8,12). Among these are phorbol diesters, which are known to promote tumor formation in the 2stage mouse skin carcinogenesis system (4). Recently, we have been studying a number of biochemical and cellular events associated with the differentiation induced in HL60 cells by PMA3 (11-14, 19). Cells both susceptible and resis tant to PMA-induced cell differentiation were used. The resistant cells, which exhibit varying degrees of resistance to PMA-induced cell differentiation, were obtained after successive treat ments of the HL-60 cells with increasing concentrations of PMA. A stepwise development of resistance to the cytotoxic effect of certain chemicals, particularly metabolite analogues, has been observed in other mammalian cells (3). One of the mechanisms responsible for such a resistance (e.g., methotrexate resistance) is gene amplification associated with a specific chromosomal 1Research sponsored by the Office of Health and Environmental Research, United States Department of Energy, under Contract W-7405-ENG-26 and W-31109-ENG-38 and by National Cancer Institute Contract Y01-CP-70222. 2To whom requests for reprints should be addressed. 3The abbreviations used are: PMA, phorbol-12-myristate-13-acetate (12-Otetradecanoyl-phorbol-13-acetate); HSRs, homogeneously staining regions; DM, double minute chromatin bodies. Received November 8, 1982; accepted August 17, 1983. change (2). This change involves an elongation of specific chro mosomal locations known as HSRs. Conclusive evidence for the presence of amplified genes in the HSRs was provided by the in situ hybridization technique (15, 16). However, in some cells, these authors could also localize amplified genes in extra chro mosomal chromatin bodies known as DM. It is therefore possible that susceptibility to PMA-induced cell differentiation could in volve specific chromosomal abnormalities associated with gene amplification. To investigate this possibility, we performed cytogenetic analyses in the susceptible HL-60 line and in a number of sublines that exhibit different degrees of resistance to PMAinduced cell differentiation. The chromosomal changes observed were then related to the appearance of markers of cell differen tiation after PMA treatment. MATERIALS AND METHODS Cell Culture and Differentiation. Our HL-60 leukemic cells, termed here the parent cell line, were originally provided by Dr. Robert C. Gallo (National Cancer Institute, Bethesda, Md.) and were maintained for more than 1 year in culture in Roswell Park Memorial Institute Medium 1640 supplemented with 20% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 ^g/ml). Cells were seeded at 2 x 106 cells/100-mm Retri dish 24 hr prior to treatment with PMA. Sublines that showed resistance to PMA-induced cell differentiation were coded by a number corresponding to the frequency of times the cells were cultured in the presence of increasing concentrations of PMA during the selection process (e.g., R-7, R-13, R-18, R-35, R-47, and R-50). Determinations of differentiation markers were performed as described previously (12, 13). The various resistant HL-60 sublines were subcultured for at least 3 to 4 weeks in the absence of PMA prior to their use in these studies. Cytogenetic Analysis. Exponentially growing cultures were harvested with 0.075 M KCI and fixed with Carney's fixatives. Air-dried cytological preparations were prepared and either stained with Giemsa and analyzed for distribution of chromosomes and DM or treated with trypsin-Giemsa to determine the specific chromosomal alterations (18). RESULTS The HL-60 parent line and the various sublines yielded similar cell densities after 3 days of incubation (6.8 to 9.0 x 106 cells/ dish). The lysozyme activities were also similar (2 to 4 //g equivalents/107 cells), and the cultures contained a similar frac tion of morphologically mature cells (9 to 15%) (Table 1). Incubation of the HL-60 cells and R-7, R-13, and R-18 sublines with 3 x 10~10or 10~9 M PMA for 2 days, initiated 1 day after cell seeding, resulted in a dose-dependent decrease in the cell densities and in an increase in the percentage of morphologically mature cells. This treatment had no detectable effect on R-47 and R-50 cells and a limited effect on R-35 cells (Table 1). Cells