The HL60 Cell Line: a Model System for Studying Human Myeloid Cell Differentiation G.D

Br. J. Cancer (1988), 58, Suppl. IX, 41-45 ,'. The Macmillan Press Ltd., 1988 The HL60 cell line: A model system for studying human myeloid cell differentiation G.D. Birnie The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 IBD, UK. Summary The HL60 cell line was established in 1977 from a patient with acute myeloid leukaemia. The cells largely resemble promyelocytes but can be induced to differentiate terminally in vitro. Some reagents cause HL60 cells to differentiate to granulocyte-like cells, others to monocyte/macrophage-like cells. The HL60 cell genome contains an amplified c-myc proto-oncogene; c-myc mRNA levels are correspondingly high in undifferentiated cells but decline rapidly following induction of differentiation. These features have made the HL60 cell line an attractive model for studies of human myeloid cell differentiation. This review summarizes the major properties of HL60 cells, describes some aspects of the regulation of gene expression in differentiating HL60 cells, including a novel interaction between transcriptional and post-transcriptional controls, and discusses the possible involvement of c-myc in proliferation and differentiation. The HL60 cell line originated from a female patient with been largely deleted (Wolf & Rotter, 1985), and one allele of acute myeloid leukaemia (Collins et al., 1977). The leukae- the GM-CSF gene on chromosome 5q21-q23 is rearranged mia was first diagnosed as acute promyelocytic leukaemia and partly deleted (Huebner et al., 1985). In addition, N-ras (FAB class M3), but a recent re-evaluation of the original mutated in codon 61 (Bos et al., 1984) has been rescued specimens from the patient indicated that FAB class M2 from HL60 DNA by transfection of NIH/3T3 cells (Murray (acute myeloblastic leukaemia with differentiation) would et al., 1983). However, the first genetic alteration observed in have been a more accurate description of the patient's HL60 cells (and the one generating most interest) is the disease (Dalton et al., 1988). The culture of peripheral blood amplification of DNA sequences encompassing the c-myc leukocytes from this patient in conditioned medium resulted gene (Collins & Groudine, 1982). In both early passage in the development of a growth-factor-independent immortal HL60 cells and in the peripheral blood leukocytes of the cell line with distinct myeloid characteristics (Gallagher et patient from whom the cell line was derived the c-myc gene al., 1979). Some of the properties of HL60 cells have made was amplified about 30-fold (Dalla-Favera et al., 1982). this cell line an attractive model for studies of differentiation Later, several sublines of HL60 cells in which the degree of in general, and human myeloid cell differentiation in particu- amplification of the c-myc gene differs were described lar, and an extensive literature (more than 700 papers) has (Graham et al., 1985). Morphological, histochemical and accumulated in the past 10 years. Despite this, the molecular immunological analyses (Graham et al., 1985), and clono- events underlying, and the mechanisms controlling, myeloid genic analyses (Donti et al., 1988), confirmed the identity of cell differentiation remain largely obscure. This brief review these sublines as HL60, but the extent to which the describes the salient features of the HL60 cell line and some c-myc-homologous sequences are amplified in the sublines novel aspects of the control of gene expression in differen- varies from 4-fold to 30-fold (Graham et al., 1985; Donti et tiating HL60 cells. It also attempts to summarize some of al., 1988). Moreover, the relative abundance of c-myc RNA the too often contradictory data and conflicting conclusions in the cells of each subline is directly proportional to the myc regarding the role of the c-myc proto-oncogene in differen- gene copy number (Graham et al., 1985). This led to the tiation, as deduced from studies of induced differentiation of conclusion that high levels of the c-myc gene and its HL60 cells and other established cell lines. transcript are not necessary for the maintenance of the HL60 cell phenotype. However, the possibility remains that over- expression of c-myc due to the amplification of the gene did Characteristics of HL60 cells have a role in the establishment of the HL60 cell line, a hypothesis with some attraction because of the known HL60 cells grow in suspension culture with a doubling time transforming activity of combinations of c-myc and a that can vary from 20h to 45h, depending on the subline. mutated rasH gene (Land et al., 1983; Lee et al., 1985; Kohl All sublines display a myeloblastic/promyelocytic morpho- & Ruley, 1987). logy: large, blast-like cells with characteristic large, rounded In normal cells the c-myc gene has been localized to nuclei containing 2-4 distinct nucleoli, and a basophilic chromosome 8q24 (Neel et al., 1982). In HL60 cells with a cytoplasm with azurophilic granules. Cultures of HL60 cells 30-fold amplification of the c-myc gene, the c-myc sequences comprise 90-95% of cells with this morphology; the remain- have been located in an abnormal banded region on chromo- ing cells display morphologies resembling those of more some 8q24 (Nowell et al., 1983) or on a marker chromosome mature myeloid cells (mainly myelocytes, with some neutro- (M3q+) (Wolman et al., 1985). In one of the sublines in phils and monocytes). The majority of the cells carry a which amplification of the c-myc gene is much reduced the variety of cell surface antigens characteristic of immature residual c-myc DNA is located on a novel chromosome, 4q+ myeloid cells as shown by their reaction with an extensive (Donti et al., 1988). These data emphasise, first, the mobility panel of monoclonal antibodies (Graham et al., 1985). Many of the genomic region containing the c-myc gene and, histochemical markers characteristic of myeloid lineage cells, second, the fluidity of much of the genome in the HL60 cell most notably myeloperoxidase and acid phosphatase, are line. also present, as well as receptors for insulin, transferrin and complement (see Tsiftsoglou & Robinson, 1985; Collins, 1987). Induced of HL60 cells Cytogenetic analysis of HL60 cells shows the occurrence differentiation of many karyotypic abnormalities, including monosomy, HL60 cultures comprise maturation-arrested cells many of trisomy and tetrasomy, and a variety of chromosomal trans- whose properties are similar to those of myeloblasts and locations (Wolman et al., 1985; Donti et al., 1988). On the promyelocytes. In all cultures of HL60 cells, however, the molecular genetic level it has been found, for example, that block in differentiation is spontaneously overcome in a small the p53 gene on chromosome 17pl3 (Isobe et al., 1986) has proportion of cells (5-10%) which display morphological 42 G.D. BIRNIE and other characteristics of more mature myeloid cells. The myeloperoxidase activity and the appearance of non-specific property of HL60 cells that has probably attracted most esterase activity (a monocyte/macrophage marker) in all interest is the ability of a variety of agents to increase the adherent cells. In addition, markedly elevated levels of proportion of cells differentiating in vitro to as great as 90%, lysozyme can be detected in the medium within 16-24h after and the capacity of cloned cell populations to differentiate initiation of differentiation. Changes in cell surface antigens either to granulocyte-like or to monocyte/macrophage-like characteristic of maturation along the monocyte/macrophage cells depending on the nature of the inducing agent (Fontana pathway also occur (Graham et al., 1985). et al., 1981; see Collins, 1987). Thus, polar-planar com- Some of the confusion surrounding the interpretation of pounds such as dimethyl sulphoxide (DMSO), and other data from experiments with HL60 cells undoubtedly arises compounds as diverse as retinoic acid and actinomycin D, all from a lack of uniformity in the response of HL60 cells to induce differentiation to granulocytes, while 1,25-dihydroxy- different inducers that have the same overall effect. For vitamin D3, phorbol esters and sodium butyrate induce example, our clones of cells differentiate more slowly when monocyte/macrophage differentiation. The bipotency of a induced with retinoic acid than with DMSO; moreover, a very large proportion (at least 80%) of the cells in HL60 higher proportion are resistant to induction with retinoic cultures is not in doubt (Fontana et al., 1981; Fibach et al., acid (40%) than with DMSO (10%). With other clones it 1982), but it does cause difficulty in the assigning of the has been reported that retinoic acid is just as effective an myeloblastic/promyelocytic HL60 cell to an appropriate inducer as DMSO (Breitman et al., 1980). Many other place in the classical scheme of myelomonocytic differen- qualitative and quantitative differences between inducers tiation (Figure 1), or perhaps casts doubt on some aspects of have been reported, some of which have been summarized this scheme (Fibach et al., 1982). by Collins (1987). However, the extent to which differences The course of the differentiation induced by any of these are due to differences between inducers or to differences agents is accompanied by a large number of changes in the between sublines of HL60 cells is unclear. In all but a few cells (Tsiftsoglou & Robinson, 1985; Collins, 1987), and is instances the effects of the different inducers have not been easily monitored by morphological, histochemical and compared directly on the same clone of cells. immunological criteria. Thus, incubation with DMSO or retinoic acid leads, over a period of 5 days, to a progressive decrease in the size of HL60 cells and condensation of Changes in gene expression with differentiation of HL60 cells nuclear material with the appearance of kidney-shaped As expected, the marked morphological and other changes nuclei characteristic of the myelocyte and, later, lobed nuclei seen when HL60 cells are induced to differentiate are characteristic of banded and segmented neutrophils.

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