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 /-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 ). 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 . The reflections of large qualitative and quantitative changes in nuclear/cytoplasmic ratio decreases, and the cytoplasm the relative abundances of many mRNAs. Colbert et al. becomes more diffuse. In parallel with these changes there (1983) analysed the mRNAs of differentiated and undifferen- occur marked changes in histochemistry, including decreased tiated HL60 cells by one-dimensional gel electrophoretic myeloperoxidase activity and the appearance of cells capable separation of the products of cell-free translations. Even this of reducing nitroblue tetrazolium (a granulocyte marker) relatively insensitive method revealed large differences in (Collins, 1987). A large number of changes in cell surface mRNA populations between induced and uninduced cells, antigens can also be detected (Graham et al., 1985), all of and between HL60 granulocytes and HL60 . them characteristic of increased maturity of the cells. In Davis et al. (1987) and Mitchell (1987) screened recombinant contrast, addition of a phorbol ester such as 12-0-tetra- cDNA libraries constructed from poly(A)' RNA isolated decanoyl-1-phorbol- 13-acetate (TPA) to cultures of HL60 from induced and uninduced HL60 cell cultures, and identi- cells causes almost all of the cells to clump and adhere to the fied recombinant clones homologous to a large number of substrate within 24h, then spread out and acquire a spindle- mRNAs that change markedly in abundance during differen- shaped morphology and prominent pseudopodia. Some tiation. A surprising observation of Davis et al. (1987) was nuclear condensation occurs, but the nuclei remain rounded. the large proportion of these clones that contained highly Histochemical changes that occur include a decrease in repeated sequences (including Alu sequences), and the even

Stem Cell

Common Precursor

(myelomonoblast?) "I IN Proliferation

Myeloblast Monoblast and

Promyelocyte Promonocyte- Differentiation MyelocyteI

Metamyelocyte Maturation

4- Granulocyte Monocyte without

Macrophage Division

Figure 1 Granulocyte and monocyte/macrophage lineages. HUMAN MYELOID CELL DIFFERENTIATION 43 higher proportion that seemed to be members of extensive NUC POL NUC POL NUC POL gene families. These observations require confirmation and A-A+A-A+A A- A+A-A+ A-A+ A-A further investigation since they clearly could be of consider- able significance in terms of the nature of the genes whose expression is differentiation-linked. Studies of the mechanisms regulating changes in the expression of individual genes during the differentiation of HL60 cells have revealed the operation of two that are novel in differentiating cells. First, the decrease in the relative abundance of c-myc mRNA following the initiation of differentiation is due to a two-phase down-regulation of transcription. In uninduced HL60 cells there is a partial block to transcript elongation at the end of exon 1 of the c-myc gene that is quickly and greatly enhanced by induction of differentiation with DMSO (Bentley & Groudine, 1986; Eick & Bornkamm, 1986; Siebenlist et al., 1988). Some time later the down-regulation is consolidated by a decrease in the rate of initiation of transcription (Siebenlist et al., 1988). The onset of this second phase is correlated with dramatic 1100 Bases o changes in chromatin structure, revealed by DNAase I hypersensitive-site analysis, and with increasing resistance to reversal of the effects of DMSO on c-myc RNA levels by removal of the inducing agent (Siebenlist et al., 1988). Down-regulation of the expression of c-myb in murine B cells appears to be due to a similar transcription elongation block (Bender et al., 1987). Second, changes in the relative abundance of one mRNA during retinoic acid-induced differentiation of HL60 cells have been ascribed to the concomitant operation of controls at transcriptional and post-transcriptional levels (Graham & Birnie, 1988). The protein encoded by this mRNA has not been identified. The rate of transcription of the gene increases during the course Uninduced RA-Induced TPA-lnduced of differentiation, yet the relative abundance of the mRNA Figure 2 Northern blot analyses of RNAs from HL60 cells. in whole cell RNA decreases. Moreover, a marked change in Nuclear (NUC) and polysomal (POL) poly(A)+ and poly(A)- the location of the bulk of the mRNA occurs following RNAs were isolated from uninduced HL60 cells and from HL60 differentiation. In uninduced cells most of the gene tran- cells induced with retinoic acid (RA) for 5 days or with TPA for 3 days, size-fractionated by gel electrophoresis, blotted to nitro- scripts are located in nuclear RNA whereas, in differentiated cellulose and hybridized with pCG56 DNA labelled with 32P_ cells, almost all are polysome-associated (Figure2). These dCTP by nick translation. Amount of RNA per lane: A', 0.5,ug; data were interpreted as evidence that the expression of this A-, 10pg. (Reproduced with permission from Graham and gene is regulated by an interaction between transcriptional Birnie, 1988.) and post-transcriptional controls, the latter including nucleo- cytoplasmic transport of the mRNA and stability of the mRNA in the cytoplasm (Graham & Birnie, 1988). et al., 1988). A direct comparison of the time-courses of DMSO-induced and TPA-induced differentiation showed there to be potentially significant differences between them A role for c-myc in differentiation and/or proliferation? (Mitchell et al., 1988). Treatment of HL60 cells with DMSO Several lines of evidence suggest that the protein encoded by leads to an extremely rapid reduction in c-myc RNA to c-myc is intimately involved in controlling gene expression, undetectable levels within 1-1.5 h. Differentiation markers in particular, some of the genes that regulate differentiation are much slower to appear: for example, after 48 h only 50% and proliferation. The protein is localized in the nucleus, and of the cells are committed to terminal differentiation (as appears to be a DNA-binding protein (Persson & Leder, determined by clonogenicity) and less than 20% have the 1984; Evan & Hancock, 1985) although as yet no sequence capacity to reduce nitroblue tetrazolium (a granulocyte specificity for the binding has been found. The c-myc protein marker). On the other hand, within 24 h 80% of TPA- can act in concert with activated c-rasH to transform rat treated HL60 cells are committed to differentiate terminally, embryo fibroblasts (Land et al., 1983; Kohl & Ruley, 1987) and 90% contain a monocyte marker (non-specific esterase). and baby rat kidney cells (Kohl & Ruley, 1987). Augmented Despite this, the rate of decrease in c-myc mRNA abundance expression of normal c-myc (driven by a viral long terminal is slower than in DMSO-treated cells: after an initial, rapid repeat) is sufficient to allow transformation of rat embryo two-fold increase the abundance of c-myc RNA declines to cells with an activated c-rasH: co-expression of c-myc with undetectable levels over the next 8 h. In contrast, others have the activated oncogene results in transformation apparently reported that the rate at which c-myc RNA levels decrease because the c-myc protein reverses the c-rasH_induced inhibi- following the induction of granulocytic differentiation with tion of proliferation (Lee et al., 1985). DMSO is very slow (Filmus & Buick, 1985), much slower A causal link between c-myc expression and differentiation than that following monocytic differentiation with phorbol has been inferred from experiments which measured the rate ester (Ely et al., 1987), but in both cases the decrease at which the relative abundance of c-myc mRNA changes in parallels the appearance of differentiation markers, and the cells after they are induced to differentiate in vitro (Lachman loss of clonogenic potential (Filmus & Buick, 1985; Ely et & Skoultchi, 1984; Dean et al., 1986; Collins, 1987). Marked al., 1987). However, the rapid but transient loss of c-myc decreases in c-myc RNA levels in HL60 cells in response to mRNA that follows addition of a phorbol ester to a initiation of differentiation have been recorded in many differentiation-resistant clone of HL60 cells is not sufficient instances, though the striking biphasic response of c-myc to by itself to trigger terminal differentiation in these cells (Ely induction of differentiation in murine erythroleukaemia cells et al., 1987). (Lachman & Skoultchi, 1984) is either absent or greatly Manipulation of the abundance of c-myc protein in HL60 attenuated in HL60 cells (McCachren et al., 1986; Siebenlist cells also affects the proliferation and differentiation of these 44 G.D. BIRNIE cells (Yokoyama & Imamoto, 1987; Holt et al., 1988; and useful for studies of the nature and control of changes Wickstrom et al., 1988). About 30% of the normal amount that occur when immature cells differentiate to cells with of c-myc protein is present in clones of HL60 cells that have more mature characteristics. Moreover, there is every indica- been transfected with an SV40-promoter-driven c-myc gene tion that much information regarding the regulation of in the anti-sense orientation and subsequently selected for changes in gene expression that accompany differentiation high levels of anti-sense myc mRNA (Yokoyama & can yet be gleaned from experiments with HL60 cells. For Imamoto, 1987). These clones have a doubling time three to example, the ability to cause HL60 cells to switch lineages four times longer than the parental cells; they contain a high opens up many possibilities of exploring how the mecha- proportion of cells expressing monocyte markers though they nisms controlling the expression of lineage-specific genes are are not terminally differentiated since they are still dividing. affected by a change in commitment from one lineage to a The use of a more direct method of causing a specific and related one. However, a question of fundamental importance marked decrease in intracellular myc protein levels, which remains (McCulloch, 1987): to what extent can conclusions should obviate some at least of the uncertainties of gene from observations with HL60 cells (and other established transfer techniques, has recently been described (Holt et al., leukaemic cell lines) be extrapolated to explain the molecular 1988; Wickstrom et al., 1988). Incubation of HL60 cells with bases of normal and malignant haematopoiesis? an oligodeoxynucleotide complementary to the first 15 bases Although they display many of the features of normal of the myc protein coding sequence causes the formation of promyelocytes, HL60 cells differ from their putative normal an intracellular DNA-RNA duplex that results in a 50-90% counterparts in many respects (Parmley et al., 1987). Not decrease in the level of the cells' c-myc protein, a slowing of least of the differences is the capacity of HL60 cells to their proliferation rate (Holt et al., 1988; Wickstrom et al., differentiate to cells with many of the characteristics of 1988), and an increase in the number showing morphological monocytes/macrophages. Even if HL60 cells were derived and histochemical changes characteristic of granulopoietic from a less mature cell than originally thought (Dalton et differentiation (Holt et al., 1988), though the extent and al., 1988), their capacity to differentiate along this pathway degree of the changes are less marked than those occurring would still be difficult to explain in terms of the classical in response to induction of differentiation with DMSO. scheme of myelomonocytic differentiation (Figure 1). It is However, why HL60 cells treated with anti-sense c-myc recognised that leukaemic phenotypes are not perfect replicas oligomer (Holt et al., 1988) should differentiate differently to of normal ones, and that, for many reasons, leukaemic cells those transfected with an anti-sense c-myc gene (Yokoyama show some distortion of phenotype as compared to their & Imamoto, 1987) is a mystery. Gene transfer experiments putative normal counterparts (Greaves et al., 1986). How- with murine erythroleukaemia cells have led to a com- ever, whether the many deviations displayed by both plementary conclusion, viz., constitutive expression of c-myc undifferentiated and differentiated HL60 cells (Collins, 1987) prevents differentiation induced by DMSO (Coppola & Cole, are explicable in terms of lineage promiscuity, as defined by 1986). Greaves et al. (1986), or are manifestations of genetic In total the data from these and many other experiments aberrations arising from the leukaemic nature of the cells, clearly indicate that there is a strong link between the exacerbated by their establishment as an immortal cell line, expression of c-myc and proliferation, and that differen- is yet to be decided. This question is not only relevant to tiation also is related, though probably indirectly, to down- conclusions drawn from experiments with the HL60 cell line, regulation of this gene. However, the nature of these links is but is also appropriate to those deduced from other estab- quite unclear and the role(s) of c-myc protein in either lished cell lines. As McCulloch (1987) stated, the issue is an proliferation or differentiation remains wholly obscure. The important one for many reasons, not least being the elucida- simplest hypothesis is that c-myc protein is a member of an tion of the mechanism of lineage commitment (Brown et al., interacting group of factors that together fill some essential 1987), and of the processes underlying normal haemato- role in proliferating cells. Disruption of this interaction by poietic differentiation and the phenomenon of maturation inhibition of the synthesis of one of the factors, such as arrest that characterizes leukaemia. c-myc protein, causes the cells to stop proliferating, and this The resolution of this issue will require much detailed is sufficient to trigger differentiation in maturation-arrested analysis. In the meantime it is clearly necessary to bear this tumour cells. Thus, in cell lines such as HL60, which can be uncertainty in mind when tempted to extrapolate conclusions induced to differentiate in vitro, reducing the level of c-myc from experiments with HL60 cells to explain processes in protein below a critical threshold may initiate growth arrest normal and malignant haematopoiesis. and this, in turn, may be all that is required to initiate differentiation. This model is compatible with the obser- vation that the inhibition of proliferation of, for example, HL60 and K562 cells by inhibitors of DNA synthesis is sufficient by itself to induce these cells to differentiate terminally (Munroe et al., 1984; Bianchi-Scarra et al., 1986). It is a privilege to record my appreciation of John Paul's sustained It is also compatible with the suggestion that c-myc protein interest in, and support of, our studies of gene expression in acts as an intracellular mediator of the response of a cell to a differentiation. I am grateful to many colleagues who have collab- growth factor (Armelin et al., 1984; Kaczmarek et al., 1985). orated in this work, in particular S. Graham, M. Lacey, L. Mitchell, R. Neill, A. Sproul and R. Tindle, and to other colleagues, M. Conclusions Harper, K. Mills and K. Vass, who have contributed many stimulat- ing discussions. This work has been supported by grants from the Undoubtedly the HL60 cell line has proved to be interesting Cancer Research Campaign to the Beatson Institute.

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