HL60 cells halted in G1 or S phase differentiate normally

Differentiating agents regulate the proliferation and myeloid maturation of HL60 cells by mechanisms that are at least partly independent (Drayson et al., (2001), Exp. Cell Res. 266, 126-134). We have investigated whether halting HL60 cells in G1 or S phase influences their commitment to or maturation along the neutrophil and monocyte pathways. Early G1 and S phase cells were isolated separately by elutriation. Quinidine was used to block the cell cycle progression of G1 cells and aphidicolin to greatly retard the progression of S phase cells. Neutrophilic (in response to all-trans-retinoic acid) or monocytic (to 1 alpha,25-dihydroxyvitamin D(3)) differentiation were assessed by induction of CD11b, M-CSF receptor and CD14 expression, acquisition of granulocyte-colony stimulating factor responsiveness, capacities to phagocytose yeast and reduce nitroblue tetrazolium, and down-regulation of CD30 and transferrin receptor expression. The cell-cycle-blocked cells differentiated at normal rates, mostly without incorporating bromodeoxyuridine. These observations establish: (a) that neither transit through the cell cycle nor a cell's position in the cell cycle substantially influences execution of the neutrophilic and monocytic differentiation programs by HL60 cells; and (b) that individual HL60 cells are genuinely bipotent.     

Activation of extracellular signal-regulated kinases (ERKs) defines the first phase of 1,25-dihydroxyvitamin D3-induced differentiation of HL60 cells

Activation of ERK1 and ERK2 protein kinases has been implicated in diverse cellular processes, including the control of cell proliferation and cell differentiation (Marshall [1995] Cell 80:179). In human myeloblastoid leukemia HL60 cells rapid (ca. 15 min) but transient activation of ERK1/2 has been reported following induction of macrophage/monocyte differentiation by phorbol esters, or by very high (10(-6) M) concentrations of 1,25-dihydroxyvitamin D(3) (1,25D3), while retinoic acid-induced granulocytic differentiation was accompanied by sustained activation of ERK1/2. We report here that monocytic differentiation of HL60 cells induced by moderate (10(-9) to 10(-7) M) concentrations of 1,25D3 could be divided into at least two stages. In the first phase, which lasts 24-48 h, the cells continued in the normal cell cycle while expressing markers of monocytic phenotype, such as CD14. In the next phase the onset of G1 cell cycle block became apparent and expression of CD11b was prominent, indicating a more mature myeloid phenotype. The first phase was characterized by high levels of ERKs activated by phosphorylation, and these decreased as the cells entered the second phase, while the levels of p27/Kip1 increased at that time. Serum-starved or PD98059-treated HL60 cells had reduced growth rate and slower differentiation, but the G1 block also coincided with decreased levels of activated ERK1/2. The data suggest that the MEK/ERK pathway maintains cell proliferation during 1,25D3-induced monocytic differentiation of HL60 cells, but that ERK1/2 activity becomes suppressed during the later stages of differentiation, and the consequent G1 block leads to "terminal" differentiation.     

Elevated cyclin E levels, inactive retinoblastoma protein, and suppression of the p27(KIP1) inhibitor characterize early development of promyeloid cells into macrophages.

Cyclin-dependent kinase inhibitors such as p27(KIP1) have recently been shown to lead to cellular differentiation by causing cell cycle arrest, but it is unknown whether similar events occur in differentiating promyeloid cells. Hematopoietic progenitor cells undergo lineage-restricted differentiation, which is accompanied by expression of distinct maturation markers. Here we show that the classical growth factor insulin-like growth factor I (IGF-I) potently promotes vitamin D(3)-induced macrophage differentiation of promyeloid cells, as assessed by measurement of a coordinate increase in expression of the integrin alpha subunit CD11b, the CD14 lipopolysaccharide receptor, and the macrophage-specific esterase, alpha-naphthyl acetate esterase, as early as 24 h following initiation of terminal differentiation. Addition of IGF-I to cells undergoing vitamin D(3)-induced differentiation also leads to an early increase in expression of cyclin E, phosphorylation of the retinoblastoma tumor suppressor protein, and a doubling of the cell number. Early expression of CD11b (24 h) is simultaneously accompanied by inhibition in the expression of p27(KIP1). Cell cycle analysis with propidium iodide revealed that CD11b expression at 24 h following initiation of differentiation occurs at all phases of the cell cycle instead of only those cells arrested in G(0)/G(1). Similarly, development of a novel double-labeling intra- and extracellular flow-cytometric technique demonstrated that single cells expressing the mature leukocyte differentiation antigen CD11b can also incorporate the thymidine analog bromodeoxyuridine. Likewise, expression of the intracellular DNA polymerase delta cofactor/proliferating-cell nuclear antigen at 24 h is also simultaneously expressed with the surface marker CD11b, indicating that these cells continue to proliferate early in their differentiation program. Finally, at 24 h following induction of differentiation, IGF-I promoted a fourfold increase in the uptake of [(3)H]thymidine by purified populations of CD11b-expressing cells. Taken together, these data demonstrate that the initial steps associated with terminal macrophage differentiation occur concomitantly with progression through the cell cycle and that these very early differentiation events do not require the accumulation of p27(KIP1).     

Differing responses of G2-related genes during differentiation of HL60 cells induced by TPA or DMSO

Differentiation leads to the cessation of cellular proliferation, but little is known about the molecular mechanisms of growth arrest. We compared the effect of two differentiation inducers, 12-o-tetradecanoyl 13-acetate (TPA) and dimethyl sulfoxide (DMSO) on both the cell-cycle and the modulation of G2-related genes in synchronized HL60 cells. TPA treatment of HL60 cells resulted in G1 arrest within 24 h. In contrast, the cell cycling of DMSO-treated cells was initially accelerated and they progressed to the second cycle before accumulating in the G1 phase. Expression of cyclin B, cdc25, wee1 and cdc2 was studied during cell cycle arrest by Northern blot hybridization. Expression of cyclin B, cdc25 and cdc2 fluctuated in association with cell cycle progression towards the G2/M phase, while wee1 expression remained constant in untreated cells. These four genes were highly expressed in TPA-treated cells for the first 12 h, but drastic down-regulation was seen at 18 h and expression became undetectable after 24 h. In contrast, no remarked changes of gene expression were seen in DMSO-treated cells. These findings suggest that cell cycle progression along with the initial process of differentiation in response to TPA differs from the response to DMSO and that the down-regulation of cdc2 expression by TPA-treated HL60 cells contributes to endorsement of G1 arrest.     

1,25-dihydroxyvitamin D(3)-induced retardation of the G(2)/M traverse is associated with decreased levels of p34(cdc2) in HL60 cells.

Cellular differentiation of neoplastic cells after exposure to 1, 25-dihydroxyvitamin D(3) (1,25 D(3)) is accompanied by altered cell cycle regulation. In previous studies, blocks in both G(1)/S and G(2)/M checkpoints have been observed in 1,25D(3)-treated HL60 cells, but the mechanism of the 1,25D(3)-induced G(2)/M block has not been previously reported. In this study, we show by cell cycle analysis, using bromodeoxyuridine pulse-chase labeling, that the G(2)/M block in 1,25D(3)-treated HL60 cells is incomplete. We also demonstrate that although the 1,25D(3)-treated cells exhibit elevated levels of cyclin B1, Cdc25C, and Cdk7, which are positive regulators of the G(2)/M traverse, these cells have decreased protein levels of p34(cdc2) and decreased p34(cdc2) kinase activity. This provides potential mechanisms for the observed accumulation of cells in the G(2) cell cycle compartment and occasional polyploidization following treatment of HL60 cells with 1,25D(3). The data also suggest that the ability of some cells to traverse this block may be the result of cellular compensatory mechanisms responding to decreased p34(cdc2) activity by increasing the levels of other regulators of the G(2) traverse, such as cyclin B1, Cdc25C, and Cdk7.     

Cyclin-Dependent Kinase 5 Activity Enhances Monocytic Phenotype and Cell Cycle Traverse in 1,25-Dihydroxyvitamin D3-Treated HL60 Cells

The function of most cyclin-dependent kinases (Cdks) is to facilitate progression through the checkpoints of the cell cycle, but Cdk5 is known to be involved in differentiation of CNS, muscle, and lens cells, though not in the cell cycle traverse. Here we show an additional role for Cdk5, an enhancement of monocytic differentiation with abrogation of the G1 checkpoint. Human leukemia HL60 cells exposed to 1alpha,25-dihydroxyvitamin D3 (1,25D3) displayed monocytic phenotype and increased Cdk5 kinase activity. An analog of 1,25D3 which does not induce differentiation failed to upregulate Cdk5, and 1,25D3-resistant cells had reduced Cdk5 activity. Active or inactive Cdk5 was associated with cyclin D1, but only active Cdk5 exhibited threonine phosphorylation. Inhibition of Cdk5 expression by an antisense construct reduced the intensity of 1, 25D3-induced expression of CD14, a marker of monocytes, and increased the 1,25D3-induced G1 block. These findings demonstrate a novel aspect of Cdk5 activity-facilitation of the G1- to S-phase transition in cells which are approaching replicative quiescence and a concomitant enhancement of monocytic differentiation.     

Monocytic differentiation of HL60 cells induced by potent analogs of vitamin D3 precedes the G1/G0 phase cell cycle block

Abstract. Differentiation of mammalian cells is accompanied by reduced rates of proliferation and an exit from the cell cycle. Human leukemic cells HL60 present a widely used model of neoplastic cell differentiation, and acquire the monocytic phenotype when exposed to analogs of vitamin D₃ (VD₃). The maturation process is accompanied by two blocks in the cell cycle: an arrest in the G₁/G₀ phase, and a recently described G₂+ M block. In this study we have analyzed the traverse of the cell cycle phases of the well-differentiating HL60-G cells exposed to one of ten analogs of VD₃, and compared the cell cycle effects of each compound with its potency as a differentiation-inducing agent. We found that in general there was a good correlation between the effects of these compounds on the cell cycle and on differentiation, but the best cell cycle predictor of differentiation potency was the extent of accumulation of the cells in the G₂ compartment. All analogs induced a marked decrease in the mitotic index, and polynucleation of HL60 cells was produced, especially by compounds which were effective as inducers of differentiation. Time course studies showed that induction of differentiation was accompanied by a transient increase of the proportion of cells in the G₂+ M compartment, but preceded the G₁ to S, and the G₂ compartment blocks. These studies indicate that complex changes in the cell cycle traverse accompany, but do not precede, the acquisition of the monocytic phenotype by HL60 cells.     

pp75: A novel tyrosine-phosphorylated protein that heralds differentiation of HL-60 cells

The human promyeloid cell line HL-60 differentiates toward monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or dibutyryl cAMP, respectively. When nondifferentiated cells were incubated for 20 min with 2 mM H2O2 and 0.1 mM sodium orthovanadate to inhibit their protein-tyrosine-phosphatase activity (Heffetz, D., Bushkin, I., Dror, R., and Zick, Y. (1990) J. Biol. Chem. 265, 2896-2902), we found marked tyrosine phosphorylation of a single major protein of 53 kDa. Induction of differentiation of HL-60 cells was accompanied by the appearance of an additional major cytosolic tyrosine-phosphorylated protein of 75 kDa (pp75). In dibutyryl cAMP-treated cells, tyrosine phosphorylation of pp75 peaked after 24 h and then declined rapidly. In 1,25(OH)2D3-treated cells, increased tyrosine phosphorylation was detected as early as 2 h and peaked after 3 days, whereas the presence of differentiated phenotypes, assessed by the capacity of the cells to reduce nitro blue tetrazolium, was detected no earlier than 24 h. Doses of 1,25(OH)2D3 as low as 1 nM induced the appearance of pp75 at a stage where almost no differentiation measured by nitro blue tetrazolium reduction was detected. Phosphorylation of pp75 was not stimulated by adriamycin, which induced growth arrest without initiation of differentiation. pp75 could also be detected in U-937, a monocytic cell line that is more advanced in its differentiation state, and also in terminally differentiated circulating human monocytes treated with H2O2/vanadate. pp75 underwent in vitro tyrosine phosphorylation in cytosolic extracts derived from 1,25(OH)2D3-induced HL-60 cells, but not in extracts derived from uninduced cells. Our results raise the possibility that tyrosine phosphorylation of pp75 may be a common early event that heralds the differentiation of HL-60 cells into both the monocytic and granulocytic pathways.     

RB phosphorylation in sodium butyrate-resistant HL-60 cells: Cross-resistance to retinoic acid but not vitamin D3

To examine the potential coupling between inducible cellular changes in RB (retinoblastoma) tumor suppressor protein phosphorylation and ability to G0 growth arrest and differentiate, HL-60 promyelocytic leukemia cells were cultured in incremental sodium butyrate (NaB) concentrations and thereby made resistant to the growth inhibitory effects of sodium butyrate, which normally induces G0 arrest and monocytic differentiation in wild type HL-60 cells. The resistant cells were also unable to differentiate in response to NaB, indicating that a regulatory function controlling both G0 growth arrest and differentiation had been affected. The induced resistance was not genetic in origin since the cells regained the ability to G0 arrest and differentiate after being recultured in medium free of sodium butyrate for only three days. The resistant cells had similar cell cycle phase durations as the original wild type cells. The resistant cells retained the ability to both G0 arrest and differentiate in response to 1,25-dihydroxy vitamin D3 (VD3), normally an inducer of G0 arrest and monocytic differentiation in wild type cells. However, they were cross-resistant to retinoic acid (RA), another ligand for the same steroid thyroid hormone receptor family, which induces G0 arrest and myeloid differentiation in wild type cells. The ability to G0 arrest and phenotypically differentiate in response to RA were both grossly impaired. Unlike wild type cells which undergo early down-regulation and then hypophosphorylation of the RB protein when induced to differentiate, in resistant cells, hypophosphorylation of RB in response to NaB was grossly retarded. These changes in RB protein occurred faster when the cells were treated with VD3. In contrast, the changes in RB phosphorylation occurred significantly slower when the cells were treated with RA. The results suggest a coupling between the ability to G0 growth arrest and phenotypically convert and the ability to hypophosphorylate RB. © 1995 Wiley-Liss, Inc.     

Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis

Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment. The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM. Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis. While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction. Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells. Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest. In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation. Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively. However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells. In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases. Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate.     

Induction of differentiation of human leukemia cells by combinations of COX inhibitors and 1, 25-dihydroxyvitamin D3 involves Raf1 but not Erk 1/2 signaling

Differentiation therapy of cancer is being explored as a potential modality for treatment of myeloid leukemia, and derivatives of vitamin D are gaining prominence as agents for this form of therapy. Cyclooxygenase (COX) inhibitors have been reported to enhance 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation of promyeloblastic HL60 cells, but the mechanisms of this effect are not fully elucidated, and whether this potentiation can occur in other types of myeloid leukemia is not known. We found that combination treatment with 1,25D and non-specific COX inhibitors acetyl salicylic acid (ASA) or indomethacin can robustly potentiate differentiation of other types of human leukemia cells, i.e. U937, THP-1, and that ASA +/- 1,25D is effective in primary AML cultures. Increased cell differentiation is paralleled by arrest of the cells in the G1 phase of the cell cycle, and by increased phosphorylation of Raf1 and p90RSK1 proteins. However, there is no evidence that this increase in phosphorylation of Raf1 is transmitted through the ERK module of the MAPK signaling cascade. Transfection of small interfering (si) RNA to Raf1 decreased differentiation of U937 cells induced by a combination of ASA or indomethacin with 1,25D. However, phosphorylation levels of ERK1/2, though not of p90RSK, were increased when P-Raf1 levels were decreased by the siRNA, suggesting that in this system the ERK module does not function in the conventional manner. Identification of the strong antiproliferative activity of ASA/1,25D combinations associated with monocytic differentiation has implications for cancer chemoprevention in individuals who have a predisposition to myeloid leukemia.     

MicroRNAs181 regulate the expression of p27Kip1 in human myeloid leukemia cells induced to differentiate by 1,25-dihydroxyvitamin D3

Human myeloid leukemia cells exposed to 1,25-dihydroxyvitamin D3 (1,25D), a major cancer chemopreventive agent, acquire features of normal monocytes and arrest in the G1 phase of the cell cycle, due to the upregulation of p27Kip1 and p21Cip1, but the mechanism is not clear. Here evidence is provided that an exposure of HL60 and U937 cells to low (1-10 nM) concentrations of 1,25D decreases the expression of miR181a and miR181b in a concentration and time-dependent manner. Since the predicted miR181 targets include the 3’-UTR of p27Kip1, we expressed pre-miR181a in these cells, and found that the elevation of cellular miR181a levels abrogates the 1,25D-induced increase in p27Kip1 at both mRNA and protein levels. In contrast, transfection of pre-miR181a resulted in a slight elevation of p21Cip1 expression. Importantly, transfection of pre-miR181a blunted the effect of 1,25D on the expression of monocytic differentiation markers, and reduced the G1 block in 1,25D-treated cells, while transfection of anti-miR181a increased 1,25D-induced differentiation. Together, these data show that miR181a participates in 1,25D-induced differentiation of HL60 and U937 cells, and suggest that a high constitutive expression of members of miR181 family may contribute to the malignant phenotype in the myeloid lineage.     

The Src-Family Kinase Inhibitor PP2 Rescues Inducible Differentiation Events in Emergent Retinoic Acid-Resistant Myeloblastic Leukemia Cells

Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-trans retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47^phox expression. However, RA-treated RA-resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid’s biological effects in WT HL60 cells.     

A dual block to cell cycle progression in HL60 cells exposed to analogues of vitamin D,

Abstract. The physiologically active form of vitamin D₃, 1,25-dihydroxy-vitamin D₃, (1,25(OH)₂, D₃), induces differentiation of several types of myeloid leukaemia cells. The acquisition of monocyte-like phenotype is accompanied by slower progression through the cell cycle, and G₁, block has been reported to be the basis of this effect. It is shown here that human promyelocytic leukaemia HL60 cells treated with analogues of vitamin D₃, which are potent inducers of monocytic differentiation, have an additional cell cycle block. Exposure to 10-⁷m 1,25(OH)₂, D₃, or 1,25-(OH)₂,-16-ene-D₃ resulted in monocytic differentiation and the expected G₁, block evident at approximately 48 h in a rapidly differentiating variant of HL60 cells (HL60-G), and at 96 h in the more slowly differentiating HL60-240 cells. In addition, a G₂,+M block was noted at approximately 72 h in HL60-G and HL60-240 cells. Exposure to vitamin D₃, analogues also markedly increased the number of dikaryons, suggesting that cytokinesis was impaired more than karyokinesis. Treatment with a third analogue 25-hydroxy-16,23-diene-D₃, produced little differentiation and had minimal effects on the cell cycle parameters. These findings indicate that vitamin D₃, analogues regulate cell proliferation by control of the transition of G₁, and G₂,+M phases, reminiscent of the cdc2/CDK2 type of cell cycle control.     

Polyomavirus small t antigen prevents retinoic acid-induced retinoblastoma protein hypophosphorylation and redirects retinoic acid-induced G0 arrest and differentiation to apoptosis

Polyomavirus small t antigen (ST) impedes late features of retinoic acid (RA)-induced HL-60 myeloid differentiation as well as growth arrest, causing apoptosis instead. HL-60 cells were stably transfected with ST. ST slowed the cell cycle, retarding G2/M in particular. Treated with RA, the ST transfectants continued to proliferate and underwent apoptosis. ST also impeded the normally RA-induced hypophosphorylation of the retinoblastoma tumor suppressor protein consistent with failure of the cells to arrest growth. The RA-treated transfectants expressed CD11b, an early cell surface differentiation marker, but inducible oxidative metabolism, a later and more mature functional differentiation marker, was largely inhibited. Instead, the cells underwent apoptosis. ST affected significant known components of RA signaling that result in G0 growth arrest and differentiation in wild-type HL-60. ST increased the basal amount of activated ERK2, which normally increases when wild-type cells are treated with RA. ST caused increased RARalpha expression, which is normally down regulated in RA-treated wild-type cells. The effects of ST on RA-induced myeloid differentiation did not extend to monocytic differentiation and G0 arrest induced by 1,25-dihydroxy vitamin D3, whose receptor is also a member of the steroid-thyroid hormone superfamily. In this case, ST abolished the usually induced G0 arrest and retarded, but did not block, differentiation without inducing apoptosis, thus uncoupling growth arrest and differentiation. In sum, the data show that ST disrupted the normal RA-induced program of G0 arrest and differentiation, causing the cells to abort differentiation and undergo apoptosis.