Evolution of large extrachromosomal elements in HL-60 cells during culture and the associated phenotype alterations.

In the HL-60 sublines that were isolated after a long-term continuous culture, abnormally stained or abnormally banded regions on chromosomes replaced extrachromosomal double minutes. The c-MYC gene is amplified in these structures. We followed the c-MYC gene loci during a consecutive passage by using FISH, and have found a large extrachromosomal element (LEE) that preexisted at the earliest passage in a very small fraction of cells. No chromosomal integration of c-MYC sequences was observed in up to 225 passages. The LEEs persistently evolved during culture and were not excluded from the nucleus. In the LEE-positive cells, the spontaneous differentiation was blocked and the granulocytic differentiation that was induced by treatment with dimethyl sulfoxide was reversed by withdrawal of the drug. The c-MYC gene integration into LEEs is unlikely to lead to these phenotypes. The reversibility might be related to the reversible c-MYC down-regulation during the early phase of the drug treatment of HL-60 cells at early cell passages.     

Granulocytic differentiation of HL-60 cells, both spontaneous and drug-induced, might require loss of extrachromosomal DNA encoding a gene(s) not c-MYC.

Treatment of HL-60 cells with drugs induces granulocytic differentiation and c-MYC down-regulation that is irreversible and associated with loss of DNase I-hypersensitive sites in c-MYC promoter. The expression of these phenotypes requires a slow process that appears to accompany a loss of c-MYC copies in double minutes via micronuclei. However, the drug treatment induced c-MYC down-regulation very early, though only reversibly. Here we show that we can resolve this paradox by assuming a gene(s) in other extrachromosomal, acentromeric DNA. Treatment with drugs might induce no down-regulation of this gene, but its complete elimination via micronuclei might be necessary and sufficient for the expression of the above phenotypes. Loss of c-MYC copies is unavoidable because the exclusion of extrachromosomal DNAs via micronuclei is at random. This conclusion is based on the observation of a substantial number of c-MYC copies in certain differentiated cells, irrespective of whether the differentiation was induced with drugs or without.     

Nuclear structure and gene activity in human differentiated cells

The nuclear arrangement of the ABL, c-MYC, and RB1 genes was quantitatively investigated in human undifferentiated HL-60 cells and in a terminally differentiated population of human granulocytes. The ABL gene was expressed in both cell types, the c-MYC gene was active in HL-60 cells and down-regulated in granulocytes, and expression of the RB1 gene was undetectable in HL-60 cells but up-regulated in granulocytes. The distances of these genes to the nuclear center (membrane), to the center of the corresponding chromosome territory, and to the nearest centromere were determined. During granulopoesis, the majority of selected genetic structures were repositioned closer to the nuclear periphery. The nuclear reposition of the genes studied did not correlate with the changes of their expression. In both cell types, the c-MYC and RB1 genes were located at the periphery of the chromosome territories regardless of their activity. The centromeres of chromosomes 8 and 13 were always positioned more centrally within the chromosome territory than the studied genes. Close spatial proximity of the c-MYC and RB1 genes with centromeric heterochromatin, forming the chromocenters, correlated with gene activity, although the nearest chromocenter of the silenced RB1 gene did not involve centromeric heterochromatin of chromosome 13 where the given gene is localized. In addition, the role of heterochromatin in gene silencing was studied in retinoblastoma cells. In these differentiated tumor cells, one copy of the RB1 gene was positioned near the heterochromatic chromosome X, and reduced RB1 gene activity was observed. In the experiments presented here, we provide evidence that the regulation of gene activity during important cellular processes such as differentiation or carcinogenesis may be realized through heterochromatin-mediated gene silencing.     

Double-minute chromatin bodies in HL-60 leukemia cells sensitive and resistant to differentiation inducing agents

We studied the chromosome characteristics of HL-60 promyelocytic leukemia cells sensitive and resistant to differentiation inducing agents (DI). The karyotypic analysis of sensitive (HL-60 S) and resistant (HL-60 R) cells revealed the presence of identical chromosome abnormalities such as loss of chromosomes 5, 9, 10, 14, 16, 17 and X; and gain of chromosome 18. HL-60 S and HL-60 R cells also share five common markers. The difference between the two cell lines consisted essentially of the loss of an unidentifiable chromosome segment in the HL-60 R cell line. In addition, the two sublines showed marked differences in the content of double-minute chromatin bodies (DM), which were abundant in HL-60 S but rarely found in HL-60 R cells. Contrary to a previous report by others, there was no evidence of chromosome rearrangement of the DM as homogeneously stained regions (HSR) or abnormally banding regions (ABR) in the resistant HL-60 R cells. The presence of DM as an expression of gene amplification may be of relevance in the determination of susceptibility of HL-60 cells to DI.     

Alterations in insulin receptor kinase activity during differentiation of HL-60 cells

Differentiation of the human promyelocytic leukemia cell line HL-60 into monocytes or macrophages is associated with increased expression of cell surface insulin receptors, while differentiation of these cells into granulocytes is associated with receptor loss. Here we demonstrate that differentiation of HL-60 cells into monocytes or granulocytes induced by 1;25(OH)2vitD3 or Bt2cAMP, respectively, has no major effect on the specific activity of the insulin receptor kinase (IRK). By contrast, when HL-60 cells are incubated with a combination of 1;25(OH)2vitD3 and Bt2cAMP, their differentiation into adherent macrophages-like cells is accompanied by a 50% reduction in the specific activity of IRK. These findings suggest that acquisition or loss of insulin receptors during differentiation of HL-60 involves selective alterations in the functional aspects of these receptors. Our results also implicate the generation of specific regulatory signals that inhibit IRK activity when HL-60 cells are stimulated with a combination of 1;25(OH)2vitD3 and Bt2cAMP.     

C/EBPalpha inactivation in FAK-overexpressed HL-60 cells impairs cell differentiation

We previously demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as oxidative stress, ionizing radiation and TNF-receptor-induced ligand (TRAIL) compared with vector-transfected (HL-60/Vect) cells. Here, we show that HL-60/FAK cells are highly resistant to all-trans retinoic acid (ATRA)-induced differentiation, whereas original HL-60 or HL-60/Vect cells are sensitive. Treatment with ATRA at 1 muM for 5 days markedly inhibited the proliferation and increased the expression of differentiation markers (CD38, CD11b) in HL-60/Vect cells, but showed no such effect in HL-60/FAK cells. Electrophoretic mobility shift assay (EMSA) using an oligonucleotide for the c/EBP consensus binding sequence showed that c/EBPalpha was activated in ATRA-treated HL-60/Vect cells but not in HL-60/FAK cells, indicating that c/EBPalpha activation by ATRA was impaired in HL-60/FAK cells. In addition, the association of retinoblastoma protein (pRb) and c/EBPalpha after treatment with ATRA was seen in HL-60/Vect cells but not in HL-60/FAK cells. Further, hyperphosphorylation of pRb was observed in HL-60/FAK cells. Finally, the introduction of FAK siRNA into HL-60/FAK cells resulted in the recovery of sensitivity to ATRA-induced differentiation, confirming that the inhibition of HL-60/FAK differentiation resulted from both the induction of pRb hyperphosphorylation and the inhibition of association of pRb and c/EBPalpha.     

Disparate effects of activators of protein kinase C on HL-60 promyelocytic leukemia cell differentiation

In previously published studies (Kreutter, D., Caldwell, A. B., and Morin, M. J. (1985) J. Biol. Chem. 260, 5979-5984), we demonstrated that the activation of the calcium- and phospholipid-dependent protein kinase C by phorbol esters was dissociable from the induction of monocytic differentiation by these agents in HL-60 promyelocytic leukemia cells. We have now compared the effects of two related diterpenes (mezerein and 12-O-tetradecanoylphorbol-13-acetate) and two cell-permeable diacylglycerols (1-oleoyl-2-acetoylglycerol and 1,2-dioctanoylglycerol) on the induction of differentiation in HL-60 cells. Each of these agents activated protein kinase C in vitro and stimulated the phosphorylation of a number of identical proteins in intact HL-60 cells. Exposure to either of the diterpenes at nanomolar concentrations resulted in an inhibition of cell growth and the induction of qualitatively distinct types of monocytic maturation in HL-60 cells. Conversely, neither of the two diacylglycerols was found to be a potent or efficacious inducer of differentiation, as measured by increases in cell adhesion, nonspecific esterase activity, or phagocytosis, even at growth-inhibitory concentrations. However, concurrent exposure of HL-60 cells to both 1,2-dioctanoylglycerol and the calcium ionophore A23187, at concentrations which were without maturational activity when used separately, resulted in measurable increases in both protein phosphorylation and in the fraction of cells expressing a differentiated phenotype. Taken together, these results suggest that specific biochemical effects associated with 12-O-tetradecanoylphorbol-13-acetate, in addition to the activation of protein kinase C, may be important determinants for the induction of leukemia cell differentiation.     

Regulated expression of the Mac-1, LFA-1, p150,95 glycoprotein family during leukocyte differentiation

The regulation of Mac-1, LFA-1, and p150,95 expression during leukocyte differentiation was examined. LFA-1 was present on almost all cell types studied. Both Mac-1 and p150,95 were present on the more mature cells of the myelomonocytic series, but only p150,95 was detected on some B cell lines and cloned cytotoxic T lymphocytes. Phorbol myristate acetate (PMA) stimulation of B chronic lymphocytic leukemia cells dramatically increased p150,95 expression. The resultant Mac-1, LFA-1, p150,95 phenotype resembled hairy cell leukemia, a B cell plasmacytoid leukemia. The promonocytic cell line U937 and the promyeloblastic cell line HL-60 expressed only LFA-1. Monocytic differentiation of U937 cells was stimulated by PMA, and induced the concomitant expression of Mac-1 and p150,95, with more p150,95 induced than Mac-1. Granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulation of U937 cells gave similar results. PMA-stimulated monocytic differentiation of the HL-60 cell line also induced expression of both Mac-1 and p150,95. The number of p150,95 molecules on PMA-stimulated U937 and HL-60 cells were 5 X 10(5) and 3 X 10(5), respectively. Retinoic acid stimulated myeloid differentiation of HL-60 cells and induced expression of both Mac-1 and p150,95. These cells acquired a Mac-1, LFA-1, p150,95 profile that resembled that of granulocytes, with more Mac-1 than p150,95 induced. GM-CSF stimulation of HL-60 cells induced a similar Mac-1 and p150,95 phenotype. The contributions of Mac-1, LFA-1, and p150,95 to aggregation of PMA-differentiated U937 cells were assessed. Monoclonal antibodies to the beta subunit and the LFA-1 alpha subunit, but not those to p150,95 or Mac-1 alpha subunit, inhibited this homotypic adherence.     

Sodium Valproate Facilitates the Propagation of Granulocytic Ehrlichiae (Anaplasma phagocytophilum) in HL-60 Cells

As already shown, some inducers of the differentiation of promyelocytic cells along the granulocytic pathway, such as dimethylsulphoxide (DMSO) or all-trans retinoic acid, can enhance propagation of granulocytic ehrlichiae in HL-60 cell cultures. This study was conducted to prove whether sodium valproate, a salt of di-n-propylacetic acid (VPA) known to trigger cellular differentiation in several solid and hematopoietic malignancies is similarly efficient in ehrlichial cultures. Two cell lines derived from HL-60, that is, low-passage undifferentiated HL-60 (HL-60F) and high-passage HL-60 spontaneously differentiated towards monocytic phenotype (HL-60J) were grown in RPMI 1640 medium supplemented with 10% FBS. The respective HL-60F and HL-60J IC₅₀-values for NaVPA were estimated to be 0.8 and 2.2 mM under these culture conditions; to stimulate the differentiation, the respective doses of 0.3 and 1.2 mM were then applied. When the NaVPA-treated cells of both lines were challenged with an ehrlichial laboratory strain (HGE), maintained in splenectomized NMRI mice, the respective 1–2 and ≤0.1% primary infection rates in HL-60F and HL-60J cultures were observed 3 days post-inoculation. In comparison, only rare (≤0.1%) infected HL-60F and no infected HL-60J cells were recorded under the same experimental conditions in untreated control cultures. HGE continuously propagated in NaVPA-supplemented HL-60F cultures remained infectious to mice at least up to the 95th passage (12 months). NaVPA can thus facilitated continuous propagation of granulocytic ehrlichiae in cell cultures without a substantial loss of infectiveness. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of immunoglobulin lambda light chain by the promyelocytic cell line HL-60

The HL-60 cell line, established from a patient with acute promyelocytic leukemia, can be induced to undergo differentiation along the granulocyte or monocyte/macrophage line, depending on the particular inducer that is used. In this communication we provide evidence that HL-60 cells also have B lymphoid characteristics because by flow cytometry and clonal excess calculations, these cells are found to express immunoglobulin (Ig) lambda light chains on their surface. Furthermore, HL-60 cells contain poly(A)+ RNA that hybridizes with a DNA fragment encoding the constant region of Ig lambda chains and comigrates with lambda mRNA on RNA blots. Treatment of HL-60 cells with a phorbol ester that induces monocyte/macrophage differentiation resulted in the loss of surface Ig lambda chains and lambda RNA.     

Low-dose ionizing radiation effects on differentiation of HL-60 cells

The biological effects of low-dose radiation have attracted attention, but data are currently insufficient to fully understand the beneficial role of the phenomenon. In the present study, we have investigated the effects of low doses of gamma-irradiation alone and in combination with all-trans-retinoic acid (RA) on proliferation, apoptosis and differentiation of the human promyelocytic leukemia HL-60 cells. Changes in cell behavior and protein expression were determined with the use of light and fluorescent microscopy, immunocytochemical and Western blot analysis. Low-dose irradiation with 1–100 cGy caused a dose-dependent inhibition of HL-60 cell proliferation, and induced apoptosis and differentiation to granulocytes with an increase in the number of CD15-positive cells. Pre-irradiation with 1–100 cGy for 24 h before treatment with RA promoted apoptosis but did not impair RA-induced differentiation. Both processes were associated with a decrease in the expression of the proliferating cell nuclear antigen (PCNA), BCL-2, c-MYC, and changes in both cytosolic and nuclear levels of protein tyrosine-phosphorylation as well as protein kinase C alpha or beta isoforms. These results demonstrate the beneficial role of low-dose irradiation in modulating leukemia cell proliferation, differentiation and apoptosis.     

Barbiturates enhance retinoic acid or 1,25-dihydroxyvitamin D3-induced differentiation of leukemia HL-60 cells.

In the presence of 1 nM retinoic acid (RA), pentobarbital markedly enhanced differentiation of HL-60 cells to granulocytic cells. In the absence of RA, pentobarbital by itself did not induce cell differentiation. Similarly, pentobarbital enhanced the action of 1,25-dihydroxyvitamin D3 to induce differentiation of HL-60 cells into monocyte/macrophage lineage. The potency of various barbiturates to enhance cell differentiation was closely correlated with their activity to inhibit protein kinase C of HL-60 cells. In contrast to staurosporine, however, barbiturates did not affect the action of differentiation inducers of other types such as dimethyl sulfoxide, dibutyryl cyclic AMP or actinomycin D.     

Altered expression profiles of microRNAs during TPA-induced differentiation of HL-60 cells

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that regulate gene expression through translational repression by base-pairing with partially complementary mRNAs. The expression of a set of miRNAs is known to be regulated developmentally and spatially, and is involved in differentiation or cell proliferation in several organisms. However, the expression profiles of human miRNAs during cell differentiation remain largely unknown. In an effort to expand our knowledge of human miRNAs, we investigated miRNAs during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of human leukemia cells (HL-60) into monocyte/macrophage-like cells. Several hundred RNAs ranging from 18 to 26 nucleotides were isolated from HL-60 cells with or without TPA-induction, and subsequently characterized by sequencing, database searching, and expression profiling. By removing non-miRNA sequences, we found three novel and 38 known miRNAs expressed in HL-60 cells. These miRNAs could be further classified into subsets of miRNAs that responded differently following TPA induction, either being up-regulated or down-regulated, suggesting the importance of regulated gene expression via miRNAs in the differentiation of HL-60 cells.     

Expression of heat shock genes during differentiation of mammalian osteoblasts and promyelocytic leukemia cells.

The progressive differentiation of both normal rat osteoblasts and HL-60 promyelocytic leukemia cells involves the sequential expression of specific genes encoding proteins that are characteristic of their respective developing cellular phenotypes. In addition to the selective expression of various phenotype marker genes, several members of the heat shock gene family exhibit differential expression throughout the developmental sequence of these two cell types. As determined by steady state mRNA levels, in both osteoblasts and HL-60 cells expression of hsp27, hsp60, hsp70, hsp89 alpha, and hsp89 beta may be associated with the modifications in gene expression and cellular architecture that occur during differentiation. In both differentiation systems, the expression of hsp27 mRNA shows a 2.5-fold increase with the down-regulation of proliferation while hsp60 mRNA levels are maximal during active proliferation and subsequently decline post-proliferatively. mRNA expression of two members of the hsp90 family decreases with the shutdown of proliferation, with a parallel relationship between hsp89 alpha mRNA levels and proliferation in osteoblasts and a delay in down-regulation of hsp89 alpha mRNA levels in HL-60 cells and of hsp89 beta mRNA in both systems. Hsp70 mRNA rapidly increases, almost twofold, as proliferation decreases in HL-60 cells but during osteoblast growth and differentiation was only minimally detectable and showed no significant changes. Although the presence of the various hsp mRNA species is maintained at some level throughout the developmental sequence of both osteoblasts and HL-60 cells, changes in the extent to which the heat shock genes are expressed occur primarily in association with the decline of proliferative activity. The observed differences in patterns of expression for the various heat shock genes are consistent with involvement in mediating a series of regulatory events functionally related to the control of both cell growth and differentiation.     

Differentiation of human monocytic cell lines confers susceptibility to Bacillus anthracis lethal toxin

Anthrax lethal toxin (LT) is comprised of protective antigen and lethal factor. Lethal factor enters mammalian cells in a protective antigen-dependent process and cleaves mitogen-activated protein kinase kinases. Although LT has no observable effect on many cell types, it causes necrosis in macrophages derived from certain mouse strains and apoptosis in activated mouse macrophages. In this study, we observed that LT treatment of three different human monocytic cell lines U-937, HL-60 and THP-1 did not induce cell death. Cells did become susceptible to the toxin, however, after differentiation into a macrophage-like state. Treatment with LT resulted in decreased phosphorylation of p38, ERK1/2 and JNK in both undifferentiated and differentiated HL-60 cells, suggesting that the change in susceptibility does not result from differences in toxin delivery or substrate cleavage. Death of differentiated HL-60 cells was accompanied by chromosome condensation and DNA fragmentation, but was not inhibited by the pan-caspase inhibitor Z-VAD-FMK. In addition, we observed that the macrophage differentiation process could be inhibited by LT. Our results indicate that LT-mediated death of mouse and human macrophages may occur through distinct processes and that the differentiation state of human cells can determine susceptibility or resistance to LT.     

Optimization of nb-4 and hl-60 differentiation for use in opsonophagocytosis assays

Production of effective vaccine formulations is dependent on the availability of assays for the measurement of protective immune responses. The development and standardization of in vitro human cell-based assays for functional opsonophagocytic antibodies require critical evaluation and optimization of the preparation of cells for the assay. We report evaluation of a number of protocols with two continuous cell lines (NB-4 and HL-60) for the provision of differentiated cells for use in functional assays. Flow cytometric analysis of CD11b antigen expression, as a marker of differentiation, indicated that all-trans-retinoic acid (ATRA) gave improved differentiation (>80% of cells differentiated at 96 h) when compared with dimethylformamide (DMF) (<60% of cells differentiated at 96 h). Morphological changes during differentiation toward a neutrophil-like phenotype were assessed by scanning electron microscopy. HL-60 and NB-4 cells treated with ATRA showed more spreading and flattening than cells treated with DMF, further evidence that they may have achieved a more differentiated phenotype. The number of cell divisions in culture appeared to be critical because cell lines maintained in exponential growth for >40 passages failed to express CD11b antigen or show morphological changes associated with differentiation after exposure to either differentiation-inducing reagent. Late-passage cells also demonstrated increased tolerance to DMF. Our results indicated that ATRA supplemented with vitamin D(3) and granulocyte colony-stimulating factor affords robust, rapid, and reproducible differentiation of both cell types.     

Activation of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase during the differentiation of HL-60 cells induced by 12-O-tetradecanoylphorbol-13-acetate

We previously reported that the synthesis of NeuAc(alpha 2-3)Gal(beta 1-4)GlcCer (GM3) ganglioside was preferentially enhanced during the differentiation of HL-60 cells into a monocyte/macrophage lineage induced by 12-O-tetradecanoylphorbol-13-O-acetate (TPA). Since exogenously added GM3 ganglioside was shown to be able to induce the differentiation of HL-60 cells into the monocyte/macrophage lineage in a synthetic medium, the functional role of the GM3 ganglioside increase during the differentiation of HL-60 cells has become the subject of much interest. In the present study, we investigated the activity of CMP-NeuAc:lactosylceramide sialyltransferase, which catalyzes the synthesis of GM3 ganglioside from lactosylceramide, in cells undergoing differentiation induced by two different reagents, TPA and 1 alpha,25-dihydroxy-vitamin D3, which induce the differentiation of HL-60 cells into the monocyte/macrophage lineage through different modes of action. We showed that the activation of CMP-NeuAc:lactosylceramide sialyltransferase and the increase in GM3 ganglioside were not related to the differentiated lineage but to the specific action of TPA, i.e. activation of protein kinase C.