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At least 163 records · Page 9Linked to original sources
PubMed2004

Tight junctions in differentiating ameloblasts and odontoblasts differentially express ZO-1, occludin, and claudin-1 in early odontogenesis of rat molars.

Little is known about the expression of associated proteins during the assembly of tight junctions (TJs). We studied the distribution of ZO-1, occludin, and claudin-1 between differentiating ameloblasts and odontoblasts in molar tooth germs from 1- to 3-day-old rats by confocal laser scanning microscopy. Immunoreactivity for ZO-1 was strong at proximal and distal junctional complexes of differentiating ameloblasts, while it was weak and punctuate at the distal region of differentiating odontoblasts. Occludin was immunoreactive at distal and proximal complexes of early differentiating ameloblasts and at distal regions of differentiating odontoblasts. However, in more advanced stages, occludin was only evident at the proximal complex of ameloblasts. Claudin-1 was strongly detected at the proximal complex but it was weak at distal complex of late differentiating ameloblasts. Thus, our results showed that ZO-1, occludin, and claudin-1 are differentially expressed as TJs assemble for regulating polarity and/or paracellular permeability in differentiating ameloblasts and odontoblasts.

Ameloblasts↗
PubMed1989

Differential expression of N-myc and c-src proto-oncogenes during neuronal and schwannian differentiation of human neuroblastoma cells.

Neuroblastoma (NB) cells can be induced to differentiate bidirectionally into either neuronal or schwannian cells by different inducers. However, the underlying mechanisms are poorly understood. We examined the expression of N-myc and c-src genes in 3 human NB-cell lines during either neuronal or schwannian differentiation in vitro, since proto-oncogenes are considered to play a pivotal role in regulating cell proliferation and differentiation. Decreased N-myc expression and increased c-src expression were observed during neuronal differentiation by retinoic acid, polyprenoic acid (E5166) and dibutyryl cyclic AMP, whereas the expression of N-myc and c-src genes was considerably reduced during schwannian differentiation by bromodeoxyuridine, demonstrating that the expression of N-myc and c-src genes was regulated independently in the bipolar differentiation processes of NB cells. Our results suggest that enhanced N-myc expression might be closely linked to the undifferentiated phenotypes of NB cells, that c-src expression might be related to the neuronal differentiation of NB cells, and that N-myc and c-src genes might be regulated independently in the determination of the bipolar differentiation of NB cells.

Cell Differentiation↗
PubMed2004

Differential response to keratinocyte growth factor receptor and epidermal growth factor receptor ligands of proliferating and differentiating intestinal epithelial cells.

The expression of the keratinocyte growth factor receptor (KGFR) has been analyzed on intestinal epithelial Caco-2 cells upon confluence-induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal growth factor receptor (EGFR), was up-modulated in post-confluent differentiated cultures compared with the pre-confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up-regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF-10, whereas they were not stimulated by the EGFR ligands TGFalpha and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post-confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up-modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands.

Antibodies, Monoclonal↗
PubMed1997

Differential inhibition of telomerase activity during induction of differentiation in hematopoietic, melanoma, and glioma cells in culture.

Accumulating evidence indicates that telomerase activity is stringently repressed in normal human somatic cells but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity may play a role in carcinogenesis and immortalization. Recently, down-regulation of telomerase activity by induction of differentiation has been reported for cells of pre-myelocytic and myelocytic leukemia as well as embryonic carcinoma. To gain further insight about the regulation of telomerase activity following induction of differentiation, telomerase activity was examined in a human hematopoietic progenitor cell line (D2), a melanoma cell line (CM73-36) and a glioma cell line (Ast812) before and after addition of differentiation inducing agents. The state of differentiation was assessed by growth inhibition and cell morphological maturation. Telomerase activity was assayed by a PCR-based telomeric repeat amplification protocol (TRAP). Our data show that telomerase activity was inhibited only in differentiation-induced D2 cells but not in differentiation-induced melanoma and glioma cells. A model for the differential inhibition of telomerase activity following induction of differentiation in different cancer cells will be presented.

Brain Neoplasms↗
PubMed1995

Independent mechanisms are utilized for the coordinate and transient accumulation of two differentiation-specific mRNAs during differentiation of Naegleria gruberi amoebae into flagellates.

During the differentiation of Naegleria gruberi amoebae into flagellates, four differentiation-specific (DS) mRNAs are transiently and coordinately accumulated. Three of the four DS mRNAs, Class II, III, and IV, encode alpha-tubulin, beta-tubulin, and flagellar calmodulin, respectively. The protein product of the Class I mRNA has not been identified. We examined the effects of inhibition of protein synthesis on transcription and accumulation of beta-tubulin mRNA and Class I mRNA to understand the mechanism of coordinate regulation. Inhibition of protein synthesis at the beginning of differentiation completely blocked transcription of the beta-tubulin gene. Addition of cycloheximide at 30 or 40 min after initiation of differentiation inactivated transcription of the beta-tubulin gene in less than 10 min as judged by nuclear run-on experiments. However, once differentiation had proceeded for more than 50 min, inhibition of protein synthesis did not inactivate transcription of beta-tubulin mRNA was more active in cycloheximide-treated cells than in control cells. Cycloheximide treatment at the initiation of the differentiation also blocked transcription of the Class I gene. However, addition of the drug after 30 min had no significant effect on the transcription of the Class I gene. Cycloheximide treatment also increased the half-lives of beta-tubulin and Class I mRNA drastically. These data suggest that: (1) the transient accumulation of the two DS mRNAs during differentiation are regulated by changing both the rate of transcription and the stability of the mRNAs; (2) protein synthesis is required for the transcriptional and post-transcriptional regulations; (3) the transcriptional regulation mechanisms of the beta-tubulin gene and that of the Class I gene are distinct; and (4) the transcription of the beta-tubulin gene is regulated by different mechanisms during differentiation.

Animals↗
PubMed2000

Evolving mechanisms of morphogenesis: on the interplay between differential adhesion and cell differentiation.

Differential cell adhesion, mediated by e.g. integrin and cadherins/catenines, plays an important role in morphogenesis and it has been shown that there is intimate cross-talk between their expression and modification, and inter-cellular signalling, cell differentiation, cell growth and apoptosis. In this paper, we introduce and use a formal model to explore the morphogenetic potential of the interplay between these processes. We demonstrate the formation of interesting morphologies. Initiated by cell differentiation, differential cell adhesion leads to a long transient of cell migrations, e.g. engulfing and intercalation of cells and cell layers. This transient can be sustained dynamically by further cell differentiation, and by cell growth/division and cell death which are triggered by the (also long range) forces (stretching and squeezing) generated by the cell adhesion. We study the interrelation between modes of cell differentiation and modes of morphogenesis. We use an evolutionary process to zoom in on gene-regulation networks which lead to cell differentiation. Morphogenesis is not selected for but appears as a side-effect. The evolutionary dynamics shows the hallmarks of evolution on a rugged landscape, including long neutral paths. We show that a combinatorially large set of morphologies occurs in the vicinity of a neutral path which sustains cell differentiation. Thus, an almost linear molecular phylogeny gives rise to mosaic evolution on the morphological level.

Animals↗
PubMed1990

Effects of mevinolin, an inhibitor of cholesterol synthesis, on the morphology and function of differentiating and differentiated rat adrenocortical cells in primary culture.

Mevinolin, an inhibitor of cholesterol synthesis, was used to study the effect of endogenous cholesterol synthesis on the morphology and function of differentiating and differentiated fetal rat adrenocortical cells grown in primary culture. Upon adrenocorticotrophic hormone (ACTH) stimulation under conditions in which endogenous cholesterol synthesis was inhibited but exogenous (lipoprotein) cholesterol was available, the cells differentiated normally from glomerulosa-like to fasciculata-like cells; the steroid hormone secretion was maximally induced. Under conditions in which cholesterol synthesis was maximally inhibited by mevinolin and the cells had no access to exogenous cholesterol, the cells did not differentiate into fasciculata-like cells; the ACTH-induced steroid response was highly suppressed under these conditions. The addition of either human low-density lipoprotein (LDL) or high-density lipoprotein (HDL3) to the culture medium restored the ACTH-induced differentiation and steroid secretion. Thus, in the absence of exogenous cholesterol, endogenous cholesterol synthesis was a prerequisite for differentiation. In cultures grown in the presence of exogenous cholesterol and ACTH with mevinolin-inhibited cholesterol synthesis and high steroid output, an increase in cytoplasmic lipids was evident, suggesting upregulation of LDL and HDL receptors. The results also demonstrated that induction of phenotypic differentiation from glomerulosa-like into fasciculata-like cells can proceed in the presence of a cholesterol synthesis inhibitor like mevinolin; this differentiation in the absence of endogenous cholesterol synthesis is accompanied by the appearance of cytoplasmic cholesterol ester droplets, typical of fasciculata cells.

Acyl Coenzyme A↗
PubMed1998

Differential effects of two protein tyrosine kinase inhibitors, tyrphostin and genistein, on human bone cell proliferation as compared with differentiation.

Protein tyrosyl phosphorylation is a key determinant of cell proliferation and differentiation. The aim of this study was to test the hypothesis that the signal transduction pathway(s) responsible for human bone cell proliferation may involve different groups of protein tyrosine kinase (PTKs) as compared with that for differentiation. To achieve this, we investigated the effects of two structurally different PTK inhibitors, viz, tyrphostin A51 and genistein, on the proliferation ([3H]thymidine incorporation) and differentiation [alkaline phosphatase (ALP) specific activity and collagen synthesis] of two normal human bone cell types: mandible-derived and vertebra-derived bone cells. Tyrphostin A51 and genistein each markedly reduced cellular tyrosyl phosphorylation level (assessed by Western analysis using a commercial anti-phosphotyrosine antibody and the enhanced chemiluminescence detection assay), confirming that these two effectors are potent PTK inhibitors in human bone cells. Regarding bone cell proliferation, tyrphostin A51 (5-30 microM) caused, a dose-dependent inhibition of basal [3H]thymidine incorporation of both human bone cell types. In contrast, genistein (5-20 microM), not only did not inhibit, but significantly stimulated [3H]thymidine incorporation of these same cell types in a dose-dependent, biphasic manner, with the optimal stimulatory dose between 10 and 20 microM. These effects on cell proliferation were confirmed by cell number counting. In addition, whereas the mitogenic activity of 10 ng/ml epidermal growth factor (EGF) on human mandible-derived bone cells was completely abolished by 5-30 microM tyrphostin A51, genistein at 5-30 microM enhanced the EGF-induced bone cell proliferation in an additive manner. With respect to bone cell differentiation, tyrphostin A51 and genistein each significantly increased basal ALP specific activity and collagen synthesis in human bone cells. In summary, (1) PTKs are involved in human bone cell proliferation and differentiation; (2) tyrphostin A51 inhibited both basal and EGF-induced cell proliferation, thus tyrphostin-sensitive PTKs are involved in basal and EGF-induced human bone cell proliferation; (3) genistein stimulated basal proliferation and enhanced EGF-mediated cell proliferation, suggesting that genistein-sensitive PTKs may play an inhibitory role in human bone cell proliferation; and (4) these differential effects of PTK inhibitors on human bone cell proliferation and differentiation are independent of basal differentiation status of the cells.

Alkaline Phosphatase↗
PubMed1984

Biological mechanisms for the loss of the differentiated phenotype by non-terminally differentiated adipocytes.

The differentiation of 3T3 T proadipocyte stem cells is controlled at two related yet distinct states in the G1 phase of the cell cycle. They are designated GD and GD'. GD is the G1 state at which cells must growth arrest prior to differentiation, and GD' is the G1 state at which non-terminal differentiation occurs. Cells arrested at the GD and GD' states have distinct characteristics; yet cells at both states can mediate the integrated control of cellular proliferation and differentiation. In this paper we report on studies designed to further characterize the relationship of these two states, specifically to determine whether non-terminally differentiated GD'-arrested cells can be induced to lose the adipocyte phenotype and revert to the GD state. We report that retinoic acid (RA) and methyl isobutyl xanthine (MIX) can induce non-terminally differentiated GD'-arrested cells to lose the adipocyte phenotype without undergoing DNA synthesis. Such cells that have lost the adipocyte phenotype are also shown to remain in the G1 phase of the cell cycle and to reacquire most of the characteristics of GD-arrested cells. Most importantly, they demonstrate the capacity to redifferentiate without DNA synthesis. We therefore conclude that when non-terminally differentiated GD'-arrested cells are induced to lose the adipocyte phenotype they do indeed revert to the GD state and they thereby become more responsive to environmental influences which can further regulate the integrated control of cellular proliferation and differentiation.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Differential induction and intracellular localization of SCG10 messenger RNA is associated with neuronal differentiation.

The differentiation of neurons involves the establishment of distinct molecular compartments which regulate neuronal shape and function. This requires targeting of specific gene products to growth-associated regions of the neuron. We have investigated the temporal and spatial regulation of SCG10 gene expression during neuronal differentiation. There are two SCG10 messenger RNAs, 1 and 2 kg in length, which encode the same growth-associated protein. These messenger RNAs were found to be differentially regulated during the onset of neurite outgrowth in early rat cerebellum development. In PC12 cells, the two SCG10 messenger RNAs were shown to be differentially induced by nerve growth factor. Regulation of the 2 kb messenger RNA, but not the 1 kb messenger RNA, is dependent on the differentiation of PC12 cells, indicating that post-transcriptional regulation of SCG10 expression during neurite outgrowth. Spatial regulation of the 2 kb SCG10 messenger RNA distribution during brain development was examined by in situ hybridization. The 2 kb messenger RNA was found to be localized to the neuronal pole where outgrowth was occurring, within differentiating neurons in vivo. Intracellular localization of SCG10 messenger RNA was also observed in differentiating primary cultured neurons, with the 2 kb messenger RNA transported into growing neurites during the development of neuronal polarity. In neurons which had developed polarity, the 2 kb SCG10 messenger RNA was consistently found in the cell body and axon. This study demonstrates both temporal and spatial post-transcriptional regulation of SCG10 expression which is associated with neurite outgrowth. The directed transport and positional translation of SCG10 messenger RNA provide a potential mechanism for protein targeting and the creation of molecular compartments during neuronal differentiation.

Amino Acid Sequence↗
PubMed2006

Differentiation stage-specific analysis of gene function with inducible short hair-pin RNA in differentiating embryonic stem cells.

Cell differentiation is regulated by spatial and temporal coordination of gene expressions. Previously, we have established an embryonic stem (ES) cell differentiation system that can trace early cardiovascular developmental process in vitro. Here we show that tetracycline-induced short hair-pin RNA (shRNA) expression in differentiating ES cells successfully suppressed stage-specific genes for differentiation and modified cell fates. We established ES cell lines carrying shRNA gene driven by tRNA(val) promoter with tetracycline operator sequences (tet-ON system). When expression of vascular endothelial growth factor receptor-2 (VEGFR2) gene, a vascular progenitor and mesoderm marker and an essential gene for endothelial cell (EC) differentiation, was suppressed by shRNA in early ES cell differentiation, appearance of VEGFR2(+) mesoderm cells was substantially reduced. Suppression of VEGFR2 expression at mesoderm stage almost completely inhibited EC differentiation from VEGFR2(+) mesoderm cells. This novel experimental system, thus, can selectively determine stage-specific roles of genes in differentiation in vitro.

Cell Differentiation↗
PubMed2004

ERK signaling pathway is differentially involved in erythroid differentiation of K562 cells depending on time and the inducing agent.

K562 cells can be induced to differentiate along the erythroid lineage by a variety of chemical compounds, including hemin, butyrate, cisplatin and ara-C. Differential signaling through MAP kinases has been suggested to be involved in this differentiation process. We have investigated the involvement of ERK activation/inhibition in hemin-, butyrate-, cisplatin- and ara-C-induced erythroid differentiation using the K562 cell line. ERK activity decreased for 2-4h after administration of either inducing agent. ERK was then activated by hemin and cisplatin, while ERK phosphorylation remained decreased during incubation with butyrate and ara-C. There was no activation of JNK or p38. The MEK-1 inhibitors UO126 or PD98059 induced erythroid differentiation in K562 cells and acted additively with butyrate. Inhibition of MEK-1 reduced the hemoglobin accumulation by hemin and cisplatin; erythroid differentiation by ara-C was unchanged. The results suggest that inhibition of signaling through ERK in K562 cells may be needed to enter the erythroid differentiation process, while after initiation both activation and inhibition of signaling through ERK enhance erythroid differentiation, which, however, is dependent on the inducing compound.

Butyrates↗
PubMed2005

Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation.

Inactivation of beta-catenin in mesenchymal progenitors prevents osteoblast differentiation; inactivation of Lrp5, a gene encoding a likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone formation. These observations indicate that Wnt signaling controls osteoblast differentiation and suggest that it may regulate bone formation in differentiated osteoblasts. Here, we study later events and find that stabilization of beta-catenin in differentiated osteoblasts results in high bone mass, while its deletion from differentiated osteoblasts leads to osteopenia. Surprisingly, histological analysis showed that these mutations primarily affect bone resorption rather than bone formation. Cellular and molecular studies showed that beta-catenin together with TCF proteins regulates osteoblast expression of Osteoprotegerin, a major inhibitor of osteoclast differentiation. These findings demonstrate that beta-catenin, and presumably Wnt signaling, promote the ability of differentiated osteoblasts to inhibit osteoclast differentiation; thus, they broaden our knowledge of the functions Wnt proteins have at various stages of skeletogenesis.

Animals↗
PubMed2007

Differential role of two VDR coactivators, DRIP205 and SRC-3, in keratinocyte proliferation and differentiation.

Cell programs such as proliferation and differentiation involve the selective activation and repression of gene expression. The vitamin D receptor (VDR), through 1,25(OH)(2)D(3), controls the proliferation and differentiation of keratinocytes. Previously, we have identified two VDR binding coactivator complexes. In proliferating keratinocytes VDR bound preferentially to the DRIP complex, whereas in differentiated keratinocytes the SRC complex was preferred. We proposed that different coactivators are required for sequential gene regulation in the transition from proliferation to differentiation. Here we examined the roles of DRIP205 and SRC-3 in this transition. Silencing of DRIP205 and VDR caused hyperproliferation of keratinocytes, demonstrated by increased XTT and BrdU incorporation. SRC-3 silencing, on the other hand, did not have an effect on proliferation. In contrast, SRC-3 as well as DRIP205 and VDR silencing blocked keratinocyte differentiation as shown by decreased expression of keratin 1 and filaggrin. These results are consistent with the differential localization of DRIP205 and SRC-3 in skin. These results indicate that DRIP205 is required for keratinocyte proliferation. Both DRIP205 and SRC-3 are required for the keratinocyte differentiation. These results support the concept that the selective use of coactivators by VDR underlies the selective regulation of gene expression in keratinocyte proliferation and differentiation.

Cell Differentiation↗
PubMed2005

Differential gene expression pattern of neurotrophins and their receptors during neuronal differentiation of rat bone marrow stromal cells.

Neural-like cells derived from bone marrow stromal cells (BMSC) have potential usefulness in repair of the CNS injuries or diseases. The functional recovery mediated by these cells, however, depends on secretion of specific growth factors and their designated receptors. In the present study, we have investigated the expression profile of neurotrophins NGF, BDNF and NT-3 and their high-affinity (TrkA, TrkB, TrkC) and common low-affinity (p75NTR) receptors before and during neural differentiation of rat BMSCs by RT-PCR. Results indicate that NGF and BDNF but not NT-3 are expressed in both un-differentiated as well as neurally differentiated BMSCs. In contrast, the expression of TrkA and TrkB is restricted to neurally differentiated cells, while TrkC is not expressed in these cells either before or after differentiation. Interestingly, p75NTR expression is absent in un-differentiated cells but is initiated upon the induction of neural differentiation, and then shut off in fully differentiated neuron-like cells.

Animals↗
PubMed2004

Differential signalling mechanisms predisposing primary human skeletal muscle cells to altered proliferation and differentiation: roles of IGF-I and TNFalpha.

To gain a clearer insight into the mechanisms of skeletal muscle cell growth, differentiation and maintenance, we have developed a primary adult human skeletal muscle cell model. Cells were cultured from biopsies of rectus muscle from the anterior abdominal wall of patients undergoing elective surgery. Under differentiating conditions, all cultures formed myotubes, irrespective of initial myoblast number. Stimulation with both IGF-I and tumour necrosis factor alpha (TNFalpha) increased cellular proliferation but while IGF-I subsequently increased myoblast differentiation, via both hyperplasia and hypertrophy, TNFalpha inhibited the initiation of differentiation, but did not induce apoptosis. Addition of IGF-I stimulated both the MAP kinase and the phosphatidylinositide 3-kinase (PI 3-kinase) signalling pathways while treatment with TNFalpha preferentially led to MAP kinase activation although with a very different profile of activation compared to IGF-I. Data using the MEK inhibitor UO126 showed MAP kinase activity is not only needed for cellular proliferation but is also necessary for both the initiation and the progression of primary human myoblast differentiation. The PI 3-kinase pathway is also involved in differentiation, but activation of this pathway could not relieve inhibition of differentiation by TNFalpha or UO126. Our results show that the controlled temporal and amplitude of activation of multiple signalling pathways is needed for successful myoblast differentiation.

Adult↗
PubMed2002

Differential gene expression in retinoic acid-induced differentiation of acute promyelocytic leukemia cells, NB4 and HL-60 cells.

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation t(15;17), which results in the fusion of the promyelocytic leukemia gene (PML) and retinoic acid receptor alpha gene (RARalpha). APL can be effectively treated with the cell differentiation inducer all-trans retinoic acid (ATRA). NB4 cells, an acute promyelocytic leukemia cell line, have the t(15;17) translocation and differentiate in response to ATRA, whereas HL-60 cells lack this chromosomal translocation, even after differentiation by ATRA. To identify changes in the gene expression patterns of promyelocytic leukemia cells during differentiation, we compared the gene expression profiles in NB4 and HL-60 cells with and without ATRA treatment using a cDNA microarray containing 10,000 human genes. NB4 and HL-60 cells were treated with ATRA (10(-6)M) and total RNA was extracted at various time points (3, 8, 12, 24, and 48h). Cell differentiation was evaluated for cell morphology changes and CD11b expression. PML/RARalpha degradation was studied by indirect immunofluoresence with polyclonal PML antibodies. Typical morphologic and immunophenotypic changes after ATRA treatment were observed both in NB4 and HL-60 cells. The cDNA microarray identified 119 genes that were up-regulated and 17 genes that were down-regulated in NB4 cells, while 35 genes were up-regulated and 36 genes were down-regulated in HL60 cells. Interestingly, we did not find any common gene expression profiles regulated by ATRA in NB4 and HL-60 cells, even though the granulocytic differentiation induced by ATRA was observed in both cell lines. These findings suggest that the molecular mechanisms and genes involved in ATRA-induced differentiation of APL cells may be different and cell type specific. Further studies will be needed to define the important molecular pathways involved in granulocytic differentiation by ATRA in APL cells.

Cell Differentiation↗
PubMed1998

Establishment of a stable HL60 subline having the potential for monocytic differentiation using granulocyte-macrophage colony-stimulating factor: possible use for the study of monocytic differentiation and oxidative stress.

A stable HL60 subline having the potential for monocytic differentiation was established by use of GM-CSF. HL60, a human promyelocytic cell line, has frequently been employed for research in the fields of monocytic differentiation and atherosclerosis because of its potential to differentiate into either granulocytes or monocytes. However, HL60 are frequently seen to change their phenotype during long-term culture. To date, many sublines or variants of HL60 cells have been established. However, most of them display diminished or complete loss of activities that characterize parental cells. The present study was conducted to establish a stable HL60 subline with the potential for monocytic differentiation. Firstly, a single HL60 cell was isolated by limiting dilution, and was successfully proliferated by incubation with GM-CSF. Secondly, from this population, cells were selected that had the ability to generate superoxide after VD-induced monocytic differentiation. Cells obtained in this manner (designated HL60/DU-1) exhibited expression of CD14 and CD11b and suppression of CD3 expression after monocytic differentiation. NBT positivity showed a consistent level of over 971% after a 6-day challenge with VD throughout the experimental period of 12 months. HL60/DU-1 cells, which were cryopreserved in liquid nitrogen for 6 months, thawed and re-cultured, exhibited over 97% NBT positivity. Carvedilol and probucol, which exhibit antioxidative activity, inhibited superoxide release from the differentiated HL60/DU-1 cells. HL60/DU-1 cell line is a promising model for the study of monocytic differentiation and the effects of oxygen radicals.

Calcitriol↗