Induced Differentiation Of HL-60 Cells Research Articles

Ａ環修飾活性型ビタミンＤ３の合成と活性

We have systematically synthesized 2α-functionalized 1α, 25-dihydroxyvitamin D3 [1α, 25 (OH) 2D3] 1 based on a convergent method using Pd-catalyzed alkylative cyclization with the A-ring precursor enynes and the CD-ring bromoolefin 5 in the following three categories : 2α-alkyl, 2α-hydroxyalkyl, and 2α-hydroxyalkoxyl derivatives, in order to study the structure-activity relationships of the natural hormone 1α, 25 (OH) 2D3, particularly on the A-ring.First, the 2-methyl analogues 2 were designed and all eight possible diastereomers on the A-ring stereochemistry at C 1, C 2, and C 3 were synthesized from the A-ring precursor enynes 22a-h, which were prepared from methyl (S) -and (R) -3-hydroxy-2-methylpropionate. Biological activities including affinities to vitamin D receptor (VDR) and vitamin D binding protein (DBP), elevation of serum Ca level, induction of HL-60 cell differentiation, and apoptosis are discussed.Next, on the basis of biological activities of the 2α-methyl derivative, the other types of 2α-functional groups were introduced stereoselectively into the 1α, 25 (OH) 2D3 skeleton using the A-ring precursor enynes derived from D-xylose or D-glucose.Five of these synthetic 2α-modified analogues showed higher VDR binding affinity than that of the natural hormone. Docking studies of the synthetic ligands to VDR based on Moras' X-ray results and remarkably high biological activities are described.

Capsaicin potentiates 1,25-dihydoxyvitamin D 3- and all- trans retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells

Human promyelocytic leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2D 3] or all- trans retinoic acid, respectively. In this study, the effect of capsaicin, an active component of the red pepper of the genus Capsocum, on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 5–30 μg/ml capsaicin for 72 h inhibited cell proliferation and induced a small increase in cell differentiation. Interestingly, synergistic induction of HL-60 cell differentiation was observed when capsaicin was combined with either 5 nM 1,25-(OH) 2D 3 or 50 nM all- trans retinoic acid. Flow cytometric analysis indicated that combinations of 1,25-(OH) 2D 3 and capsaicin stimulated differentiation predominantly to monocytes whereas combinations of all- trans retinoic acid and capsaicin stimulated differentiation predominantly to granulocytes. Capsaicin enhanced protein kinase C activity in 1,25-(OH) 2D 3- and all- trans retinoic acid-treated HL-60 cells. In addition, inhibitors for protein kinase C [bisindolylmaleimide (GF-109203X), chelerythrine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7)] and an inhibitor for extracellular signal-regulated kinase [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (PD-098059)] significantly inhibited HL-60 cell differentiation induced by capsaicin in combination with either 1,25-(OH) 2D 3 or all- trans retinoic acid. These results indicate that capsaicin potentiates 1,25-(OH) 2D 3- or all- trans retinoic acid-induced HL-60 cell differentiation and that both protein kinase C and extracellular signal-regulated kinase are involved in the cell differentiation synergistically enhanced by capsaicin.

(23S)- and (23R)-25-dehydro-1α-hydroxyvitamin D 3-26,23-lactone function as antagonists of vitamin D receptor-mediated genomic actions of 1α,25-dihydroxyvitamin D 3

We synthesized various analogues of 1α,25-(OH) 2D 3-26,23-lactone and examined the effects of them on HL-60 cell differentiation using the evaluation system of the genomic action of 1α,25-(OH) 2D 3. We found that (23S)- and (23R)-25-dehydro-1α-OH-D 3-26,23-lactone (TEI-9647 and TEI-9648) strongly bound to the VDR, but did not induce HL-60 cell differentiation. Intriguingly, TEI-9647 and TEI-9648 did inhibit that induced by 1α,25-(OH) 2D 3, whereas they did not suppress that caused by retinoic acid or TPA. On the contrary, the similar 25-dehydrated 24-dehydro analogues, TEI-D1807 and TEI-D1808, weakly but significantly induced HL-60 cell differentiation, never showing inhibitory effect on HL-60 cell differentiation induced by 1α,25-(OH) 2D 3. In other experiments, TEI-9647 and TEI-9648 markedly suppressed 25-OH-D 3-24-hydroxylase gene expression induced by 1α,25-(OH) 2D 3 in HL-60 cells. TEI-9647 also inhibited the heterodimer formation between VDR and RXRα, and the VDR interaction with co-activator SRC-1 according to the results obtained from the mammalian two-hybrid system in Saos-2 cells. Taking all these results into consideration, we reached a manifest conclusion that TEI-9647 and TEI-9648 are the specific and first antagonists of 1α,25-(OH) 2D 3 action, specifically VDR-VDRE mediated genomic action.

Extracellular ATP-dependent suppression of proliferation and induction of differentiation of human HL-60 leukemia cells by distinct mechanisms

Extracellular ATP suppressed the growth of HL-60 leukemia cells and induced their differentiation as revealed by N-formyl-methionyl-leucyl-phenylalanine-induced β-glucuronidase release. ATP degraded to ADP, AMP, and adenosine, and the effect of ATP on cell growth was mimicked by these metabolites added to the cultures. The stable analog α,β-methylene ATP, however, had only a weak inhibitory effect on cell growth. Adenine nucleotide-induced growth suppression was reversed by uridine, suggesting the involvement of intracellular pyrimidine starvation secondary to adenosine accumulation. Consistent with this, ATP induced intracellular starvation of pyrimidine nucleotides, and this effect was also prevented by pretreatment of cells with uridine. The order of effectiveness of ATP-induced differentiation of HL-60 cells, unlike that for growth suppression, was ATP > ADP > AMP, and adenosine had no effect. Furthermore, uridine had no effect and the stable analog, α,β-methylene ATP also induced HL-60 cell differentiation, suggesting that differentiation was due to ATP per se. We tested the hypothesis that ATP-induced differentiation arises from activation of adenylyl cyclase by the novel P2Y11 receptor using the cell-permeable inhibitor of protein kinase A, Rp-CPT-cAMPS (8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphorothioate, Rp isomer). Rp-CPT-cAMPS (1–100 μM) prevented ATP-induced differentiation of HL-60 cells as assessed by fMLP-induced β-glucuronidase release. However, Rp-CPT-cAMPS did not prevent ATP-induced growth suppression. Taken together, the data indicate that extracellular ATP suppresses HL-60 growth and induces their differentiation by distinct mechanisms. Growth suppression arises from adenosine generation and consequent pyrimidine starvation. Differentiation arises, at least in part, from a distinct mechanism involving the activation of cell surface P2 receptors coupled to cAMP generation and activation of protein kinase A.

EFFICACY OF MICROTUBULE-ACTIVE DRUGS FOLLOWED BY KETOCONAZOLE IN HUMAN METASTATIC PROSTATE CANCER CELL LINES

Once a relapse occurs following primary endocrine treatment, metastatic prostate cancer is one of the most therapy-resistant human neoplasms. Ketoconazole is used for complete androgen deprivation, and recent data suggest it has direct activity against prostate cancer cells. LNCaP, DU145, and PC3 cells, human prostate cancer cell lines, and HL60, a human leukemia cell line, were lysed and soluble proteins were harvested. Cells were plated in 96-well flat bottom plates and then exposed to the pharmacological agents, ketoconazole, vinblastine and paclitaxel. DNA synthesis was monitored by 3H-thymidine incorporation. We demonstrate that ketoconazole exerts a cytostatic effect on a panel of human prostate cancer cell lines, with IC50 of 4 to 5 microg./ml., 12 microg./ml., and 25 microg./ml. for LNCaP, PC3/PC3M, and DU145 cells, respectively. On the other hand, using microtubule-active drugs, vinblastine and paclitaxel, we found that PC3M and PC3 cells were more resistant than either DU145 or LNCaP cells. This resistance was associated with a lesser degree of Raf-1 and Bcl-2 phosphorylation following exposure to microtubule-active drugs. Combinations of microtubule-active drugs with ketoconazole were a beneficial treatment in DU145 cancer cells. Furthermore, ketoconazole blocked recovery of all the prostate cancer cell lines following 24 hours-pulse treatment with vinblastine. Pulse-administration of vinblastine followed by continuous administration of ketoconazole warrants investigation in the treatment of hormone-independent metastatic prostate cancer.

Augmentation of methylprednisolone-induced differentiation of myeloid leukemia cells by serine/threonine protein phosphatase inhibitors

To elucidate the roles of serine/threonine protein phosphatases type 1 (PP1) and type 2A (PP2A) in methylprednisolone-induced differentiation of HL60 cells into granulocytes and K562 cells into monocytes, we examined the effect of serine/threonine protein phosphatase inhibitors, okadaic acid and Cal-A on the proliferation/ differentiation of HL60 and K562 cells. Okadaic acid and Cal-A augmented methylprednisolone induced granulocytic differentiation and cell death of HL60 cells and monocytic differentiation and cell death of K562 cells in different dose ranges, respectively. These data suggest an important role of PP1 and PP2A in the mechanism leading to differentiation of leukemic cells.

Cell cycle specific induction of HL-60 cell differentiation and apoptosis by mycophenolic acid

HL-60 cells undergo terminal differentiation and apoptosis in response to different types of sub-toxic and toxic perturbations respectively. The mechanism by which cells sense different amounts of perturbation to activate pathways that lead to the engagement of a relevant biological response is not known. The response of HL-60 cells to treatment with the immunosuppressant mycophenolic acid (MPA), a specific inhibitor of dGTP/GTP-synthesis, allowed quantitation of a metabolic perturbation which triggered a cellular response. 1.5 microM MPA induced 38% terminal differentiation to CD14 positive, early monocyte-like cells and 22% cell death by apoptosis, whereas 3 microM MPA induced 70% apoptosis but no differentiation. Despite the difference in biological outcomes, 72 h exposure to both 1.5 microM and 3 microM MPA caused a similar ( approximately 75%) depletion of total GTP levels. Cells synchronized by centrifugal elutriation were treated with MPA. Elutriated cells were overall less sensitive to the effects of MPA but 3 microM MPA induced significantly less apoptosis and more differentiation in an elutriation-enriched G1-population than in a population normally distributed in the cell cycle, suggesting that the effects of MPA in S-phase may subsequently lead to cell death. However, analysis of apoptosis by using a terminal deoxynucleotidyltransferase assay and measurement of bromodeoxyuridine incorporation showed that apoptosis was engaged in G1. Analysis of the phosphorylation status of the retinoblastoma protein demonstrated that Rb was hypophosphorylated prior to apoptosis and that in apoptotic cells, separated by flow cytometry, Rb protein was absent, presumably due to proteolysis. The loss of Rb protein did not appear to permit transit to S-phase, and was not accompanied by an expression of c-Myc. Surprisingly, therefore, an antimetabolite inducing a loss of GTP brought about cell death by apoptosis in the G1 phase of the cell cycle.

Synergistic induction of HL60 cell differentiation by ketoconazole and 1-desoxy analogues of vitamin D3.

The goal of differentiation therapy is to induce cancer cells to stop proliferating and to express characteristics of normal cells. Vitamin D analogues, such as the deltanoids, are being evaluated as differentiation agents in the treatment of several human cancers (e.g., myeloid leukemias); however, these compounds have a tendency to produce hypercalcemia in patients receiving therapy. A combination of a differentiation-inducing deltanoid with a compound that blocks entry of calcium into cells (e.g., ketoconazole) may offer a new approach to differentiation therapy and address the problem of hypercalcemia. We investigated whether various ketoconazole-deltanoid combinations would alter cellular differentiation or intracellular calcium homeostasis in comparison with deltanoids used alone. Cultured human leukemia HL60 cells were treated with ketoconazole-deltanoid combinations. Markers of differentiation (expression of CD11b and CD14 antigens and of non-specific esterase) were measured by flow cytometry and cytochemistry; cell cycle distribution was measured by flow cytometry of propidium iodide-stained cells. Expression of differentiation-related genes was assessed by northern blotting and immunoblotting, and changes in intracellular calcium homeostasis were monitored by fluorescence analysis of fura-2-containing cells. Ketoconazole strongly potentiated the differentiating activity of the deltanoids, which exhibited low potency when used alone. Ketoconazole-deltanoid combinations had little effect on HL60 cell-cycle distribution, although the cells did stop proliferating and they differentiated. Ketoconazole-deltanoid combinations produced only minor changes in intracellular calcium homeostasis compared with changes produced by 1,25-dihydroxyvitamin D3, either alone or in combination with ketoconazole. These results suggest that ketoconazole may be useful in combination with vitamin D analogues in the differentiation therapy for myeloid leukemias.

Adenosine metabolism during phorbol myristate acetate-mediated induction of HL-60 cell differentiation: changes in expression pattern of adenosine kinase, adenosine deaminase, and 5'-nucleotidase.

Adenosine has potent immunosuppressive activity. Since the source of adenosine and the mechanism of its release in the immune system is largely unknown and may vary according to cell type, we have evaluated the relationship between adenosine metabolism and the enzymatic activities and mRNA levels of adenosine-metabolizing enzymes in myeloid and lymphoid cell lines. Induction of HL-60 cell differentiation along the macrophage lineage by PMA resulted in a reduction in the activities of adenosine deaminase (ADA), adenosine kinase (AK), and inosine monophosphate-specific cytosolic 5'-nucleotidase and an elevation of ecto-5'-nucleotidase (ecto-5'-NT). These changes were accompanied by an elevation of ecto-5'-NT mRNA and a decrease in ADA and AK mRNAs in a time-dependent fashion. Comparison of AK and ADA mRNA levels in several other leukemic cell lines revealed generally similar responses to PMA with much stronger suppression in immature T cells than in B cells. The metabolism of adenosine either through phosphorylation (AK) or deamination (ADA) was reduced in PMA-stimulated cells. Furthermore, the cumulative changes in enzyme expression resulted in a 2.5-fold increase in intracellular adenosine formation in PMA-stimulated cells. The inhibition of AK by 5'-iodotubercidin further increased adenosine formation by 6-fold over that in untreated cells. In accord with the increase in ecto-5'-NT activity, extracellular AMP dephosphorylation increased dramatically, but there was no increase in extracellular ATP degradation. These results indicate that a coordinated shift in adenosine-metabolizing enzyme levels during PMA-induced HL-60 cell differentiation is accompanied by a decrease in adenosine uptake and an increase in adenosine release.

Differentiation induction of HL60 cells by 1,25(OH)2D3, all trans retinoic acid, rTGF-beta2 and their combinations.

1,25 Dihydroxyvitamin D3 (D3), all trans retinoic acid (atRA) and the cytokine rTGF-beta2 are growth and differentiation modulators of promyelocytic leukemia. D3 gives rise to a functional monocytic cell population whereas single atRA therapy induces granulocytic cell features. rTGF-beta2 reduces HL60 cell proliferation but has no differentiating capacity. Combination treatment demonstrates additive effects between either D3 and atRA or D3 and rTGF-beta2, resulting in a cell population with mixed characteristics since individual cells exhibit both monocytic as granulocytic cell features. The capacity of single and combined treatments to induce a permanent differentiation was investigated. Therefore, cells were preincubated with the drugs during six days, test drugs were removed and cell number was monitored. The total cell count of populations treated with single agents remains constant for only a few days and then increases rapidly. rTGF-beta2 cooperated with D3 in inducing a long-lasting differentiation state (3 weeks). Addition of atRA to this combination did not significantly alter proliferation or differentiation, but some cells underwent apoptosis. Therefore, a total and permanent differentiation of leukemic cells may be achieved by repeated exposure to a combination of differentiation inducing agents.

Polyenylidene thiazolidinedione derivatives with retinoidal activities.

Several polyenylidene thiazolidine or 2-thioxo-4-thiazolidinone derivatives were synthesized and their retinoidal activities were examined in terms of the differentiation-inducing ability towards human promyelocytic leukemia HL-60 cells and inhibitory effect on interleukin (IL)-1 alpha-induced IL-6 production in MC3T3-E1 cells. Compounds containing a trimethylcyclohexenyl ring induced HL-60 cell differentiation with weaker activity than retinoic acid (1a) by one or two orders of magnitude. The thiazolidinedione derivatives (2, 5, 7) showed stronger activity than the corresponding 2-thioxo-4-thiazolidinone derivatives (3, 6, 8). The effects of a retinoid antagonist (LE540) and synergists (retinoid X receptor (RXR) agonists, HX600 or HX630) on the activities of thiazolidine derivatives indicate that these compounds elicit their activities through the nuclear retinoic acid receptors (RARs). All the thiazolidines examined also inhibited IL-1 alpha-induced IL-6 production with IC50 values of 10 nM order. The retinoidal activities of the thiazolidines are significant, considering that replacement of the carboxylic acid in retinoid structures with bioisosteric functional groups is generally ineffective, as seen in the structure-activity relationships of retinoidal benzoic acids.

Modulation of Protein Kinase C Activity and Calcium-Sensitive Isoform Expression in Human Myeloid Leukemia Cells by Bryostatin 1: Relationship to Differentiation and Ara-C-Induced Apoptosis

Previous studies have shown that pretreatment of human myeloid leukemia cells (HL-60) with the protein kinase C (PKC) activator bryostatin 1 potentiates ara-C-induced apoptosis. To test the hypothesis that this capacity stems from down-regulation of PKC activity and/or Ca2+-dependent (group-I; cPKC) isoform expression, comparisons were made between the effects of this agent and the stage-2 tumor promoter mezerein under conditions favoring either cellular differentiation or drug-induced apoptosis. Twenty-four-hour pretreatment of HL-60 cells with 10 nMbryostatin 1, which does not induce differentiation in this cell line, led to a profound reduction in membrane and cytosolic PKC activity, decreased expression of cPKC isoforms (α, βI, βII, γ), and a marked increase in ara-C induced apoptosis. In contrast, 10 nMmezerein, which induces HL-60 cell differentiation, was less effective in down-regulating membrane and cytosolic PKC activity as well as α, βI, and γ cPKC isoform expression, and failed to potentiate ara-C-related apoptosis. The effects of bryostatin 1 were dominant to those of mezerein, in that the combination resulted in down-regulation of PKC activity and expression and potentiation of ara-C-induced apoptosis, but not cellular maturation. However, coadministration of the Ca2+ionophore A23187 (250 nM) restored bryostatin 1’s differentiating ability while antagonizing its capacity to augment apoptosis, despite failing to reverse bryostatin 1-induced down-regulation of PKC activity and cPKC isoform expression. Furthermore, pretreatment of differentiation-responsive monocytic leukemia cells (U937) with bryostatin 1 substantially reduced PKC activity and cPKC isoform expression, but exerted minimal effects on ara-C-related apoptosis. In contrast, exposure of U937 cells to bryostatin 1afterara-C dramatically increased apoptosis, a phenomenon that did not occur in differentiation-unresponsive HL-60 cells. Collectively, these observations indicate that down-regulation of total assayable PKC activity and cPKC expression by bryostatin 1 are insufficient, by themselves, to account for potentiation of leukemic cell apoptosis, at least under conditions in which differentiation occurs. They also provide further evidence that a reciprocal and highly schedule-dependent relationship exists between leukemic cell differentiation and drug-induced apoptosis.

Enzymatic Pattern of Aldehyde Metabolism during HL-60 Cell Differentiation

A number of metabolic changes, including modification of different enzyme activities, are linked to the acquisition of differentiated phenotype in HL-60 cells. Enzymes metabolizing aldehydes contribute to maintaining the intracellular steady-state concentration of aldehydes derived from lipid peroxidation. 4-Hydroxynonenal is one of the most important aldehydes produced by this process, and it is able to inhibit proliferation and induce differentiation of HL-60 human leukemic cells. We have now demonstrated that, after induction of HL-60 cell differentiation by 4-hydroxynonenal or DMSO, glutathione transferase activity increases in parallel to the degree of differentiation, whereas aldehyde dehydrogenase, aldehyde reductase and alcohol dehydrogenase are not affected by differentiation induction. Moreover, in 4-hydroxynonenal- or DMSO-treated cells, the concentration of reduced glutathione decreases five days after treatment. The rise of glutathione transferase activity, as well as the decrease of reduced glutathione, are possibly linked to the increase of detoxification capability of differentiated cells.

A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation.

Examination of the interactions involving transcription factor E2F activity during cell growth and terminal differentiation suggests distinct roles for Rb family members in the regulation of E2F accumulation. The major species of E2F in quiescent cells is a complex containing the E2F4 product in association with the Rb-related p130 protein. As cells enter the cell cycle, this complex disappears, and there is a concomitant accumulation of free E2F activity of which E2F4 is a major component. E2F4 then associates with the Rb-related p107 protein as cells enter S phase. Rb can be found in interactions with each E2F species, including E2F4, during G1, but there appears to be a limited amount of Rb with respect to E2F, likely due to the maintenance of most Rb protein in an inactive state by phosphorylation. A contrasting circumstance can be found during the induction of HL60 cell differentiation. As these cells exit the cell cycle, active Rb protein appears to exceed E2F, as there is a marked accumulation of E2F-Rb interactions, involving all E2F species, including E2F4, which is paralleled by the conversion of Rb from a hyperphosphorylated state to a hypophosphorylated state. These results suggest that the specific ability of Rb protein to interact with each E2F species, dependent on concentration of active Rb relative to accumulation of E2F, may be critical in cell-growth decisions.

Effects of cAMP and cGMP elevating agents on HL-60 cell differentiation.

Previous studies have demonstrated low percentage of HL-60 cell differentiation with theophylline. The present study demonstrate that millimolar concentrations of the non-selective phosphodiesterase inhibitors theophylline, caffeine and isobutyl-methylxanthine all inhibit growth, induce substantial differentiation and elevation of both cAMP and cGMP in HL-60 cells. Selective inhibition of cAMP hydrolysis by Ro20-1724 was without effect. The guanylate cyclase stimulator sodium nitroprusside, which increased cGMP only poorly and also increased cAMP, produced growth inhibition but no differentiation. We put forward the hypothesis that elevation of both cAMP and cGMP above a critical level is necessary for significant cyclic nucleotide induced HL-60 cell differentiation.

Dexamethasone modifies the functional responses of the granulocytic differentiating HL-60 cells

We have studied the effect of dexamethasone on the granulocytic differentiation of the human promyelocytic cell line HL-60 induced by treatment with retinoic acid (RA) or dimethyl sulphoxide (DMSO). Dexamethasone potentiated the immunophenotypic and functional parameters associated with the granulocytic differentiation induced by RA, including changes in CD11b and CD71 expression, inhibition of cell proliferation, enhancement of secretory and oxidative responses and increase of the phospholipase C (PLC), phospholipase A2 (PLA2) and phospholipase D (PLD) activities. However, dexamethasone had selective effects on several parameters of DMSO-induced cell differentiation. Dexamethasone inhibited the DMSO-induced increase of CD11b cell surface expression as well as the oxidative response and PLD activation triggered by 4 beta-phorbol 12-myristate 13-acetate. Nevertheless, dexamethasone potentiated the receptor-mediated PLC activation and the receptor-mediated secretory and oxidative responses in DMSO-treated cells. Unlike RA-treated HL-60 cells, the DMSO-treated cells contained high values of activatable PLA2 activity which were not affected by dexamethasone. Thus dexamethasone affected differently functional parameters and effector systems of granulocytic HL-60 cells, depending on the differentiation agent used. Dexamethasone by itself did not induce HL-60 cell differentiation, but enhanced the receptor- and non-receptor-mediated secretory responses and induced the appearance of stimulated PLA2 activity in undifferentiated HL-60 cells. These data provide evidence for the selective modulation of functional responses by dexamethasone through alterations in signalling processes.

Characteristics and partial purification of a novel cytosolic, magnesium-independent, neutral sphingomyelinase activated in the early signal transduction of 1 alpha,25-dihydroxyvitamin D3-induced HL-60 cell differentiation.

Treatment of HL-60 cells with a 1 alpha,25-dihydroxyvitamin D3 induces activation of a neutral sphingomyelinase (SMase), resulting in a decrease in sphingomyelin (SM) levels and an increase in ceramide levels in a proposed "sphingomyelin cycle" of cell regulation (Okazaki, T., Bell, R., and Hannun, Y. (1989) J. Biol. Chem. 264, 19076-19080). Cell-permeable synthetic ceramides induce HL-60 cell differentiation toward a monocytic lineage without conversion to sphingosine, suggesting that ceramide is a lipid mediator of cell differentiation (Okazaki, T., Bielawska, A., Bell, R., and Hannun, Y. (1990) J. Biol. Chem. 265, 15823-15831). In this study, we investigated a novel SMase that was activated 2-2.5 h after treatment of cells with 1 alpha,25-dihydroxyvitamin D3. The activated SMase was localized to the cytosolic fraction. It was inhibited by copper, ferric iron, and zinc and showed optimal activity at pH 7.5. A mixed micellar assay was developed for the enzyme, with optimal activity achieved at 12 mol% SM in Triton X-100 mixed micelles and at 20 mol% SM in deoxycholate micelles. The activity was modestly enhanced by phosphatidic acid, phosphatidylserine, or phosphatidylinositol, but not by other major phospholipids. Purification was performed by chromatography on DEAE anion-exchange, Q-Sepharose Fast Flow, hydroxylapatite, sphingosylphosphocholine affinity, and Superose 12 gel filtration columns. Two peaks of activity with molecular masses of 45 and 95 kDa were resolved by gel filtration chromatography on Superose 12. The specific activities of the purified 45- and 95-kDa enzymes were 2780 and 2790 nmol/mg/h, respectively. These data identify a novel cytosolic, magnesium-independent, neutral SMase(s) that is activated during cell differentiation.

C-myc protein expression during cell cycle phases in differentiating HL-60 cells.

We examined c-myc protein expression in cell cycle phases during differentiation induction of HL-60 cells by flow cytometry using an indirect immunofluorescence method. In exponentially proliferating HL-60 cells, c-myc protein was expressed in a cell cycle dependent manner. During the differentiation induction of HL-60 cells with dimethylsulfoxide (DMSO), c-myc protein was rapidly down-regulated in the G1/0 specific phase prior to the appearance of differentiation associated markers. Our results indicate that c-myc protein functions in the G1/0 specific phase in cellular differentiation, and the rapid down-regulation of c-myc protein in G1/0 phase is closely associated with initial differentiation programs.

Effect of substituting fluorine for hydrogen at C-26 and C-27 on the side chain of 1,25-dihydroxyvitamin D 3

Previous reports have demonstrated that introduction of fluorine atoms at C-26 and C-27 of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) results in the potentiation of various aspects of some biological activities. The higher biological activities of 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D 3 (26,27-F 6-1,25-(OH) 2D 3) were accounted for in part by a decrease in metabolic inactivation via the 26-and 27-hydroxylation pathways. In addition to 26,27-F 6-1,25-(OH) 2D 3 not being hydroxylated in the 26 and 27 positions, it did not undergo 24-hydroxylation despite a significant induction by 26,27-F 6-1,25-(OH) 2D 3 of 24-hydroxylase activity in the HL-60 cell system. Another fluorinated vitamin D 3 analog, 26,26,26,27,27,27-hexafluoro-1α-hydroxyvitamin D 3 (26,27-F 6-1α-OH-D 3) may not undergo 25-hydroxylation as efficiently as 1α-OH-D 3 in vivo because a rise in serum 26,27-F 6-1,25-(OH) 2D 3 levels after injection of 26,27-F 6-1α-OH-D 3 was delayed significantly with a much smaller amplitude. Furthermore, 26,26,26,27,27,27-hexafluoro-1,23( S),25-trihydroxyvitamin D 3 retained full activity in the induction of HL-60 cell differentiation even after 23( S)-hydroxylation, in contrast to 1,23( S),25-(OH) 3D 3. These data suggested that substitution of fluorines for hydrogens at C-26 and at C-27 positions may result in alteration in chemical reactivity and/or conformation of C-23, C-24 and C-25 positions of the 1,25(OH) 2D 3 molecule.

Defective translocation of protein kinase C in multidrug-resistant HL-60 cells confers a reversible loss of phorbol ester-induced monocytic differentiation

Previous studies have demonstrated that human HL-60 myeloid leukemia cells differentiate in response to phorbol esters. This event is associated with induction of the c-jun early response gene and appearance of a monocytic phenotype. The present studies have examined the effects of vincristine-selected, multidrug resistance on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 cell differentiation. The results demonstrate that multidrug-resistant HL-60 cells, designated HL-60/vinc, fail to respond to TPA with an increase in c-jun transcripts or other phenotypic characteristics of monocytic differentiation. By contrast, treatment of HL-60/vinc cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases, induces c-jun transcription, growth arrest, and expression of the c-fms gene. Studies were also performed with an HL-60/vinc revertant (HL-60/vinc/R) line that has regained partial sensitivity to vincristine. The finding that HL-60/vinc/R cells respond to TPA with induction of a monocytic phenotype, but not c-jun expression, suggests that c-jun induction is not obligatory for monocytic differentiation. Other studies further demonstrate that the jun-B and fra-1 genes are induced by TPA in both HL-60/vinc and HL-60/vinc/R cells, whereas c-fos expression is attenuated in the HL-60/vinc line. Since TPA activates protein kinase C (PKC), we examined translocation of PKC from the cytosol to the membrane fraction. Although HL-60 and HL-60/vinc/R cells demonstrated translocation of PKC activity, this subcellular redistribution was undetectable in HL-60/vinc cells. Activity of the mitogen-activated protein kinase family with associated phosphorylation of c-Jun Y-peptide was markedly diminished in TPA-treated HL-60/vinc cells, but not in response to okadaic acid. Taken together, these findings suggest that vincristine resistance confers insensitivity to TPA-induced differentiation and can include defects in PKC-mediated signaling events and induction of jun/fos early response gene expression.