Induction of HL-60 cell differentiation by water-soluble and nitrogen-containing conjugates of retinoic acid and retinol

Retinoids induce the promyelocytic cell line, HL-60, to differentiate along the granulocytic pathway in vitro. A number of water-soluble and nitrogen-containing retinoids were synthesized in our laboratory [retinoyl-glucose (RAGL), retinyl-glucose (ROGL), retinoyl-adenosine (RADS), retinoyl-adenine (RAD), retinoyl-beta-glucuronide (beta RAG), and retinoyl-alpha-glucuronide (alpha RAG)]. These retinoids (10(-5) to 10(-8) M), as well as retinoic acid (RA) and retinol (ROL), were tested for their ability to induce the differentiation of HL-60 cells in vitro and to affect cell growth and viability during a 24- to 72-h incubation period. Differentiation was assessed by measuring the percentage of cells expressing the Mac-1 antigen on their cell surfaces. RA and the conjugates of RA were all quite active in inducing HL-60 cell differentiation, whereas ROL and ROGL had much less activity at equimolar concentrations. beta RAG, alpha RAG, RADS, and RAD were less toxic, whereas the glucose conjugates of retinol and retinoic acid (ROGL and RAGL) were both considerably more toxic than either RA or ROL at equimolar concentrations. All retinoids affected cell growth in a dose-dependent fashion. At 24 h, free RA or ROL was not detected in the cells after incubation with any of the retinoid conjugates.     

Magnolol and honokiol enhance HL-60 human leukemia cell differentiation induced by 1,25-dihydroxyvitamin D3 and retinoic acid

Magnolol (MG) and honokiol (HK), two lignans showing anti-inflammatory and anti-oxidant properties and abundantly available in the medicinal plants Magnolia officinalis and M. obovata, were found to enhance HL-60 cell differentiation initiated by low doses of 1,25-dihydroxyvitamin D3 (VD3) and all-trans-retinoic acid (ATRA). Cells expressing membrane differentiation markers CD11b and CD14 were increased from 4% in non-treated control to 8-16% after being treated with 10-30 microM MG or HK. When added to 1 nM VD3, MG or HK increased markers expressing cells from approximately 30% to 50-80%. When either MG or HK was added to 20 nM ATRA, only CD11b, but not CD14, expressing cells were increased from 9% to 24-70%. Under the same conditions, adding MG or HK to VD3 or ATRA treatment further enlarged the G0/G1 cell population and increased the expression of p27(Kip1), a cyclin-dependent kinase inhibitor. Pharmacological studies using PD098059 (a MEK inhibitor), SB203580 (a p38 MAPK inhibitor) and SP600125 (a JNK inhibitor) suggested that the MEK pathway was important for VD3 and ATRA-induced differentiation and also its enhancement by MG or HK, the p38 MAPK pathway had a inhibitory effect and the JNK pathway had little influence. It is evident that MG and HK are potential differentiation enhancing agents which may allow the use of low doses of VD3 and ATRA in the treatment for acute promyelocytic leukemia.     

Ca2+ and phorbol ester synergistically induce HL-60 differentiation

Exposure of HL-60 cells to subthreshold concentrations of TPA caused monocytic differentiation only when cells were cotreated with the Ca2+ ionophore A23187. Phorbol ester dose-response curves for growth arrest and enzymatic markers of differentiation were shifted to lower concentrations when the ionophore was present. Expression of a monocyte/granulocyte cell surface antigen also occurred only when cells were treated with both agents. Similar effects were seen with other active but not inactive phorbol esters and with another Ca2+ ionophore. The Ca2+ component of phosphoinositide-based signalling may thus play a role in HL-60 differentiation.     

Synergistic action of tiazofurin and retinoic acid on differentiation and colony formation of HL-60 leukemia cells

Tiazofurin and retinoic acid synergistically induced differentiation and inhibited colony formation in HL-60 human promyelocytic leukemia cells in cell culture. The synergism was the result of different mechanisms of action, since the effect of tiazofurin, unlike that of retinoic acid, was prevented by addition of guanosine. Since it has been shown that tiazofurin down-regulated the expression of c-Ki-ras oncogene, and retinoic acid that of the myc oncogene, the joint impact of these drugs is of clinical interest particularly in end-stage leukemia where the therapeutic usefulness of tiazofurin has recently been demonstrated.     

Differential cellular distribution of retinoic acid during staurosporine potentiation of retinoic acid-induced granulocytic differentiation in human leukemia HL-60 cells

Pretreatment of cells with staurosporine, a protein kinase C (PKC) inhibitor, was found to potentiate the granulocytic differentiation induced by a brief (2 h) retinoic acid treatment. By cell cycle analysis, staurosporine was found to have little effect on the cell cycle. Retinoic acid was distributed equally in the nuclei (40%) and in the plasma membrane (40%) of staurosporine-pretreated cells while less than 20% of retinoic acid was found in the membrane of control non-staurosporine-pretreated cells during the retinoic acid-induced differentiation. These results indicate that the enhancing effect of staurosporine may be somehow associated with the localization of retinoic acid in the plasma membrane of the cell. -1This work is dedicated to Prof. Harris Busch (Baylor College of Medicine, Tex., USA) for his 33 wonderful years at Baylor and 50 years in medicine.     

Transglutaminase activity increases in HL60 cells induced to differentiate with retinoic acid and TPA but not with DMSO

HL60 cells induced to differentiate into myeloid cells by retinoic acid exhibited a 300-fold increase in transglutaminase (TGase) activity which peaked on day 5. HL60 cells induced to differentiate into monocytes by a phorbol ester tetradecanoylphorbol-12-myristate-13-acetate (TPA) had a greater than 840-fold increase in TGase activity on day 7. In contrast, cells induced to differentiate along the myeloid pathway by dimethyl sulfoxide (DMSO) exhibited no increase in TGase activity. Elevation of TGase activity appears to be characteristic of monocyte differentiation and retinoic acid-induced myeloid differentiation but not of myeloid differentiation in response to DMSO.     

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.     

Combined effects of aphidicolin and retinoic acid on proliferation and differentiation of human leukaemic (HL-60) cells

The relationships between replicative DNA synthesis and retinoic acid (RA)-induced differentiation of human promyelocytic leukaemic (HL-60) cells are evaluated with the use of Aphidicolin, a specific and reversible inhibitor of DNA polymerase alpha (alpha). Addition of a sublethal concentration of Aphidicolin (0.4 microM) in culture for 3 days suppresses DNA synthesis to a similar level of the resting stage (day 8) in control cultures. DNA synthesis is reactivated to the level observed in the growing stage of control cultures once Aphidicolin is removed after 3 days in culture. The level of DNA synthesis at the early stage of RA-induction (day 3) is suppressed by only 17% when compared to control cultures. The inhibitory effect of Aphidicolin on DNA synthesis in both control cultures and RA-induced cell cultures is similar. However, no reactivation of DNA synthesis is observed after removal of Aphidicolin on day 3 from RA-induced cell cultures. Flow cytometric analysis of DNA content on day 3 reveals that cells accumulate in G1 and early S phases of the cell cycle after exposure to Aphidicolin with or without RA. Of interest is the fact that, while Aphidicolin alone did not induce cells to differentiate, neither did it interfere with RA-induced cell differentiation (the rate of RA-induced cell differentiation in the presence of Aphidicolin is similar to that of RA-treated cultures in the absence of Aphidicolin). These results suggest that the combined use of Aphidicolin and RA may inhibit leukaemic cell proliferation more effectively without causing severe cytotoxicity and without interfering with RA-induced cell differentiation.     

Realgar-induced differentiation is associated with MAPK pathways in HL-60 cells

The clinical efficacy and safety of realgar (arsenic sulfide, As(4)S(4)) in the treatment of acute promyelocytic leukemia in China have given rise to an upsurge in research on the underlying mechanism. We prepared realgar nanoparticles (RNPs) to examine their effect on the differentiation of HL-60 cells. Treatment with RNPs at 6 microM for 72 h induced cell differentiation that was assessed by morphological change, NBT reductive ability, and elevation of CD11b expression at both mRNA and protein levels. The RNP-induced differentiation was synergized, enhanced and suppressed by the inhibition of p38 MAPK, JNK and ERK pathways, respectively. Our findings demonstrate that MAPK signaling pathways are closely related to the RNP-induced differentiation in HL-60 cells.     

Control of HL-60 monocytic differentiation. Different pathways and uncoupled expression of differentiation markers

Control of expression of the terminally differentiated phenotype was studied using the human promyelocytic leukemia cell line HL-60. Three known inducers of HL-60 monocytic differentiation were compared: 1,25-dihydroxy vitamin D3, tetradecanoylphorbol acetate (TPA), and sodium butyrate. At concentrations where all three inducers resulted in similar courses of G1/0-specific growth arrest, the kinetics of appearance of certain differentiation markers typically characteristic of mature monocytic cells was determined. The markers were inducible oxidative metabolism, non-specific esterase activity, and the cell surface determinants Mo1, My4, and Mo2. The results indicate that: Regulation of the expression of these markers during induced monocytic differentiation is not controlled in common. The three monocytic inducers do not induce the same metabolic cascade leading to differentiation. Similar states of differentiation could thus be reached by different pathways apparently due to the fact that control of expression of different differentiation markers was not tightly coupled.     

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.     

Immunochemical evidence that three protein kinase C isozymes increase in abundance during HL-60 differentiation induced by dimethyl sulfoxide and retinoic acid

Activity of the Ca2+/phospholipid-dependent protein kinase C has been shown to increase during differentiation of the human promyelocytic leukemia cell line HL-60 by dimethyl sulfoxide and retinoic acid (Zylber-Katz, E., and Glazer, R. I. (1985) Cancer Res. 45, 5159-5164). Antipeptide antibodies were prepared that specifically recognize the alpha, beta, and gamma isozymes of protein kinase C in rat brain cytosol and HL-60 cell extracts. The three isozymes do not share a common tissue distribution pattern. The gamma enzyme is abundant in brain but a relatively minor component in HL-60 cells; the opposite is true for the alpha enzyme. All three isozymes increase at least 2-fold in abundance in HL-60 cells exposed to 1.2% dimethyl sulfoxide for 48 h. The increase in abundance of the alpha and beta isoforms reaches 7- and 5-fold, respectively, by 96 h without further increase in the abundance of the gamma isozyme. Similarly, all three isozymes increase at least 1.5-fold in abundance after 48 h and 3-fold after 96 h with 1 microM retinoic acid. No further increase in the abundance of any of the isozymes is seen between 96 and 144 h of incubation with retinoic acid. The increase in protein kinase C activity is not limited to the cytosolic forms of the enzyme; a parallel increase in membrane-associated protein kinase C is also observed during differentiation. Approximately 10% of total protein kinase C activity is membrane-associated in both control and differentiating cells. These studies provide the first immunochemical evidence that all three protein kinase C isozymes increase during HL-60 cell differentiation, and they suggest that the increase in the isozyme levels may be coordinately regulated.     

Human myelogenous leukemia cell line HL-60 cells resistant to differentiation induction by retinoic acid. Decreased content of glycosphingolipids and granulocytic differentiation by neolacto series gangliosides

We have recently reported that neolacto series gangliosides (NeuAc-nLc) are increased during granulocytic differentiation of human myelogenous leukemia cell line HL-60 cells induced by retinoic acid and that HL-60 cells are differentiated into mature granulocytes when the cells are cultivated with NeuAc-nLc (Nojiri, H., Kitagawa, S., Nakamura, M., Kirito, K., Enomoto, Y., and Saito, M. (1988) J. Biol. Chem. 263, 7443-7446). In contrast to these wild-type-HL-60 cells, HL-60 cells resistant to differentiation induction by retinoic acid showed a markedly decreased content of gangliosides, especially NeuAc-nLc, and did not show any increase in the content of gangliosides when cultivated with retinoic acid. Neutral glycosphingolipids, the precursors of gangliosides, were not accumulated in these resistant cells. When retinoic acid-resistant HL-60 cells were cultivated in the presence of NeuAc-nLc, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was dependent on the concentration of gangliosides and was accompanied by inhibition of cell growth. Wild-type HL-60 cells differentiated by NeuAc-nLc showed the changes in ganglioside composition, which were similar to those in wild-type HL-60 cells differentiated by retinoic acid; among the gangliosides changed, 2----3 sialylparagloboside and 2----3 sialylnorhexaosylceramide were increased. These findings suggest (a) that the synthesis of particular NeuAc-nLe molecules is an important step for retinoic acid-induced granulocytic differentiation and this step could be bypassed or replaced by exogenous NeuAc-nLc, and (b) that the defective synthesis of particular NeuAc-nLc molecules is responsible for the failure of differentiation induction in retinoic acid-resistant HL-60 cells by retinoic acid.     

Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.     

Neolacto-series gangliosides induce granulocytic differentiation of human promyelocytic leukemia cell line HL-60

Neolacto-series gangliosides having linear poly-N-acetyl-lactosaminyl oligosaccharide structure have been demonstrated to be increased characteristically during granulocytic differentiation of human promyelocytic leukemia cell line HL-60 cells induced by dimethyl sulfoxide or retinoic acid (Nojiri, H., Takaku, F., Tetsuka, T., Motoyoshi, K., Miura, Y., and Saito, M. (1984) Blood 64, 534-541). When HL-60 cells were cultured in the presence of neolacto-series gangliosides prepared from mature granulocytes, the cells were found to be differentiated into mature granulocytes on the basis of the changes of morphology, surface membrane antigens, nonspecific esterase activity, and the activity of phagocytosis and respiratory burst. The differentiation of cells was dependent on the concentration of gangliosides and accompanied with inhibition of cell growth. These findings suggest that the particular ganglioside molecules play an important role in regulation of cell differentiation and that the appearance of neolacto-series gangliosides on cell surface membrane not only triggers the differentiation but also determines the direction of differentiation in HL-60 cells.     

Suppression of ethanolamine-containing glycerophospholipid synthesis in HL-60 cells during retinoic acid-induced differentiation.

Synthesis and degradation of glycerophospholipids in HL-60 cells and retinoic acid (RA)-treated HL-60 cells were examined. The synthesis of each subclass of ethanolamine-containing glycerophospholipids was extremely suppressed in RA-treated HL-60 cells, while that of other glycerophospholipids was not seriously affected. A pulse-chase experiment revealed that about 88% of 1,2-diacyl and 28% of 1-alkenyl-2-acyl glycerophosphoethanolamine were degraded during 4 days in RA-treated HL-60 cells. These characteristics of metabolism observed in RA-treated HL-60 cells might be responsible for the change of subclass composition of ethanolamine-containing glycerophospholipids in HL-60 cells during differentiation to granulocytes.