Control of differentiation and apoptosis of human myeloid leukemia cells by cytokinins and cytokinin nucleosides, plant redifferentiation-inducing hormones.

We examined the effects of various adenine analogues on the growth and differentiation of human myeloid leukemia HL-60 cells. Some of these analogues inhibit growth and induce nitroblue tetrazolium reducing activity in HL-60 cells. Cytokinins such as kinetin, isopentenyladenine, and benzyladenine were very effective in inducing nitroblue tetrazolium reduction and morphological changes in the cells into mature granulocytes. On the other hand, cytokinin ribosides such as kinetin riboside, isopentenyladenosine, and benzylaminopurine riboside were the most potent for growth inhibition and apoptosis. Cytokinin ribosides greatly reduced the intracellular ATP content and disturbed the mitochondrial membrane potential and the accumulation of reactive oxygen species, whereas cytokinins did not. When the cells were incubated with cytokinin ribosides in the presence of O(2)(-) scavenger, antioxidant or caspase inhibitor, apoptosis was significantly reduced and differentiation was greatly enhanced. These results suggest that both cytokinins and cytokinin ribosides can induce granulocytic differentiation of HL-60 cells, but cytokinin ribosides also induce apoptosis prior to the differentiation process.     

Design and synthesis of photolabile caged cytokinin

Cytokinins are phytohormones that regulate diverse developmental processes throughout the life of a plant. trans-Zeatin, kinetin, benzyladenine and dihydrozeatin are adenine-type cytokinins that are perceived by the AHK cytokinin receptors. Endogenous cytokinin levels are critical for regulating plant development. To manipulate intracellular cytokinin levels, caged cytokinins were designed on the basis of the crystal structure of the AHK4 cytokinin receptor. The caged cytokinin was photolyzed to release the cytokinin molecule inside the cells and induce cytokinin-responsive gene expression. The uncaging of intracellular caged cytokinins demonstrated that cytokinin-induced root growth inhibition can be manipulated with photo-irradiation. This caged cytokinin system could be a powerful tool for cytokinin biology.     

Chemical modification of santonin into a diacetoxy acetal form confers the ability to induce differentiation of human promyelocytic leukemia cells via the down-regulation of NF-kappaB DNA binding activity

Many sesquiterpene lactone compounds either induce or enhance the cell differentiation of human leukemia cells. However, we reported in a previous study that santonin, a eudesmanolide sesquiterpene lactone, exerts no effects on the differentiation of leukemia cells. In this report, to evaluate the possibility of chemically modifying santonin into its derivatives with differentiation inducing activity, we synthesized a series of santonin derivatives, and determined their effects on cellular differentiation in the human promyelocytic leukemia HL-60 cell system. A diacetoxy acetal derivative of santonin (DAAS) was found to induce significant HL-60 cell differentiation in a dose-dependent manner, whereas santonin in its original form did not. The HL-60 cells were differentiated into a granulocytic lineage when exposed to DAAS. In addition, the observed induction in cell differentiation closely correlated with the levels of NF-kappaB DNA binding activity inhibited by DAAS. Both Western blot analyses and kinase inhibitor studies determined that protein kinase C, ERK, and phosphatidylinositol 3-kinase were upstream components of the DAAS-mediated inhibition of NF-kappaB binding activity in HL-60 leukemia cells. The results of this study indicate that santonin can, indeed, be chemically modified into a derivative with differentiation inducing abilities, and suggest that DAAS might prove useful in the treatment of neoplastic diseases.     

Immediate up-regulation of the calcium-binding protein S100P and its involvement in the cytokinin-induced differentiation of human myeloid leukemia cells

Cytokinins are important purine derivatives that act as redifferentiation-inducing hormones to control many processes in plants. Cytokinins such as isopentenyladenine (IPA) and kinetin are very effective at inducing the granulocytic differentiation of human myeloid leukemia HL-60 cells. We examined the gene expression profiles associated with exposure to IPA using cDNA microarrays and compared the results with those obtained with other inducers of differentiation, such as all-trans retinoic acid (ATRA), 1 alpha,25-dihydroxyvitamin D3 (VD3) and cotylenin A (CN-A). Many genes were up-regulated, and only a small fraction were down-regulated, upon exposure to the inducers. IPA and CN-A, but not ATRA or VD3, immediately induced the expression of mRNA for the calcium-binding protein S100P. The up-regulation of S100P was confirmed at the protein expression level. We also examined the expression of other S100 proteins, including S100A8, S100A9 and S100A12, and found that IPA preferentially up-regulated S100P at the early stages of differentiation. IPA-induced differentiation of HL-60 cells was suppressed by treatment with antisense oligonucleotides against S100P, suggesting that S100P plays an important role in cell differentiation.     

Synthesis and biological evaluation of 7-azaindole derivatives, synthetic cytokinin analogues

Cytokinins, N6-substituted adenine derivatives, are plant hormones playing important roles in various processes in plant development. Furthermore, cytokinins and their derivatives are able to control mammalian cell apoptosis and differentiation. The aim of our study was the synthesis of 7-azaindole derivatives as cytokinin analogues with the Hartwig-Buchwald coupling reaction in order to evaluate their biological properties on human myeloblastic leukaemia cells (HL-60 cell line). All these compounds presented a cytotoxic activity on HL-60 cells especially the 4-phenylaminopyrrolo[2,3-b]pyridine and the 4-phenethylaminopyrrolo[2,3-b]pyridine.     

Hormonal Control of Differentiation of Shoots, Roots and Embryos in Leaf and Stem Cultures of Petunia inflata and Petunia hybrida

Factors influencing adventitious bud and root development, callus induction and embryogenesis were investigated in stem and leaf cultures of Petunia inflata R. E. Fries and Petunia hybrida cv. Cascade and cv. Rose du ciel grown on a synthetic nutrient medium. Indoleacetic acid caused limited callus development and root formation whereas naphthaleneacetic acid Induced abundant roots. 2,4-Dichlorophenoxyacetic acid promoted callus growth and differentiation of embryos which eventually developed into plantlets. Cytokinins such as benzyladenine, zeatin and kinetin induced bud development. A combination of auxins and cytokinins caused an interaction which was manifested in altered morphogenetic response. Thus 2,4-dichlorophenoxyacetic acid in conjunction with benzyladenine caused suppression of bud development and retarded differentiation of embryos. Likewise, when benzyladenine was used with indoleacetic acid root development was totally inhibited and abundant buds were produced.     

Induction of differentiation of HL-60 cells by protein kinase C inhibitor, K252a

To clarify the role of protein kinase C and protein kinase A in cell proliferation and differentiation, the effects of K252a and its derivatives (K252b, KT5720), which have different inhibitory activity to these protein kinases, on the proliferation and differentiation of HL-60 cells were investigated. The proliferation and DNA synthesis of the HL-60 cells were inhibited by K252a in a dose dependent manner. However, K252b and KT5720 which are more specific inhibitors of protein kinase C or protein kinase A, respectively, had no observable effect on cell proliferation. K252a (40nM) enhanced the differentiation of HL-60 cells induced by 1,25(OH)2D3, retinoic acid and DMSO. K252b and KT5720 did not affect 1,25(OH)2D3-induced differentiation. K252a significantly inhibited the differentiation induced by PMA. These results demonstrate that K252a but not its derivatives can function as an antitumor drug and enhancer of the differentiation induced by various inducers.     

Retinoic acid selectively activates the ERK2 but not JNK/SAPK or P38 map kinases when inducing myeloid differentiation

Summary Among the three major mitogen-activated protein kinase (MAPK) cascades—the extracellular signal regulated kinase (ERK) pathway, the c-JUN N-terminal/stress-activated protein kinase (JNK/SAPK) pathway, and the reactivating kinase (p38) pathway—retinoic acid selectively utilizes ERK but not JNK/SAPK or p38 when inducing myeloid differentiation of HL-60 human myeloblastic leukemia cells. Retinoic acid is known to active ERK2. The present data show that the activation is selective for this MAPK pathway. JNK/SAPK or p38 are not activated by retinoic acid. Presumably because it activates relevant signaling pathways including MAPK, the polyoma middle T antigen, as well as certain transformation defective mutants thereof, is known to promote retinoic acid-induced differentiation, although the mechanism of action is not well understood. The present results show that consistent with the selective involvement of ERK2, ectopic expression of either the polyoma middle T antigen or its dl23 mutant, which is defective for PLCγ and PI-3 kinase activation, or the Δ205 mutant, which in addition is also weakened for activation of src-like kinases, caused no enhanced JNK/SAPK or p38 kinase activity that promoted the effects of retinoic acid. However, all three of these polyoma antigens are known to enhance ERK2 activation and promote differentiation induced by retinoic acid. Polyoma-activated MAPK signaling relevant to retinoic acid-induced differentiation is thus restricted to ERK2 and does not involve JNK/SAPK or p38. Taken together, the data indicate that among the three parallel MAPK pathways, retinoic acid-induced HL-60 myeloid differentiation selectively depends on activating ERK but not the other two MAPK pathways, JNK/SAPK or p38, with no apparent cross talk between pathways. Furthermore, the striking ability of polyoma middle T antigens to promote retinoic acid-induced differentiation appears to utilize ERK, but not JNK/SPK or p38 signaling.     

Tumor necrosis factor induced DNA fragmentation of HL-60 cells

Tumor necrosis factor (TNF) induces differentiation of HL-60 cells, with only slight effects upon proliferation and little or no cytotoxicity. TNF induced cytotoxicity of other target cell lines has been associated with DNA fragmentation. To assess whether TNF-induced DNA fragmentation might also contribute to HL-60 differentiation, studies were performed using a [3H]-dThd release assay. Between 1 and 2 hours of culture, significant [3H]-dThd release was induced by TNF at concentrations of 10 U/ml and greater. This response was blocked by inhibiting energy metabolism, but not by several inhibitors of cell surface signal transduction, protein or RNA synthesis, or free radical scavengers. DNA electrophoresis of the released DNA disclosed a wide range of low molecular weight fragments. It is possible that TNF-induced DNA fragmentation contributes to HL-60 differentiation.     

Exogenous Ca2+ Inhibits Senescence-Retarding Effect of Cytokinins in Detached Rice Leaves

Cytokinins and Ca2+ singly retarded senescence of detached rice leaves. When Ca2+ was applied together with cytokinins, the effectiveness of cytokinins was significantly reduced. Ca2+ and cytokinins did not stimulate ethylene production synergistically, ruling out the possibility that ethylene was involved in the inhibition of cytokinin-induced senescence- retarding effect by Ca2+. The experiment with specific compounds known to increase (Ca ionophore A23187), or decrease (EGTA, LaCl3, Verapamil, chlorpromazine) cytosolic Ca2+ level indicated that the elevated cytosolic Ca2+ retards senescence.     

Introduction of deoxyribonucleoside triphosphates into intact cells by electroporation

A simple method, employing high-voltage electric discharge (electroporation), was developed to introduce phosphorylated nucleosides into the cytoplasm of viable cells. HL-60 leukemia cells permeabilized by this technique remained viable and incorporated deoxyribonucleoside triphosphates into nuclear DNA. Furthermore, DNA synthesis was depressed for at least 24 h in HL-60 cells made permeable to 1-beta-D-arabinosylcytosine 5'-triphosphate by this methodology. Electroporation was found to be applicable to the permeabilization of a wide variety of cell lines in culture to nucleotides, suggesting that this methodology may be useful for the introduction into intact cells of a wide variety of molecules that are not normally transported effectively.     

Guanine analog-induced differentiation of human promyelocytic leukemia cells and changes in queuine modification of tRNA.

Treatment of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient human promyelocytic leukemia (HL-60) cells with 6-thioguanine results in growth inhibition and cell differentiation. 6-Thioguanine is a substrate for the tRNA modification enzyme tRNA-guanine ribosyltransferase, which normally catalyzes the exchange of queuine for guanine in position 1 of the anticodon of tRNAs for asparagine, aspartic acid, histidine, and tyrosine. During the early stages of HGPRT-deficient HL-60 cell differentiation induced by 6-thioguanine, there was a transient decrease in the queuine content of tRNA, and changes in the isoacceptor profiles of tRNA(His) indicate that 6-thioguanine was incorporated into the tRNA in place of queuine. Reversing this structural change in the tRNA anticodon by addition of excess exogenous queuine reversed the 6-thioguanine-induced growth inhibition and differentiation. Similar results were obtained when 8-azaguanine (another inhibitor of queuine modification of tRNA that can be incorporated into the anticodon) replaced 6-thioguanine as the inducing agent. The data suggest a primary role for the change in queuine modification of tRNA in mediating the differentiation of HGPRT-deficient HL-60 cells induced by guanine analogs.     

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.     

A nuclear cAMP binding protein in retinoic acid-treated HL-60 cells

A cAMP binding protein was detected in HL-60 cells using photoaffinity labeling with 8-azido [32P]cAMP. The binding protein was found in a 0.35 M NaCl nuclear protein extract from untreated HL-60 cells and from the HL-60 cells induced to mature with retinoic acid. While the quantity of the cAMP binding protein did not change following the induced differentiation, a second form of the subunit, altered in charge, was present at 3 and 5 days after retinoic acid treatment. The findings indicate that the regulatory subunit of the type II cAMP-dependent protein kinase could be involved in nuclear functions associated with human myeloid cell differentiation.     

Characterization of the metabolic forms of 6-thioguanine responsible for cytotoxicity and induction of differentiation of HL-60 acute promyelocytic leukemia cells

HL-60 human acute promyelocytic leukemia cells that lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity have been developed by mutagenization and selection. These cells exhibited markedly decreased sensitivity to the cytotoxic action of 6-thioguanine (TG) and, in contrast to parental HL-60 cells, had the capacity to undergo terminal granulocytic differentiation after treatment with this purine antimetabolite. Analysis of extracellular and intracellular metabolites of TG revealed negligible metabolism of TG in these HGPRT- HL-60 cells. These findings are consistent with the concept that inhibition of cellular replication requires generation of analog nucleotide and suggest that TG itself is capable of initiation of differentiation. 6-Thioguanosine (TGuo) had limited activity, while beta-2'-deoxythioguanosine (dTGuo) was inactive, as an inducer of maturation of HGPRT- HL-60 cells. These cells converted relatively large amounts of the nucleosides to the free base TG; the simultaneous exposure of cells to 8-aminoguanosine (AGuo), an inhibitor of purine nucleoside phosphorylase activity, decreased the degradation of TGuo and dTGuo to TG and promoted the intracellular accumulation of TG nucleotides, presumably through the action of nucleoside kinase activities. In a double mutant deficient in both HGPRT and deoxycytidine kinase (DCK) activities, dTGuo was devoid of cytotoxicity and was an effective inducer of maturation. The potency of dTGuo as an inducer in this system was not significantly affected by the presence of AGuo. These results suggested that dTGuo itself was also an active initiator of maturation. Thus, induction of differentiation appeared to be due to the free base, TG, as well as its deoxynucleoside form, dTGuo, whereas the formation of TG nucleotides appeared to antagonize maturation and produce cytotoxicity.     

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.     

Effect of AML serum on normal human myeloid differentiation and hexamethylene bisacetamide-induced granulocytic differentiation of the human HL-60 promyelocytic leukaemic cell line.

The effect of serum from 32 AML patients on the normal human myeloid differentiation and the hexamethylene-bisacetamide induced granulocytic differentiation of HL-60 promyelocytic leukaemic cell line was studied. Nonadherent normal mononuclear marrow cells were cultured in vitro at a concentration of 5 x 10(5) cells/ml for 6 days with each of the 32 AML sera. Ten normal human AB sera were used as control. The results showed an inhibitory activity on both morphological and functional differentiation of normal human myeloid immature marrow cells by 29 out of the 32 AML sera tested. These 29 AML sera were added to cultures of HL-60 (2.5 x 10(5)/ml) leukaemia cell line which incorporated 2 mM hexamethylene-bisacetamide for 6 days. The results showed no significant inhibition of hexamethylene-bisacetamide induced granulocytic differentiation by any of the 29 AML sera. The efficacy of hexamethylene-bisacetamide in inducing differentiation in the presence of inhibitory factors suggests a possible role in the treatment of AML patients.     

The role of interferon-gamma in induction of differentiation of human myeloid leukemia cell lines, ML-1 and HL-60

Highly purified natural interferon-gamma (IFN-gamma) induced differentiation having characteristics that are associated with the human promyelocytic leukemia cell line, HL-60. Monoclonal antibody to INF-gamma neutralized its activity. However, the natural IFN-gamma had almost no inducing activity in ML-1, a human myeloblastic leukemia cell line. Similar results were obtained using recombinant IFN-gamma. Mitogen stimulated human leukocyte conditioned medium (LCM) induced differentiation of both ML-1 and HL-60 cells. After treatment of LCM with monoclonal antibody to IFN-gamma, LCM activity was reduced more than 50% in ML-1 cells, and 80% in HL-60 cells. Even if IFN-gamma was eliminated from LCM by affinity chromatography, the LCM induced differentiation of ML-1 and HL-60 cells, but IFN-gamma markedly enhanced the ML-1 cell differentiation induced by IFN-gamma free LCM. The results suggest that leukocytes produce differentiation inducing factor(s) other than IFN-gamma, and that IFN-gamma is both an inducer and an enhancer of induction of human myelogenous leukemia cells.