Differentiation Of Human Leukemia Cells Research Articles

Calcitriol Derivatives with Two Different Side Chains at C-20. V. Potent Inhibitors of Mammary Carcinogenesis and Inducers of Leukemia Differentiation

Calcitriol is implicated in many cellular functions including cellular growth and differentiation, thus explaining its antitumor effects. It was shown that gemini, the calcitriol derivative containing two side chain at C20, is also active in gene transcription with enhanced antitumor activity. We have now further optimized both the A-ring and the two side chains. The chemical structures of the resulting 18 geminis were correlated with biological activities. Those containing the 1alpha-fluoro A-ring are the least active. Those featuring 23-yne and 23(E) side-chains are generally more active in human breast cancer cell growth inhibition and human leukemia cell differentiation induction than their 23(Z) counterparts. On the basis of these evaluations, we selected as lead compound a 20(R) gemini, related to calcitriol in terms of it is A-ring, where one side chain was modified by introduction of a 23-yne function and replacement of the geminal methyl groups with trifluoromethyl groups, the other created by extension of C21 with a 3-hydroxy-3-trideuteromethyl-4,4,4-trideutero-butyl moiety.

Oral small-molecule tyrosine kinase inhibitor midostaurin (PKC412) inhibits growth and induces megakaryocytic differentiation in human leukemia cells

Midostaurin (PKC412), a small-molecule multiple tyrosine kinase inhibitor, has been shown to suppress the growth of various tumor cells. Since kinases inhibited by midostaurin are involved in megakaryocytic differentiation, we hypothesized that this novel target therapeutic might have a role in the treatment of human leukemia cells. Hence, we examined the effect of midostaurin on human erythroleukemia cells and evaluated potential mechanisms. Midostaurin inhibited the growth of both K562 and HEL cells in dose- and time-dependent manner. Morphological changes such as enlarged contours, multipolarity of mitotic spindles, and multinucleation of megakaryocytes were noted in both cell lines treated by midostaurin. A smaller population of apoptotic cells was also observed. DNA histogram revealed polyploid and hypoploid populations of midostaurin-treated cells. Midostaurin treatment enhanced the surface expression of the megakaryocytic marker CD61; in contrast, the erythroid marker glycophorin A expression was decreased. At optimal conditions for inducing megakaryocytic differentiation, midostaurin upregulated the expression and signaling of c-Mpl, a thrombopoietin receptor-encoding gene, in HEL cells. These results indicate that midostaurin can inhibit growth; induce megakaryocytic differentiation; and to a lesser extent, cause apoptosis in HEL cells. This effect might involve the expression and signaling of c-Mpl.

Gene expression profiles in differentiating leukemia cells induced by methyl jasmonate are similar to those of cytokinins and methyl jasmonate analogs induce the differentiation of human leukemia cells in primary culture

Jasmonates are potent lipid regulators in plants that play pivotal roles in their biological activities. Methyl jasmonate (MJ) is very effective at inducing the myelomonocytic differentiation of human myeloid leukemia HL-60 cells. We examined the gene expression profiles associated with exposure to MJ using cDNA microarrays, and compared the results with those obtained with other inducers of differentiation, such as all-trans retinoic acid (ATRA), 1alpha,25-dihydroxyvitamin D(3) (VD(3)), isopentenyladenine (IPA) and cotylenin A (CN-A). Many genes were upregulated, and only a small fraction was downregulated, upon exposure to the inducers. MJ, IPA and CN-A, but not ATRA or VD(3), immediately induced the expression of mRNA for the calcium-binding protein S100P. The gene expression profile induced by MJ resembled that induced by IPA, suggesting that these inducers share many common signal transduction systems for inducing the differentiation of leukemia cells. Methyl 4,5-didehydrojasmonate was about 30 times more potent than MJ and the natural form of the stereoisomer was more effective than the unnatural isomer. It significantly stimulated both the functional and morphological differentiation of leukemia cells that had been freshly isolated from patients with hematological malignancies. Jasmonate derivatives may be promising therapeutic agents for differentiation therapy of leukemia.

Pectenotoxin-2 represses telomerase activity in human leukemia cells through suppression of hTERT gene expression and Akt-dependent hTERT phosphorylation

In this study, we found that pectenotoxin-2 (PTX-2) decreased cell viability and inhibited telomerase activity with downregulation of hTERT expression in human leukemia cells. PTX-2 treatment also reduced c-Myc and Sp1 gene expression and DNA binding activity. Further chromatin immunoprecipitation assay demonstrated that PTX-2 attenuated the binding of c-Myc and Sp1 to the regulatory regions of hTERT. We also observed that PTX-2 treatment attenuated the phosphorylation of Akt, thereby reducing the phosphorylation and nuclear translocation of hTERT. We concluded that PTX-2 suppressed telomerase activity through the transcriptional and post-translational suppression of hTERT and this process precedes cellular differentiation of human leukemia cells. Structured summaryMINT-6742762:hTERT (uniprotkb:O14746) physically interacts (MI:0218) with AKT (uniprotkb:P31749) by anti bait coimmunoprecipitation (MI:0006)

Induction of differentiation of human leukemia cells by combinations of COX inhibitors and 1, 25-dihydroxyvitamin D3 involves Raf1 but not Erk 1/2 signaling

Differentiation therapy of cancer is being explored as a potential modality for treatment of myeloid leukemia, and derivatives of vitamin D are gaining prominence as agents for this form of therapy. Cyclooxygenase (COX) inhibitors have been reported to enhance 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation of promyeloblastic HL60 cells, but the mechanisms of this effect are not fully elucidated, and whether this potentiation can occur in other types of myeloid leukemia is not known. We found that combination treatment with 1,25D and non-specific COX inhibitors acetyl salicylic acid (ASA) or indomethacin can robustly potentiate differentiation of other types of human leukemia cells, i.e. U937, THP-1, and that ASA +/- 1,25D is effective in primary AML cultures. Increased cell differentiation is paralleled by arrest of the cells in the G1 phase of the cell cycle, and by increased phosphorylation of Raf1 and p90RSK1 proteins. However, there is no evidence that this increase in phosphorylation of Raf1 is transmitted through the ERK module of the MAPK signaling cascade. Transfection of small interfering (si) RNA to Raf1 decreased differentiation of U937 cells induced by a combination of ASA or indomethacin with 1,25D. However, phosphorylation levels of ERK1/2, though not of p90RSK, were increased when P-Raf1 levels were decreased by the siRNA, suggesting that in this system the ERK module does not function in the conventional manner. Identification of the strong antiproliferative activity of ASA/1,25D combinations associated with monocytic differentiation has implications for cancer chemoprevention in individuals who have a predisposition to myeloid leukemia.

Epigenetic regulation of telomerase in retinoid-induced differentiation of human leukemia cells

Changes in the promoter methylation of hTERT, the gene that encodes telomerase, a ribonucleoprotein responsible for replacing telomeric repeats, have been demonstrated in differentiating cells where hTERT is inhibited, suggesting epigenetic regulation of hTERT. All-trans retinoic acid (ATRA) induces differentiation in human leukemia cells and has had significant clinical success treating promyelocytic leukemia in what is termed 'differentiation therapy'. It is thought that the inhibition of telomerase is a target of retinoids and is closely tied to the differentiated phenotype. This study demonstrates the epigenetic changes associated with ATRA-induced inhibition of telomerase activity, including the hypoacetylation and hypermethylation of the hTERT promoter. Further, we have found changes in the differential expression of the three DNA methyltransferases during ATRA-induced differentiation of HL60 human leukemia cells. These results suggest that alteration of DNA methylation may play a role in the activation of telomerase in cancer cells and that epigenetic mechanisms may represent a target for differentiation therapy mechanisms. We propose that epigenetic changes in the hTERT promoter represent a stable locking mechanism in the retionoid-induced suppression of telomerase activity.

Induction of differentiation of myeloid leukemia cells in primary culture in response to lithocholic acid acetate, a bile acid derivative, and cooperative effects with another differentiation inducer, cotylenin A

Lithocholic acid (LCA) acetate induced the differentiation of human leukemia cells. Treatment with a combination of LCA acetate and cotylenin A, an inducer of the differentiation of leukemia cells, was more effective than that with LCA acetate or cotylenin A alone at inducing monocytic differentiation. LCA acetate activated mitogen-activated protein kinase (MAPK) before inducing differentiation. Cotylenin A did not activate MAPK, suggesting that cotylenin A has a different mode of action. The cooperative effects of LCA acetate and cotylenin A on inducing differentiation were, at least partly, due to the enhancement of LCA acetate-induced MAPK activation by cotylenin A.

Verticinone induces cell cycle arrest and apoptosis in immortalized and malignant human oral keratinocytes

Although verticinone, a major alkaloid isolated from the bulbus of Fritillaria ussuriensis, has been shown to induce differentiation in human leukemia cells, the exact mechanism of this action is not completely understood in cancer cells. Verticinone was used to conduct growth and apoptosis-related experiments for two stages of oral cancer on immortalized human oral keratinocytes (IHOKs) and primary oral cancer cells (HN4). The procedures included MTT assay, three-dimensional (3-D) raft cultures, Western blotting, cell cycle analysis, nuclear staining and cytochrome c expression related to the apoptosis signaling pathway. Verticinone inhibited the proliferation of immortalized and malignant oral keratinocytes in a dose- and time-dependent manner. In 3-D organotypic culture, verticinone-treated cells were less mature than the control cells, displaying low surface keratinization and decreased epithelial thickness. The major mechanism by which verticinone inhibits growth appears to be induced apoptosis and G(0)G(1) cell cycle arrest. This finding is supported by the results of the cell cycle analysis, FITC-Annexin V staining, DNA fragmentation assay and Hoechst 33258 staining. Furthermore, the cytosolic level of cytochrome c was increased, while the expression of Bcl-2 protein was gradually down-regulated and Bax was up-regulated, accompanied by caspase-3 activation. The data suggests that verticinone may induce apoptosis through a caspase pathway mediated by mitochondrial damage in immortalized keratinocytes and oral cancer cells.

Fibroblast nemosis arrests growth and induces differentiation of human leukemia cells

Interactive signaling between cancer cells and stroma plays an important role in determining tumor development. We recently found tumor cell-derived factors to induce fibroblast clustering, and that the high amounts of hepatocyte growth factor/scatter factor (HGF/SF) produced by these cell-cell contact-activated fibroblasts enhanced the invasiveness of c-Met-expressing cancer cells. We now observed that leukemia cells lacking c-Met respond to this novel type of fibroblast activation, nemosis, with growth arrest and differentiation to a dendritic cell-like phenotype. This effect was counteracted by introduction of c-Met expression into these cells. Moreover, those leukemia cell lines harboring properly processed c-Met showed no effect in response to nemosis. We propose this effect to be mediated by nemosis-derived cytokine signals, and present the potential candidates induced in the nemotic fibroblasts: interleukins-1, -6, -8, -11, leukemia inhibitory factor and granulocyte-macrophage-colony-stimulating factor. Our results emphasize the role of activated stromal fibroblasts in controlling progression of certain hematologic malignancies in a c-Met expression-dependent manner.

PI 3-K signaling pathway suppresses PMA-induced expression of p21WAF1/Cip1 in human leukemia cells

Despite the understanding of the importance of phosphoinositide 3-kinase (PI 3-K) signaling pathway in the regulation of cellular proliferation, little is known about its role during phorbol 12-myristate 13-acetate (PMA)-induced differentiation in human leukemia cells. Here, we report a novel finding that PI 3-K inhibition by LY294002 significantly increases p21WAF1/Cip1 expression in PMA-stimulated human leukemia cells NB4 and THP1. LY294002 potentiated expression of p21WAF1/Cip1 via a p53-independent mechanism and did not affect mitogen activated protein kinase (MAPK) activation. Electrophoretic mobility shift (EMSA) experiments revealed that blocking of PI 3-K was associated with increased binding of transcription factor Sp1 to the PMA-responsive sites on the p21WAF1/Cip1 promoter. Pretreatment with rapamycin, an inhibitor of mTOR kinase, decreased the expression of p21WAF1/Cip1 protein in PMA-stimulated NB4 cells. The level of PMA-induced p21WAF1/Cip1 protein expression was lower in NB4 cells overexpressing wild type protein kinase C zeta (PKC zeta) compared to those transfected with empty vector or with kinase inactive PKC zeta. Sp1 binding to the p21WAF1/Cip1 promoter was completely lost in a wild type PKC zeta overexpressing and PMA-stimulated NB4 cells. We demonstrate that PI 3-K signaling pathway suppresses PMA-induced expression of p21WAF1/Cip1 in human leukemia cells, and that this effect is partly mediated by PKC zeta.

Pyrrolo[2,3-b]Pyridinic Derivatives Induce Cell Growth Inhibition of the Myeloblastic HL-60 Cell Line.

Cytokins, N6-subsitued adenine derivatives play an important role in many different processes in plant development (Mok et al, Annu Rev Plant. Physiol Plant Mol Biol 2001, 52:89–118), including the cell growth and division control, the vegetable cell differentiation with auxin and the storage of various metabolites as alkaloids (Yahia et al, Plant Science 1998, 133:9–15). Furthermore, several studies suggest that cytokinins and their purin derivated are able to control mammalian cell apoptosis and differentiation of human leukemic cells (HL-60 myeloblastic cell line) into mature granulocytes (Ishii et al, Cell Growth Differ 2002, 13: 19–26). All these compounds exert their biological activity via Cyclin-Dependant Kinase (CDK) inhibition and particulary CDK1 and CDK2. The aim of our study was the synthesis of new 7-azaindole derivates as cytokinin analogues and the evaluation of their biological effects on HL-60 cells.Eight analogues of 7-azaindole were prepared by the condension of (N-methyl-)4-chloropyrrolo[2,3-b]pyridine with corresponding amines using palladium-catalyzed reaction (Hartwig et al, Angew Chem IN Ed 1998, 37: 2046–2067). The four derivates from 1H-pyrrolo[2,3-b]pyridine were 4-phenylamino-7-azaindole, 4-benzylamino-7-azaindole, 4-phenethylamino-7-azaindole and 4-phenylpiperazylamino-7-azaindole. The four derivates from 1-methyl-pyrrolo[2,3-b]pyridine were 4-phenylamino-N-methyl-7-azaindole, 4-benzylamino-N-methyl-7-azaindole, 4-phenetylamino-N-methyl-7-azaindole and 4-phenylpiperazylamino-N-methyl-7-azaindole. HL-60 cells were exposed to three concentrations of these compounds (10–100–500 μM) during 72h at 37°C. The number of viable cells was determined by Trypan blue exclusion, and the cell cycle was assessed by propidium iodide staining followed by flow cytometric analysis. All these compounds decreased the number of viable cells. The compounds of the NH serie were more active than their methyl-analogues, especially 4-phenylamino-N-methyl-7-azaindole and 4-phenethylamino-7-azaindole which presented an estimated IC50 of 2 μM. Moreover, when used at 10 μM, 4-phenylamino-N-methyl-7-azaindole induced apoptosis whereas 4-phenethylamino-7-azaindole promoted inhibition of the cell cycle without pro-apoptotic effect.These results suggest that cytokinin analogues derived from 1H-pyrrolo[2,3-b]pyridine may present interesting therapeutic potential as cytostatic agents. Further studies will clarify their biological effects on leukemic cells.

Vitamin D Receptor Signaling of Monocytic Differentiation in Human Leukemia Cells: Role of MAPK Pathways in Transcription Factor Activation

Among the many important physiological functions of the activated vitamin D receptor (VDR) is the signaling of monocytic differentiation, first demonstrated by conversion of malignant myeloid leukemia cells to nonproliferating cells with mature monocyte/macrophage appearance. However, the understanding of how 1, 25-dihydroxyvitamin D3 (1,25D) signals monocytic differentiation is still developing. Recent advances summarized here include the role of the principal "mitogen-activated protein kinase" (MAPK) pathways, their potential downstream target the CCAAT/enhancer binding protein beta (C/EBP beta), cell cycle related proteins, and cyclin-dependent kinase 5 (Cdk5) in 1,25D-induced differentiation. The precise steps by which activated VDR signals differentiation are incompletely understood in any of the cell types known to respond to 1,25D. We have focused our studies on HL60 cells, a widely available cell line derived from a patient with promyeloblastic leukemia, with the goal of achieving as clear a picture as possible with the currently available tools. In this model, outlined in Fig. 1, a plausible sequence of events is presented, with the caveats that these are not the only pathways activated by liganded VDR, and that several other pathways, also operative, remain to be convincingly demonstrated. The details of the scheme will be discussed in the sections below.

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-κB 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.

Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells

Many proteins require the binding of trace metals such as Ca, Fe, Cu, or Zn, which may modulate their structure, function, or activity. To determine if there were any overall changes in metalloprotein distribution or metal concentration during the process of macrophage differentiation we induced human myeloid HL-60 leukemia cells with phorbol 12-myristate 13-acetate (PMA) and quantitatively mapped their metal content using hard X-ray fluorescence micro-analysis. We found a transient increase in the zinc content of HL-60 cell nuclei during the early stages of differentiation induction. This finding was confirmed by spectrofluorometry in HL-60 cells and extended to U-937 leukemia cells. A role for protein kinase C-β (PKC-β) in this process was established by examining zinc content in an HL-60 variant, HL-525, which is PKC-β deficient, and in HL-525 cells in which PKC-β was restored by stable overexpression. Chemical chelation of both Cu and Zn served to inhibit macrophage differentiation in HL-60 cells, indicating a requirement for these metals during this process. Finally, we demonstrate that growth of HL-60 cells in a low-zinc environment removes their susceptibility to PMA-induced differentiation, and that this capacity can be partially restored by the addition of exogenous zinc.

Tumor Necrosis Factor-a Enhances Human Leukemia Cell Differentiation Induced by DMSO, but Not Retinoic Acid.

One of the human leukemia treatment methods is to differentiate leukemia cells into mature cells. Because differentiated cells lose their proliferative and tumor-forming abilities, differentiation inducers may be useful for the treatment of leukemia. Differentiation of leukemia cells has been studied using HL60 cells, a human promyelocytic leukemia cell line, which can be differentiated into granulocyte-like or monocyte/macrophage-like cells by various pharmacological agents such as dimethyl sulfoxide (DMSO), retinoic acid and phorbol myristic acetate (PMA). We previously reported that nuclear factor - kB (NF-kB) activation plays the important role in DMSO-induced differentiation of HL60 cells. Thus, we hypothesized that NF-kB activators could enhance DMSO-induced differentiation of HL60 cells. Here we examine whether tumor necrosis factor-a (TNF-a), a potent NF-kB inducer, enhance DMSO-induced differentiation of HL60 cells. TNF-a was found to enhance HL60 cell differentiation induced by DMSO. CD11b, a differentiation marker, was increased in 0.5 % DMSO-treated cells compared to control cells. When TNF-a was added to the same condition, CD11b expression was further enhanced in a dose and a time dependent manners. We also found that nitro blue tetrazolium (NBT) reducing activity, a marker for granulocytic differentiation, was further increased in DMSO plus TNF-a treated cells compared to only DMSO- treated cells. However, TNF-a alone had no effect on CD11b expression and NBT reducing activity. The enhancement of DMSO-induced HL60 differentiation by TNF-a was offset by NF-kB inhibition. Interestingly, retinoic acid- induced differentiation of HL60 cells showed no enhancing effect of TNF--a on the differentiation. These findings indicate that TNF--a might affect only NF-kB dependent differentation of HL60 cells. Taken together, we demonstrated that TNF-a enhances DMSO-induced differentiation of HL60 cells by stimulating NF-kB activation. Our results suggest that NF-kB inducers such as TNF-a are useful for the treatment of leukemia in combination with DMSO.

Translational study of vitamin D differentiation therapy of myeloid leukemia: effects of the combination with a p38 MAPK inhibitor and an antioxidant

Human myeloid leukemia cell lines are induced to terminal differentiation into monocyte lineage by 1,25-dihydroxyvitamin D3 (1,25D3) or its analogs (deltanoids). However, translation of these findings to the clinic is limited by calcemic effects of deltanoids. Strategies to overcome this problem include combination of deltanoids with other compounds to induce differentiation at lower, noncalcemic, deltanoid concentrations. We previously showed that either carnosic acid, an antioxidant, or SB202190, a p38 MAPK inhibitor, increase the potency of 1,25D3 in the HL60 cell line. Here, we report that simultaneous addition of both these agents further increases differentiation potency of deltanoids in this cell line and in freshly obtained leukemic cells ex vivo. Activity of MAPK pathways showed that increased differentiation was associated with enhanced activity of JNK pathway in all responding cell subtypes. Our studies suggest that patients with CML or AML subtypes M2 and M4, but not M1, M3 or M4eo, are particularly suitable for this combination therapy. We conclude that the established cell line HL60 presents a good model for some, but not all, subtypes of myeloid leukemia, and that the JNK pathway plays an important role in monocytic differentiation of human leukemic cells ex vivo, as well as in vitro.

Autocrine signaling of platelet-derived growth factor regulates disabled-2 expression during megakaryocytic differentiation of K562 cells

Platelet-derived growth factor (PDGF) is involved in megakaryocytopoiesis and is secreted into the culture medium during megakaryocytic differentiation of human leukemic cells. We investigate whether PDGF plays a role in the regulation of the adapter protein Disabled-2 (DAB2) that expresses abundantly in platelets and megakaryocytes. Western blot analysis revealed that conditioned medium from 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated, megakaryocytic differentiating K562 cells upregulated DAB2 expression. DAB2 induction and megakaryocytic differentiation was abrogated when cells were co-treated with the PDGF receptor inhibitor STI571 or when the conditioned medium was derived from TPA-plus STI571-treated cells. Although the level of PDGF mRNA was not altered by STI571, an approximate 44% decrease in PDGF in the conditioned medium was observed. Consistent with these findings, interfering PDGF signaling by PDGF neutralization antibody or dominant negative PDGF receptors attenuated DAB2 expression. Accordingly, transfection of an expression plasmid encoding secreted PDGF upregulated DAB2. This study shows for the first time that PDGF autocrine signaling regulates DAB2 expression during megakaryocytic differentiation.

Cooperation between antioxidants and 1,25‐dihydroxyvitamin D3 in induction of leukemia HL60 cell differentiation through the JNK/AP‐1/Egr‐1 pathway

Vitamin D derivatives have demonstrated anti-cancer activity, but their clinical use is precluded by hypercalcemia. Previously, we found that carnosic acid potentiates differentiation of human leukemia cells induced by low concentrations of 1alpha,25-dihydroxyvitamin D(3) (1,25D(3)). In this study, we investigated if this effect is a general property of antioxidants, and whether there is a common mechanism whereby antioxidants potentiate monocytic differentiation. We found that all antioxidants tested enhanced differentiation-related cell cycle arrest induced by a low (1 nM) concentration of 1,25D(3). Addition of antioxidants to 1,25D(3) activated the JNK pathway as indicated by increased phosphorylation of c-jun and ATF-2, although each compound alone had a minimal effect. Antioxidants also enhanced the 1,25D(3)-induced AP-1 DNA binding and transactivation ability. Expression of Egr-1 and c-fos was increased by combinations of antioxidants and 1,25D(3), in parallel with the activation of the JNK pathway. The potentiation of differentiation by antioxidants was inhibited by JNK inhibitor SP600125 and a dominant negative JNK 1/2 construct, and Egr-1 and c-fos expression was proportionally decreased, suggesting that JNK pathway regulates these transcription factors. While potentiating the prodifferentiation effect of 1,25D(3), antioxidants did not promote the elevation of basal levels of intracellular calcium by 1,25D(3). The results indicate that JNK-AP1 pathway has an important role in the potentiation of 1,25D(3)-induced differentiation by antioxidants, and regulates expression of Egr-1 and c-fos. Combinations of antioxidants with 1,25D(3) should be further evaluated for use in cancer chemoprevention and therapy.

Ligation of CD44 by A3D8 Monoclonal Antibody Induces Terminal Differentiation of THP-1 Leukemia Cells by Promoting Cross-Talk between Extracellular Signal-Regulated Kinase and Phosphoinositide-3 Kinase/Akt Signaling Pathway.

Ligation of CD44 with anti-CD44 monoclonal antibody A3D8 induces terminal differentiation of human leukemia cells. However, the underlying molecular mechanisms remain largely unknown. In this study, we examined the importance of MEK/ERK, p38 MAPK, and phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in A3D8-induced monocytic differentiation of THP-1 leukemia cells. THP-1 cells displayed cytologic changes typical of mature monocytes and an increased expression of monocyte-specific antigen CD14 (49% ± 4%) and of myeloid-specific antigen CD11b (68% ± 6%) after 3 days of A3D8 treatment. The level of CD15 was also increased. The increase in the expression of these differentiation antigens was dose- and time-dependent. We found that CD44 ligation with A3D8 led to rapid and sustained activation of the essential kinases in the extracellular signal-regulated kinase pathway such as phospho-Raf-1, phospho-MEK1/2, and phospho-ERK1/2. However, the total levels of these kinases were not affected during the course of differentiation. Ser473 Akt phosphorylation was also observed shortly after the cells were exposed to A3D8 and sustained thereafter. In contrast, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and C-Jun N-terminal kinase were not increased by CD44 ligation. Pretreatment of cells with 20mM of MEK1 inhibitor PD98059 abrogated the A3D8-induced activation of MEK1/2 and ERK1/2 and potently inhibited the THP-1 differentiation. However, a p38 MAPK inhibitor SB203580 did not abolished the CD44 ligation-induced terminal differentiation of THP-1 cells. Pretreatment of cells with PI3-K inhibitor LY294002 resulted in a nearly complete inhibition of A3D8-induced terminal differentiation. Overexpression of dominant-negative Akt also reduced the A3D8-induced THP-1 differentiation, suggesting that A3D8-induced THP-1 differentiation is coupled to PI3-K/Akt activation. Surprisingly, the pharmacological inhibition of PI3-K blocked not only A3D8-induced Akt activation but also A3D8-induced activation of Raf, MEK and ERK pathway. By contrast, pretreatment of cells with PD98059 did not inhibit the A3D8-induced Akt activation, suggesting that PI3-K/Akt is the upstream of Raf/MEK/ERK pathway in A3D8-induced terminal differentiation of THP-1 leukemia cells. Taken together, our findings demonstrated that the cross-talk between the activation of A3D8-inducible PI3-K/Akt pathway and the Raf/MEK/ERK pathway in THP-1 cell exists, and plays a critical role during CD44 ligation-induced THP-1 differentiation.

Magnolol and honokiol enhance HL-60 human leukemia cell differentiation induced by 1,25-dihydroxyvitamin D 3 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 D 3 (VD 3) 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 μM MG or HK. When added to 1 nM VD 3, 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 VD 3 or ATRA treatment further enlarged the G 0/G 1 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 VD 3 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 VD 3 and ATRA in the treatment for acute promyelocytic leukemia.