19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.     

Effects of prostaglandin E2 and all-trans retinoic acid combination on induced differentiation of human acute promyelocytic leukemia NB4 cells

OBJECTIVE: A human promyelocytic leukemia (APL) cell line NB4 was used to demonstrate the synergistic effects between all-trans retinoic acid (ATRA) and prostaglandin E2 (PGE2) on growth inhibition and cytodifferentiation induction. METHODS: NB4 cells were cultured in the presence of either ATRA or PGE2 as a single agent or in combinations at various ratio. Cell growth was measured and myeloid differentiation was tested on consecutive days over the whole course of culture. RESULTS: PGE2 and ATRA synergistically induced the myeloid differentiation of NB4 cells. In comparison with ATRA alone, the combination of PGE2 with ATRA caused an almost 20-fold decrease of effective concentration of ATRA. CONCLUSION: The combination of ATRA and PGE2 at an appropriate ratio may provide a convenient oral regimen of combined differentiation therapy for a better clinical outcome in APL patients.     

Bronchial responsiveness to ultrasonic fog in occupational asthma due to toluene diisocyanate

Accumulating evidence suggests that prothymosin alpha has an as yet undefined intracellular, perhaps intranuclear, function related to cell proliferation. Prothymosin alpha mRNA and/or peptide levels increase when cells are stimulated to proliferate. Because proliferation and differentiation events are often inversely correlated, we examined prothymosin alpha gene expression during proliferation and differentiation of HL-60 myeloid leukemia cells. Steady-state levels of prothymosin alpha mRNA, which are high in exponentially growing HL-60, decrease within hours after induction of HL-60 to differentiate along the neutrophil pathway with dimethylsulfoxide (DMSO) or along the macrophage lineage with either tetradecanoylphorbol acetate (TPA) or bryostatin 1. The decline in prothymosin alpha mRNA in response to these differentiation signals parallels that of c-myc mRNA under the same conditions. We then determined whether the downregulation of prothymosin alpha and c-myc mRNA were due to differentiation or cessation or proliferation. Recombinant human gamma-interferon induces monocytic differentiation of HL-60, but permits continued proliferation, and, under these conditions, expression of prothymosin alpha, as well as of c-myc, mRNA remains elevated. We conclude that prothymosin alpha and c-myc expression are coregulated in differentiating HL-60 and that their expression correlates with the proliferative state of HL-60 cells, rather than with the differentiated state.     

Induction of thrombospondin-1 by all-trans retinoic acid modulates growth and differentiation of HL-60 myeloid leukemia cells

Thrombospondin-1 (TSP), a multifunctional extracellular matrix protein, modulates human hematopoietic stem cell adherence and thus may play a role in blood cell proliferation and/or differentiation. The expression of TSP was studied in the human myeloid leukemia cell line, HL-60, upon differentiation into monocytes by phorbol-13-monoacetate (PMA) or into granulocytes by all-trans retinoic acid (RA). HL-60 cells cultured under serum-free conditions constitutively secreted low amounts of TSP into the cultured medium, approximately 13 ng/10(6) cells/24 h. PMA used at 4 x 10(-8) M did not significantly modulate TSP secretion over a 24 h period. In contrast, RA at 10(-7) M induced a 5- to 10-fold increase in TSP secreted by HL-60 cells during their differentiation into granulocytes over a 5 day period. The role of secreted TSP in RA-dependent cessation of growth and differentiation was examined using blocking anti-TSP antibodies. In the presence of the polyclonal anti-TSP antibody R5 (25 microg/ml), growth of RA-treated HL-60 cells was maintained at control levels for up to 3 days and a concomitant delay in granulocytic differentiation was observed. Moreover, the addition of soluble TSP (0.5-5 microg/ml) to untreated HL-60 cells decreased their growth and promoted their differentiation in a dose-dependent manner. Using a neutralizing antibody to transforming growth factor beta (TGF-beta) or purified TGF-beta1 we further demonstrated that the effects of TSP were not mediated through activation of latent TGF-beta. These studies indicate that TSP decreases the proliferation and promotes the differentiation of HL-60 cells.     

Neplanocin A, a potent inhibitor of S-adenosylhomocysteine hydrolase, potentiates granulocytic differentiation of acute promyelocytic leukemia cells induced by all-trans retinoic acid

Several neplanocin A analogs were synthesized and their growth-inhibiting and differentiation-inducing activities on myelogenous leukemia cells were examined. An adenosine kinase-ineffective analog of neplanocin A was effective in inducing differentiation, suggesting that phosphorylation of the nucleoside is not essential for inducing the differentiation of leukemia cells. Neplanocin A induced functional and morphological differentiation of HL-60 cells, but did not effectively induce differentiation of NB4, a cell line derived from a leukemia patient with t(15;17). However, these cells have been known to undergo granulocytic differentiation upon treatment with all-trans retinoic acid (ATRA), and are used as a model for differentiation therapy in acute promyelocytic leukemia. Preexposure of NB4 cells to low concentrations of neplanocin A greatly enhanced the ATRA-induced differentiation of the cells, whereas representative antileukemic drugs such as cytosine arabinoside and daunomycin did not enhance this differentiation. A clinical strategy that combines intermittent treatment with neplanocin A analogs and a low dose of ATRA may increase the clinical response and decrease the adverse effects of ATRA.     

Ligation of cell surface CD38 protein with agonistic monoclonal antibody induces a cell growth signal in myeloid leukemia cells

CD38 is expressed during early stages of differentiation in normal and leukemic myeloid cells. Recently, CD38 has been shown to participate in intracellular signal transduction pathways following its ligation with CD38-specific mAbs. In this study we report that ligation of CD38 by one such agonistic mAb (IB4) induced proliferation of cultured leukemic cells in vitro. In HL-60, KG-1A, NB4, and OCI-AML-3 myeloid leukemia cell lines, IB4 mAb induced an increase in the proliferating cell fraction as determined by cell number, clonogenic assay, and flow cytometric analysis. The presence of Ab caused a dose-dependent increase in the number of CFU and an increase in cell divisions. HL-60-Dox cells (a HL-60-doxorubicin-resistant cell line), which have no detectable CD38 expression, failed to respond to IB4 mAb. The effect of CD38 ligation on cell growth was also evaluated in freshly isolated leukemic cells from patients with acute myelogenous leukemia (AML). A significant increase in the proliferating cell fraction (S+G2M) was observed in 50% of the patients incubated with IB4 mAb. In five of the six AML patients, anti-CD38 mAb stimulated the proliferation of AML colony-forming cells. These results suggest that ligation of CD38 can induce the proliferation of leukemic cells and may play a role in the propagation of leukemic cell clones in certain cohorts of AML patients.     

Phorbol ester causes ligand-independent activation of the androgen receptor

The effect of differentiating doses of all-trans retinoic acid (ATRA, 10(-6) M) and vitamin D3 (10(-7) M) was investigated on the nuclear levels of endogenous ceramide and protein kinase C-zeta (PKC-zeta) catalytic activity in HL-60 myeloid cells. ATRA induced a parallel increase of ceramide and catalytically active PKC-zeta into the nuclear compartment of HL-60 cells (peak at 72 h). On the other hand, vitamin D3 increased the levels of nuclear ceramide and PKC-zeta activity to a lesser extent and with a delayed kinetics compared to ATRA (peak at 96 h). Pretreatment of HL-60 cells with high pharmacological concentrations of exogenously-added C2-ceramide (10(-6) M) completely blocked the ATRA-mediated activation of nuclear PKC-zeta. Exogenous C2-ceramide (10(-6) M) also inhibited the granulocytic differentiation induced by ATRA, whereas it did not affect monocytic differentiation mediated by vitamin D3. Transient transfection experiments performed with a plasmid construct containing a constitutively active mutated form of the PKC-zeta cDNA fused in 3' to a fluorescent tag protein (pEGFP-PKC-zeta) demonstrated that the overexpression of catalytically active PKC-zeta was not accompanied by the appearance of a differentiated morphology. These findings suggest that nuclear PKC-zeta is necessary but not sufficient to induce granulocytic differentiation of HL-60 myeloid malignant cells.     

Autocrine regulation of matrix metalloproteinase-9 gene expression and secretion by tumor necrosis factor-alpha (TNF-alpha) in NB4 leukemic cells: specific involvement of TNF receptor type 1

Matrix metalloproteinases have been reported to be involved in tumor cell invasion and metastasis. Dissemination of malignant cells in acute myeloid leukemia (AML) may be mediated by similar mechanisms. Here, we report, that the t(15/17)+ acute promyelocytic leukemia (APL) cell line NB4 constitutively expresses and releases the proenzyme form of matrix metalloproteinase-9 (MMP-9, 92 kDa type IV collagenase/gelatinase, gelatinase B), as well as tissue inhibitor of metalloproteinases-1 (TIMP-1). Both proteins were identified by N-terminal amino acid sequence analysis after purification using gelatin Sepharose affinity chromatography. Whereas 12-O-tetradecanoylphorbol-13 acetate (TPA) increased both MMP-9 and TIMP-1 mRNA levels, tumor necrosis factor-alpha (TNF-alpha) stimulated only MMP-9 gene expression in a dose- and time-dependent manner. Neutralizing monoclonal antibodies (MoABs) to TNF-alpha (anti-TNF-alpha) decreased the constitutive and TPA-dependent expression of MMP-9 but did not influence TIMP-1 expression, either in unstimulated or in TPA-treated NB4 cells. FACS analyses showed that NB4 cells express both TNF receptor 1 (TNF-R1) and TNF-R2 to a similar extent. Blocking MoABs against TNF-R 1 (anti-TNF-R1) decreased the constitutive expression of MMP-9, whereas anti-TNF-R2 had almost no effect. Our results show, that in NB4 cells the expression of MMP-9 but not of TIMP-1 is maintained by autocrine stimulation with TNF-alpha. Thus, leukemic cells may be enabled to leave the bone marrow and infiltrate peripheral tissues by a dysfunction in the regulation of the MMP-9:TIMP-1 equilibrium, possibly triggered through autostimulation by TNF-alpha.     

Effect of acute acipimox administration on the rates of lipid and glycogen synthesis in cachectic tumor-bearing rats

2'-Deoxycoformycin (dCF), a specific and potent inhibitor of adenosine deaminase, has demonstrated significant antitumor effect on lymphoid malignancies. The drug induced functional and morphologic differentiation of myeloid leukemia cells in combination with 2'-deoxyadenosine (dAd), but not dCF alone. NB4, a cell line derived from a patient with t(15; 17) acute promyelocytic leukemia (APL) underwent granulocytic differentiation when treated with all-trans retinoic acid (ATRA) or dCF plus dAd, but not with cytosine arabinoside. Pre-exposure of NB4 cells to ATRA greatly potentiated differentiation induced by dCF plus dAd, but pretreatment with dCF plus dAd before exposure to ATRA was less effective. Differentiation of NB4 cells was effectively induced by clinically applicable concentrations of dCF in combination with dAd. These findings may provide useful information about induction of differentiation in vivo.     

Fibronectin-mediated cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation. The signaling role of protein kinase C-beta

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-beta-deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor alpha5 beta1 integrin. HL-525 cells, which constitutively display high levels of surface alpha5 beta1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that alpha5 beta1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.     

Prospective study of mutant frequencies at the hprt and T-cell receptor gene loci in pediatric cancer patients during chemotherapy

With the use of three different hematopoietic cell lineages, the downregulation of telomerase activity was found to be a general response to the induction of differentiation. The decrease in telomerase activity occurred as early as 24 h when HL-60 and K562 cells were cultured in the presence of 1alpha, 25 dihydroxyvitamin D3 (VD3), all-trans-retinoic acid (ATRA) and hemin, and completely disappeared after 3 days. On the other hand, MEG-01 cells showed a marked inhibition of telomerase activity after 6 days of culture with 12-0-tetradecanoylphorbal 13-acetate (TPA). The analysis of telomeric DNA in the HL-60 cells and K562 cells demonstrated no detectable loss of telomeric DNA with cellular differentiation, with a loss of telomerase activity. The repression of telomerase is a common molecular event during leukemic cell differentiation.     

The role of vitamin D derivatives and retinoids in the differentiation of human leukaemia cells

The capabilities of 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3), and two novel vitamin D analogues, EB1089 and KH1060, to induce the differentiation of two established leukaemia cell lines, U937 and HL-60, were assessed alone or in combination with the retinoid compounds, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA). The vitamin D derivatives acted to increase the differentiation of U937 and HL-60 cell cultures in a dose-dependent manner, as determined by nitroblue tetrazolium (NBT) reduction, with EB1089 and KH1060 being more effective than the native hormone. As an additional index of leukaemic cell differentiation, induction of expression of the phenotypic cell surface antigen, CD14, and the beta2-integrins, CD11b and CD18 by the vitamin D and retinoid compounds were monitored using fluorescence activated cell sorting (FACS) analyses. Following 96-hr treatment of U937 and HL-60 cells with 5 x 10(-10) M of the vitamin D derivatives, a striking increase in CD14 antigen expression was apparent, indicating the promotion by these compounds of a monocyte/macrophage lineage of cells. CD11b and CD18 antigen expression were also raised above control levels. In contrast, both retinoid compounds used at the higher concentration of 1 x 10(-8) M were not effective inducers of CD14 antigen expression. However, CD11b and CD18 were both readily increased in U937 and HL-60 cell cultures. Treatment of U937 cell cultures with the vitamin D compounds and the retinoids resulted in cooperative effects on induction of differentiation, with correlation by both NBT reduction and FACS analyses of CD14 antigen expression. The presence of 9-cis RA or ATRA appeared to contribute to the further increase of CD14 in these cells. HL-60 cell cotreatment with these compounds also displayed enhanced cooperative effects in phagocytic function by NBT reduction. However, analysis of CD14 revealed a dramatic diminution in HL-60 cells treated with the combinations of the vitamin D derivatives and the retinoids. Assessment of HL-60 cell morphology treated with these combinations demonstrated the presence of a mixed population of monocytes and granulocytes. CD11b and CD18 antigen expression was also enhanced in both cell lines with cotreatment. The ability of EB1089 and KH1060 to induce leukaemic cell differentiation may provide an additional option for therapeutic use alone or together with other differentiation agents such as 9-cis RA or ATRA.     

New developments in hematopoietic stem cell expansion

In the present study we investigated the effects of various doses of gamma-irradiation, followed by induction of granulocytic differentiation with all-trans-retinoic acid (ATRA), on proliferative rate, differentiation capability and oxidative metabolism of leukaemic cells from two different myeloid leukaemia cell lines, HL-60 and PLB-985. Regarding the effects of such combined treatment on the proliferative capabilities of HL-60 and PLB-985 cell lines, we showed that their growth kinetics were similar after 2 Gy gamma-irradiation combined with ATRA. However, with doses >2 Gy, the behaviour of the cell lines differed largely. Indeed, HL-60 appeared to be more radiosensitive than PLB-985 regarding cell viability and proliferation. Besides, whatever dose of irradiation (2, 5 or 10 Gy) was applied, ATRA was still able to induce differentiation of HL-60 and PLB-985 into granulocytes that retained the capacity to produce superoxide anion. The results of these in vitro studies suggest that leukaemia cell lines retain their ability to respond to ATRA, a granulocytic-differentiating inducer following high doses of irradiation. This may have implications for the use of radiation therapy in combination with ATRA for the treatment of extramedullary infiltrations of myeloid leukaemias in humans.