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The biological effects of low-dose radiation have attracted attention, but data are currently insufficient to fully understand the beneficial role of the phenomenon. In the present study, we have investigated the effects of low doses of gamma-irradiation alone and in combination with all-trans-retinoic acid (RA) on proliferation, apoptosis and differentiation of the human promyelocytic leukemia HL-60 cells. Changes in cell behavior and protein expression were determined with the use of light and fluorescent microscopy, immunocytochemical and Western blot analysis. Low-dose irradiation with 1–100 cGy caused a dose-dependent inhibition of HL-60 cell proliferation, and induced apoptosis and differentiation to granulocytes with an increase in the number of CD15-positive cells. Pre-irradiation with 1–100 cGy for 24 h before treatment with RA promoted apoptosis but did not impair RA-induced differentiation. Both processes were associated with a decrease in the expression of the proliferating cell nuclear antigen (PCNA), BCL-2, c-MYC, and changes in both cytosolic and nuclear levels of protein tyrosine-phosphorylation as well as protein kinase C alpha or beta isoforms. These results demonstrate the beneficial role of low-dose irradiation in modulating leukemia cell proliferation, differentiation and apoptosis.

The modifications of intracellular redox balance leads to important cellular changes in many cell types. Here, a causal relationship among redox state, granulocytic differentiation induced by all-trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) and apoptosis have been studied in the human acute promyelocytic leukaemia HL-60 cells. The modulation of intracellular reactive oxygen species levels by D: , L: -buthionine-(S, R) sulfoximide (BSO), and N: -acetyl-L: -cysteine (NAC) caused inducer- and time-dependent or stage-specific effects on HL-60 cell growth inhibition, differentiation and subsequent apoptosis. The presence of BSO during the commitment stage suppressed RA-but not dbcAMP-mediated differentiation, while NAC inhibited both. BSO alone and in combination with RA or dbcAMP-induced apoptosis, which was prevented by NAC in dbcAMP-but not in RA-treated cells. Using protein kinase C inhibitor, calphostin C, cross-talk effects between the intracellular redox state and PKC signalling was identified by demonstrating inducer-dependent changes in cell differentiation or apoptosis, which were associated with the changes in DNA-NF-kappaB binding activity. These observations suggest a critical role of redox state in determining HL-60 cell behaviour and provide new insights into the complex effects of redox perturbations on the intracellular signalling network via the involvement of PKC and NF-kappaB.

Acute promyelocytic leukemia KG1 cells with t(11;17) PLZF-RARalpha respond poorly to the differentiation inducer all-trans retinoic acid (RA), and the reason for the RA resistance is the recruitment of histone deacetylase by PLZF-RARalpha. Here, we demonstrate that histone deacetylase inhibitors (HDACIs), FK228, BML-210, phenyl butyrate, and vitamin B3, in different combinations with RA, act as KG1 cell growth inhibitors. Partial differentiation to granulocytes was induced by 3 micromol/L RA, and its combination with HDAC inhibitors did not enhance RA-induced but potentiated apoptosis. HDACIs induced accumulation of hyperacetylated histone H4. Chromatin immunoprecipitation analysis has revealed phenyl butyrate and its combinations with RA and vitamin B3 cause histone H4 acetylation in the p21 promoter regions corresponding to p53 and/or Sp1 sites. This was coincident with the activation of the transcription factor p53-binding activity to the p21 promoter in electrophoretic mobility shift assay. The results indicate the possibility of using the combination of agents for therapeutic strategy in RA-resistant acute myeloid leukemia to produce both differentiation and apoptosis.

FK228 (depsipeptide) is a novel histone deacetylase inhibitor (HDACI) that has shown therapeutical efficacy in clinical trials for malignant lymphoma. In this article, we examined in vitro effects of FK228 on human leukemia cell lines, NB4 and HL-60. FK228 alone (0.2-1 ng/mL) inhibited leukemia cell growth in a dose-dependent manner and induced death by apoptosis. FK228 had selective differentiating effects on two cell lines when used for 6 h before induction of granulocytic differentiation by retinoic acid (RA) or in combination with RA. These effects were accompanied by a time- and dose-dependent histone H4 hyper-acetylation or histone H3 dephosphorylation and alterations in DNA binding of NF-kappaB in association with cell death and differentiation. Pifithrin-alpha (PFT), an inhibitor of p53 transcriptional activity, protected only NB4 cells with functional p53 from FK228-induced apoptosis and did not interfere with antiproliferative activity in p53-negative HL-60 cells. In NB4 cells, PFT inhibited p53 binding to the p21 (Waf1/Cip1) promotor and induced DNA binding of NF-kappaB leading to enhanced cell survival. Thus, beneficial effects of FK228 on human promyelocytic leukemia may be exerted through the induction of differentiation or apoptosis via histone modification and selective involvement of transcription factors, such as NF-kappaB and p53.

Histone deacetylase inhibitors have a potent role in the strategy for the treatment of leukemias. BML-210 (N-(2-Aminophenyl)-N' phenyloctanol diamine) is the novel histone deacetylase inhibitor, and its mechanism of action has not been characterized. In this study, we examined the in vitro effects of BML-210 on the human leukemia cell lines (NB4, HL-60, THP-1, and K562). We found that BML-210 inhibits the growth of all cell lines and promotes apoptosis in a dose- and time-dependent manner. BML-210 alone induces HL-60 and K562 cell differentiation (up to 30%) to granulocytes and erythrocytes, respectively, and in combination with differentiation agents - all-trans retinoic acid and hemin, markedly potentates it. Those treatments cause G1 arrest and histone H4 acetylation, affects transcription factor NF-kappaB and Sp1 binding activity to their consensus sequences, the p21 or the FasL promoters, and influences expression of Sp1, NF-kappaB, p21 and FasL. These findings suggest that BML-210 could be a promising antileukemic agent to induce apoptosis and to modulate differentiation through the modulation of histone acetylation and gene expression.

The molecular mechanisms of the cellular response to different apoptotic effectors are only partially understood. Herein, the role of transcription factors, Sp1 and NFkappaB in differentiation-related and etoposide-induced apoptosis was examined in a number of human leukemia cell lines (HL-60, NB4, HEL, THP-1, K562). This was investigated with respect to the recruitment of one cell-cycle regulating gene, p21 and one cell death gene, FasL. Using electrophoretic mobility shift assay (EMSA), we consistently observed Sp1 and NFkappaB binding activity to the promoter of either gene during cell differentiation and the decrease associated with apoptosis upon long-term treatment with differentiation inducers in HL-60, NB4 and HEL cells. By contrast, Sp1 and NFkappaB binding capacities were lost in all myeloid cell lines undergoing etoposide-induced fast apoptosis. This effect was eliminated by the broad-spectrum caspase inhibitor, benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethylketone, thus restoring transcription factors' binding activity. However, sustained NFkappaB binding to the FasL promoter was noticed in apoptosis undergoing HEL cells treated by etoposide. Our results suggest that p21 and FasL gene activation is required for myeloid leukemia cell survival or maturation but not for cell death via Sp1 and NFkappaB as regulators of these genes. The findings also support the idea of a common mechanism for cellular responses to different apoptotic effectors in malignant hematopoietic cell lines.

Treatment of human acute promyelocytic leukemia cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest and differentiation toward monocytes, which subsequently die by apoptosis. However, the relationship between terminal differentiation and apoptosis remains unclear. Here we have studied Sp1 and nuclear factor kappaB (NF-kappaB) transcription factor activity in controlling promoters of cell cycle-regulating (p21/WAF1/CIP1) and cell death (FasL) genes during monocytic differentiation and apoptosis of the human acute promyelocytic leukemia cell lines NB4 and HL-60. Using the electrophoretic mobility shift assay, we observed that PMA treatment of NB4 cells caused an early response in Sp1 binding to the p21 and FasL promoters at 8 h. The firmly adherent cell phenotype, characteristic of differentiated cells, retained Sp1-binding activity to either promoter, but it was often lost completely in detached, apoptotic cells. The association of Sp1 with the p21 promoter during monocytic differentiation correlated with the levels of expressed p21 in the cytoplasmic fraction, as detected by immunoblotting. In HL-60 cells, very weak or no Sp1 binding to either promoter was observed. Low NF-kappaB affinity for its consensus sites and to the FasL promoter was characteristic of apoptotic cells. The results of this study suggest a positive role of Sp1 and NF-kappaB, as regulators of p21 and FasL genes, in leukemic cell survival and monocytic differentiation and a negative role in apoptotic cell death.

A main shortcoming of using HL-60 cells as a model of granulocyte-macrophage differentiation is that some cells in the differentiating population undergo apoptosis. To address this issue, we have identified which tyrosine-phosphorylated proteins are involved in apoptosis and differentiation, respectively. HL-60 cells were induced specifically to undergo apoptosis with 68 microM etoposide, and to undergo granulocytic differentiation with 1 microM retinoic acid (RA). The corresponding two-dimensional electrophoretic maps of tyrosine-phosphorylated proteins from treated cells were compared. In the 8 h etoposide-treated HL-60 cell population, 83% of the cells were apoptotic. In the 120 h RA-treated cells, 50% of the cells were apoptotic. Eighteen cytosolic and nuclear tyrosine-phosphorylated proteins were found in both the 8 h etoposide- and the 120 h RA-treated cells, but not in the proliferating HL-60 cell population. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry analyses suggested that some of the proteins may be involved in signal transduction pathways (NFkappaB, GTP-binding protein, protein disulfide isomerase, Cyclophilin A), others in cell transcriptional and translational control (hnRNP H, hnRNP L, Hsp60, Hp1, Hcc-1, 26S proteasome beta-subunit, ATP synthase beta-chain), and a third group in cell cytoskeleton organization and receptor cycling (profilin, caveolin-1). An understanding of signal transduction in apoptosis initiation by screening for tyrosine-phosphorylated proteins associated with apoptosis may provide new targets for the treatment of leukemia.

Therapeutic strategies targeting histone deacetylase (HDAC) inhibition have become promising in many human malignancies. Belinostat (PXD101) is a hydroxamate-type HDAC inhibitor tested in phase I and II clinical trials in solid tumors and hematological cancers. However, little is known about the use of belinostat for differentiation therapy against acute myelogenous leukemia. Here, we characterize the antileukemia activity of belinostat as a single drug and in combination with all-trans-retinoic acid (RA) in promyelocytic leukemia HL-60 and NB4 cells. Belinostat exerted dose-dependent growth-inhibitory or proapoptotic effects, promoting cell cycle arrest at the G0/G1 or the S transition. Apoptosis was accompanied by activation of caspase 3, degradation of PARP-1, and cell cycle-dependent changes in the expression of survivin, cyclin E1, and cyclin A2. Belinostat induced a dose-dependent reduction in the expression of EZH2 and SUZ12, HDAC-1, HDAC-2, and histone acetyltransferase PCAF (p300/CBP-associated factor). Belinostat increased acetylation of histone H4, H3 at K9 and H3 at K16 residues in a dose-dependent manner, but did not reduce trimethylation of H3 at K27 at proapoptotic doses. Combined treatment with belinostat and RA dose dependently accelerated and reinforced granulocytic differentiation, accompanied by changes in the expression of CD11b, C/EBPα (CCAAT/enhancer binding protein-α), and C/EBPε. Our results concluded the usefulness of belinostat, as an epigenetic drug, for antileukemia and differentiation therapy.

Aberrant DNA methylation is a critical epigenetic process involved in gene expression of tumor cells. Diverse DNA methyltransferase inhibitors are being studied as potential anticancer drugs, and there is interest in developing novel and more effective DNMTIs. We evaluated zebularine, a stable and low-toxic cytidine analog, effects on human promyelocytic leukemia cell lines, NB4 and KG1. Zebularine caused a dose- and time-dependent NB4 and KG1 cell growth inhibition, did not induce myeloid differentiation but triggered concentration-dependent apoptosis as manifested by procaspase-3 and PAR-1 cleavage and the occurrence of early apoptosis detected by Annexin-V-propidium iodide. Zebularine co-treatment with all-trans retinoic acid (RA) at pharmacological dose (1 μM for NB4 cells) and higher (3 μM for KG1 cells) increased granulocytic differentiation in both cell lines. Pretreatment for 24 or 48 h with zebularine before the treatment with different doses of RA alone or RA with histone deacetylase inhibitors, phenyl butyrate, and BML-210, resulted in significant acceleration and enhancement of differentiation and cell cycle arrest at G0/1. Zebularine alone or in sequential combination with RA decreased expression of DNMT1, caused fast and time-dependent expression of pan-cadherin and partial demethylation of E-cadherin but not tumor suppressor p15. When used in combination with RA, zebularine increased expression of both genes transcript and protein. Zebularine induced regional chromatin remodeling by local histone H4 acetylation and histone H3-K4 methylation in promoter sites of methylated E-cadherin and also in the promoter of unmethylated p21 as evidenced by chromatin immunoprecipitation assay. Our results extend the spectrum of zebularine effects and the evaluation its utility in acute myeloid leukemia therapy based on epigenetics.

DNA topoisomerase inhibitors induce a specific signaling cascade that promotes an active apoptotic caspase-dependent cell death process. However, little is known about the initial signals elicited by these agents. In the present study, we compared apoptosis in HL-60 cells treated either with the chemotherapeutic drug etoposide (VP16) alone or combined with the broad caspase inhibitor ZVAD.fmk. Apoptosis was assessed by changes in cell morphology and agarose gel electrophoresis of extracted cell DNA. We found that ZVAD.fmk prevents VP16-induced DNA fragmentation and the appearance of an increased number of apoptotic cells in the culture. We also compared the effects of etoposide alone or together with the pan-caspase inhibitor ZVAD.fmk on proliferating cell nuclear antigen, Bcl-2, and actin expression in human promyelocytic leukemia HL-60 cells. In addition, we screened for proteins that were initially upregulated in a caspase-dependent manner. Indeed, some proteins were induced in the cytoplasm and subsequently accumulated in the nuclei after etoposide treatment. This process was slightly inhibited by the caspase inhibitor ZVAD.fmk. We suggest that these proteins are associated with the induction of specific signaling cascades that characterize the apoptotic cell death process.

Two-dimensional electrophoretic analysis was used to assess quantitative and qualitative changes in the expression and tyrosine phosphorylation of cytoplasmic proteins of proliferating, differentiating HL-60 cells and mature human blood neutrophils. The total tyrosine phosphorylation level of cytoplasmic proteins appeared approximately constant during the pre-commitment period, i.e., 6–24 h after induction of differentiation by 700 nM all-trans retinoic acid. At the time of granulocytic phenotype formation (48–120 h), the total level of tyrosine phosphorylation of cytoplasmic proteins increased significantly. Tyrosine phosphorylation of cytoplasmic proteins in matured blood neutrophils was significantly lower than that of cytoplasmic proteins of HL-60 cells differentiated for 96 h with retinoic acid. Immunoblotting with anti-Erk2 and anti-phosphotyrosine monoclonal IgG2bk antibodies showed that Erk2 was expressed and tyrosine-phosphorylated at different levels in HL-60 proliferating cells and in cells at all stages of differentiation. Our data showed that tyrosine phosphorylation of cytoplasmic proteins in differentiating HL-60 cells changes dramatically during the period of phenotype formation and is accompanied by increasing activity of Erk2. An increasing number of apoptotic cells appeared in the differentiating HL-60 cell population during the granulocyte maturation stage (48–120 h of differentiation). The appearance at this time of differentiation of a new set of tyrosine-phosphorylated cytoplasmic proteins (also distinctive for apoptotic HL-60 cells mediated by etoposide) together with an increasing number of apoptotic cells in the differentiating population strongly suggests that these proteins are associated with the apoptotic process.