Improved coupling between proliferation-arrest and differentiation-induction in ML-1 human myeloblastic leukemia cells

Proliferation and differentiation are coupled in normal cells and are aberrant in leukemia cells. The studies reported here were aimed at more effectively coupling proliferation-arrest and differentiation-induction in a human myeloblastic leukemia cell line (ML-1). This was accomplished by using reduced serum conditions in conjunction with a differentiation-inducing agent: cells were first incubated in reduced serum [0.3% fetal bovine serum (FBS)] instead of standard conditions (7.5% FBS) and, second, exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The effects of this protocol were as follows: first, cell proliferation was slowed and cells accumulated in G0/G1 phase of the cell cycle; this occurred with only a minimal decrease in viability [to approximately 88-92% (0.3% FBS) from greater than or equal to 96% (7.5% FBS)]. Second, the induction of differentiation was accelerated; this allowed the time of exposure to TPA to be decreased. Acceleration of induction was very pronounced when cells were maintained in 0.3% FBS both before and during exposure to TPA, with TPA at concentrations above the minimum sufficient for induction but below those causing significant cytotoxicity; as little as 1 hour of TPA exposure resulted in near-maximal induction (approximately 80%) with this protocol, compared to the greater than or equal to 1 day required with previous standard protocols. In sum, conditions that slow ML-1 cell proliferation (0.3% FBS) enhance TPA-induced differentiation, substantially narrowing the time frame of induction; these conditions should be useful for studying the molecular mechanisms that underlie the induction process.     

Differentiation-inducing and cytotoxic effects of tumor necrosis factor and interferon-gamma in myeloblastic ML-1 cells

The effects of the tumor necrosis factor (TNF), and a second pleiotropic cytokine interferon-gamma (IFN), were examined in a line of human myeloblastic leukemia cells (ML-1). By itself, TNF causes ML-1 to differentiate along the monocytic pathway. The cells exhibit an increase in Fc receptors and acquire the morphological characteristics of maturing phenotype. They remain viable and continue to proliferate (at greater than or equal to 50% of the control growth rate) even with 10(2)-10(4) units/ml TNF. IFN alone has similar effects, causing an increase in Fc receptors but little cytotoxicity. In contrast to either cytokine alone, the combination of TNF plus IFN causes a cessation of proliferation and extensive cell death. Cytotoxicity occurs in a synergistic fashion; it requires the simultaneous presence of both cytokines, occurring with concurrent but not sequential exposure. These different responses, differentiation (TNF alone) and cytotoxicity (TNF + IFN), occur with a similar range of doses (approximately 10(2)-10(4) units/ml) and in a similar time frame (beginning on day 2). In other cell types, IFN can augment either the differentiation-inducing or the cytotoxic effect of TNF. In ML-1, the combined application of TNF plus IFN results in a shift from differentiation to cytotoxicity.     

Identification of transferrin as a progression factor for ML-1 human myeloblastic leukemia cell differentiation

We have previously demonstrated (Guan X.-P., Hromchak, R. A., and Bloch, A. (1989) Cancer Commun. 1, 111-115) that ML-1 human myeloblastic leukemia cells differentiate to monocyte/macrophage-like cells by the sequential action of competence and progression factors. Tumor necrosis factor-alpha, transforming growth factor-beta, and the phorbol ester tetradecanoylphorbol acetate were found to induce competence, whereas a 77-kDa glycoprotein (DF77) isolated from mitogen-stimulated human leukocyte-conditioned medium initiated progression. In this communication we show DF77 to be an isoform of human transferrin. Hemin or soluble iron complexes did not induce differentiation progression, suggesting that the participation of transferrin in ML-1 cell differentiation may not be related to its iron-carrying capacity.     

Synergism of tumor necrosis factor and interferon-gamma in induction of differentiation of human myeloblastic leukemic ML-1 cells

Natural or recombinant human tumor necrosis factor (TNF) induced NBT-reducing activity of ML-1 cells in a dose-dependent manner. Interferon-gamma (IFN-gamma) induced NBT-reducing activity only marginally. However, when IFN-gamma was combined with TNF, induction of NBT-reducing activity was remarkably increased. IFN-alpha or -beta had almost no effect on the induction of NBT-reducing activity of ML-1 cells, either alone or in combination with TNF. Treatment with both TNF and IFN-gamma synergistically enhanced morphological changes, growth inhibition and activity of Fc receptors, and NBT reduction in ML-1 cells, but not phagocytic activity. The TNF treated cells were classified as macrophage-like by morphology, and by lineage-specific alpha-naphthyl acetate esterase stain. The results indicate that combinations of TNF and IFN-gamma act synergistically in the induction of differentiation of human myeloblastic ML-1 cells.     

Okadaic acid suppresses TPA-induced differentiation by stimulating G1/S transition in human myeloblastic leukaemia ML-1 cells

The association between the phosphorylation status of the retinoblastoma protein, pRb and changes in cell cycle control caused by either protein kinase C (PKC) or protein kinase A (PKA) stimulation was evaluated in human myeloblastic leukaemia ML-1 cells. TPA-induced PKC activation resulted in dephosphorylation of pRb and subsequently induced ML-1 differentiation based on morphological changes and CD14 expression. In the present study, we showed that inhibition of protein phosphatases (PP-1 and PP-2a) prevented the TPA-induced differentiation in ML-1 cells. Preinhibition of PP-1 and PP-2a activities with 1–100 nM okadaic acid dose-dependently blunted the decrease in the phosphorylation status of pRb obtained with TPA and overrode cell cycle arrest. PKA stimulation with 8-chlorophenylthio-cAMP (100 µM) decreased cell proliferation by 65% and the distribution of cells in the G₁ phase significantly increased from 38% to 83% concomitant with a 34% decline in the number of cells present in the S phase. In addition, PKA stimulation significantly decreased the pRb phosphorylation status but did not elicit CD14 expression, indicating that cAMP-induced dephosphorylation of pRb cannot by itself trigger differentiation in ML-1 cells.     

Growth factor-mediated K+ channel activity associated with human myeloblastic ML-1 cell proliferation

ML-1 cell proliferation is dependent on the presence of serumgrowth factors. Removing serum from the culture medium results ingrowth arrest and promotes differentiation. In this study, we foundthat a 4-aminopyridine-sensitiveK⁺ channel was highly expressed inproliferating ML-1 cells and significantly diminished inG₁-arrested ML-1 cells induced by serum deprivation but was restored within 30 min in these cells withaddition of 10% fetal bovine serum (FBS) or 5 ng/ml epidermal growthfactor (EGF). Intracellular adenosine 3',5'-cyclicmonophosphate (cAMP) levels, but not guanosine 3',5'-cyclicmonophosphate, were significantly increased in serum-deprived cellsstimulated by FBS or EGF, and the effects of FBS and EGF on the channelactivation were mimicked by exogenous cAMP. In inside-out patches,K⁺ channel activity wassignificantly increased by the cAMP-dependent protein kinase catalyticsubunit, whereas the effect of EGF on K⁺ channel activation was blockedby Rp-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphothioate. Together, our resultsdemonstrate that serum growth factors stimulateK⁺ channel activity inproliferation of ML-1 cells through protein kinase-inducedphosphorylation and suggest an important molecular mechanism for serumgrowth factor-stimulated mitogenesis in ML-1 cells.

     

Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

The diterpene triepoxide triptolide is a major active component of Tripterygium wilfordii Hook F, a popular Chinese herbal medicine with the potential to treat hematologic malignancies. In this study, we investigated the roles of triptolide in apoptosis and cell signaling events in human leukemia cell lines and primary human leukemia blasts. Triptolide selectively induced caspase-dependent cell death that was accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. Furthermore, we found that triptolide dramatically induced ROCK1 cleavage/activation and MLC and MYPT phosphorylation. ROCK1 was cleaved and activated by caspase-3, rather than RhoA. Inhibiting MLC phosphorylation by ML-7 significantly attenuated triptolide-mediated apoptosis, caspase activation, and cytochrome c release. In addition, ROCK1 inhibition also abrogated MLC and MYPT phosphorylation. Our in vivo study showed that both ROCK1 activation and MLC phosphorylation were associated with the tumor growth inhibition caused by triptolide in mouse leukemia xenograft models. Collectively, these findings suggest that triptolide-mediated ROCK1 activation and MLC phosphorylation may be a novel therapeutic strategy for treating hematological malignancies.     

Isolation and purification of protein kinase C from human leukemia ML-1 cells phosphorylation of human leukemia RNA polymerase II in vitro

Protein kinase C (PKC) was purified to near homogeneity from human leukemia ML-1 cells. The purified enzyme showed a single polypeptide band of 80 kDa on SDS-polyacrylamide gel after electrophoresis, and was totally dependent on Ca2+/phospholipid for activity. Diacylglycerol and the tumor-promoting on Ca2/phospholipid for activity. Diacylglycerol and the tumor-promoting phorbol esters stimulated the enzyme activity. Autophosphorylation of PKC purified from phenyl-Sepharose column showed both 80- and 37 kDa polypeptides. Further fractionation of PKC on a hydroxyapatite column revealed two peaks of enzyme activity, indicating that there may be two different forms of protein kinase C present in human leukemia cells. The purified PKC was used to phosphorylate RNA polymerase II of human leukemia cells in vitro and the autoradiogram showed that RNA polymerase II large subunits (240, 220 and 150 kDa) were phosphorylated in a time-dependent manner.     

All-trans retinoic acid induces apoptosis in acute myeloblastic leukaemia cells

The effect of all-trans retinoic acid (ATRA) on leukaemia cell differentiation, proliferation and induction of apoptosis was studied by using autonomously growing blast cells isolated from eight patients with acute myeloblastic leukaemia (AML) either at diagnosis ( n=4) or at relapse (n=4). No morphological or functional differentiation induced by ATRA was observed in any of the cases studied. In cell cultures, inhibition of leukaemia cell growth by ATRA was obvious, especially in the case of clonogenic cells, and it was both time- and concentration-dependent. Induction of apoptosis was more difficult to achieve. The cells retained over 90% viability in suspension when the ATRA exposure at any of the concentrations studied was 48 h or less. When the time of exposure to ATRA was longer than 48 h, the viability of the cells decreased in a concentration-dependent manner. Apoptosis was observed morphologically in each of the AML cases with 10-5 to 10-8 M ATRA, if the incubation time of cells in ATRA was 72 h. The percentage of apoptotic cells increased with increasing ATRA concentrations from 12± 9% of 10-8 M ATRA to 78±12% of 10-5 M ATRA. The DNA electrophoretic method was able to detect apoptosis in all the AML samples exposed to 10-7 and 10-6 ATRA for 48 h and occasionally in cases where lower concentrations and longer exposure time were used. In conclusion, the present study shows that it is possible to induce apoptotic leukaemia cell death in vitro with ATRA in AML, and this effect is dependent on both concentration and exposure time.     

Retinoic-acid-induced apoptosis in leukemia cells

Retinoic acid (RA) cures more than 75% of patients with acute promyelocytic leukemia (APL). Here, we review the various anti-cancer activities of retinoids and rexinoids, alone and in combination with other drugs, with emphasis on the RA-dependent induction of a cancer-cell-selective apoptosis signaling pathway to which multiple anti-cancer signals converge. These findings identify the TRAIL (tumor-necrosis-factor-related apoptosis-inducing ligand) pathway as a central cell-autonomous anti-cancer weapon that can act independently of the immune system.     

Membrane potential in human myeloid leukemia cell line ML-1: responsiveness of granulocytic and monocytic differentiated cells.

The membrane potential responsiveness of human myeloid leukemia cells (ML-1 line) was studied with the voltage sensitive fluorescent dye diS-C3-(5). The experimental procedure used in this study enabled us to assess the magnitude of the membrane potential change in cells treated with ouabain, 12-0-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), relative to the membrane potential in the untreated control. Inhibition of the Na, K-ATPase by ouabain was followed by a (20 +/- 4) mV depolarization. In undifferentiated homogeneous cell population TPA caused a (19.4 +/- 4.4) mV depolarization while FMLP had virtually no effect. Cells in which granulocytic or monocytic differentiation was induced by retinoic acid or 1,25-dihydroxyvitamin D3 exhibited under the effect of TPA a (57.8 +/- 7.1) mV and (34.8 +/- 10.9) mV depolarization, respectively. A very small transient depolarization was also observed up on treating of the cells with FMLP. The changes in the membrane potential responsiveness in the induced cells are obviously connected with the cell differentiation.     

RGD peptide-induced apoptosis in human leukemia HL-60 cells requires caspase-3 activation

RGD motif-containing peptides have been used in various studies of cell adhesion and growth. We report that RGD triggered apoptosis at a concentration of 1 mmol/L, whereas RAD-containing peptides failed to induce apoptosis in HL-60 cells. RGD-treated cells revealed internucleosomal DNA fragmentation. Western blot reveals caspase-3 activation in RGD peptide-treated cells. A caspase-3 inhibitor z-VAD-FMK completely blocked the apoptosis, but a caspase-1 inhibitor (Ac-YVAD-CMK) and caspase-2 inhibitor (z-VDVAD-FMK) did not block the apoptosis, suggesting that caspase-3 might have a critical role in the execution process of apoptosis induced by RGD. RGD peptides have been used extensively to inhibit tumor metastasis. Our results should help in further understanding the RGD peptide-induced apoptosis, which is important since RGD peptides have a potential role in therapies of the future. This revised version was published online in July 2006 with corrections to the Cover Date.     

Arsenic trioxide induces apoptosis of myeloid leukemia cells by activation of caspases

The primary objective of this study was to determine whether caspases are involved in arsenic trioxide(ATO)-induced apoptosis of human myeloid leukemia cells. A secondary objective was to determine whether apoptosis induced by ATO compared with VP-16 is differentially affected by an activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA), which has been reported to inhibit apoptosis induced by some chemotherapeutic agents. NB4 and HL60 cells were incubated with ATO in the presence and absence of the caspase protease inhibitors Z-VAD.fmk or Y-VAD. cho. Apoptosis was assessed by morphology, DNA laddering and flow cytometry. Poly (ADP-ribose) polymerase (PARP) cleavage was used as a marker for the activation of caspases. PARP cleavage occurred during ATO-induced apoptosis in both NB4 and HL60 cells. Z-VAD.fmk, a broad-spectrum inhibtor, could block ATO-induced apoptosis and PARP cleavage, whilst Y-VAD. cho, a selective inhibitor of caspase 1, had no such effect. PMA pre-incubation for up to 8 hours under conditions known to activate PKC had no effect on either ATO- or VP-16-induced apoptosis. We conclude that in cultured myeloid leukemia cells ATO-induced apoptosis is executed by caspases from the distal, PARP-cleaving part of the activation cascade and that PKC activation has no effect on apoptosis induced by either ATO or VP-16 in these cells.     

Chrysin-induced apoptosis is mediated through caspase activation and Akt inactivation in U937 leukemia cells

Chrysin is a natural, biologically active compound extracted from many plants, honey, and propolis. It possesses potent anti-inflammation, anti-cancer, and anti-oxidation properties. The mechanism by which chrysin initiates apoptosis remains poorly understood. In the present report, we investigated the effect of chrysin on the apoptotic pathway in U937 human promonocytic cells. We show that chrysin induces apoptosis in association with the activation of caspase 3 and that Akt signal pathway plays a crucial role in chrysin-induced apoptosis in U937 cells. Furthermore, we have shown that inhibition of Akt phosphorylation in U937 cells by the specific PI3K inhibitor, LY294002 significantly, enhanced apoptosis. Overexpression of a constitutively active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase 3, and PLC-gamma1 cleavage by chrysin. Together, these findings suggest that the Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to chrysin and raise the possibility that combined interruption of chrysin and PI3K/Akt-related pathways may represent a novel therapeutic strategy in hematological malignancies.     

The effects of recombinant CSF-1 on the blast cells of acute myeloblastic leukemia in suspension culture

Recombinant hemopoietic colony-stimulating factors (CSFs), including GM-CSF, G-CSF and IL-3, have been shown to be effective stimulators of both self-renewal and terminal differentiation of blast stem cells in acute myeloblastic leukemia (AML). We have examined the activity of a fourth growth factor, recombinant CSF-1 (or M-CSF), on the growth of leukemic blasts in culture. CSF-1 was found to be active on some, but not all, blast populations. In sensitive cells, CSF-1 often stimulated the production of adherent blast cells incapable of division. This observation leads us to suggest that CSF-1 may be useful in the treatment of selected cases of AML.