Effect of Dimethyl Sulfides on the Induction of Apoptosis in Human Leukemia Jurkat Cells and HL-60 Cells

Organosulfur compounds have been established to possess anticancer effects. To provide a better understanding of the biological function of dimethyl sulfides, dimethyl monosulfide (Me₂S), dimethyl disulfide (Me₂S₂), dimethyl trisulfide (Me₂S₃) and dimethyl tetrasulfide (Me₂S₄) were used as experimental materials to investigate their effects on apoptosis induction in human leukemia Jurkat cells and HL-60 cells. Treatment with 20 μM dimethyl sulfides for 24 h decreased the viability of both cells. The cell viability-reducing effect of these sulfides was in the following order: Me₂S₄ ≈ Me₂S₃ > Me₂S₂ ≈ Me₂S for Jurkat cells and Me₂S₄ > Me₂S₃ > Me₂S₂ ≈ Me₂S for HL-60 cells. Me₂S₃ and Me₂S₄ significantly induced DNA fragmentation and caspase-3 activation. The addition of GSH or NAC completely suppressed the sulfide-induced apoptosis. Our results indicate that dimethyl sulfides with a larger number of sulfur atoms more strongly induced apoptosis in both human leukemia cells via ROS production and caspase-3 activation.     

Apoptosis photoinduction by C60 fullerene in human leukemic T cells

The viability of normal (Wistar rat thymocytes) and transformed (human leukemia Jurkat cells) T cells after UV/Vis irradiation in the presence of pristine C60 fullerene was studied. The data obtained have shown that C60 fullerene exhibits cytotoxic effect against transformed T lymphocytes when combined with UV/Vis irradiation using mercury-vapor lamp (320-600 nm). C60 fullerene photocytotoxicity was not detected in thymocytes. C60-dependent photoinduced apoptosis of Jurkat cells was confirmed by DNA fragmentation and caspase-3 activation. No substantial increase of caspase-3 activation was observed in thymocytes treated with C60 fullerene plus irradiation, while antileukemic agent cytosine arabinoside was shown to induce caspase-3 activation both in Jurkat cells and thymocytes. The data obtained may be useful for development of photosensitizers for photodynamic therapy with selective action on leukemia cells.     

A novel lignan composition from Cedrus deodara induces apoptosis and early nitric oxide generation in human leukemia Molt-4 and HL-60 cells.

AP9-cd, a standardized lignan composition from Cedrus deodara consisting of (-)-wikstromal, (-)-matairesinol, and dibenzyl butyrolactol, showed cytotoxicity in several human cancer cell lines reported earlier. An attempt was made in this study to investigate the mechanism of cell death in human leukemia Molt-4 and HL-60 cells. It inhibited Molt-4 cell proliferation with 48-h IC(50) of approximately 15 microg/ml, increased sub-G0 cell fraction with no mitotic block, produced apoptotic bodies and induced DNA ladder formation. Flow cytometric analysis of annexinV-FITC/PI-stained cells showed time-related increase in apoptosis and post-apoptotic necrosis. All these biological end-points indicated cell death by apoptosis. Further, initial events involved massive nitric oxide (NO) formation within 4 h with subsequent late appearance of peroxides in cells; measured by flow cytometry using specific fluorescent probes. Persistently high levels of NO and peroxide appeared to decrease mitochondrial membrane potential (Psi(mt)) which was recovered by cyclosporin A in Molt-4 cells. AP9-cd caused 2-fold activation of caspase-3 in Molt-4 and 5-fold activation in HL-60 cells. Also caspases-8 and -9 were activated in HL-60 cells. Ascorbate suppressed the enhanced caspases activities indicating a pro-oxidant effect of AP9-cd. Further, caspase-3 activation correlated with NO generation that was partially impaired by nitric oxide synthase (NOS) inhibitors and ascorbate suggesting a role of pro-oxidant species in caspase-3 activation. AP9-cd produced no cytotoxicity in primary rat hepatocyte culture at the concentrations used. The studies indicated that AP9-cd mediated early NO formation leads to caspases activation, peroxide generation, and mitochondrial depolarization which may be responsible for mitochondrial-dependent and -independent apoptotic pathways involved in the killing of leukemia cells by AP9-cd.     

Differential expression of cytosolic activation factors for NADPH oxidase in HL-60 leukemic cells

Activation of NADPH oxidase in undifferentiated HL-60 leukemic cells and in HL-60 cells differentiated along the myeloid pathway with dibutyryl cyclic AMP (dbcAMP) or dimethyl sulfoxide (Me2SO) was studied. Upon stimulation with a calcium ionophore, a phorbol ester, arachidonic acid or gamma-hexachlorocyclohexane, Me2SO-differentiated HL-60 cells generated superoxide (O2-) at higher rates than dbcAMP-differentiated cells. Undifferentiated cells generated O2- only at low rates upon stimulation with the above agents. In cell-free systems, NADPH oxidase activity was reconstituted by combining membranes of undifferentiated or dbcAMP- or Me2SO-differentiated HL-60 cells, cytosol of Me2SO-differentiated cells and arachidonic acid. This basal O2- formation was enhanced several-fold by guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]), a potent activator of guanine nucleotide-binding proteins. In contrast, cytosol of dbcAMP-differentiated cells reconstituted O2- formation only in the presence of GTP[gamma S], and cytosol of undifferentiated cells was inactive. Submaximally stimulatory amounts of cytosolic protein of Me2SO- and dbcAMP-differentiated cells synergistically stimulated O2- formation in the presence but not in the absence of GTP[gamma S]. We conclude that differentiations of HL-60 cells with Me2SO and dbcAMP are not equivalent with respect to activation of NADPH oxidase and that two cytosolic activation factors are involved in the regulation of this effector system.     

Apigeninidin induces apoptosis through activation of Bak and Bax and subsequent mediation of mitochondrial damage in human promyelocytic leukemia HL-60 cells

Treatment of human promyelocytic leukemia HL-60 cells with apigeninidin could induce cytotoxicity (IC₅₀ = ～80 μM), along with apoptotic sub-G₁ cells, TUNEL-positive apoptotic DNA fragmentation, activation of the multidomain pro-apoptotic Bcl-2 proteins (Bak and Bax), mitochondrial membrane potential (Δψ_m) loss, release of mitochondrial cytochrome c and AIF into the cytoplasm, activation of caspase-9, -3, -8, and -7, and cleavage of PARP and lamin B. These induced apoptotic events were accompanied by decrease of Bcl-2 level and increase of Bak and Bax levels. Apigeninidin-induced sub-G₁ cells and activation of Bak and Bax were also detected in human acute leukemia Jurkat T cells, but not in Jurkat T cells overexpressing Bcl-2. Pretreatment of HL-60 cells with the pan-caspase inhibitor z-VAD-fmk reduced significantly apigeninidin-induced sub-G₁ cells and caspase cascade activation, whereas it failed to suppress Bak and Bax activations, Δψ_m loss, and release of mitochondrial cytochrome c and AIF. None of FADD and caspase-8 deficiencies affected the sensitivity of Jurkat T cells to apigeninidin-induced cytotoxicity. These results demonstrated that apigeninidin-induced apoptosis was mediated by activation of Bak and Bax, mitochondrial damage and resultant release of not only cytochrome c, causing caspase cascade activation, but also caspase-independent death effector AIF in HL-60 cells.     

Quercetin-induced apoptosis of HL-60 cells by reducing PI3K/Akt

To explore the effect and mechanism of quercetin on proliferation and apoptosis of leukemia cells, and provide a theoretical basis for its clinical application. HL-60 leukemia cell lines was treated with different dose quercetin, the proliferation activity of leukemia cells was assessed by MTT method; the morphological changes of apoptosis of HL-60 cells, including nuclear condensation and DNA fragmentation, were observed by Hoechst 33258 fluorescence staining, the apoptosis rate and caspase 2,3 activation were assessed by flow cytometry, and the cell signal pathway including phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (pAkt), Bcl-2, Bax were detected by western blotting. Quercetin could significantly decrease the proliferation activity of HL-60 cells through the blockade of G(0)/G(1) phase, and induce the apoptosis of HL-60 cells in a time- and dose-dependent manner. Quercetin caused leukemia cells apoptosis by decreasing the protein expression of PI3K and Bax, the inhibitory phosphorylation of Akt, the decreased levels of Bcl-2 protein and increased activations of caspase-2 and -3, and increased poly(ADP-ribose) polymerase cleavage. Our results indicate that the apoptotic processes caused by quercetin are mediated by the decrease of pAkt and Bcl-2 levels, the increase of Bax level, and the activation of caspase families in HL-60 cells.     

Photodynamic therapy induces caspase-3 activation in HL-60 cells

Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. In this study, we show for the first time that photodynamic therapy (PDT), using benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) as the photosensitizer, induces the complete cleavage and subsequent activation of caspase-3 (CPP32/Yama/Apopain) but not caspase-1 (ICE) in human promyelocytic leukemia HL-60 cells. Poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of DNA dependent protein kinase (DNA PK(CS)) were cleaved within 60 min of light activation of BPD-MA. The general caspase inhibitor Z-Asp-2,6 dichlorobenzoyloxymethylketone (Z-Asp-DCB) blocked PARP cleavage while the serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and N-tosyl-lysyl chloromethyl ketone (TLCK) blocked the cleavage of caspase-3 suggesting that they act upstream of caspase-3 activation. All three inhibitors were able to block DNA fragmentation that was induced by treatment with BPD-MA followed by light application. These studies demonstrate that protease activity, particularly that of caspase-3, is triggered in HL-60 cells treated with lethal levels of BPD-MA and visible light.     

Hydroxylation at C4' or C6 is essential for apoptosis-inducing activity of flavanone through activation of the caspase-3 cascade and production of reactive oxygen species

Previous studies demonstrated that hydroxyl groups play important roles in the antioxidative activities of flavonoids; however, the importance of structurally related hydroxylation in their apoptosis-inducing activities is still undefined. In the present study, flavanone with hydroxylation at C4' and C6 had a significant cytotoxic effect in human leukemia HL-60 cells accompanied by the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, characteristics of apoptosis. The replacement of a hydroxyl group (OH) by a methoxyl (OCH3) group at C4' or C6 attenuated the apoptotic effect in cells, and there was no significant cytotocity of flavanone or flavanone with OH or OCH3 in C7-treated HL-60 cells. Induction of enzyme activity of caspase-3 and -9, but not caspase-1 and -8, accompanied by release of cytocrome C from mitochondria to cytosol and the appearance of cleaved of PARP (85 kDa), D4-GDI (23 kDa), and caspase-3 (p17/p15) fragments, was identified in 4'-OH- or 6-OH- flavanone-treated HL-60 cells. Caspase-3 and -9 inhibitors Ac-DEVD-FMK and Ac-LEHD-FMK, but not caspase-1 and -8 inhibitors Ac-YVAD-FMK and Ac-LETD-FMK, attenuated 4'-OH- or 6-OH-flavanone-induced cell death. And, inhibition of capsase-9 activity by Ac-LEHD-FMK suppresses caspase-3 protein procession induced by 4'-OH- and 6-OH-flavanone, indicative of caspase-9 activation locating upstream of caspase-3. A decrease in the antiapoptotic protein Mcl-1 and increases in the pro-apoptotic proteins Bax and Bad were found in 4'-OH- or 6-OH-flavanone-treated HL-60 cells. Induction of endogenous ROS production was detected in 4'-OH- or 6-OH-flavanone-treated HL-60 cells by the DCHF-DA assay. Antioxidants such as N-acetylcysteine (NAC), catalase (CAT), superoxide dismutase (SOD), and allopurinol (ALL), but not pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited 4'-OH- or 6-OH-flavanone-induced ROS production, with blocking of the apoptosis induced by 4'-OH- or 6-OH-flavanone. The apoptosis-inducing activity of 4'-OH- or 6-OH-flavanone was also observed in another leukemia cell line (Jurkat), but was not found in mature monocytic cells (THP-1) and normal human polymorphonuclear neutrophils (PMNs). This suggests that hydroxylation at C4' or C6 is important to the apoptosis-inducing activities of flavanone through ROS production, and that activation of the caspase-3 cascade, downstream of caspase-9 activation, is involved.     

Myeloperoxidase-dependent caspase-3 activation and apoptosis in HL-60 cells: protection by the antioxidants ascorbate and (dihydro)lipoic acid

The heme enzyme myeloperoxidase (MPO) has recently been implicated in hydrogen peroxide H(2)O(2)-induced apoptosis of HL-60 human leukemia cells. The purpose of this study was to investigate the molecular mechanism(s) of MPO-mediated apoptosis, in particular caspase-3 activation, and to determine the effects of the antioxidants ascorbate and (dihydro)lipoic acid. Incubation of HL-60 cells (1 x 10(6) cells/ml media) with H(2)O(2) (0-200 microM) resulted in dose-dependent stimulation of caspase-3 activity, DNA fragmentation, and morphological changes associated with apoptosis. Caspase-3 activity, DNA fragmentation and apoptosis were maximal at approximately 50 microM H(2)O(2). Pre-incubation of the cells with the MPO-specific inhibitor 4-aminobenzoic acid hydrazide (ABAH) and the heme enzyme inhibitor 3-aminotriazole (100 microM each) resulted in complete and partial inhibition, respectively, of intracellular MPO, caspase-3 activity, and apoptosis following addition of 50 microM H(2)O(2). Enhancement of cellular antioxidant status by pre-incubation of the cells with dehydro-ascorbic acid and lipoic acid, which are reduced intracellularly to ascorbate and dihydrolipoic acid, respectively, afforded protection against caspase-3 activation and apoptosis following addition of H(2)O(2). Addition of high concentrations of H(2)O(2) (200 microM) to cells pre-incubated with lipoic acid, however, resulted in cytotoxicity. Overall, our data indicate that MPO-derived oxidants, rather than H(2)O(2) itself, are involved in caspase-3 activation and apoptosis in HL-60 cells, and the antioxidants ascorbate and (dihydro)lipoic acid inhibit caspase-3 activation and apoptosis in these cells, likely via scavenging the MPO-derived oxidants.     

β-Elemene piperazine derivatives induce apoptosis in human leukemia cells through downregulation of c-FLIP and generation of ROS

β-Elemene is an active component of the herb medicine Curcuma Wenyujin with reported antitumor activity. To improve its antitumor ability, five novel piperazine derivatives of β-elemene, 13-(3-methyl-1-piperazinyl)-β-elemene (DX1), 13-(cis-3,5-dimethyl-1-piperazinyl)-β-elemene (DX2), 13-(4-ethyl-1-piperazinyl)-β-elemene (DX3), 13-(4-isopropyl-1-piperazinyl)-β-elemene (DX4) and 13-piperazinyl-β-elemene (DX5), were synthesized. The antiproliferative and apoptotic effects of these derivatives were determined in human leukemia HL-60, NB4, K562 and HP100-1 cells. DX1, DX2 and DX5, which contain a secondary amino moiety, were more active in inhibiting cell growth and in inducing apoptosis than DX3 and DX4. The apoptosis induction ability of DX1 was associated with the generation of hydrogen peroxide (H(2)O(2)), a decrease of mitochondrial membrane potential (MMP), and the activation of caspase-8. Pretreatment with the antioxidants N-acetylcysteine and catalase completely blocked DX1-induced H(2)O(2) production, but only partially its activation of caspase-8 and induction of apoptosis. HL-60 cells were more sensitive than its H(2)O(2)-resistant subclone HP100-1 cells to DX1-induced apoptosis. The activation of caspase-8 by these compounds was correlated with the decrease in the levels of cellular FLICE-inhibitory protein (c-FLIP). The proteasome inhibitor MG-132 augmented the decrease in c-FLIP levels and apoptosis induced by these derivatives. FADD- and caspase-8-deficient Jurkat subclones have a decreased response to DX1-induced apoptosis. Our data indicate that these novel β-elemene piperazine derivatives induce apoptosis through the decrease in c-FLIP levels and the production of H(2)O(2) which leads to activation of both death receptor- and mitochondrial-mediated apoptotic pathways.     

Gold(I) complexes with thiosemicarbazones: cytotoxicity against human tumor cell lines and inhibition of thioredoxin reductase activity

Complexes [Au(H2Ac4DH)Cl]?MeOH (1) [Au(H₂2Ac4Me)Cl]Cl (2) [Au(H₂2Ac4Ph)Cl]Cl?2H₂O (3) and [Au(H₂2Bz4Ph)Cl]Cl (4) were obtained with 2-acetylpyridine thiosemicarbazone (H2Ac4DH), its N(4)-methyl (H2Ac4Me) and N(4)-phenyl (H2Ac4Ph) derivatives, as well as with N(4)-phenyl 2-benzoylpyridine thiosemicarbazone (H2Bz4Ph). The compounds were cytotoxic to Jurkat (immortalized line of T lymphocyte), HL-60 (acute myeloid leukemia), MCF-7 (human breast adenocarcinoma) and HCT-116 (colorectal carcinoma) tumor cell lines. Jurkat and HL-60 cells were more sensitive than MCF-7 and HCT-116 cells. Upon coordinating to the gold(I) metal centers in complexes (2) and (4), the cytotoxic activity of the H2Ac4Me and H2Bz4Ph ligands increased against the HL-60 and Jurkat tumor cell lines. 2 was more active than auranofin against both leukemia cells. Most of the studied compounds were less toxic than auranofin to peripheral blood mononuclear cells (PBMC). All compounds induced DNA fragmentation in HL-60 and Jurkat cells indicating their pro-apoptotic potential. Complex (2) strongly inhibited the activity of thioredoxin reductase (TrxR), which suggests inhibition of TrxR to be part of its mechanism of action.     

Induction of apoptosis in HL-60 cells by photochemically generated hydroxyl radicals

Reactive oxygen species (ROS), especially hydroxyl radicals are postulated to mediate apoptosis of the cell. Here we demonstrate that hydroxyl radicals generated selectively by photolysis of a photo-Fenton reagent, N,N'-bis(2-hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthaldiimide (NP-III), induce apoptosis in HL-60 (human promyelocytic leukemia) cells involving the activation of caspase-3.     

Involvement of protein kinase C-beta and ceramide in tumor necrosis factor-alpha-induced but not Fas-induced apoptosis of human myeloid leukemia cells.

The role of protein kinase C-beta (PKC-beta) in apoptosis induced by tumor necrosis factor (TNF)-alpha and anti-Fas monoclonal antibody (mAb) in the human myeloid HL-60 leukemia cell line was studied by using its variant HL-525, which is deficient in PKC-beta. In contrast to the parental HL-60 cells, HL-525 is resistant to TNF-alpha-induced apoptosis but sensitive to anti-Fas mAb-induced apoptosis. Both cell types expressed similar levels of the TNF-receptor I, whereas the Fas receptor was detected only in HL-525 cells. Transfecting the HL-525 cells with an expression vector containing PKC-beta reestablished their susceptibility to TNF-alpha-induced apoptosis. The apoptotic effect of TNF-alpha in HL-60 and the transfectants was abrogated by fumonisin, an inhibitor of ceramide generation, and by the peptide Ac-YVAD-BoMK, an inhibitor of caspase-1 and -4. Supplementing HL-525 cells with exogenous ceramides bypassed the PKC-beta deficiency and induced apoptosis, which was also restrained by the caspase-1 and -4 inhibitor. The apoptotic effect of anti-Fas mAb in HL-525 cells was abrogated by the antioxidants N-acetylcysteine and glutathione and by the peptide z-DEVD-FMK, an inhibitor of caspase-3 and -7. We suggest that TNF-alpha-induced apoptosis involves PKC-beta and then ceramide and, in turn, caspase-1 and/or -4, whereas anti-Fas mAb-induced apoptosis utilizes reactive oxygen intermediates and, in turn, caspase-3 and/or -7.     

Caspase-8 mediates caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis of HL-60 cells.

We reported previously that singlet oxygen, generated by irradiation of rose bengal with visible light, induced apoptosis in human promyelocytic leukemia HL-60 cells. However, the mechanism of apoptosis caused by this reactive oxygen species is unclear. In this study, we demonstrate that singlet oxygen induced caspase-3 activation and Z-DEVD-FMK, a caspase-3 inhibitor, blocked apoptosis induction, while caspase-1 activity was not detectable and the caspase-1 inhibitor Z-YVAD-FMK had a very limited effect on apoptosis. This suggests that the activation of caspase-3 by singlet oxygen is essential for the commitment of cells to undergo apoptosis. Further studies showed that singlet oxygen induced an increase in caspase-8 activity and a reduction in mitochondrial cytochrome c. Time course analysis indicated that the cleavage of caspase-8 precedes that of caspase-3. In addition, blockade of caspase-8 by Z-IETD-FMK inhibited cleavage of pro-caspase-3 and prevented loss of mitochondrial cytochrome c. These results suggest that caspase-8 mediates caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis in HL-60 cells.     

Docosahexaenoic acid induces apoptosis via the Bax-independent pathway in HL-60 cells

We attempted to determine whether docosahexaenoic acid (DHA)-induced apoptosis is mediated via the Bax-mediated pathway in human myeloid leukemia HL-60 cells. DHA-induced apoptosis was confirmed by morphological analysis and caspase-3 activation. But, cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition (MPT), did not inhibit DHA-induced Bax translocation to mitochondria or caspase-3 activation. These data suggest that DHA can induce apoptosis via the Bax-independent pathway.     

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.     

2-(4-Methylphenyl)-1,3-selenazol-4-one induces apoptosis by different mechanisms in SKOV3 and HL 60 cells

We examined the ability of the synthetic selenium compound, 2-(4-methylphenyl)-1,3-selenazol-4-one (hereafter designated 3a), to induce apoptosis in a human ovarian cancer cell line (SKOV3) and a human leukemia cell line (HL-60). Flow cytometry showed that 3a treatment induced apoptosis in both cell lines to degrees comparable to that of the positive control, paclitaxel. Apoptosis was measured by PS externalization, DNA fragmentation and decreased mitochondrial membrane potential (MMP). However, analysis of the mechanism of action revealed differences between the responses of the two cell lines. Treatment with 3a arrested the cell cycle and induced caspase-3 activation in HL-60 cells, but not in SKOV3 cells. In contrast, 3a treatment induced apoptosis through translocation of AIF, a novel pro-apoptotic protein, in SKOV3 cells, but not in HL-60 cells. Collectively, our data demonstrated that 3a induced apoptosis in both cell lines, but via different action mechanisms.     

The novel triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid induces apoptosis of human myeloid leukemia cells by a caspase-8-dependent mechanism.

The oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a multifunctional molecule that induces growth inhibition and differentiation of human myeloid leukemia cells. The present studies demonstrate that CDDO treatment results in apoptosis of U-937 and HL-60 myeloid leukemia cells. Similar to 1-beta-D-arabinofuranosylcytosine (ara-C), another agent that inhibits growth and induces apoptosis of these cells, CDDO induced the release of mitochondrial cytochrome c and activation of caspase-3. Overexpression of Bcl-X(L) blocked cytochrome c release, caspase-3 activation, and apoptosis in ara-C-treated cells. By contrast, CDDO-induced release of cytochrome c, and activation of caspase-3 were diminished only in part by Bcl-X(L). In concert with these findings, we demonstrate that CDDO, but not ara-C, activates caspase-8 and thereby caspase-3 by a cytochrome c-independent mechanism. The results also show that CDDO-induced cytochrome c release is mediated by caspase-8-dependent cleavage of Bid. These findings demonstrate that CDDO induces apoptosis of myeloid leukemia cells and that this novel agent activates an apoptotic signaling cascade distinct from that induced by the cytotoxic agent ara-C.     

Commitment of neutrophilic differentiation and proliferation of HL-60 cells coincides with expression of transferrin receptor. Effect of granulocyte colony stimulating factor on differentiation and proliferation.

To examine the regulatory mechanisms of proliferation and maturation in neutrophilic lineage cells, we have tried to sort dimethyl sulfoxide (Me(2)SO)-treated HL-60 cells into transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells. Differentiated Trf-R(-) cells expressed more formyl-Met-Leu-Phe receptor (fMLP-receptor) and ability of O-(2) genaration, as markers of differentiation, than Trf-R(+) cells, and Trf-R(-) cell differentiation was markedly accelerated by the incubation with granulocyte colony stimulating factor (G-CSF). On the other hand, Trf-R(+) cells had a tendency to proliferate rather than differentiate, and proliferation was enhanced by G-CSF. These results indicate that Trf-R expression coincides with the commitment to proliferate or differentiate of HL-60 cells, and G-CSF accelerates these commitments. G-CSF-induced tyrosine phosphorylation of STAT 3 in Trf-R(-) cells much more than in Trf-R(+) cells. Protein 70 S6 kinase expression was higher in Trf-R(+) cells than in Trf-R(-) cells. Furthermore, p70 S6 kinase was hyperphosphorylated by G-CSF in Trf-R(+) cells, but not in Trf-R(-) cells. Rapamycin, an inhibitor of p70 S6 kinase activity, inhibited G-CSF-dependent proliferation of Trf-R(+) cells and increased fMLP-R expression on these cells. These results suggest that commitment to proliferation and differentiation in Me(2)SO-treated HL-60 cells is preprogrammed and correlated with Trf-R expression, and G-CSF potentiates the cellular commitment. STAT 3 may promote differentiation of Me(2)SO-treated HL-60 cells into neutrophils, while p70 S6 kinase may promote proliferation and negatively regulate neutrophilic differentiation.