Cell death initiated by 3-deazaadenosine in HL-60 cells is apoptosis and is partially inhibited by homocysteine.

Cell death initiated by the adenosine analog 3-deazaadenosine (c3 Ado) was studied in human promyelocytic leukemia HL-60 cells. A rapid decrease in cell number was seen after 4-hr exposure to 50-100 microM c3 Ado. The dominating mode of cell death was apoptosis as demonstrated by condensation and fragmentation of the nucleus, formation of apoptotic bodies and endonucleolytic degradation of DNA. Four hour treatment with 100 microM c3 Ado resulted in a reduction of early S-phase cells, and appearance of cells with a lower DNA and protein content than that of the G1 population. Whereas 25 and 50 microM c3 Ado only initiated apoptosis in S-phase cells, 75 and 100 microM c3 Ado also initiated apoptosis in G1- and G2 + M-phase cells, suggesting different mechanisms for cell death at different concentrations. Apoptosis initiated by 100 microM c3 Ado was completely inhibited by 1 mM ZnCl2. Addition of homocysteine thiolactone (Hcy) partly inhibited cell death by c3 Ado. Light microscopic examination of cultures treated with 100 microM c3 Ado and 1 mM Hcy showed nuclear condensation and fragmentation consistent with the first stage in apoptosis, however, only a minor formation of apoptotic bodies took place in these cultures compared to that observed in cultures treated with 100 microM c3 Ado alone. The modifying action of Hcy on c3 Ado initiated apoptosis in HL-60 cells and this suggests that c3 Ado and 3-deazaadenosylhomocysteine (c3 AdoHcy) interact with different targets during initiation and progression of cell death in this cell line.

Elevation of cyclic AMP levels in HL-60 cells accumulated in G1 or G2 by transmethylation inhibitors.

Effects of the transmethylation inhibitors 3-deazaadenosine (c3Ado) and 3-deaza-(+/-)-aristeromycin (c3Ari) on cell cycle and cyclic AMP (cAMP) concentrations in human promyelocytic leukemia cells (HL-60) were studied by flow cytometry and radioimmunoassay techniques. Previously described cell cycle accumulations, after incubation with drugs (25 microM) for two cell doublings (36 hr), were localized to G1 and G2 after incubation with c3Ado and c3Ari, respectively. cAMP levels were elevated in cells treated with c3Ado (35%) and c3Ari (92%) for 36 hr. Addition of the phosphodiesterase (PDE) inhibitor theophylline, increased cAMP levels further, while cAMP responsiveness to the beta-adrenergic stimulator isoproterenol was attenuated after c3Ado and c3Ari incubation. Homocysteine thiolactone (Hcy) alone reduced cell growth slightly (5%) and increased cAMP levels (17%). Hcy increased the growth inhibitory effects of c3Ado, while no modulating effect was seen in combination with c3Ari, nor did Hcy counteract the effects on the cell cycle perturbations. The results suggest that c3Ado- and c3Ari-induced cell cycle accumulation is, at least in part, mediated through cAMP elevation, possibly due to PDE inhibition secondary to S-adenosyl-homocysteine hydrolase inhibition and S-adenosyl-homocysteine build-up.

Morphological modifications of apoptosis in HL-60 cells: effects of homocysteine and cytochalasins on apoptosis initiated by 3-deazaadenosine.

Using electron microscopy, confocal laser scanning microscopy and measurements of intact DNA we have studied the morphology and DNA degradation of human leukaemia HL-60 cells undergoing drug initiated apoptosis. Apoptosis was initiated by 100 microM 3-deazaadenosine (c3Ado), 25 microM c3Ado plus 1 mM homocysteine thiolactone (Hcy) and 100 microM c3Ado plus 5 micrograms/ml cytochalasin B (CB). Two different phenotypes of apoptotic cells (APC), blebbed and non-blebbed, were present in the cultures. Blebbed APC dominated in cultures exposed to c3Ado, whereas most APC in cultures treated with c3Ado plus Hcy and all the APC in cultures treated with c3Ado plus CB displayed a non-blebbed phenotype. A more pronounced reduction of the chromatin/cytoplasm ratio, lower volume fractions of uncondensed chromatin and higher volume fractions of highly condensed chromatin (micronuclei) were found in cultures exposed to c3Ado and c3Ado plus Hcy when compared with cultures exposed to c3Ado plus CB. A partial inhibition of c3Ado apoptosis by CB was confirmed by measurements of intact DNA. The inhibitory effect of CB was not reproducible by CE, indicating that CB exerts its effect by an actin independent mechanism. Both blebbed and non-blebbed APC displayed nuclear fragmentation, segregation of organelles and cytoplasmic vesiculation, suggesting that the differences between the phenotypes were restricted to the cytoplasmic membrane. We were not able to demonstrate the presence of F-actin by fluorescein isothiocyanate-phalloidin staining in blebbed APC nor in non-blebbed APC in cultures treated with c3Ado plus Hcy. Non-blebbed APC in cultures treated with c3Ado plus CB displayed foci of F-actin at the internal part of the cytoplasmic membrane. This suggests that F-actin is preserved by the mechanism by which CB inhibits blebbing, and may indicate that blebbing of the cytoplasmic membrane during apoptosis is associated with F-actin deficiency rather than a result of actin-myosin interactions.

A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F-actin and DNA.

Drug-initiated apoptosis of human leukemia HL-60, THP-1, and U-937 cells was studied via multiparameter flow cytometry and cell sorting. A new flow cytometric method that allows both identification and quantitation of apoptotic cells and estimation of their cell cycle specificity is presented. The method is based on paraformaldehyde fixation followed by staining of F-actin and DNA with fluorescein isothiocyanate (FITC)-phalloidin and propidium iodide (PI), respectively. Bivariate green fluorescence (F-actin) vs. side scatterplots of HL-60 cells treated with 10 microM etoposide for 4 h showed two cell populations, one with high green fluorescence and low side scatter and one with low green fluorescence and high side scatter. Sorting revealed cells with intact nuclei in the high green fluorescence/low side scatter population and cells with fragmented nuclei in the low green fluorescence/high side scatter population, demonstrating that the cells in the latter population were apoptotic. Exposure of HL-60 cells to 10 microM etoposide for 4 h resulted in S-phase selective apoptosis, whereas 5 micrograms/ml cycloheximide initiated apoptosis mainly in G0/G1-phase and S-phase cells. The apoptotic response of HL-60 cells to 20 GY gamma-irradiation was selective for S-phase and G2 + M-phase cells. The present method offers the opportunity to estimate the cell cycle distributions of both the apoptotic and the nonapoptotic cell populations, which is especially valuable when apoptosis occurs in association with cell cycle perturbations. A similar shift from one to two cell populations in green fluorescence vs. side scatter-plots, similar to that observed for HL-60 cells, was observed in the THP-1 and U-937 cell lines secondary to etoposide treatment.

Apoptosis and transmethylation metabolites in HL-60 cells.

To investigate the role of transmethylation metabolites in initiation of apoptosis in human leukemia HL-60 cells exposed to 3-deazaadenosine (c3 Ado) as single agent and c3Ado plus homocysteine thiolactone (Hcy), S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy) and 3-deazaadenosylhomocysteine (c3AdoHcy), were measured by HPLC in HL-60 cells exposed to 1-100 microM c3Ado as single agent and 1 to 100 microM c3Ado plus 1 mM Hcy. 3-deaza-(+/-)-aristeromycin (c3Ari), a more specific and potent S-adenosylhomocysteine hydrolase inhibitor compared with c3Ado, was used to inhibit synthesis of c3AdoHcy. AdoMet increased to maximum values 1.5- and 2.3-fold control in cultures treated with c3Ado as single agent and c3Ado plus Hcy, respectively. AdoHcy did not change in cultures treated with c3Ado as single agent, but increased to maximum values 2.0-fold control when Hcy was added. The synthesis of c3AdoHcy was favored by Hcy, and 35- to 70-fold higher levels of c3AdoHcy were found in cultures treated with c3Ado plus Hcy vs. c3Ado as single agent. The amounts of c3AdoHcy in cultures treated with c3Ado at concentrations initiating apoptosis did not exceed c3AdoHcy levels in cultures treated with c3Ado plus Hcy at concentrations not initiating apoptosis. Pretreatment of c3Ado plus Hcy cultures with c3Ari diminished c3AdoHcy and almost completely abrogated apoptosis. Exposure of cells to 100 microM c3Ari as single agent resulted in an increase in AdoHcy to 8.4-fold control but no changes in AdoMet and no initiation of apoptosis. Our findings indicate that c3Ado as single agent exerts its apoptosis-initiating effect through a nontransmethylase-related bio-chemical action. c3AdoHcy seems to be related to biochemical events initiating apoptotic cell death of HL-60 cells when c3Ado and Hcy are combined.

Homocysteine increases the relative number of apoptotic cells and reduces the relative number of apoptotic bodies in HL-60 cells treated with 3-deazaadenosine.

The effect of exogenous homocysteine thiolactone (Hcy) on apoptosis initiated by 3-deazaadenosine (c3Ado) was studied in human leukemia HL-60 cells. Flow cytometric analysis allowed evaluation of the relative number of apoptotic cells (APC) to apoptotic bodies (APB) with a sub G0/G1 DNA content. Addition of 1 mM Hcy to HL-60 cells exposed to 5 to 100 microM c3Ado was followed by a large increase in the number of APC and a simultaneous decrease in the number of APB. The effects of Hcy on both the formation of APC and APB was dose-dependent; however, Hcy concentrations above 250 microM were required for inhibition of APB formation to take place. Fluorescence microscopic examination of unfixed cells stained with acridine orange demonstrated different morphology of APC between cultures treated with c3Ado or c3Ado plus Hcy. Whereas APC in cultures treated with 100 microM c3Ado displayed pronounced cytoplasmic membrane blebbing, only minor blebbing was displayed by APC in cultures treated with 25 microM c3Ado and 1 mM Hcy. Extensive nuclear fragmentation was observed in APC regardless of Hcy addition. By cell sorting we demonstrate the presence of APC with the same DNA content as viable G0/G1 and S-phase cells in cultures treated with 25 microM c3Ado and 1 mM Hcy, indicating that cells in all cell cycle phases undergo apoptosis in these cultures. Neither the formation of APC nor APB in apoptosis initiated by cycloheximide or dactinomycin were influenced by 1 mM Hcy. The Hcy effects on c3Ado apoptosis were abrogated in part by 3-deaza-(+/-)-aristeromycin, a more specific and potent inhibitor of S-adenosylhomocysteine hydrolase.(ABSTRACT TRUNCATED AT 250 WORDS)