Simultaneous multi-parameter observation of Harringtonine-treating HL-60 cells with both two-photon and confocal laser scanning microscopy

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluorescent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simultaneously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.     

Thioredoxin reductase is one of the selenoproteins in both promyelocytic and granulocytic HL-60 cells

Human leukemia promyelocytic HL-60 cells differentiate into granulocytes when cultured with 1.25% dimethyl sulfoxide for 3 d. The radioactive Na₂ ⁷⁵SeO₃ incorporation and the amount of total proteins were interrelated in both promyelocytic and granulocytic HL-60. Promyelocytic cells had four times higher⁷⁵Se incorporation and 34% more protein synthesis than the granulocytic cells on the fifth culturing day. The enzyme activities of glutathione peroxidase (GSH-Px, E.C. 1.11.1.9) and thioredoxin reductase (TrxR, E.C. 1.6.4.5) in both types of cells increased significantly and approached steady stage on the third day. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) analysis and autoradiography of the proteins from the cells revealed three proteins with molecular weights of57, 28, and 21 kDa, respectively. These three⁷⁵Se-labeled proteins were present in both types of cells. The proteins from HL-60 cells were separated by DEAE-Sepharose and 2′5′-ADP-Sepharose columns. The purified 57-kDa protein had TrxR activity of 0.744 Μmol 5′-thionitrobenzoic acid (TNB) formed/min/mg protein and two isoelectric points at pH 5.9 and 6.0. These results suggest that TrxR is one of the selenoproteins in both promyelocytic and granulocytic HL-60 cells.     

Antibiofilm action of a toluidine blue O-silver nanoparticle conjugate on Streptococcus mutans: a mechanism of type I photodynamic therapy

The objective of this study was to evaluate the anti-biofilm efficacy of photodynamic therapy by conjugating a photosensitizer (TBO) with silver nanoparticles (AgNP). Streptococcus mutans was exposed to laser light (630 nm) for 70 s (9.1 J cm^?2) in the presence of a toluidine blue O–silver nanoparticle conjugate (TBO–AgNP). The results showed a reduction in the viability of bacterial cells by 4 log₁₀. The crystal violet assay, confocal laser scanning microscopy and scanning electron microscopy revealed that the TBO–AgNP conjugates inhibited biofilm formation, increased the uptake of propidium iodide and leakage of the cellular constituents, respectively. Fluorescence spectroscopic studies confirmed the generation of OH^? as a major reactive oxygen species, indicating type I phototoxicity. Both the conjugates down-regulated the expression of biofilm related genes compared to TBO alone. Hence TBO–AgNP conjugates were found to be more phototoxic against S. mutans biofilm than TBO alone.     

Phospholipase A2 has a role in proliferation but not in differentiation of HL-60 cells

The role of phospholipase A₂ (PLA₂) and its metabolite arachidonic acid (AA) in the proliferation and differentiation of HL-60 cells was investigated. Addition of either 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) or retinoic acid (RA) to HL-60 cells for 2 h inhibited PMA-stimulated PLA₂ activity measured by [³H]AA release. The inhibitor of PLA₂ activity, p-bromophenacyl bromide (BPB), significantly inhibited the proliferation of HL-60 cells and of fibroblast L929 and Swiss 3T3 cells in a dose-dependent manner. The effect of BPB on proliferation is probably through its inhibitory effect on PLA₂ activity, since the same doses of BPB which inhibited proliferation also inhibited PLA₂ activity determined by [³H]AA release. The importance of PLA₂ activity for cell growth was further supported by the effect of two other PLA₂ inhibitors, AACOCF₃ and scalaradial, which inhibited HL-60 proliferation in a dose-dependent manner. BPB, AACOCF₃ and scalaradial significantly increased the doubling time to 32.4 h, 34.0 h and 31.8 h, respectively, compared with 24.6 h in the control. The inhibitory effect of BPB on HL-60 proliferation was reversed by addition of exogenous free AA to HL-60 cells, indicating the importance of this metabolite for the proliferation process. This reversible effect is specific for AA since it was not achieved by other fatty acids like linolenic acid (LA) or oleic acid (OA). Addition of free AA to HL-60 cells did not induce differentiation, as expected. Although BPB, AACOCF₃, or scalaradial inhibited proliferation, they did not induce differentiation nor affect the differentiation induced by 1,25(OH)₂D₃ or RA. These results implicate that PLA₂ activity has no regulatory role in differentiation of HL-60 cells. The differential effect of PLA₂ inhibitors on proliferation and differentiation of HL-60 cells suggests that these two processes function under different regulatory mechanisms.     

Changes in the Susceptibility of 12-O-Tetradecanoylphorbol 13-Acetate (TPA)-Treated HL-60 Cells to Staphylococcal Leukocidin

Susceptibility of human promyelocytic leukemia HL-60 cells to staphylococcal leukocidin following treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined. TPA treatment for 6 hr rendered the cells very resistant transiently to leukocidin. There was no change in binding of leukocidin to the cells, but leukocidin-induced ⁴⁵CaCl₂ influx, phospholipase A₂ and C activities were inhibited. Further incubation with TPA rendered the cells sensitive again and then more sensitive than original HL-60 cells following increase of the binding, and leukocidin-induced activities described above appeared again. Those cells treated with TPA for more than 18 hr started to differentiate to macrophages morphologically and functionally. These data suggest that the differentiated cells were more sensitive than original HL-60 cells because of increased binding of leukocidin and that treatment of TPA for 6 hr may transiently impair the signal transduction system of leukocidin after binding of leukocidin to the specific receptor of the cell membrane. Using these TPA-treated cells, it was shown in this report that calcium influx, phospholipase A₂ and C activities were important to induce cytotoxic action of leukocidin after binding of leukocidin to specific receptors on the cells.     

Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis

Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC₅₀ and MBRC₅₀) of dextranase were 2 U ml^?1 and 5 U ml^?1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.     

Differentiation of HL-60 promyelocytic leukemia cells is accompanied by a modification of magnesium homeostasis

Magnesium homeostasis in HL-60 promyelocytic leukemia cells was compared to that in neutrophyl-like HL-60 cells obtained by 1.3% DMSO treatment. Magnesium homeostasis was studied by the characterization of magnesium efflux, the identification of intracellular magnesium pools, and the regulation of intracellular ionized Mg²⁺. In both undifferentiated and neutrophyl-like HL-60 cells, magnesium efflux occurred via the Na-Mg antiporter which was inhibited by imipramine and stimulated by db cAMP and forskolin. Receptor-mediated signals such as ATP, IFN-α, or PGE1, which can trigger cAMP-dependent magnesium efflux, were ineffective in undifferentiated HL-60 cells but induced 60–70% increase of magnesium efflux in neutrophyl-like HL-60 cells. Selective membrane permeabilization by the cation ionophore A23187 induced a large magnesium release when cells were treated with rotenone. In both cell populations, the addition of glucose to rotenone-treated cells restored magnesium release to the control level. Permeabilization by 0.005% digitonin provoked the release of 90% cell total magnesium in both cell types. Intracellular [Mg²⁺]_i was 0.15 and 0.26 mM in undifferentiated and neutrophyl-like HL-60 cells, respectively. Stimuli that triggered magnesium efflux, such as db cAMP in undifferentiated and IFN-α in neutrophyl-like HL-60 cells, induced a slow but consistent increase of [Mg²⁺]_i which was independent from Ca²⁺movements. Overall, these data indicate that magnesium homeostasis is regulated by receptor-mediated magnesium efflux which was modified during differentiation of HL-60 cells. Stimulation of magnesium efflux is paralleled by an increase of [Mg²⁺]_i which reflects a release of magnesium from the bound cation pool. J. Cell. Biochem. 71:441–448, 1998. © 1998 Wiley-Liss, Inc.     

Alkyllysophospholipid ET-18-OCH3 acts as an activator of protein kinase C in HL-60 cells

HL-60 cells are very sensitive to the cytotoxic action of ether lipids. Several hypotheses have been proposed to explain this cytotoxicity. We investigated the influence of the alkylphospholipid ET-18-OCH₃ on the activity of protein kinase C. HL-60 cells were incubated with ET-18-OCH₃ at a concentration of 20 μg/ml for 4 h. After the incubation the membrane fraction of the HL-60 cells was isolated and the activity of protein kinase C was determined while it was still associated with the membrane, using the synthetic peptide substrate [Ser²⁵]-protein kinase C (19–31) as a protein kinase C specific substrate. The activity of the membrane-bound protein kinase C was increased in HL-60 cells treated with ET-18-OCH₃ compared to untreated HL-60 cells. The increase in protein kinase C activity was not a consequence of translocation and appeared to be additive to the effect of the phorbol ester 12-myristate 13-acetate. In contrast, solubilized protein kinase C from HL-60 cells could be inhibited or stimulated in vitro by ET-18-OCH₃, dependent on the mode of addition of ET-18-OCH₃ and phospholipids.     

1,2-Di-O-α-linolenoyl-3-O-β-galactosyl-sn-glycerol as a Superoxide Generation Inhibitor from Perilla frutescens var. crispa

Using a superoxide (O₂ ^?) generation assay system with differentiated HL-60 cells, 1,2-di-O-α-linolenoyl-3-O-β-galactosyl-sn-glycerol (DLGG) was identified as an O₂ ^? generation inhibitor from Perilla frutescens var. crispa (a local variety, kida-chirimen shiso). DLGG suppressed the O₂ ^? level in a dose-dependent manner with an IC₅₀ value of 21 μM, comparable to those of rosmarinic acid (RoA, IC₅₀=29 μM) and caffeic acid (CA, IC₅₀=30 μM). While RoA and CA also dose-dependently inhibited O₂ ^? generation in a xanthine-xanthine oxidase system, DLGG had no effect in the same system. Thus DLGG appeared to decrease the O₂ ^? level in the HL-60 assay system by mechanisms different from those of RoA and CA, which appeared to act as O₂ ^? scavengers.     

Spicamycin,a New Differentiation Inducer of Mouse Myeloid Leukemia Cells (Ml) and Human Promyelocytic Leukemia Cells (HL-60)

A new antibiotic was obtained from the culture broth of Streptomyces alanosinicus 879-MT₃, and the name spicamycin was given. Spicamycin had a marked effect on the induction of differentiation of human promyelocytic leukemia cells (HL-60) as well as mouse myeloid leukemia cells (Ml). Its structure was elucidated by degradative studies and ¹H and ¹³C NMR spectral analysis as shown in Fig. 1.     

Electric field-induced changes in agonist-stimulated calcium fluxes of human HL-60 leukemia cells

The mechanism of biological effects of extremely-low-frequency electric and magnetic fields may involve induced changes of Ca²⁺ transport through plasma membrane ion channels. In this study we investigated the effects of externally applied, low-intensity 60 Hz electric (E) fields (0.5 V/m, current density 0.8 A/m²⁺) on the agonist-induced Ca²⁺ fluxes of HL-60 leukemia cells. The suspensions of HL-60 cells received E-field or sham exposure for 60 min and were simultaneously stimulated either by 1 μM ATP or by 100 μM histamine or were not stimulated at all. After E-field or sham exposure, the responses of the intracellular calcium levels of the cells to different concentrations of ATP (0.2–100 μM) were assessed. Compared with control cells, exposure of ATP-activated cells to an E-field resulted in a 20–30% decrease in the magnitude of [Ca²⁺]_i elevation induced by a low concentration of ATP (<1 μM). In contrast, exposure of histamine-activated HL-60 cells resulted in a 20–40% increase of ATP-induced elevation of [Ca²⁺]_i. E-field exposure had no effect on non-activated cells. Kinetic analysis of concentration-response plots also showed that compared with control cells, exposure to the E-field resulted in increases of the Michaelis constant, K_m, value in ATP-treated cells and of the maximal [Ca²⁺]_i peak rise in histamine-treated HL-60 cells. The observed effects were reversible, indicating the absence of permanent structural damages induced by acute 60 min exposure to electric fields. These results demonstrate that low-intensity electric fields can alter calcium distribution in cells, most probably due to the effect on receptor-operated Ca²⁺ and/or ion channels. Bioelectromagnetics 19:366–376, 1998. © 1998 Wiley-Liss, Inc.     

PKC and AMPK regulation of Kv1.5 potassium channels

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K⁺ current (I_Kur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4–2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems.     

Induction ofMRP/GS-X pump and cellular resistance to anticancer prostaglandins

We provide evidence that the expression of the humanMRP/GS-X pump encoded by theMRP (multidrug resistance associated protein) gene is induced by cisplatin in human leukemia HL-60/R-CP (cisplatin-resistant) cells and modulates cell growth inhibition by Δ⁷-prostaglandin A₁ (PGA₁) methyl ester. TheMRP mRNA level in HL-60/R-CP cells increased remarkably after a 24-h incubation with 20 μM cisplatin; interestingly, however, no amplification of theMRP gene was detected. In cisplatin-sensitive HL-60 cells, which express theMRP/GS-X pump at low levels, c-myc expression was substantially supressed by Δ⁷-PGA₁ methyl ester and the cell cycle was arrested in G1 phase. By contrast, in HL-60/R-CP cells overexpressing theMRP/GS-X pump, c-myc expression and cell proliferation were much less affected by Δ⁷-PGA₁ methyl ester. This suggests that induction of theMRP/GS-X pump may confer on cancer cells resistance to anticancer prostaglandins and that the resistance mechanism may involve the increased efflux of PG-glutathione conjugates, as active intermediates, from the cells via theMRP/GS-X pump.     

Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunonoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.     

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

Summary The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10⁻⁸ M 1α,25(OH)₂D₃ alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)₂D₃ was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)₂D₃ might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.     

Transmembrane potential responses during HL-60 promyelocyte differentiation

Myeloid cells, including granulocytes and monocyte/macrophages, are important in disease-associated inflammatory reactions. These cells come from a common progenitor, the promyelocyte. The human promyelocytic cell line, HL-60, can be induced to terminally differentiate into granulocytes or monocyte/macrophages in a controlled fashion providing a model to study various aspects of myelomonocytic differentiation. The expression of several ion channels is controlled in HL-60 cells in a differentiation specific pattern. The purpose of this study was to determine if lineage-specific ion channel expression during HL-60 differentiation resulted in differences in functional responses to external stimuli. This was investigated by examining transmembrane potential responses in HL-60 promyelocytes, HL-60-derived polymorphonuclear cells (PMNs), and monocytes to various stimuli using the transmembrane potential sensitive dye, diSBAC₂-(3). Exposure of HL-60 promyelocytes to ionomycin or ATP produced a membrane hyperpolarization. Studies using ion substitutions and ion channel blockers indicate that the hyperpolarization was mediated by K_Ca channels. During HL-60 promyelocyte differentiation to PMNs, the membrane potential response to ionomycin and ATP shifted from a hyperpolarization to a depolarization over 7 days. Conversely, HL-60-derived monocytes exhibited a membrane hyperpolarization in response to ionomycin and ATP. HL-60-derived monocytes also exhibit a Cl⁻ conductance specifically induced by ATP. Lineage-specific expression of ion channels during HL-60 cell differentiation is important in determining the transmembrane potential response of these cells. This may be translated into functional responses of various myelomonocytic cells during disease-associated inflammatory reactions. © 1996 Wiley-Liss, Inc.     

Increased mutant frequencies in the HPRT gene locus of leukemia HL-60 cells treated with succinylacetone

Succinyl acetone (SA) was initially identified in the urine of patients with tyrosinemia type I, an autosomally recessive inherited disease. SA has been used to downregulate the activity of myeloperoxidase (MPO) through its specific inhibition of heme biosynthesis and to investigate the biological properties of MPO in the human myeloid leukemic (HL-60) cell line. The goal of this study is to evaluate the mutagenic potential of SA by determining the frequencies of somatic mutations in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) reporter gene in HL-60 cells following treatment with the chemical. Treatments of HL-60 cells with 500 μmol/L SA for 72 h, a condition generally used to inhibit the MPO activity, resulted in a significantly increased HPRT mutant frequency (HPRT-Mf), compared with the control of untreated cells (47.25 × 10^-6 versus 7.5 × 10^-6, respectively, p <0.01). Treatment of the cells with lower doses of SA also led to an increase in HPRT-Mf but this was significant only with 200 μmol/L (28.67 × 10^-6, p<0.05) and not with doses lower than 100 μmol/L (p0.05), compared with the control of untreated cells (7.5 × 10^-6). These data show a dose–response increase in HPRT-Mf in HL-60 cells treated with SA, suggesting that this chemical causes mutations in the HPRT locus in these cells either directly or indirectly through its inhibition of the MPO activity.     

In-vitro cytotoxic and genotoxic effects of arsenic trioxide on human leukemia (HL-60) cells using the MTT and alkaline single cell gel electrophoresis (Comet) assays

Although arsenic trioxide (ATO) has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by ATO in a human leukemia (HL-60) cell line using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. HL-60 cells were treated with different doses of ATO for 24 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that ATO significantly (P < 0.05) reduced the viability of HL-60 cells in a dose-dependent manner, showing a LD₅₀ value of 6.4 ± 0.6 μg/mL. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HL-60 cells associated with ATO exposure. We observed a significant increase (P < 0.05) in comet tail-length, tail arm and tail moment, as well as in percentages of DNA cleavage at all doses tested, showing an evidence of ATO-induced genotoxic damage in HL-60 cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by heavy metals like arsenic. Taken together, our findings suggest that ATO exposure significantly (P < 0.05) reduces cellular viability and induces DNA damage in HL-60 cells as assessed by MTT and alkaline single cell gel electrophoresis assays, respectively.     

Histone H2AX Phosphorylation after Cell Irradiation with UV-B: Relationship to Cell Cycle Phase and Induction of Apoptosis

Damage to DNA that engenders double-strand breaks (DSBs) triggers phosphorylation of histone H2AX on Ser-139. Expression of phosphorylated H2AX (_H2AX) can be revealed immunocytochemically; the intensity of ?H2AX immunofluorescence (IF) measured by cytometry was reported to correlate with the frequency of DSBs induced by X-ray radiation or by DNA damaging antitumor drugs. The aim of the present study was to measure expression of ?H2AX following exposure of HeLa and HL-60 cells to a wide range of doses of UV-B light (6.1 J/m²-3.45 kJ/m²) and using multiparameter flow and laser scanning cytometry (LSC) to correlate DNA damage with cell cycle phase and induction of apoptosis. In both cell lines, the highest degree of H2AX phosphorylation induced by UV was seen in S-phase cells, particularly during early portion of S. In cells that did not replicate DNA (G₁, G₂ and M) the degree of H2AX phosphorylation was markedly lower than that in S-phase cells, and was strongly UV dose-dependent. Furthermore, the level of UV-induced &gamma;H2AX in G₁, G₂ and M was much higher in HeLa- than in HL-60-cells. Apoptotic cells become apparent >2h after exposure to UV and exhibited nearly an order of magnitude higher intensity of &gamma;H2AX IF than that initially induced by UV; predominantly S-phase cells underwent apoptosis. While the suppression of DNA replication aphidicolin prevented the induction of H2AX phosphorylation by UV in most S phase cells, it had no effect on a small cohort of cells that appeared to be entering S-phase, that expressed very high levels of &gamma;H2AX. Furthermore, aphidicolin itself induced &gamma;H2AX in early-S phase cells. The induction of &gamma;H2AX by UV was inhibited, but the incidence of apoptosis increased, by 5 mM caffeine, a known inhibitor of PI-3-related kinases. The data are consistent with the notion that H2AX phosphorylation observed throughout S phase reflects formation of DSBs due to the collision of replication forks with the UV-induced primary DNA lesions. Induction of &gamma;H2AX in GG₁, GG₂ and M is likely a response to the primary DSBs generated during UV exposure and/or DNA repair. It is unclear why the latter process was more pronounced in HeLa than in HL-60 cells.