A 1,4-dihydropyridine derivative reduces DNA damage and stimulates DNA repair in human cells in vitro

Compounds of the 1,4-dihydropyridine (1,4-DHP) series have been shown to reduce spontaneous, alkylation- and radiation-induced mutation rates in animal test systems. Here we report studies using AV-153, the 1,4-DHP derivative that showed the highest antimutagenic activity in those tests, to examine if it modulates DNA repair in human peripheral blood lymphocytes and in two human lymphoblastoid cell lines, Raji and HL-60. AV-153 caused a 50% inhibition of growth (IC50) of Raji and HL-60 cells at 14.9+/-1.2 and 10.3+/-0.8mM, respectively, but did not show a cytotoxic effect at concentrations <100 microM. Alkaline single-cell gel electrophoresis (comet) assays showed that AV-153 reduced the number of DNA strand breaks in untreated cells and also in cells exposed to 2 Gy of gamma-radiation, 100 microM ethylmethane sulfonate (EMS), or 100 microM H2O2. DNA damage was reduced by up to 87% at AV-153 concentrations between 1 and 10nM, and a positive dose-effect relationship was seen between 0.01 and 1 nM. Comparison of the kinetics of DNA strand-break rejoining in the presence and absence of AV-153 revealed a considerable influence on the rate of repair. In view of the resemblance of this compound's structure to that of dihydronicotinamide, a substrate for poly(ADP-rybose)polymerase, the modulation of DNA repair by AV-153 could involve an influence on poly(ADP)ribosylation.     

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.     

3-Hydrogenkwadaphnin targets inosine 5'-monophosphate dehydrogenase and triggers post-G1 arrest apoptosis in human leukemia cell lines

3-Hydrogenkwadaphnin (3-HK) is a recently characterized daphnane-type compound isolated from Dendrostellera lessertii with high anti-tumor activity in animal models. Herein, we report on time- and dose-dependent effects of this compound on growth, differentiation, IMPDH inhibition, cell cycle and apoptosis of a panel of human leukemia cell lines (HL-60, K562 and Molt4). The drug decreased the growth of leukemia cells in less than 24 h of treatment. However, longer exposure times and/or higher concentrations were required to promote cell apoptosis. Cell cycle analysis revealed the accumulation of cells in their G1 phase as early as 12 h after drug exposure but sub-G1 population was recorded after 24 h. Occurrence of apoptosis was constantly accompanied by morphological (staining with DNA-binding dyes) and biochemical (DNA fragments) variations among drug-treated cells. Despite these observations, non-activated normal human PBL were insensitive to the drug action. In addition, treatment of PHA-activated PBL, K562, Molt4 and HL-60 cells with a single dose of the drug for 24 h led to the inhibition of IMPDH activity by almost 37, 38, 44 and 50%, respectively. In contrast, no difference in IMPDH activities were seen between normal PBL and the drug treated PBL cells. Restoration of the depleted GTP concentration by exogenous addition of guanosine (25-50 microM) reversed the drug effects on cell growth, DNA fragmentation and apoptosis. Furthermore, the drug effects were potentiated by exogenous addition of hypoxanthine to the drug-treated cells. Reduction of the drug potency on the non-proliferative (retinoic acid treated) HL-60 cells by almost 40%, compared to the proliferative cells, clearly shows type II IMPDH as one of the main targets of the drug. These results suggest that 3-HK may be a powerful candidate for treatment of leukemia.     

Single-strand breaks, cell cycle arrest and apoptosis in HL-60 and LLCPK1 cells exposed to 1,2-dibromo-3-chloropropane

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK₁, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30–300 µmol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK₁ cells (100 µmol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 µmol/L. Flow cytometric analysis showed that DBCP (1–10 µmol/L) exposure for 24 h caused an accumulation of LLCPK₁ cells in the G₂/M-phase. In HL-60 cells the accumulation in G₂/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK₁ cells exposed to 100–500 µmol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G₁ cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK₁ cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G₂/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.     

Pivotal role of reactive oxygen species as intracellular mediators of hyperthermia-induced apoptosis

The effects of cellular antioxidant capacity on hyperthermia (HT)-induced apoptosis and production of antiapoptotic heat shock proteins (HSPs) were investigated in HL-60 cells and in HL-60AR cells that are characterized by an elevated endogenous catalase activity. Exposure of both cell lines to 43 degrees C for 1 h initiated apoptosis. Apoptosis peaked at 3-6 h after heat exposure in the HL-60 cells. Whereas HL-60AR cells were partially protected against HT-induced apoptosis at these early time points, maximal levels of apoptosis were detected later, i.e. 12-18 h after heat exposure. This differential induction of apoptosis was directly correlated to the induction of the antiapoptotic HSP27 and HSP70. In particular, in the HL-60 cells HSP27 was significantly induced at 12-18 h after exposure to 43 degrees C when apoptosis dropped. In contrast, coinciding with the late onset of apoptosis in HL-60AR cells at that time HL-60AR cells lacked a similar HSP response. In line with the higher antioxidant capacity HL-60AR cells accumulated reactive oxygen species to a lesser degree than HL-60 cells after heat treatment. Protection from HT-induced apoptosis as well as diminished heat-induced HSP27 expression was also observed after cotreatment of HL-60 cells with 43 degrees C and catalase but not with superoxide dismutase. These data emphasize the pivotal role of reactive oxygen species for HT induced pro- and antiapoptotic pathways.     

Inhibition of protein synthesis sensitizes thermotolerant cells to heat shock induced apoptosis

Hyperthermia is a potent inducer of apoptosis in many cell lines. A brief exposure to mildly elevated temperatures elicits a transient state of augmented resistance to subsequent thermal stress. Here we show that a hyperthermic treatment of 43°C for 1 h is sufficient to induce apoptosis in the cell line HL-60. This observation is based on morphologic evaluation and on comet assay results (an extremely sensitive method of detecting and quantifying apoptotic DNA fragmentation in individual cells). The thermotolerance phenomenon was also verified in the same manner by giving the cells a brief 30 min sub-lethal heat conditioning treatment at 43°C followed by a 6 h incubation time prior to the administration of a lethal heat load (43°C for 1 h). We observed a dramatic decrease in resultant apoptoses in the thermotolerized cells in comparison to unconditioned cells. We assessed the necessity of de novo protein synthesis in the protective phenomenon. When the conditioned cells were given a cycloheximide treatment prior to heat conditioning we saw a sensitization of the conditioned cells to secondary thermal injury. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selective cytotoxicity of withaphysalins in myeloid leukemia cell lines versus peripheral blood mononuclear cells

Withaphysalins are C(28)-steroidal lactones structurally based on the ergostane skeleton that possess antiproliferative activity against tumor cell lines. In the present study, the antileukemic actvity of withaphysalin O (1), M (2), and N (3) isolated from Acnistus arborescens, against two leukemic cell lines, HL-60 and K562, was evaluated, and the cytotoxicity compared with the effects on peripheral blood mononuclear cells (PBMC). All tested compounds reduced the number of viable cells of the tumor cell lines after 24 h of exposure, except for compound 2 against the K562 cell line. The reduction was time-and concentration-dependent, and the IC(50) values ranged from 0.7 to 3.5 microM after 72 h of incubation. In addition to the growth inhibitory properties, the drugs decreased DNA synthesis after 24 h of drug exposure evaluated by the 5-bromo-2 -deoxyuridine incorporation method. None of the tested compounds reduced the number of PBMC (IC(50)>20 microM) after 72 h of incubation, in contrast to doxorubicin that decreased viable cells and increased non-viable cells even after 24 h of incubation. Morphological analysis of treated cells using hematoxylin/eosin staining indicated the presence of necrotic cells for all tested compounds in HL-60, confirmed by the use of acridine orange/ethidium bromide staining. In addition to necrotic cells, K562 cells showed morphological alterations consistent with apoptosis.     

UV-B Irradiated Cell Lines Execute Programmed Cell Death in Various Forms

We investigated changes typical for apoptosis in various cell lines after UV-B irradiation. Using established methods for detection of apoptosis we demonstrate changes of cellular morphology, phosphatidylserine (PS) exposure, ollgonucleosomal DNA fragmentation and generation of hypochrome nuclei. To isolated high-molecular-weight (hmwt) DNA fragments we engaged a new method avoiding pulse field gel electrophoresis. Most UV-B irradiated cell lines showed oligonucleosomal DNA fragmentation, hypochrome nuclei, morphological changes, annexin-V binding and positive TUNEL reaction. However, no oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. Whereas HaCaT cells displayed all other changes typical for apoptosis, Raji cells were TUNEL negative, formed low amounts of hmwt DNA and showed an 'atypically' low hypochrome shift. Nevertheless, UV-B irradiated Raji cells excluded propidium iodide (PI), bound annexin-V and stopped proliferation. This suggests that Raji cells underwent growth arrest with exposure of PS being the only feature of apoptosis. However, in the presence of phagocytes expressing the phosphatidylserine receptor these cells would share the removal pathway with apoptotic cells. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation or chromatin condensation is not suitable to exclude programmed (apoptotic?) cell death.     

Cytotoxic activities of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma cells by induction of apoptosis

Coriolus versicolor (CV), also known as Yunzhi, is one of the commonly used Chinese medicinal herbs. Although recent studies have demonstrated its antitumour activities on cancer cells in vitro and in vivo, the exact mechanism is not fully elucidated. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized aqueous ethanol extract prepared from Coriolus versicolor on a B-cell lymphoma (Raji) and two human promyelocytic leukemia (HL-60, NB-4) cell lines using a MTT cytotoxicity assay, and to test whether the mechanism involves induction of apoptosis. Cell death ELISA was employed to quantify the nucleosome production resulting from nuclear DNA fragmentation during apoptosis. The present results demonstrated that CV extract at 50 to 800 microg/ml dose-dependently suppressed the proliferation of Raji, NB-4, and HL-60 cells by more than 90% (p < 0.01), with ascending order of IC50 values: HL-60 (147.3 +/- 15.2 microg/ml), Raji (253.8 +/- 60.7 microg/ml) and NB-4 (269.3 +/- 12.4 microg/ml). The extract however did not exert any significant cytotoxic effect on normal liver cell line WRL (IC50 > 800 microg/ml) when compared with a chemotherapeutic anticancer drug, mitomycin C (MMC), confirming the tumour-selective cytotoxicity. Nucleosome productions in HL-60, NB-4 and Raji cells were significantly increased by 3.6-, 3.6- and 5.6-fold respectively upon the treatment of CV extract, while no significant nucleosome production was detected in extract-treated WRL cells. The CV extract was found to selectively and dose-dependently inhibit the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.     

DNA ligase I mRNA and enzyme levels in human hematopoietic cells under dimethyl sulfoxide-induced growth-arrest and differentiation.

About half the activity level of DNA ligase I in cycling human lymphoblastoid cells (Raji and Akata) remained in the cells arrested at G1 by a 4-day treatment with 1.5% dimethyl sulfoxide (DMSO), and one-third the enzyme activity in actively growing promyelocytic leukemia cells HL-60 was detected in the terminally differentiated cells after DMSO-treatment. In contrast, DNA ligase I mRNA was negligible in the G1-arrested Raji and differentiated HL-60 cells. The steady-state mRNA level was increased 9 h after release from DMSO in the G1-arrested Raji cells and reached a maximum at 18 h. These results indicate that gene expression of human DNA ligase I, but not activity level of the enzyme, is closely correlated with activity of cell proliferation.     

Evidence for a G2 checkpoint in p53-independent apoptosis induction by X-irradiation.

The p53 tumor suppressor gene is thought to be required for the induction of programmed cell death (apoptosis) initiated by DNA damage. We show here, however, that the human promyelocytic leukemia cell line HL-60, which is known to be deficient in p53 because of large deletions in the p53 gene, can be induced to undergo apoptosis following X-irradiation. We demonstrate that the decision to undergo apoptosis in this cell line appears to be made at a G2 checkpoint. In addition, we characterize an HL-60 variant, HCW-2, which is radioresistant. HCW-2 cells display DNA damage induction and repair capabilities identical to those of the parental HL-60 cell line. Thus, the difference between the two cell lines appears to be that X-irradiation induces apoptosis in HL-60, but not in HCW-2, cells. Paradoxically, HCW-2 cells display high levels of expression of bax, which enhances apoptosis, and no longer express bcl-2, which blocks apoptosis. HCW-2 cells' resistance to apoptosis may be due to the acquisition of expression of bcl-XL, a bcl-2-related inhibitor of apoptosis. In summary, apoptosis can be induced in X-irradiated HL-60 cells by a p53-independent mechanism at a G2 checkpoint, despite the presence of endogenous bcl-2. The resistance shown by HCW-2 cells suggests that bcl-XL can block this process.     

NK4拮抗HGF促进肿瘤细胞凋亡的生物学效应

观察NK4通过拮抗肝细胞生长因子（HGF）诱导不同肿瘤细胞凋亡,研究其生物学作用及分子机制.以足叶乙甙（VP-16）诱导凋亡,分别或经HGF蛋白、NK4蛋白处理5种肿瘤细胞（B细胞淋巴瘤细胞系Raji、人急性粒细胞白血病细胞系HL-60、宫颈癌细胞系HeLa、前列腺癌细胞系PC-3、非小细胞肺癌细胞系A549）,采用流式细胞术（FCM）、吖啶橙 (AO) 染色法、苏木素 伊红（HE）染色法定量观察5种肿瘤细胞的凋亡情况,并进行相关分析. FCM发现,经VP-16处理5种肿瘤细胞凋亡率均显著高于对照组(P<0.001),而HGF+VP-16组凋亡率明显下降(P<0.01),HGF+NK4+VP-16组细胞凋亡率均明显高于HGF+VP-16组(P<0.05). AO染色和HE染色结果也证实,5种肿瘤细胞经VP-16处理后凋亡率均显著增高 (P<0.001,P<0.001),而HGF+VP-16组细胞凋亡率均明显低于VP-16组(P<0.001,P<0.01), HGF+NK4+VP-16组细胞凋亡率均明显高于HGF+VP-16组(P<0.001,P<0.05).此外,发现NK4+VP-16组、HGF+ NK4+VP-16组、VP-16组等3组间凋亡率无统计学差异(P>0.05). 以上结果提示,HGF蛋白可抵抗凋亡诱导剂VP-16的作用, 明显降低细胞凋亡;NK4通过竞争性抑制HGF从而促进肿瘤细胞的凋亡,具有潜在的肿瘤治疗价值.     

Estragole: a weak direct-acting food-borne genotoxin and potential carcinogen

We evaluated the genotoxicity of the food-flavouring agent estragole in V79 cells using the sister chromatid exchange (SCE) assay and the alkaline comet assay. Unexpectedly, we observed an increase in SCE without an exogenous biotransformation system (S9) and a decrease in its presence. Positive results were also observed in the alkaline comet assay without S9, indicating DNA strand breakage. To ascertain repair of damage, we performed the comet assay in V79 cells after two hours of recovery, and observed a reduction of the genotoxic response. Estragole did not produce strand breaks in plasmid DNA in vitro. We then evaluated the formation of DNA adducts in V79 cells by use of the (32)P-postlabelling assay and detected a dose-dependent formation of DNA adducts, which may be responsible for its genotoxicity. We then assayed estragole in the comet assay with two CHO cell lines, a parental AA8 cell line, and an XRCC1-deficient cell line, EM9. Results confirmed the genotoxicity of estragole without biotransformation in both cell lines, although the genotoxicity in EM9 cells compared with that in AA8 cells was not significantly different, suggesting that the XRCC1 protein is not involved in the repair of estragole-induced lesions. Estragole induces apoptosis, but only with high doses (2000μM), and after long treatment periods (24h). Overall, our results suggest that estragole, besides being metabolized to genotoxic metabolites, is a weak direct-acting genotoxin that forms DNA adducts.     

Mycoplasmal lipoproteins induce toll-like receptor 2- and caspases-mediated cell death in lymphocytes and monocytes

Lipoproteins of Mycoplasma salivarium and Mycoplasma fermentans preferentially induced necrotic cell death in lymphocytic cell lines, MOLT-4 and Raji, and in one monocytic cell line, THP-1, whereas they preferentially induced apoptotic cell death in another monocytic cell line, HL-60. These findings were also supported by ultrastructural observations by the use of scanning and transmission electron microscopes and by agarose gel electrophoresis of the genomic DNA. The lipoproteins activated caspase-3 in both MOLT-4 and HL-60 cells, which was assessed by the cleavage of the synthetic substrate DEVD-pNA and the endogenous substrate poly(ADP-ribose) polymerase. The cytotoxicity to MOLT-4 and HL-60 cells was inhibited by various caspase inhibitors, Ac-DMQD-CHO, Ac-IETD-CHO, and Z-VAD-FMK. The cytotoxicity was also partially suppressed by the monoclonal antibody to Toll-like receptor 2. Thus this study demonstrated that mycoplasmal lipoproteins induce caspases-dependent necrotic and apoptotic cell death in lymphocytes and monocytes/macrophages, which is partially induced by TLR2-mediated signaling.     

DNA damage and apoptosis in the immature mouse cerebellum after acute exposure to a 1 mT, 60 Hz magnetic field.

Several recent studies have reported that whole-body exposure of rodents to power frequency magnetic fields (MFs) can result in DNA single- and double-strand breaks in the brains of these animals. The current study was undertaken to investigate whether an acute 2h exposure of a 1 mT, 60 Hz MF could elicit DNA damage, and subsequently apoptosis, in the brains of immature (10-day-old) mice. DNA damage was quantitated at 0, 2, 4, and 24h after exposure using the alkaline comet assay. Apoptosis was quantitated in the external granule cell layer (EGCL) of the immature mouse cerebellum at 0 and 24h after exposure to MF by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. While increased DNA damage was detected by tail ratio at 2h after MF exposure, no supporting evidence of increased DNA damage was detected by the other parameters. In addition, no similar differences were observed using these parameters at any of the other post-exposure times. No increase in apoptosis was observed in the EGCL of MF-exposed mice, when compared to sham mice. Taken together, these results do not support the hypothesis that acute MF exposure causes DNA damage in the cerebellums of immature mice.     

DNA damage and repair in human peripheral blood lymphocytes following treatment with microcystin-LR

The purpose of this study was to find a possible explanation of the inconsistency of data regarding the genotoxicity of microcystin-LR (MC-LR). We compared the results of the comet assay with the results of the analysis of chromosome aberrations and apoptosis. In order to investigate the influence of MC-LR on DNA damage in human lymphocytes, cells were treated with MC-LR at different concentrations (1, 10 and 25 microg/ml) for 6, 12, 18 and 24 h. Analyses of Olive Tail Moment (OTM) as an indicator of DNA damage showed that MC-LR treatment induced DNA damage in a time-dependent manner, reaching its maximum after 18 h. The lowest values of OTM were observed after 24 h. MC-LR had no effect on the frequency of chromosome aberrations in lymphocytes. Since some data available in the literature indicate that apoptosis may lead to overestimated or false positive results regarding the genotoxicity of mutagens in the comet assay, we measured the frequency of late apoptotic cells by use of the comet assay and the frequency of early apoptotic cells with the TUNEL method. The comet assay results revealed that the highest level of apoptosis was observed after 24 h and the lowest after 18 h. The comparison of the frequency of apoptotic cells determined by the comet assay with DNA damage (OTM) examined by the comet assay revealed a statistically significant, negative correlation. The TUNEL results showed that the frequency of apoptotic cells progressively increased in a dose- and time-dependent manner. The comparison of the frequency of apoptotic cells determined by TUNEL method with DNA damage (OTM) examined by the comet assay showed a significant positive correlation for lymphocytes treated with MC-LR for 6, 12 and 18 h. Therefore, our findings indicate that microcystin-LR-induced DNA damage observed in the comet assay may be related to the early stages of apoptosis due to cytotoxicity but not genotoxicity. In addition, we examined the DNA repair kinetics in lymphocytes following treatment with microcystin-LR and ionizing radiation. Our results indicate that MC-LR has an inhibiting effect on the repair of radiation-induced damage.     

Antioxidant response induced by serum withdrawal protects HL-60 cells against inhibition of NAD(P)H:quinone oxidoreductase 1

We have previously shown that inhibition of NAD(P)H:quinone acceptor oxidoreductase 1 with dicoumarol decreases growth and viability of HL-60 cells in the absence of serum. Here we demonstrate that culturing HL-60 cells in serum-free medium in the presence of dicoumarol results in a significant potentiation of apoptosis. However, when cells were preincubated for 24 h without serum before they were treated with dicoumarol, the effect of the inhibitor on cell growth and death was much lower. We have investigated cellular changes induced in HL-60 cells by removal of serum that could account for protection against the effects of dicoumarol. Serum removal induced significant increases of NAD(P)H:quinone acceptor oxidoreductase 1, particularly at 32 h after serum withdrawal. Total amounts of ubiquinone in cells were unchanged but, its reduction state paralleled the observed increase in quinone reductase activity. Levels of the antiapoptotic protein Bcl-2 were also significantly increased after serum removal. Our results indicate that removal of serum evokes an antioxidant protective response that make HL-60 cells less sensitive to cell death induced by inhibition of NAD(P)H:quinone acceptor oxidoreductase 1 with dicoumarol.     

Apoptosis and clonogenic survival in three tumour cell lines exposed to gamma rays or chemical genotoxic agents

We compared the extent to which apoptosis is induced and clonogenicity reduced in three tumour cell lines - the human melanoma Me45 and promyelocytic leukaemia HL-60, and the rat rhabdomyosarcoma R1 - after exposure to the anticancer drugs etoposide and cis-platinum or to gamma radiation; each induces different types of DNA damage. Cells which readily underwent apoptosis did not necessarily show a correlated loss of clonogenicity; for example, Me45 cells showed the highest sensitivity to all three agents in clonogenic assays but much lower levels of apoptotic cells than R1 or HL-60 cells. These results show that the efficiency of the eradication of clonogenic cells by genotoxic agents does not solely depend on the induction of apoptotic processes, and suggest that the induction of apoptosis and suppression of clonogenicity are independent processes.     

Effect of UV-C mediated oxidative stress in leukemia cell lines and its relation to ubiquinone content

UV-C radiation is able to impair cellular functions by directly damaging DNA, and by inducing an increased formation of reactive oxygen species that leads to a condition of oxidative stress. In this study we evaluated different responses to UV insult of two leukemia cell lines, HL-60 and Raji, and the relationship with their CoQ10 content. DNA damage was monitored by means of the alkaline single cell gel electrophoresis (Comet assay); intracellular levels of ROS, mitochondrial depolarization and cell viability was measured by flow cytometry. Raji cells appeared more resistant to the UV insult; moreover, they did not show any increase in ROS content and the extent of mitochondrial depolarisation was much lower than in HL 60 cell line. Raji cells also contained significantly higher levels of CoQ10 and their ability to incorporate and to reduce exogenous CoQ10 added to the culture medium was remarkably elevated compared with HL 60.