| Micronuclei (MN) are small chromatin-containing bodies that are found in the cytoplasm of a small percentage of dividing mammalian cells. A micronucleus (the name means 'small nucleus') is generally believed to be formed in anaphase when chromosome fragments or whole chromosomes fail to segregate into the daughter cells. However, MN were also reported to form during interphase, due to disruptions in chromatin remodeling, or oncogene amplification. Scoring of micronuclei (MN) is widely used to monitor genomic instability and genotoxic exposure. In humans, MN scoring in cultured peripheral lymphocytes and exfoliated buccal cells is commonly conducted for the purpose of genotoxicity testing and biomonitoring. Though MN assay is now recognized as one of the most important assays in genetic toxicology, the mechanism governing MN remains to be identified.Another well-established and very sensitive marker of DNA damage is the accumulation of the phosphorylated form of histone H2AX (y-H2AX) at the sites of DNA double-strand breaks (DSBs). Formation of DSBs activated the ATM/ATR pathway, which then caused a rapid phosphorylation of histone H2AX at serine139in chromatin domains surrounding DSBs. It is believed that phosphorylation of H2AX plays a critical role in recruiting DNA repair factors to the damaged site and facilitate DNA repair. With antibodies against the phosphorylated form of H2AX (y-H2AX), DSBs can be easily visualized and quantified under a microscope. Discrete nuclear y-H2AX foci have become a sensitive and reliable marker of DSBs. In addition to their occurrence as discrete nuclear foci, y-H2AX signals can occupy a whole nucleus in a diffuse and uniform manner in cells experiencing replication stress, in cells exposed to high concentration of NaCl or in cells exposed to hypoxia. Therefore, the y-H2AX assay serves as a very sensitive tool measuring DSBs after cells are exposed to DNA-damaging agents.Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen. Examples include O2·-(superoxide radical), OH'(hydroxyl radical), NO (nitric oxide) and H2O2(hydrogen peroxide). ROS can be generated by endogenous sources and exogenous sources. There is a positive feedback between ROS and persistent DNA damages. Increased ROS production can lead to DNA damage. Increasing evidence suggests that persistent DNA damage (due for example to defective DNA repair pathways) also causes oxidative stress. Thus taken together, ROS increase leads to the formation of DNA breaks, which in turn favor further ROS production.We observed a novel type of MN with uniform labeling of y-H2AX, termed as MN-γ-H2AX (+). In this study, we characterized the formation of MN-γ-H2AX (+) in various types/lines of mammalian cells and studied the factors that contribute to their formation.PART ONE Replication stress induces micronuclei comprising aggregated DNA double-strand breaksWe observed MN coated with y-H2AX signals in a series of mammalian cells. We hypothesized that MN-γ-H2AX (+) are associated with DNA replication stress. We tested this hypothesis by examining the frequency of MN-γ-H2AX (+) under different conditions that interfere with DNA replication. We examined the frequency of MN-γ-H2AX (+) after MCF-7cells were treated with DNA replication stressors and in mutants with DNA replication defects.1. MN can be divided into MN-γ-H2AX (+) and MN-γ-H2AX (-). By immunofluorescence staining of γ-H2AX, MN appear in two forms:1) γ-H2AX signals, if present, usually occupy the majority or the whole micronucleus and usually display a diffuse, and often uniform, pattern throughout the whole MN. We designated those MN coated with y-H2AX signals for at least half of the whole MN as MN-γ-H2AX (+), and the others as MN-γ-H2AX (-).2) The spontaneous frequencies of MN-γ-H2AX (+) vary among different cell lines, in the range from less than1×10-2to4×10-2. In some cell lines, the MN-γ-H2AX (+) accounted for nearly half of the total MN.2. We determined the frequency of MN-γ-H2AX (+) in MCF-7cells which were treated with agents that cause replication stress.1) MCF-7cells were analyzed for cell cycle distribution by flow cytometry after treated with hydroxyurea, aphidicolin and thymidine. The results showed that all the agents can cause S phase arrest. The percentages of cells in S phase were increased to60%, from30%in the control.2) Three agents (hydroxyurea, aphidicolin and thymidine) that can cause S phase arrest all significantly induced the formation MN-γ-H2AX (+). The fold increase in frequency is more pronounced for MN-γ-H2AX (+) than for MN-γ-H2AX (-).3) Paclitaxel, which inhibits the disassembly of microtubules, only induced MN-γ-H2AX (-).3. To gain further insight into the association of MN-γ-H2AX (+) with DNA replication stress, we subjected cells to different drugs (hydroxyurea, aphidicolin, thymidine and paclitaxel) for24h, examined the frequencies of MN-γ-H2AX (+) at different time points (0h,6h,24h,48h,72h,96h) after the drugs were washed out. 1) During an extended time course after the drugs were washed out, the frequencies of MN-γ-H2AX (+) were found to be elevated at all time points till72h. They dropped at96h.2) Cells were classified into three types by the distribution of γ-H2AX signals in nuclei and type1and type2cells are associated with replication stress. By examining the distribution of the two types of MN in three types of cells at different time points following release from replication stress, we concluded that at MN-γ-H2AX (+) were formed during S phase.4. We surveyed cells with nuclear projections or blebs that may later give rise to MN in mouse skin fibroblasts at5th passage. We encountered numerous instances in which y-H2AX signals were being sequestered and were moving toward the tips. Such movements caught in snap shots probably reflect the steps leading to the formation of MN-γ-H2AX (+).5. We examined the frequency of MN-γ-H2AX (+) in mutants with DNA replication defects. In cells that were depleted of RPA1by RNAi, and therefore were encountering DNA replication stress, the frequency of MN-γ-H2AX (+) is significantly increased.In this part, we presented several lines of evidence suggesting that the MN-γ-H2AX (+) can be preferentially induced by replication stress. Thus, unlike MN that are formed due to failures of chromosomes or chromosome fragments to segregate into daughter cells during anaphase, MN-γ-H2AX (+) formation probably involves active clustering and disposal of DNA DSBs before the onset of mitosis. Examination of MN-γ-H2AX (+) may provide a more accurate evaluation of the various genotoxic agents as well the genomic instability caused by defects in DNA replication and repair.PART TWOReactive oxygen species (ROS) can preferentially induce MN-γ-H2AX (+)It was well known that ROS can cause DNA damage. In order to test whether ROS influence the formation of MN-γ-H2AX (+), we examined the frequency of MN-γ-H2AX (+) in cells treated with oxidant H2O2and/or antioxidant N-acetylcysteine (NAC) and in mutant cells with defective antioxidant genes.1. We examined the frequency of MN-γ-H2AX (+) after cells were treated with the hydrogen peroxide (H2O2), and with or without the presence of antioxidant N-acetylcysteine (NAC).1) We subjected U2OS cells to different doses of H2O2(50μM,100μM,150μM) for different durations (2h,6h,12h,24h), and examined the frequencies of the MN-γ-H2AX (+) at48h after H2O2was washed out. The results indicated that H2O2could induce MN-γ-H2AX (+) in a time-and dose-dependent manner.2) The increase in the frequencies of U2OS cells containing MN-γ-H2AX (+) after H2O2treatment could be prevented by incubation with the antioxidant NAC. These results suggested that ROS can induce MN-γ-H2AX (+).2. We examined the frequency of MN-γ-H2AX (+) in cells with varying function of antioxidant p53. Results as followed:1) The frequency of MN-γ-H2AX (+) was decreased after treatment with nutlin-3(p53pathway activator).2) The frequency of MN-γ-H2AX (+) was increased after treatment with pifithrin-a (p53inhibitor).3) In U2OS cells in which p53was silenced by shRNA and in p53null mouse skin fibroblasts (MSFs), there was an elevation in the frequency of MN-γ-H2AX (+).4) In U2OS-mtp53cells and MSF-p53mt, the frequency of MN-γ-H2AX (+) was decreased by incubation with the antioxidant NAC when compared with control cells.3. SESN1is one of the p53-regulated antioxidant genes. In order to investigate whether p53influences the formation of MN-γ-H2AX (+) via upregulating SESN1, we treated U2OS cells with SESN1siRNA. In cells that were depleted of SESN1by RNAi, the frequency of MN-γ-H2AX (+) was increased. 4. It was recently reported that depletion of p400increased intracellular ROS levels. In U2OS cells that were depleted of p400by RNAi, the frequency of MN-γ-H2AX (+) was significantly increased. This result further suggested that ROS induces MN-γ-H2AX (+).In conclusion, the frequency of MN-γ-H2AX (+) was increased after treatment with H2O2and in mutants with reduced expression of antioxidant genes (p53, SESN1and p400). Such increase can be offset by incubation with antioxidant NAC. These results suggested that ROS can preferentially induce MN-γ-H2AX (+).
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