The roles of kinetochore of micronucleus in mitosis of HeLa cells: a live cell imaging study.

BACKGROUND: Micronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability. However, the roles of kinetochore of MN in mitosis have not been completely addressed. METHODS: The HeLa CENP B-GFP H2B-mCherry cells are applied to address these questions via the long-term live-cell imaging. In the cells, the kinetochore-positive micronucleus (K+MN) contained CENP B-GFP, while the kinetochore-negative micronucleus (K-MN) did not. RESULTS: K-MN-bearing cells produced much more chromosome fragments than did MN-free cells. Most of the chromosome fragments eventually merged into K-MNi. K+MN-bearing cells yielded more kinetochore-positive lagging chromosomes (K+LCs) and K+MNi than MN-free cells did. The results suggested the differences in the fates of K+MNi and K-MNi in mitosis. The cycle of K-MN → Chromosome fragment → K-MN may occur in generations of K-MN-bearing cells, while part of K+MNi might reincorporate into the main nucleus. The K+MN-bearing cells prolonged significantly duration of mitosis compared with MN-free cells. The presence of micronuclei, regardless of K-MN and K+MN, enhanced apoptosis cell death. And K+MN-bearing cells were inclined to apoptosis more than K-MN-bearing cells. The results suggested differences in fates between K-MN-bearing and K+MN-bearing cells. CONCLUSIONS: Kinetochore determined the fates of micronuclei. Kinetochore in micronuclei indirectly prolonged the duration of mitosis. Kinetochore enhanced cytotoxicity of micronuclei. Our data are direct evidences showing the roles of kinetochore of micronucleus in mitosis of HeLa cells.

A Simple and Rapid Turn On ESIPT Fluorescent Probe for Colorimetric and Ratiometric Detection of Biothiols in Living Cells.

Biothiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play a key role in an extensive range of physiological processes and biological functions. Therefore, the selective and sensitive detection of intracellular thiols is important for revealing cellular function. In this study, ethyl 2-(4-(acryloyloxy)-3-formylphenyl)-4-methylthiazole-5-carboxylate (NL-AC) was designed and synthesized as a colorimetric and ratiometric fluorescent probe that can be utilized to rapidly, sensitively and selectively detect biothiols in physiological media. The fluorescence intensity of this probe using the three target biothiols at a concentration of 20 equiv. of the probe increased by approximately 6~10-fold in comparison to that without the biothiols in aqueous solution. The limits of detection (LOD) for Cys, Hcy and GSH were 0.156, 0.185, and 1.838 µM, respectively. In addition, both 1H-NMR and MS analyses suggested the mechanism of fluorescence sensing to be excited-state intramolecular proton transfer (ESIPT). The novel colorimetric and ratiometric probe is structurally simple and offers detection within 20 min. Furthermore, this probe can be successfully applied in bioimaging. The results indicate high application potential in analytical chemistry and diagnostics.

Differences in the origins of kinetochore-positive and kinetochore-negative micronuclei: A live cell imaging study.

Micronuclei (MNi) are extensively used to evaluate genotoxicity and chromosomal instability. Classification of kinetochore-negative (K-MNi) and kinetochore-positive micronuclei (K+MNi) improves the specificity and sensitivity of the micronucleus (MN) test; however, the fundamental differences in the origins of K-MNi and K+MNi have not been addressed due to the limitations of traditional methods. In the current study, HeLa CENP B-GFP H2B-mCherry cells were constructed in which histone 2B (H2B) and centromere protein B (CENP B) were expressed as fusion proteins to monomeric Cherry (mCherry) and EGFP, respectively. MNi were identified using H2B-mCherry; K+MN contained CENP B-GFP, while K-MN did not. Long-term live cell imaging was conducted to examine MN formation in the dual-color fluorescent HeLa cells. The results suggested that K-MNi were derived from kinetochore-negative displaced chromosomes (K-DCs), kinetochore-negative lagging chromosomes (K-LCs) and fragments of broken chromosome bridges (CBs) during late mitotic stages. The results also indicated that K+MNi are derived from kinetochore-positive displaced chromosomes (K+DCs), kinetochore-positive lagging chromosomes (K+LCs), and fragments of broken CBs. Different aberrant chromosomes emerged during mitosis at different frequencies and developed into K-MNi and/or K+MNi in the daughter cells at different rates. K+LCs formed K+MNi at a higher frequency than K+DCs, and K-LCs formed K-MNi at a higher rate than K-DCs; however, broken CBs transformed into K-MNi and/or K+MNi. In summary, these results show that K-MNi and K+MNi have different origins in HeLa cells and that each mechanism of MN formation contributes differently to the overall number of K-MNi and K+MNi.

The fate of micronucleated cells post X-irradiation detected by live cell imaging.

Micronuclei are closely related to DNA damage. The presence of micronuclei in mammalian cells is a common phenomenon post ionizing radiation. The level of micronucleation in tumor cells has been used to predict prognosis after radiotherapy in many cancers. In order to understand how irradiation-induced micronuclei affect cell fate, we performed extensive long-term live cell imaging on X-irradiated nasopharyngeal carcinoma (NPC) cells. To visualize the dynamics of micronuclei more clearly, chromosomes were stably labeled with red fluorescent protein (RFP) by targeting to human histone H2B. Initially, significantly more micronuclei were observed in radiosensitive cells than in radioresistant cells post irradiation. Additionally, cells with micronuclei were found to be more likely to die or undergo cell cycle arrest when compared with micronucleus-free cells after irradiation, and the more micronuclei the cells contained the more likely they would die or undergo arrest. Moreover, micronucleated cells showed predisposition to produce daughter cells with micronuclei through chromosome lagging. Fluorescence in situ hybridization using human pan-centromeric probes revealed that about 70% of these micronuclei and lagging chromosomes did not contain centromeric signals. Finally, DNA damage was more severe and p38 stress kinase activity was higher in micronucleated cells than in micronucleus-free cells as shown by phospho-H2AX and phospho-p38 immunofluorescence staining. Altogether, our observations indicated that the presence of micronuclei coupled with activated DNA damage response could compromise the proliferation capacity of irradiated cells, providing the evidence and justification for using micronucleus index as a valuable biomarker of radiosensitivity.

Activated toxicity of diesel particulate extract by ultraviolet a radiation in mammalian cells: role of singlet oxygen.

BACKGROUND: Diesel exhaust [diesel exhaust particles (DEPs) and their extracts (DPE)] and ultraviolet A radiation (UVA) are two ubiquitous environmental factors that have been identified as essential risk factors for various benign or malignant human diseases, either alone or in combination with other agents. OBJECTIVES: We aimed to investigate the synergistic effects of DPE and UVA at low-dose exposures in human-hamster hybrid (AL) cells and their underlying mechanisms. METHODS: We exposed exponentially growing AL cells to DPE and/or UVA radiation with or without reactive oxygen species (ROS) quenchers and then assayed the cells for survival, mutation induction, apoptosis, and micronucleus generation. In addition, using a singlet oxygen (1O2) trapping probe, 2,2,6,6-tetramethyl-4-piperidone, coupled with electron paramagnetic resonance spectroscopy, we determined the production of 1O2. RESULTS: Treatment of AL cells with DPE+UVA induced significant cytotoxic and genotoxic damage. In contrast, we found no significant damage in cells treated with either UVA or DPE alone at the same doses. Mutation spectra of CD59- mutants showed that treatment with DPE+UVA easily induces multilocus deletions. Sodium azide significantly inhibited both cellular and DNA damage induced by DPE+UVA treatment, whereas other ROS inhibitors had little protecting effect. Furthermore, we found a significant increase of 1O2 in the cells that received DPE+UVA treatment. CONCLUSION: These findings suggest that UVA activated the genotoxicity and cytotoxicity of DPE in mammalian cells and that 1O2 played an important role in these processes.

Enhanced radiation damage in irradiated and non-irradiated bystander regions by co-exposure to myosmine.

It is important to evaluate the health effects of radiation concurrent exposure to chemicals in our daily life. Myosmine, an alkaloid in tobacco plants and various edibles and staple foods, has been considered as a co-genotoxic agent in vitro. In the present study, the damage induced by radiation concurrent exposure to myosmine was assessed in human primary cell line AG1522. Myosmine at 5 or 10 mM for 3 h treatment induced a significantly dose-dependent increase in micronucleus (MN) frequencies, but not for 1 mM. However, 1 mM myosmine distinctly enhanced MN frequencies in both irradiated and non-irradiated bystander regions after different doses (0.2, 1 and 10 cGy) of alpha-particle irradiation. Treatment with c-PTIO, a nitric oxide (NO) scavenger, the induced fractions of MN frequencies were dramatically inhibited both in 1 cGy alpha-particle irradiated and non-irradiated bystander regions with or without myosmine treatment. Moreover, 1mM myosmine treatment distinctly enhanced gamma-H2AX foci formation in both 1 cGy alpha-particle irradiated and non-irradiated bystander regions. These data indicated that myosmine effectively enhanced the low dose alpha-particle-induced DNA damage in both irradiated and non-irradiated bystander regions and nitric oxide played a very important role in such process.

Radiation-induced bystander effects enhanced by elevated sodium chloride through sensitizing cells to bystander factors.

Radiation-induced bystander effects (RIBE) have been demonstrated to occur widely in various cell lines. However, very little data is available on the genotoxic effects of RIBE combined with other factor(s). We reported previously that with a low dose of alpha-particle irradiation, the fraction of gamma-H2AX foci-positive cells in non-irradiated bystander cells was significantly increased under elevated NaCl culture conditions. In this study, we further investigated the functional role of NaCl in the enhancement of RIBE using a specially designed co-culture system and micronucleus (MN) test. It was shown that the MN frequency was not increased significantly by elevated NaCl (9.0 g/L) alone or by medium exposure. However, with 1.0 cGy alpha-particle irradiation, the induced MN frequency increased significantly in both irradiated and non-irradiated bystander regions. Additional studies showed that elevated NaCl made the non-irradiated bystander cells more vulnerable to bystander factors. Furthermore, it was found that the induced MN frequency in cells both in irradiated and non-irradiated bystander regions was weakened when the hypertonic medium was changed to normotonic medium for 2h before irradiation. Such observations were quite similar to the co-effect of NaCl and hydrogen peroxide (H(2)O(2)), indicating that elevated NaCl might sensitize non-irradiated cells to bystander factors-induced oxidative stress.

Elevated sodium chloride concentrations enhance the bystander effects induced by low dose alpha-particle irradiation.

Previous studies have shown that high NaCl can be genotoxic, either alone or combined with irradiation. However, little is known about the relationship between environmental NaCl at elevated conditions and radiation-induced bystander effects (RIBE). RIBE, which has been considered as non-targeted bystander responses, has been demonstrated to occur widely in various cell lines. In the present study, RIBE under the elevated NaCl culture condition was assessed in AG 1522 cells by both the induction of gamma-H2AX, a reliable marker of DNA double-strand break (DSB) for the early process (<1h post irradiation), and the generation of micronuclei (MN), a sensitive marker for relative long process of RIBE. Our results showed that in the absence of irradiation, NaCl at elevated concentration such as 8.0, 9.0 and 10.0g/L did not significantly increase the frequency of gamma-H2AX foci-positive cells and the number of foci per positive cell comparing with that NaCl at a normal concentration (6.8g/L). However, with 0.2cGy alpha-particle irradiation, the induced fraction of gamma-H2AX foci-positive cells and the number of induced gamma-H2AX foci per positive cell were significantly increased in both irradiated and adjacent non-irradiated regions. Similarly, the induction of MN by 0.2cGy alpha-particle irradiation also increased with the elevated NaCl concentrations. With N(G)-methyl-l-arginine, an inhibitor of nitric oxide synthase, the induced fraction of foci-positive cells was effectively inhibited both in 0.2cGy alpha-particle irradiated and adjacent non-irradiated regions under either normal or elevated NaCl conditions. These results suggested that the cultures with elevated NaCl medium magnified the damage effects induced by the low dose alpha-particle irradiation and nitric oxide generated by irradiation was also very important in this process.