Identification of cellular responses to low-dose radiation by the profiling of phosphorylated proteins in human B-lymphoblast IM-9 cells.

PURPOSE: The aim of this study was to explore the potential for radiation-specific signaling of various LDIR-induced effects in human B-lymphoblast IM-9 cells. MATERIALS AND METHODS: Human lymphoblast IM-9 cells were exposed to ionizing radiation at 0.1 and 2 Gy using a 137Cs γ-irradiator at a dose rate of 0.8 Gy/min. Cell viability and DNA fragmentation were determined using MTT assay and TUNEL assay at 24 h after irradiation. Profiling of protein phosphorylation by radiation was identified using a phospho-antibody array at 4 h after irradiation and Dataset of the profiling was analyzed by IPA. RESULTS: Cell survival and apoptotic signaling were not affected by 0.1 Gy of radiation, whereas 2 Gy induced cellular damage. The analysis of low-dose ionizing radiation (LDIR) or high-dose ionizing radiation (HDIR)-specific responses by IPA generated different results. Various cell maintenance functions were only apparent following the analysis of increased protein phosphorylation by LDIR, whereas several cancer formation- and development-related functions were only detected following the analysis of increased protein phosphorylation by HDIR. CONCLUSIONS: The LDIR-induced protein phosphorylation patterns might be involved in various cell survival responses or cellular maintenance functions, which provide important insight into our understanding of the different effects of LDIR and HDIR.

Ionizing Radiation Induces Altered Neuronal Differentiation by mGluR1 through PI3K-STAT3 Signaling in C17.2 Mouse Neural Stem-Like Cells.

Most studies of IR effects on neural cells and tissues in the brain are still focused on loss of neural stem cells. On the other hand, the effects of IR on neuronal differentiation and its implication in IR-induced brain damage are not well defined. To investigate the effects of IR on C17.2 mouse neural stem-like cells and mouse primary neural stem cells, neurite outgrowth and expression of neuronal markers and neuronal function-related genes were examined. To understand this process, the signaling pathways including PI3K, STAT3, metabotrophic glutamate receptor 1 (mGluR1) and p53 were investigated. In C17.2 cells, irradiation significantly increased the neurite outgrowth, a morphological hallmark of neuronal differentiation, in a dose-dependent manner. Also, the expression levels of neuronal marker proteins, ß-III tubulin were increased by IR. To investigate whether IR-induced differentiation is normal, the expression of neuronal function-related genes including synaptophysin, a synaptic vesicle forming proteins, synaptotagmin1, a calcium ion sensor, γ-aminobutyric acid (GABA) receptors, inhibitory neurotransmitter receptors and glutamate receptors, excitatory neurotransmitter receptors was examined and compared to that of neurotrophin-stimulated differentiation. IR increased the expression of synaptophysin, synaptotagmin1 and GABA receptors mRNA similarly to normal differentiation by stimulation of neurotrophin. Interestingly, the overall expression of glutamate receptors was significantly higher in irradiated group than normal differentiation group, suggesting that the IR-induced neuronal differentiation may cause altered neuronal function in C17.2 cells. Next, the molecular mechanism of the altered neuronal differentiation induced by IR was studied by investigating signaling pathways including p53, mGluR1, STAT3 and PI3K. Increases of neurite outgrowth, neuronal marker and neuronal function-related gene expressions by IR were abolished by inhibition of p53, mGluR-1, STAT3 or PI3K. The inhibition of PI3K blocked both p53 signaling and STAT3-mGluR1 signaling but inhibition of p53 did not affect STAT3-mGluR1 signaling in irradiated C17.2 cells. Finally, these results of the IR-induced altered differentiation in C17.2 cells were verified in ex vivo experiments using mouse primary neural stem cells. In conclusion, the results of this study demonstrated that IR is able to trigger the altered neuronal differentiation in undifferentiated neural stem-like cells through PI3K-STAT3-mGluR1 and PI3K-p53 signaling. It is suggested that the IR-induced altered neuronal differentiation may play a role in the brain dysfunction caused by IR.

Ionizing radiation induces neuronal differentiation of Neuro-2a cells via PI3-kinase and p53-dependent pathways.

PURPOSE: The influence of ionizing radiation (IR) on neuronal differentiation is not well defined. In this study, we investigated the effects of IR on the differentiation of Neuro-2a mouse neuroblastoma cells and the involvement of tumor protein 53 (p53) and mitogen-activated protein kinases (MAPK) during this process. MATERIALS AND METHODS: The mouse neuroblastoma Neuro-2a cells were exposed to (137)Cs γ-rays at 4, 8 or 16 Gy. After incubation for 72 h with or without inhibitors of p53, phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) and other kinases, the neuronal differentiation of irradiated Neuro-2a cells was examined through analyzing neurite outgrowth and neuronal maker expression and the activation of related signaling proteins by western blotting and immunocytochemistry. Mouse primary neural stem cells (NSC) were exposed to IR at 1 Gy. The change of neuronal marker was examined using immunocytochemistry. RESULTS: The irradiation of Neuro-2a cells significantly increased the neurite outgrowth and the expression of neuronal markers (neuronal nuclei [NeuN], microtubule-associated protein 2 [Map2], growth associated protein-43 [GAP-43], and Ras-related protein 13 [Rab13]). Immunocytochemistry revealed that neuronal class III beta-tubulin (Tuj-1) positive cells were increased and nestin positive cells were decreased by IR in Neuro-2a cells, which supported the IR-induced neuronal differentiation. However, the IR-induced neuronal differentiation was significantly attenuated when p53 was inhibited by pifithrin-α (PFT-α) or p53-small interfering RNA (siRNA). The PI3K inhibitor, LY294002, also suppressed the IR-induced neurite outgrowth, the activation of p53, the expression of GAP-43 and Rab13, and the increase of Tuj-1 positive cells. The increase of neurite outgrowth and Tuj-1 positive cells by IR and its suppression by LY294002 were also observed in mouse primary NSC. CONCLUSION: These results suggest that IR is able to trigger the neuronal differentiation of Neuro-2a cells and the activation of p53 via PI3K is an important step for the IR-induced differentiation of Neuro-2a cells.

Prolonged expression of senescence markers in mice exposed to gamma-irradiation.

Although ionizing radiation is known to induce cellular senescence in vitro and in vivo, its long-term in vivo effects are not well defined. In this study, we examined the prolonged expression of senescence markers in mice irradiated with single or fractionated doses. C57BL/6 female mice were exposed to 5 Gy of γ-rays in single or 5, 10, 25 fractions. At 2, 4, and 6 months after irradiation, senescence markers including mitochondrial DNA (mtDNA) common deletion, p21, and senescence-associated ß-galactosidase (SA ß-gal) were monitored in the lung, liver, and kidney. Increases of mtDNA deletion were detected in the lung, liver, and kidney of irradiated groups. p21 expression and SA ß-gal staining were also increased in the irradiated groups compared to the non irradiated control group. Increases of senescence markers persisted up to 6 months after irradiation. Additionally, the extent of mtDNA deletion and the numbers of SA ß-gal positive cells were greater as the number of radiation fractions increased. In conclusion, our results showed that ionizing radiation, especially that delivered in fractions, can cause the persistent upregulation of senescence marker expression in vivo. This should be considered when dealing with chronic normal tissue injuries caused by radiation therapy or radiation accidents.

Chronic effects of single and fractionated γ-irradiation on an impairment of Th1-related immune response.

PURPOSE: We already reported that levels of interferon (IFN)-γ have been shown to be markedly reduced in mice seven weeks after irradiation, resulting in a T helper (Th) 1/Th2 imbalance. To investigate whether the single or fractionated γ-irradiation induced an immune imbalance, we analysed the Th1-related immune response profile until six months after the fractionated whole-body irradiation. METHODS AND MATERIALS: Mice were exposed to γ-rays at a fractionated 5 Gy cumulative dose for five weeks. At two, four and six months later from the first exposure, experiments were performed. Cell populations in the spleen, the production of IFN-γ, interleukin (IL)- 4 and IL-12p70, natural killer (NK) cell activity and the expression of IL-12 receptors, signal transducer and activator of transcription (STAT) 4 and suppressors of cytokine signaling (SOCS) 3 were detected. RESULTS: The IFN-γ was lower in the mice exposed by all irradiation conditions than in normal control mice, but the IL-4 had increased in all the irradiated mice. To investigate Th1 profile, NK cell activity, IL-12p70 level and its receptor expression was confirmed. In all fractionated irradiation groups, the NK cell activity as well as the absolute numbers of NK cells was much decreased. Also, all the irradiated mice showed a lower IL-12p70 level. However, the expression of IL-12 receptor ß2 was lower in the irradiated mice except the 0.2 Gy × 10 mice group. The phosphoylated STAT4 was lower in all the irradiated mice. This suppression was associated with an overexpression of SOCS3. CONCLUSIONS: The fractionated whole-body irradiations of a dose of 5 Gy appear to be the down-regulation of the Th1-like immune response. These changes, in turn, maintain an immunological imbalance that persists in the long term.

Propolis inhibits the proliferation of human leukaemia HL-60 cells by inducing apoptosis through the mitochondrial pathway.

Propolis, a natural product derived from plant resins collected by honeybees, has been reported to exert a wide spectrum of biological functions. This research aimed at investigating the effect of propolis on the proliferation of human leukaemia HL-60 cells and whether propolis might induce apoptosis in HL-60 cells. The results showed dose- and time-dependent decreases in the proliferation of HL-60 cells treated with propolis (above 3 microg mL(-1) of propolis). Further studies revealed that the anti-proliferative effects of propolis were caused by inducing apoptosis. Agarose electrophoresis of genomic DNA of HL-60 cells treated with propolis showed the ladder pattern typical for apoptotic cells. Propolis induced the activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase in HL-60 cells. Propolis also induced the release of cytochrome c from mitochondria to cytosol. Taken together, these findings demonstrate that the inhibitory effect of propolis on HL-60 cell proliferation is caused by inducing apoptosis through the mitochondrial pathway.