Kwang Hee Yang

Ret finger protein 2 enhances ionizing radiation-induced apoptosis via degradation of AKT and MDM2.

Ret finger protein 2 (RFP2), a gene frequently deleted in multiple tumor types, encodes a protein with a RING finger, B-box, and coiled-coil domain that belongs to the RBCC/TRIM protein family. Although RBCC proteins are involved in diverse cellular processes such as apoptosis, proliferation, differentiation, and transcriptional regulation, the biological function of RFP2 has not been well defined. Here, we demonstrate that overexpression of RFP2 in cells induced apoptosis through proteasomal degradation of MDM2 and AKT. The expression of RFP2, which possesses RING domain-dependent E3 ubiquitin ligase activity, was increased by ionizing radiation dose- and time-dependently, and RFP2 overexpression induced cell death with increased expression of apoptotic molecules (p53, p21, and Bax). These results depended on the E3 ubiquitin ligase activity of RFP2 because mutant RFP2, which contains a mutated RING domain, failed to drive apoptosis compared with wild-type RFP2. We observed that RFP2 formed a complex with MDM2, a negative regulator of the p53 tumor suppressor, and AKT, a regulator of apoptosis inhibition at the cellular level. Additionally, we found that the interaction of RFP2 with MDM2 and AKT resulted in ubiquitination and proteasomal degradation of MDM2 and AKT in vivo and in vitro. Thus, these data suggest that irradiation causes RFP2 overexpression, which enhances ionizing radiation-induced apoptosis by increasing p53 stability and decreasing AKT kinase activity through MDM2 and AKT degradation.

Genome-wide analysis of low-dose irradiated male Drosophila melanogaster with extended longevity.

Ionizing radiation generates oxidative stress, which is thought to be a major cause of aging. Although living organisms are constantly exposed to low levels of radiation, most studies examining the effect of radiation have focused on accelerated aging and diminished life span that result from high-dose radiation. On the other hand, several studies have suggested that low-dose radiation enhances the longevity of Drosophila melanogaster. Therefore, investigation of the biological effects of low-dose radiation could contribute to a more comprehensive understanding of the aging process. In this study, microarray and quantitative real time-PCR were used to measure genome-wide changes in transcript levels in low-dose irradiated fruit flies that showed enhanced longevity. In response to radiation, approximately 13% of the genome exhibited changes in gene expression, and a number of aging-related genes were significantly regulated. These data were compared with quantitative trait loci affecting life-span to identify candidate genes involved in enhanced longevity induced by low-dose radiation. This genome-wide survey revealed novel information about changes in transcript levels in low-dose irradiated flies and identified 39 new candidate genes for molecular markers of extended longevity induced by ionizing radiation. In addition, this study also suggests a mechanism by which low-dose radiation extends longevity.

Phosphorylation of CLK2 at Serine 34 and Threonine 127 by AKT Controls Cell Survival after Ionizing Radiation

AKT phosphorylates components of the intrinsic cell survival machinery and promotes survival to various stimuli. In the present study, we identified CDC-like kinase 2 (CLK2) as a new substrate of AKT activation and elucidated its role in cell survival to ionizing radiation. AKT directly binds to and phosphorylates CLK2 on serine 34 and threonine 127, in vitro and in vivo. CLK2 phosphorylation was detected in HeLa cells overexpressing active AKT. In addition, we demonstrated that ionizing radiation induces CLK2 phosphorylation via AKT activation. In contrast, the suppression of endogenous AKT expression by siRNA inhibited CLK2 phosphorylation in response to 2 gray of γ-ray or insulin. Furthermore, we examined the effect of CLK2 on the survival of irradiated CCD-18Lu cells overexpressing Myc-CLK2. CLK2 overexpression significantly increased cell growth and inhibited cell death induced by 2 gray. The role of CLK2 in cell survival to ionizing radiation was dependent on the phosphorylation of serine 34 and threonine 127. Our results suggest that AKT activation controls cell survival to ionizing radiation by phosphorylating CLK2, revealing an important regulatory mechanism required for promoting cell survival.

A critical role for AKT activation in protecting cells from ionizing radiation-induced apoptosis and the regulation of acinus gene expression

Although AKT activation leads to the activation of various pathways related to cell survival, the roles of AKT in modulating cellular responses induced by ionizing radiation in normal human cells remain unclear. Here we show that low-dose radiation of 0.05Gy did not affect cell death, but high-dose radiation (> 0.2Gy) induced apoptosis through the activation of caspases and acinus cleavage. Ionizing radiation induced acinus phosphorylation via AKT activation. Thus, we examined the effect of AKT activation on radiation-induced cell death using CCD-18Lu cells transduced with a retroviral vector expressing constitutively active AKT (CA-AKT). The overexpression of CA-AKT rendered the cells resistant to ionizing radiation and prevented the proteolytic cleavage of acinus via phosphorylation. In addition, overexpression of CA-AKT resulted in the upregulation of acinus expression by activation of the NF-kappaB pathway. On the other hand, suppression of endogenous AKT expression by siRNA resulted in the reduction of acinus expression and enhanced the radiation-induced apoptosis in both CCD-18Lu and IM-9 cells. Our results suggest that AKT activation inhibits cell death during radiation-induced apoptosis through the regulation of phosphorylation and expression of acinus. The AKT/NF-kappaB/acinus pathway functions as one of the important regulatory mechanisms required for modulating ionizing radiation sensitivity.

Role of AKT and ERK pathways in controlling sensitivity to ionizing radiation and adaptive response induced by low-dose radiation in human immune cells.

Despite many studies of the effect of ionizing radiation, biological mechanisms of action might differ greatly depend on dose, dose rate, and cell type. This study was performed to explore the effects of low- and high-dose radiation in human immune cell lines. We examined cell sensitivity after irradiation with 0.05, 0.1, or 2Gy in two normal cell lines and three tumor cell lines. Low-dose radiation of 0.05 and 0.1Gy had no effect on cell survival in any tested cell line, with the exception of IM-9 cells, whose viability was transiently increased. However, IM-9 and C1R-sB7 cells were very sensitive to high-dose radiation-induced cell death, whereas Jurkat and JM1 cells showed moderate sensitivity, and THP-1 cells were completely resistant. This radiosensitivity was correlated with basal AKT activation, which is induced by phosphorylation. In radiosensitive IM-9 cells, priming with chronic low-dose irradiation blocked cell death induced by high-dose radiation challenge via inhibition of caspase activation and PARP cleavage. AKT phosphorylation was not altered in IM-9 cells, but ERK phosphorylation was greatly elevated immediately after chronic low-dose irradiation. Taken together, our results suggest that the different responses of normal and tumor cells to low-dose and high-dose radiation depend on AKT activation, which is regulated by protein phosphatase 2 (PP2A). In radiosensitive normal cells lacking basal AKT activity, chronic low-dose radiation increases activation of the ERK pathway, which plays an important role in the adaptive response to radiation, providing a very important insight into understanding the effects of ionizing radiation on health.

The effects of low-dose ionizing radiation in the activated rat basophilic leukemia (RBL-2H3) mast cells

Background: The effect of ionizing radiation in mast cells is not well known. Results: Low-dose ionizing radiation that did not induce cell toxicity inhibited mediator release through the suppression of receptor expression. Conclusion: Low-dose ionizing radiation regulates mast cell activation. Significance: This is the first evidence of the effect of low-dose radiation in the activated mast cell. Mast cells play important roles in many biological responses, such as those during allergic diseases and inflammatory disorders. Although laser and UV irradiation have immunosuppressive effects on inflammatory diseases by suppressing mast cells, little is known about the effects of γ-ionizing radiation on mast cells. In this study, we investigated the effects of γ-ionizing radiation on RBL-2H3 cells, a convenient model system for studying regulated secretion by mast cells. Low-dose radiation (<0.1 gray (Gy)) did not induce cell death, but high-dose radiation (>0.5 Gy) induced apoptosis. Low-dose ionizing radiation significantly suppressed the release of mediators (histamine, β-hexosaminidase, IL-4, and tumor necrosis factor-α) from immunoglobulin E (IgE)-sensitized RBL-2H3 cells. To determine the mechanism of mediator release inhibition by ionizing radiation, we examined the activation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, PKCs, and MAPK, and intracellular free calcium concentrations ([Ca2+]i). The phosphorylation of signaling molecules following stimulation of high-affinity IgE receptor I (FcϵRI) was specifically inhibited by low-dose ionizing radiation (0.01 Gy). These results were due to the suppression of FcϵRI expression by the low-dose ionizing radiation. Therefore, low-dose ionizing radiation (0.01 Gy) may function as a novel inhibitor of mast cell activation.

The Inhibitory Effects of Low-Dose Ionizing Radiation in IgE-Mediated Allergic Responses

Ionizing radiation has different biological effects according to dose and dose rate. In particular, the biological effect of low-dose radiation is unclear. Low-dose whole-body gamma irradiation activates immune responses in several ways. However, the effects and mechanism of low-dose radiation on allergic responses remain poorly understood. Previously, we reported that low-dose ionizing radiation inhibits mediator release in IgE-mediated RBL-2H3 mast cell activation. In this study, to have any physiological relevance, we investigated whether low-dose radiation inhibits allergic responses in activated human mast cells (HMC-1(5C6) and LAD2 cells), mouse models of passive cutaneous anaphylaxis and the late-phase cutaneous response. High-dose radiation induced cell death, but low-dose ionizing radiation of <0.5 Gy did not induce mast cell death. Low-dose ionizing radiation that did not induce cell death significantly suppressed mediator release from human mast cells (HMC-1(5C6) and LAD2 cells) that were activated by antigen-antibody reaction. To determine the inhibitory mechanism of mediator released by low-dose ionizing radiation, we examined the phosphorylation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, and protein kinase C, as well as the intracellular free Ca2+ concentration ([Ca2+]i). The phosphorylation of signaling molecules and [Ca2+]i following stimulation of FcεRI receptors was inhibited by low dose ionizing radiation. In agreement with its in vitro effect, ionizing radiation also significantly inhibited inflammatory cells infiltration, cytokine mRNA expression (TNF-α, IL-4, IL-13), and symptoms of passive cutaneous anaphylaxis reaction and the late-phase cutaneous response in anti-dinitrophenyl IgE-sensitized mice. These results indicate that ionizing radiation inhibits both mast cell-mediated immediate- and delayed-type allergic reactions in vivo and in vitro.

Chronic low-dose radiation inhibits the cells death by cytotoxic high-dose radiation increasing the level of AKT and acinus proteins via NF-κB activation

Abstract Purpose: This study explored the effects of low-dose and low-dose-rate irradiation in human lung fibroblast CCD-18Lu cells and examined the role of AKT (protein kinase B, PKB) in cellular responses. Materials and methods: We examined cell survival after chronic low-dose irradiation (0.01 Gy or 0.05 Gy) with challenging high-dose (2 or 10 Gy) irradiation. We examined the effect of AKT activation on cell survival after chronic low-dose radiation using transduced cells with retroviral vector expressing constitutively active AKT (CA-AKT). Results: Chronic low-dose priming irradiation increased cells viability against the challenging high-dose irradiation. Irradiation at 0.05 Gy increased cellular levels of AKT and acinus long form (L) and short form (S). The chronic low-dose radiation promoted cells proliferation in the exogenously expressed CA-AKT cells. It also increased nuclear factor-kappa B (NF-κB) activity in a biphasic induction pattern. Suppression of NF-κB activation by mutant form of inhibitor of kappa B alpha (IκBαM) antagonized the radiation-induced expression of AKT and acinus L and S. Conclusions: Chronic low-dose radiation increases the levels of AKT and acinus proteins via NF-κB activation, and the NF-κB/AKT pathway responding to chronic low-dose irradiation plays an important role in the radiation adaptive response.

TOPORS Modulates H2AX Discriminating Genotoxic Stresses

H2AX plays an important role in chromatin reorganization implicated in DNA repair and apoptosis under various DNA damaging conditions. In this study, the interaction between TOPORS (topoisomerase I‐binding protein) and H2AX was verified using mammalian cell extracts exposed to diverse DNA damaging stresses such as ionizing radiation, doxorubicin, camptothecin, and hydrogen peroxide. In vitro assays for ubiquitination revealed that TOPORS functions as a novel E3 ligase for H2AX ubiquitination. TOPORS was found to be dissociated from H2AX proteins when cells were exposed to oxidative stress, but not replication‐inducing DNA damaging stress. The protein stability of H2AX was decreased when TOPORS was ectopically expressed in cells, and oxidative stresses such as hydrogen peroxide and ionizing radiation induced recovery of the H2AX protein level. Therefore, these biochemical data suggest that TOPORS plays a key role in the turnover of H2AX protein, discriminating the type of DNA damaging stress.

Intrinsic radiosensitivity correlated with radiation-induced ROS and cell cycle regulation

Ionizing radiation (IR) can induce multiple cellular responses which determine the fate of cells, including chromosomal aberrations, genetic mutations, cell cycle arrest, apoptosis, and cell differentiation. IR also enhances the production of reactive oxygen species (ROS) in a variety of cells, causing DNA damage and activating several proteins involved in the DNA repair system. The balance between DNA damage and repair is a key determinant of intrinsic radiation sensitivity. In this study, we investigated the survival rate in different types of cell lines (HUVEC, Bud-8, CCD-18Lu and Jurkat) after irradiation at various doses of γ-radiation, and compared the radiationinduced ROS generation with cell viability. We also measured micronuclei frequency in the irradiated cells in order to verify the cytogenetic analysis by radiation. In addition, we analyzed the cellular level of several proteins related to the cell cycle in order to characterize the correlation between the intrinsic radiosensitivity and cell cycle regulation. We found that the intrinsic viability of irradiated cells was correlated with the radiation-induced ROS production as well as the function of the organ of origination. We also observed increased cell cycle protein expression in radiationsensitive cells compared to radiation-resistant cells. These results suggest that intrinsic cellular sensitivity to irradiation is dependent on ROS generation and cell cycle regulation.