Byung Uk Lim

Neutralization epitopes on the antigenic domain II of the Orientia tsutsugamushi 56-kDa protein revealed by monoclonal antibodies

Monoclonal antibodies (MoAbs) reactive with the authentic Orientia tsutsugamushi 56-kDa protein were generated. MoAb FS10 and FS15 showed in vitro, as well as, in vivo neutralizing activity upon O. tsutsugamushi infection. Deletion mutants of the gene for 56-kDa protein of O. tsutsugamushi Boryong were expressed to map the binding region. FS10 and FS15 are bound to amino acids (aa) located in an antigenic domain II, at residues 140-160 and 187-214, respectively. Computer modeling indicated that aa 146-153 were important for antigenicity against FS10. A sequence for aa 142-150 was highly homologous between oriential strains. These results suggest that the antigenic determinant for neutralizing MoAbs is an epitope within aa 140-160. Furthermore, this region may be important for the adhesion/invasion or intracellular survival of O. tsutsugamushi within host cells.

Endoplasmic Reticulum Stress-Induced JNK Activation Is a Critical Event Leading to Mitochondria-Mediated Cell Death Caused by β-Lapachone Treatment

Background β-lapachone (β-lap) is a bioreductive agent that is activated by the two-electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although β-lap has been reported to induce apoptosis in various cancer types in an NQO1-dependent manner, the signaling pathways by which β-lap causes apoptosis are poorly understood. Methodology/Principal Findings β-lap-induced apoptosis and related molecular signaling pathways in NQO1-negative and NQO1-overexpressing MDA-MB-231 cells were investigated. Pharmacological inhibitors or siRNAs against factors involved in β-lap-induced apoptosis were used to clarify the roles played by such factors in β-lap-activated apoptotic signaling pathways. β-lap leads to clonogenic cell death and apoptosis in an NQO1- dependent manner. Treatment of NQO1-overexpressing MDA-MB-231 cells with β-lap causes rapid disruption of mitochondrial membrane potential, nuclear translocation of AIF and Endo G from mitochondria, and subsequent caspase-independent apoptotic cell death. siRNAs targeting AIF and Endo G effectively attenuate β-lap-induced clonogenic and apoptotic cell death. Moreover, β-lap induces cleavage of Bax, which accumulates in mitochondria, coinciding with the observed changes in mitochondria membrane potential. Pretreatment with Salubrinal (Sal), an endoplasmic reticulum (ER) stress inhibitor, efficiently attenuates JNK activation caused by β-lap, and subsequent mitochondria-mediated cell death. In addition, β-lap-induced generation and mitochondrial translocation of cleaved Bax are efficiently blocked by JNK inhibition. Conclusions/Significance Our results indicate that β-lap triggers induction of endoplasmic reticulum (ER) stress, thereby leading to JNK activation and mitochondria-mediated apoptosis. The signaling pathways that we revealed in this study may significantly contribute to an improvement of NQO1-directed tumor therapies.

Influence of Environmental pH on G2-Phase Arrest Caused by Ionizing Radiation

Abstract Park, H. J., Lee, S. H., Chung, H., Rhee, Y. H., Lim, B. U., Ha, S. W., Griffin, R. J., Lee, H. S., Song, C. W. and Choi, E.;thK. Influence of Environmental pH on G2-Phase Arrest Caused by Ionizing Radiation. Radiat. Res. 159, 86–93 (2003). We investigated the effects of an acidic environment on the G2/M-phase arrest, apoptosis, clonogenic death, and changes in cyclin B1-CDC2 kinase activity caused by a 4-Gy irradiation in RKO.C human colorectal cancer cells in vitro. The time to reach peak G2/M-phase arrest after irradiation was delayed in pH 6.6 medium compared to that in pH 7.5 medium. Furthermore, the radiation-induced G2/M-phase arrest decayed more slowly in pH 6.6 medium than in pH 7.5 medium. Finally, there was less radiation-induced apoptosis and clonogenic cell death in pH 6.6 medium than in pH 7.5 medium. It appeared that the prolongation of G2-phase arrest after irradiation in the acidic environment allowed for greater repair of radiation-induced DNA damage, thereby decreasing the radiation-induced cell death. The prolongation of G2-phase arrest after irradiation in the acidic pH environment appeared to be related at least in part to a prolongation of the phosphorylation of CDC2, which inhibited cyclin B1-CDC2 kinase activity.

Apoptosis of Endothelial Cell Line ECV304 Persistently Infected with Orientia tsutsugamushi

Endothelial cells are major targets of Orientia tsutsugamushi. To examine the consequences of the infection of endothelial cells with O. tsutsugamushi, we used human endothelial cell line ECV304. Persistent infection was established and infected cultures could be maintained for over seven months without the addition of normal cells. The heavily infected cells became round and floated in the culture medium, harboring large numbers of organisms inside them. Some of the infected ECV304 cells showed features of apoptotic cells, as determined by the terminal deoxytransferase‐mediated dUTP nick end‐labeling reaction and DNA fragmentation. We also found that O. tsutsugamushi increased transcription of the mRNAs of proinflammatory cytokines such as IL‐6 and IL‐8. These results show the first evidence of in vitro‐persistent infection by O. tsutsugamushi, which may be related to in vivo persistence reported previously.

Hyperthermia improves therapeutic efficacy of doxorubicin carried by mesoporous silica nanocontainers in human lung cancer cells

Purpose: We investigated the use of hyperthermia to improve the anti-cancer efficacy of doxorubicin (DOX)-loaded mesoporous silica nanocontainer Si-SS-CD-PEG. The hypothesis was that heat stimulates glutathione-mediated degradation of cyclodextrin gatekeeper, thereby causing the release of DOX from the carrier and DOX-induced cell death. Materials and methods: The release of DOX from DOX-loaded Si-SS-CD-PEG suspended in PBS containing glutathione (GSH) was studied by assessing the changes in DOX fluorescence intensity. The effect of heating at 42°C on the release of DOX from the intracellular carriers was determined with confocal microscopy. The extents of clonogenic and apoptotic cell death caused by DOX-loaded Si-SS-CD-PEG were determined. Results: The release of DOX from DOX-loaded Si-SS-CD-PEG in PBS occurred only when GSH presented in the suspension, and heating at 42°C slightly increased the release of DOX from the carriers. Heating significantly elevated the GSH content in A549 cells and increased the release of DOX from the internalised carriers. Heating the cancer cells treated with the carriers at 42°C markedly increased the clonogenic death and apoptosis. The GSH content in A549 cells was greater than that in L-132 cells, and A549 cells were far more sensitive than L-132 cells to DOX-loaded Si-SS-CD-PEG at both 37°C and 42°C. Conclusions: Hyperthermia increased the GSH-mediated release of DOX from DOX-loaded Si-SS-CD-PEG. Furthermore, hyperthermia markedly elevated the GSH content in cancer cells, thereby increasing the release of DOX from the internalised carriers and potentiating the DOX-induced clonogenic and apoptotic cell death.

Orientia tsutsugamushi Inhibits Apoptosis of Macrophages by Retarding Intracellular Calcium Release

ABSTRACT Orientia tsutsugamushi shows both pro- and antiapoptotic activities in infected vertebrate cells. Apoptosis of THP-1 cells induced by beauvericin was inhibited by O. tsutsugamushi infection. Beauvericin-induced calcium redistribution was significantly reduced and retarded in cells infected with O. tsutsugamushi. Antiapoptotic activities of O. tsutsugamushi in infected cells are most probably due to inhibition of the increase in the cytosolic calcium concentration.

Radiosensitization of tumor cells by modulation of ATM kinase

Purpose: To elucidate the relationship between the radiation-induced activation of ataxia telangiectasia mutated (ATM) kinase, G2 arrest and the caffeine-induced radiosensitization. Method: RKO cells (human colorectal cancer cells) and ATM kinase over-expressing RKO/ATM cells were used. The cellular radiosensitivity was determined with clonogenic survival assay and the cell cycle progression, including G2 arrest, was studied with flow cytometry. The activity of ATM kinase, check point 2 (Chk2) kinase and cycline B1/cell division cycle 2 (Cdc2) kinase was investigated. The radiosensitivity of RKO xenografts grown in nude mice was studied. Results: RKO/ATM cells were radioresistant as compared with RKO cells. There was a greater increase in ATM kinase activity and G2 arrest in RKO/ATM cells than in RKO cells. Caffeine also sensitized both RKO cells and RKO/ATM cells to radiation. The caffeine treatment suppressed the radiation-induced activation of ATM kinase, suppressed the activation of Chk2 kinase and inhibited the accumulation of cells in G2 phase. The activity of cycline B1/Cdc2 kinase increased earlier but decayed rapidly in the presence of caffeine. Caffeine enhanced radiation-induced growth delay of RKO xenografts. Conclusions: Caffeine inhibited the radiation-induced activation of ATM kinase, thereby preventing the accumulation of cells in G2 phase. Consequently, radiosensitivity of cells increased in the presence of caffeine both in vitro and in vivo.

Anti-cancer effect of bio-reductive drug β-lapachon is enhanced by activating NQO1 with heat shock

Purpose: Bio-reduction/activation of anti-cancer drug β-lapachone (β-lap) is mediated by NAD(P)H: Quinone oxidoreductase (NQO1). We investigated the feasibility of using mild temperature hyperthermia to increase the anti-cancer effect of β-lap by up-regulating NQO1 expression. Methods: NQO1 expression in FSaII fibrosarcoma of C3H mice and A549 human lung cancer cells was evaluated with Western blot analysis and immunostaining of cells at different times after water-bath heating. Clonogenic cell survival method was used to determine the sensitivity of cells to heating, β-lap, and in combination. The growth of FSaII tumors in the right hind legs of C3H mice was studied after heating the tumors at 42°C for 1 h with water bath, an i.p. injection of β-lap to host mice or an i.p. injection of β-lap 24 h after heating the tumors. Results: Heating at 42°C for 1 h significantly increased the expression of NQO1 in the cancer cells with a maximum increase occurring 8–24 h after heating. The sensitivity of cancer cells to β-lap treatment progressively increased until 24 h after heating most likely due to the increase in NQO1 expression. Heating the FSaII tumors at 42°C for 1 h and treating the host mice with an i.p. injection of 50 mg/kg β-lap 24 h after the tumor heating was far more effective than heating alone or β-lap treatment alone to suppress the tumor growth. Conclusion: Mild temperature heat shock elevates the NQO1 expression in cancer cells, which in turn markedly increases the sensitivity of the cells to the bioreductive drug β-lap in vitro and in vivo.

Induction of apoptosis by carboplatin and hyperthermia alone or combined in WERI human retinoblastoma cells

This paper investigated the induction of apoptosis and perturbation of cell cycle progression caused by carboplatin (CPt) and hyperthermia alone or combined in WERI human retinoblastoma cells in vitro . An incubation of the cells with 25 or 50 µ m of CPt at 37°C caused apoptosis, which progressively increased during the 24-72 h treatment. Hyperthermia at 42.5°C for 1 h induced apoptosis, which became significant from 24 h after the heating. Heating the cells in the presence of CPt and subsequent incubation with CPt was far more effective than treating the cells with hyperthermia or CPt treatment alone in inducing apoptosis in the WERI cells, indicating that the combination of these two modalities is potentially useful for the treatment of retinoblastoma. It appeared that the apoptosis in WERI cells caused by hyperthermia and CPt occurs during G1 phase. An interesting observation was that caspase 9 activation preceded the release of cytochrome C from mitochondria during apoptosis in WERI cells, contrary to the general notion that caspase 9 is activated by cytochrome C.

Aurora-A Contributes to Radioresistance by Increasing NF-κB DNA Binding

Abstract Aurora-A, a serine/threonine kinase that is overexpressed in certain human cancer cell lines, plays an important role in mitotic progression. Aurora-A has also been reported to be involved in the activation of nuclear factor kappa B (NF-&kgr;B). The purpose of the present study was to identify the role of Aurora-A in the radiation-induced activation pathway of NF-&kgr;B. Wild-type and Aurora-A knockdown (Aurora-AKD) HeLa cells were irradiated with 4 Gy of &ggr; rays and the EMSA, luciferase reporter gene assay and immunoblot analysis were performed. The siRNA-based gene knockdown and overexpression system was adopted to elucidate the role of Aurora-A in radiation-induced NF-&kgr;B pathway activation. The clonogenic survival study indicated that Aurora-AKD cells and the wild-type cells transfected with Aurora-A siRNA or RelA/p65 siRNA were more radiosensitive than the wild-type cells. In both the wild-type and Aurora-AKD cells, radiation caused I&kgr;B kinase-mediated phosphorylation, degradation of I&kgr;B&agr; and phosphorylation of RelA/p65. The nuclear translocation of RelA/p65 was also similar in the wild-type and Aurora-AKD cells. However, RelA/p65-DNA binding was markedly suppressed in Aurora-AKD cells compared to that in wild-type cells. It was concluded that Aurora-A enhances the binding of NF-&kgr;B to DNA, thereby increasing the gene transcription by NF-&kgr;B and decreasing the radiosensitivity of the cells.