Ji-Seon Lee

Wip1 directly dephosphorylates gamma-H2AX and attenuates the DNA damage response

The integrity of DNA is constantly challenged throughout the life of a cell by both endogenous and exogenous stresses. A well-organized rapid damage response and proficient DNA repair, therefore, become critically important for maintaining genomic stability and cell survival. When DNA is damaged, the DNA damage response (DDR) can be initiated by alterations in chromosomal structure and histone modifications, such as the phosphorylation of the histone H2AX (the phosphorylated form is referred to as gamma-H2AX). gamma-H2AX plays a crucial role in recruiting DDR factors to damage sites for accurate DNA repair. On repair completion, gamma-H2AX must then be reverted to H2AX by dephosphorylation for attenuation of the DDR. Here, we report that the wild-type p53-induced phosphatase 1 (Wip1) phosphatase, which is often overexpressed in a variety of tumors, effectively dephosphorylates gamma-H2AX in vitro and in vivo. Ectopic expression of Wip1 significantly reduces the level of gamma-H2AX after ionizing as well as UV radiation. Forced premature dephosphorylation of gamma-H2AX by Wip1 disrupts recruitment of important DNA repair factors to damaged sites and delays DNA damage repair. Additionally, deletion of Wip1 enhances gamma-H2AX levels in cells undergoing constitutive oncogenic stress. Taken together, our studies show that Wip1 is an important mammalian phosphatase for gamma-H2AX and shows an additional mechanism for Wip1 in the tumor surveillance network.

Senescent growth arrest in mesenchymal stem cells is bypassed by Wip1-mediated downregulation of intrinsic stress signaling pathways.

Human mesenchymal stem cells (hMSCs) have been widely studied as a source of primary adult stem cells for cell therapy because of their multidifferentiation potential; however, the growth arrest (also known as “premature senescence”) often found in hMSCs cultured in vitro has been a major obstacle to the in‐depth characterization of these cells. In addition, the inability to maintain constant cell growth hampers the development of additional genetic modifications aimed at achieving desired levels of differentiation to specific tissues; however, the molecular mechanisms that govern this phenomenon remain unclear, with the exception of a few studies demonstrating that induction of p16INK4a is responsible for this senescence‐like event. Here, we observed that the premature growth arrest in hMSCs occurs in parallel with the induction of p16INK4a, following abrogation of inhibitory phosphorylation of retinoblastoma protein. These stress responses were concurrent with increased formation of reactive oxygen species (ROSs) from mitochondria and increased p38 mitogen‐activated protein kinase (MAPK) activity. The introduction of Wip1 (wild‐type p53 inducible phosphatase‐1), a well‐studied stress modulator, significantly lowered p16INK4a expression and led to p38 MAPK inactivation, although it failed to affect the levels of ROSs. Moreover, the suppression of stress responses by Wip1 apparently extended the life span of hMSCs, compared with control conditions, while maintaining their multilineage differentiation potential. Based on these results, we suggest that senescent growth arrest in hMSCs may result from activation of stress signaling pathways and consequent onset of stress responses, due in part to ROS production during prolonged in vitro culture. STEM CELLS 2009;27:1963–1975

Comment on Saeednia et al.'s strong designated verifier signature scheme

In 1996, Jakobsson et al. proposed a designated verifier signature scheme in which only one specified person, called a designated verifier, can be convinced of the validity of the signature and the identity of the signer. This is possible by giving the designated verifier the ability to simulate a signature him/herself in an indistinguishable way. Therefore, the other third party cannot determine whether the signature is from the signer or the designated verifier. However, in some circumstances, the third party may be convinced that a signature intended for the designated verifier is actually generated by the signer. In 2003, Saeednia et al. proposed a strong designated verifier signature scheme to overcome this problem. However, we found that Saeednia et al.s scheme would reveal the identity of the signer if the secret key of this signer is compromised. In this paper, we provide a new strong designated verifier signature scheme that provides signer ambiguity, even if the secret key of the signer is compromised. We also analyze the proposed scheme.

The modulation of the oxidative stress response in chondrocytes by Wip1 and its effect on senescence and dedifferentiation during in vitro expansion

Obtaining a sufficient number of cells ex vivo for tissue regeneration, which are appropriate for cartilage repair, requires improved techniques for the continuous expansion of chondrocytes in a manner that does not change their innate characteristics. Rapid senescence or dedifferentiation during in vitro expansion results in loss of chondrocyte phenotype and the formation of fibrous cartilage replacement tissue, rather than hyaluronic cartilage, after transplantation. As demonstrated in the current study, wild-type p53-inducible phosphatase (Wip1), a well-established stress modulator, was highly expressed in early-passage chondrocytes, but declined rapidly during in vitro expansion. Stable Wip1-expressing chondrocytes generated by microporation were less susceptible to the onset of senescence and dedifferentiation, and were more resistant to oxidative stress. The increased resistance of Wip1 chondrocytes to oxidative stress was due to modulation of p38 mitogen-activated protein kinase (MAPK) activity. Importantly, chondrocytes expressing Wip1 maintained their innate chondrogenic properties for a longer period of time, resulting in improvements in cartilage regeneration after transplantation. Chondrocytes from Wip1 knockout (Wip1(-/-)) mice were defective in cartilage regeneration compared with those from wild-type mice. Thus, Wip1 expression represents a potentially useful mechanism by which a chondrocyte phenotype can be retained during in vitro expansion through modulation of cellular stress responses.

Generation of Cancerous Neural Stem Cells Forming Glial Tumor by Oncogenic Stimulation

Neural stem cells in the brain have been shown to be ‘cells of origin’ of certain brain cancers, most notably astrocytomas and medulloblastoma. In particular, in a mouse model, the targeting of genetic modifications for astrocytoma-relevant tumor suppressors to neural stem cells causes malignant astrocytoma to arise, thereby suggesting that astrocytoma is derived from neural stem cells. However, it remains to be determined whether this important finding is reproducible in humans. Herein, we generated cancerous neural stem cells by introducing a set of oncogenes to human fetal neural stem cells (hfNSCs). Serial genetic modification with v-myc for immortalization and consequent H-Ras for oncogenic stimulation with viral gene delivery proved sufficient to induce the transformation of hfNSCs. The resultant F3.Ras cells evidenced a variety of the hallmarks of brain cancer stem cells and most importantly were tumorigenic, forming brain cancers consisting of both a large number of differentiated and a very few undifferentiated populations of cells in an in vivo mouse model. On the contrary, oligodendrocytes derived from the v-myc expressing parent neural stem cells were not transformed by H-Ras, which suggests that neural stem cells may be more susceptible to cancerous transformation by a combination of oncogenes. We also determined that v-myc expressing fetal neural stem cells were defective in p53 response upon the introduction of H-Ras; this finding suggests that an insufficient p53-dependent tumor suppressive mechanism would be associated with high oncogenic susceptibility to H-Ras introduction.

Accelerated wound healing by S-methylmethionine sulfonium: evidence of dermal fibroblast activation via the ERK1/2 pathway.

S-Methylmethionine sulfonium (SMMS) is a derivative of the amino acid methionine, and is synthesized in a variety of plants. SMMS is widely referred to as vitamin U because of its potent therapeutic effect on gastrointestinal ulceration. Skin wounds are accompanied by mucosal erosion and share similar histopathological aspects with gastric ulcers, so it is plausible that SMMS may promote skin wound healing. In animal models, topical administration of SMMS for a given period of time, to both physical and chemical wounds, facilitated wound closure and promoted re-epithelialization compared with a control. In addition, single SMMS treatment was sufficient to promote the growth of human dermal fibroblasts (hDFs) as well as the migration of hDFs, which are indispensable steps for skin wound healing. The promotion of hDF proliferation and migration resulted from considerable activation of ERK1/2 by SMMS, and inhibition of ERK activity by a chemical inhibitor significantly abrogated both the promoted proliferation and migration of hDFs. Therefore, we concluded that SMMS facilitated the repair process of skin damage by activation of dermal fibroblasts, which suggests that SMMS has potential as a skin wound-healing agent.

Tumor necrosis factor-inducible gene 6 promotes liver regeneration in mice with acute liver injury

IntroductionTumor necrosis factor-inducible gene 6 protein (TSG-6), one of the cytokines released by human mesenchymal stem/stromal cells (hMSC), has an anti-inflammatory effect and alleviates several pathological conditions; however, the hepatoprotective potential of TSG-6 remains unclear. We investigated whether TSG-6 promoted liver regeneration in acute liver failure.MethodsThe immortalized hMSC (B10) constitutively over-expressing TSG-6 or empty plasmid (NC: Negative Control) were established, and either TSG-6 or NC-conditioned medium (CM) was intraperitoneally injected into mice with acute liver damage caused by CCl4. Mice were sacrificed at 3 days post-CM treatment.ResultsHigher expression and the immunosuppressive activity of TSG-6 were observed in CM from TSG-6-hMSC. The obvious histomorphological liver injury and increased level of liver enzymes were shown in CCl4-treated mice with or without NC-CM, whereas those observations were markedly ameliorated in TSG-6-CM-treated mice with CCl4. Ki67-positive hepatocytic cells were accumulated in the liver of the CCl4 + TSG-6 group. RNA analysis showed the decrease in both of inflammation markers, tnfα, il-1β, cxcl1 and cxcl2, and fibrotic markers, tgf-β1, α-sma and collagen α1, in the CCl4 + TSG-6 group, compared to the CCl4 or the CCl4 + NC group. Protein analysis confirmed the lower expression of TGF-β1 and α-SMA in the CCl4 + TSG-6 than the CCl4 or the CCl4 + NC group. Immunostaining for α-SMA also revealed the accumulation of the activated hepatic stellate cells in the livers of mice in the CCl4 and CCl4 + NC groups, but not in the livers of mice from the CCl4 + TSG-6 group. The cultured LX2 cells, human hepatic stellate cell line, in TSG-6-CM showed the reduced expression of fibrotic markers, tgf-β1, vimentin and collagen α1, whereas the addition of the TSG-6 antibody neutralized the inhibitory effect of TSG-6 on the activation of LX2 cells. In addition, cytoplasmic lipid drops, the marker of inactivated hepatic stellate cell, were detected in TSG-6-CM-cultured LX2 cells, only. The suppressed TSG-6 activity by TSG-6 antibody attenuated the restoration process in livers of TSG-6-CM-treated mice with CCl4.ConclusionsThese results demonstrated that TSG-6 contributed to the liver regeneration by suppressing the activation of hepatic stellate cells in CCl4-treated mice, suggesting the therapeutic potential of TSG-6 for acute liver failure.

Strong Designated Verifier Signature Scheme with Message Recovery

In this paper, we propose a strong designated verifier signature scheme with message recovery mechanism. In designated verifier signature scheme, the designated verifier can simulate a signature which is indistinguishable from the signature by the real signer. Our scheme guarantees that only the designated verifier can recover the message, verify the signature, and be convinced of the real signer. If the verifier reveals his secret key, then anyone can recover the message and verify the signature. However, still no one can be convinced that who the real signer is. The security assumption of our scheme only depends on the discrete logarithm problem.

Key factors for e-commerce business success

For the key factors of competitiveness or key success factors for an e-commerce business, there have been many studies and papers to examine and clarify the causes and backgrounds of success in e-commerce marketplace. Though these trials and attempts have covered remarkably broad factors, most of them have weakness and limitations in that focusing relatively narrow perspectives applicable to e-commerce service, applications-level. To resolve these limitations and show broaden perspectives to be considered in clarifying the key success factors for an e-commerce corporate, this paper adopts the concepts and methodologies used in strategic management field such as value chain analysis and balanced scorecard (BSC). By adopting those concepts and methodologies, we try to provide the corporate-level key success factors for e-commerce business. The key success factors for an e-commerce business suggested in this paper are composed of three perspectives: tangible assets, intangible assets and processes. By reflecting many aspects to be considered in clarifying essential competitiveness factors, we give corporate a thorough lookout for key success factors of an e-commerce and suggest a guideline to follow in the course of obtaining competitive advantage in e-commerce industry

Off-target response of a Wip1 chemical inhibitor in skin keratinocytes

BACKGROUND The wild type p53 inducible phosphatase (Wip1) plays an important role in modulating not only stress responses by various environmental stresses, but when overexpressed it also impairs the intrinsic tumor surveillance networks that are frequently found in a number of cancers including skin cancers. As a result, using a pharmacological inhibitor of Wip1 has been suggested to be a novel chemotherapeutic approach to recover the innate tumor surveillance in a variety of cancers. OBJECTIVE We studied the effect of a pharmacological inhibitor of Wip1 in skin keratinocytes, under a ultra-violet (UV) stress condition. METHODS A human keratinocyte cell line or human epidermal keratinocytes were exposed to UV, with or without the sole commercially available chemical inhibitor of Wip1, CCT007093; subsequently, we determined the diverse stress responses, including apoptosis and the activation of stress signaling. RESULTS We demonstrate that the Wip1 inhibitor unexpectedly attenuated the UV-mediated apoptotic response in skin keratinocytes, as a consequence of attenuated JNK activation and reduced H2AX phosphorylation in both, skin keratinocytes and a Wip1-null cell model. On the other hand, the loss of Wip1 expression, either by knockout or knockdown in mice or human keratinocytes respectively, promoted apoptosis and potentiated H2AX phosphorylation following UV treatment. Of note, CCT007093 treatment appeared to promote apoptosis in breast cancer cells and skin transformed keratinocytes that ectopically expressed Wip1, demonstrating that the effect of CCT007093 differs based on the level of Wip1 expression. CONCLUSION Thus, our studies suggest that the development of a more potent and specific Wip1 inhibitor is necessary to achieve the desired chemotherapeutic potential and to avoid off-target effects.