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Dai-Wu Seol

Publications and source records attributed to Dai-Wu Seol.

18 recordsLinked to original sources
PubMed2007

A split enhanced green fluorescent protein-based reporter in yeast two-hybrid system.

We have developed a novel reporter system involving a yeast two-hybrid assay, which utilizes the reconstitution of the split EGFP reporter in order to characterize the relevant protein-protein interactions. To our knowledge, this study represents the first application of the split EGFP system as a read-out in a yeast two-hybrid assay. In comparison with the existing two-hybrid system, the bait and prey vectors were improved with regard to the reporter and the replication control element. As a result, the reconstituted EGFP has been observed to evidence a restored fluorescence upon protein-protein interactions in yeast, thereby allowing for the characterization of its interactor. The use of a split EGFP reporter has some salient advantages. Firstly, no substrates are required for the production of fluorescence. Secondly, low copy number plasmids may help to solve the protein toxicity problem, via the reduction of expression. Thirdly, this technique may prove useful in overcoming the autoactivation problem, due to the fact that the read-out of the yeast two-hybrid system is transcription-independent. Collectively, our results showed that the split EGFP reporter system might potentially be applied in yeast two-hybrid assays for the high-throughput screening of protein-protein interactions, with a simple and direct fluorescent read-out.

Escherichia coli↗
PubMed2006

Generation of a novel proform of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein that can be reactivated by matrix metalloproteinases.

TRAIL has been suggested to induce the cell death in various tumor cells but not in normal cells; however, several studies have provided the evidence that TRAIL can induce the cell death in some normal cells including human normal hepatocytes, suggesting that TRAIL may show hepatic toxicity in human. In this study, we designed a pro-form of TRAIL (sTRAIL:IL-18) in that soluble TRAIL (sTRAIL) is fused to IL-18, and a matrix metalloproteinases (MMPs) cleavage site is introduced at the connecting site. We showed that sTRAIL:IL-18 has significantly diminished the killing activity in HeLa cells but regains the activity by releasing the free sTRAIL through MMP-2-mediated cleavage. In addition, the killing activity of sTRAIL:IL-18 was significantly increased in HeLa cells when active MMP-2 was produced by TNF-alpha. Taken together, the data suggested that the sTRAIL:IL-18 can be reactivated at the specialized areas where MMPs are pathologically produced.

Apoptosis↗
PubMed2006

Characterization of cell clones stably transfected with short form caspase-9: apoptotic resistance and Bcl-XL expression.

Caspases play important roles in the initiation and progression of apoptosis. In experimental models of ATP depletion, we have demonstrated the activation of caspase-9, -8, and -3, which is followed by the development of apoptotic morphology. To determine the specific contribution of caspase-9 to ATP depletion-induced apoptosis, we transfected renal epithelial cells with its endogenous dominant-negative inhibitor caspase-9S. Two cell clones with stable transfection were obtained. These clones expressed caspase-9S, and the cytosol isolated from these cells was resistant to cytochrome c-induced caspase activation in vitro. The clones were then examined for ATP depletion-induced apoptosis. Compared with the wild-type cells, the caspase-9S clones were markedly resistant to apoptosis in this model. Caspase activation was also inhibited. Surprisingly, these clones also showed significantly less cytochrome c release during ATP-depletion. Moreover, Bax translocation to mitochondria was inhibited, suggesting that these clones were resistant to apoptosis not only at the cytosolic caspase activation level but also at the upstream mitochondrial level. To gain insights into the mitochondrial resistance, we analyzed the expression of Bcl-2 family proteins. While the expression of Bax, Bak, and Bcl-2 was comparable to the wild-type cells, the selected clones showed specific up-regulation of Bcl-XL, an anti-apoptotic protein. We conclude that the selected clones were resistant to apoptosis at two levels. In the cytosol, they expressed dominant negative caspase-9, and at the mitochondria they up-regulated Bcl-XL.

Adenosine Triphosphate↗
PubMed2006

Intra-articular injection of recombinant TRAIL induces synovial apoptosis and reduces inflammation in a rabbit knee model of arthritis.

We demonstrated previously that local, intra-articular injection of an adenoviral vector expressing human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a rabbit knee model of inflammatory arthritis stimulated synovial apoptosis and reduced inflammation. To examine whether intra-articular injection of recombinant chimeric human TRAIL protein (rTRAIL) also induces apoptosis of proliferating rabbit synovium and reduces inflammation, we used an experimental rabbit arthritis model of rheumatoid arthritis, induced by intra-articular introduction of allogeneic fibroblasts genetically engineered to secrete human IL-1beta. Analysis of synovium isolated from the rabbits treated with intra-articular injection of rTRAIL, relative to saline control, showed areas of extensive acellular debris and large fibrous regions devoid of intact cells, similar to adenoviral mediated TRAIL gene transfer. Extensive apoptosis of the synovial lining was demonstrated using TUNEL analysis of the sections, corresponding to the microscopic findings in hematoxylin and eosin staining. In addition, leukocyte infiltration into the synovial fluid of the inflamed knee joints following rTRAIL treatment was reduced more than 50% compared with the saline control. Analysis of the glycosaminoglycan synthetic rate by cultured cartilage using radiolabeled sulfur and cartilage histology demonstrated that rTRAIL did not adversely affect cartilage metabolism and structure. Analysis of serum alanine aminotransferase showed that intra-articular injection of rTRAIL did not have adverse effects on hepatic function. These results demonstrate that intra-articular injection of rTRAIL could be therapeutic for treating pathologies associated with rheumatoid arthritis.

Animals↗
PubMed2005

Detection of Bax protein conformational change using a surface plasmon resonance imaging-based antibody chip.

We describe an antibody chip technology that uses a surface plasmon resonance (SPR) imaging system to examine the conformational change of a protein. In this study, we used Bax protein, a pro-apoptotic member of the Bcl-2 family of proteins, as a model protein to investigate the conformational alteration triggered by a TNF-related apoptosis-inducing ligand (TRAIL), a potent inducer of apoptosis. To develop the antibody chip for detecting the Bax conformational change, we immobilized Bax monoclonal antibody 6A7, which recognizes only a conformationally changed Bax protein on a gold surface. The resultant immobilized Bax antibodies provided specific and accurate measurements of the active conformation-specific epitope in the apoptotic cancer cells treated with the TRAIL; these measurements corresponded to the data obtained by immunoprecipitation analysis using an active conformation-specific Bax antibody (6A7). The results of our study indicated that TRAIL-induced Bax structural change could be monitored quickly and simply using an SPR imaging system, thus demonstrating the potential for using such a system for the analysis of conformational properties of target proteins.

Antibodies↗
PubMed2005

Cisplatin inactivation of caspases inhibits death ligand-induced cell death in vitro and fulminant liver damage in mice.

Cisplatin is a platinum-containing chemotherapeutic drug that has been widely used to treat various human cancers. It acts by forming inter- and intracross-links of DNA, which is believed to be a major cause for its therapeutic efficacy. However, little attention has been paid to the effect of cisplatin on death ligand-induced cell death. Here we demonstrate that cisplatin inhibits death ligand-induced cell death in cell lines in a p53-independent manner. This inhibitory effect of cisplatin on cell death is direct, whereby cisplatin forms a complex with caspases leading to their inactivation. The cisplatin-caspase complex is reversed by the addition of reducing agent dithiothreitol, and caspase activity is regained. In addition, cisplatin shows a death-inhibition effect in in vivo animal models of fulminant liver damage induced by Fas activation and lipopolysaccharide-induced liver shock mediated by tumor necrosis factor-alpha. Together, we demonstrate that cisplatin inhibits cell death induced by death ligands in cell lines and in mice through caspase inactivation.

Animals↗
PubMed2005

Wortmannin elevates tumor necrosis factor-related apoptosis-inducing ligand sensitivity in LNCaP cells through down-regulation of IAP-2 protein.

BACKGROUND AND AIM: TNF-related apoptosis-inducing ligand (TRAIL), which is a member of the tumor necrosis factor (TNF) family, has been shown to effectively induce tumor apoptosis without any detectable cytotoxic side effects. But recent studies have shown that LNCaP cells are resistant to TRAIL, because LNCaP cells express the constitutively active Akt/protein kinase B. Therefore this study examined the effect of Wortmannin, a PI-3 kinase inhibitor, and the intracellular mechanisms to TRAIL-induced apoptosis. METHODS: LNCaP cells, which are resistant to apoptotic induction of TRAIL alone, were exposed to Wortmannin for 3 h and treated with the recombinant TRAIL protein for an additional 3 h. RESULTS: TRAIL alone induced apoptosis in approximately 10% of the cells, whilst pretreatment with Wortmannin increased the TRAIL-induced apoptosis to 45%. Wortmannin alone had no effect on the tumor cell viability, but enhanced the TRAIL-induced apoptosis in a dose-dependent manner and completely blocked Akt phosphorylation. The blocking of the Akt phosphorylation decreased the IAP-2 protein expression level, which is believed to mediate the enhanced TRAIL-induced apoptosis. CONCLUSION: Wortmannin elevates the TRAIL sensitivity in LNCaP cells by regulating the phosphorylation of Akt and IAP-2 protein expression.

Androstadienes↗
PubMed2004

The secretable form of trimeric TRAIL, a potent inducer of apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine molecule of the TNF family. Soluble recombinant TRAIL has been shown to induce apoptosis in a wide variety of cancer cells in vitro and to specifically limit tumor growth without damaging normal cells and tissues in vivo. These results suggest a strong potential of TRAIL as an anticancer therapy. Here we report an artificial TRAIL gene that expresses and secretes trimeric TRAIL into the culture supernatant. This novel TRAIL gene is composed of three functional elements, including a secretion signal, a trimerization domain, and an apoptosis-inducing moiety of TRAIL gene sequence. The expression vectors delivering this TRAIL gene produced secretable forms of trimeric TRAIL proteins. These TRAIL proteins showed greater apoptotic activity than the known TRAIL protein that does not contain an additional trimerization domain. Our data suggest that the gene therapy using our artificial TRAIL gene may be used as an anticancer therapy.

Amino Acid Sequence↗
PubMed2004

Hypoxia inhibits tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by blocking Bax translocation.

The hypoxic environment in solid tumors results from oxygen consumption by rapid proliferation of tumor cells. Hypoxia has been shown to facilitate the survival of tumor cells and to be a cause of malignant transformation. Hypoxia also is well known to attenuate the therapeutic activity of various therapies in cancer management. These observations indicate that hypoxia plays a critical role in tumor biology. However, little is known about the effects of hypoxia on apoptosis, especially on apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent apoptosis inducer that has been shown to specifically limit tumor growth without damaging normal cells and tissues in vivo. To address the effects of hypoxia on TRAIL-induced apoptosis, HCT116 human colon carcinoma cells were exposed to hypoxic or normoxic conditions and treated with soluble TRAIL protein. Hypoxia dramatically inhibited TRAIL-induced apoptosis in HCT116 cells, which are highly susceptible to TRAIL in normoxia. Hypoxia increased antiapoptotic Bcl-2 family member proteins and inhibitors of apoptosis proteins. Interestingly, these hypoxia-increased antiapoptotic molecules were decreased by TRAIL treatment to the levels lower than those of the untreated conditions, suggesting that hypoxia inhibits TRAIL-induced apoptosis via other mechanisms rather than up-regulation of these antiapoptotic molecules. Additional characterization revealed that hypoxia significantly inhibits TRAIL-induced translocation of Bax from the cytosol to the mitochondria in HCT116 and A549 cells, with the concomitant inhibition of cytochrome c release from the mitochondria. Bax-deficient HCT116 cells were completely resistant to TRAIL regardless of oxygen content, demonstrating a pivotal role of Bax in TRAIL-induced apoptotic signaling. Thus, our data indicate that hypoxia inhibits TRAIL-induced apoptosis by blocking Bax translocation to the mitochondria, thereby converting cells to a Bax-deficient state.

Antineoplastic Agents↗
PubMed2004

Carbon monoxide inhibits T lymphocyte proliferation via caspase-dependent pathway.

T lymphocyte activation and proliferation is involved in many pathological processes. We have recently shown that carbon monoxide (CO), an enzymatic product of heme oxygenase-1 (HO-1), confers potent antiproliferative effects in airway and vascular smooth muscle cells. The purpose of this study was to determine whether CO can inhibit T lymphocyte proliferation and then to determine the mechanism by which CO can modulate T lymphocyte proliferation. In the presence of 250 parts per million CO, CD3-activated T lymphocyte proliferation was, remarkably, inhibited by 80% when compared with controls. We observed that the antiproliferative effect of CO in T lymphocytes was independent of the mitogen-activated protein kinase or cGMP signaling pathways, unlike what we demonstrated previously in smooth muscle cells. We demonstrate that CO inhibited caspase-3 and caspase-8 expression and activity, and caspase inhibition with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK pan-caspase inhibitor) blocked T lymphocyte proliferation. Furthermore, in caspase-8-deficient lymphocytes, the antiproliferative effect of CO was markedly attenuated, further supporting the involvement of caspase-8 in the antiproliferative effects of CO. CO also increased the protein level of p21(Cip1), and CO-mediated inhibition of caspase activity is partially regulated by p21(Cip1). Taken together, these data suggest that CO confers potent antiproliferative effects in CD3-activated T lymphocytes and that these antiproliferative effects in T lymphocytes are mediated by p21(Cip1)-dependent caspase activity, in particular caspase-8, independent of cGMP and mitogen-activated protein kinase signaling pathways.

Animals↗
PubMed2004

IFN-gamma enhances TRAIL-induced apoptosis through IRF-1.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family and a potent inducer of apoptosis. TRAIL has been shown to effectively limit tumor growth in vivo without detectable cytotoxic side-effects. Interferon (IFN)-gamma often modulates the anticancer activities of TNF family members including TRAIL. However, little is known about the mechanism. To explore the mechanism, A549, HeLa, LNCaP, Hep3B and HepG2 cells were pretreated with IFN-gamma, and then exposed to TRAIL. IFN-gamma pretreatment augmented TRAIL-induced apoptosis in all these cell lines. A549 cells were selected and further characterized for IFN-gamma action in TRAIL-induced apoptosis. Western blotting analyses revealed that IFN-gamma dramatically increased the protein levels of interferon regulatory factor (IRF)-1, but not TRAIL receptors (DR4 and DR5) and pro-apoptotic (FADD and Bax) and anti-apoptotic factors (Bcl-2, Bcl-XL, cIAP-1, cIAP-2 and XIAP). To elucidate the functional role of IRF-1 in IFN-gamma-enhanced TRAIL-induced apoptosis, IRF-1 was first overexpressed by using an adenoviral vector AdIRF-1. IRF-1 overexpression minimally increased apoptotic cell death, but significantly enhanced apoptotic cell death induced by TRAIL when infected cells were treated with TRAIL. In further experiments using an antisense oligonucleotide, a specific repression of IRF-1 expression abolished enhancer activity of IFN-gamma for TRAIL-induced apoptosis. Therefore, our data indicate that IFN-gamma enhances TRAIL-induced apoptosis through IRF-1.

Adenoviridae↗
PubMed2003

TRAIL, a mighty apoptosis inducer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a membrane-bound cytokine molecule that belongs to the family of tumor necrosis factor (TNF). TRAIL has been shown to be a potent apoptosis inducer in a wide variety of cancer cells in vitro and to limit tumor growth efficiently in vivo without damaging normal tissues. These features have focused considerable attention on TRAIL as a potential therapeutic agent to treat human cancers. Recent data also suggest the implication of TRAIL in a natural defense mechanism since its abrogation results in certain autoimmune disorders. This review will summarize recent progress in TRAIL research, including understanding of apoptotic signaling, regulation of TRAIL action, and possible therapeutic applications.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Upregulation of FLIP(S) by Akt, a possible inhibition mechanism of TRAIL-induced apoptosis in human gastric cancers.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in some, but not all cancer cells. To assess the regulation of TRAIL-resistance in the human gastric cancer cells, we examined TRAIL sensitivity, TRAIL receptor expression, and intracellular signaling events induced by TRAIL. All the gastric cancer cell lines tested were susceptible to TRAIL to some extent, except for SNU-216 cell line, which was completely resistant. TRAIL receptor expression was not related to the TRAIL-sensitivity. Of the cell lines tested, SNU-216 showed the highest level of constitutively active Akt and the short form of FLICE inhibitory protein (FLIP(S)). Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or with the protein synthesis inhibitor cycloheximide induced a suppression of constitutive Akt activation in SNU-216 cells and a concomitant decrease in the expression of FLIP(S). The reduction of Akt activity by LY294002 affected the transcriptional level of FLIP(S), but not the mRNA stability. As a result, LY294002 or cycloheximide significantly enhanced TRAIL-induced apoptosis. Moreover, the overexpression of constitutively active Akt in the TRAIL-sensitive cell line, SNU-668, rendered the cell line resistant to TRAIL. In addition, infection of the same cell line with retrovirus expressing FLIP(S) completely inhibited TRAIL-induced apoptosis by blocking the activation of caspase-8. Therefore, our results suggest that Akt activity promotes human gastric cancer cell survival against TRAIL-induced apoptosis via upregulation of FLIP(S), and that the cytotoxic effect of TRAIL can be enhanced by modulating the Akt/FLIP(S) pathway in human gastric cancers.

Adenocarcinoma↗
PubMed2002

Regulation of Akt by EGF-R inhibitors, a possible mechanism of EGF-R inhibitor-enhanced TRAIL-induced apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine and a potent inducer of apoptosis. Epidermal growth factor (EGF) signaling is well known to involve in tumor survival and overexpression of EGF receptor (EGF-R) attributes to decreased responsiveness to many available therapies in cancer treatment. We investigated whether EGF-R inhibitors enhance TRAIL-induced apoptosis. We exposed A549 cells to Genistein, PD153035, and PD158780 for 12h and then treated with recombinant TRAIL protein. TRAIL alone induced 25% cell death after a 3-h treatment, but in cells pretreated with EGF-R inhibitors, TRAIL induced cell death to more than 70% after 3h treatment. Genistein enhanced TRAIL-induced apoptosis in a time- and dose-dependent manner. Western blot analyses showed that pretreatment with Genistein down-regulated the protein levels of total Akt and phosphorylated active Akt. Genistein also decreased the protein level of Bcl-XL that is regulated by Akt. These molecules are well characterized to act against induction of apoptotic cell death. Therefore, our data suggest that EGF-R inhibitor may sensitize A549 cells to TRAIL-induced apoptosis by regulating expression of these proteins. EGF-R inhibitors may play an important role in the anti-cancer activity of TRAIL protein, especially in TRAIL-resistant tumors that arise by expressing constitutively active Akt.

Apoptosis↗
PubMed2002

IFN-gamma inhibition of TRAIL-induced IAP-2 upregulation, a possible mechanism of IFN-gamma-enhanced TRAIL-induced apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine molecule of TNF family and a potent inducer of apoptosis. The anticancer activities of TNF family members are often modulated by interferon (IFN)-gamma. Thus, we investigated whether IFN-gamma enhances TRAIL-induced apoptosis. We exposed HeLa cells to IFN-gamma for 12 h and then treated with recombinant TRAIL protein. No apoptosis was induced in cells pretreated with IFN-gamma, and TRAIL induced 25% cell death after 3 h treatment. In HeLa cells pretreated with IFN-gamma, TRAIL induced cell death to more than 70% at 3 h, indicating that IFN-gamma pretreatment sensitized HeLa cells to TRAIL-induced apoptosis. We investigated molecules that might be regulated by IFN-gamma pretreatment that would affect TRAIL-induced apoptosis. Western blotting analyses demonstrated that TRAIL treatment increased the level of IAP-2 protein and IFN-gamma pretreatment inhibited the upregulation of IAP-2 protein by TRAIL protein. Our data indicate that TRAIL can signal to activate both apoptosis induction and antiapoptotic mechanism, at least, through IAP-2 simultaneously. IFN-gamma or TRAIL treatment alone did not change expression of other pro- or antiapoptotic proteins such as DR4, DR5, FADD, Bax, IAP-1, XIAP, Bcl-2, and Bcl-XL. Our findings suggest that IFN-gamma may sensitize HeLa cells to TRAIL-induced apoptosis by preventing TRAIL-induced IAP-2 upregulation, and IFN-gamma may play a role in anticancer therapy of TRAIL protein through such mechanism.

Apoptosis↗
PubMed2002

Hypoxia inhibition of apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Hypoxia is a common environmental stress. Particularly, the center of rapidly growing solid tumors is easily exposed to hypoxic conditions. Thus, tumor cell response to hypoxia plays an important role in tumor progression as well as tumor therapy. However, little is known about hypoxic effect on apoptotic cell death. To examine the effects of hypoxia on TRAIL-induced apoptosis, human lung carcinoma A549 cells were exposed to hypoxia and treated with TRAIL protein. Hypoxia significantly protected A549 cells from apoptosis induced by TRAIL. Western blotting analysis demonstrated that hypoxia increased expression of antiapoptotic proteins such as Bcl-2, Bcl-XL, and IAP family members. The increase of these antiapoptotic molecules is believed to play an hypoxia-mediated protective role in TRAIL-induced apoptosis. Our findings suggest that an increase of antiapoptotic proteins induced by hypoxia may regulate the therapeutic activity of TRAIL protein in cancer therapy.

Apoptosis↗
PubMed2002

Tumor necrosis factor-related apoptosis-inducing ligand induces caspase-dependent interleukin-8 expression and apoptosis in human astroglioma cells.

Among the tumor necrosis factor (TNF) family of cytokines, FasL and TNF-related apoptosis-inducing ligand (TRAIL) are known to induce cell death via caspase activation. Recently, other biological functions of these death ligands have been postulated in vitro and in vivo. It was previously shown that Fas ligation induces chemokine expression in human glioma cells. In this study, we investigated whether the TRAIL-DR5 system transduces signals similar to those induced by other TNF family ligands and receptors. To address this issue, two human glioma cell lines, CRT-MG and U87-MG, were used, and an agonistic antibody against DR5 (TRA-8) and human recombinant TRAIL were used to ligate DR5. We demonstrate that DR5 ligation by either TRAIL or TRA-8 induces two functional outcomes, apoptosis and expression of the chemokine interleukin-8 (IL-8); the nonspecific caspase inhibitor Boc-D-Fmk blocks both TRAIL-mediated cell death and IL-8 production; the caspase 3-specific inhibitor z-DEVD-Fmk suppresses TRAIL-mediated apoptosis but not IL-8 induction; caspase 1- and 8-specific inhibitors block both TRAIL-mediated cell death and IL-8 production; and DR5 ligation by TRAIL mediates AP-1 and NF-kappaB activation, which can be inhibited by caspase 1- and 8-specific inhibitors. These findings collectively indicate that DR5 ligation on human glioma cells leads to apoptosis and that the activation of AP-1 and NF-kappaB leads to the induction of IL-8 expression; these responses are dependent on caspase activation. Therefore, the TRAIL-DR5 system has a role not only as an inducer of apoptotic cell death but also as a transducer for proinflammatory and angiogenic signals in human brain tumors.

Antibodies, Monoclonal↗