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Infection of hepatitis B virus (HBV) increase the incidence of chronic liver disease and hepatocellular carcinoma (HCC). The hepatitis B viral x (HBx) protein encoded by the HBV genome contributes to the pathogenesis of HCC and thus, negative regulation of HBx is beneficial for the alleviation of the disease pathogenesis. MARCH5 is a mitochondrial E3 ubiquitin ligase and here, we show that high MARCH5 expression levels are correlated with improved survival in HCC patients. MARCH5 interacts with HBx protein mainly accumulated in mitochondria and targets it for degradation. The N-terminal RING domain of MARCH5 was required for the interaction with HBx, and MARCH5H43W lacking E3 ligase activity failed to reduce HBx protein levels. High expression of HBx results in the formation of protein aggregates in semi-denaturing detergent agarose gels and MARCH5 mediates the elimination of protein aggregates through the proteasome pathway. HBx-induced ROS production, mitophagy, and cyclooxygenase-2 gene expression were suppressed in the presence of high MARCH5 expression. These results suggest MARCH5 as a target for alleviating HBV-mediated liver disease.

TNF receptor-associated death domain (TRADD) is an essential mediator of TNF receptor signaling, and serves as an adaptor to recruit other effectors. TRADD has been shown to cycle between the cytoplasm and nucleus due to its nuclear localization (NLS) and export sequences (NES). However, the underlying function of nuclear TRADD is poorly understood. Here we demonstrate that cytoplasmic TRADD translocates to DNA double-strand break sites (DSBs) during the DNA damage response (DDR). Deficiency of TRADD or its sequestration in cytosol leads to accumulation of γH2AX-positive foci in response to DNA damage, which is reversed by nuclear TRADD expression. TRADD facilitates non-homologous end-joining (NHEJ) by recruiting NHEJ repair factors 53BP1 and Ku70/80 complex, whereas TRADD is dispensable for homologous recombination (HR) repair. Finally, an impaired nuclear localization of TRADD triggers cell death through the persistent activation of JNK and accumulation of reactive oxygen species (ROS). Thus, our findings suggest that translocation of TRADD to DSBs into the nucleus contributes to cell survival in response to DNA damage through an activation of DNA damage repair.

Toxic epidermal necrolysis (TEN) is a severe adverse drug reaction involving extensive keratinocyte death in the epidermis. Histologically, the skin from TEN patients exhibits separation at the dermo-epidermal junction and accompanying necrosis of epidermal keratinocytes. Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is an essential part of the cellular machinery that executes "programmed", or "regulated", necrosis and has a key role in spontaneous cell death and inflammation in keratinocytes under certain conditions. Here we show that RIP3 expression is highly upregulated in skin sections from TEN patients and may therefore contribute to the pathological damage in TEN through activation of programmed necrotic cell death. The expression level of mixed lineage kinase domain-like protein (MLKL), a key downstream component of RIP3, was not significantly different in skin lesions of TEN. However, elevated MLKL phosphorylation was observed in the skin from TEN patients, indicating the presence of RIP3-dependent programmed necrosis. Importantly, in an in vitro model of TEN, dabrafenib, an inhibitor of RIP3, prevented RIP3-mediated MLKL phosphorylation and decreased cell death. Results from this study suggest that the high expression of RIP3 in keratinocytes from TEN patients potentiates MLKL phosphorylation/activation and necrotic cell death. Thus, RIP3 represents a potential target for treatment of TEN.

Although loss of Sirt1 leads to chromosome aneuploidy, which accounts for higher tumor susceptibility, the molecular mechanisms remain unclear. Herein, we demonstrate that Sirt1 directly regulates Plk1, of which activity is critical for mitotic progression and spindle dynamics. Depletion or inhibition of Sirt1 significantly perturbs the formation of the mitotic spindle, leading to defective chromosome segregation. Elevated depolymerization of the mitotic spindle following loss of Sirt1 was associated with the deregulation of Plk1 activity. Thus, we conclude that Sirt1 may contribute to a mitotic regulator that controls spindle dynamics through Plk1 activity, resulting in fine-tuning of Plk1 dependent microtubule dynamics.

// Soo Ah Jeong 1, 2, * , In Young Kim 1, 2, * , A Reum Lee 1, 2 , Mi Jin Yoon 1, 2 , Hyeseong Cho 1, 2 , Jong-Soo Lee 3 , Kyeong Sook Choi 1, 2 1 Department of Biochemistry, Ajou University School of Medicine, Suwon, Korea 2 Graduate Program of Cancer Biology, Department of Biomedical Sciences, Ajou University School of Medicine, Suwon, Korea 3 Department of Life Science, Ajou University, Suwon, Korea * These authors have contributed equally to this work Correspondence to: Kyeong Sook Choi, e-mail: kschoi@ajou.ac.kr Keywords: CDDO-Me, endoplasmic reticulum, Ca 2+ , c-FLIP L , apoptosis Received: January 23, 2015 Accepted: May 12, 2015 Published: May 25, 2015 ABSTRACT The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-C28-methyl ester (CDDO-Me) is considered a promising anti-tumorigenic compound. In this study, we show that treatment with CDDO-Me induces progressive endoplasmic reticulum (ER)-derived vacuolation in various breast cancer cells and ultimately kills these cells by inducing apoptosis. We found that CDDO-Me–induced increases in intracellular Ca 2+ levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca 2+ levels, CDDO-Me markedly increased the generation of reactive oxygen species (ROS). Interestingly, there exists a reciprocal positive-regulatory loop between Ca 2+ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me. In addition, CDDO-Me rapidly reduced the protein levels of c-FLIP L (cellular FLICE-inhibitory protein) and overexpression of c-FLIP L blocked CDDO-Me–induced cell death, but not vacuolation. These results suggest that c-FLIP L downregulation is a key contributor to CDDO-Me–induced apoptotic cell death, independent of ER-derived vacuolation. Taken together, our results show that ER-derived vacuolation via Ca 2+ influx and ROS generation as well as caspase activation via c-FLIP L downregulation are responsible for the potent anticancer effects of CDDO-Me on breast cancer cells.

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is an essential part of the cellular machinery that executes "programmed" or "regulated" necrosis. Here we show that programmed necrosis is activated in response to many chemotherapeutic agents and contributes to chemotherapy-induced cell death. However, we show that RIP3 expression is often silenced in cancer cells due to genomic methylation near its transcriptional start site, thus RIP3-dependent activation of MLKL and downstream programmed necrosis during chemotherapeutic death is largely repressed. Nevertheless, treatment with hypomethylating agents restores RIP3 expression, and thereby promotes sensitivity to chemotherapeutics in a RIP3-dependent manner. RIP3 expression is reduced in tumors compared to normal tissue in 85% of breast cancer patients, suggesting that RIP3 deficiency is positively selected during tumor growth/development. Since hypomethylating agents are reasonably well-tolerated in patients, we propose that RIP3-deficient cancer patients may benefit from receiving hypomethylating agents to induce RIP3 expression prior to treatment with conventional chemotherapeutics.

Hepatocyte growth factor (HGF) and its receptor, Met, regulate skeletal muscle differentiation. In the present study, we identified a novel alternatively spliced isoform of Met lacking exon 13 (designated Δ13Met), which is expressed mainly in human skeletal muscle. Alternative splicing yielded a truncated Met having extracellular domain only, suggesting an inhibitory role. Indeed, Δ13Met expression led to a decrease in HGF-induced tyrosine phosphorylation of Met and ERK phosphorylation, as well as cell proliferation and migration via sequestration of HGF. Interestingly, in human primary myoblasts undergoing differentiation, Δ13Met mRNA and protein levels were rapidly increased, concomitantly with a decrease in wild type Met mRNA and protein. Inhibition of Δ13Met with siRNA led to a decreased differentiation, whereas its overexpression potentiated differentiation of human primary myoblasts. Furthermore, in notexin-induced mouse injury model, exogenous Δ13Met expression enhanced regeneration of skeletal muscle, further confirming a stimulatory role of the isoform in muscle cell differentiation. In summary, we identified a novel alternatively spliced inhibitory isoform of Met that stimulates muscle cell differentiation, which confers a new means to control muscle differentiation and/or regeneration.

α-Mangostin (α-MG), one of the active substances in Garcinia mangostana, has been shown to exhibit anti-cancer effects in various cancer cell types. α-MG treatment induces G1 arrest in cancer cell models through the induction of cyclin-dependent kinase inhibitors (CDKIs) and the subsequent loss of CDK activity. However, outside its role in the p53-p21CIP1 axis, the precise molecular mechanisms underlying the effect of α-MG on cell cycle arrest remain unclear. In this study, we observed that α-MG inhibits the proliferation of HCT116 cells in a dose-dependent manner. Interestingly, although the loss of p53 rescued the α-MG effect on cell cycle arrest, in agreement with previous reports, p21Cip1 expression was only marginally delayed in the absence of p53 after α-MG treatment. Instead, we found that the activation of p38 mitogen activated protein kinase (MAPK) and the subsequent downregulation of Bmi-1 also contributed to the induction of p16Ink4a, which is responsible for G1 arrest upon α-MG treatment. These findings indicate that α-MG exerts cytostatic effects on colon cancer cells by inducing G1 arrest via the p38MAPK-p16INK4a axis.

The signal-responsive E3 ubiquitin ligase pellino 1 (PELI1) regulates TLR and T cell receptor (TCR) signaling and contributes to the maintenance of autoimmunity; however, little is known about the consequence of mutations that result in upregulation of PELI1. Here, we developed transgenic mice that constitutively express human PELI1 and determined that these mice have a shorter lifespan due to tumor formation. Constitutive expression of PELI1 resulted in ligand-independent hyperactivation of B cells and facilitated the development of a wide range of lymphoid tumors, with prominent B cell infiltration observed across multiple organs. PELI1 directly interacted with the oncoprotein B cell chronic lymphocytic leukemia (BCL6) and induced lysine 63-mediated BCL6 polyubiquitination. In samples from patients with diffuse large B cell lymphomas (DLBCLs), PELI1 expression levels positively correlated with BCL6 expression, and PELI1 overexpression was closely associated with poor prognosis in DLBCLs. Together, these results suggest that increased PELI1 expression and subsequent induction of BCL6 promotes lymphomagenesis and that this pathway may be a potential target for therapeutic strategies to treat B cell lymphomas.

ObjectivesHepatitis B virus (HBV) is a member of the hepadnavirus family. The HBV genome contains four genes designated as S, C, P, and X. The HBV X (HBx) gene encodes for a 16.5-kDa regulatory protein that enhances HBV replication and exerts multifunctional activities. The aim of this study is to describe the rapid and easy purification of HBx using ELP (elastin-like polypeptide) fusion protein.MethodsThe ELP–HBx fusion protein was overexpressed in Escherichia coli. Environmental sensitivity was demonstrated via turbidity and dynamic light scattering as a function of temperature. HBx was purified as an ELP fusion protein. ELPs are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition. ELP follows in temperature and salt consistency, precipitation, and solution repetition (inverse transition cycling) with polypeptide, where it purifies the protein in a simple manner.ResultsFusion proteins underwent supramolecular aggregation at 40 °C in 1 M NaCl and slowly resolubilized at subphysiologic temperatures. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity to the commercial HBx.ConclusionThis study suggests that HBx can be purified rapidly and easily using inverse transition cycling, and that this method can be applied in determination of HBx 3D structures and HBx stability study.

Growth factors accelerate G0 to S progression in the cell cycle, however, the roles of growth factors in other cell cycle phases are largely unknown. Here, we show that treatment of HeLa cells with hepatocyte growth factor (HGF) at G2 phase induced the G2/M transition delay as evidenced by FACS analysis as well as by mitotic index and time-lapse analyses. Growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) also induced G2/M transition delay like HGF. HGF treatment at G2 phase causes a delayed activation of cyclin B1-associated kinase and a diminished nuclear translocation of cyclin B1. Either U0126, a MAPK kinase (MEK) inhibitor, or kinase-dead mutant of ribosomal S6 kinase (RSK) abolished the delay. Additionally, knockdown of RSK1, but not RSK2, with siRNA abrogated the delay, indicating that the extracellular-regulated protein kinase (ERK)-RSK1 mediates the HGF-induced delay. We further found that the delay in G2/M transition of cells expressing oncogenic HGF receptor, M1268T, was abolished by RSK1 knockdown. Intriguingly, we observed that HGF induced chromosomal segregation defects, and depletion of RSK1, but not RSK2, aggravated these chromosomal aberrations. Taken together, the ERK-RSK1 activation by growth factors delays G2/M transition and this might be required to maintain genomic integrity during growth factor stimulation.

Multifunctional activities of the hepatitis B virus X-protein (HBx) in cells have been largely implicated in the development of liver cancer; one of these activities is the loss of p53 function by sequestering p53 in the cytoplasm. We have previously found that doxorubicin increased the p53 levels in cells containing p53-binding HBx protein and restored the p53-mediated transcriptional activity that was suppressed by HBx. Here, we investigated the mechanism underlying p53 reactivation. We found that six phosphorylation sites of the Serine residues of p53 were efficiently phosphorylated in HBx-expressing ChangX-34 cells, suggesting that the binding of HBx to the p53 protein does not interfere with the phosphorylation of p53 by signaling kinases. In addition, doxorubicin caused a dramatic reduction of Hdm2 mRNA and protein levels in cells expressing HBx. Intriguingly, reactivation of p53 was accompanied with a nuclear accumulation of p53 and the phosphorylated p53 at Serine15 was only detected in nuclear fraction, but not in cytosolic fraction of doxorubicin-treated ChangX-34 cells. Functional restoration of the p53 protein in HBx-expressing cells occurs according to the dual effects of doxorubicin: a significant reduction of Hdm2 expression and a nuclear accumulation of the phosphorylated p53 protein. Thus, proper usage of doxorubicin as an effective antitumor agent may be reevaluated and can be extended to tumors primarily caused by infection of DNA tumor viruses.

Mitochondria serve as platforms for innate immunity. The mitochondrial antiviral signalling (MAVS) protein forms aggregates that elicit robust type-I interferon induction on viral infection, but persistent MAVS signalling leads to host immunopathology; it remains unknown how these signalling aggregates are resolved. Here we identify the mitochondria-resident E3 ligase, MARCH5, as a negative regulator of MAVS aggregates. March5+/− mice and MARCH5-deficient immune cells exhibit low viral replication and elevated type-I interferon responses to RNA viruses. MARCH5 binds MAVS only during viral stimulation when MAVS forms aggregates, and these interactions require the RING domain of MARCH5 and the CARD domain of MAVS. MARCH5, but not its RING mutant (MARCH5H43W), reduces the level of MAVS aggregates. MARCH5 transfers ubiquitin to Lys7 and Lys500 of MAVS and promotes its proteasome-mediated degradation. Our results indicate that MARCH5 modulates MAVS-mediated antiviral signalling, preventing excessive immune reactions., RNA viral infections trigger an immune response mediated by the formation of aggregates of the MAVS protein. Here the authors show that the mitochondrial protein MARCH5 modulates this response by transferring ubiquitin to MAVS aggregates, thus promoting their proteasomal degradation.

Hepatocyte chromosome polyploidization is an important feature of liver development and seems to be required for response to liver stress and injury signals. However, the question of how polyploidization can be tightly regulated in liver growth remains to be answered. Using a conditional knockout mouse model, liver-specific depletion of Ssu72 protein phosphatase was found to result in impairment in regulation of polyploidization. Interestingly, the aberrant polyploidization in Ssu72-depleted mice was associated with impaired liver damage response and increased markers of liver injury and seemed to mimic the phenotypic features of liver diseases such as fibrosis, steatosis, and steatohepatitis. In addition, depletion of Ssu72 caused deregulation of cell cycle progression by overriding the restriction point of the cell cycle and aberrantly promoting DNA endoreplication through G2/M arrest. Conclusion: Ssu72 plays a substantial role in the maintenance of hepatic chromosome homeostasis and would allow monitoring of liver function. (Hepatology 2016;63:247–259)

Genetic instability is intimately associated with tumour development. In particular, liver cancers associated with hepatitis B virus (HBV) exhibit high genetic instability; however, our understanding of the underlying molecular mechanisms remains limited. In this study, we found that γ-H2AX, a marker of DNA double-strand breaks (DSBs), and the levels of phospho-Chk2 (p-Chk2, the activated form) were significantly elevated in HBV-associated hepatocellular carcinomas and neighbouring regenerating nodules. Likewise, introduction of the pHBV or pMyc-HBx plasmids into cells induced accumulation of γ-H2AX foci and increased the p-Chk2 level. In these cells, inhibitory phosphorylation of Cdc25C phosphatase (Ser(216)) and CDK1 (Tyr(15)) was elevated; consequently, cell-cycle progression was delayed at G2/M phase, suggesting that activation of the ATM-Chk2 pathway by the HBV X protein (HBx) induces cell-cycle delay. Accordingly, inhibition of ataxia telangiectasia mutated (ATM) by caffeine or siRNA abolished the increase in the p-Chk2 level and restored the delayed CDK1 kinase activity in ChangX cells. We also found that cytoplasmic HBx, but not nuclear HBx, induced reactive oxygen species (ROS) production and led to the accumulation of γ-H2AX foci and the increased p-Chk2 level. Together, these data indicate that HBx-induced ROS accumulation induces DNA damage that activates the ATM-Chk2 pathway. Our findings provide insight into the mechanisms of HBV pathogenesis.

Cetuximab, a chimeric monoclonal antibody developed for targeting the Epidermal Growth Factor Receptor (EGFR), has been intensively used to treat cancer patients with metastatic colorectal cancer and head and neck cancer. Intact immunoglobulin G (IgG) antibody like cetuximab, however, has some limitations such as high production cost and low penetration rate from vasculature into solid tumor mass due to its large size. In attempt to overcome these limitations, we engineered cetuximab to create single chain variable fragments (scFv-CH3; Minibody) that were expressed in bacterial system. Among three engineered minibodies, we found that MI061 minibody, which is composed of the variable heavy (V-H) and light (V-L) region joined by an 18-residue peptide linker, displays higher solubility and better extraction properties from bacterial lysate. In addition, we validated that purified MI061 significantly interferes ligand binding to EGFR and blocks EGFR's phosphorylation. By using a protein microarray composed of 16,368 unique human proteins covering around 2,400 plasma membrane associated proteins such as receptors and channels, we also demonstrated that MI061 only recognizes the EGFR but not other proteins as compared with cetuximab. These results indicated that engineered MI061 retains both binding specificity and affinity of cetuximab for EGFR. Although it had relatively short half-life in serum, it was shown to be highly significant anti-tumor effect by inhibiting ERK pathway in A431 xenograft model. Taken together, our present study provides compelling evidence that engineered minibody is more effective and promising agent for in vivo targeting of solid tumors.

EGFR mutations and ALK translocation are considered mutually exclusive. We found that a portion of NSCLCs have EGFR and ALK co-alteration. When using sensitive assays for EGFR, the rate of co-alteration increased up to 15.4% of ALK-positive cases. These patients were sensitive to ALK inhibitors, but not to gefitinib. The clinical relevance of finding co-alteration with sensitive methods was discussed. Background Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) translocation are considered mutually exclusive in nonsmall-cell lung cancer (NSCLC). However, sporadic cases having concomitant EGFR and ALK alterations have been reported. The present study aimed to assess the prevalence of NSCLCs with concomitant EGFR and ALK alterations using mutation detection methods with different sensitivity and to propose an effective diagnostic and therapeutic strategy. Patients and methods A total of 1458 cases of lung cancer were screened for EGFR and ALK alterations by direct sequencing and flourescence in situ hybridization (FISH), respectively. For the 91 patients identified as having an ALK translocation, peptide nucleic acid (PNA)-clamping real-time PCR, targeted next-generation sequencing (NGS), and mutant-enriched NGS assays were carried out to detect EGFR mutation. Results EGFR mutations and ALK translocations were observed in 42.4% (612/1445) and 6.3% (91/1445) of NSCLCs by direct sequencing and FISH, respectively. Concomitant EGFR and ALK alterations were detected in four cases, which accounted for 4.4% (4/91) of ALK-translocated NSCLCs. Additional analyses for EGFR using PNA real-time PCR and ultra-deep sequencing by NGS, mutant-enriched NGS increased the detection rate of concomitant EGFR and ALK alterations to 8.8% (8/91), 12.1% (11/91), and 15.4% (14/91) of ALK-translocated NSCLCs, respectively. Of the 14 patients, 3 who were treated with gefitinib showed poor response to gefitinib with stable disease in one and progressive disease in two patients. However, eight patients who received ALK inhibitor (crizotinib or ceritinib) showed good response, with response rate of 87.5% (7/8 with partial response) and durable progression-free survival. Conclusions A portion of NSCLC patients have concomitant EGFR and ALK alterations and the frequency of co-alteration detection increases when sensitive detection methods for EGFR mutation are applied. ALK inhibitors appear to be effective for patients with co-alterations.

Although the hepatitis B virus X protein (HBx) is thought to play a causative role in the development of hepatocellular carcinoma, it is not yet known whether interfering with HBx function may affect the cellular transformation of HBx-expressing tumor cells. To address this question, we adopted an intracellular antibody fragment expression approach to block the function of HBx. Expression of a single-chain variable fragment (scFv) specific to HBx (designated as H7scFv) inhibited HBx-dependent cellular transactivation. Furthermore, H7scFv suppressed the growth of HBx-expressing tumor cells in both soft agar and nude mice. The suppressive effect of H7scFv on tumorigenicity appeared not to be mediated by inhibition of HBx-induced growth stimulation since the growth rate of these cells was not affected significantly by H7scFv expression. In conclusion, these data suggest that the HBx-dependent transformed phenotype is reversible and that HBx may be a good molecular target for the treatment of HBV-related tumors.

Multinucleated cells resulted from mitosis defect have been noted in pathophysiological states of the cells such as inflammation, senescence and cancer. Since oxidative stress has been known to correlate with these pathophysiological conditions, we tested the effect of H2O2 on the cell cycle progression and formation of multinucleated cells. H2O2 induced a significant delay in cell cycle progression in Chang liver cells. Interestingly, H2O2 actively induced hyperamplification of centrosomes (n>or=3) and multipolar spindle formation during mitosis and subsequently increased the generation of multinucleated cells. A significant increase of the phospho-ERK level was observed upon H2O2 treatment but PD98059, an MEK1/2 inhibitor, didn't reduce the frequency of cells with hyperamplified centrosomes. On the other hand, treatment of either H2O2 or adriamycin increased intracellular ROS levels and multinucleated cells, which were significantly suppressed by antioxidants, N-acetylcysteine and PDTC. Thus, our results suggest that oxidative stress can trigger centrosome hyperamplification and multinucleated cell formation, which may promote pathophysiological progression.

Conditionally immortalized hepatocytes (CIH) established with a gene for the temperature-sensitive mutant of the T antigen (tsT) have characteristics to stop proliferating and to differentiate at nonpermissive temperatures (37-39 degrees C) due to inactivation of the T antigen. Therefore, they may be a good alternative to primary hepatocytes for experimental investigations or clinical applications. Deinduction of the T antigen results in a transient increase of p53 in these cells, leading to reexpression of normal senescence because of the telomere attrition occurring during the early stages of immortalization. To determine this T antigen dependency for the maintenance of immortality, a type of rat CIH was cultured continuously at 39 degrees C. The frequency of occurrence of T-antigen-independent clones ranged from 0.053% to 0.093%. These clones maintained the temperature-sensitive property of the T antigen; nevertheless, they were able to progress to the S phase and proliferate without undergoing apoptosis at 39 degrees C as at 33 degrees C, a permissive temperature. The temperature-sensitive point mutation of tsT was not affected in these clones and the T antigen was functioning properly. The integrity of the p53 pathway was also maintained from the point of Western blot analysis of p21. Although the telomerase continued to be expressed and the telomere length was maintained, marked chromosomal damage could not be avoided in these cells. It is a plausible explanation that this escape phenomenon from conditional immortalization may be related to the change of other genes involved in cell cycles, which have yet to be elucidated. In conclusion, CIH could lose their temperature-sensitive characteristics without the change of tsT, itself, and the T antigen is not always necessary to maintain their immortality. Therefore, the results obtained from experimental investigations using these cells should be interpreted carefully, and unpredictable phenotypic changes should also be taken into consideration when using them in clinical applications.

Autocrine stimulation via coexpression of hepatocyte growth factor (HGF) and its receptor (Met) has been reported in many human sarcomas, but few in carcinomas. In this report, we found that one gastric cancer cell line, SNU-484, among 11 gastric cell lines tested has an autocrine HGF- Met stimulation. RT-PCR, ELISA and scattering assay using MDCK cells revealed that SNU-484 cells secreted a significant amount of active HGF (about 1.25 +/- 0.41 ng/24 h/10(6) cells) into conditioned medium. Resultantly, Met in this cell line was constitutively phosphorylated. Neutralizing antibodies against HGF reduced the tyrosine phosphorylation of Met, resulting in the inhibition of cell proliferation and migration (P

Ligand activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been reported to induce growth inhibition and apoptosis in various cancers including hepatocellular carcinoma (HCC). However, the effect of hepatitis B virus X protein (HBx) on PPARgamma activation has not been characterized in hepatitis B virus (HBV)-associated HCC. Herein, we demonstrated that HBx counteracted growth inhibition caused by PPARgamma ligand in HBx-associated HCC cells. We found that HBx bound to DNA binding domain of PPARgamma and HBx/PPARgamma interaction blocked nuclear localization and binding to recognition site of PPARgamma. HBx significantly suppressed a PPARgamma-mediated transactivation. These results suggest that HBx modulates PPARgamma function through protein-protein interaction.

Hepatitis B virus (HBV) X protein (HBx) is thought to be involved in the development of liver cancer and alteration of cellular HBx level may influence the pathological progression of HBV-induced liver diseases. We found that the cellular levels of HBx mRNA transcript and protein in cells were greatly enhanced by adriamycin, a topoisomerase II inhibitor. Up-regulation of HBx mRNA by adriamycin was also observed in HBx transgenic mice, which was accompanied with a significant increase of VEGF mRNA, the downstream target of HBx. When we investigated the underlying mechanism, we found that half-life of HBx mRNA in HBx-expressing Chang cells was about 3 h, but was prolonged to >6 h in the presence of adriamycin. Moreover, half-life of rapidly degrading HBx protein was determined as about 15 min however, it remained almost constant until 60 min in the presence of adriamycin. These results provide the first evidence that the cellular level of HBx gene can be increased at the post-transcriptional level.

Hepatitis B virus (HBV) X protein (HBx) plays an essential role in development of HBV-associated hepatocellular carcinoma (HCC). Recently, we reported that HBx induces Fas Ligand (FasL) expression, which may help HCC cells to evade host-immune surveillance. The aim of this study was to investigate the role of HBx in expression of Nur77, an orphan nuclear receptor implicated in the upregulation of FasL. When Chang X-34 expressing HBx under the control of a doxycycline-inducible promoter was examined, induction of Nur77 was observed following HBx expression. Blocking of Nur77 function by introduction of an antisense or a dominant negative mutant Nur77 significantly inhibited the induction of FasL, indicating that Nur77 plays critical roles in FasL expression. Further, a high-level expression of transcripts and DNA binding of Nur77 were observed in the HBV-integrated cell lines established from HCC patients that express HBx. These results suggested that Nur77 may contribute to leading the HBx-induced Fas/FasL signaling pathway which eliminates invading Fas-expressing lymphocytes.

Hepatocellular carcinoma (HCC) generally shows chemoresistant features to anticancer agents. Paclitaxel has been clinically used in the treatment of various cancers. However, effect of paclitaxel on HCC has not been adequately addressed. Here, we found two categories of hepatoma cells in response to paclitaxel. Paclitaxel effectively decreased the cell viability of SNU475, Hep3B, and SNU387 HCC cells and Chang liver cells (death prone). In contrast, the other five hepatoma cell lines (SNU449, SNU398, SUN368, SNU354, and HepG2 cells) were resistant to paclitaxel (death reluctant). In response to paclitaxel, Bcl-2 was highly phosphorylated in death-prone cells, whereas much less Bcl-2 was phosphorylated in death-reluctant cells. Cotreatment with SP600125, an inhibitor JNK, significantly reduced the phosphorylated Bcl-2 in death-prone cells and caused a significant reduction in cell death. The reduced cell death was due to prohibition into mitotic entry as evidenced by low cyclin B1/Cdk1 kinase activity. In death-reluctant cells, inbuild-phospho-JNK levels were high but no longer activated in response to paclitaxel. We found that paclitaxel combined with caffeine or UCN-01, inhibitors of G2 DNA damage checkpoint, was able to partially overcome resistance to paclitaxel in these cells. Thus our data provide the molecular basis of paclitaxel resistance in hepatoma cells, and appropriate combination therapy may increase treatment efficacy.

Thymidine kinase (TK) activity was examined during the development of preimplantation mouse embryos. TK activity was increased approximately 20-fold from day 2 embryos (2-cell) to day 5 embryos (late blastocyst). TK activity did not change along with the progression into S-phase of the first and the second cell cycles but increased sharply at S-phase of the third cell cycle. Analysis of TK mRNA with a reverse transcription-polymerase chain reaction (RT-PCR) method showed that the level of TK mRNA was low in ovulated eggs and 1-cell embryos and was hardly detectable in day 2 embryos (2-cell), but sharply increased in day 3 embryos (mixture of 5- to 8-cell and morula). The functional role of 5'-flanking sequence of TK gene was also investigated in preimplantation embryos after microinjection with the DNA construct of 5'-flanking sequence of TK (2.4 kb) linked to bacterial lacZ gene (TK2.5lacZ) into the pronucleus of 1-cell and subsequently by histochemical staining with X-gal. beta-Galactosidase activity was first detected in day 3 embryos (8-cell), and 30% of embryos were stained with X-gal in day 4 and day 5 embryos, respectively. These results show that an increase in TK activity occurred after 2-cell stage, and this increase was primarily due to the embryonic activation of TK gene expression. Also, it appears that the 5'-flanking sequence of TK may directly regulate the TK gene expression at the transcriptional level during preimplantation murine development.

Wound healing requires re-epithelialization from the wound margin through keratinocyte proliferation and migration, and some growth factors are known to influence this process. In the present study, we found that the co-treatment with hepatocyte growth factor (HGF) and TGF-beta1 resulted in enhanced migration of HaCaT cells compared with either growth factor alone, and that N-acetylcysteine, an antioxidant agent, was the most effective among several inhibitors tested, suggesting the involvement of reactive oxygen species (ROS). Fluorescence-activated cell sorter analysis using 2,7-dichlorofluorescein diacetate (DCF-DA) dye showed an early (30 min) as well as a late (24 h) increase of ROS after scratch, and the increase was more prominent with the growth factor treatment. Diphenyliodonium (DPI), a potent inhibitor of NADPH oxidase, abolished the increase of ROS at 30 min, followed by the inhibition of migration, but not the late time event. More precisely, gene knockdown by shRNA for either Nox-1 or Nox-4 isozyme of gp91phox subunit of NADPH oxidase abolished both the early time ROS production and migration. However, HaCaT cell migration was not enhanced by treatment with H((2))O((2)). Collectively, co-treatment with HGF and TGF-beta1 enhances keratinocyte migration, accompanied with ROS generation through NADPH oxidase, involving Nox-1 and Nox-4 isozymes.

The Hepatitis B virus (HBV) is a causative agent of acute chronic hepatitis, cirrhosis, and hepatocarcinoma. The Hepatitis B virus X protein (HBx) has pleiotypic functions in the regulation of proliferation and apoptosis. It has been suggested that the anti-inflammatory drug sulfasalazine, which is commonly used to treat rheumatoid arthritis and inflammatory bowel disease, inhibits nuclear factor NF-kappaB and induces cell death in HBx-expressing liver cells. In this study, we demonstrate that sulfasalazine induces cell death via apoptosis in HBx-expressing liver cells, as evidenced by characteristic changes in nuclear morphology, cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3 and caspase-9, and activation of caspase-3. We also demonstrate that inhibition of NF-kappaB by siRNA fails to induce apoptosis of HBx-expressing liver cells, indicating that sulfasalazine modulates apoptosis of HBx-expressing cells in an NF-kappaB-independent manner.

When cells are deprived of iron, their growth is invariably inhibited. However, the mechanism involved remains largely unclear. Recently, we have reported that subcytotoxic concentration of deferoxamine mesylate (DFO), an iron chelator, specifically inhibited transition of Chang cell, a normal hepatocyte cell line, from G1 to S phase, which was accompanied by irreversible appearance of senescent biomarkers. To investigate factors responsible for the irreversible arrest, we examined mitochondrial activities because they require several irons for their proper structure and function. After exposure to 1 M DFO, total cellular ATP level was irreversibly decreased with concurrent disruption of mitochondrial membrane potential (DeltaPsim), implying that it might be one of the crucial factors involved in the arrest. DFO did not directly inhibit the mitochondrial respiratory activities in vitro. Among the respiratory activities, complex II activity was specifically inhibited through a down-regulation of the expression of its iron-sulfur subunit. We also observed that mitochondrial morphology was drastically changed to highly elongated form. Our results suggest that mitochondrial function is sensitive to cellular iron level and iron deprivation might be involved in inducing the senescent arrest. In addition, complex II, which is a part of both oxidative phosphorylation and the Krebs cycle, could be one of the critical factors that regulate mitochondrial function by responding to iron levels.

HBx (hepatitis B virus X) viral oncoprotein is a multifunctional protein of which the cellular level may be one of the important factors in determining HBV-mediated pathological progression of liver diseases, chronic hepatitis, and hepatocellular carcinoma. Our previous work revealed that adriamycin, a chemotherapeutic agent, caused a marked increase in the intracellular level of HBx by retarding its rapid degradation. In the present study, modulation of HBx expression was found to be confined to adriamycin but not to other chemotherapeutic agents, cisplatin and 5-fluorouracil. Interestingly, adriamycin caused a rapid increase of reactive oxygen species (ROS) and its accumulation continued until 24h. In contrast, two other agents had little effect on ROS generation, suggesting the possible involvement of ROS in the HBx regulation. In fact, direct addition of H(2)O(2) to the cells significantly increased the level of HBx protein in HBx-expressing ChangX-34 cells as well as in hepatitis B virus-related hepatoma cells, PLC/PRF/5 and HepG2.2.15 cells. Furthermore, antioxidants, N-acetyl-cysteine and pyrrolidinedithiocarbamate (PDTC), completely abolished the increase of HBx protein induced by adriamycin, indicating that adriamycin modulates the intracellular HBx level via ROS generation. Together, these findings provide a novel aspect of HBx regulation by cellular ROS level. Therefore, intracellular microenvironments generating ROS such as severe inflammation may aggravate the pathogenesis of liver disease by accumulating the HBx level.

Background/Aims : The hepatitis B virus X protein (HBx), a major viral transactivator, is implicated in hepatic inflammation, since it induces many pro-inflammatory cytokines at transcriptional level. The aim of this study was to investigate role of HBx in expression of interleukin 18 (IL-18), a newly identified cytokine that up-regulates Fas ligand (FasL) expression. Methods : Chang X-34 that expressing HBx under the control of a doxycycline-inducible promoter, and hepatitis B virus (HBV)-integrated hepatoma cell lines were examined for IL-18 expression by Northern and Western blotting analysis. To test the role of IL-18 produced by hepatoma cells, FasL expression was examined by flow cytometry after treatment with neutralizing anti-IL-18 antibodies. Further, IL-18 expression was examined in the liver tissues of HBx-transgenic mice. Results : Induction of IL-18 following HBx expression in Chang X-34 and the pattern of IL-18 expression in HBV-integrated cell lines, implicated that HBx transcriptionally induces IL-18 expression. Neutralizing anti-IL-18 antibodies blocked the expression of FasL, suggesting that IL-18 plays a critical role in FasL expression. Further, IL-18 expression in the HBx-transgenic liver, was correlated with the degree of hepatitis. Conclusions : Our results demonstrated that HBx induces IL-18 expression in liver, which may be associated with hepatic injury by amplifying FasL expression during HBV infection.

Iron is essential for cellular proliferation in all organisms. When deprived of iron, the growth of cells is invariably inhibited. However, the mechanism involved remains largely unclear. In the present study, we have observed that subcytotoxic concentrations of desferroxamine mesylate (DFO), an iron chelator, specifically inhibited the transition from G1 to S-phase of Chang cells, a hepatocyte cell line. This was accompanied by the appearance of senescent biomarkers, such as enlarged and flattened cell morphology, senescence-associated beta-galactosidase activity and reduced expression of poly(ADP-ribose) polymerase. Concomitantly, p27Kip1 (where Kip is kinase-inhibitory protein) was induced markedly, whereas other negative cell-cycle regulators, such as p21Cip1 (where Cip is cyclin-dependent kinase-interacting protein), p15INK4B and p16INK4A (where INK is inhibitors of cyclin-dependent kinase 4), were not, implying its association in the G1 arrest. Furthermore, the induction of p27Kip1 was accompanied by an increased level of transforming growth factor beta1 (TGF-beta1) mRNA. When neutralized with an anti-(TGF-beta1) antibody, p27Kip1 induction was completely abolished, indicating that TGF-beta1 is the major inducer of p27Kip1. Finally, DFO-induced senescence-like arrest was found to be independent of p53, since cell-cycle arrest was still observed with two p53-negative cell lines, Huh7 and Hep3B cells. In conclusion, DFO induced senescence-like G1 arrest in hepatocyte cell lines and this was associated with the induction of p27Kip1 through TGF-beta1, but was independent of p53.

We earlier reported that N-carbamoyl-L-glutamate (CG) plus L-arginine (Arg) protected normal and 70% hepatectomized rats from intoxication by a lethal or sub-lethal dose of ammonium acetate, respectively. In the present study, the protective effect of these compounds on cirrhotic rats was assessed.CG plus Arg were administered prior to the injection of a sub-lethal dose of ammonium acetate into dimethylnitrosamine-induced cirrhotic rats. Control rats were given phosphate-buffered saline (PBS) instead of the mixture. The behavior of the rats was monitored until the time of sacrifice. Blood ammonia level, blood urea nitrogen (BUN) and liver carbamoylphosphate synthetase I (CPS I) activity were determined.Pretreatment of rats with the mixture of CG plus Arg could significantly lower the blood ammonia level (P0.05), increase the activity of CPS I (P0.05), improve abnormal behavior associated with ammonia intoxication (P0.05), and increase BUN (P0.05), as compared with the PBS-injected control group. There were significantly close correlations between (1) the increase of CPS I activity; (2) the improvement of abnormal behavior; (3) the increase of BUN; and (4) the decrease of the blood ammonia level.A mixture of CG plus Arg could protect rats with liver cirrhosis from acute ammonia intoxication.

Hepatitis B virus X (HBx) protein is a multifunctional protein that exerts dual activity on cell proliferation and death. Although HBx is thought to be a major determinant that leads to hepatocellular carcinoma, its pathophysiological role in humans remains to be established. Attempts have been made to evaluate the role of HBx in liver specimens derived from patients with chronic B viral hepatitis and hepatocellular carcinoma. Among 25 paired liver specimens of hepatocellular carcinoma and corresponding nontumour liver tissues, HBx mRNA was hardly detected and was significantly lower than other HBV transcripts. An immunohistochemical study demonstrated that expression of HBx protein was also lower than other HBV gene products. Interestingly, however, expression of HBx protein changed with the progression of chronic hepatitis. HBx was expressed in 5.0% of patients with chronic hepatitis without cirrhosis but increased to 44.8% in chronic hepatitis with cirrhosis. In contrast, only one (3.7%) of 27 hepatocellular carcinomas showed HBx positivity whereas 29.6% of surrounding nontumour tissues was still HBx-positive. These results suggest that HBx may play a major role at the promotion stage of carcinogenesis. Noticeably, HBx-positive cells were preferentially localized in the periportal region of chronic hepatitis or periphery of cirrhotic nodules where high necroinflammatory activity was accompanied. We found a positive correlation between HBx expression and periportal inflammatory activity (P < 0.001). Thus, HBx may potentiate cell destruction and regeneration of liver that provide an opportunity for the accumulation of genetic mutations, which contribute to multistep hepatocarcinogenesis.

Activating mutations in the Met receptor tyrosine kinase, both germline and somatic, have been identified in human papillary renal cancer. Here we report a novel germline missense Met mutation, P1009S, in a patient with primary gastric cancer. The dosage of the mutant Met DNA was elevated in the tumor when compared to its matched normal DNA. Therefore, as with hereditary renal papillary cancer, the mutant Met allele may also be selectively duplicated in the tumor. Different from previously reported Met mutations, which occur in the tyrosine kinase domain, this missense mutation is located at the juxtamembrane domain, and is not constitutively activated. However, following treatment with HGF/SF, the P1009S mutant Met protein, expressed in NIH3T3 cells, displays increased and persistent tyrosine phosphorylation compared to the wild-type Met. Importantly, these cells also form colonies in soft agar, and are highly tumorigenic in athymic nude mice. A second nucleotide change in this region of Met, T1010I, was found in a breast cancer biopsy and a large cell lung cancer cell line. Although this previously reported 'polymorphism' did not stimulate NIH3T3 cell growth in soft agar, it was more active than the wild-type Met in the athymic nude mice tumorigenesis assay, suggesting that it may have effects on tumorigenesis. Met has been shown to be highly expressed in human gastric carcinoma cell lines, and our results raise the possibility that activating missense Met mutations could contribute to tumorigenesis of gastric cancer.

We previously reported the protective effect of N-carbamoyl-L-glutamate plus L-arginine in rats given a lethal dose (LD99.9) of ammonium acetate (Kim, S. et al., Proc. Natl. Acad. Sci. 69, 3530-3533, 1971; ref.1). The present study was undertaken to find out whether the same compounds could also be effective even after the functional mass of the liver was significantly reduced. Thus, the protective effect of these compounds in 70% partial hepatectomized rats following the injection of sublethal dose of ammonium acetate was assessed. The mixture could significantly decrease blood ammonia level compared with PBS-injected control group. In addition, abnormal behaviors observed in the control rats were significantly improved. The protective effect on the behavioral change seemed to be closely related with their effect on blood ammonia level, showing a strong correlation between the blood ammonia level and the behavioral score. The findings provide a rational basis for the clinical use of N-carbamoyl-L-glutamate plus L-arginine in the prevention and treatment of hyperammonemia encountered in liver diseases.

The present study attempts to examine the possible involvement of retinoic acid (RA) in the regulation of gonadotropin-releasing hormone (GnRH) release and gene expression in the rat hypothalamic fragments and GT1-1 neuronal cells in vitro. During a short-term period (2h), RA (0.01-1 microM) increased GnRH release in a dose-related manner. Time-course experiments showed that RA rapidly increased GnRH release by 30 min in both cells. RA-induced GnRH release was slowly attenuated in the next incubation period in hypothalamic fragments, but rapidly returned to control levels in GT-1 cells. In hypothalamic fragments, GnRH mRNA levels decreased, but in GT1-1 cells, no change in GnRH mRNA levels was observed. We then extended the incubation time to see any changes in GnRH mRNA levels by RA in GT1-1 cells. In a long term (up to 48 h), RA increased GnRH mRNA levels in a dose- and time-related manner. Significant increase in GnRH mRNA levels by RA (at higher than 10 nM) was observed within 12h. Transient transfection experiments with a luciferase reporter vector containing more than 3 kb of the rat GnRH 5'-flanking region (-3002 to +88) revealed that RA also increased the rat GnRH promoter activity in a similar dose-and time-dependent manner, suggesting that increases in GnRH mRNA levels are attributable, at least in part, to the enhanced gene transcription. The promoter analysis with the 5'-deletional constructs demonstrated that cis-elements responsible for the RA action may reside within -1640/-1438 of the rat GnRH promoter, where multiple direct or palindromic arrangements of the AGGTCA-related sequences exist. We also showed that GT1-1 cells as well as the hypothalamic tissues express mRNA for multiple subtypes of retinoid receptors, and that reporter plasmids with three copies of the strong retinoic acid response element (RARE) were activated by 80 folds upon treatment with RA in GT1-1 cells, suggesting that retinoid receptors in GT1-1 cells are functional. Taken together, the present study strongly suggests that RA is an important regulator of the GnRH neurons.