Hyeseong Cho

A novel germ line juxtamembrane Met mutation in human gastric cancer

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.

Mitochondrial Fission and Fusion Mediators, hFis1 and OPA1, Modulate Cellular Senescence

The number and morphology of mitochondria within a cell are precisely regulated by the mitochondrial fission and fusion machinery. The human protein, hFis1, participates in mitochondrial fission by recruiting the Drp1 into the mitochondria. Using short hairpin RNA, we reduced the expression levels of hFis1 in mammalian cells. Cells lacking hFis1 showed sustained elongation of mitochondria and underwent significant cellular morphological changes, including enlargement, flattening, and increased cellular granularity. In these cells, staining for acidic senescence-associated β-galactosidase activity was elevated, and the rate of cell proliferation was greatly reduced, indicating that cells lacking hFis1 undergo senescence-associated phenotypic changes. Reintroduction of the hFis1 gene into hFis1-depleted cells restored mitochondrial fragmentation and suppressed senescence-associated β-galactosidase activity. Moreover, depletion of both hFis1 and OPA1, a critical component of mitochondrial fusion, resulted in extensive mitochondrial fragmentation and markedly rescued cells from senescence-associated phenotypic changes. Intriguingly, sustained elongation of mitochondria was associated with decreased mitochondrial membrane potential, increased reactive oxygen species production, and DNA damage. The data indicate that sustained mitochondrial elongation induces senescence-associated phenotypic changes that can be neutralized by mitochondrial fragmentation. Thus, one of the key functions of mitochondrial fission might be prevention of the sustained extensive mitochondrial elongation that triggers cellular senescence.

Loss of MARCH5 mitochondrial E3 ubiquitin ligase induces cellular senescence through dynamin-related protein 1 and mitofusin 1

Mitochondria constantly divide and combine through fission and fusion activities. MARCH5, a mitochondrial E3 ubiquitin ligase, has been identified as a molecule that binds mitochondrial fission 1 protein (hFis1), dynamin-related protein 1 (Drp1) and mitofusin 2 (Mfn2), key proteins in the control of mitochondrial fission and fusion. However, how these interactions control mitochondrial dynamics, and cellular function has remained obscure. Here, we show that shRNA-mediated MARCH5 knockdown promoted the accumulation of highly interconnected and elongated mitochondria. Cells transfected with MARCH5 shRNA or a MARCH5 RING domain mutant displayed cellular enlargement and flattening accompanied by increased senescence-associated β-galactosidase (SA-β-Gal) activity, indicating that these cells had undergone cellular senescence. Notably, a significant increase in Mfn1 level, but not Mfn2, Drp1 or hFis1 levels, was observed in MARCH5-depleted cells, indicating that Mfn1 is a major ubiquitylation substrate. Introduction of Mfn1T109A, a GTPase-deficient mutant form of Mfn1, into MARCH5-RNAi cells not only disrupted mitochondrial elongation, but also abolished the increase in SA-β-Gal activity. Moreover, the aberrant mitochondrial phenotypes in MARCH5-RNAi cells were reversed by ectopic expression of Drp1, but not by hFis1, and reversion of the mitochondria morphology in MARCH5-depleted cells was accompanied by a reduction in SA-β-Gal activity. Collectively, our data indicate that the lack of MARCH5 results in mitochondrial elongation, which promotes cellular senescence by blocking Drp1 activity and/or promoting accumulation of Mfn1 at the mitochondria.

Hepatitis B Virus X Protein Enhances Transcriptional Activity of Hypoxia-inducible Factor-1α through Activation of Mitogen-activated Protein Kinase Pathway

Hepatitis B virus X protein (HBx) of the hepatitis B virus was strongly implicated in angiogenesis and metastasis during hepatocarcinogenesis. Here, we explored the possibility of cross-talk between HBx and hypoxia-inducible factor-1α (HIF-1α), a potent transcriptional inducer of angiogenic factors. First, we showed that stability of HIF-1α protein was increased by HBx in HBx-inducible Chang liver cells as well as in transient HBx expression system of non-hepatic cells. Immunofluorescence studies revealed that the HBx-induced HIF-1α was partially translocated into the nucleus in majority of cells while additional CoCl2-induced hypoxic condition caused complete nuclear translocation. Second, HBx induced both phosphorylation of HIF-1α and activation of p42/p44 mitogen-activated protein kinases (MAPKs), which were synergistically enhanced in the presence of CoCl2. Furthermore, HBx enhanced transcriptional activity of HIF-1α in the reporter genes encoding hypoxia response element or VEGF promoter. Either treatment of MEK inhibitor PD98059 or coexpression of dominant-negative MAPK mutants abolished the HBx-induced transcriptional activity and protein stability as well as nuclear translocation of HIF-1α, suggesting that HBx activates HIF-1α through MAPK pathway. Third, the association of HIF-1α with von Hippel-Lindau was decreased but the association with CREB-binding protein was enhanced in the presence of HBx, suggesting the molecular mechanism by which HBx enhances the protein stability and transactivation function of HIF-1α. Finally, we demonstrated that expression of HIF-1α and vascular endothelial growth factor was increased in the liver of HBx-transgenic mice, suggesting that the cross-talk between HIF-1α and HBx may lead to transcriptional activation of HIF-1α target genes, which play a critical role in hepatocarcinogenesis.

Methylation-dependent loss of RIP3 expression in cancer represses programmed necrosis in response to chemotherapeutics.

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.

NF-κB activation by hepatitis B virus X (HBx) protein shifts the cellular fate toward survival

In this paper, we examined the cellular effect of hepatitits B virus X (HBx) in ChangX-34 cells, inducible HBx-expressing cells. High expression of HBx protein in ChangX-34 cells resulted in approximately three-fold increase in DNA synthesis and did not show apoptotic changes. Expression of HBx in these cells was accompanied by the NF-kappaB-mediated transcription. Interestingly, inhibition of NF-kappaB activity either by treatment with sulfasalazine, a specific inhibitor of NF-kappaB, or by expressing IkappaBalpha super-repressor significantly increased cell death in ChangX-34 cells but had no influence on parental Chang cells. Thus, the activation of NF-kappaB in HBx-expressing cells may play a critical role in shifting the balance toward cell survival.

Hepatitis B Virus X Protein Induces Perinuclear Mitochondrial Clustering in Microtubule- and Dynein-Dependent Manners

ABSTRACT The hepatitis B virus (HBV) X protein (HBx) is thought to play a key role in HBV replication and the development of liver cancer. It became apparent that HBx induces mitochondrial clustering at the nuclear periphery, but the molecular basis for mitochondrial clustering is not understood. Since mitochondria move along the cytoskeleton as a cargo of motor proteins, we hypothesized that mitochondrial clustering induced by HBx occurs by an altered intracellular motility. Here, we demonstrated that the treatment of HBx-expressing cells with a microtubule-disrupting drug (nocodazole) abrogated mitochondrial clustering, while the removal of nocodazole restored clustering within 30 to 60 min, indicating that mitochondrial transport is occurring in a microtubule-dependent manner. The addition of a cytochalasin D-disrupting actin filament, however, did not measurably affect mitochondrial clustering. Mitochondrial clustering was further studied by observations of HBV-related hepatoma cells and HBV-replicating cells. Importantly, the abrogation of the dynein activity in HBx-expressing cells by microinjection of a neutralizing anti-dynein intermediate-chain antibody, dynamitin overexpression, or the addition of a dynein ATPase inhibitor significantly suppressed the mitochondrial clustering. In addition, HBx induced the activation of the p38 mitogen-activated protein kinase (MAPK) and inhibition of the p38 kinase activity by SB203580-attenuated HBx-induced mitochondrial clustering. Taken together, HBx activation of the p38 MAPK contributed to the increase in the microtubule-dependent dynein activity. The data suggest that HBx plays a novel regulatory role in subcellular transport systems, perhaps facilitating the process of maturation and/or assembly of progeny particles during HBV replication. Furthermore, mitochondrion aggregation induced by HBx may represent a cellular process that underlies disease progression during chronic viral infection.

Interferon γ stimulates β-secretase expression and sAPPβ production in astrocytes

Neurons, but not astrocytes, are known as the major source of Abeta, because astrocytes express low levels of putative beta-secretase (BACE). Astrocytes near senile plaque cores show enhanced levels of BACE protein expression, however, suggesting that astrocytes can contribute to Abeta production under pathological conditions. To investigate factors that stimulate BACE protein expression in astrocytes, we tested the effects of interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) on BACE protein expression in U373MG astrocytoma cells and primary astrocyte cultures from Tg2576 mouse brains. BACE protein expression and sAPPbeta production were dramatically increased, without changes in holo APP levels, following IFN-gamma treatment in both cell types. AG490, which is a blocker of IFN-gamma-induced STAT signaling, decreased IFN-gamma-induced BACE protein expression and sAPPbeta production in a dose-dependent manner. These results show that astrocytes are capable of expressing BACE and producing sAPPbeta in response to certain stimulating factors, and IFN-gamma is one such factor.

Expression of hepatitis B virus X protein is closely correlated with the high periportal inflammatory activity of liver diseases

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.

Complex II Defect via Down-regulation of Iron-Sulfur Subunit Induces Mitochondrial Dysfunction and Cell Cycle Delay in Iron Chelation-induced Senescence-associated Growth Arrest

Mitochondria play a pivotal role as an ATP generator in aerobically growing cells, and their defects have long been implicated in the cellular aging process, although its detailed underlying mechanisms remain unclear. Recently, we found that, in the cellular senescent process of Chang cells induced by desferroxamine mesylate, an iron chelator, a significant decrease of intracellular ATP level was accompanied by decline in complex II activity, which preceded acquisition of the senescent phenotype. In the present study, we investigated the mechanism of how the mitochondrial ATP productivity was damaged by iron chelation and how complex II defect was involved in the senescent arrest. The ATP loss was irreversible and accompanied by sustained collapse of mitochondrial membrane potential (Δψm), but the ATP loss itself did not seem to be essential in progression to the senescent arrest. The Δψm disruption was due to decreased mitochondrial respiration, which was primarily associated with the defective complex II activity. Furthermore, we found that the declined activity of complex II was mainly due to down-regulation of protein expression of the iron-sulfur subunit, which was associated with the irreversibility of the arrest. Finally, we demonstrated that specific inhibition of complex II with 2-thenoyltrifluoroacetone induced overall delay of the cell cycle, suggesting that the delayed arrest by desferroxamine mesylate might be in part due to inhibition of complex II activity. Taken together, our results suggest that complex II might be considered as one of the primary factors to regulate mitochondrial respiratory function by responding to the cellular iron level, thereby influencing cellular growth.