CPEB2-activated axonal translation of VGLUT2 mRNA promotes glutamatergic transmission and presynaptic plasticity.

BACKGROUND: Local translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity. METHODS: Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles. RESULTS: Electrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles. CONCLUSIONS: We identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment.

The Prognostic Value of Cytokeratin and Sal-Like Protein 4 Expression in Hepatocellular Carcinoma and Intra-Hepatic Cholangiocarcinoma in Taiwan.

Background: We previously reported that modulation of cytokeratin18 induces pleomorphism of liver cells, higher cell motility, and higher drug sensitivity to sorafenib treatment of hepatoma cells. These relationships were established by in vitro experiments. The aim of this study was to determine the in vivo association between cytokeratin expression and tumor behavior, as well as cancer stem cells of hepatocellular carcinoma and intra-hepatic cholangiocarcinoma in Taiwan. Methods: Cytokeratins and sal-like protein 4 expression was determined in 83 hepatocellular carcinoma and 30 intra-hepatic cholangiocarcinoma specimens by immunohistochemistry. The relationship between cytokeratins and sal-like protein 4 expression with hepatitis virus infection, clinicopathologic factors, and survival was analyzed. Further, the correlation among cytokeratins and sal-like protein 4 expression was studied. Results: In addition to cytokeratin8/18, the expression of cytokeratin7/19 and sal-like protein 4 was noted in hepatocellular carcinoma; however, only cytokeratin19 expression had a significant correlation with poor overall survival and poor disease-free survival. The expression of cytokeratins and sal-like protein 4 was not correlated with hepatitis virus infection. The expression of cytokeratin19, but not 7, 8, and 18, was correlated with sal-like protein 4 expression in hepatocellular carcinoma. Cytokeratin7 expression was decreased and the sal-like protein 4 expression was absent in all 30 intra-hepatic cholangiocarcinoma cases. The expression of cytokeratins had not statistically significant correlation with overall and disease-free survival in patients with intra-hepatic cholangiocarcinoma. Conclusions: The expression of cytokeratin19 was associated with sal-like protein 4 expression, as well as poor overall and disease-free survival in hepatocellular carcinoma patients in Taiwan.

Transient knockdown-mediated deficiency in plectin alters hepatocellular motility in association with activated FAK and Rac1-GTPase.

BACKGROUND: Plectin is one of the cytolinker proteins that play a crucial role in maintaining the integrity of cellular architecture. It is a component of desmosome complexes connecting cytoskeletal proteins and trans-membrane molecules. In epithelial cells, plectin connects cytokeratins and integrin α6ß4 in hemidesmosomes anchoring to the extracellular matrix. In addition to the function of molecular adherent, plectin has been reported to exhibit functions affecting cellular signals and responsive activities mediated by stress, cellular migration, polarization as well as the dynamic movement of actin filaments. Plectin deficiency in hepatocellular carcinoma results in abnormal expression of cytokeratin 18 and disassembled hemidesmosome. Therefore, it is hypothesized that the plectin deficiency-mediated collapse of cytoskeleton may modulate cellular motility that is associated with consequent metastatic behaviors of cancer cells. METHODS AND RESULTS: The cellular motility of plectin-deficient Chang liver cells generated by transient knockdown were analyzed by trans-well migration assay and the results revealed a higher migration rate. The confocal microscopy also demonstrated less organized and more polarized morphology as well as more focal adhesion kinase activity in comparison with that of the mock Chang liver cells. Furthermore, plectin-knockdown in Chang liver cells was associated with a higher activity of Rac1-GTPase in accordance with the results of the Rac1 pull-down assay. The immunohistochemical assay on human hepatocellular carcinoma showed that the expression of focal adhesion kinase was increased in the invasive front of tumor. CONCLUSION: Plectin-deficient human hepatic cells exhibit higher cell motility associated with increase in focal adhesion kinase activity that are comparable to the properties of invasive hepatocellular carcinoma.

The Innovative Role of Nuclear Receptor Interaction Protein in Orchestrating Invadosome Formation for Myoblast Fusion.

BACKGROUND: Nuclear receptor interaction protein (NRIP) is versatile and engages with various proteins to execute its diverse biological function. NRIP deficiency was reported to cause small myofibre size in adult muscle regeneration, indicating a crucial role of NRIP in myoblast fusion. METHODS: The colocalization and interaction of NRIP with actin were investigated by immunofluorescence and immunoprecipitation assay, respectively. The participation of NRIP in myoblast fusion was demonstrated by cell fusion assay and time-lapse microscopy. The NRIP mutants were generated for mechanism study in NRIP-null C2C12 (termed KO19) cells and muscle-specific NRIP knockout (NRIP cKO) mice. A GEO profile database was used to analyse NRIP expression in Duchenne muscular dystrophy (DMD) patients. RESULTS: In this study, we found that NRIP directly and reciprocally interacted with actin both in vitro and in cells. Immunofluorescence microscopy showed that the endogenous NRIP colocalized with components of invadosome, such as actin, Tks5, and cortactin, at the tips of cells during C2C12 differentiation. The KO19 cells were generated and exhibited a significant deficit in myoblast fusion compared with wild-type C2C12 cells (3.16% vs. 33.67%, p < 0.005). Overexpressed NRIP in KO19 cells could rescue myotube formation compared with control (3.37% vs. 1.00%, p < 0.01). We further confirmed that NRIP directly participated in cell fusion by using a cell-cell fusion assay. We investigated the mechanism of invadosome formation for myoblast fusion, which depends on NRIP-actin interaction, by analysing NRIP mutants in NRIP-null cells. Loss of actin-binding of NRIP reduced invadosome (enrichment ratio, 1.00 vs. 2.54, p < 0.01) and myotube formation (21.82% vs. 35.71%, p < 0.05) in KO19 cells and forced NRIP expression in KO19 cells and muscle-specific NRIP knockout (NRIP cKO) mice increased myofibre size compared with controls (over 1500 µm2, 61.01% vs. 20.57%, p < 0.001). We also found that the NRIP mRNA level was decreased in DMD patients compared with healthy controls (18 072 vs. 28 289, p < 0.001, N = 10 for both groups). CONCLUSIONS: NRIP is a novel actin-binding protein for invadosome formation to induce myoblast fusion.

Dysregulated proteostasis network in neuronal diseases.

Long-term maintenance of synaptic connections is important for brain function, which depends on varying proteostatic regulations to govern the functional integrity of neuronal proteomes. Proteostasis supports an interconnection of pathways that regulates the fate of proteins from synthesis to degradation. Defects in proteostatic signaling are associated with age-related functional decline and neurodegenerative diseases. Recent studies have advanced our knowledge of how cells have evolved distinct mechanisms to safely control protein homeostasis during synthesis, folding and degradation, and in different subcellular organelles and compartments. Neurodegeneration occurs when these protein quality controls are compromised by accumulated pathogenic proteins or aging to an irreversible state. Consequently, several therapeutic strategies, such as targeting the unfolded protein response and autophagy pathways, have been developed to reduce the burden of misfolded proteins and proved useful in animal models. Here, we present a brief overview of the molecular mechanisms involved in maintaining proteostatic networks, along with some examples linking dysregulated proteostasis to neuronal diseases.

MicroRNA-92a-CPEB3 axis protects neurons against inflammatory neurodegeneration.

Neuroinflammation causes neuronal injury in multiple sclerosis (MS) and other neurological diseases. MicroRNAs (miRNAs) are important modulators of neuronal stress responses, but knowledge about their contribution to neuronal protection or damage during inflammation is limited. Here, we constructed a regulatory miRNA-mRNA network of inflamed motor neurons by leveraging cell type-specific miRNA and mRNA sequencing of mice undergoing experimental autoimmune encephalomyelitis (EAE). We found robust induction of miR-92a in inflamed spinal cord neurons and identified cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) as a key target of miR-92a-mediated posttranscriptional silencing. We detected CPEB3 repression in inflamed neurons in murine EAE and human MS. Moreover, both miR-92a delivery and Cpeb3 deletion protected neuronal cultures against excitotoxicity. Supporting a detrimental effect of Cpeb3 in vivo, neuron-specific deletion in conditional Cpeb3 knockout animals led to reduced inflammation-induced clinical disability in EAE. Together, we identified a neuroprotective miR-92a-Cpeb3 axis in neuroinflammation that might serve as potential treatment target to limit inflammation-induced neuronal damage.

Synemin down-regulation in human hepatocellular carcinoma does not destabilize cytoskeletons in vivo.

Synemin is a large intermediate filament protein that has been identified in all types of muscle cells. It plays a role in human muscle diseases; however, the role of synemin in tumor cell transformation has rarely been investigated. Because hepatocellular carcinoma cells are morphologically different from normal human hepatocytes, we hypothesized that altered synemin expression and cytoskeletal disorganization might underlie this pleomorphic transformation. To test this hypothesis, we studied synemin expression in hepatocellular carcinoma and liver tissues by immunohistochemistry and immunoblotting. In addition, we analyzed the expression level and organization of all cytoskeletal elements after synemin knock-down in human Chang liver cells. Previously we found that plectin knock-down in human Chang liver cells causes a reduction in cytokeratin 18 expression with effects on intermediate filament disorganization and altered cellular morphology. In this study we also compared the effects of synemin knock-down and plectin knock-down on the cytoskeleton expression and organization. The results revealed that synemin expression was down-regulated in human hepatocellular carcinoma compared with normal liver, which is similar to the plectin expression. Surprisingly, the expression of cytoskeletal elements (cytokeratin 18, actin and tubulin) was not influenced by synemin knock-down in human Chang liver cells. The organization of cytoskeletal networks was also unaltered after synemin knock-down. In conclusion, both plectin and synemin are down-regulated in human hepatocellular carcinoma in vivo and transformed human liver cell in vitro. However, the mechanism of cell transformation caused by synemin knock-down is different from that of plectin knock-down. Plectin, but not synemin, knock-down provoked liver cell transformation via suppressing cytokeratin 18 expression and disrupting intermediate filament networks. Synemin knock-down did not influence the cytoskeleton expression and organization of human Chang liver cells.

Cytokeratin 18-mediated disorganization of intermediate filaments is induced by degradation of plectin in human liver cells.

Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork that helps maintain the uniform size and shape of cells. As cells of hepatocellular carcinoma are morphologically different from healthy human hepatocytes, we hypothesized that plectin deficiency and cytoskeletal disorganization underlies this pleomorphic transformation. To test this hypothesis we induced apoptosis as the most accessible pathway for creating plectin deficiency status in vivo. We analyzed expression levels and organization of plectin and other cytoskeletal elements, including intermediate filaments, microfilaments, and microtubules, after staurosporine-induced apoptosis in human Chang liver cells. The results revealed the expression of plectin and cytokeratin 18 were downregulated in hepatocellular carcinoma tissues in vivo. The expression of actin and tubulin, however, were not altered. In vitro analysis indicated that plectin and cytokeratin 18 were cleaved following staurosporine-treatment of human Chang liver cells. Time course experiments revealed that plectin was cleaved 2h earlier than cytokeratin 18. The organization of plectin and cytokeratin 18 networks collapsed after staurosporine-treatment. Conclusively, degradation of plectin induced by staurosporine-treatment in liver cells resulted in cytoskeleton disruption and induced morphological changes in these cells by affecting the expression and organization of cytokeratin 18.

Plectin deficiency on cytoskeletal disorganization and transformation of human liver cells in vitro.

Plectin is a versatile cytoplasmic cross-linking protein that connects intermediate filaments to microfilaments, microtubules, and membrane adhesion sites. The cross-linking functions of plectin help organize the cytoskeleton into a stable meshwork important for maintaining uniformity in cell size and shape. As cells of hepatocellular carcinoma are morphologically different from normal human hepatocytes, we hypothesized that altered plectin expression and cytoskeletal organization underlies this pleomorphic transformation. To test this hypothesis, we analyzed expression levels and organization of all cytoskeletal elements, including intermediate filaments, microfilaments, and microtubules, after plectin knockdown in human Chang liver cells. We found that expression of cytokeratin 18, but not actin or tubulin, was downregulated by suppression of plectin protein. Furthermore, cytokeratin networks were partially collapsed and actin-rich stress fibers were increased. The organization of microtubule networks, by contrast, was unaltered. These findings support our hypothesis that, via effects on cytoskeletal organization, plectin deficiency might play an important role in the transformation of human liver cells.

Sorafenib combined with dasatinib therapy inhibits cell viability, migration, and angiogenesis synergistically in hepatocellular carcinoma.

PURPOSE: Sorafenib is a multikinase inhibitor used for treatment of advanced hepatocellular carcinoma. Sorafenib resistance may be related to Src-induced cell migration and angiogenesis, which are regulated by cancer stem cell activation and release of vascular endothelial growth factor. Dasatinib is a Src inhibitor that inhibits Src phosphorylation and suppresses Src-associated cell migration and angiogenesis. This study investigated whether combined treatment with dasatinib can overcome sorafenib resistance. METHODS: Hepatoma cell lines were used for sorafenib and/or dasatinib treatment. Cell viability, cell migration, molecular expressions, and release of vascular endothelial growth factor by hepatoma cells were evaluated. Hepatoma cell culture medium was applied on human umbilical vein endothelial cells to monitor angiogenesis promoted by the hepatoma cells. RESULTS: Sorafenib and dasatinib combined therapy suppressed cell viability of hepatoma cells synergistically. Dasatinib suppressed sorafenib-induced cell migration via inhibiting sorafenib-induced Src/FAK phosphorylation, cell-to-cell contact and cancer stem cell activation. Culture medium from Chang liver and PLC/PRF/5 cells suppressed angiogenesis of human umbilical vein endothelial cells with any treatment, whereas sorafenib-treated medium of HepG2 cells induced angiogenesis. This sorafenib-induced angiogenesis was then suppressed by dasatinib. Vascular endothelial growth factor released from hepatoma cells was also inhibited by combined treatment. CONCLUSION: Src/FAK phosphorylation and cancer stem cell activation inducing cell migration and angiogenesis may be the key factors of sorafenib resistance. Sorafenib and dasatinib combined treatment suppresses cell migration and angiogenesis by inhibiting the Src/FAK phosphorylation, cell-to-cell contact, cancer stem cell activation, and release of vascular endothelial growth factor.

The Roles Of Angiogenesis And Cancer Stem Cells In Sorafenib Drug Resistance In Hepatocellular Carcinoma.

BACKGROUND: An increasing number of studies support cancer stem cells as the reason for chemoresistance to sorafenib therapy in hepatocellular carcinoma (HCC), but the mechanism is still unclear. In this study, the mechanism of sorafenib resistance in cancer stem cells was examined by in vitro experiments and xenograft mouse model. METHODS: The expression of cancer stem cell markers in the Chang liver cell line and PLC/PRF/5 and HepG2 hepatoma cell lines were compared by immunoblot assay before and after sorafenib treatment in vitro. As a xenograft mouse model, subcutaneous injection of hepatoma cells followed by sorafenib therapy was performed in NU/NU mice. The effects of sorafenib therapy on tumor growth and cancer stem cell markers were studied. Angiogenesis associated with cancer stem cells was studied by immunoblot and immunohistochemistry assay. RESULTS: The expression of cancer stem cell markers was higher in PLC/PRF/5 and HepG2 cells than Chang liver cells, indicating that these hepatoma cells had more stemness-related characteristics. The cancer stem cell markers were upregulated in the hepatoma cell lines following sorafenib treatment in vitro. In the xenograft model, tumors from PLC/PRF/5 and HepG2 cells with high E-cadherin expression were more resistance to sorafenib therapy. However, the expression of cancer stem cell markers was not significantly different after sorafenib therapy in these tumors. Furthermore, we found that sorafenib therapy induced angiogenesis within tumors from high E-cadherin expressing hepatoma cells. CONCLUSION: The mechanism of chemoresistance in sorafenib therapy in HCC may be the tumor angiogenesis associated with high E-cadherin expression in cancer stem cells.

The influence of plectin deficiency on stability of cytokeratin18 in hepatocellular carcinoma.

Intermediate filaments are important in building the cellular architecture. Previously we found cytokeratin18 was modulated in human hepatocellular carcinoma. Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork, which can maintain the uniform size and shape of hepatocytes. Because the cells of hepatocellular carcinoma were morphologically different from the hepatocytes, we speculated that expression of plectin and organization of intermediate filament might play roles in the pleomorphism of hepatocellular carcinoma cells. In this paper, we studied the plectin expression of hepatocellular carcinoma and liver tissues by immunohistochemistry and immunoblot. The results revealed that plectin was deficient and cytokeratin18 was modulated in hepatocellular carcinoma. Furthermore, we knockdown the plectin mRNA in Chang cells, the result revealed the plectin was deficient and the organization of cytokeratin18 was altered. Conclusively, this study offers a hypothesis that plectin deficient might play an important role in the tumorigenesis of hepatocellular carcinoma.

Possible relation between histone 3 and cytokeratin 18 in human hepatocellular carcinoma.

BACKGROUND: Previously we found some low molecular weight proteins identified as histone in hepatocelluar carcinoma. Our objective was to clarify whether the coimmunoprecipitation of histone and cytokeratin 18 was an artifact or not. MATERIALS AND METHODS: Histone 3 and cytokeratin 18 were investigated in three cases of human hepatocellular carcinoma and one case of normal liver tissue. Nuclei of the tissues were isolated; the proteins inside the nuclei were analyzed by Western blot. RESULTS: The results revealed histone was co-immunoprecipitated with cytokeratin 18 in hepatocellular carcinoma. It was speculated that modulation of the cytoskeleton in human hepatocellular carcinoma might disturb the organization of the nucleoskeleton. The unstable nucleoskeleton might further cause instability and fragility of nuclei, thus possibly exposing the histone and co-immunoprecipitating it with cytokeratin 18. CONCLUSION: The evidence might indicate that expression of histone 3 was highly related to modulation of cytokeratin 18 and might play an important role in tumorigenesis of hepatocellular carcinoma.

Degradation of plectin with modulation of cytokeratin 18 in human liver cells during staurosporine-induced apoptosis.

BACKGROUND: Hepatoma cells are morphologically different from those of the normal liver. Intermediate filaments (IFs) are important in building the cellular architecture and maintaining the outline of cells. Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork, which can maintain the uniform size and shape of hepatocytes. Apoptosis might be the most possible pathway for creating plectin deficiency in the in vivo state. MATERIALS AND METHODS: Apoptosis was induced by staurosporine (STS) treatment in liver cells. The protein expression of cytokeratin 18 (CK18) and plectin as well as the morphology of the liver cells and the distribution of CK18 and plectin in the cells was studied after STS treatment. RESULTS: Plectin was cleaved in the liver cells during apoptosis and CK18 was modulated. Morphological changes were observed in the liver cells. CONCLUSION: By affecting the organization of IFs, plectin might play an important role in the pleomorphism of hepatoma cells and even the tumorigenesis of hepatoma.

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment.

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.

Cytokeratin 18-associated Histone 3 Modulation in Hepatocellular Carcinoma: A Mini Review.

Unstable cytokeratins are associated with tumor transformation in the development of human hepatocellular carcinoma. We previously demonstrated that the cytokeratin 18 was modulated and that a histone H3-specific modification occured, among members of the histone family, during the development of human hepatocellular carcinoma. Evidence suggested that the modification of histone H3 was highly correlated with the modulation of cytokeratin 18 and probably plays an important role in tumorigenesis of hepatocytes. Aberrant expression of histone deacetylase leading to imbalance between acetylation and deacetylation of histones may exhibit regulatory roles in tumor transformation. Recently we found that overexpression of histone deacetylase-1 and hypoacetylation of histone H3 were associated with hepatocellular carcinoma. The underlying roles of histone H3 modulation are discussed in this mini-review article.

Cell Pleomorphism and Cytoskeleton Disorganization in Human Liver Cancer.

BACKGROUND/AIM: Nucleoskeleton maintains the framework of a cell nucleus that is required for a variety of nuclear functions. However, the nature of nucleoskeleton structure has not been yet clearly elucidated due to microscopy visualization limitations. Plectin, a nuclear pore-permeable component of cytoskeleton, exhibits a role of cross-linking between cytoplasmic intermediate filaments and nuclear lamins. Presumably, plectin is also a part of nucleoskeleton. Previously, we demonstrated that pleomorphism of hepatoma cells is the consequence of cytoskeletal changes mediated by plectin deficiency. In this study, we applied a variety of technologies to detect the cytoskeletons in liver cells. MATERIALS AND METHODS AND RESULTS: The images of confocal microscopy did not show the existence of plectin, intermediate filaments, microfilaments and microtubules in hepatic nuclei. However, in the isolated nuclear preparation, immunohistochemical staining revealed positive results for plectin and cytoskeletal proteins that may contribute to the contamination derived from cytoplasmic residues. Therefore, confocal microscopy provides a simple and effective technology to observe the framework of nucleoskeleton. Accordingly, we verified that cytoskeletons are not found in hepatic cell nuclei. Furthermore, the siRNA-mediated knockdown of plectin in liver cells leads to collapsed cytoskeleton, cell transformation and pleomorphic nuclei. CONCLUSION: Plectin and cytoskeletons were not detected in the nuclei of liver cells compared to the results of confocal microscopy. Despite the absence of nuclear plectin and cytoskeletal filaments, the evidence provided support that nuclear pleomorphism of cancer cells is correlated with the cytoplasmic disorganization of cytoskeleton.

Hypoacetylation in association with histone 3 modulation in human hepatocellular carcinoma.

BACKGROUND: Aberrant histone deacetylase expression may cause imbalance between acetylation and deacetylation of histone and play roles in tumor transformation. We found that histone 3 was modulated in human hepatocellular carcinoma. We determined if histone 3 modulation is related to the aberrant expression of histone deacetylase. MATERIALS AND METHODS: We analyzed human liver and hepatocellular carcinoma tissues and fibroblast and fibrosarcoma cell lines for the expression of histone 3, histone deacetylase 1 and acetylated histone 3 using immunohistochemistry, western blot and immunofluorescence. RESULTS: Histone deacetylase 1 and histone 3 were more strongly detected in hepatocellular carcinoma tissue and fibrosarcoma cells than in liver tissues and fibroblast cells, respectively. However, acetylated histone 3 was more strongly expressed in normal liver and fibroblast cells and less expressed in hepatocellular carcinoma and fibrosarcoma cells. CONCLUSION: Histone deacetylase 1 overexpression and hypoacetylation of histone 3 might play critical roles in the modulation of histone 3 in human hepatocellular carcinoma.

Laparoscopy-assisted billroth I gastrectomy for ectopic pancreas in the prepyloric region.

Ectopic pancreatic tissue is an uncommon developmental anomaly. The condition mostly occurs in the gastrointestinal tract and is usually asymptomatic. It rarely causes symptoms of inflammation, bleeding and perforation, and has potential for malignant change. Though it is an uncommon condition, cases of ectopic pancreas have been reported worldwide. Preoperative diagnosis of ectopic pancreas is challenging because of its nonspecific symptoms and signs. Owing to the revolution of minimally invasive surgery, submucosal tumors of the stomach can be resected by laparoscopic techniques. We have earlier reported on a case of ectopic pancreas in the stomach treated by robotics-assisted laparoscopic wedge resection. Herein, we report a case of ectopic pancreas in the prepyloric region of the stomach. A 44-year-old female presented with a two-week history of epigastralgia with radiation to the back. She received endoscopy check-up which disclosed a mass in the stomach. By endoscopic findings, a submucosal lesion in the prepyloric region with umbilical folding on the mucosa was identified. The umbilical folding on the mucosa hint the orifice of the duct of ectopic pancreas into the gastric mucosa suggestive of ectopic pancreas. Contrast-enhanced abdominal computed tomography showed a 5 cm cystic mass with heterogeneous content. To sum it up, the patient was diagnosed as ectopic pancreas in the stomach. She underwent laparoscopy-assisted antrectomy with Billroth I anastomosis (excision of the antrum and prepyloric region with reconstruction of gastrointestinal continuity by gastroduodenostomy) and had an uneventful hospitalization course. The histopathology of the resected tumor demonstrated ectopic pancreatic tissue in the gastric wall. To the best of our knowledge, excision of gastric ectopic pancreas using laparoscopy-assisted antrectomy with Billroth I anastomosis has never been reported in the literature.

Pleomorphism of cancer cells with the expression of plectin and concept of filament bundles in human hepatocellular carcinoma.

Intermediate filaments are important in building the architecture of liver cells and are proposed to interact with other cellular components. Among intermediate filament associated proteins, plectin is a versatile cytoskeletal linkage protein which has been shown to interact with a variety of cytoskeletal structures. Intermediate filament and plectin might play some roles in tumorigenesis of human hepatocellular carcinoma since cells of hepatocellular carcinoma were morphologically different from normal liver. Plectin exhibited wide distribution spectrum among various tissues, however, it was poorly investigated in human liver and hepatoma tissues. In this paper, we studied the plectin expression in 18 cases of human hepatocellular carcinoma and normal hepatocytes by immunohistochemistry. The results revealed that plectin expression was deficient in human hepatocellular carcinoma and was probably through post-translational modification. Many 0.4 to 0.8 microm-thick keratin bundles were found in intermediate filament extracts of liver and hepatoma tissues. These bundles were greater in diameter about 40 to 80 times of single intermediate filament. We speculated that intermediate filament organized into "filament bundles" to maintain the shape of normal cells. In cancer cells, plectin was deficient and the irregularly loosened filament bundles could cause pleomorphism of cancer cells.