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 2 h 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.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

RNF8 is a nuclear protein having an N-terminal forkhead-associated (FHA) domain and a C-terminal RING-finger (RF) domain. Depletion of RNF8 caused cell growth inhibition and cell cycle arrest at not only S but also G2/M phases. In addition, cell death was frequently observed in RNF8-depleted cells. Analyses of time-lapse microscopy revealed that the cells died in mitosis and interphase. To elucidate the RNF8 function in M phase, the Plk1 content in RNF8-depleted cells was examined. The amount of RNF8 decreased time-dependently, whereas Plk1 reciprocally increased by transfection of RNF8 siRNA. Protein contents of RNF8 and Plk1 among various cell lines were also compared. RNF8 in normal cell lines was much higher than that in many cancer cell lines. Conversely, Plk1 in normal cell lines was lower than in cancer cell lines. These results suggest that RNF8 is downregulated in many cancer cells and inversely correlated with Plk1.     

Role of Twist in vasculogenic mimicry formation in hypoxic hepatocellular carcinoma cells in vitro

Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive human tumor cells to form matrix-rich networks de novo when cultured on a three-dimensional matrix, thus mimicking embryonic vasculogenesis. Some studies have shown that tumor hypoxia can promote tumor cells to form vessel-like tubes in vitro and express genes associated with VM. Although, the mechanisms involved in hypoxia-induced VM remain elusive, we hypothesized that the epithelial–mesenchymal transition (EMT) regulator Twist may play a major role in hypoxia-induced VM. We investigated this hypothesis in vitro by pretreating hepatocellular carcinoma cells under hypoxic conditions. Following the hypoxia treatment, the cells formed typical pipe-like VM networks. Moreover, the expression of VM markers was increased. Hypoxia-induced VM was accompanied by the increased expression of Twist. Twist siRNA reversed the effects of hypoxia on VM. These results suggest that the overexpression of Twist correlates to hypoxia-induced VM in hepatocellular carcinoma cells.     

Mutation in keratin 18 induces mitochondrial fragmentation in liver-derived epithelial cells

Microtubules (MTs) and microfilaments (MFs) are known to modulate mitochondrial morphology, distribution and function. However, little is known evidence about the role of intermediate filaments (IFs) in modulating mitochondria except desmin. To investigate whether or not the IFs regulate mitochondrial morphology, distribution, and function, we manipulated the IFs of cultured epithelial cells to express a mutant keratin 18 (K18). In contrast to the filamentous expression of wild K18, mutant K18 induced aggregation of K8/18, showing no fine IF network in the cells. In mutant K18-transfected cells, the mitochondria were fragmented into small spheroids, although they were observed as mitochondrial fibers in un-transfected or wild K18-transfected cells. Fluorescence recovery after photobleaching of fluorescence-labeled mitochondria was markedly less in the mutant K18-transfected cells, although a significant recovery was confirmed in wild K18-transfected cells. These findings suggest that the IFs are important for the maintenance of normal mitochondrial structures.     

GalNAcβ1,3-linked paragloboside carries the epitope of a sperm maturation-related glycoprotein that is recognized by the monoclonal antibody MC121

The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54 kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with β-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa, MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL. Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc + Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV³GalNAcβ-nLc₄Cer² sequence.     

Role of p53/FAK association and p53 phosphorylation in staurosporine-mediated apoptosis: Wild type versus mutant p53-R175H

A novel survival role of focal adhesion kinase (FAK) that involves its nuclear translocation and direct association with p53 has been demonstrated. Here we examined the relationship between the p53/FAK interaction and Ser46 phosphorylation of p53 (p-p53^Ser46) in the apoptotic regulation of human esophageal squamous cell carcinoma (HOSCC) cell lines, expressing either wild type (wt) p53 or mutant (mt) p53-R175H. In contrast to the wt p53 cell lines, the mt p53-R175H cell line was resistant to staurosporine (STS)-mediated detachment and caspase-3 activation. Furthermore, despite the resistance of mt p53-R175H to Ser46 phosphorylation, both wt and mt HOSCC cells translocate FAK into the nucleus and maintain the p53/FAK interaction post STS treatment. These findings provide unique insight into how tumor cells harboring the R175H mutant may resist chemotherapeutic intervention.

Structured summary

MINT-7294020: FAK (uniprotkb:Q05397) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by anti-bait coimmunoprecipitation (MI:0006)     

Quantitative aspects of endocytic activity in lipid-mediated transfections.

Variation in transfection efficiency observed in different cell-types is poorly understood. To investigate the influence of endocytic activity on lipid-mediated transfections, we have monitored both the processes in 12 different cell-types. The endocytic activity shows a strong positive correlation (P < 0.01), with transfection efficiency. Treatment with wortmannin resulted in cell-type-dependent inhibition of transfection. Studies on M-phase cells by confocal microscopy show that compared to interphase cells, uptake of cationic liposomes was substantially reduced. In addition, transfection efficiency of cells in mitotic phase was inhibited by >70% compared to controls. Our study based on several cell-types demonstrates for the first time that quantitative aspects of endocytosis have decisive influence on the overall process of lipid-mediated transgene expression.     

Inhibition of protein synthesis and JNK activation are not required for cell death induced by anisomycin and anisomycin analogues

Anisomycin was identified in a screen of clinical compounds as a drug that kills breast cancer cells (MDA16 cells, derived from the triple negative breast cancer cell line, MDA-MB-468) that express high levels of an efflux pump, ABCB1. We show the MDA16 cells died by a caspase-independent mechanism, while MDA-MB-468 cells died by apoptosis. There was no correlation between cell death and either protein synthesis or JNK activation, which had previously been implicated in anisomycin-induced cell death. In addition, anisomycin analogues that did not inhibit protein synthesis or activate JNK retained the ability to induce cell death. These data suggest that either a ribosome-ANS complex is a death signal in the absence of JNK activation or ANS kills cells by binding to an as yet unidentified target.     

Reflux induces DNA strand breaks and expression changes of MMP1+9+14 in a human miniorgan culture model

Gastroesophageal reflux disease has been implicated in the pathogenesis of adenocarcinoma of the oesophagus. The same applies to laryngopharyngeal reflux (LPR) and squamous cell cancer of the head and neck, but so far, this link has not been proven. The impact of low pH and bile acids has not been studied extensively in cells other than oesophageal cancer cell lines and tissue. The aims of this study were to investigate the pathogenic potential of reflux and its single components on the mucosa of the upper respiratory tract. We measured DNA stability in human miniorgan cultures (MOCs) and primary epithelial cell cultures (EpCs) in response to reflux by the alkaline comet assay. As matrix metalloproteinases (MMPs) are involved in extracellular matrix remodelling processes and may contribute to cancer progression, we studied the expression of MMP1, -9, and -14 in MOCs, EpC, UM-SCC-22B, and FADU_DD. DNA strand breaks (DNA-SBs) increased significantly at low pH and after incubation with human or artificial gastric juice. Single incubation with glycochenodeoxycholic acid also showed a significant increase in DNA-SBs. In epithelial cell cultures, human gastric juice increased the number of DNA-SBs at pH 4.5 and 5.5. Artificial gastric juice significantly up regulated the gene expression of MMP9. Western blot analysis confirmed the results of gene expression analysis, but the up regulation of MMP1, -9, and -14 was donor-specific. Reflux has the ability to promote genomic instability and may contribute to micro environmental changes suitable for the initiation of malignancy. Further functional gene analysis may elucidate the role of laryngopharyngeal reflux in the development of head neck squamous cell carcinoma (HNSCC).     

Bisphenol A and its methylated congeners inhibit growth and interfere with microtubules in human fibroblasts in vitro

Bisphenol A (BPA), a monomer of polycarbonate plastics and epoxy resins, has previously been reported to induce micronuclei containing whole chromosomes in Chinese hamster V79 cells. In the present study, the aneuploidogenic potential of BPA was investigated in cultured human AG01522C fibroblasts. In contrast to the known aneugens diethylstilbestrol (DES) and 17beta-estradiol, which caused mitotic arrest and the induction of kinetochore-positive micronuclei, BPA did not induce micronuclei and inhibited the proliferation of AG01522C cells in G2 phase and probably also in G1 phase. Fluorescence microscopy of the BPA-treated cells after immunofluorescent staining of microtubules revealed structural abnormalities of the cytoplasmic microtubule complex (CMTC): densely stained rings and loops of tubulin were observed, which increased in number with increasing BPA concentration and were more stable against low temperature than normal microtubules. The mechanisms of the growth inhibition and the interference with microtubules elicited by BPA in AG01522C cells are presently unknown. The formation of rings and loops in the CMTC of AG01522C cells was also observed with two congeners of BPA carrying one and two, respectively, additional methyl groups in ortho-position to the phenolic hydroxyl group at each aromatic ring. However, in contrast to BPA itself, these congeners of BPA behaved "DES-like" by inducing mitotic arrest and kinetochore-positive micronuclei in AG01522C cells.     

Binding of a Naja naja venom acidic phospholipase A2 cognate complex to membrane-bound vimentin of rat L6 cells: Implications in cobra venom-induced cytotoxicity

An acidic phospholipase A₂ enzyme (NnPLA₂-I) interacts with three finger toxins (cytotoxin and neurotoxin) from Naja naja venom to form cognate complexes to enhance its cytotoxicity towards rat L6 myogenic cells. The cytotoxicity was further enhanced in presence of trace quantity of venom nerve growth factor. The purified rat myoblast cell membrane protein showing interaction with NnPLA₂-I was identified as vimentin by LC-MS/MS analysis. The ELISA, immunoblot and spectrofluorometric analyses showed greater binding of NnPLA₂-I cognate complex to vimentin as compared to the binding of individual NnPLA₂-I. The immunofluorescence and confocal microscopy studies evidenced the internalization of NnPLA₂-I to partially differentiated myoblasts post binding with vimentin in a time-dependent manner. Pre-incubation of polyvalent antivenom with NnPLA₂-I cognate complex demonstrated better neutralization of cytotoxicity towards L6 cells as compared to exogenous addition of polyvalent antivenom 60–240 min post treatment of L6 cells with cognate complex suggesting clinical advantage of early antivenom treatment to prevent cobra venom-induced cytotoxicity. The in silico analysis showed that 19–22 residues, inclusive of Asp48 residue, of NnPLA₂-I preferentially binds with the rod domain (99–189 and 261–335 regions) of vimentin with a predicted free binding energy (ΔG) and dissociation constant (K_D) values of ?12.86 kcal/mol and 3.67 × 10^?10 M, respectively; however, NnPLA₂-I cognate complex showed greater binding with the same regions of vimentin indicating the pathophysiological significance of cognate complex in cobra venom-induced cytotoxicity.