Crumbs interacts with moesin and beta(Heavy)-spectrin in the apical membrane skeleton of Drosophila

The apical transmembrane protein Crumbs is necessary for both cell polarization and the assembly of the zonula adherens (ZA) in Drosophila epithelia. The apical spectrin-based membrane skeleton (SBMS) is a protein network that is essential for epithelial morphogenesis and ZA integrity, and exhibits close colocalization with Crumbs and the ZA in fly epithelia. These observations suggest that Crumbs may stabilize the ZA by recruiting the SBMS to the junctional region. Consistent with this hypothesis, we report that Crumbs is necessary for the organization of the apical SBMS in embryos and Schneider 2 cells, whereas the localization of Crumbs is not affected in karst mutants that eliminate the apical SBMS. Our data indicate that it is specifically the 4.1 protein/ezrin/radixin/moesin (FERM) domain binding consensus, and in particular, an arginine at position 7 in the cytoplasmic tail of Crumbs that is essential to efficiently recruit both the apical SBMS and the FERM domain protein, DMoesin. Crumbs, Discs lost, betaHeavy-spectrin, and DMoesin are all coimmunoprecipitated from embryos, confirming the existence of a multimolecular complex. We propose that Crumbs stabilizes the apical SBMS via DMoesin and actin, leading to reinforcement of the ZA and effectively coupling epithelial morphogenesis and cell polarity.     

Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. In addition, it has been shown that HP1 is also involved in telomere capping in Drosophila. Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila. By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes. Because the puffs are the cytological phenotype of intense gene activity, we did a detailed analysis of the heat shock-induced expression of the HSP70 encoding gene in larvae with different doses of HP1 and found that HP1 is positively involved in Hsp70 gene activity. These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.     

Decreased expression of the ARID1A gene is associated with poor prognosis in primary gastric cancer

Background

The ARID1A gene encodes adenine-thymine (AT)-rich interactive domain-containing protein 1A, which participates in chromatin remodeling. ARID1A has been showed to function as a tumor suppressor in various cancer types. In the current study, we investigated the expression and prognosis value of ARID1A in primary gastric cancer. Meanwhile, the biological role of ARID1A was further investigated using cell model in vitro.

Methodology/Principal Findings

To investigate the role of ARID1A gene in primary gastric cancer pathogenesis, real-time quantitative PCR and western blotting were used to examine the ARID1A expression in paired cancerous and noncancerous tissues. Results revealed decreased ARID1A mRNA (P = 0.0029) and protein (P = 0.0015) expression in most tumor-bearing tissues compared with the matched adjacent non-tumor tissues, and in gastric cancer cell lines. To further investigate the clinicopathological and prognostic roles of ARID1A expression, we performed immunohistochemical analyses of the 224 paraffin-embedded gastric cancer tissue blocks. Data revealed that the loss of ARID1A expression was significantly correlated with T stage (P = 0.001) and grade (P = 0.006). Consistent with these results, we found that loss of ARID1A expression was significantly correlated with poor survival in gastric cancer patients (P = 0.003). Cox regression analyses showed that ARID1A expression was an independent predictor of overall survival (P = 0.029). Furthermore, the functions of ARID1A in the proliferation and colony formation of gastric cell lines were analyzed by transfecting cells with full-length ARID1A expression vector or siRNA targeting ARID1A. Restoring ARID1A expression in gastric cancer cells significantly inhibited cell proliferation and colony formation. Silencing ARID1A expression in gastric epithelial cell line significantly enhanced cell growth rate.

Conclusions/Significance

Our data suggest that ARID1A may play an important role in gastric cancer and may serve as a valuable prognostic marker and potential target for gene therapy in the treatment of gastric cancer.     

Reduced expression of netrin-1 is associated with fetal growth restriction

Two series of fluorescent molecules were synthesized by acylation of dansyl ethylenediamine and phenylalanine dansyl ethylenediamine with one of either acetyl (C(2)), hexanyl (C(6)), cyclohexanecarbonyl (C(7)), myristyl (C(14)), or palmityl (C(16)) groups and examined for entry and localization in Chinese Hamster Ovary (CHO) cells in tissue culture. Gross total fluorescence retention and cellular microscopic fluorescence patterns were analyzed. In both series, molecules with myristyl or palmityl groups entered cells. Only in the phenylalanine series did hexyl and cyclohexanecarbonyl modification enable entry. Consistent with a mechanism of passive diffusion, entry of compounds into cells was neither energy dependent nor endocytosis linked. Acylated molecules were observed to localize in cytoplasm and not enter nuclei or associate with lipophilic plasma membranes.     

Reduced neuron-specific expression of the TAF1 gene is associated with X-linked dystonia-parkinsonism

X-linked dystonia-parkinsonism (XDP) is a movement disorder endemic to the Philippines. The disease locus, DYT3, has been mapped to Xq13.1. In a search for the causative gene, we performed genomic sequencing analysis, followed by expression analysis of XDP brain tissues. We found a disease-specific SVA (short interspersed nuclear element, variable number of tandem repeats, and Alu composite) retrotransposon insertion in an intron of the TATA-binding protein-associated factor 1 gene (TAF1), which encodes the largest component of the TFIID complex, and significantly decreased expression levels of TAF1 and the dopamine receptor D2 gene (DRD2) in the caudate nucleus. We also identified an abnormal pattern of DNA methylation in the retrotransposon in the genome from the patient's caudate, which could account for decreased expression of TAF1. Our findings suggest that the reduced neuron-specific expression of the TAF1 gene is associated with XDP.     

Differential expression of lysosomal associated membrane protein (LAMP-1) during mammalian spermiogenesis

The mammalian acrosome is a secretory vesicle of mature sperms that plays an important role in fertilization. Recent evidence had pointed out that some components found at endosomes in somatic cells are associated with the developing acrosome during the early steps of spermiogenesis. Moreover, the mammalian acrosome contains many enzymes found within lysosomes in somatic cells. In this work, we studied the dynamics of some components of the endosome/lysosome system, as a way to understand the complex membrane trafficking circuit established during spermatogenesis. We show that the cation independent-mannose-6-phosphate receptor (CI-MPR) is transiently expressed in the cytoplasm of mid-stage spermatids (steps 5-11). On the other hand, gamma-adaptin, an adaptor molecule of a complex involved in trafficking from the Golgi to lysosomes, was expressed in cytoplasmic vesicles only in pachytene and Cap-phase spermatids (steps 1-5). Our major finding is that the lysosomal protein LAMP-1 is differentially expressed during spermiogenesis. LAMP-1 appears late in spermatogenesis (Acrosome-phase) contrasting with LAMP-2, which is present throughout the complete process. Both proteins appear to be associated with cytoplasmic vesicles and not with the developing acrosome. None of the studied proteins is present in epididymal spermatozoa. Our results suggest that the CI-MPR could be involved in membrane trafficking and/or acrosomal shaping during spermiogenesis.     

Phosphatidylinositol 4-phosphate kinase is associated with the membrane skeleton in human erythrocytes

Phosphatidylinositol 4-phosphate kinase was eluted from human erythrocyte stroma by three separate and distinct techniques which are known to disrupt the membrane skeleton. In addition, this kinase was found to be associated with the intact skeletons prepared by Triton X-100 extraction of stroma. Phosphatidylinositol 4-phosphate kinase which has been extracted from the membrane is a freely soluble protein with poor enzymatic activity toward added phosphatidylinositol-4-phosphate; however, the enzyme was shown to reassociate with skeleton-depleted stroma and then regain full enzymatic activity toward stromal bound substrate.     

The expression of carbonic anhydrases II and IV in the human pancreatic cancer cell line (Capan 1) is associated with bicarbonate ion channels

Summary— Human pancreatic ductal cells of the Capan 1 cell line differentiate progressively during growth. After the exponential growth phase, the cells elongate and become polarized with their apical poles covered by microvilli and separated from the basolateral pole by tight junctions. In this stationary phase, they form domes, which are thought to result from the exchange of water and electrolytes. In this study, we demonstrated, using patch-clamp techniques, that HCO₃^? ions exit via the g350 high conductance anionic channel we observed recently at the Capan 1 cell surface. This g350 channel was thought to be either a Cl^?/HCO₃^? antiport or a simple HCO₃^? channel. The stilbene derivatives 4-acetamido-4 isothiocyano-2-2′-disulfonic acid (SITS) and 4,4′ diisothiocyano stilbene-2,2′ disulfonic acid (DIDS) reduced both the number of domes and the Cl^? and HCO₃^? flux through the g350 channel. Moreover, using histochemical, immunocytochemical and biochemical methods we showed that Capan 1 cells express a specific pattern of carbonic anhydrases (CA). Two types of CA were detected: the CA II isozyme mainly localized in the cytoplasm, but also found beneath the inner leaflet of the apical plasma membrane, and the CA IV isozyme localized on the outer leaflet of the apical plasma membrane and microvilli. Their molecular masses were 30 (CA II) and 55 kDa (CA IV), respectively. They were expressed continuously during the exponential growth phase, although their activity increased greatly during the stationary phase. Inhibition of dome formation by acetazolamide indicated the existence of a direct relationship between dome formation and CA. Characteristic structures with a central electron-dense core surrounded by a light halo were observed on the surface of cell membranes using histochemical and immunocytochemical methods. These structures were thought to represent a channel, corresponding possibly to CA IV. Our observations suggest that Capan 1 cells, despite their neoplasic transformation, produce HCO₃^? ions in the same way as normal human pancreatic ductal cells. Capan 1 cells in culture may therefore represent a suitable model for studying pancreatic duct HCO₃^? secretion at the cellular and molecular levels.     

High expression of LASS2 is associated with unfavorable prognosis in patients with ovarian cancer

Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), is a gene isolated from a human liver complementary DNA library. In this study, we found that LASS2 protein level was positively related to International Federation of Gynecology and Obstetrics (FIGO) stage and LASS2-negative tumors showed significant association with longer disease-free survival (DFS) and overall survival (OS) in ovarian cancer patients. The heterogeneous expression of LASS2 had been exhibited in diverse ovarian cancer cells. A significantly lower messenger RNA (mRNA) and protein level of LASS2 was seen in 3AO cell compared with those in other types of ovarian cancer cells. Meanwhile, the mRNA and protein levels of LASS2 in ES-2 and NIH:OVCAR-3 cells were obviously higher. LASS2 overexpression in 3AO cell could promote migration, invasion, and metastasis abilities in vitro and in vivo, while LASS2 knockdown in ES-2 and NIH:OVCAR-3 cells had the opposite effects. The oncogenic capacity of LASS2 in ovarian cancer may be mediated by increased expression of YAP/TAZ. It is indicated that lowering the expression of LASS2 is likely to serve as an unprecedented approach for the treatment of ovarian cancer.     

Downregulation of ERp57 expression is associated with poor prognosis in early-stage cervical cancer

Abstract

Objective: We investigated the clinical significance of ERp57 in the progression of cervical cancer.

Methods: mRNA and protein expression of ERp57 in cervical neoplasias were examined.

Results: ERp57 mRNA expression was significantly decreased in cervical cancers. Immunohistochemistry revealed that ERp57 expression in 123 cervical cancers was down-regulated compared to cervical intraepithelial neoplasias or normal tissues (p?<?0.001). Low ERp57 expression was significantly associated with worse overall survival (HR?=?12.19, p?=?0.018).

Conclusions: Low ERp57 expression independently predicts a poor outcome for patients with cervical cancer, supporting the notion that ERp57 may be a promising novel cancer target.     

Exonuclease 1 expression is associated with clinical progression,metastasis, and survival prognosis of prostate cancer

Prostate cancer (PCa) is the most prevalent malignancy and the second leading cause of cancer-related deaths in the male population in western countries, and we explored the association between exonuclease 1 (EXO1) expression and clinical progression, metastasis (Met), and survival prognosis of PCa. EXO1 expression of high/low-metastatic patient-derived xenografts model was investigated and clinical correlation and prognosis outcomes were validated. EXO1 in high-metastatic models was significantly increased compared with low-metastatic lines. In memorial sloan-kettering cancer center (MSKCC) cohort, EXO1 expression positively correlated with PCa Met, and patients with high EXO1 had poor biochemical recurrence-free survival in primary PCa cohort. Validation in The Cancer Genome Atlas primary cohort indicated EXO1 expression was significantly associated with lymph node Met and disease-free survival. The overexpression of EXO1 is significantly associated with PCa poor survival outcome, and is a promising biomarker for PCa, especially for primary PCa. A prospective study is clearly needed to validate these findings.     

Overexpression of GLUT1 in colorectal cancer is independently associated with poor prognosis

Background: To investigate the expression of glucose transporter 1 (GLUT1) in colorectal cancer (CRC) and its relationship to clinicopathological variables. Methods: The expression of GLUT1 in 163 primary tumors together with the corresponding normal mucosa, and 36 liver metastases was examined using real-time PCR. Results: The mean value of GLUT1 was higher in primary tumors (50.390 ± 68.648) than in the corresponding normal mucosa (20.437 ± 28.703, p<0.0001), while there was no significant difference in GLUT1 expression between CRC and liver metastasis (50.390 ± 68.648 vs 52.277 ± 52.482, p=0.190). In CRCs, GLUT1 expression was higher in poorly differentiated than in well and moderately differentiated tumors (p=0.022), and higher in stage III + IV than in stage I + II tumors (p=0.035). The patients with high-expressed GLUT1 had a worse prognosis than those with low-expressed GLUT1 independently of gender, age, tumor site, stage and differentiation (p=0.026, RR 2.737, 95% CI 1.126-6.651) in stage I-III CRCs. In liver metastasis, GLUT1 expression was higher in larger tumors than in smaller ones (p=0.025). Conclusions: Overexpression of GLUT1 in stage I-III CRCs was independently associated with poor prognosis.     

Expression of the membrane complement regulatory protein CD59 (protectin) is associated with reduced survival in colorectal cancer patients

It has been known for some time that the immune system can recognise growing tumours, and that tumours may respond by modulation of molecules, which make them resistant to further attack. Expression, over-expression, or loss of these molecules may function as markers of tumour progression and prognosis. Among such molecules are the membrane-bound complement regulatory proteins (mCRP), which protect cells from bystander attack by autologous complement. These include CD59 (protectin), which prevents formation of the MAC complex in the terminal stages of complement activation. In the present study, we evaluated immunohistochemical expression of CD59 in a series of over 460 well-characterised colorectal cancers using tissue microarrays (TMA), and related this information to known tumour and patient variables and to survival. The CD59 expression was observed in 69 (15%) of cases overall, and was significantly associated with tumour grade. In contrast, no associations were noted with tumour site, stage or histological type. On survival analysis, a further correlation was observed between expression of CD59 by the colorectal tumours and a reduction in disease-specific patient survival. This observation was strongest for patients with early stage disease. However, a negative impact on survival was also seen in those patients with late stage disease. These results indicate that TMA linked to good clinicopathological databases with good long term follow up are useful tools for determining new prognostic indicators that can be used in future patient management. Immune surveillance may result in immune–editing that induces variable expression of a range of target antigens, and these may be useful prognostic markers. This study has identified CD59 expression as a marker of poor prognosis in colorectal cancer patients.This article is a symposium paper from the "Robert Baldwin Symposium: 50 years of Cancer Immunotherapy", held in Nottingham, Great Britain, on 30th June 2005.     

Reduced intimal thickening following alpha(v)beta3 blockade is associated with smooth muscle cell apoptosis

The adhesion integrin alpha(v)beta3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of alpha(v)beta3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of alpha(v)beta3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of alpha(v)beta3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of alpha(v)beta3. At both 2 and 4 wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of alpha(v)beta3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing alpha(v)beta3. These findings may explain the reduction in restenosis observed clinically following beta3 integrin blockade.     

Abundant expression of 150-kDa oxygen-regulated protein in mouse pancreatic beta cells is correlated with insulin secretion

The 150-kDa oxygen-regulated protein (ORP150) is a member of glucose-regulated proteins (GRPs), which are induced by stressful conditions such as oxygen or glucose deprivation. Here we investigated the highly abundant expression of ORP150 in mouse pancreas and its relationship with insulin secretion. Immunohistochemical analysis revealed that ORP150 expression was restricted to islets, especially to beta cells. The beta cell-specific expression was also observed in a mouse insulinoma cell line, MIN6, which secretes insulin in response to increased glucose concentration. Furthermore, ORP150 in islets dramatically diminished by fasting, concomitant with reduction of the serum insulin level. These results strongly suggest the role for ORP150 in insulin secretion.     

A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39. Other (62, 51, and 25 kD) quantitatively minor peripheral proteins also interact with IP39 on the nitrocellulose overlays, and the possible significance of this binding is discussed.