Loss of Ep-CAM (CO17-1A) expression predicts survival in patients with gastric cancer

Preoperative staging of gastric cancer is difficult and not optimal. The TNM stage is an important prognostic factor, but it can only be assessed reliably after surgery. Therefore, there is need for additional, reliable prognostic factors that can be determined preoperatively in order to select patients who might benefit from (neo) adjuvant treatment. Expression of immunohistochemical markers was demonstrated to be associated with tumour progression and metastasis. The expression of p53, CD44 (splice variants v5, v6 and v9), E-cadherin, Ep-CAM (CO17-1A antigen) and c-erB2/neu were investigated in tumour tissues of 300 patients from the Dutch Gastric Cancer Trial, investigating the value of extended lymphadenectomy compared to that of limited lymphadenectomy). The expression of tumour markers was analysed with respect to patient survival. Patients without loss of Ep-CAM-expression of tumour cells (19%) had a significantly better 10-year survival (P<0.0001) compared to patients with any loss: 42% (s.e.=7%) vs 22% (s.e.=3%). Patients with CD44v6 (VFF18) expression in more than 25% of the tumour cells (69% of the patients) also had a significantly better survival (P=0.01) compared to patients with expression in less than 25% of the tumour cells: 10 year survival rate of 29% (s.e.=3%) vs 19% (s.e.=4%). The prognostic value of both markers was stronger in stages I and II, and independent of the TNM stage. Ep-CAM and CD44v6-expression provides prognostic information additional to the TNM stage. Loss of Ep-CAM-expression identifies aggressive tumours especially in patients with stage I and II disease. This information may be helpful in selecting patients suitable for surgery or for additional treatment pre- or postoperatively

Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving

A Genome-Wide Approach to Discovery of Small RNAs Involved in Regulation of Virulence in Vibrio cholerae

Small RNAs (sRNAs) are becoming increasingly recognized as important regulators in bacteria. To investigate the contribution of sRNA mediated regulation to virulence in Vibrio cholerae, we performed high throughput sequencing of cDNA generated from sRNA transcripts isolated from a strain ectopically expressing ToxT, the major transcriptional regulator within the virulence gene regulon. We compared this data set with ToxT binding sites determined by pulldown and deep sequencing to identify sRNA promoters directly controlled by ToxT. Analysis of the resulting transcripts with ToxT binding sites in cis revealed two sRNAs within the Vibrio Pathogenicity Island. When deletions of these sRNAs were made and the resulting strains were competed against the parental strain in the infant mouse model of V. cholerae colonization, one, TarB, displayed a variable colonization phenotype dependent on its physiological state at the time of inoculation. We identified a target of TarB as the mRNA for the secreted colonization factor, TcpF. We verified negative regulation of TcpF expression by TarB and, using point mutations that disrupted interaction between TarB and tpcF mRNA, showed that loss of this negative regulation was primarily responsible for the colonization phenotype observed in the TarB deletion mutant

PDZ domains and their binding partners: structure, specificity, and modification

PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins. They regulate multiple biological processes such as transport, ion channel signaling, and other signal transduction systems. This review discusses the structural characterization of PDZ domains and the use of recently emerging technologies such as proteomic arrays and peptide libraries to study the binding properties of PDZ-mediated interactions. Regulatory mechanisms responsible for PDZ-mediated interactions, such as phosphorylation in the PDZ ligands or PDZ domains, are also discussed. A better understanding of PDZ protein-protein interaction networks and regulatory mechanisms will improve our knowledge of many cellular and biological processes

MicroRNA miR-328 Regulates Zonation Morphogenesis by Targeting CD44 Expression

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion

Epigenetic regulation of CD44 in Hodgkin and non-Hodgkin lymphoma

<p>Abstract</p> <p>Background</p> <p>Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a hallmark of cancer. To assay its extent in human lymphoma, methylation of 24 TSG was analyzed in lymphoma-derived cell lines as well as in patient samples.</p> <p>Methods</p> <p>We screened for TSG methylation using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in 40 lymphoma-derived cell lines representing anaplastic large cell lymphoma, Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma and mantle cell lymphoma (MCL) as well as in 50 primary lymphoma samples. The methylation status of differentially methylated <it>CD44 </it>was verified by methylation-specific PCR and bisulfite sequencing. Gene expression of <it>CD44 </it>and its reactivation by DNA demethylation was determined by quantitative real-time PCR and on the protein level by flow cytometry. Induction of apoptosis by anti-CD44 antibody was analyzed by annexin-V/PI staining and flow cytometry.</p> <p>Results</p> <p>On average 8 ± 2.8 of 24 TSG were methylated per lymphoma cell line and 2.4 ± 2 of 24 TSG in primary lymphomas, whereas 0/24 TSG were methylated in tonsils and blood mononuclear cells from healthy donors. Notably, we identified that <it>CD44 </it>was hypermethylated and transcriptionally silenced in all BL and most FL and DLBCL cell lines, but was usually unmethylated and expressed in MCL cell lines. Concordant results were obtained from primary lymphoma material: <it>CD44 </it>was not methylated in MCL patients (0/11) whereas <it>CD44 </it>was frequently hypermethylated in BL patients (18/29). In cell lines with <it>CD44 </it>hypermethylation, expression was re-inducible at mRNA and protein levels by treatment with the DNA demethylating agent 5-Aza-2'-deoxycytidine, confirming epigenetic regulation of <it>CD44</it>. CD44 ligation assays with a monoclonal anti-CD44 antibody showed that CD44 can mediate apoptosis in CD44⁺lymphoma cells. <it>CD44 </it>hypermethylated, CD44^-lymphoma cell lines were consistently resistant towards anti-CD44 induced apoptosis.</p> <p>Conclusion</p> <p>Our data show that <it>CD44 </it>is epigenetically regulated in lymphoma and undergoes <it>de novo </it>methylation in distinct lymphoma subtypes like BL. Thus <it>CD44 </it>may be a promising new epigenetic marker for diagnosis and a potential therapeutic target for the treatment of specific lymphoma subtypes.</p