Examining Soft and Hard Attributes of Health Care Service Quality and Their Impacts on Patient Satisfaction and Loyalty.

BACKGROUND AND OBJECTIVES: Many studies have confirmed the influences of various service quality dimensions on patient satisfaction and loyalty, but no existing theoretical model accounts for variation in how different types of patients evaluate service quality's soft and hard attributes. This research gap may cause problems for administrators needing to decide how to distribute resources appropriately across multiple departments. Therefore, this study establishes a theoretical model of the differences between inpatients' and outpatients' evaluations of hard and soft qualities and compares such evaluations' influences on patient satisfaction and loyalty. Also, to supplement statistical analysis and respond to scholars' calls for more mixed-methods studies of health care quality, this research incorporates analysis of online reviews to provide a holistic, close to real-time picture of patients' service experience perceptions. METHODS: This study's survey sample comprised 292 inpatients and 137 outpatients from a Taiwanese hospital. We used partial least squares structural equation modeling to test the hypothetical model and importance-performance map analysis to identify factors that were significant to the service process but performed poorly. Finally, we used a text-mining technique to scrape 536 reviews posted on Google Maps, and Leximancer Portal to perform automated content and sentiment analyses on those data, as a means of mapping the critical concepts and themes that influenced patient experiences. RESULTS: This study's analyses support the ideas that both hard and soft qualities are critical dimensions of service quality, and that each has different influences on inpatients' and outpatients' satisfaction and loyalty. Specifically, the sampled inpatients strongly valued the hard qualities of the hospital but were not satisfied with it. On the other hand, soft qualities attracted outpatients' attention and influenced their satisfaction and loyalty. In addition, content analysis revealed that soft qualities were the main reason patients left comments, whether positive or negative. Waiting time emerged as another critical element in triggering patients' unfavorable reviews. CONCLUSIONS: Patient population type, whether inpatient or outpatient, has been found to impact perceptions of service quality within health care institutions. As such, health care administrators should be cognizant of this phenomenon and make informed and tailored decisions when addressing quality within their respective services. Emphasis on the development of both interpersonal and professional skills among health care personnel may prove beneficial in enhancing the patient experience and ultimately fostering positive online reviews.

Patient-Derived Organoid Serves as a Platform for Personalized Chemotherapy in Advanced Colorectal Cancer Patients.

Background: Addition of oxaliplatin to adjuvant 5-FU has significantly improved the disease-free survival and served as the first line adjuvant chemotherapy in advanced colorectal cancer (CRC) patients. However, a fraction of patients remains refractory to oxaliplatin-based treatment. It is urgent to establish a preclinical platform to predict the responsiveness toward oxaliplatin in CRC patients as well as to improve the efficacy in the resistant patients. Methods: A living biobank of organoid lines were established from advanced CRC patients. Oxaliplatin sensitivity was assessed in patient-derived tumor organoids (PDOs) in vitro and in PDO-xenografted tumors in mice. Based on in vitro oxaliplatin IC50 values, PDOs were classified into either oxaliplatin-resistant (OR) or oxaliplatin-sensitive (OS) PDOs. The outcomes of patients undergone oxaliplatin-based treatment was followed. RNA-sequencing and bioinformatics tools were performed for molecular profiling of OR and OS PDOs. Oxaliplatin response signatures were submitted to Connectivity Map algorithm to identify perturbagens that may antagonize oxaliplatin resistance. Results: Oxaliplatin sensitivity in PDOs was shown to correlate to oxaliplatin-mediated inhibition on PDO xenograft tumors in mice, and parallelled clinical outcomes of CRC patients who received FOLFOX treatment. Molecular profiling of transcriptomes revealed oxaliplatin-resistant and -sensitive PDOs as two separate entities, each being characterized with distinct hallmarks and gene sets. Using Leave-One-Out Cross Validation algorithm and Logistic Regression model, 18 gene signatures were identified as predictive biomarkers for oxaliplatin response. Candidate drugs identified by oxaliplatin response signature-based strategies, including inhibitors targeting c-ABL and Notch pathway, DNA/RNA synthesis inhibitors, and HDAC inhibitors, were demonstrated to potently and effectively increase oxaliplatin sensitivity in the resistant PDOs. Conclusions: PDOs are useful in informing decision-making on oxaliplatin-based chemotherapy and in designing personalized chemotherapy in CRC patients.

Structure-guided antibody cocktail for prevention and treatment of COVID-19.

Development of effective therapeutics for mitigating the COVID-19 pandemic is a pressing global need. Neutralizing antibodies are known to be effective antivirals, as they can be rapidly deployed to prevent disease progression and can accelerate patient recovery without the need for fully developed host immunity. Here, we report the generation and characterization of a series of chimeric antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Some of these antibodies exhibit exceptionally potent neutralization activities in vitro and in vivo, and the most potent of our antibodies target three distinct non-overlapping epitopes within the RBD. Cryo-electron microscopy analyses of two highly potent antibodies in complex with the SARS-CoV-2 spike protein suggested they may be particularly useful when combined in a cocktail therapy. The efficacy of this antibody cocktail was confirmed in SARS-CoV-2-infected mouse and hamster models as prophylactic and post-infection treatments. With the emergence of more contagious variants of SARS-CoV-2, cocktail antibody therapies hold great promise to control disease and prevent drug resistance.

Identification of COVID-19 B-cell epitopes with phage-displayed peptide library.

BACKGROUND: Coronavirus disease 19 (COVID-19) first appeared in the city of Wuhan, in the Hubei province of China. Since its emergence, the COVID-19-causing virus, SARS-CoV-2, has been rapidly transmitted around the globe, overwhelming the medical care systems in many countries and leading to more than 3.3 million deaths. Identification of immunological epitopes on the virus would be highly useful for the development of diagnostic tools and vaccines that will be critical to limiting further spread of COVID-19. METHODS: To find disease-specific B-cell epitopes that correspond to or mimic natural epitopes, we used phage display technology to determine the targets of specific antibodies present in the sera of immune-responsive COVID-19 patients. Enzyme-linked immunosorbent assays were further applied to assess competitive antibody binding and serological detection. VaxiJen, BepiPred-2.0 and DiscoTope 2.0 were utilized for B-cell epitope prediction. PyMOL was used for protein structural analysis. RESULTS: 36 enriched peptides were identified by biopanning with antibodies from two COVID-19 patients; the peptides 4 motifs with consensus residues corresponding to two potential B-cell epitopes on SARS-CoV-2 viral proteins. The putative epitopes and hit peptides were then synthesized for validation by competitive antibody binding and serological detection. CONCLUSIONS: The identified B-cell epitopes on SARS-CoV-2 may aid investigations into COVID-19 pathogenesis and facilitate the development of epitope-based serological diagnostics and vaccines.

Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44.

BACKGROUND: Serglycin (SRGN), previously recognized as an intracellular proteoglycan involved in the storage processes of secretory granules, has recently been shown to be upregulated in several solid tumors. We have previously shown that SRGN in non-small cell lung cancer (NSCLC) promotes malignant phenotypes in a CD44-dependent manner and increased expression of SRGN predicts poor prognosis of primary lung adenocarcinomas. However, the underlying mechanism remains to be defined. METHODS: Overexpression, knockdown and knockout approaches were performed to assess the role of SRGN in cell motility using wound healing and Boyden chamber migration assays. SRGN devoid of glycosaminoglycan (GAG) modification was produced by site-directed mutagenesis or chondroitinase treatment. Liquid chromatography/tandem mass spectrometry was applied for quantitative analysis of the disaccharide compositions and sulfation extent of SRGN GAGs. Western blot and co-immunoprecipitation analyses were performed to determine the expression and interaction of proteins of interest. Actin cytoskeleton organization was monitored by immunofluorescence staining. RESULTS: SRGN expressed by NSCLC cells is readily secreted to the extracellular matrix in a heavily glycosylated form attached with mainly chondroitin sulfate (CS)-GAG chains, and to a lesser extent with heparin sulfate (HS). The CS-GAG moiety serves as the structural motif for SRGN binding to tumor cell surface CD44 and promotes cell migration. SRGN devoid of CS-GAG modification fails to interact with CD44 and has lost the ability to promote cell migration. SRGN/CD44 interaction promotes focal adhesion turnover via Src-mediated paxillin phosphorylation and disassembly of paxillin/FAK adhesion complex, facilitating cell migration. In support, depletion of Src activity or removal of CS-GAGs efficiently blocks SRGN-mediated Src activation and cell migration. SRGN also promotes cell migration via inducing cytoskeleton reorganization mediated through RAC1 and CDC42 activation accompanied with increased lamellipodia and filopodia formation. CONCLUSIONS: Proteoglycan SRGN promotes NSCLC cell migration via the binding of its GAG motif to CD44. SRGN/CD44 interaction induces Rho-family GTPase-mediated cytoskeleton reorganization and facilitates Src-mediated focal adhesion turnover, leading to increased cell migration. These findings suggest that targeting specific glycans in tumor microenvironment that serve as ligands for oncogenic pathways may be a potential strategy for cancer therapy.

ITPKA Gene Body Methylation Regulates Gene Expression and Serves as an Early Diagnostic Marker in Lung and Other Cancers.

INTRODUCTION: Despite recent advances in cancer therapy, the overall 5-year survival rate of patients with lung cancer remains low. The aim of our study was to search for novel markers for early diagnosis in patients with lung cancer. METHODS: Complementary DNA microarray analysis was performed in primary lung adenocarcinomas and cell lines to search for differentially expressed genes, followed by in vivo and in vitro tumorigenic assays to characterize the oncogenic potential of the candidate genes. Gene body methylation was analyzed by 450K methylation array, bisulfite sequencing, and quantitative methylation-specific polymerase chain reaction assays. In silico analysis of The Cancer Genome Atlas data set was also performed. RESULTS: Inositol-trisphosphate 3-kinase A gene (ITPKA), a kinase with limited tissue distribution, was identified as a potential oncogene. We showed that ITPKA expression is up-regulated in many forms of cancers, including lung and breast cancers, and that overexpressed ITPKA contributes to tumorigenesis. We also demonstrated that ITPKA expression is regulated by epigenetic DNA methylation of ITPKA gene body through modulation of the binding of SP1 transcription factor to the ITPKA promoter. ITPKA gene body displayed low or absent levels of methylation in most normal tissue but was significantly methylated in malignant tumors. In lung cancer, ITPKA gene body methylation first appeared at the in situ carcinoma stage and progressively increased after invasion. CONCLUSIONS: ITPKA is a potential oncogene that it is overexpressed in most tumors, and its overexpression promotes tumorigenesis. ITPKA gene body methylation regulates its expression and thus serves as a novel and potential biomarker for early cancer detection.

Loss of PTPRM associates with the pathogenic development of colorectal adenoma-carcinoma sequence.

Identification and functional analysis of genes from genetically altered chromosomal regions would suggest new molecular targets for cancer diagnosis and treatment. Here we performed a genome-wide analysis of chromosomal copy number alterations (CNAs) in matching sets of colon mucosa-adenoma-carcinoma samples using high-throughput oligonucleotide microarray analysis. In silico analysis of NCBI GEO and TCGA datasets allowed us to uncover the significantly altered genes (p ≤ 0.001) associated with the identified CNAs. We performed quantitative PCR analysis of the genomic and complementary DNA derived from primary mucosa, adenoma, and carcinoma samples, and confirmed the recurrent loss and down-regulation of PTPRM in colon adenomas and carcinomas. Functional characterization demonstrated that PTPRM negatively regulates cell growth and colony formation, whereas loss of PTPRM promotes oncogenic cell growth. We further showed that, in accordance to Knudson's two-hit hypothesis, inactivation of PTPRM in colon cancer was mainly attributed to loss of heterozygosity and promoter hypermethylation. Taken together, this study demonstrates a putative tumor suppressive role for PTPRM and that genetic and epigenetic alterations of PTPRM may contribute to early step of colorectal tumorigenesis.

Label-free quantitative proteomics and N-glycoproteomics analysis of KRAS-activated human bronchial epithelial cells.

Mutational activation of KRAS promotes various malignancies, including lung adenocarcinoma. Knowledge of the molecular targets mediating the downstream effects of activated KRAS is limited. Here, we provide the KRAS target proteins and N-glycoproteins using human bronchial epithelial cells with and without the expression of activated KRAS (KRAS(V12)). Using an OFFGEL peptide fractionation and hydrazide method combined with subsequent LTQ-Orbitrap analysis, we identified 5713 proteins and 608 N-glycosites on 317 proteins in human bronchial epithelial cells. Label-free quantitation of 3058 proteins (≥2 peptides; coefficient of variation (CV) ≤ 20%) and 297 N-glycoproteins (CV ≤ 20%) revealed the differential regulation of 23 proteins and 14 N-glycoproteins caused by activated KRAS, including 84% novel ones. An informatics-assisted IPA-Biomarker® filter analysis prioritized some of the differentially regulated proteins (ALDH3A1, CA2, CTSD, DST, EPHA2, and VIM) and N-glycoproteins (ALCAM, ITGA3, and TIMP-1) as cancer biomarkers. Further, integrated in silico analysis of microarray repository data of lung adenocarcinoma clinical samples and cell lines containing KRAS mutations showed positive mRNA fold changes (p < 0.05) for 61% of the KRAS-regulated proteins, including biomarker proteins, CA2 and CTSD. The most significant discovery of the integrated validation is the down-regulation of FABP5 and PDCD4. A few validated proteins, including tumor suppressor PDCD4, were further confirmed as KRAS targets by shRNA-based knockdown experiments. Finally, the studies on KRAS-regulated N-glycoproteins revealed structural alterations in the core N-glycans of SEMA4B in KRAS-activated human bronchial epithelial cells and functional role of N-glycosylation of TIMP-1 in the regulation of lung adenocarcinoma A549 cell invasion. Together, our study represents the largest proteome and N-glycoproteome data sets for HBECs, which we used to identify several novel potential targets of activated KRAS that may provide insights into KRAS-induced adenocarcinoma and have implications for both lung cancer therapy and diagnosis.

Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas.

BACKGROUND: Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale. METHODOLOGY/PRINCIPAL FINDINGS: By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells. CONCLUSIONS/SIGNIFICANCE: This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.

SOCS6, down-regulated in gastric cancer, inhibits cell proliferation and colony formation.

Members of the suppressor of cytokine-induced signaling (SOCS) family are negative regulators of cytokine signaling pathways. By mRNA differential display, we showed that SOCS6 was frequently down-regulated in gastric cancer (GC). Our data showed that allelic loss and promoter hypermethylation may account for the major mechanisms leading to SOCS6 inactivation. Ectopic expression of SOCS6 suppressed cell growth and colony formation, in part through eliciting intrinsic apoptotic pathway, accompanied with decreased mitochondrial membrane potential. Taken together, this study provides molecular and functional data supporting the importance of loss-of-function of SOCS6 as a frequent event in gastric tumorigenesis.

Genomic organization and functional characterization of the promoter for the human suppressor of cytokine signaling 6 gene.

In this study, we report the expression and genomic structure of the gene encoding human suppressor of cytokine signaling 6 (SOCS6), and the characterization of the functional promoter region. The human SOCS6 gene, spanning 40 kb on chromosome 18q22.2, is composed of two exons separated by an intron of 35 kb. Two transcripts are ubiquitously expressed, and both encode the full-length open reading frame of SOCS6. A primer extension assay revealed that the major transcription initiation site is located 469 bp upstream the ATG codon. Luciferase promoter analysis demonstrated that the 5'-flanking region is able to drive transcription, and the CpG-rich sequences near the transcription initiation site are important for the TATA-less SOCS6 promoter activity. Analogous to SOCS1 and SOCS3, which are down-regulated in several human cancers, SOCS6 is expressed at lower levels in carcinomas of stomach and colon. We demonstrated that hypermethylation of the SOCS6 promoter is one of the mechanisms for the epigenetic regulation of SOCS6 expression. Firstly, in vitro methylation of the reporter promoter plasmid significantly suppressed the promoter activity. Secondly, SOCS6 expression in vivo was enhanced by treating cells with a methyltransferase inhibitor. The SOCS6 gene from various species shares significant homology in amino acid sequences, transcription factor binding motifs in promoter regions and the two-exon genomic structure, suggesting that the SOCS6 gene is highly conserved.

Acetylation and activation of STAT3 mediated by nuclear translocation of CD44.

Expression of the type I transmembrane glycoprotein CD44 has recently been recognized as a signature for cancer stem cells. In this study, we demonstrate that CD44, once engaged, is internalized and translocated to the nucleus, where it binds to various promoters, including that of cyclin D1, leading to cell fate change through transcriptional reprogramming. In regulating cyclin D1 expression, the internalized CD44 forms a complex with STAT3 and p300 (acetyltransferase), eliciting STAT3 acetylation at lysine 685 and dimer formation in a cytokine- and growth factor-independent manner. A bipartite nuclear localization signal (NLS) was mapped to the cytoplasmic tail of CD44, which mediates its nuclear translocation. Expression of CD44(NLS) mutant sequesters STAT3 in cytosol. In the nucleus, the acetylated STAT3 dimer remains associated with CD44 and binds to the cyclin D1 promoter, leading to increased cyclin D1 expression and cell proliferation. This study describes a novel function for CD44 in transcriptional modulation through nuclear translocation of the internalized CD44 and complex formation with transcription factors.

CD44 engagement promotes matrix-derived survival through the CD44-SRC-integrin axis in lipid rafts.

CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation. By using a series of CD44 mutants and pharmacological inhibitors selectively targeted to various cellular pathways, we show in this study that engagement of CD44 induces lipid raft coalescence to facilitate a CD44-Src-integrin signaling axis in lipid rafts, leading to increased matrix-derived survival. Palmitoylation of the membrane-proximal cysteine residues and carboxyl-terminal linkage to the actin cytoskeleton both contribute to raft targeting of CD44. The enrichment of integrin beta1 in lipid rafts is tightly coupled to CD44 ligation-elicited lipid raft reorganization and associated with temporally delayed endocytosis. Through the interaction with the CD44 carboxyl-terminal ankyrin domain, Src is cotranslocated to lipid rafts, where it induces integrin activation via an inside-out mechanism. Collectively, this study demonstrates an important role of the dynamic raft reorganization induced by CD44 clustering in eliciting the matrix-derived survival signal.

Osteopontin promotes integrin activation through outside-in and inside-out mechanisms: OPN-CD44V interaction enhances survival in gastrointestinal cancer cells.

Osteopontin (OPN) and splice variants of CD44 (CD44(V)) have independently been identified as markers for tumor progression. In this study, we show that both OPN and CD44(V) are frequently overexpressed in human gastric cancer and that OPN-engaged CD44(V) ligation confers cells an increased survival mediated through integrin activation. First, we show that OPN treatment confers cells an increased resistance to UV-induced apoptosis. The OPN-mediated antiapoptosis is dependent on the expression of the variant exon 6 (V6)- or V7-containing CD44 as shown by overexpression of individual CD44(V) in gastric AZ521 cells that express no or very low level of endogenous CD44 and by knockdown of the constitutively expressed V6-containing CD44 isoforms in colon HT29 cells. Although OPN also interacts with RGD integrins, OPN-RGD sequence is dispensable for OPN-mediated antiapoptosis. OPN-induced antiapoptosis is mainly attributed to the engagement of CD44(V) isoforms and the relay of an inside-out signaling via Src activity, leading to robust integrin activation. Furthermore, OPN-elicited antiapoptosis was observed when cells were plated on fibronectin but not on poly-D-lysin, and preincubation of cells with anti-integrin beta(1) antibody to block integrin-extracellular matrix (ECM) interaction or ectopic expression of the dominant-negative forms of focal adhesion kinase to block ECM-derived signal abolished OPN-induced survival, suggesting that OPN-elicited antiapoptotic function is propagated from matrix transduced by integrin. Taken together, we showed that OPN-CD44(V) interaction promotes ECM-derived survival signal mediated through integrin activation, which may play an important role in the pathogenic development and progression of gastric cancer.

Quantitative proteomic and genomic profiling reveals metastasis-related protein expression patterns in gastric cancer cells.

Gastric cancer is a leading cause of death worldwide, and patients have an overall 5-year survival rate of less than 10%. Using quantitative proteomic techniques together with microarray chips, we have established comprehensive proteome and transcriptome profiles of the metastatic gastric cancer TMC-1 cells and the noninvasive gastric cancer SC-M1 cell. Our qualitative protein profiling strategy offers the first comprehensive analysis of the gastric cancer cell proteome, identifying 926 and 909 proteins from SC-M1 and TMC-1 cells, respectively. Cleavable isotope-coded affinity tagging analysis allows quantitation of a total of 559 proteins (with a protein false-positive rate of <0.005), and 240 proteins were differentially expressed (>1.3-fold) between the SC-M1 and TMC-1 cells. We identified numerous proteins not previously associated with gastric cancer. Notably, a large subset of differentially expressed proteins was associated with tumor metastasis, including proteins functioning in cell-cell and cell-extracellular matrix (cell-ECM) adhesion, cell motility, proliferation, and tumor immunity. Gene expression profiling by DNA microarray revealed differential expression (of >2-fold) of about 1000 genes. The weak correlation observed between protein and mRNA profiles highlights the important complementarities of DNA microarray and proteomics approaches. These comparative data enabled us to map the disease-perturbed cell-cell and cell-ECM adhesion and Rho GTPase-mediated cytoskeletal pathways. Further validation of a subset of genes suggests the potential use of vimentin and galectin 1 as markers for metastasis. We demonstrate that combining proteomic and genomic approaches not only provides a rapid, robust, and sensitive platform to elucidate the molecular mechanisms underlying gastric cancer metastasis but also may identify candidate diagnostic markers and therapeutic targets.

Rho/Rhotekin-mediated NF-kappaB activation confers resistance to apoptosis.

Rhotekin (RTKN), the gene coding for the Rho effector, RTKN, was shown to be overexpressed in human gastric cancer (GC). In this study, we further showed that RTKN is expressed at a low level in normal cells and is overexpressed in many cancer-derived cell lines. The function of RTKN as an effector protein in Rho GTPase-mediated pathways regulating apoptosis was investigated. By transfection and expression of RTKN in cells that expressed endogenous RTKN at a low basal level, we showed that RTKN overexpression conferred cell resistance to apoptosis induced by serum deprivation or treatment with sodium butyrate, and the increased resistance correlated to the level of RTKN. Conversely, reducing RTKN expression by small interfering RNAs greatly sensitized cells to apoptosis. The RTKN-mediated antiapoptotic effect was blocked by the nuclear factor-kappaB (NF-kappaB) inhibitors, curcumin or parthenolide, but not by the phosphatidylinositol 3'-OH-kinase inhibitor, LY294002, or the MAP kinase inhibitor, PD98059. Reporter gene assays and electrophoretic mobility shift assay confirmed that RTKN overexpression led to constitutive activation of NF-kappaB through the phosphorylation of IkappaB by IKKbeta. By using the RTKN truncation mutants, we showed that RTKN mediated Rho activity eliciting signaling pathway to activate NF-kappaB, with a concomitant induction of expression of the NF-kappaB antiapoptotic genes, cIAP-2, BCl-xL, A1, and A20. Consistent with these data, RTKN-expressing cells showed increased chemoresistance to 5-fluorouracil and paclitaxol, and the resistance was greatly attenuated by NF-kappaB inhibitor. In conclusion, overactivated Rho/RTKN/NF-kappaB signaling pathway through overexpression of RTKN may play a key role in gastric tumorigenesis by conferring cells resistance to apoptosis, and this signaling pathway may serve as an important target for novel therapeutic approaches to the treatment of human GC.

Overexpression of rho effector rhotekin confers increased survival in gastric adenocarcinoma.

Like many epithelial-derived cancers, gastric cancer (GC) results from a multistep tumorigenic process. However, the detailed mechanisms involved in GC formation are poorly characterized. Using an ordered differential display method, we have identified rhotekin (RTKN), the gene coding for the Rho effector, RTKN, as one of the genes differentially expressed in human GC. Northern analysis using human multiple tissue blots showed that RTKN is predominantly expressed in the kidney and spinal cord, and, to a lesser degree, in the thyroid, tongue, liver, brain, prostate, trachea, and stomach. RT-PCR analysis confirmed that RTKN was overexpressed in most (5/7; 71%) GC examined. By analyzing the Stanford Microarray Database for the expression profiles of gastric tissues, we also found a progressional increase in RTKN expression in nonneoplastic mucosa, GC, and then lymph node metastases (p < 0.005 by Jonckheere-Terpstra test), suggesting that RTKN expression correlates with GC progression. The role of RTKN in the pathogenic development of GC was investigated by transfection and expression of RTKN in AGS gastric cells, which express endogenous RTKN at a low basal level. Flow-cytometric analysis showed that RTKN-transfected AGS cells were significantly more resistant than vector-transfected cells to apoptosis upon treatment with sodium butyrate. To explore the mechanisms underlying RTKN-mediated cell survival, a reporter assay was performed. Since the NF-kappaB activation is known to promote cell survival and Rho GTPase may lead to NF-kappaB activation, we transfected AGS cells with the RTKN expression vector along with a pNF-kappaB-Luc reporter plasmid. Our results showed that overexpression of RTKN induced robust activation of NF-kappaB, and RTKN-mediated NF-kappaB activation was suppressed significantly by C3 transferase, an inhibitor of the small GTPase Rho. We conclude that Rho/RTKN-mediated NF-kappaB activation leading to cell survival may play a key role in gastric tumorigenesis. This study provides original documentation for the overrepresentation of the Rho GTPase effector rhotekin in human cancer and its links to cancer formation.

Interaction of Saccharomyces Cdc13p with Pol1p, Imp4p, Sir4p and Zds2p is involved in telomere replication, telomere maintenance and cell growth control.

Telomeres are the physical ends of eukaryotic chromosomes. They are important for maintaining the integrity of chromosomes and this function is mediated through a number of protein factors. In Saccharomyces cerevisiae, Cdc13p binds to telomeres and affects telomere maintenance, telomere position effects and cell cycle progression through G(2)/M phase. We identified four genes encoding Pol1p, Sir4p, Zds2p and Imp4p that interact with amino acids 1-252 of Cdc13p using a yeast two-hybrid screening system. Interactions of these four proteins with Cdc13p were through direct protein-protein interactions as judged by in vitro pull-down assays. Direct protein-protein interactions were also observed between Pol1p-Imp4p, Pol1p-Sir4p and Sir4p-Zds2p, whereas no interaction was detected between Imp4p-Sir4p and Zds2p-Imp4p, suggesting that protein interactions were specific in the complex. Pol1p was shown to interact with Cdc13p. Here we show that Zds2p and Imp4p also form a stable complex with Cdc13p in yeast cells, because Zds2p and Imp4p co-immunoprecipitate with Cdc13p, whereas Sir4p does not. The function of the N-terminal 1-252 region of Cdc13p was also analyzed. Expressing Cdc13(252-924)p, which lacks amino acids 1-252 of Cdc13p, causes defects in progressive cell growth and eventually arrested in the G(2)/M phase of the cell cycle. These growth defects were not caused by progressive shortening of telomeres because telomeres in these cells were long. Point mutants in the amino acids 1-252 region of Cdc13p that reduced the interaction between Cdc13p and its binding proteins resulted in varying level of defects in cell growth and telomeres. These results indicate that the interactions between Cdc13(1-252)p and its binding proteins are important for the function of Cdc13p in telomere regulation and cell growth. Together, our results provide evidence for the formation of a Cdc13p-mediated telosome complex through its N-terminal region that is involved in telomere maintenance, telomere length regulation and cell growth control.