A functional regulatory variant of MYH3 influences muscle fiber-type composition and intramuscular fat content in pigs.

Muscle development and lipid accumulation in muscle critically affect meat quality of livestock. However, the genetic factors underlying myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western commercial breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle of KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and cotransfection assays showed that the structural variant in the 5'-flanking region of MYH3 abrogated the binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of MYH3 among pig populations worldwide indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role in improving pork quality in current breeding programs.

Export of membrane proteins from the Golgi complex to the primary cilium requires the kinesin motor, KIFC1.

Microtubule-based motors contribute to the efficiency and selectivity of Golgi exit and post-Golgi transport of membrane proteins that are targeted to distinct compartments. Cytoplasmic dynein moves post-Golgi vesicles that carry rhodopsin toward the base of the connecting cilium in photoreceptor cells; however, the identity of the motors that are involved in the vesicular trafficking of ciliary membrane proteins in nonphotoreceptor cells remains unclear. Here, we demonstrate that the minus end-directed kinesin KIFC1 (kinesin family member C1) is required for both ciliary membrane protein transport and serum starvation-induced ciliogenesis in retinal pigmented epithelial 1 cells. Although KIFC1 is known as a mitotic motor that is sequestered in the nucleus during interphase, KIFC1 immunoreactivity appeared in the Golgi region after serum starvation. Knockdown of KIFC1 inhibited the export of ciliary receptors from the Golgi complex. KIFC1 overexpression affected the Golgi localization of GMAP210 (Golgi microtubule-associated protein 210) and IFT20 (intraflagellar transport 20), which are involved in membrane protein transport to cilia. Moreover, KIFC1 physically interacted with ASAP1 (ADP-ribosylation factor GTPase-activating protein with SH3 domain, ankyrin repeat and PH domain 1), which regulates the budding of rhodopsin transport carriers from the Golgi complex, and KIFC1 depletion caused Golgi accumulation of ASAP1. A decrease in the centrosomal levels of IFT20 and TTBK2 (τ-tubulin kinase 2) was associated with ciliogenesis defects in KIFC1-depleted cells. Our results suggest that KIFC1 plays roles in the Golgi exit of ciliary receptors and in the recruitment of ciliogenesis regulators.-Lee, S.-H., Joo, K., Jung, E. J., Hong, H., Seo, J., Kim, J. Export of membrane proteins from the Golgi complex to the primary cilium requires the kinesin motor, KIFC1.

Curcumin induces ER stress-mediated apoptosis through selective generation of reactive oxygen species in cervical cancer cells.

Prolonged accumulation of misfolded or unfolded proteins caused by cellular stress, including oxidative stress, induces endoplasmic reticulum stress, which then activates an unfolded protein response (UPR). ER stress is usually maintained at higher levels in cancer cells as compared to normal cells due to altered metabolism in cancer. Here, we investigated whether curcumin is ER stress-mediated apoptosis in cervical cancer cells, and ROS increased by curcumin are involved in the process as an upstream contributor. Curcumin inhibited proliferation of cervical cancer cells (C33A, CaSki, HeLa, and ME180) and induced apoptotic cell death. Curcumin activated ER-resident UPR sensors, such as PERK, IRE-1α, and ATF6, and their downstream-signaling proteins in cervical cancer cells, but not in normal epithelial cells and peripheral blood mononuclear cells (PBMCs). CHOP, a key factor involved in ER stress-mediated apoptosis, was also activated by curcumin. CHOP decreased the ratio of anti-apoptotic protein Bcl-2 to pro-apoptotic protein Bax expression, and subsequently increased the apoptotic population of cervical cancer cells. Furthermore, curcumin elevated levels of intracellular reactive oxygen species (ROS) in cervical cancer cells, but not in normal epithelial cells. Scavenging ROS resulted in inhibition of ER stress and partially restored cell viability in curcumin-treated cancer cells. Collectively, these observations show that curcumin promotes ER stress-mediated apoptosis in cervical cancer cells through increase of cell type-specific ROS generation. Therefore, modulation of these differential responses to curcumin between normal and cervical cancer cells could be an effective therapeutic strategy without adverse effects on normal cells.

Genome-wide QTL analysis of meat quality-related traits in a large F2 intercross between Landrace and Korean native pigs.

BACKGROUND: We conducted a genome-wide linkage analysis to identify quantitative trait loci (QTL) that influence meat quality-related traits in a large F2 intercross between Landrace and Korean native pigs. Thirteen meat quality-related traits of the m. longissimus lumborum et thoracis were measured in more than 830 F2 progeny. All these animals were genotyped with 173 microsatellite markers located throughout the pig genome, and the GridQTL program based on the least squares regression model was used to perform the QTL analysis. RESULTS: We identified 23 genome-wide significant QTL in eight chromosome regions (SSC1, 2, 6, 7, 9, 12, 13, and 16) (SSC for Sus Scrofa) and detected 51 suggestive QTL in the 17 chromosome regions. QTL that affect 10 meat quality traits were detected on SSC12 and were highly significant at the genome-wide level. In particular, the QTL with the largest effect affected crude fat percentage and explained 22.5% of the phenotypic variance (F-ratio = 278.0 under the additive model, nominal P = 5.5 × 10(-55)). Interestingly, the QTL on SSC12 that influenced meat quality traits showed an obvious trend for co-localization. CONCLUSIONS: Our results confirm several previously reported QTL. In addition, we identified novel QTL for meat quality traits, which together with the associated positional candidate genes improve the knowledge on the genetic structure that underlies genetic variation for meat quality traits in pigs.

Genome-wide association analysis to identify SNP markers affecting teat numbers in an F2 intercross population between Landrace and Korean native pigs.

Most reproductive traits have low heritability and are greatly affected by environmental factors. Teat number and litter size are traits related to the reproduction ability of pigs. To identify quantitative trait loci (QTLs) for teat number traits, a genome-wide association study (GWAS) was conducted using an F2 intercross between Landrace and Korean native pigs. Genotype analysis was performed using the porcine SNP 60 K beadchip. The GWAS was performed using a mixed-effects model and linear regression approach. When a genome-wide threshold was determined using the Bonferroni method (P = 1.61 × 10(-6)), 38 single nucleotide polymorphism (SNP) markers in pig chromosome 7 (SSC7) were significantly associated with three teat number traits (total teat number, left teat number, and right teat number). Among these, SNPs in 5 genes (HDDC3, LOC100156276, LOC100155863, ANPEP, SCAMP2) were selected for further study based primarily on their statistical significance. A significant association was detected in SCAMP2 g.25280 G>A for total teat number (P = 2.0 × 10(-12)), HDDC3 g.1319 G>A SNP for left teat number (P = 2.3 × 10(-7)), and SCAMP2 g.14198 G>A for right teat number (P = 4.7 × 10(-12)). These results provide valuable information about the selective breeding for desirable teat numbers in pigs.

QTL analysis of clinical-chemical traits in an F2 intercross between Landrace and Korean native pigs.

Clinical-chemical traits are essential when examining the health status of individuals. The aim of this study was to identify quantitative trait loci (QTL) and the associated positional candidate genes affecting clinical-chemical traits in a reciprocal F(2) intercross between Landrace and Korean native pigs. Following an overnight fast, 25 serum phenotypes related to clinical-chemical traits (e.g., hepatic function parameters, renal function parameters, electrolyte, lipids) were measured in >970 F(2) progeny. All experimental samples were subjected to genotyping analysis using 165 microsatellite markers located across the genome. We identified eleven genome-wide significant QTL in six chromosomal regions (SSC 2, 7, 8, 13, 14, and 15) and 59 suggestive QTL in 17 chromosomal regions (SSC 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, and 18). We also observed significant effects of reciprocal crosses on some of the traits, which would seem to result from maternal effect, QTL on sex chromosomes, imprinted genes, or genetic difference in mitochondrial DNA. The role of genomic imprinting in clinical-chemical traits also was investigated. Genome-wide analysis revealed a significant evidence for an imprinted QTL in SSC4 affecting serum amylase levels. Additionally, a series of bivariate linkage analysis provided strong evidence that QTL in SSC 2, 13, 15, and 18 have a pleiotropic effect on clinical-chemical traits. In conclusion, our study detected both novel and previously reported QTL influencing clinical-chemical traits in pigs. The identified QTL together with the positional candidate genes identified here could play an important role in elucidating the genetic structure of clinical-chemical phenotype variation in humans and swine.

A missense mutation (c.1963A

Serum Ca(++) levels play important roles in the humoral immunity. The aim of this study was to detect quantitative trait loci and the associated positional candidate genes affecting baseline serum Ca(++) concentrations. A genome-wide association study was conducted in an F(2) intercross population between Landrace and Korean native pigs using the porcine single nucleotide polymorphism (SNP) 60 K beadchip and the PLINK program based on linear regression. Data used in the study included 410 F(2) pigs. All experimental animals were genotyped with 36,613 SNP markers located throughout the pig autosomes. We identified a strong association between a SNP marker on chromosome 7 and serum Ca(++) levels (DIAS0002191, genomic control-corrected P = 7.7 × 10(-5)). The position of DIAS0002191 was closely located to SLA class III region containing the C2 gene encoding the complementary component 2 protein, a protein which is important in the humoral immune responses. De novo sequencing of the porcine C2 gene revealed a missense mutation [c.1963A

Suicide on YouTube:Factors engaging viewers to a selection of suicide-themed videos.

Visual social media platforms can function as both facilitators and intervenors of concerning behaviors. This study focused on one of the health concerns worldwide, a leading cause of death related to mental health-suicide-in the context of a dominant visual social media platform, YouTube. This study employed content analysis method to identify the factors predicting viewer responses to suicide-themed content from the perspectives of who's, what's, and how's of suicide-themed videos. The results of the hierarchical multiple regression showed that the characteristics of content provider and content expression were more significant predictors of viewer engagement than were the characteristics of the message. These findings have implications for not only platform service providers but also diverse groups of individuals who participate in online discussions on suicide. YouTube has the potential to function as a locus for open discussion, education, collective coping, and even the diagnosis of suicidal ideation.

Reduced expression and homozygous deletion of annexin A10 in gastric carcinoma.

Annexins (ANXs) are a family of calcium and phospholipid binding proteins that have been implicated in diverse important biological and physiological process. ANX A10 (ANXA10) is a member of this family, though little is known about its functions. In the present study, array based comparative genomic hybridization (CGH) was used to screen DNA copy number change in gastric cancer cell lines and the results obtained were compared with oligonucleotide microarray data. DNA loss of the ANXA10 locus in chromosome 4q33 was found in several gastric cancer cell lines by array based CGH and these cell lines showed decreased ANXA10 expression by oligonucleotide microarray analysis. Functional analysis using siRNA and full-length cDNA transfection in gastric cancer cell lines demonstrated that ANXA10 regulates gastric cancer cell proliferation. Of the 585 primary gastric carcinoma tissues examined, ANXA10 expression at the protein level was found to be reduced in 289 (49.4%) cases. Quantitative real-time PCR analysis validated loss of DNA at the ANXA10 locus in gastric carcinomas with reduced ANXA10 expression. By univariate survival analysis, lack of ANXA10 expression was associated with poor survival (p = 0.016). These results suggest that ANXA10 inactivation by chromosomal deletion may play a role during gastric cancer progression.

Comparative analysis of DNA methylation between primary and metastatic gastric carcinoma.

Metastasis is a multi-step process involving many biomolecular changes and DNA methylation is one such molecular change. Although differences in DNA methylation have been reported in matched primary and metastatic mammary carcinoma, no such differences have been reported in gastric carcinoma. Accordingly, to investigate whether DNA methylation profiles in metastatic gastric carcinoma differ from those of their primary counterparts, we investigated the DNA methylation of eleven genes, ADAM23, CDH1, FHIT, FLNC, GSTP1, ITGA4, LOX, RUNX3, THBS1, TIMP3, and UCHL1 in 74 matched human primary gastric carcinomas, lymph node metastases, non-neoplastic gastric mucosal, and uninvolved lymph node tissues by utilizing methylation-specific PCR. Seven of these genes (ADAM23, FLNC, ITGA4, LOX, RUNX3, TIMP3, and UCHL1) showed cancer-specific methylation, and three (CDH1, FHIT, and THBS1) showed cancer-unrelated methylation. GSTP1 was rarely methylated in any tissue type. Of the seven genes that showed cancer-specific methylation, FLNC was more frequently methylated in metastatic gastric carcinomas than in their primary counterparts (p=0.004). In addition, the average number of methylated genes in metastatic tumors was greater than that in primary tumors (p=0.004). The high-methylation group (cases with three or more genes methylated in primary tumors) was found to contain more women (p=0.031) and diffuse type tumors by Lauren classification (p=0.022). DNA methylation profiles were not found to affect prognosis. We suggest that promoter methylation of FLNC may be involved in the lymph node metastasis of gastric carcinoma and that the DNA methylation statuses of metastatic tumors should be considered in node-positive gastric carcinoma.

Mobile Health Management Platform-Based Pulmonary Rehabilitation for Patients With Non-Small Cell Lung Cancer: Prospective Clinical Trial.

BACKGROUND: Lung cancer patients experience various symptoms during treatment. Although pulmonary rehabilitation is an effective way to improve these symptoms, a medical environment of limited availability makes it difficult to provide seamless and adequate rehabilitation for lung cancer patients. OBJECTIVE: This study aimed to investigate the effects of a personalized pulmonary rehabilitation program using real-time mobile patient health data for patients with non-small cell lung cancer. METHODS: We conducted a prospective clinical trial in 64 patients with non-small cell lung cancer aged between 20 and 80 years at a large tertiary hospital in Seoul, South Korea. A 12-week personalized pulmonary rehabilitation program, called efil breath, was administered to determine the effectiveness of the newly developed rehabilitation app. Participants were randomly allocated to the fixed exercise or fixed-interactive exercise group (which received the personalized program). We measured changes in 6-minute walk distance (6MWD) and dyspnea (modified Medical Research Council [mMRC] score) at 6 weeks; and quality of life and service satisfaction at 12 weeks. We used the paired t test to analyze the variables. RESULTS: Patients used the newly developed mobile health pulmonary rehabilitation app and a real-time patient monitoring website. In all participants, significant changes were observed in 6MWD at 12 weeks from a mean of 433.43m (SD 65.60) to 471.25m (SD 75.69; P=.001), and mMRC from a mean score of 0.94 (0.66) to 0.61 (SD 0.82; P=.02). The intervention significantly improved their quality of life (EuroQol-visual analog scale [EQ-VAS]) compared with baseline (mean score 76.05, SD 12.37 vs 82.09, SD 13.67, respectively; P=.002). CONCLUSIONS: A personalized mobile health-based pulmonary rehabilitation app for recording and monitoring real-time health data of patients with non-small cell lung cancer can supplement traditional health care center-based rehabilitation programs. This technology can encourage improvement of physical activity, dyspnea, and quality of life.

An mHealth Management Platform for Patients with Chronic Obstructive Pulmonary Disease (efil breath): Randomized Controlled Trial.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is one of the major morbidities in public health, and the use of mHealth technology for rehabilitation of patients with COPD can help increase physical activity and ameliorate respiratory symptoms. OBJECTIVE: This study aimed to develop a comprehensive rehabilitation management platform to improve physical activity and quality of life in patients with COPD. METHODS: The study comprised the following 2 stages: (1) a pilot stage in which a prototype app was developed; and (2) a fully-fledged platform development stage in which 2 apps and 1 COPD patient monitoring website were developed. We conducted a randomized clinical trial to investigate the efficacy of the apps developed in the second stage of the study. In addition, two 12-week exercise regimens (fixed and fixed-interactive) were tested for the trial. The clinical parameters of the respiratory function and patient global assessment (PGA) of the app were obtained and analyzed. Notably, Android was the chosen operating system for apps. RESULTS: We developed 2 COPD rehabilitation apps and 1 patient monitoring website. For the clinical trial, 85 patients were randomized into the following 3 groups: 57 were allocated to the 2 intervention groups and 28 to the control group. After 6 weeks, the COPD assessment test scores were significantly reduced in the fixed group (P=.01), and signs of improvement were witnessed in the fixed-interactive group. In addition, the PGA score was moderate or high in all aspects of the user experience of the apps in both intervention groups. CONCLUSIONS: A well-designed mobile rehabilitation app for monitoring and managing patients with COPD can supplement or replace traditional center-based rehabilitation programs and achieve improved patient health outcomes. TRIAL REGISTRATION: ClinicalTrials.gov NCT03432117; https://clinicaltrials.gov/ct2/show/NCT03432117 (Archived by WebCite at http://www.webcitation.org/71Yp0P64a).

Lytic induction and apoptosis of Epstein-Barr virus-associated gastric cancer cell line with epigenetic modifiers and ganciclovir.

Epstein-Barr virus (EBV) is an oncogenic herpes virus. EBV gene transcription is regulated by an epigenetic mechanism to establish a persistent infection and to evade the host immune system. We found that low concentrations of epigenetic modifying agents, 5-aza-2'-deoxycytidine (5-aza-CdR) or trichostatin A (TSA), induced the expression of BMRF1, BZLF1, and BRLF1 genes, which are found in the lytic form of the virus, in an EBV-positive gastric cancer cell line. This effect did not involve PI3 kinase, MAP/ERK kinase, protein kinase C delta, or p38 MAPK signaling pathway. The cytotoxic effect of ganciclovir (GCV) was enhanced after the lytic induction by epigenetic modifiers, and the combination of GCV and epigenetic modifiers induced apoptosis, which is dependent on caspases. In conclusion, the combination of GCV with 5-aza-CdR or TSA might be a useful therapeutic strategy for EBV-induced human gastric cancer.

Evaluation of HER-2 gene status in gastric carcinoma using immunohistochemistry, fluorescence in situ hybridization, and real-time quantitative polymerase chain reaction.

HER-2 gene amplification and the overexpression of HER-2 protein have been observed in various solid tumors, including gastric carcinomas. HER-2 gene amplification has attracted research attention since the development of the new therapeutic agent trastuzumab. Here, we evaluated HER-2 status in the surgically resected tissues of 248 gastric carcinoma cases using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and real-time quantitative polymerase chain reaction (q-PCR) and compared the results. In addition, we compared clinicopathologic characteristics with the presence of HER-2 gene amplification and with protein overexpression. Among the 248 cases, 56 (22.6%) cases showed HER-2 overexpression (2+ or 3+) by IHC and 19 cases (7.7%) showed HER-2 gene amplification by FISH. Four (2.1%) of the 192 cases negative (0 or 1+) by IHC showed amplification by FISH, whereas 15 (26.8%) of the 56 cases with HER-2 protein overexpression showed HER-2 amplification by FISH. The correlation between IHC and FISH results was statistically significant (P < .001). HER-2 protein overexpression and HER-2 gene amplification were common in cases with a well- or moderately differentiated histology according to the World Health Organization classification (P < .001) and in cases of intestinal type by the Lauren classification (P < .001). Real-time q-PCR results showed that calculated HER-2/GAPDH ratios were higher in amplified cases with 100.0% sensitivity and 96.9% specificity using FISH results as the standard. Measurements of HER-2 expression by FISH and real-time q-PCR and of HER-2 protein by IHC were found to be highly concordant at determining HER-2 status in gastric carcinoma.

Role and prognostic significance of proapoptotic proteins in Epstein-Barr virus-infected gastric carcinomas.

BACKGROUND: The purposes of the present study were to evaluate the role and the prognostic values of proapoptotic proteins involved in the death receptors (Fas and TRAIL receptors) and mitochondrial pathways (Bax) in Epstein-Barr virus (EBV)-infected gastric carcinomas. MATERIALS AND METHODS: Fifty-five EBV-infected gastric carcinomas were identified by in situ hybridization for EBV-encoded small RNAs. Immunohistochemistry was performed for Fas, Fas-ligand, FADD, TRAIL, DR4, DR5 and Bax. Apoptotic indices (AIs) were determined using TUNEL assay and assessed. RESULTS: No remarkable differences in protein expressions were observed between EBV-infected gastric carcinomas and conventional gastric carcinomas. Bax positivity tended to be associated with higher AI (p = 0.068), whereas Fas and FADD positivities were related to lower AI (p = 0.006 and 0.059, respectively). Proteins involved in TRAIL pathways showed no statistical significant relationship with AI. TNM stage and Fas and FADD expressions were related to overall survival (p < 0.05), but TNM stage was the only independent prognostic factor. CONCLUSION: Apoptosis in EBV-infected gastric carcinomas probably occurs via the mitochondrial pathway through Bax, rather than via the death receptor pathways. Fas and FADD expressions, and pathological tumor stage (TNM stage) may be the prognostic factors.

Expression of E-cadherin and beta-catenin in the adenoma-carcinoma sequence of ampulla of Vater cancer.

BACKGROUND/AIMS: Ampullary carcinoma is uncommon but provides a good model for adenoma-carcinoma sequence. During the adenoma-carcinoma transition, the tumor cells should acquire the ability to invade. The E-cadherin-catenin complex connects the adjacent epithelial cells at the zona adherens, and this adhesion interferes with the tumor cell invasion. METHODOLOGY: 111 cases of ampullary carcinoma were investigated with E-cadherin and beta-catenin expression with immunohistochemistry and the result was compared with their clinicopathologic and survival results. Forty-nine (44.1%) cases were associated with adenomatous component. RESULTS: Expressional loss of E-cadherin was detected in 3 (6.1%) adenomas and 73 (65.8%) carcinomas, and the expressional loss was significantly associated with tumor cell differentiation (p<0.05) and survival (p<0.05) in carcinoma. In beta-catenin immunostaining, 4 (8.2%) adenomas and 45 (40.5%) carcinomas showed abnormal staining patterns either as nuclear staining or as a loss of membrane staining. The cases with membranous loss of beta-catenin expression were correlated with poor survival rate. CONCLUSIONS: Alteration of E-cadherin and beta-catenin is a late event during the adenoma-carcinoma sequence in ampullary neoplasms, and the loss of membranous expression of both E-cadherin and beta-catenin is closely correlated with less differentiated histology and poor prognosis.

Energy Expenditures for Activities of Daily Living in Korean Young Adults: A Preliminary Study.

OBJECTIVE: To investigate the energy expenditure (EE) of Korean young adults based on activities refined to a deskbound lifestyle. METHODS: Sixty-four healthy office workers aged between 25 and 46 years participated in this study. EE was expressed as metabolic equivalent of task (MET). Participants were evaluated in terms of their EE during physical activities of sleeping (n=22), typing (n=37), folding laundry (n=34), dishwashing (n=32), studying (n=18), mopping (n=35), walking (n=33), stair climbing (n=23), and running (n=29). Volume of oxygen consumption was measured by indirect calorimetry K4b(2) (COSMED). The results were compared to the established Compendium MET. RESULTS: The MET of activities were: sleeping, 1.24±0.43; typing, 1.35±0.25; folding laundry, 1.58±0.51; dishwashing, 2.20±0.51; studying, 2.11±0.90; mopping, 2.72±0.69; walking at 4 km/hr, 3.48±0.65; stair climbing of five stories, 6.18±1.08; and running at 8 km/hr, 7.57±0.57. The values of typing and mopping were similar to those in the Compendium, whereas those of sleeping, folding laundry, dishwashing, studying, walking, stair climbing and running were different. CONCLUSION: To our knowledge, this estimation of EE in MET during activities of daily living is the first data of young adults in Korea. These data could be used as a reference to modify the guidelines of physical activities for the age group examined in this study.

Cell-cycle regulators, bcl-2 and NF-kappaB in Epstein-Barr virus-positive gastric carcinomas.

The mechanism by which Epstein-Barr virus (EBV) contributes to the carcinogenesis of gastric mucosa remains unanswered. In this study, the role of cell-cycle regulators (p53, p21, p27, p16, cyclin D1, Rb), bcl-2 and NF-kappaB p65 (Rel A) was evaluated. Immunohistochemistry for these proteins was performed in EBV-positive (n=55) and EBV-negative gastric carcinomas (n=72). The bcl-2 protein by western blot and EBV transcripts using RT-PCR were studied in cell lines. The p27 loss, p16 loss, cyclin D1 expression and NF-kappaB nuclear positivity were more frequent in EBV-positive gastric carcinomas than those in EBV-negative gastric carcinomas, while p53 overexpression seldom occurred in EBV-positive carcinomas (p<0.001). EBV-positive gastric carcinoma showed unique p53 immunostaining (heterogeneous, weak to moderate, focal staining), and rare bcl-2 positivity (1 case). Western blot showed bcl-2 to be irrespective of EBV status in stomach cancer cell lines. However, bcl-2 was highly expressed in EBV-positive lymphoma or EBV-positive lymphoblastoid cell lines. The BARF1 transcript was confirmed in both EBV-positive stomach cancer and EBV-positive lymphoma, suggesting tissue type-specific bcl-2 activation by BARF1. The pathological tumor stage was the only independent prognostic factor. A small size of tumor, p16 preservation and NF-kappaB nuclear positivity were associated with a good prognosis in univariate analysis (p<0.05). p27, p16, cyclin D1 and NF-kappaB may be associated with oncogenesis in EBV-positive gastric carcinomas. EBV-positive gastric carcinomas showed infrequent p53 overexpression, wild-type p53 stabilization and rare bcl-2 involvement. The characteristic expression of proteins may relate to both EBV and tissue type.

Expression of family A melanoma antigen in human gastric carcinoma.

BACKGROUND: Since the members of the MAGE (melanoma antigen) gene family have been reported to be expressed in tumor cells but not in normal tissues, they have been considered as targets for tumor-specific immunotherapy. MATERIALS AND METHODS: The expression pattern of MAGE-A genes and their expression mechanisms were investigated in 10 gastric cancer cell lines and 1,097 gastric carcinoma specimens by RT-PCR, IHC, Western blot and MSP. RESULTS: MAGE-A1, -A2 and -A3 gene transcripts were detected in 1, 3 and 4 of 10 gastric cancer cell lines, respectively. In those cases in which the mRNA expression of MAGE-A2 or -A3 was detected, the promoters of the corresponding genes were hypomethylated. MAGE-A protein expression was detected in 30% (3/10) of the cell lines and 15.8% (173 out of 1,097) of the carcinoma specimens. Promoter hypomethylation of the MAGE-A2 or -A3 genes correlated with their expression in primary gastric cancer tissue and gastric cancer cell lines. MAGE-A protein expression was associated with tumor invasiveness (p=0.002), lymph node metastasis (p<0.001), advanced pathologic stage (p<0.001) and a worse prognosis (p<0.005). CONCLUSION: MAGE-A protein expression occurred due to promoter hypomethylation in a minor subset of gastric cancers, and MAGE-A expression increased during the progression of the gastric cancer.

Prognostic significance of glutathione peroxidase 1 (GPX1) down-regulation and correlation with aberrant promoter methylation in human gastric cancer.

BACKGROUND: This study aimed at examining the association of gene silencing and promoter methylation of glutathione peroxidase 1 (GPX1) and glutathione peroxidase 3 (GPX3) in gastric cancer cells and determined the clinical significance of GPX1 and GPX3 expression loss in gastric cancer tissue. MATERIALS AND METHODS: Analysis of mRNA expression was carried out by reverse transcription-polymerase chain reaction (RT-PCR). Methylation of the GPX1 promoter region was analyzed by bisulfite sequencing, and that of the GPX3 promoter region was analyzed by methylation-specific PCR (MSP). Tissue microarray-based immunohistochemistry of GPX1 and GPX3 in 1,163 resected gastric cancer specimens was performed to assess the associations with clinicopathological parameters. RESULTS: Reduced GPX1 and GPX3 mRNA expression was associated with promoter methylation in gastric cancer cell lines. A correlation between DNA promoter methylation and loss of GPX1 expression was noted in 16 gastric cancer tissue samples (p=0.005). Loss of GPX1 and GPX3 proteins was found in 24.4% and 30.8% of gastric cancer tissues. Loss of GPX1 expression was significantly associated with advanced gastric cancer (p=0.039) and lymphatic invasion (p=0.010); loss of GPX3 expression was associated with advanced gastric cancer (p<0.001) and lymph node metastasis (p<0.001). Kaplan-Meier analysis showed that low expression of GPX1 was associated with poor cancer-specific survival (p=0.010). CONCLUSION: Data from this study implicate aberrant hypermethylation of promoter regions of GPX1 and GPX3 as a mechanism for down-regulation of GPX1 and GPX3 mRNA expression in gastric cancer cells. Loss of GPX1 expression was associated with aggressiveness and poor survival in patients with gastric cancer.