Role of Gasdermin E in the Biogenesis of Apoptotic Cell-Derived Exosomes.

The gasdermins are a family of pore-forming proteins that has recently been suggested to play a central role in pyroptosis. In this study, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell-derived exosomes. In apoptotic human HeLa and HEK293 cells, GSDMA, GSDMC, GSDMD, and GSDME increased the release of apoptotic exosomes. GSDMB and DFNB59, in contrast, negatively affected the release of apoptotic exosomes. GSDME at its full-length and cleaved forms was localized in the exosomes and exosomal membrane. Full-length and cleaved forms of GSDME are suggested to increase Ca2+ influx to the cytosol through endosomal pores and thus increase the biogenesis of apoptotic exosomes. In addition, the GSDME-mediated biogenesis of apoptotic exosomes depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins, that is, annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of apoptotic exosomes begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that GSDME-bearing tumors released apoptotic exosomes to induce inflammatory responses in the in vivo mouse 4T1 orthotropic model of BALB/c breast cancer. The data indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

Update on CD164 and LMX1A genes to strengthen their causative role in autosomal dominant hearing loss.

Novel hearing loss (HL) genes are constantly being discovered, and evidence from independent studies is essential to strengthen their position as causes of hereditary HL. To address this issue, we searched our genetic data of families with autosomal dominant HL (ADHL) who had been tested with high-throughput DNA sequencing methods. For CD164, only one pathogenic variant in one family has so far been reported. For LMX1A, just two previous studies have revealed its involvement in ADHL. In this study we found two families with the same pathogenic variant in CD164 and one family with a novel variant in LMX1A (c.686C>A; p.(Ala229Asp)) that impairs its transcriptional activity. Our data show recurrence of the same CD164 variant in two HL families of different geographic origin, which strongly suggests it is a mutational hotspot. We also provide further evidence for haploinsufficiency as the pathogenic mechanism underlying LMX1A-related ADHL.

Natural course of residual hearing preservation with a slim, modiolar cochlear implant electrode array.

PURPOSE: Understanding residual hearing preservation and its natural course following cochlear implantation is important for developing rehabilitation strategies for hearing loss. However, non-uniform evaluation criteria and varying surgical skills pose challenges in fair comparison of the effect of different electrodes on residual hearing preservation. We compared the effect of a slim modiolar electrode (SME) and a slim straight electrode (SSE), implanted by a single surgeon, on progression of residual hearing using different parameters, based on cross-sectional and longitudinal audiological analyses. METHODS: Patients with preoperative low-frequency pure-tone average (LFPTA) ≤85 dB at 250 and 500 Hz and who underwent minimally traumatic surgical techniques were included. The progression of residual hearing using threshold shifts, hearing preservation rate according to the HEARRING classification, and maintenance of functional low-frequency hearing potentially qualifying for a hybrid stimulation was analyzed up to five time points throughout the 1-year follow-up period. RESULTS: Threshold shifts and hearing preservation rates according to the HEARRING classification of the electrodes were comparable from 3 months through 12 months postoperatively. Maintenance of functional low-frequency hearing, required for the usage of a hybrid stimulation, was similar for both electrodes. A substantial proportion of implantees with SME use a hybrid stimulation, resulting in long-term use. However, a difference in the pattern of postoperative residual hearing preservation between the two electrodes is possible, probably due to differences in their physical characteristics and location. Specifically, correlation analysis exhibited that significantly less tight modiolar proximity negatively affect the residual hearing preservation, albeit only at 3 months postoperatively, among patients with the SME. CONCLUSION: Collectively, both SME and SSE implantation showed favorable residual hearing preservation. Our findings further refine the recently proposed hearing preservation with the SME and suggest that the physical characteristics and location of electrodes, in terms of electrode-to-modiolus distance, could affect loss of acoustic hearing in various ways.

Molecular aetiology of ski-slope hearing loss and audiological course of cochlear implantees.

PURPOSE: A challenge for patients with ski-slope hearing loss is that hearing aids do not adequately amplify the mid-to-high frequencies necessary for speech perception and conversely, cochlear implant (CI) may damage low-frequency hearing. We aimed to describe the clinical profile of patients with ski-slope hearing loss, with a special focus on aetiology of such hearing loss and audiological course of low-frequency hearing after CI. METHODS: We recruited hearing-impaired patients who visited a tertiary referral centre and met the criteria for ski-slope hearing loss patients from 2015 to 2021. Genetic testing was performed in all ski-slope hearing loss patients unless refused. Baseline audiograms of patients who continued to use hearing aids or who finally underwent CIs were reviewed. As for CI patients, outcome and hearing preservation rate were rigorously analysed. RESULTS: Of 46 recruited patients with ski-slope hearing loss, 45 agreed to undergo genetic testing and causative variants were identified in 17 (37.8%) patients. The TMC1, MYO7A, and TMPRSS3 variants were the most common, while LRTOMT was newly identified as a causative gene. Twenty-five patients eventually received CI, while 13 continued to wear the hearing aid and 8 patients did not ever try hearing aids. CI in ski-slope hearing loss led to immediate and sufficient improvement of sentence recognition by as early as 3 months, however, the duration of hearing loss was inversely correlated with the sentence recognition score. The average hearing preservation rate (using the HEARRING classification) after CI was 53.0% (SD 30.0) and 45.6% (SD 31.1) at 1 year. Seventy-nine percent of implantees maintained functional low-frequency hearing (better than 85 dB at 250 and 500 Hz) eligible for electric-acoustic stimulation (EAS). A trend was found that patients with hair cell stereocilia-associated genetic variants may have a slightly better preservation, albeit with no statistical significance. CONCLUSION: Detection rate of a molecular genetic aetiology of ski-slope hearing loss appears to be lower than other type of hearing loss reported in the literature. Especially with short hearing loss duration, CI in ski-slope hearing loss leads to immediate and sufficient speech improvement, while preserving functional low-frequency hearing eligible for EAS as many as in 79%. A certain genetic aetiology might be associated with a trend towards better low-frequency hearing preservation.

AICAR upregulates ABCA1/ABCG1 expression in the retinal pigment epithelium and reduces Bruch's membrane lipid deposit in ApoE deficient mice.

The etiology of age-related macular degeneration (AMD) is diverse; however, recent evidence suggests that the lipid metabolism-cholesterol pathway might be associated with the pathophysiology of AMD. The ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1, are essential for the formation of high-density lipoprotein (HDL) and the regulation of macrophage cholesterol efflux. The failure of retinal or retinal pigment epithelium (RPE) cholesterol efflux to remove excess intracellular lipids causes morphological and functional damage to the retina. In this study, we investigated whether treatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMP-activated protein kinase (AMPK) activator, improves RPE cholesterol efflux and Bruch's membrane (BM) lipid deposits. The protein and mRNA levels of ABCA1 and ABCG1 in ARPE-19 cells and retinal and RPE/choroid tissue from apolipoprotein E-deficient (ApoE-/-) mice were evaluated after 24 weeks of AICAR treatment. The cholesterol efflux capacity of ARPE-19 cells and the cholesterol-accepting capacity of apoB-depleted serum from mice were measured. The thickness of the BM and the degree of lipid deposition were evaluated using electron microscopy. AICAR treatment increased the phosphorylation of AMPK and the protein and mRNA expression of ABCA1 and ABCG1 in vitro. It promoted cholesterol efflux from ARPE-19 cells and upregulated the protein and mRNA levels of ABCA1 and ABCG1 in the retina and RPE in vivo. ApoB-depleted serum from the AICAR-treated group showed enhanced cholesterol-accepting capacity. Long-term treatment with AICAR reduced BM thickening and lipid deposition in ApoE-/- mice. In conclusion, AICAR treatment increased the expression of lipid transporters in the retina and RPE in vivo, facilitated intracellular cholesterol efflux from the RPE in vitro, and improved the functionality of HDL to accept cholesterol effluxed from the cell, possibly via AMPK activation. Collectively, these effects might contribute to the improvement of early age-related pathologic changes in the BM. Pharmacological improvement of RPE cholesterol efflux via AMPK activation may be a potential treatment strategy for AMD.

Arsenic trioxide attenuates STAT-3 activity and epithelial-mesenchymal transition through induction of SHP-1 in gastric cancer cells.

BACKGROUND: We investigated the effect of arsenic trioxide (ATO) for inhibition of signal transducer and activator of transcription 3 (STAT3) and epithelial-mesenchymal transition (EMT) in gastric cancer cells, and the role of SH2 domain-containing phosphatase-1 (SHP-1) during this process. METHODS: We used AGS cells, which showed minimal SHP-1 expression and constitutive STAT3 expression. After treatment of ATO, cellular migration and invasion were assessed by using wound closure assay, Matrigel invasion assay and 3-D culture invasion assay. To validate the role of SHP-1, pervanadate, a pharmacologic phosphatase inhibitor, and SHP-1 siRNA were used. Xenograft tumors were produced, and ATO or pervanadate were administered via intraperitoneal (IP) route. RESULTS: Treatment of ATO 5 and 10 µM significantly decreased cellular migration and invasion in a dose-dependent manner. Western blot showed that ATO upregulated SHP-1 expression and downregulated STAT3 expression, and immunofluorescence showed upregulation with E-cadherin (epithelial marker) and downregulation of Snail1 (mesenchymal marker) expression by ATO treatment. Anti-migration and invasion effect and modulation of SHP-1/STAT3 axis by ATO were attenuated by pervanadate or SHP-1 siRNA. IP injection of ATO significantly decreased the xenograft tumor volume and upregulated SHP-1 expression, which were attenuated by co-IP injection of pervanadate. CONCLUSION: Our data suggest that ATO inhibits STAT3 activity and EMT process by upregulation of SHP-1 in gastric cancer cells.

Epigenetic regulation and anti-tumorigenic effects of SH2-containing protein tyrosine phosphatase 1 (SHP1) in human gastric cancer cells.

SH2-containing protein tyrosine phosphatase 1 (SHP1) is an important negative regulator in cytokine-mediated signal transduction and cell cycling. Recent studies have demonstrated that SHP1 promoter methylation is frequently observed in gastric adenocarcinoma tissues. In this in vitro study, we attempted to reveal promoter hypermethylation and to investigate effects of SHP1 in gastric carcinoma cell lines. We observed that both gene and protein expression of SHP1 were negative in 8 of 10 gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-638, SNU-719, MKN-28, MKN-45, AGS). Methylation-specific PCR (MSP) showed a methylation-specific band only in the 10 gastric cancer lines. Bisulfite pyrosequencing in AGS, MKN-28, and SNU-719 cells indicated that methylation frequency was as high as 94.4, 92.6, and 94.5 %, respectively, in the three cell lines. Treatment of SNU-719, MKN-28, and AGS cells with 5-Aza-2'-deoxycytidine (5-Aza-dc) led to re-expression of SHP1 in these cells. Introduction of exogenous SHP1 in SNU-719 and MKN-28 cells by transient transfection substantially downregulated protein expression of constitutive phosphor-Janus kinase 2 (JAK2) (tyrosine 1007/1008) and phosphor-signal transducers and activators of transcription 3 (STAT3) (tyrosine 705), which in turn decreased expression of STAT3 target genes including those encoding cyclin D1, MMP-9, VEGF-1, and survivin. Induction of SHP1 significantly inhibited cell proliferation, migration and invasion in SNU-719 and MKN-28 cells. Taken together, epigenetic silencing of SHP1 is frequently caused by promoter hypermethylation in gastric carcinoma cells. Overexpression of SHP1 downregulates the JAK2/STAT3 pathway to modulate various target genes and inhibit cell proliferation, migration, and invasion in gastric cancer cells.

The roles of HOXB7 in promoting migration, invasion, and anti-apoptosis in gastric cancer.

BACKGROUND AND AIM: The aim of this study was to compare HOXB7 expression level between gastric cancer and non-cancerous gastric tissues. Additionally, the functional effects of HOXB7, including its pro-migration or invasion and anti-apoptosis roles, were evaluated in gastric cancer cells. METHODS: Both gene and protein expression levels of HOXB7 were examined in gastric cancer cell lines, and HOXB7 expression was compared between primary or metastatic gastric cancer tissues and chronic gastritis or intestinal metaplasia tissues. Functional studies included a wound healing assay, a Matrigel invasion assay, and an Annexin-V assay were performed, and Akt/PTEN activity was measured by western blotting. RESULTS: Both gene and protein expression levels of HOXB7 could be clearly detected in various gastric cancer cell lines except MKN-28 cell. HOXB7 expression was significantly higher in primary or metastatic gastric cancer tissues than in chronic gastritis or intestinal metaplasia tissues. HOXB7 knockdown led to inhibition of cell invasion and migration, had an apoptotic effect, downregulated phosphor-Akt, and upregulated PTEN in AGS and SNU-638 cells. Reinforced expression of HOXB7 caused the opposite effects in MKN-28 and MKN-45 cells. CONCLUSION: Our study suggests that HOXB7 has an oncogenic role in gastric cancer, which might be related to the modulation of Akt/PTEN activity to induce cell migration/invasion and anti-apoptotic effects.

Choroidal vascularity index of patients with coronary artery disease.

We investigated the changes in subfoveal choroidal thickness and choroidal vascularity index (CVI) and their relationship with the severity of coronary artery stenosis in patients with cardiovascular risk factors and symptoms suggestive of coronary artery disease (CAD). Ninety patients who underwent coronary angiography (CAG) for evaluation of their coronary artery status and cardiac symptoms were included. Forty-two patients showed no evidence of CAD; 31 patients had one to two vessel disease; and 17 had a triple vessel disease. There were no significant differences in the subfoveal choroidal thickness among the three groups; however, the CVI in the triple vessel disease group was lower than those in the other groups. The CVI values were good predictors of the presence of triple-vessel disease (p = 0.020). Multivariate logistic regression analysis results revealed that male sex (odds ratio 5.4, p = 0.049), hypertension (odds ratio 4.9, p = 0.017), and CVI (%, odds ratio 0.8, p = 0.016) were significant factors associated with the presence of triple vessel disease. Although CVI may not be a sensitive marker for detecting early changes in the coronary artery, it may be helpful in indicating severe CAD.

Efficacy of cochlear implants in children with borderline hearing who have already achieved significant language development with hearing aids.

There are still debates about timing and effectiveness of cochlear implants (CI) in pediatric subjects with significant residual hearing who do not belong to traditional indication of CI. In this study, we aimed to investigate the outcomes of CI, specifically on improvement of pronunciation, among hearing-impaired children already with a substantial degree of language skills as evaluated by Categories of Auditory Perception (CAP) scores or sentence score. Our cohort comprised pediatric CI recipients from July 2018 through October 2020. Among them, cases with CAP scores of 5 or 6 preoperatively were defined as "borderline cases". We investigated prevalence and etiologies, and compared speech evaluation data preoperatively and postoperatively at three time points (3, 6 and 9-12 months after implantation). Among 86 pediatric CI recipients, 13 subjects (15.12%) had language development that reached CAP scores of 5 or 6 before implantation. Postoperative speech evaluation data 6 months after implantation revealed significant improvement of pronunciation (Urimal Test of Articulation and Phonation scores: UTAP), Infant-Toddler Meaningful Auditory Integration Scale (IT-MAIS) and word perception scores, but not of CAP and sentence perception scores. Notably, the significant improvement of pronunciation based on UTAP scores outstripped that of other speech parameters and this continued steadily up to one-year postoperatively. The result of the study serves as evidence for what to expect from cochlear implantation in hearing-impaired children who have already achieved a substantial degree of language development in terms of CAP scores or sentence perception scores, preoperatively.

The spread of pandemic H1N1 2009 by age and region and the comparison among monitoring tools.

This report describes the pattern of the spread of the pandemic H1N1 2009 and compares 3 monitoring tools until the 57th week or January 31, 2010. The 1st week was from December 28th, 2008 to January 3rd, 2009. A total of 740,835 patients were reported to be infected with pandemic H1N1 2009 and 225 patients were reported to have died of pandemic H1N1 2009. The number of patients aged from 7 to 12 was the largest (183,363 patients in total) but the virus spread and then was suppressed most quickly among the children between 13 and 18. The region-determinant incidence of patients showed diverse patterns according to regions. The peak of the ILI per thousand was at the 45th week, the number of antiviral prescriptions reached its peak at the 44th week, and the peak based on reported patients was the 46th week. As of February 3 2010, the outbreak passed through the peak and has gradually subsided. Now it is time for the government and the academic world to review this outbreak, efficacy of vaccination, and further preparation and response for the next pandemic.

Inter-ocular and inter-visit differences in ocular biometry and refractive outcomes after cataract surgery.

This study aimed to determine whether inter-ocular differences in axial length (AL), corneal power (K), and adjusted emmetropic intraocular lens power (EIOLP) and inter-visit differences in these ocular biometric values, measured on different days, are related to refractive outcomes after cataract surgery. We retrospectively reviewed 279 patients who underwent phacoemulsification. Patients underwent ocular biometry twice (1-4 weeks before and on the day of surgery). Patients were divided into three groups: group S (similar inter-ocular biometry in different measurements; n = 201), group P (inter-ocular differences persisted in the second measurement; n = 37), and group D (inter-ocular difference diminished in the second measurement; n = 41). Postoperative refractive outcomes (mean absolute errors [MAEs]) were compared among the groups. Postoperative MAE2, based on second measurement with reduced inter-ocular biometry difference, was smaller than that calculated using the first measurement (MAE1) with borderline significance in group D (MAE1, 0.49 ± 0.45 diopters vs. MAE2, 0.41 ± 0.33 diopters, p = 0.062). Postoperative MAE2 was greater in group P compared to the other two groups (p = 0.034). Large inter-ocular biometry differences were associated with poor refractive outcomes after cataract surgery. These results indicate that measurements with smaller inter-ocular differences were associated with better refractive outcomes in cases with inter-visit biometry differences.

Author Correction: Inter-ocular and inter-visit differences in ocular biometry and refractive outcomes after cataract surgery.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Timeliness of national notifiable diseases surveillance system in Korea: a cross-sectional study.

BACKGROUND: With the increase of international travels, infectious disease control is gaining a greater importance across regional borders. Adequate surveillance system function is crucial to prevent a global spread of infectious disease at the earliest stage. There have been limited reports on the characteristics of infectious disease surveillance in Asia. The authors studied the timeliness of the Korean National Notifiable Disease Surveillance System with regard to major notifiable diseases from 2001 to 2006. METHODS: Six notifiable infectious diseases reported relatively frequently were included in this study. Five diseases were selected by the criteria of reported cases > 100 per year: typhoid fever, shigellosis, mumps, scrub typhus, and hemorrhagic fever with renal syndrome. In addition, dengue fever was also included to represent an emerging disease, despite its low number of cases. The diseases were compared for the proportion notified within the recommended time limits, median time lags, and for the cumulative distribution of time lags at each surveillance step between symptom onset and date of notification to the Korea Centers for Disease Control and Prevention (KCDC). RESULTS: The proportion of cases reported in time was lower for disease groups with a recommended time limit of 1 day compared with 7 days (60%-70% vs. > 80%). The median time from disease onset to notification to KCDC ranged between 6 and 20 days. The median time from onset to registration at the local level ranged between 2 and 15 days. Distribution of time lags showed that main delays arose in the time from onset to diagnosis. There were variations in timeliness by disease categories and surveillance steps. CONCLUSION: Time from disease onset to diagnosis generally contributed most to the delay in reporting. It is needed to promote public education and to improve clinical guidelines. Rapid reporting by doctors should be encouraged, and unification of recommended reporting time limit can be helpful. Our study also demonstrates the utility of the overall assessment of time-lag distributions for disease-specific strategies to improve surveillance.

Inhibition of STAT3 in gastric cancer: role of pantoprazole as SHP-1 inducer.

BACKGROUND: We investigated the inhibitory effect of pantoprazole on signal transducer and activator of transcription 3 (STAT3) activity and invasiveness of gastric adenocarcinoma cells, and the role of SH2-containing protein tyrosine phosphatase 1 (SHP-1) in mediating role. METHODS: We used AGS and MKN-28 cells because of reduced SHP-1 and preserved p-STAT3 expression. Western blot, wound closure assay, Matrigel invasion assay and 3-D culture invasion assay were performed. Pharmacologic inhibitor of SHP-1 and siRNA were used for validation of the role of SHP-1. RESULTS: We observed that pantoprazole at 40, 80, and 160 µg/ml upregulated SHP-1 and downregulated p-STAT3 expression in a dose-dependent manner in AGS and MKN-28 cells. Furthermore, pantoprazole significantly downregulated mesenchymal markers (Snail1 and vimentin), upregulated epithelial marker (E-cadherin), and inhibited migration and invasion of AGS and MKN-28 cells. To validate the role of SHP-1 in inhibition of STAT3 activity by pantoprazole in gastric cancer cells, we performed pharmacologic inhibition (pervanadate) or knockdown of SHP-1 before pantoprazole treatment, which significantly attenuated the suppression of p-STAT3 and anti-migration and invasion effect by pantoprazole in AGS cells. In xenograft tumor model, tumor volume was significantly reduced by intraperitoneal injection of pantoprazole, with upregulation of SHP-1 and downregulation of p-STAT3, which were attenuated by concomitant injection of pervanadate. CONCLUSION: Our data suggest that the inhibitory effect of pantoprazole on cellular migration and invasion might be through inducing SHP-1 in gastric cancer cells.

Antitumorigenic effect of plumbagin by induction of SH2-containing protein tyrosine phosphatase 1 in human gastric cancer cells.

A recent study reported that plumbagin downregulated the activity of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway to show various antitumor effects in multiple myeloma cells. We aimed in this in vitro study to demonstrate the inhibition of JAK2/STAT3 pathway by plumbagin through inducing SH2-containing protein tyrosine phosphatase 1 (SHP1) expression in the MKN-28 gastric cancer cell line. We performed western blot analysis to measure SHP1, phosphor-JAK2/STAT3 level, and observed that plumbagin induced SHP1 expression and simultaneously downregulated phosphor-JAK2/STAT3 in MKN-28 cells, with negative SHP1 expression. This effect was consistent when JAK2/STAT3 signaling was activated by interleukin-6 (IL-6), and ameliorated when cells were treated with prevanadate, a protein tyrosin phosphatase inhibitor. Furthermore, plumbagin significantly reduced gene expression of cyclin D1, vascular endothelial growth factor (VEGF)-1, Bcl-xL, survivin and matrix metalloproteinase-9 (MMP-9), known target products of STAT3 activation in gastric carcinogenesis by reverse transcription-polymerase chain reaction (RT-PCR). Several functional studies such as water soluble tetrazolium salt-1 (WST-1) assay, wound closure assay, Matrigel invasion assay and Annexin V assay were also performed, and we validated the functional effect of plumbagin for inhibition of cell proliferation, migration and invasion, and induction of apoptosis. Collectively, our findings suggest that plumbagin is a potential regulator of cellular growth, migration, invasion and apoptosis by inhibiting both constitutive and inducible STAT3 activity through induction of SHP1 in gastric cancer cells.

CpG island promoter hypermethylation of Ras association domain family 1A gene contributes to gastric carcinogenesis.

Methylation rates of the Ras association domain family 1A gene (RASSF1A) have been variously reported as between 7.5 and 66.7% in gastric carcinoma tissues. The role of this gene in gastric cancer also remains to be fully elucidated. The present study aimed to investigate whether promoter hypermethylation of RASSF1A occurs in gastric adenocarcinoma tissues and gastric cancer cell lines, and to determine the effects of RASSF1A in gastric carcinoma cell lines. The results showed a methylation­specific band only in SNU­719, MKN28 and AGS human gastric cancer cells (indicating full methylation), none of which exhibited RASSF1A expression. By contrast, SNU­16, MKN­45 and KATO­III human gastric carcinoma cells exhibited methylation as well as unmethylation­specific bands (indicating partial methylation), and all displayed positive or weakly positive expression of RASSF1A. Bisulfite sequencing in AGS and SNU­719 cells revealed that virtually all CpG sites were densely methylated. When SNU­719, MKN­28 and AGS cells were treated with the demethylating agent 5­aza­2'­deoxycytidine, RASSF1A gene expression was restored and the methylation­specific polymerase chain reaction pattern was altered in all three cell lines. Transfection of a plasmid expressing RASSF1A into AGS and SNU­719 cells significantly inhibited cell proliferation. Exogenous RASSF1A also reduced the expression of cyclin D1 and phospho­retinoblastoma protein, and increased that of p27 as demonstrated by western blot analysis. Furthermore, RASSF1A expression was significantly reduced (P=0.048) and the methylation rate was elevated in gastric adenocarcinoma tissues, compared with those in adjacent healthy intestinal metaplasia (34.6 vs. 66.7%, P=0.029). The present study indicated that epigenetic silencing of RASSF1A is frequently caused by promoter hypermethylation in gastric cancer cell lines as well as in gastric adenocarcinoma tissues, which may contribute to gastric carcinogenesis.