Gastric Mucosal Atrophy Impedes Housekeeping Gene Methylation in Gastric Cancer Patients / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Helicobacter pylori infection induces phenotype-stabilizing methylation and promotes gastric mucosal atrophy that can inhibit CpG-island methylation. Relationship between the progression of gastric mucosal atrophy and the initiation of CpG-island methylation was analyzed to delineate epigenetic period for neoplastic transformation. MATERIALS AND METHODS: Normal-appearing gastric mucosa was biopsied from 110 H. pylori–positive controls, 95 H. pylori–negative controls, 99 gastric cancer patients, and 118 gastric dysplasia patients. Gastric atrophy was assessed using endoscopic-atrophic-border score. Methylation-variable sites of eight CpG-island genes adjacent to Alu (CDH1, ARRDC4, PPARG, and TRAPPC2L) or LTR (MMP2, CDKN2A, RUNX2, and RUNX3) retroelements and stomach-specific TFF3 gene were analyzed using radioisotope-labeled methylation-specific polymerase chain reaction. RESULTS: Mean ages of H. pylori–positive controls with mild, moderate, and severe atrophy were 51, 54, and 65 years and those of H. pylori–associated TFF3 overmethylation at the three atrophic levels (51, 58, and 63 years) tended to be periodic. Alu-adjacent overmethylation (50 years) was earlier than TFF3 overmethylation (58 years) in H. pylori–positive controls with moderate atrophy. Cancer patients with moderate atrophy showed late Alu-adjacent (58 years) overmethylation and frequent LTR-adjacent overmethylation. LTR-adjacent overmethylation was frequent in cancer (66 years) and dysplasia (68 years) patients with severe atrophy. CONCLUSION: Atrophic progression is associated with gastric cancer at moderate level by impeding the initiation of Alu-adjacent methylation. LTR-adjacent methylation is increased in cancer patients and subsequently in dysplasia patients.

Periodic Methylation Patterns in the Background Mucosa of Gastric Cancer

BACKGROUND/AIMS: Gastrointestinal glandular stem cells renew every 8 years. New stem cells with impeded housekeeping gene methylation have unstable phenotypes and are prone to transform into malignant cells. Age-related changes in methylation in the gastric mucosa were evaluated to define the period of cancer-prone stem cell replacement. MATERIALS AND METHODS: Endoscopic biopsy specimens of normal-appearing gastric mucosa were obtained from 148 Helicobacter pylori-negative controls, 124 H. pylori-positive controls, and 69 gastric cancer patients with closed-type mucosal atrophy. Methylation-variable sites of two stomach-specific genes (TFF2 and TFF3) and four housekeeping genes (CDH1, ARRDC4, MMP2, and CDKN2A) were analyzed using radioisotope-labeled methylation-specific polymerase chain reaction. Age-related methylation was evaluated depending on the gastric mucosal atrophy at 2-year intervals. RESULTS: TFF2 methylation peaked periodically at 40 to 41, 48 to 49, 56 to 57, and 64 to 65 years of age in H. pylori-negative controls. Periodic peaks of TFF2 methylation were also found in H. pylori-positive controls. Housekeeping-gene methylation troughed at 48 to 49, 56 to 57, and 68 to 69 years of age in cancer patients. Trough methylation of CDH1 and ARRDC4 was lower in cancer patients than in H. pylori-positive controls. CONCLUSIONS: Methylation peaks of stomach-specific TFF2 in controls and methylation troughs of housekeeping genes in cancer patients were found every 8 years. Periodic methylation patterns may be used to identify individuals at high risk for gastric cancer.

DNA Methylation as Surrogate Marker For Gastric Cancer

Stomach cancer remains, stubbornly, highly prevalent in East Asia. Still, stomach cancer has few biomarkers by which it can be predicted. Helicobacter pylori infection, a known carcinogen of stomach cancer, usually goes undetected prior to cancer diagnosis, due to the poor mucosal environments that its related gastric atrophy causes. We propose, herein, an endoscopic-biopsy-based cancer-predicting DNA methylation marker. We semi-quantitatively examined the methylation-variable sites near the CpG-island margins by radioisotope-labeling methylation-specific polymerase chain reaction in association with H. pylori, which increases age-related over-methylation in CpG islands of gastric mucosa. These age-related methylation patterns of the transitional-CpG sites are proposed as useful surrogate markers for stomach cancer. It would be helpful for setting the optimal screening interval for high-risk subjects as well as for estimating the prognosis and the predictability for recurrence of early gastric cancer in patients having undergone endoscopic submucosal dissection. New screening-interval guidelines for gastric cancer should be suggested considering individual risk based on age, severity of atrophy, H. pylori status, and DNA methylation pattern.

DNA Methylation Patterns of Ulcer-Healing Genes Associated with the Normal Gastric Mucosa of Gastric Cancers

Recent evidence suggests that gastric mucosal injury induces adaptive changes in DNA methylation. In this study, the methylation status of the key tissue-specific genes in normal gastric mucosa of healthy individuals and cancer patients was evaluated. The methylation-variable sites of 14 genes, including ulcer-healing genes (TFF1, TFF2, CDH1, and PPARG), were chosen from the CpG-island margins or non-island CpGs near the transcription start sites. The healthy individuals as well as the normal gastric mucosa of 23 ulcer, 21 non-invasive cancer, and 53 cancer patients were examined by semiquantitative methylation-specific polymerase chain reaction (PCR) analysis. The ulcer-healing genes were concurrently methylated with other genes depending on the presence or absence of CpG-islands in the normal mucosa of healthy individuals. Both the TFF2 and PPARG genes were frequently undermethylated in ulcer patients. The over- or intermediate-methylated TFF2 and undermethylated PPARG genes was more common in stage-1 cancer patients (71%) than in healthy individuals (10%; odds ratio [OR], 21.9) and non-invasive cancer patients (21%; OR, 8.9). The TFF2-PPARG methylation pattern of cancer patients was stronger in the older-age group (> or =55 yr; OR, 43.6). These results suggest that the combined methylation pattern of ulcer-healing genes serves as a sensitive marker for predicting cancer-prone gastric mucosa.

Relationship of Glomerular Basement Membrane Alterations to Epithelial Cell Structure and Clinical Parameters in Alport Syndrome

PURPOSE: This study was performed to evaluate the relationship between glomerular basement membrane (GBM) alterations to epithelial cell (EpC) structure and renal function in Alport Syndrome (AS) patients. METHODS: Fifteen patients diagnosed with AS (4-26yrs) were examined. The GBM in AS was categorized as: C1) normal, C2) minor alterations (widening of lamina rara interna or externa without lamina densa change), C3) nonspecific splitting of lamina densa, C4) basket-weaving pattern of lamina densa splitting. The length of each GBM portion along the epithelial side was measured on the systematically obtained electron microscopic photographs. Furthermore to obtain an objective assessment of the degree of glomerular EpC foot process change, the number of slit pores along 10 microm of peripheral GBM in each category was obtained. RESULTS: The percentage of normal GBM portion (C1) correlated inversely with daily protein excretion (g/day/m2, P <0.05) and sum of the percentage of abnormal GBM portion (C2+C3+C4) had direct correlation with daily protein excretion (g/day/m2, P <0.05). There were no significant relationships between the percentages of other categories of GBM alterations and creatinine clearance or protein excretion. There were no significant relationships between of creatinine clearance in relation to normal GBM(C1) portion as well as that in relation to sum of the percentage of abnormal GBM portion (C2+C3+C4). GBM abnormality did not correlate with age at biopsy. CONCLUSION: The extent of GBM structural abnormality is related to proteinuria in AS but the epithelial response is uniform even though the GBM ultrastructural lesions are not.

DNA Methylation and Expression Patterns of Key Tissue-specific Genes in Adult Stem Cells and Stomach Tissues

CpG-island margins and non-island-CpG sites round the transcription start sites of CpG-island-positive and -negative genes are methylated to various degrees in a tissue-specific manner. These methylation-variable CpG sites were analyzed to delineate a relationship between the methylation and transcription of the tissue-specific genes. The level of tissue-specific transcription was estimated by counting the number of the total transcripts in the SAGE (serial analysis of gene expression) database. The methylation status of 12 CpG-island margins and 21 non-island CpG sites near the key tissue-specific genes was examined in pluripotent stromal cells obtained from fat and bone marrow samples as well as in lineage-committed cells from marrow bulk, stomach, colon, breast, and thyroid samples. Of the 33 CpG sites examined, 10 non-island-CpG sites, but none of the CpG-island margins were undermethylated concurrent with tissue-specific expression of their nearby genes. The net methylation of the 33 CpG sites and the net amount of non-island-CpG gene transcripts were high in stomach tissues and low in stromal cells. The present findings suggest that the methylation of the non-island-CpG sites is inversely associated with the expression of the nearby genes, and the concert effect of transitional-CpG methylation is linearly associated with the stomach-specific genes lacking CpG-islands.

Chromosomal Losses are Associated with Hypomethylation of the Gene-Control Regions in the Stomach with a Low Number of Active Genes

Transitional-CpG methylation between unmethylated promoters and nearby methylated retroelements plays a role in the establishment of tissue-specific transcription. This study examined whether chromosomal losses reducing the active genes in cancers can change transitional-CpG methylation and the transcription activity in a cancer-type-dependent manner. The transitional-CpG sites at the CpG-island margins of nine genes and the non-island-CpG sites round the transcription start sites of six genes lacking CpG islands were examined by methylation-specific polymerase chain reaction (PCR) analysis. The number of active genes in normal and cancerous tissues of the stomach, colon, breast, and nasopharynx were analyzed using the public data in silico. The CpG-island margins and non-island CpG sites tended to be hypermethylated and hypomethylated in all cancer types, respectively. The CpG-island margins were hypermethylated and a low number of genes were active in the normal stomach compared with other normal tissues. In gastric cancers, the CpG-island margins and non-island-CpG sites were hypomethylated in association with high-level chromosomal losses, and the number of active genes increased. Colon, breast, and nasopharyngeal cancers showed no significant association between the chromosomal losses and methylation changes. These findings suggest that chromosomal losses in gastric cancers are associated with the hypomethylation of the gene-control regions and the increased number of active genes.

Methylation and Chromosomal Losses in Squamous Cell Carcinoma of the Head and Neck / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation in many human cancers. Another concern with regards to CpG methlation is unilateral chromosomal losses in head and neck cancer. In this study, we investigated the extent of chromosomal losses and the status of CpG methylation in head and neck cancer in relation with clinicopathologic factors. SUBJECTS AND METHOD: Both normal mucosa and tumor tissue samples were secured from 17 cases to a total of 34 samples to be examined with a methylation- specific PCR on 15 cancer-linked genes. A total of 29 cases were analyzed for PCR-based loss of heterozygosity (LOH) using a panel of 41 microsatellite markers on 8 chromosomes. RESULTS: The pattern of methylation changes between the paired normal mucosa and tumor site was variable. Of the total of 206 cases examined for the methylation status of non-CpG island, 34 cases showed hypomethylation changes, 26 cases hypermethylation changes, and 31 cases no methylation changes. Regions containting CpG islands had 8 cases showing hypomethylation changes, 17 cases hypermethylation changes, and 31 cases of no methylation changes. The relationship between methylation and lymph node invasion revealed that, in the event of lymph node invasion, p16 downstream 0.7 kbp, p16 upstream 1.0 kbp, and hMLH1 upstream 1.0 kbp showed hypomethylation, whereas BGLAP upstream 4.5 kbp, Runx3 upstream 1.7 kbp, KIAA downstream 0.4 kbp showed hypermethylation. However, the rest of the genes were not changed. In 29 tumor foci, a LOH was found most frequently on the chromosomes 3p, 8p, 9p, and 13q. Interestingly, although other previous reports have not reported the detection of 8p chromosomal loss in head and neck cancer, this study frequently detected 8p chromosomal loss. Chromosomal loss yielded an overall mean value of 4.79+/-2.2 per tumor focus. A special relationship could not be drawn based on the relationship between the methylation and LOH. But in several genes such as p16 and hMLH1, there were differences between the hypomethylation. Genetic instability was raised when hypomethylation increased. CONCLUSION: This study showed that the head and neck cancer and its progression generally need the proper level of chromosomal losses to accomplish cancer progression or development. Methylation pattern and LOH might be important rules and target event in head and neck cancer. In the future, experiments to find the point of genetic modification will help the way to prevent the cancer.

Relationship Between the Extent of Chromosomal Losses and the Pattern of CpG Methylation in Gastric Carcinomas

The extent of unilateral chromosomal losses and the presence of microsatellite instability (MSI) have been classified into high-risk (high- and baseline-level loss) and low-risk (low-level loss and MSI) stem-line genotypes in gastric carcinomas. A unilateral genome-dosage reduction might stimulate compensation mechanism, which maintains the genomic dosage via CpG hypomethylation. A total of 120 tumor sites from 40 gastric carcinomas were examined by chromosomal loss analysis using 40 microsatellite markers on 8 chromosomes and methylation analysis in the 13 CpG (island/non-island) regions near the 10 genes using the bisulfite-modified DNAs. The high-level-loss tumor (four or more losses) showed a tendency toward unmethylation in the Maspin, CAGE, MAGE-A2 and RABGEF1 genes, and the other microsatellite-genotype (three or fewer losses and MSI) toward methylation in the p16, hMLH1, RASSF1A, and Cyclin D2 genes (p<0.05). The non-island CpGs of the p16 and hMLH1 genes were hypomethylated in the high-level-loss and hypermethylated in the non-high-level-loss sites (p<0.05). Consequently, hypomethylation changes were related to a high-level loss, whereas the hypermethylation changes were accompanied by a baseline-level loss, a low-level loss, or a MSI. This indicates that hypomethylation compensates the chromosomal losses in the process of tumor progression.

Periodic Explosive Expansion of Human Retroelements Associated with the Evolution of the Hominoid Primate

Five retroelement families, L1 and L2 (long interspersed nuclear element, LINE), Alu and MIR (short interspersed nuclear element, SINE), and LTR (long terminal repeat), comprise almost half of the human genome. This genome-wide analysis on the time-scaled expansion of retroelements sheds light on the chronologically synchronous amplification peaks of each retroelement family in variable heights across human chromosomes. Especially, L1s and LTRs in the highest density on sex chromosomes Xq and Y, respectively, disclose peak activities that are obscured in autosomes. The periods of young L1, Alu, LTR, and old L1 peak activities calibrated based on sequence divergence coincide with the divergence of the three major hominoid divergence as well as early eutherian radiation while the amplification peaks of old MIR and L2 account for the marsupial-placental split. Overall, the peaks of autonomous LINE (young and old L1s and L2s) peaks and non-autonomous SINE (Alus and MIRs) have alternated repeatedly for 150 million years. In addition, a single burst of LTR parallels the Cretaceous-Tertiary (K-T) boundary, an exceptional global event. These findings suggest that the periodic explosive expansions of LINEs and SINEs and an exceptional burst of LTR comprise the genome dynamics underlying the macroevolution of the hominoid primate lineage.

Seroepidemiological Survey of Hantaan Virus Infection in Sera of Patients at the Kangnam St. Mary Hospital in Korea from 1998 to 2002

Sera of patients visited at the Kangnam St. Mary Hospital in Seoul were collected randomly at the Department of Clinicopathology from January, 1998 to December, 2002. Specimens were collected two twice a month, in a 15-day interval, and 100 specimens were collected at a time. Specimens test in duplicate, and/or displaying antinuclear antibody reaction were excluded from the seroepidemiological analyses. Detection of antibodies to Hantaan virus, an etiologic agent of hemorrhagic fever with renal syndrome (HFRS), was done by indirect immunofluorescent antibody (IFA) technique. Out of 11,361 sera tested, 445 cases (3.9%) showed specific antibody to Hantaan virus. Sexual difference was not noted. Annual incidence of HFRS cases showed a 3 year-periodicity. In the monthly incidence analysis, two peaks of incidence were appeared in the male cases, the first peak in March and the second in August. Female cases showed a single peak in October. The age distribution showed that 64.9% of the sero-positive cases were from 40 to 69 years of age. Peak age-group was in the 6th decade. Each decade of age-group showed diverse patterns of annual and monthly incidences. These results suggest the incidence of HFRS shows a periodicity and a unique pattern in each age group.

Differentiation of Osteoblast Progenitor Cells from Human Umbilical Cord Blood

BACKGROUND: Human umbilical cord bloods, which could be taken during the delivery are utilized as a source of hematopoietic stem cells. Also in cord blood, there are several kinds of stem cells such as endothelial and mesenchymal stem cells. METHODS: We isolated the mesenchymal stem cells from human umbilical cord bloods and confirmed the differentiation of these cells into osteoblast progenitor cells. The mesenchymal stem cells derived from umbilical cord blood have the ability to differentiate into specific tissue cells, which is one of characteristics of stem cells. These cells were originated from the multipolar shaped cells out of adherent cells of the umbilical cord blood mononuclear cell culture. RESULTS: The mesenchymal stem cells expressed cell surface antigen CD13, CD90, CD102, CD105, alpha-smooth muscle actin and cytoplasmic antigen vimentine. Having cultrued these cells in bone formation media, we observed the formation of extracellular matrix and the expression of alkaline phosphatase and of mRNA of cbfa-1, ostoecalcin and type I collagen. CONCLUSION: From these results we concluded that the cells isolated from the umbilical cord blood were mesenchymal stem cells, which we could differentiate into osteoblast when cultured in bone formation media. In short, it is suggested that these cells could be used as a new source of stem cells, which has the probability to alternate the embryonic stem cells.

Differentiation of Osteoblast Progenitor Cells from Human Umbilical Cord Blood

BACKGROUND: Human umbilical cord bloods, which could be taken during the delivery are utilized as a source of hematopoietic stem cells. Also in cord blood, there are several kinds of stem cells such as endothelial and mesenchymal stem cells. METHODS: We isolated the mesenchymal stem cells from human umbilical cord bloods and confirmed the differentiation of these cells into osteoblast progenitor cells. The mesenchymal stem cells derived from umbilical cord blood have the ability to differentiate into specific tissue cells, which is one of characteristics of stem cells. These cells were originated from the multipolar shaped cells out of adherent cells of the umbilical cord blood mononuclear cell culture. RESULTS: The mesenchymal stem cells expressed cell surface antigen CD13, CD90, CD102, CD105, alpha-smooth muscle actin and cytoplasmic antigen vimentine. Having cultrued these cells in bone formation media, we observed the formation of extracellular matrix and the expression of alkaline phosphatase and of mRNA of cbfa-1, ostoecalcin and type I collagen. CONCLUSION: From these results we concluded that the cells isolated from the umbilical cord blood were mesenchymal stem cells, which we could differentiate into osteoblast when cultured in bone formation media. In short, it is suggested that these cells could be used as a new source of stem cells, which has the probability to alternate the embryonic stem cells.

Effects of Topical 2% Dorzolamide(Trusopt(R)) in Q-switched Nd:YAG Laser Induced Glaucoma Rabbits

The purpose of this study is to evaluate the effects of 2% dorzolamide(Trusopt(R)) on the ocular blood flow and retinal microcirculation. To creat an experimental glaucoma model in rabbits and to study the effects of elevated intraocular pressure(IOP), ocular blood flow, retinal effects of elevated intraocular pressure(IOp), ocular blood flow, retinal microcirculation on rabbits eyes, we treated trabecular meshwork of 6 adult pigmented rabbits with Q-switched Nd;YAG laser. And then we investigated the IOP lowering effect, ocular blood flow, and the microcirculation on retina of 2% dorzolamide(Trusopt(R)) in experimental glaucoma model. The IOP, ocular blood flow and the microcirculation were measured with applanation pneumotonography(Alcon, Texas), pneumotonometric probe linked to Langham ocular blood foow system(OBF, Blue mountain) and Heidelberg Retina Flowmeter(HRF). During sustained IOP elevation, 2% dorzolamide(Trusopt(R)) was instilled in one eye and normal saline in the fellow eye. The IOP and ocular blood flow were measured 1, 2, 4 and 8 hours after instillation. The retinal microcirculation was measured 2 and 8 hours after instillation. The retinal microcirculation was measured 2 and 8 hours after instillation at 200micrometer apart from the superior optic disc margin. There were statistically significant reductions in IOP in both 2% dorzolamide(Trusopt(R)) and normal saline-treated eyes(P0/1). From the above results, we concluded that 2% dorzolamide(Trusopt(R)) reduced the IOP but did not alter ocular blood flow and peripapillary retinal microcirculation.

An Effects of Needling Revision for Failing Filteration Blebs after Trabeculectomy

We investigated the outcome of needling revision with adjunctive subconjunctival 5-fluorouracil(5-FU) injection performed on 20 eyes of 20 consecutive glaucoma patients with failed filtering blebs. Follow-up time was 44.5+/-53.9 weeks from the first needling revision and 31.8+/-43.5 weeks from the last needling procedures. We divided the patients into the success group and the failed group depending on the change of the intraocular pressure(IOP) after needling, and analysed the multi-factors which influenced the success rate of needling by logistic regression analysis. Thirteen(65%) of the eyes were classified treatment success after 1.6+/-0.8 needling revision, with a mean intraocular presure(IOP) of 19.8+/-1.9mmHg on 1.1+/-0.9 medications, significantly lower mean IOP and number of medications than before the procedure(28.1+/-15.7mmHg [P<0.01, paired t-test]). Needling revision with adjunctive 5-FU appears to be a safe and effective means of reestablished filtration.

An Effects of Contact Transscleral Diode Laser Cyclophotocoagulation in Glaucoma Patients

We observed the changes of IOP after contact transscleral cyclophotocoagualtion with Diode laser which emit 810mm beam and we tride to investigatethe suitable level and range of energy when Diode laser was applied to the refractory glaucoma patients. Fifteen eyes underwent contact transscleral cyclophotocoagulation with energy 3J(Group A, 6eyes), 4J(Group B, 9eyes) separately and intraocular pressure(IOP) was measured at 1 day, 1 week, 1 month, 2 month, 3 month, 4month, 5month, 6month, postoperatively. The intraocular pressure(IOP) decreasing rate was 83.02% in A group and 64.72% in B group. The success rate was 50% in A group and 66.7% in B group at 6 month postoperatively. It is suggested that contact transscleral cyclophotocoagulation with Diode laser si useful in lowering IOP in refractory glaucoma patients.