Polymorphisms in PRKCDBP, a Transcriptional Target of TNF-alpha, Are Associated With Inflammatory Bowel Disease in Korean

BACKGROUND/AIMS: Emerging data indicate that polymorphic sequence variations in the tumor necrosis factor alpha (TNF-alpha) gene may affect its production, and be associated with the risk of inflammatory bowel disease (IBD). PRKCDBP is a putative tumor suppressor gene and a transcriptional target of TNF-alpha. The aim of this case-control study is to explore the possible association of single nucleotide polymorphisms (SNPs) in PRKCDBP with the development of IBD in Koreans. METHODS: Genotyping analysis of four SNPs of PRKCDBP [rs35301211 (G210A), rs11544766 (G237C), rs12294600 (C797T), and rs1051992 (T507C)] was performed on 170 ulcerative colitis (UC),131 Crohn's disease (CD) patients, and 100 unrelated healthy controls using polymerase chain reaction and restriction fragment length polymorphism. RESULTS: Heterozygous configuration of three SNPs (G210A, G237C, and C797T) was very rare in both patients and healthy controls. However, allele frequencies of the T507C SNP showed a significant difference between UC patients and controls (P=0.037). The CC genotype of the T507C SNP was identified in 46.6% (61 of 131) of CD and 49.4% (84 of 170) of UC patients, but only in 33.0% (33 of 100) of healthy controls. Furthermore, CC homozygosity was more prevalent than TC heterozygosity in both CD and UC patients versus controls (P=0.016; gender-adjusted odds ratio [aOR], 2.16; 95% confidence interval [CI], 1.16-4.04 and P=0.009; aOR, 2.09; 95% CI, 1.193.64; respectively) CONCLUSIONS: Our results suggest that the T507C SNP in PRKCDBP, a TNF-alpha-inducible gene, might be associated with susceptibility to IBD (particularly UC) development in Koreans.

Characterization of Gp41 Polymorphisms in the Fusion Peptide Domain and T-20 (Enfuvirtide) Resistance-Associated Regions in Korean HIV-1 Isolates

HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naive Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.

Saccharomyces boulardii Reduced Intestinal Inflammation in Mice Model of 2,4,6-trinitrobencene Sulfonic Acid Induced Colitis: Based on Microarray / 대한소화기학회지

BACKGROUND/AIMS: Saccharomyces boulardii has been reported to be beneficial in the treatment of inflammatory bowel disease. The aim of this work was to evaluate the effect of S. boulardii in a mice model of 2,4,6-trinitrobencene sulfonic acid (TNBS) induced colitis and analyze the expression of genes in S. boulardii treated mice by microarray. METHODS: BALB/c mice received TNBS or TNBS and S. boulardii treatment for 4 days. Microarray was performed on total mRNA form colon, and histologic evaluation was also performed. RESULTS: In mice treated with S. boulardii, the histological appearance and mortality rate were significantly restored compared with rats receiving only TNBS. Among 330 genes which were altered by both S. boulardii and TNBS (>2 folds), 193 genes were down-regulated by S. boulardii in microarray. Most of genes which were down-regulated by S. bouardii were functionally classified as inflammatory and immune response related genes. CONCLUSIONS: S. boulardii may reduce colonic inflammation along with regulation of inflammatory and immune responsive genes in TNBS-induced colitis.

Expression and mutational analysis of TGF-beta/Smads signaling in human cervical cancers / 부인종양

OBJECTIVE: To define the molecular basis of TGF-beta1 function in cervical carcinogenesis, we explored the expression and mutational status of TGF-beta1, TGF-beta1 receptors, and Smads, the regulators of the TGF-beta1 signaling pathway, in human cervical cancers. METHODS: Expression of TGF-beta1, TGF-beta1 receptors, and Smads transcripts were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR), and sequence alteration was analyzed using RT-PCR-single-strand conformation polymorphism (SSCP) analysis. Genomic levels of TGF-beta1, TGF-beta1 receptors and Smads was also measured by quantitative genomic PCR. RESULTS: Abnormal overexpression of TGF-beta1 and abnormal reduction of type II TGF-beta1 receptor were identified in 36% (18 of 50) and 20% (10 of 50) of cervical cancer tissues, respectively. 22% (11 of 50) in Smad2 and 14% (7 of 50) in Smad4 revealed tumor specific mRNA reduction less than a half of normal means. In addition, no evidence for sequence alterations of the gene was found by RT-PCR-SSCP analysis. CONCLUSION: Our study demonstrates that disruption of TGF-beta/Smad signaling pathway exist in human cervical cancer, suggesting that abnormal expressions of the member of TGF-beta/Smad signaling pathway might contribute to the malignant progression of human cervical tumors via suppressing the tumor suppression function of TGF-beta1 1's tumor suppression function.

Role of 5'-CpG island hypermethylation of the FHIT gene in cervical carcinoma / 부인종양

OBJECTIVE: The abnormal expression of fragile histidine triad (FHIT) gene has been frequently reported in a variety of epithelial malignancies including cervical carcinoma. Furthermore, in a recent study it was proposed that transcriptional inactivation of FHIT, as a consequence of aberrant 5'-CpG island methylation, plays an important role in the carcinogenesis of human cervical carcinoma. The authors sought to determine whether abnormal FHIT transcription occurs in human cervical carcinoma, and if so, whether this abnormal expression is associated with aberrant 5'-CpG island methylation. In addition, the clinical significance of FHIT inactivation was investigated in Korean women with cervical cancer. METHODS: To examine for abnormal transcripts of the FHIT gene, quantitative RT-PCR, genomic DNA-PCR and nonisotopic RT-PCR-SSCP analysis were performed using the standard method. The methylation status was determined by methylation specific PCR and bisulfite DNA sequencing. RESULTS: The FHIT gene was down-regulated in 15 of 58 (25.9%) cervical carcinomas. FHIT promoter hypermethylation was detected in 15 of 15 (100%) abnormally expression in cervical carcinomas. Bisulfite DNA sequencing confirmed these findings and a significant correlation was found between CpG site hypermethylation and low FHIT expression. However, no significant correlation was found between reduced FHIT expression and clinicopathological characteristics. CONCLUSION: In this study, FHIT inactivation in cervical cancer was found to be strongly correlated with 5'-CpG island hypermethylation rather than a genetic alteration. Furthermore, no significant relation was found between a lack of FHIT expression and the prognostic factors of cervical cancer in our Korean cohort.

Frequent epigenetic inactivation of XAF1 (XIAP-associated factor 1), a candidate tumor suppressor, by aberrant promoter hypermethylation in human cervical carcinomas / 대한산부인과학회지

OBJECTIVE: X-linked inhibitor of apoptosis (XIAP) is the most potent member of IAP family that exerts antiapoptotic effects by interfering with activities of caspases. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. We explore the candidacy of XAF1, Smac/DIABLO and HtrA2 as a tumor suppressor in cervical carcinogenesis and determine the mechanisms of altered XAF1 expression. METHODS: We investigated the expression and mutation status of the genes in 64 cervical cancer tissues, 5 cervical cancer cell lines and 10 normal cervical tissues. RESULTS: XAF1 transcript was not expressed or extremely low levels in 40% (2/5) of cancer cell line and in 31% (20/64) of primary carcinomas whereas Smac/DIABLO and HtrA2 are normally expressed in all cells. As somatic mutations of the gene was not detected, expression of XAF1 transcript was reactivated in all nonexpressor cell lines after 5-aza-2-deoxycytidine treatment. Bisulfite DNA analysis for CpG sites in the promoter region revealed a strong association between CpG sites hypermethylation and gene silencing. CONCLUSION: XAF1 undergoes epigenetic silencing in a considerable proportion of cervical carcinomas by aberrant promoter hypermethylation rather than genetic alterations, and closely associated with reduced gene expression. Although additional studies are required to determine the biological significance of XAF1 inactivation, it will be valuable to examine the expression status of XAF1 could be a clinically useful marker for cancer treatment.

Expression and Mutation Analysis of RKIP (Raf-1 Kinase Inhibitor Protein) in Human Gastric Cancer

PURPOSE: RKIP (Raf kinase inhibitor protein) is a novel candidate tumor suppressor, known to inhibit the MAPK signaling by interfering with the MEK phosphorylation by Raf-1. The aim of this study was to investigate the expression of RKIP and analyze the pattern of inactivation and mutation of the RKIP gene in human gastric cancer. METHODS: To explore if RKIP inactivation is implicated in gastric tumorigenesis, an expression analysis on the transcription and protein expression levels and a mutational analysis of RKIP were performed in 15 human gastric cancer cell lines and 92 primary carcinoma tissues. RESULTS: Abnormal reduction of the level of RKIP expression was frequently detected in the cancer cell lines and primary tumor tissues, at both the transcript and protein levels. Moreover, the expression level of RKIP in the tumor cells was inversely correlated with the level of Erk phosphorylation, indicating that RKIP plays a key role in the regulation of the Raf-MEK-Erk signaling pathway in human gastric cells. While the expression of the RKIP transcript was not re-activated in low expressor cells by treatment with the demethylating agent 5'Aza-dC, the genomic RKIP was detected at low levels in many cancer cell lines, suggesting that an abnormal reduction of level of RKIP expression in tumors might be caused by allelic deletion of the gene rather than transcriptional silencing due to aberrant DNA hypermethylation. A loss of heterozygosity study, using an intragenic polymorphic marker, revealed that approximately 21% of the gastric cancers harbored allelic loss of the RKIP gene. CONCLUSION: Collectively, this study has demonstrated that RKIP is a tumor suppressor, whose expression is frequently downregulated by allelic deletion in human gastric cancers. This study also suggests that an altered expression of RKIP might contribute to the development of gastric cancer via abnormal elevation of the Raf-Erk signaling pathway.

Elevated Expression of p73 and Its Relation to VEGF and TSP-1 Production in Cervical Carcinoma / 대한산부인과학회지

OBJECTIVE: Measure the over-expression of p73 and analyze as the prognostic as well as angiogenic factor of cervical cancer by comparing the degree of expression of VEGF and TSP-1 by RT-PCR. METHODS: 7 normal and 37 cervical cancer specimens were put through RT-PCR and the expression of p73, VEGF and TSP-1 were measured. After immunohistochemical staining, the number of microvessels was counted. With the level of expression, investigated the relationship with the clinicopathological characteristics and the number of microvessels. RESULTS: 57% of cancer tissues showed abnormally high levels of p73 mRNA. In quantitative genomic DNA PCR, the p73 was over-expressed in the transcription level. Through allotyping with Sty I polymorphism, the over-expression of p73 was due to the transcription activity of the silent allele. In RT-PCR-SSCP analysis of over-expressed specimens, sequence alterations was not seen. In 73%, VEGF was over-expressed while TSP-1 was under-expressed in 35%. There was no association between the number of microvessels with the over-expression of p73 and VEGF, but inversely associated with the under-expression of TSP-1. There was no correlation between the over-expression of p73 and the clinicopathological characteristics. The over-expression of p73 coincided 80% with the over-expression of VEGF, and 40% with the under-expression of TSP-1. CONCLUSION: These data support the expression of p73 was increased in cervical cancer tissues and was associated with the over-expression of the VEGF but not associated with the under-expression of TSP-1. The biological and clinical significance of the over-expression of p73 should be studied further in the future.

Expression of Vascular Endothelial Growth Factor and Thrombospondin-1 mRNA Related to Microvessel Density in Invasive Cervical Carcinomas / 대한산부인과학회지

OBJECTIVE: Vascular Endothelial Growth Factor (VEGF) is a potent stimulator of angiogenesis in solid tumors. Thrombospondin-1 (TSP-1) has inhibitory role in cancer cell proliferation and metastasis. To analyze the correlation with expression of VEGF and TSP-1 including microvessel density (MVD), the levels of VEGF/TSP-1 mRNA expression and microvessel count (MVC) were estimated in patients with invasive cervical carcinomas. METHODS: From 1996 to 1999, 37 carcinomas and 7 normal cervical tissues were collected, frozen and stored at -70 degrees C until used. The levels of VEGF and TSP-1 mRNAs were determined by quantitative RT-PCR. MVD was assessed by immunostaining for factor VIII-related antigen. The results are expressed as the largest number of microvessels present within a single x 40 field, and counted at x 100 field. RESULTS: Quantitative RT-PCR analysis demonstrated abnormally increased VEGF mRNA expression levels (>0.66) in 14 (37.8%) of 37 cervical carcinomas comparing to control groups (mean: 0.32+/-0.09) and abnormally low TSP-1 mRNA expression levels (<0.72) in 13 (35.1%) of 37 cervical carcinomas comparing to control groups (mean: 0.51+/-0.07). MVC was higher in tumors showing decreased expression of TSP-1 (but not statistically) (p<0.18) and overexpression of VEGF (p<0.05). When VEGF overexpression was accompanied with reduced TSP-1 expression, the microvessel density showed significantly increased pattern (p<0.05). CONCLUSION: Our study demonstrates that reduced expression of TSP-1 mRNAs and overexpression of VEGF mRNAs may be an important contributing factor in cervical carcinomas. Moreover, the inversed correlation of VEGF and TSP-1 mRNA expression can be an evidence of angiogenic role in cervical carcinomas.

Saccharomyces boulardii Activates Expression of Peroxisome Proliferator-activated Receptor-gamma in HT-29 Cells / 대한소화기학회지

BACKGROUND/AIMS: Saccharomyces boulardii (S. boulardii) has been reported to be beneficial in the treatment of inflammatory bowel disease, however, little is known about its mechanism of action. Peroxisome proliferator- activated receptor-gamma (PPAR-gamma) is recently found to regulate inflammation in intestinal epithelial cells. We hypothesized that the anti-inflammatory effects of S. boulardii are mediated, in part, through PPAR-gamma. To test this hypothesis, we examined the ability of S. boulardii to modulate the expression of PPAR-gamma in human colon cells. METHODS: Effects of S. boulardii on survival and proliferation of HT-29 human colon cells were assessed by MTT and [3H]thymidine incorporation assays. PPAR-gamma expression was assessed by Western blot and RT-PCR. Induction of interleukin-8 (IL-8) expression by tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), or lipopolysaccharide (LPS) was assessed by RT-PCR. RESULTS: S. boulardii did not affect viability and proliferation of HT-29 cells. S. boulardii up-regulated PPAR-gamma expression at both mRNA and protein levels. Pretreatment of HT-29 cells with S. boulardii blocked PPAR-gamma down-regulation by TNF-alpha, IL-1beta, or LPS, whereas it ameliorated IL-8 response to these proinflammatory factors. CONCLUSIONS: S. boulardii stimulates PPAR-gamma expression and reduces response of human colon cells to proinflammatory cytokines.

Frequent Epigenetic Inactivation of XAF1 by Promotor Hypermethylation in Human Colon Cancers / 대한소화기학회지

BACKGROUND/AIMS: X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family that exerts antiapoptotic effects. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. We explored the candidacy of XAF1, Smac/DIABLO and HtrA2 as a tumor suppressor in colonic carcinogenesis. METHODS: Expression and mutation status of the genes in 10 colorectal carcinoma cell lines and 40 primary tumors were examined by quantitative PCR analysis. RESULTS: XAF1 transcript was not expressed or present at extremely low levels in 60% (6/10) of cancer cell lines whereas Smac/DIABLO and HtrA2 are normally expressed in all cell lines examined. Tumor-specific loss or reduction of XAF1 was also found in 35% (14/40) of matched tissue sets obtained from the same patients. XAF1 transcript was reactivated in all the low expressor cell lines by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Moreover, bisulfite DNA sequencing analysis for 34 CpG sites in the promoter region revealed a strong association between hypermethylation and gene silencing. Restoration of XAF1 expression resulted in enhanced apoptotic response to etoposide and 5-flurouracil, whereas knockdown of XAF1 expression by siRNA transfection significantly inhibited chemotherapeutic drug-induced apoptosis. CONCLUSIONS: XAF1 undergoes epigenetic gene silencing in a considerable proportion of human colon cancers by aberrant promoter hypermethylation, suggesting that XAF1 inactivation might be implicated in colonic tumorigenesis.

TSP-1 mRNA Expression in Invasive Cervical Cancer: Correlate with Angiogenesis and Clinicopathological Features / 대한산부인과학회잡지

OBJECTIVE: Acquisition of a proangiogenic environment is essential to the cervical cancer growth, invasion and metastasis, and the angiogenic phenotype in cervical cancer is strongly associated with clinical outcome. However, the regulation of the metastatic process in cervical cancer has not been well defined. Thrombospondin-1 (TSP-1) is a representative angiogenesis suppressor whose loss or reduced expression has been frequently observed in many types of human neoplasms. In this study, we examined whether expression of TSP-1 is associated with clinicopathological features, including microvessel density and evaluated its prognostic significance in patients with cervical cancer. METHODS: The expression and mutation status of TSP-1 was examined by quantitative RT- and genomic PCR and RT-PCR-SSCP analysis and microvessel density was performed using immunohistochemical staining in 7 normal cervix and 37 cervical cancers. RESULTS: All normal cervix tissues express easily detetable levels of TSP-1 transcript in range of 1.41-1.62 (mean 1.51 +/- 0.07). In contrast to normal tissue, mRNA expression of TSP-1 in primary cancer was detected in range of 0.51-1.69 (mean 1.03 +/- 0.36), and 35.1% (13 of 37) of carcinomas expressed abnormally low levels of TSP-1 (p<0.05). Moreover, abnormal reduction of TSP-1 expression was more frequently observed in IIa-IIb cancer (60%, 6 of 10) compared to Ib cancer (25.9%, 7 of 27) (p<0.05). None of carcinoma tissues we tested showed abnormal reduction of TSP-1 gene level and no evidences for sequence alterations leading to amino acid substitution were identified, indicating that allelic deletion or mutational alteration of TSP-1 might be a rare event in cervical carcinogenesis. Microvessel density was significantly higher in tumors showing decreased expression of TSP-1 (abnormal low group: 11.3 +/- 5.06, others: 6.64 +/- 7.15) (p<0.05). To detect the possible deletion of the gene and the presence of sequence alteration in TSP-1 transcripts, we performed quantitative genomic PCR and RT-PCR-SSCP analysis. However, none of carcinoma tissues we tested showed abnormal reduction of TSP-1 gene level and no evidence for sequence alterations leading to amino acid substitution were identified. CONCLUSION: Our study demonstrates that abnormal reduction of TSP-1 mRNA expression is frequent in cervical cancer and correlates with the malignant progression of cervical cancers. Our data also show that allelic deletion or mutational alteration of TSP-1 is rare in cervical cancers, suggesting that abnormal reduction of TSP-1 mRNA expression in cervical cancers might be caused by altered transcriptional down regulation of the gene, such as epigenetic gene silencing. The inverse correlation between TSP expression and microvessel density also indicates that decreased TSP-1 expression might be associated with an angiogenic phenotype in cervical cancer.

Role of TGF-beta signaling and Ski/SnoN mRNA expression in cervical carcinomas / 대한산부인과학회잡지

OBJECTIVE: TGF-beta signaling is dependent on the heterodimerization of the type II TGF-beta receptor (TbetaR-II) with the type I TGF-beta receptor (TbetaR-I). which mediate intracellular signals through Smad proteins. Whereas physiologic concentrations of SnoN and Ski allow a feedback regulation of TGF-beta signaling, deregulation of SnoN or Ski expression leads to total inhibition of TGF-beta signaling and of the tumor suppressors Smad2 and Smad4, which can explain the role of SnoN and Ski as oncogenes. In order to identify possible molecular mechanisms responsible for TGF-beta resistance, the author investigated the mutation and expression of TGF-beta1, its receptors, Ski/SnoN in cervical carcinomas. METHODS: From December 1995 to December 1999, 45 carcinomas and 7 normal cervical tissue specimens were obtained by surgical resection in the Kyung Hee University Medical Center. Tissue specimens were snap-forzen in liquid N2 and stored at -70 degrees C until used. Total RNA was extracted from specimens and evaluated the expression levels using densitometric analysis of quantitative RT-PCR products (TGF-beta1, Tbeta1R-I, Tbeta1R-II, Ski/SnoN), and the mutations were investigated by quantitative genomic-PCR followed by nonisotopic RT-PCR-SSCP analysis (Tbeta1R-II, Tbeta1R-I, Ski/SnoN). The abnorally expressed levels of RT-PCR products (TGF-beta1, Tbeta1R-II) were analysed for the clinicopathologic characteristics. RESULTS: Quantitative RT-PCR analysis demonstrated variable expression of TGF-beta1 mRNA (0.05-0.89) in tumors and significantly increased TGF-beta1 expression level (>0.48) in 15 of 45 samples (33.3%). There is no significant reduction of Tbeta1R-I expression (1.36) in 2 of 45 samples (4.4%), and there is no amplification of Ski/SnoN gene by quantitative genomic-PCR analysis. CONCLUSIONS: The overexpression of TGF-beta1 mRNA and the reduced or absent expression of Tbeta1R-II may be an important contributing factors, and the abnormally low genomic levels and no mutational alterations of Tbeta1R-II is caused by monoallelic deletion suggesting that Tbeta1R-II might play as a tumor suppressor of haloinsufficiency in cervical carcinomas. We could not show that high levels of Ski/SnoN expression could produce a disruption of TGF-beta signaling in cervical carcinomas.

Expression of Vascular Endothelial Growth Factor (VEGF) and its mRNA in Uterine Cervical Carcinomas / 대한산부인과학회잡지

OBJECTIVE: Angiogenesis, the formation of blood vessels by sprouting from pre-existing ones, is essential for the growth of solid tumors beyond 2-3mm in diameter and for tumor metastasis. Vascular endothelial growth factor (VEGF), is known as vascular permeability factor(VPF) and mediates vascularization and tumor-induced angiogenesis. This study examined the potential of growth, invasion, and metastasis of uterine cervical carcinomas associated with neovascularization. METHODS: From January 1996 to December 1999, at the Department of Obstetrics and Gynecology, Kyung-Hee University Hospital, 37 uterine cervical carcinomas and 7 normal cervical tissues were obtained and the samples were immediately frozen and stored at -70 degrees C. Immunohistochemical staining for VEGF was carried out to study VEGF localization, and the levels of VEGF subtype mRNAs were determined by quantitative RT-PCR in specimens. The relation between VEGF subtypes expression of cervical cancers was analysed. RESULTS: The positive staining for VEGF is seen dominantly in the cytoplasm of the cancer cells, and faintly in interstitial cells. The intensity of staining was stronger in squamous carcinomas than in adenocrcinomas, but there was no significant difference (p>0.05). Quantitative RT-PCR analysis demonstrated significantly increased VEGF121/VEGF165 mRNA expression levels (>0.56 / >0.72) in 21 (56.8%) and 15 (40.5%) of 37 cervical carcinomas comparing to control groups (mean: 0.28 / 0.36). There was no obvious relationship between VEGF121/VEGF165 mRNA expression levels and the clinical parameters examined including age, pathology, differentiation, tumor size, lymphovascular space invasion, LN involvement and invasion depth except clinical stage (p<0.05). CONCLUSIONS: The overexpression of VEGF mRNA may be an important contributing factor in cervical carcinomas. There is no significant differenece of VEGF mRNAs levels according to clinical parameters, so it seems that the expression of VEGF is involved in the promotion of angiogenesis on cervical cancer and plays an important role in early invasion.

Polymorphisms of the 5'-Flanking Region of the Human Tumor Necrosis Factor-alpha Gene in Korean Patients with Crohn's Disease

PURPOSE: Recently, a key role of tumor necrosis factor (TNF) in the development of inflammatory bowel disease (IBD), especially Crohn's disease (CD), has emerged. In Japan, 3 single base pair polymorphisms in the 5'-flanking region of the TNF-alpha gene at position 1031, 863, and 857, which are related to high transcriptional promoter activity, have been identified in the Japanese CD patients. And the polymorphisms of the TNF-alpha gene at position 308, 238 have been reported in western CD patients. So, in order to find the same polymorphisms in Korean population and CD patients, the author evaluate the patients diagnosed with CD, ulcerative colitis (UC) and healthy controls (HCs). METHODS: Blood samples were obtained from 70 patients with CD, 72 patients with UC and 52 healthy controls. Polymorphisms in the TNF-alpha gene at their respective positions were analyzed by single strand conformational polymorphism (SSCP), and allele frequencies in CD and UC patients were compared with those in healthy controls. RESULTS: Allele frequencies of 1031C, 863A, and 857T in health controls were 18.3%, 8.7%, and 19.2%, respectively. Polymorphic allele frequencies of 1031C, 863A, 857T were 22.9%, 27.1%, and 24.3% in CD patients respectively. The frequencies at all 3 positions were higher in CD patients than in HCs. However, the frequency at 863A was statistically significant (P=0.000). The allele frequencies of 308A and 238A alleles were 0.7% and 3.6% in CD, 0.7% and 2.1% in UC, and 1.9% and 4.8% in HCs, respectively. The allele frequency of 1031C was significantly higher in B3 than in B2 (P=0.033). CONCLUSION: Polymorphisms of 5'-flanking region of the TNF-alpha at positions 1031 (T/C), 863 (C/A) and 857 (C/T) may be associated with susceptibility of CD.

Telomerase Activity in Gynecologic Cancers and correlation of HPV E6 gene expression in cervical cancer tissues / 대한산부인과학회잡지

OBJECT: In this study, to evaluate the putative role of telomerase in gynecologic malignancies (cervical ca, ovarian ca, endometrial ca), we measured telomerase activity in malignant gynecologic tumor tissues and normal tussues, and compared it with prognostic factors in cervical cancer. To evaluate the correlation of telomerase activity and human papillomavirus (HPV) infection in cervical cancer, the analysis of HPV E6 gene was performed. METHOD: Specimens were obtained from 51 women who underwent gynecologic radical operation and 13 normal tissues (from December 1995 to December 1996) in the Department of Obstetrics and Gynecology, Kyung-Hee Univ. Medical Center. With Telomerase PCR ELISA (Boehring Mannheim), modified TRAP (Telomere Repeat Amplication Protocol), we examined telomerase activity of 32 cervical carcinomas, 11 ovarian carcinomas, 8 endometrial carcinomas, 5 normal cervical tissues, 4 normal ovarian tissues and 4 normal endometrial tissues. The analysis of HPV E6 gene was performed by PCR amplication. We compared the abnormally high telomerase activity with prognostic factors, also compared the telomerase activity with the expression of HPV E6 gene in cervical cancer tissues. RESULT: We detected the abnormally high telomerase activity in all cervical carcinomas, 10 of 11 (90.9%) ovarian carcinomas, 6 of 8 (75.0%) endometrial carcinomas, but couldn't detect in each normal tissues. There was statistically no significant difference of telomerase activity levels according to age, clinical stage, pathology, differentiation, LN involvement, depth of invasion and tumor size except lymphovascular space invasion in cervical carcinomas (p<0.05). According to the analysis of HPV E6 gene, 29 of 32 (90.6%) in 32 cervical cancer tissues showed HPV E6 positivity. So it was considered that telomerase activation was closely related with the expression of HPV E6 gene. CONCLUSION: Telomerase activation is associated with immortalization or malignant transformation of gynecologic cancers. The expression of HPV E6 gene is considered to activate telomerase in cervical cancer.

Two dinucleotide repeat polymorphisms (AC/TG and GT/CA) in the 5' upstream region of the mouse tryptophan hydroxylase gene

Tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin biosynthesis, is primarily expressed in serotonergic neurons of the raphe nuclei. Simple tandem repeat polymorphisms, typically one to four nucleotides long, are tandemly repeated several times and often characterized by many alleles. To identify the presence of polymorphic repeats, we sequenced the 5'-upstream region of the mouse TPH gene. For the detection of any allelic variants, polymerase chain reaction, nonisotopic single-strand conformation polymophism, and DNA sequencing analyses of the tandem repeat sequences were performed using genomic DNA extracted from 60 ICR mice. Two dinucleotide repeats, 5'-(AC/TG)22-3' and 5'-(GT/CA)17-3', were identified at approximately -5.7 kb and -3.4 kb upstream from the transcriptional initiation site of the mouse TPH gene, respectively. Minor allelic variants, 5'-(AC/TG)21-3' and 5'-(GT/CA)18-3', were observed in heterozygous pairs from 3 of 60 and 1 of 60 ICR mice, respectively. The identification of these microsatellites in the mouse TPH promoter raises the possibility that identical and/or other polymorphic sequences might exist in the upstream region of the human TPH gene.

TGF-beta1 inhibition of apoptosis through the transcriptional up-regulation of Bcl-X(L) in human monocytic leukemia U937 cells

To characterize the TGF-beta1 response of monocytic leukemia cells, we analyzed the effects of TGF-beta1 on cell proliferation, differentiation, and apoptosis of human monoblastic U937 cells. Treatment of cells with TGF-beta1 in the absence of growth factors significantly enhanced cell viability. Flow cytometric analysis of DNA content and CD14 expression revealed that TGF-beta1 does not affect cell proliferation and differentiation. Consistent with these results was the finding that no transcriptional induction of Cdk inhibitors such as p21Waf1, p15Ink4b, and p27Kip1 was detected following TGF-beta1 treatment. Interestingly, however, pretreatment of TGF-beta1 significantly inhibited Fas-, DNA damage-, and growth factor deprivation-induced apoptosis. This antiapoptotic effect was totally abrogated by anti-TGF-beta1 antibody. Quantitative RT-PCR analysis demonstrated a dose- and time-dependent transcriptional up-regulation of Bcl-X(L), suggesting its implication in the TGF-1-mediated antiapoptotic pathway. We also observed elevated expression of c-Fos and PTEN/MMAC1. But, no detectable change was recognized in expression of c-Jun, Fas, Fadd, Fap-1, Bcl-2, and Bax. Taken together, our study shows that TGF-beta1 enhancement of cellular viability is associated with its antiapoptotic effect, which may result from the transcriptional up-regulation of Bcl-X(L).

Heterogeneous HBV mutants coexist in Korean hepatitis B patients

Although many hepatitis B virus (HBV) mutants have been found in all open reading frames since the precore defective mutant was initially reported, systematic investigations of diverse HBV mutant populations in hepatitis B patients have not been performed. Therefore, we examined whether heterogeneous mutant populations simultaneously exist in Korean hepatitis B patients. In order to detect hepatitis B virus mutants, we amplified a conserved core region and a surface antigen region of HBV DNA by PCR from sera of 27 Korean chronic hepatitis B patients, and then performed single strand conformational polymorphism analysis followed by DNA sequencing analysis. The results showed that heterogeneous HBV mutants in both regions were present in a single as well as in various hepatitis B patients. Sequence analysis revealed a defective interfering particle with missense mutation in the core region. We also found that two subtypes of adr and adw coexisted in a single patient. In addition, a point mutation causing a stop codon in the surface antigen region was observed. We are further analyzing the clinical implications of HBV mutants to identify their roles in the pathogenesis of chronic hepatic disorders induced by HBV.