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1.  Induction of Premalignant Host Responses by Cathepsin X/Z-Deficiency in Helicobacter Pylori-Infected Mice 
PLoS ONE  2013;8(7):e70242.
Helicobacter pylori are responsible for the induction of chronic gastric inflammation progressing to atrophy, metaplasia, and gastric cancer. The overexpression of Cathepsin X/Z (Ctsz) in H. pylori-infected mucosa and gastric cancer is mediated predominantly by an augmented migration of ctsz−/−positive macrophages and the up-regulation of Ctsz in tumor epithelium. To explore the Ctsz-function in the context of chronic inflammation and the development of preneoplastic lesions, we used Ctsz-deficient mice in a H. pylori gastritis model. Ctsz−/− and wild-type (wt) mice were infected with H. pylori strain SS1. The mice were sacrificed at 24, 36, and 50 weeks post infection (wpi). The stomach was removed, and gastric strips were snap-frozen or embedded and stained with H&E. Tissue sections were scored for epithelial lesions and inflammation. Ki-67 and F4/80 immunostaining were used to measure epithelial cell proliferation and macrophage infiltration, respectively. The upregulation of compensating cathepsins and cytokines were confirmed by Western blotting and quantitative RT-PCR. SS1-infected wt and ctsz−/− mice showed strong inflammation, foveolar hyperplasia, atrophy, and cystically-dilated glands. However, at 50 wpi, ctsz−/− mice developed significantly more severe spasmolytic polypeptide-expressing metaplasia (SPEM), showed enhanced epithelial proliferation, and higher levels of infiltrating macrophages. Induction of cytokines was higher and significantly prolonged in ctsz−/− mice compared to wt. Ctsz deficiency supports H. pylori-dependent development of chronic gastritis up to metaplasia, indicating a protective, but not proteolytic, function of Ctsz in inflammatory gastric disease.
PMCID: PMC3728094  PMID: 23936173
2.  Role of tight junction proteins in gastroesophageal reflux disease 
BMC Gastroenterology  2012;12:128.
Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD.
Eighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)].
Histopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and −2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD.
Taken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD.
PMCID: PMC3503771  PMID: 22994974
Gastroesophageal reflux disease; Tight junction; Claudins; Esophagitis; Inflammation
3.  The Transmembrane Adaptor Protein SIT Inhibits TCR-Mediated Signaling 
PLoS ONE  2011;6(9):e23761.
Transmembrane adaptor proteins (TRAPs) organize signaling complexes at the plasma membrane, and thus function as critical linkers and integrators of signaling cascades downstream of antigen receptors. We have previously shown that the transmembrane adaptor protein SIT regulates the threshold for thymocyte selection. Moreover, T cells from SIT-deficient mice are hyperresponsive to CD3 stimulation and undergo enhanced lymphopenia-induced homeostatic proliferation, thus indicating that SIT inhibits TCR-mediated signaling. Here, we have further addressed how SIT regulates signaling cascades in T cells. We demonstrate that the loss of SIT enhances TCR-mediated Akt activation and increased phosphorylation/inactivation of Foxo1, a transcription factor of the Forkhead family that inhibits cell cycle progression and regulates T-cell homeostasis. We have also shown that CD4+ T cells from SIT-deficient mice display increased CD69 and CD40L expression indicating an altered activation status. Additional biochemical analyses further revealed that suppression of SIT expression by RNAi in human T cells resulted in an enhanced proximal TCR signaling. In summary, the data identify SIT as an important modulator of TCR-mediated signaling that regulates T-cell activation, homeostasis and tolerance.
PMCID: PMC3177817  PMID: 21957439
4.  Epigenetic and Genetic Silencing of CHFR in Esophageal Adenocarcinomas 
Cancer  2010;116(17):4033-4042.
The checkpoint with Forkhead-associated domain (FHA) and Ring finger domain (CHFR) is a mitotic checkpoint protein with tumor-suppressor functions. In this study, we investigated the epigenetic and genetic mechanisms that regulate CFHR expression in esophageal adenocarcinomas (EACs). Quantitative real-time RT-PCR analysis demonstrated down-regulation of CHFR transcript in 79% of EACs (44/56) as compared to 41 normal samples (P<.001). Immunohistochemical analysis of CHFR protein expression showed absence or weak immunostaining for CHFR in 75% of EACs (56/75), as compared to normal tissue samples. We next examined the promoter DNA hypermethylation of CHFR using quantitative bisulfite pyrosequencing technology. We detected significant CHFR promoter DNA hypermethylation in 31% of tumor samples (18/58), as compared to normal samples (P<.001). Treatment of OE33 cells with 5-Aza-deoxycytidine led to reduction in the promoter DNA methylation levels with restoration of the CHFR mRNA expression, confirming promoter DNA methylation as an epigenetic mechanism regulating CHFR expression. However, we identified several EACs where the CHFR mRNA expression was silenced in absence of notable methylation. Therefore, we examined the relative DNA copy number level of CHFR, as compared to normal samples. The results confirmed a decrease or absence of the relative CHFR DNA copy number levels in 59% of tumor samples. Nine tumors showing loss of CHFR mRNA expression, in absence of promoter DNA hypermethylation, demonstrated a significant loss of relative CHFR DNA copy numbers. Taken together, our findings demonstrate that both epigenetic and genetic mechanisms are involved in silencing CHFR expression in EACs.
PMCID: PMC2930066  PMID: 20564104
methylation; copy numbers; CHFR; esophageal; Barrett's; cancer
5.  Promoter methylation of CDKN2A and lack of p16 expression characterize patients with hepatocellular carcinoma 
BMC Cancer  2010;10:317.
The product of CDKN2A, p16 is an essential regulator of the cell cycle controlling the entry into the S-phase. Herein, we evaluated CDKN2A promoter methylation and p16 protein expression for the differentiation of hepatocellular carcinoma (HCC) from other liver tumors.
Tumor and corresponding non-tumor liver tissue samples were obtained from 85 patients with liver tumors. CDKN2A promoter methylation was studied using MethyLight technique and methylation-specific PCR (MSP). In the MethyLight analysis, samples with ≥ 4% of PMR (percentage of methylated reference) were regarded as hypermethylated. p16 expression was evaluated by immunohistochemistry in tissue sections (n = 148) obtained from 81 patients using an immunoreactivity score (IRS) ranging from 0 (no expression) to 6 (strong expression).
Hypermethylation of the CDKN2A promoter was found in 23 HCCs (69.7%; mean PMR = 42.34 ± 27.8%), six (20.7%; mean PMR = 31.85 ± 18%) liver metastases and in the extralesional tissue of only one patient. Using MSP, 32% of the non-tumor (n = 85), 70% of the HCCs, 40% of the CCCs and 24% of the liver metastases were hypermethylated. Correspondingly, nuclear p16 expression was found immunohistochemically in five (10.9%, mean IRS = 0.5) HCCs, 23 (92%; mean IRS = 4.9) metastases and only occasionally in hepatocytes of non-lesional liver tissues (mean IRS = 1.2). The difference of CDKN2A-methylation and p16 protein expression between HCCs and liver metastases was statistically significant (p < 0.01, respectively).
Promoter methylation of CDKN2A gene and lack of p16 expression characterize patients with HCC.
PMCID: PMC2927998  PMID: 20569442
6.  DNA hypermethylation regulates the expression of members of the Mu-class Glutathione-S-Transferases and Glutathione Peroxidases in Barrett's adenocarcinoma 
Gut  2008;58(1):5-15.
The accumulation of reactive oxygen species and subsequent oxidative DNA damage underlie the development of Barrett's esophagus (BE) and its progression to Barrett's dysplasia (BD) and adenocarcinoma (BAC).
We systematically analyzed the promoter regions of 23 genes of the Glutathione S-transferase (GST) and Glutathione peroxidase (GPX) families. Quantitative bisulfite pyrosequencing, real-time RT-PCR (qRT-PCR), Western blot, and immunohistochemical (IHC) analysis methods were utilized in this study.
We identified 14 genes that have CpG islands around their transcription start sites; GSTs (M2-M5, A4, P1, Z1, T2, O1-O2) and GPXs (GPX1, GPX3, GPX4, GPX7). Analysis of an initial set of 20 primary samples demonstrated promoter DNA hypermethylation and mRNA down-regulation of GPX3, GPX7, GSTM2, GSTM3, and GSTM5 in more than half of the BACs samples. Further analysis of 159 primary human samples (37 normal, 11 BE, 11 BD, and 100 BACs) indicated frequent hypermethylation (≥10% methylation) of GPX3 (62%), GPX7 (67%), GSTM2 (69.1%), and GSTM3 (15%) in BACs. A significant inverse correlation between DNA methylation and mRNA expression level was shown for GPX3 (P<.0001), GPX7 (P=.002), GSTM2 (P<.0001), and GSTM5 (P=.01). Treatment of esophageal cancer cell lines with 5-Aza-2’-deoxycytidine and Trichostatin-A led to reversal of the methylation pattern and re-expression of these genes at the mRNA and protein levels. The IHC analysis of GPX3, GPX7, and GSTM2 on a tissue microarray that contained 75 BACs with normal squamous esophageal samples demonstrated an absent-to-weak staining in tumors (52% for GPX3, 57% for GPX7, and 45% for GSTM2) and a moderate-to-strong immunostaining in normal samples.
Epigenetic inactivation of members of the glutathione pathway can be an important mechanism in Barrett's tumorigenesis.
PMCID: PMC2845391  PMID: 18664505
DNA methylation; Glutathione-S-Transferases; Glutathione Peroxidases; Esophagus; Barrett's adenocarcinoma
7.  APC promoter methylation and protein expression in hepatocellular carcinoma 
We investigated the impact of promoter methylation on APC protein expression in patients with hepatocellular carcinoma (HCC).
Materials and methods
50 patients [HCC (n=19), liver metastasis (n=19), cholangiocellular cancer (n=7), and benign liver tumors (n=5)] were studied for methylation using Methylight analysis. APC mutation was investigated by protein truncation test and direct sequencing of genomic DNA. The protein expression was evaluated by immunohistochemistry and Western blot analysis.
The APC promoter was hypermethylated in 81.8% of non-cancerous liver tissue samples. All HCC samples and ten patients with liver metastasis (52.6%) exhibited APC promoter methylation. The degree of methylation was significantly higher in samples from HCC compared to the non-cancerous liver tissue samples (63.1% vs. 24.98%; p=0.001). The level of APC protein expression was significantly reduced in HCC samples compared to that of the corresponding non-tumor liver tissue (p<0.05).
Promoter methylation of the APC gene seems to be of significance in hepatocarcinogenesis and results in reduced protein expression in HCC. Interestingly, APC promoter methylation is also present in the vast majority of non-cancerous liver tissue whose (patho)physiological function remains unresolved.
PMCID: PMC2757596  PMID: 17973119
Promoter methylation; APC; Methylight assay; Protein; Hepatocellular carcinoma
8.  Early Involvement of Death-Associated Protein Kinase Promoter Hypermethylation in the Carcinogenesis of Barrett's Esophageal Adenocarcinoma and Its Association with Clinical Progression1 
Neoplasia (New York, N.Y.)  2007;9(3):236-245.
Esophageal Barrett's adenocarcinoma (BA) develops through a multistage process, which is associated with the transcriptional silencing of tumor-suppressor genes by promoter CpG island hypermethylation. In this study, we explored the promoter hypermethylation and protein expression of proapoptotic deathassociated protein kinase (DAPK) during the multistep Barrett's carcinogenesis cascade. Early BA and paired samples of premalignant lesions of 61 patients were analyzed by methylation-specific polymerase chain reaction and immunohistochemistry. For the association of clinicopathological markers and protein expression, an immunohistochemical tissue microarray analysis of 66 additional BAs of advanced tumor stages was performed. Hypermethylation of DAPK promoter was detected in 20% of normal mucosa, 50% of Barrett's metaplasia, 53% of dysplasia, and 60% of adenocarcinomas, and resulted in a marked decrease in DAPK protein expression (P < .01). The loss of DAPK protein was significantly associated with advanced depth of tumor invasion and advanced tumor stages (P < .001). Moreover, the severity of reflux esophagitis correlated significantly with the hypermethylation rate of the DAPK promoter (P < .003). Thus, we consider DAPK inactivation by promoter hypermethylation as an early event in Barrett's carcinogenesis and suggest that a decreased protein expression of DAPK likely plays a role in the development and progression of BA.
PMCID: PMC1838580  PMID: 17401463
Barrett's adenocarcinoma; Barrett's metaplasia; DAPK; reflux esophagitis; inflammation
9.  Hypermethylation and Loss of Expression of Glutathione Peroxidase-3 in Barrett's Tumorigenesis1 
Neoplasia (New York, N.Y.)  2005;7(9):854-861.
Chronic gastroesophageal reflux disease is a known risk factor for Barrett's esophagus (BE), that induces oxidative mucosal damage. Glutathione peroxidase-3 (GPx3) is a secretory protein with potent extracellular antioxidant activity. Herein, we have investigated the mRNA and protein expression of GPx3, and explored promoter hypermethylation as an epigenetic mechanism for GPx3 gene inactivation during Barrett's carcinogenesis. Quantitative real-time reverse transcription polymerase chain reaction on 42 Barrett's adenocarcinomas (BAs) revealed consistently reduced levels of GPx3 mRNA in 91% of tumor samples. GPx3 promoter hypermethylation was detected in 62% of Barrett's metaplasia, 82% of dysplasia, and 88% of BA samples. Hypermethylation of both alleles of GPx3 was most frequently seen in BA (P = .001). Immunohistochemical staining of GPx3 in matching tissue sections (normal, BE, Barrett's dysplasia, and BA) revealed a weak-to-absent GPx3 staining in Barrett's dysplasia and adenocarcinoma samples where the promoter was hypermethylated. The degree of loss of immuno-histochemistry correlated with the hypermethylation pattern (monoallelic versus biallelic). The observed high frequency of promoter hypermethylation and progressive loss of GPx3 expression in BA and its associated lesions, together with its known function as a potent antioxidant, suggest that epigenetic inactivation and regulation of glutathione pathway may be critical in the development and progression of BE.
PMCID: PMC1501938  PMID: 16229808
Barrett's; dysplasia; cancer; GPx3; ROS
10.  Selective Loss of Codon 72 Proline p53 and Frequent Mutational Inactivation of the Retained Arginine Allele in Colorectal Cancer1 
Neoplasia (New York, N.Y.)  2004;6(5):529-535.
According to recent reports, some cancer types exhibit nonrandom allele loss at codon 72 in exon 4 of the p53 gene [coding for proline (72Pro) or arginine (72Arg)]. To clarify this phenomenon for colorectal cancer and to find out if this preferential loss might have any functional significance, p53 loss of heterozygosity (LOH) and p53 mutations were investigated in a group of 61 colorectal cancers and 28 liver metastases, and were correlated with clinicopathologic factors. A comparison of a patient's blood codon 72 status with a healthy control group did not reveal an enhanced risk of developing colorectal tumors for one of the two isoforms. p53-LOH and p53 mutations were found in 62.2% and 39.4% of primary tumors, respectively, and in 57.9% and 25% of hepatic metastases, respectively. In 14 heterozygous cases showing exon 4-LOH, only the 72Pro allele was lost and the retained 72Arg was preferentially mutated. In general, p53 mutations were significantly associated with the 72Arg tumor status (P < .001). Distal tumors showed allelic losses of the p53 gene more commonly than proximal tumors (P = .054). The prevalence of 72Arg increased in frequency with higher Dukes stage (P = .056). We suggest that either the preferential loss of 72Pro or the mutation of the 72Arg in colorectal cancer and hepatic metastases is associated with malignant potential and might reflect carcinogenic exposure, particularly in the distal part of the large intestines.
PMCID: PMC1531656  PMID: 15548361
Codon 72 polymorphism; p53-LOH; p53 mutation; colorectal cancer; allelic loss
11.  Helicobacter pylori-Mediated Gastritis Induces Local Downregulation of Secretory Leukocyte Protease Inhibitor in the Antrum  
Infection and Immunity  2004;72(4):2383-2385.
Helicobacter pylori-infected subjects exhibited a strong decline in antral secretory leukocyte protease inhibitor (SLPI) levels compared to H. pylori-negative subjects and subjects from whom H. pylori had been eradicated (P = 0.002). This reduction was specific for the antrum, whereas SLPI expression in corpus and duodenum was not affected. Antral SLPI levels were inversely correlated with inflammatory scores of antrum-predominant gastritis.
PMCID: PMC375164  PMID: 15039364
12.  ATF2 knockdown reinforces oxidative stress-induced apoptosis in TE7 cancer cells 
Cancer cells showing low apoptotic effects following oxidative stress-induced DNA damage are mainly affected by growth arrest. Thus, recent studies focus on improving anti-cancer therapies by increasing apoptosis sensitivity. We aimed at identifying a universal molecule as potential target to enhance oxidative stress-based anti-cancer therapy through a switch from cell cycle arrest to apoptosis. A cDNA microarray was performed with hydrogen peroxide-treated oesophageal squamous epithelial cancer cells TE7. This cell line showed checkpoint activation via p21WAF1, but low apoptotic response following DNA damage. The potential target molecule was chosen depended on the following demands: it should regulate DNA damage response, cell cycle and apoptosis. As the transcription factor ATF2 is implicated in all these processes, we focused on this protein. We investigated checkpoint activation via ATF2. Indeed, ATF2 knockdown revealed ATF2-triggered p21WAF1 protein expression, suggesting p21WAF1 transactivation through ATF2. Using chromatin immunoprecipitation (ChIP), we identified a hitherto unknown ATF2-binding sequence in the p21WAF1 promoter. p-ATF2 was found to interact with p-c-Jun, creating the AP-1 complex. Moreover, ATF2 knockdown led to c-Jun downregulation. This suggests ATF2-driven induction of c-Jun expression, thereby enhancing ATF2 transcriptional activity via c-Jun-ATF2 heterodimerization. Notably, downregulation of ATF2 caused a switch from cell cycle arrest to reinforced apoptosis, presumably via p21WAF1 downregulation, confirming the importance of ATF2 in the establishment of cell cycle arrest. 1-Chloro-2,4-dinitrobenzene also led to ATF2-dependent G2/M arrest, suggesting that this is a general feature induced by oxidative stress. As ATF2 knockdown also increased apoptosis, we propose ATF2 as a target for combined oxidative stress-based anti-cancer therapies.
PMCID: PMC3780530  PMID: 23800081
oxidative stress-induced DNA damage; cell cycle arrest; ATF2 knockdown; increase in apoptosis sensitivities; combined treatment; p21WAF1

Results 1-12 (12)