- Email: [email protected]
Thr phosphorylation of B-catenin may regulate its abundance in native epithelia
line and 4 above. Twelve patients were alive without disease at a mean follow-up of 58 months. LOH at 17p was present in 12 patients (66%). LOH at 13q was documented in 8 patients (44%). LOH at 18q was also documented in 12 patients (66%). Seven tumors (38%) presented LOH at both 17p and 18q. All together 60 allelic losses were found (mean 3.3 / patient). None of the genetic alterations was sigmficantly correlated with resistance to CRT. Conclusions: This is the first report a high mutation rate of p53 and DCC in SCCA. Whether these alterations may contribute to resistance to CRT need to be further investigated, most likely in a multicentric trial. TSG implicated in colorectal cancer carcinogenesis seem also involved in SCCA progression.
Pancreatic samples exhibited strong cell membrane staining within the Islets of Langerhans. Liver samples exhibited strong staining in portal tract lymphocytes, Kupffer cells and endothelinm. Lymphocytes in all gastrointestinal tissues stained strongly. Tissues not incubated with anti-hENT1 mAb were not stained. Conclusion: We report a novel immunohistoebemical assay and the first known immunohistochemieal localization of hENT 1 in human gastrointestinal tissues. This localization supports functional studies that eqinlibrative NT proteins are predominately found on the basolateral aspect of epithehal cells. As the absence of hENT1 confers high level nucleoside analogue drug resistance, this assay may serve as an important predictive tool in nucleoside analogue based gastrointestinal cancer therapies. CKWW is the recipient of an AHFMR Clinical Fellowship.
M982 M979 Rapid Detection of the Mutations in the BRAF Gene Using Real-time PCR and Melting Curve Analysis Tsuneo Ikenoue
Hepatocyte growth factor (HGF) and proinflammatory cytokines induce gastrin in hepatic cells: an important link in the development of hepatocellular carcinoma (HCC) ?? Peter Konturek, Stanislaw Konturek, Felix Stickel, Karolina Bazela, Wladyslaw Bielanski, Jens F. Rehfeld, Eckhart G. Hahn, Detlef Sehuppan Background: Cirrhosis is the primary risk factor for the development of HCC. Certain growth factors such as HGF contribute to HCC development. Recently, a truphic effect of gastrin on bepatoma cells has been demonstrated implicating a role of gastrin in HCC pathogenesis. The aim of this study was to compare serum levels of progastrin, gastrin, HGF and the proinflammatory cytokines IL-8 and TNF~ in patients with HCC, liver cirrhosis without HCC and healthy controls. Methods: 39 pts with biopsy-proven HCC (61,5% alcoholic liver cirrhosis, 33,3% hepatitis C and 5,2% hepatitis B), 86 pts with cirrhosis alone and 96 ageand gender-matcbed healthy controls. Serum progastrin (P-G) and amidated gastrin were measured by RIA. HGF, ILo8 and TNFa by ELISA. For in vitro experiments, HepG2 hepatoma cells were incubated with HGF and TNFa. The expression of gastrin mRNA was assessed by RT-PCR. Results: In HCC mean basal serum levels of P-G, amidated gastrin and HGF were 173+/-20 pmol/L, 93+/-12 pmol/L and 820+/-73 pg/mL, respectively. In cirrhosis alone, the corresponding values were 1534/-25 pmolfL, 101 +/- 18 pmol/L and 560+/40 pg/mL, respectively, while the respective values in control subjects were 72.3 +/-5 and 12.4+/-1.7 pmol/L, 230+/-22 pg/mL Serum 11-8 and TNFa levels were similar in HCC and cirrhosis, and about three times higher than in healthy controls. In vitro HGF and TNFa increased gastrin mRNA expression in HepG2 cells dose-dependently. Conclusions: HGF and TNFa may contribute to HCC development through the upregulation of gastrin production in hepatocytes. Their increased expression m patients with cirrhosis and cirrhosis/ HCC imply these cytokines as potential promotors of hepatoearcinogenesis in patients with cirrhosis.
Rapid Detection of the Mutations in the BRAF Gene Using Real-time PCR and Melting Curve Analysis Tsuneo Ikenoue ~'2 Yoko Hikiha ~, Fumihiko Kanal2, Goichi Togo2, Tadashi Goto L 2, Masaynki Matsumura 1.2 Takao Kawabe2, and Masao Omata: 1. Division of Gastroenterology, The Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan 2. Department of Gastroenterology, University of Tokyo, Tokyo, Japan (Background/Aim) It has been reported that BRAF gene is mutated in 66% of melanomas and at lower frequency in various human cancers and that more than 80% of these mutation are T to A transversions at nucleotide 1796 (T1796A), leading to a substitution of valine by glutamic acid at 599 (V599E). The aim of the present study is to establish a new method for rapidly detecting the V599E mutations. (Methods) On the basis of the reported sequence of the BRAF gene, a set of primers for PCR (BRF-F in intron 14 and BRF-R in intron 15) which amplifys a 263-bp product and two kinds of probes (detection probe DP-BRF and anchor probe APBRF) were designed. The detection probe was 5' labeled with LC-Red 640 and 3' phosphorylated, and the anchor probe was 3' labeled with fluorescein isothiocyanate. The latter was located downstream of the former at a distance of 1 nucleotide. Real-time PCR and melting curve analysis was performed using a LightCycler with these primers and probes. Twelve colorectal and 8 gastric cancer cell lines were examined. (Results) LightCycler melting curve analysis could detect V599E mutant DNAs when they were present among wild-type DNAs at a levelof as little as 10%. We found the mutations in 1 of 12 (8%) colorectal cancer cell lines, but no mntaion was detected in 8 gastric cancer cell fines. (Conclusion) The real-time PCR and melting curve analysis which we established can be used as a simple method for rapidly detecting the BRAF V599E mutation. Furthermore, the analyses of gastrointestinal cancer cell lines suggest that the BRAF V599E mutation is involved in the progression of a small proportion of colorectal tumors but unlikely to be involved in gasm~ccarcinogenesis.
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Proteomie Monitoring of Hepatocellular Carcinoma using Patients' Sera Yujin Hoshida, Takayuki Kawakami, Motoyuki Otsuka, Naoya Hatano, Hisaaki Taniguchi, Shin-lchiro Nishimura, Shuntaro Obi, Shuichiro Shiina, Takao Kawabe, Masao Omata
Differential Expressions of Neuropilin-1 and VEGF-A in Barrett's esophagus, dysplasia and adenocarcinoma of the esophagus: A possible marker for the progression of espphageal adenocarcinoma Swamali Baneqee, Allan P. Weston, Rachel Cherian, Sushanta K. Banerjee
Background~Aim Hepatocelhilar carcinoma (HCC) involves numbers of heterogeneous genetic changes that hampers efficient monitoring of the status of cancer. To do this systematically, we used simultaneous quantitative analysis of protein expression profiles. Methods Using the sera of 7 patients with HCC before and after curative ablation of the tumor, we performed 2-D gel electrophoresis. From these gel images, we identified approximately 150 to 200 protein spots and quantified their densities in each gel. Averaged densities of duphcated experiments were designated as protein expression levels. Annotation of each spot was obtained from web-based public databases. Based on these data, proteins and samples were classified by unsupervised clustering methods. Proteins differently expressed after ablation were selected by supervtsed learning method. We also analyzed changes in relevance networks of proteins sharing similar expression profiles after ablation of HCC. Results Clustering of global protein expression showed that samples from same patient tended to accumulate in near cluster irrespective of the status of cancer, suggesting major influence of individual difference. Supervrsed method derived proteins with significant difference in expression levels after the treatment across all samples, including leucine-rich alpha-2-glycoprotein. Using expression profiles of these proteins, we can clearly discriminate the status with or without HCC. Leave-one-out cross validation resulted success classification rate of >80%. Comparison of relevance networks before and after ablation suggested systematic change in protein expressions according to the presence of HCC with information of post-translational modification of several glycoproteins such as apolipoproteni J. Conclusions Quantitative profiling of protein expression provides useful information to monitor the status of HCC.
BACKGROUND: Adenocarcinoma is a serious complication of Barrett's esophageal syndrome (BE). It develops sequentially from the epithelium of BE to columnar dysplasia and to carcinoma. However, the factor(s) responsible for the induction of these sequential events is(are) unclear. It is well documented that VEGF-A, and its co-receptor Neuropilin-1 (NRP1), play significant roles in the development of various cancers by increasing angiogenesis, or by protecting cancer cells from apoptotic death. AIM: The present study undertaken to deterrmne the status of VEGF-A and its co-receptor NRP-1 in pre-neoplastic and neoplastic stages of esophagus. EXPERIMENTAL DESIGN: Twenty four biopsy samples were studied. Out of 24, 14 samples were traditional Barrett and 10 samples were adenocarcinoma associated with Barrett change and dysplasia. VEGF-A and NRP-1 proteins were determined using anmunohistochemistry and mRNA by in situ hybridization using DIG-labeled VEGF-A and NRP-1 specific non-radioactive probes. RESULTS: VEGF-A protein and RNA was expressed in BE, columnar dysplasia, and adenoearcinoma specimens. However, the level of expression in pre-neoplastic specimens such as BE and columnar dysplasia, was significantly lower as compared to adenocarcinoma samples. Co-expression of both VEGF-A and NRP-1 was present in 3 BE samples (21%) and 9 adenocarcinoma samples (90%). The level of expression of NRP-1 was significantly lower in BE as compared to adenocarcinoma samples. VEGF-A and NRP-1 proteins or mRNA expressions were undetected in adjacent normal tissue sections. CONCLUSIONS: The results of this study suggest that both VEGF-A and NRP-1 may be involved in the angiogenesis related to the induction of esophageal neoplasia and its progression into adenocarcinoma.
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Frequent Allelic Losses on Chromosomes 17p and 18q in Squamous Cell Carcinoma of the Anus Pascal Gervaz, Dietrich Hahnloser, Bruce Wolff
Selective Ser/Thr Phosphorylation of B-Catenin May Regulate Its Abundance in Native Epithelia Shahid Umar, Famourou Kourouma, Andrew P. Morris, Joseph H. Sellin
Aim: Loss of heterozygosity (LOH) is one of the mechanisms responsible for inactivation of tumor suppressor genes (TSG). The molecular mechanisms involved in squamous cell carcinoma of the anus (SCCA) progression are virtually unknown. We hypothesized that some of them may contribute to resistance to chemoradiotherapy (CRT). The aim of this study was : 1) to characterize LOH on chromosomes 17p (p53), 18q (DCC), 13q (Rb), and 5q (APC) in SCCA; and 2) to determine whether specific patterns of LOH correlate with tumor recurrence after CRT. Methods: We retrieved the tumor specimen of 18 patients, all HIVnegative, who were diagnosed with SCCA in our institution between 1986 and 1999. LOH pattems were investigated with various primers in 4 loci associated with known tumorsuppressor genes: D18S35, D18S46 (DCC/Smad4); D13S270, D13S319 (Rb); D17S786, D17S513 (p53); and D5S346, D5S421 (APC). Results: There were 5 men and 13 women. The mean age was 55 (range 28-80) years. Fourteen tumors were located below the dentate
BACKGROUND: Increased [3-catenin abundance is a pivotal oncogenic stimulus in neoplastic transformation of the colon. APC mutations may be a causative factor m the increased [3catenin in many, but certainly not all, colonic polyps/tumors by preventing appropriate targeted Serfrhr phosphorylation and subsequent proteasomal degradation. We have previously shown increased [3-catenin, with its nuclear translocation and stimulation of downstream targets in a mouse model of hyperproliferation, transmissible murine colonic hyperplasia (TMCH). AIM: Determine the mechanisms regulating increased [3-catenin abundance during TMCH. METHODS: TMCH was induced by Citrobacter rodentium exposure. Westerns and immunoprecipitation (IP) studies were performed in distal colonic crypts isolated from normal and 12 days post-infected mice (at peak hyperproliferation/hyperplasia). RESULTS: APC abundance did not decrease, but increased during TMCH. Sequencing of exon 3, which encodes regulatory phosphorylation sites in [3-catenin, did not show any
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Purpose: The purpose of this work is to isolate novel, functional pancreatic cancer-tumor suppressor genes located in the genome at 12q21.31-.32 and at 12q23.2. These regions of the genome are frequently lost in pancreatic cancer (Kimura M. et al, Cancer Research. 58:2456-60, 1998). Methods: To identify functional tumor suppressor gene(s) at 12q, we have introduced normal human DNA mapping to 12q21.31-.32 and to 12q23.2 into the human pancreatic cancer cell lines, Panc-1, which contains the same allehe loss at 12q. Introduction of tumor suppressor genes may alter the oncogenic phenotype of the Panc-1 cells. We used normal human DNA cloned into Yeast Artificial Chromosomes (YACs), which were retrofitted with a mammalian cell selectable marker (NeaR). Four YAC clones were used in transfection experiments with Panc-1 cells and Neo resistant cells were selected. Results: These experiments resulted in the successful isolation of NeaR cell lines with two of the YAC clones. One of these YACs was a control (Y807E4) and the other was Y909H1, containing a portion of the DNA from 12q21.31-.32. Y909H1 did not slow the growth of cells based on the cell doubling time, and MTT assays. However, we did observe a 2 fold decrease in the ability to form colonies in soft agar in some of the Y909H1 cell isolates. We are currently replicating these experiments to determine if these results are reproducible. Two other YAC clones, one mapping to the other part of 12q21.31-32 (Y790F8) and the other mapping to 12q23.2 (Y721G9) did not yield NeaR cells. While this result could be due to technical difficulties, it is interesting that transfection was successful with two other YAC clones. Active tumor suppressor genes may inhibit the ability of cells to grow complicating their functional isolation. Repetition of these expenments is currently in progress. Conclusions: Mild tumor suppressor activity may be contained in the 1.55 MB YAC 909H1. Growth inhibitory activity may be located on Y721G9 and Y790F8 based on our inability to isolate YAC transformants. Additional experiments are necessary to confirm and extend these conclusions.
mutations. Neither axin nor GSK-36 levels were altered. IP failed to show significant alterations in association of [3-catenin with GSK-3~ or axin. GSK-3[3 activity did not change significantly during day 12 TMCH. SerfI'hr phosphorylated [3-catenin (P6 -catenin) was minimally detectable in normal crypt. At day 12, however, a dramatic increase in Thr41/ Ser45 phosphorylated [~-catenin, a putative phosphorylation site for casein kinase 1, was observed, while the primary GSK-36-phosphorylation site for degradation (Ser33/Ser37 residues), did not show increased phosphorylation, compared to control. IP with anti-p 6catenin followed by blotting with anti-ubiquitin exhibited decreased polyubiquitinated 6catenin at day 12, compared to normal. CONCLUSIONS: 1. Phosphorylation at Thr41/ Ser45 residues may he critical for [3-catenin stability in native epithelia. 2. A disruption of the APC-GSK3[3-axin mediated degradation pathway, perhaps related to a decreased ubiquinnation, may account for increased [3-catenin during TMCH. SIGNIFICANCE: In native epithelia, APC-independent mechanisms of increased [3-cateinn can lead to nuclear translocation, suggesting that Ser/Thr phosphorylated [3-catenin may be an oncogenic factor.
M985 [B-Catenin Is Consistently Overexpressed in Squamous Neoplasia of the Esophagus and Is Not Due To Altered Full-Length APC Expression Michiko lwamoto, Mark Roth, Dennis J. Ahnen Accumulation of 6-catenin in the cytosol and nucleus of colonic neoplasia occurs commonly and is thought to be due to bi-anelic trucating mutations, LOH or hypermethylation of the APC gene, all of which would lead to loss of full length APC protein. The expression of these 2 proteins is not well characterized in other GI cancers. Purpose- To assess the expression of ~-catenin and full length APC protein in the normal esophagus and in esophageal dyspLasia and cancer. Methods- 154 samples of normal esophagus, 54 samples containing squamous dysplasia and 100 samples of squamous cancer of the esophagus were stained by immunohistochemistry using a mouse monoclonal antibody to 6-catenin and a rabbit polyclonal antibody to the C-terminal 20 amino acids of the APC protein (this antibody does not detect trucated APC). Positive staining was defined as over 10% of the cells having definite immunoreactiviry. Results- Weak full length APC immunoreactwiey was found in the mature squamons cells at the lumenal surface but none was seen in the deeper layers of the normal mucnsa or in any of the dysplastic or cancer samples. 13-catenin was present, predominantly at the plasma membrane, of all the samples of normal esophagus; only faint cytoplasmic and no nuclear immunoreactivity was present in the normal esophagus. All samples of squamous dysplasLa had evidence of cytoplasmic 13-catenin immunoreactivity and 40% also had nuclear immanoreactivity. Similarly all esophageal squamons cancers had evidence of cytoplasmic t3-catenin immunoreactivity and 30% also had nuclear immunorcactivity. Conclusions- Full length APC protein is not expressed at high levels in the normal or neoplastic esophagus. 6-cateinn accumulation in the cytoplasm and nucleus occurs commonly in esophageal dysplasia and cancer. The mechanism for this accumulation is not known but it does not appear to be due to alterations in the expression of full length APC protein in these tissues. 6-catenin immunohistochemistry could he a useful addition to standard histology for the diagnosis of squamons dysplasia of the esophagus.
M988 Differential "granscriprional Regulation of Human Telomerase in a CeUular Model of Esophageal Squamous Carcinogenesis M~chael Quante, Alexander Van Werder, Gitta Goessel, Birgit Jacobmeier, Yasir Suliman, Roderick Beijershergen, Anil K. Rnstgi, Hubert E. Bhim, Oliver G. Opitz Normal human somatic cells have a limited replicative life span before undergoing senescence. Erosion of telomeric DNA is a key factor in repheative senescence. Cellular immortalization occurs as long as telomeres are maintained. In fact, most human cancer ceils including esophageal cancer cells maintain their telomeres through activation of telomerase. Little is known about the differential regulation of telomerase during the distinct steps of sqnamons carcinogenesis, the process from normal to immortalized to malignant transformed cells. Therefore, we aimed to investigate the transcriptional regulation of _hutnan telomerase in a cellular model of esophageal carcinogenesis. Cyclin D1 (EPC-D1) or hTERT (EPC-hTERT) overexpressing cells were generated by retroviral transduction from normal human esophageal sqnamons epithelial cells (EPC). Growth characteristics, telomerase activity (TRAP-Assay) and telomere length (TRF) of these cells as well as esophageal cancer cells (TE-12) were assessed regularly. Full length (1242bp) and deletion constructs of the hTERT promoter(in pGL3)were generated by a PCR-based strategy, and transiently transfected by an improved lipofectamin method. Transfection results were correlated with telomerase activity and telomere length, hTERT-promoter activity was highest in esophagcal cancer cells, even higher than SV40-promoter activity, which served as positive control. Promoter activity was about 10-40 fold increased in immortalized cells (EPC-D1/EPC-hTERT) compared to the empty vector control. In EPC D1 celia promoter activity was dependent on cellular density. Dissection of the promoter activity by deletion constructs revealed that a 5 pnme silencer region as well as core promoter region regulated hTERT promoter activity in cancer cells. Whereas the silencer region was important for promoter reguLation in EPC-hTERT ceils as well, only part of the core promoter containing the classical myc-binding site determined the promoter activity. In EPC-D1 cells the silencer region did not pLay a role in promoter regulation, and a more 3 pnme region of the core promoter containing multiple Spl sites was repressed. In summary the human TERT promoter is differentially regulated in esophageal carcinogenesis depending on the transformation/immortalization status of esophageal epithelial cells. Furthermore, the respective pathway inducing cellular immortalization seems to play an important role in the regulation.
M986 Frequent Loss of RUNX3 Gene Expression in Human Bile Duct and Pancreatic Cancer Cell Lines Manabu Wada, Shojiro Yazumi, Shigeo Takaishi, Kazunori Hasegawa, Mitsutaka Sawada, Hiroshi Ida, Chohei Sakakura, Kosei lto, Yoshiaki Ira, Tsutomu Chiba Introduction: RUNX3, a Runt domain transcription factor involved in TGF-6 signaling, is a candidate tumor suppressor gene localized in lp36, a region commonly deleted in a wide variety of human tumors, including those of the stomach, bile duct, and pancreas. Recently, it has been demonstrated that frequent inactivation of RUNX3 is found in human gastric carcinomas, and that RUNX3-null mice develop hyperplasia of the gastric mucosa through activation of cellular proliferation and suppression of apoptosis in epithelial cells. Multiple tumor suppressor pathways are known to be abrogated in bile duct and pancreatic caminomas, and disreguLation of the TGF-6/Smad signahng pathway apparently plays an important role in the pathogenesis of both pancreatic and bile duct carcinomas. In this study, we examined involvement of RUNX3 abnormalities in tumorigenesis of bile duct as well as pancreatic cancers. Material & Methods: We investigated not only expression but also methylation state of RUNX3 in human 10 bile duct and 12 pancreatic cancer cell lines. Result: Seven out of 10 (70%) of the bile duct and 9 of 12 (75%) of the pancreatic cancer cell lines exhibited no expression of RUNX3 by both Nonhem blot analysis and RT-PCR. All of the 16 cell lines that did not express RUNX3 showed methylation of the promoter CpG island of the gene, whereas the 6 cell lines with RUNX3 gene expression were not methylated or only partially methylated in the RUNX3 promoter region. Moreover, treatment with the methylation inhibitor, 5'-aza-2'-deoxycitidine, induced RUNX3 mRNA expression in all of 16 cancer cell lines that originally lacked RUNX3 expression. Finally, hemizygous deletion of RUNX3, as detected by fluorescence in situ hybridization, was found in 15 of the 16 cancer cell lines that lacked RUNX3 expression. Conclusion: We have demonstrated for the first time that RUNX3expression is frequently lost in human bile duct and pancreatic cancer celllines and that methylation of the promoter region and hemizygons deletion are responsible for inactivating the gene. Thus, inactivation of RUNX3 appears to play an important role in tumorigenesis of bile duct and pancreatic carcinomas.
M989 Minichromosome Maintenance Protein 2 Plays a Critical Role in Cellular Proliferation and Senescence Hideki Harada, Hiroshi Nakagawa, Oliver G. Opitz lmroduction: DNA replication is regulated in a carefully orchestrated fashion during discrete phases of the cell cycle. Critical to this process are the minichromosome maintenance proteins (MCM), which are licensing factors that ensure DNA replication occurs only once per cell cycle. In addition, MCM2 protein is deregulated in a variety of cancers and thought to be a useful marker in that context. We have examined the functional propenies of MCM2 protein during senescence, proliferation and transformation in esophageal cells. Methods: MCM2 RNA and protein expression was examined by RT-PCR, Northern blot and Western blot in primary human esophageal epithelial cells (EPC1 and EPC2) and esophageal cancer cell lines (TEl and 3). The chromatin-bindmg assay was employed to detect MCM2 protein bound to chromatin. [3H]Thymidine incorporation assay was done to evaluate DNA synthesis and cell proliferation. Results: The expression of MCM2 (120 kDa) was downregnlated in EPC1 and EPC2 after serial passages and absent in senescent cells, suggesting that it associates with cell proliferation. By contrast, we found that a novel lower molecular mass (60 kDa) isoform of MCM2 was upreguhted after serial passages and abundant in senescent EPC1 and EPC2. Interestingly, this 60 kDa MCM2 isoform was not expressed in TEl and TE3. We also found that there is a reciprocal relationship between the two isoforms in terms of cell confluence and call proliferation in EPC2 as analyzed by the [3H] thymidine incorporation assay. Northern blot demonstrated single transcript of MCM2 in EPC2, indicating that the alternative isoform might be induced through translational or posttranslational modifications. The chromatin-binding assay revealed that the 60 kDa isoform was bound to chromatin together with the 120 kDa MCM2 isoform, suggesting the coordinated regulation of DNA
M987 A Functional Approach for the Identification of a Pancreatic Cancer Tumor Suppressor Gene at 12q21-23 Young-Mi Park, Kay L. Pogue-Geile Background: Loss of heterozygosity studies of pancreatic cancer suggests that many unknown pancreatic cancer tumor suppressor genes are involved in pancreatic cancer. The identification of these genes is critical to the understanding and successful treatment of pancreatic cancer.
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Abstracts
Pancreatic samples exhibited strong cell membrane staining within the Islets of Langerhans. Liver samples exhibited strong staining in portal tract lymphocytes, Kupffer cells and endothelinm. Lymphocytes in all gastrointestinal tissues stained strongly. Tissues not incubated with anti-hENT1 mAb were not stained. Conclusion: We report a novel immunohistoebemical assay and the first known immunohistochemieal localization of hENT 1 in human gastrointestinal tissues. This localization supports functional studies that eqinlibrative NT proteins are predominately found on the basolateral aspect of epithehal cells. As the absence of hENT1 confers high level nucleoside analogue drug resistance, this assay may serve as an important predictive tool in nucleoside analogue based gastrointestinal cancer therapies. CKWW is the recipient of an AHFMR Clinical Fellowship.
M982 M979 Rapid Detection of the Mutations in the BRAF Gene Using Real-time PCR and Melting Curve Analysis Tsuneo Ikenoue
Hepatocyte growth factor (HGF) and proinflammatory cytokines induce gastrin in hepatic cells: an important link in the development of hepatocellular carcinoma (HCC) ?? Peter Konturek, Stanislaw Konturek, Felix Stickel, Karolina Bazela, Wladyslaw Bielanski, Jens F. Rehfeld, Eckhart G. Hahn, Detlef Sehuppan Background: Cirrhosis is the primary risk factor for the development of HCC. Certain growth factors such as HGF contribute to HCC development. Recently, a truphic effect of gastrin on bepatoma cells has been demonstrated implicating a role of gastrin in HCC pathogenesis. The aim of this study was to compare serum levels of progastrin, gastrin, HGF and the proinflammatory cytokines IL-8 and TNF~ in patients with HCC, liver cirrhosis without HCC and healthy controls. Methods: 39 pts with biopsy-proven HCC (61,5% alcoholic liver cirrhosis, 33,3% hepatitis C and 5,2% hepatitis B), 86 pts with cirrhosis alone and 96 ageand gender-matcbed healthy controls. Serum progastrin (P-G) and amidated gastrin were measured by RIA. HGF, ILo8 and TNFa by ELISA. For in vitro experiments, HepG2 hepatoma cells were incubated with HGF and TNFa. The expression of gastrin mRNA was assessed by RT-PCR. Results: In HCC mean basal serum levels of P-G, amidated gastrin and HGF were 173+/-20 pmol/L, 93+/-12 pmol/L and 820+/-73 pg/mL, respectively. In cirrhosis alone, the corresponding values were 1534/-25 pmolfL, 101 +/- 18 pmol/L and 560+/40 pg/mL, respectively, while the respective values in control subjects were 72.3 +/-5 and 12.4+/-1.7 pmol/L, 230+/-22 pg/mL Serum 11-8 and TNFa levels were similar in HCC and cirrhosis, and about three times higher than in healthy controls. In vitro HGF and TNFa increased gastrin mRNA expression in HepG2 cells dose-dependently. Conclusions: HGF and TNFa may contribute to HCC development through the upregulation of gastrin production in hepatocytes. Their increased expression m patients with cirrhosis and cirrhosis/ HCC imply these cytokines as potential promotors of hepatoearcinogenesis in patients with cirrhosis.
Rapid Detection of the Mutations in the BRAF Gene Using Real-time PCR and Melting Curve Analysis Tsuneo Ikenoue ~'2 Yoko Hikiha ~, Fumihiko Kanal2, Goichi Togo2, Tadashi Goto L 2, Masaynki Matsumura 1.2 Takao Kawabe2, and Masao Omata: 1. Division of Gastroenterology, The Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan 2. Department of Gastroenterology, University of Tokyo, Tokyo, Japan (Background/Aim) It has been reported that BRAF gene is mutated in 66% of melanomas and at lower frequency in various human cancers and that more than 80% of these mutation are T to A transversions at nucleotide 1796 (T1796A), leading to a substitution of valine by glutamic acid at 599 (V599E). The aim of the present study is to establish a new method for rapidly detecting the V599E mutations. (Methods) On the basis of the reported sequence of the BRAF gene, a set of primers for PCR (BRF-F in intron 14 and BRF-R in intron 15) which amplifys a 263-bp product and two kinds of probes (detection probe DP-BRF and anchor probe APBRF) were designed. The detection probe was 5' labeled with LC-Red 640 and 3' phosphorylated, and the anchor probe was 3' labeled with fluorescein isothiocyanate. The latter was located downstream of the former at a distance of 1 nucleotide. Real-time PCR and melting curve analysis was performed using a LightCycler with these primers and probes. Twelve colorectal and 8 gastric cancer cell lines were examined. (Results) LightCycler melting curve analysis could detect V599E mutant DNAs when they were present among wild-type DNAs at a levelof as little as 10%. We found the mutations in 1 of 12 (8%) colorectal cancer cell lines, but no mntaion was detected in 8 gastric cancer cell fines. (Conclusion) The real-time PCR and melting curve analysis which we established can be used as a simple method for rapidly detecting the BRAF V599E mutation. Furthermore, the analyses of gastrointestinal cancer cell lines suggest that the BRAF V599E mutation is involved in the progression of a small proportion of colorectal tumors but unlikely to be involved in gasm~ccarcinogenesis.
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Proteomie Monitoring of Hepatocellular Carcinoma using Patients' Sera Yujin Hoshida, Takayuki Kawakami, Motoyuki Otsuka, Naoya Hatano, Hisaaki Taniguchi, Shin-lchiro Nishimura, Shuntaro Obi, Shuichiro Shiina, Takao Kawabe, Masao Omata
Differential Expressions of Neuropilin-1 and VEGF-A in Barrett's esophagus, dysplasia and adenocarcinoma of the esophagus: A possible marker for the progression of espphageal adenocarcinoma Swamali Baneqee, Allan P. Weston, Rachel Cherian, Sushanta K. Banerjee
Background~Aim Hepatocelhilar carcinoma (HCC) involves numbers of heterogeneous genetic changes that hampers efficient monitoring of the status of cancer. To do this systematically, we used simultaneous quantitative analysis of protein expression profiles. Methods Using the sera of 7 patients with HCC before and after curative ablation of the tumor, we performed 2-D gel electrophoresis. From these gel images, we identified approximately 150 to 200 protein spots and quantified their densities in each gel. Averaged densities of duphcated experiments were designated as protein expression levels. Annotation of each spot was obtained from web-based public databases. Based on these data, proteins and samples were classified by unsupervised clustering methods. Proteins differently expressed after ablation were selected by supervtsed learning method. We also analyzed changes in relevance networks of proteins sharing similar expression profiles after ablation of HCC. Results Clustering of global protein expression showed that samples from same patient tended to accumulate in near cluster irrespective of the status of cancer, suggesting major influence of individual difference. Supervrsed method derived proteins with significant difference in expression levels after the treatment across all samples, including leucine-rich alpha-2-glycoprotein. Using expression profiles of these proteins, we can clearly discriminate the status with or without HCC. Leave-one-out cross validation resulted success classification rate of >80%. Comparison of relevance networks before and after ablation suggested systematic change in protein expressions according to the presence of HCC with information of post-translational modification of several glycoproteins such as apolipoproteni J. Conclusions Quantitative profiling of protein expression provides useful information to monitor the status of HCC.
BACKGROUND: Adenocarcinoma is a serious complication of Barrett's esophageal syndrome (BE). It develops sequentially from the epithelium of BE to columnar dysplasia and to carcinoma. However, the factor(s) responsible for the induction of these sequential events is(are) unclear. It is well documented that VEGF-A, and its co-receptor Neuropilin-1 (NRP1), play significant roles in the development of various cancers by increasing angiogenesis, or by protecting cancer cells from apoptotic death. AIM: The present study undertaken to deterrmne the status of VEGF-A and its co-receptor NRP-1 in pre-neoplastic and neoplastic stages of esophagus. EXPERIMENTAL DESIGN: Twenty four biopsy samples were studied. Out of 24, 14 samples were traditional Barrett and 10 samples were adenocarcinoma associated with Barrett change and dysplasia. VEGF-A and NRP-1 proteins were determined using anmunohistochemistry and mRNA by in situ hybridization using DIG-labeled VEGF-A and NRP-1 specific non-radioactive probes. RESULTS: VEGF-A protein and RNA was expressed in BE, columnar dysplasia, and adenoearcinoma specimens. However, the level of expression in pre-neoplastic specimens such as BE and columnar dysplasia, was significantly lower as compared to adenocarcinoma samples. Co-expression of both VEGF-A and NRP-1 was present in 3 BE samples (21%) and 9 adenocarcinoma samples (90%). The level of expression of NRP-1 was significantly lower in BE as compared to adenocarcinoma samples. VEGF-A and NRP-1 proteins or mRNA expressions were undetected in adjacent normal tissue sections. CONCLUSIONS: The results of this study suggest that both VEGF-A and NRP-1 may be involved in the angiogenesis related to the induction of esophageal neoplasia and its progression into adenocarcinoma.
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M984
Frequent Allelic Losses on Chromosomes 17p and 18q in Squamous Cell Carcinoma of the Anus Pascal Gervaz, Dietrich Hahnloser, Bruce Wolff
Selective Ser/Thr Phosphorylation of B-Catenin May Regulate Its Abundance in Native Epithelia Shahid Umar, Famourou Kourouma, Andrew P. Morris, Joseph H. Sellin
Aim: Loss of heterozygosity (LOH) is one of the mechanisms responsible for inactivation of tumor suppressor genes (TSG). The molecular mechanisms involved in squamous cell carcinoma of the anus (SCCA) progression are virtually unknown. We hypothesized that some of them may contribute to resistance to chemoradiotherapy (CRT). The aim of this study was : 1) to characterize LOH on chromosomes 17p (p53), 18q (DCC), 13q (Rb), and 5q (APC) in SCCA; and 2) to determine whether specific patterns of LOH correlate with tumor recurrence after CRT. Methods: We retrieved the tumor specimen of 18 patients, all HIVnegative, who were diagnosed with SCCA in our institution between 1986 and 1999. LOH pattems were investigated with various primers in 4 loci associated with known tumorsuppressor genes: D18S35, D18S46 (DCC/Smad4); D13S270, D13S319 (Rb); D17S786, D17S513 (p53); and D5S346, D5S421 (APC). Results: There were 5 men and 13 women. The mean age was 55 (range 28-80) years. Fourteen tumors were located below the dentate
BACKGROUND: Increased [3-catenin abundance is a pivotal oncogenic stimulus in neoplastic transformation of the colon. APC mutations may be a causative factor m the increased [3catenin in many, but certainly not all, colonic polyps/tumors by preventing appropriate targeted Serfrhr phosphorylation and subsequent proteasomal degradation. We have previously shown increased [3-catenin, with its nuclear translocation and stimulation of downstream targets in a mouse model of hyperproliferation, transmissible murine colonic hyperplasia (TMCH). AIM: Determine the mechanisms regulating increased [3-catenin abundance during TMCH. METHODS: TMCH was induced by Citrobacter rodentium exposure. Westerns and immunoprecipitation (IP) studies were performed in distal colonic crypts isolated from normal and 12 days post-infected mice (at peak hyperproliferation/hyperplasia). RESULTS: APC abundance did not decrease, but increased during TMCH. Sequencing of exon 3, which encodes regulatory phosphorylation sites in [3-catenin, did not show any
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Purpose: The purpose of this work is to isolate novel, functional pancreatic cancer-tumor suppressor genes located in the genome at 12q21.31-.32 and at 12q23.2. These regions of the genome are frequently lost in pancreatic cancer (Kimura M. et al, Cancer Research. 58:2456-60, 1998). Methods: To identify functional tumor suppressor gene(s) at 12q, we have introduced normal human DNA mapping to 12q21.31-.32 and to 12q23.2 into the human pancreatic cancer cell lines, Panc-1, which contains the same allehe loss at 12q. Introduction of tumor suppressor genes may alter the oncogenic phenotype of the Panc-1 cells. We used normal human DNA cloned into Yeast Artificial Chromosomes (YACs), which were retrofitted with a mammalian cell selectable marker (NeaR). Four YAC clones were used in transfection experiments with Panc-1 cells and Neo resistant cells were selected. Results: These experiments resulted in the successful isolation of NeaR cell lines with two of the YAC clones. One of these YACs was a control (Y807E4) and the other was Y909H1, containing a portion of the DNA from 12q21.31-.32. Y909H1 did not slow the growth of cells based on the cell doubling time, and MTT assays. However, we did observe a 2 fold decrease in the ability to form colonies in soft agar in some of the Y909H1 cell isolates. We are currently replicating these experiments to determine if these results are reproducible. Two other YAC clones, one mapping to the other part of 12q21.31-32 (Y790F8) and the other mapping to 12q23.2 (Y721G9) did not yield NeaR cells. While this result could be due to technical difficulties, it is interesting that transfection was successful with two other YAC clones. Active tumor suppressor genes may inhibit the ability of cells to grow complicating their functional isolation. Repetition of these expenments is currently in progress. Conclusions: Mild tumor suppressor activity may be contained in the 1.55 MB YAC 909H1. Growth inhibitory activity may be located on Y721G9 and Y790F8 based on our inability to isolate YAC transformants. Additional experiments are necessary to confirm and extend these conclusions.
mutations. Neither axin nor GSK-36 levels were altered. IP failed to show significant alterations in association of [3-catenin with GSK-3~ or axin. GSK-3[3 activity did not change significantly during day 12 TMCH. SerfI'hr phosphorylated [3-catenin (P6 -catenin) was minimally detectable in normal crypt. At day 12, however, a dramatic increase in Thr41/ Ser45 phosphorylated [~-catenin, a putative phosphorylation site for casein kinase 1, was observed, while the primary GSK-36-phosphorylation site for degradation (Ser33/Ser37 residues), did not show increased phosphorylation, compared to control. IP with anti-p 6catenin followed by blotting with anti-ubiquitin exhibited decreased polyubiquitinated 6catenin at day 12, compared to normal. CONCLUSIONS: 1. Phosphorylation at Thr41/ Ser45 residues may he critical for [3-catenin stability in native epithelia. 2. A disruption of the APC-GSK3[3-axin mediated degradation pathway, perhaps related to a decreased ubiquinnation, may account for increased [3-catenin during TMCH. SIGNIFICANCE: In native epithelia, APC-independent mechanisms of increased [3-cateinn can lead to nuclear translocation, suggesting that Ser/Thr phosphorylated [3-catenin may be an oncogenic factor.
M985 [B-Catenin Is Consistently Overexpressed in Squamous Neoplasia of the Esophagus and Is Not Due To Altered Full-Length APC Expression Michiko lwamoto, Mark Roth, Dennis J. Ahnen Accumulation of 6-catenin in the cytosol and nucleus of colonic neoplasia occurs commonly and is thought to be due to bi-anelic trucating mutations, LOH or hypermethylation of the APC gene, all of which would lead to loss of full length APC protein. The expression of these 2 proteins is not well characterized in other GI cancers. Purpose- To assess the expression of ~-catenin and full length APC protein in the normal esophagus and in esophageal dyspLasia and cancer. Methods- 154 samples of normal esophagus, 54 samples containing squamous dysplasia and 100 samples of squamous cancer of the esophagus were stained by immunohistochemistry using a mouse monoclonal antibody to 6-catenin and a rabbit polyclonal antibody to the C-terminal 20 amino acids of the APC protein (this antibody does not detect trucated APC). Positive staining was defined as over 10% of the cells having definite immunoreactiviry. Results- Weak full length APC immunoreactwiey was found in the mature squamons cells at the lumenal surface but none was seen in the deeper layers of the normal mucnsa or in any of the dysplastic or cancer samples. 13-catenin was present, predominantly at the plasma membrane, of all the samples of normal esophagus; only faint cytoplasmic and no nuclear immunoreactivity was present in the normal esophagus. All samples of squamous dysplasLa had evidence of cytoplasmic 13-catenin immunoreactivity and 40% also had nuclear immanoreactivity. Similarly all esophageal squamons cancers had evidence of cytoplasmic t3-catenin immunoreactivity and 30% also had nuclear immunorcactivity. Conclusions- Full length APC protein is not expressed at high levels in the normal or neoplastic esophagus. 6-cateinn accumulation in the cytoplasm and nucleus occurs commonly in esophageal dysplasia and cancer. The mechanism for this accumulation is not known but it does not appear to be due to alterations in the expression of full length APC protein in these tissues. 6-catenin immunohistochemistry could he a useful addition to standard histology for the diagnosis of squamons dysplasia of the esophagus.
M988 Differential "granscriprional Regulation of Human Telomerase in a CeUular Model of Esophageal Squamous Carcinogenesis M~chael Quante, Alexander Van Werder, Gitta Goessel, Birgit Jacobmeier, Yasir Suliman, Roderick Beijershergen, Anil K. Rnstgi, Hubert E. Bhim, Oliver G. Opitz Normal human somatic cells have a limited replicative life span before undergoing senescence. Erosion of telomeric DNA is a key factor in repheative senescence. Cellular immortalization occurs as long as telomeres are maintained. In fact, most human cancer ceils including esophageal cancer cells maintain their telomeres through activation of telomerase. Little is known about the differential regulation of telomerase during the distinct steps of sqnamons carcinogenesis, the process from normal to immortalized to malignant transformed cells. Therefore, we aimed to investigate the transcriptional regulation of _hutnan telomerase in a cellular model of esophageal carcinogenesis. Cyclin D1 (EPC-D1) or hTERT (EPC-hTERT) overexpressing cells were generated by retroviral transduction from normal human esophageal sqnamons epithelial cells (EPC). Growth characteristics, telomerase activity (TRAP-Assay) and telomere length (TRF) of these cells as well as esophageal cancer cells (TE-12) were assessed regularly. Full length (1242bp) and deletion constructs of the hTERT promoter(in pGL3)were generated by a PCR-based strategy, and transiently transfected by an improved lipofectamin method. Transfection results were correlated with telomerase activity and telomere length, hTERT-promoter activity was highest in esophagcal cancer cells, even higher than SV40-promoter activity, which served as positive control. Promoter activity was about 10-40 fold increased in immortalized cells (EPC-D1/EPC-hTERT) compared to the empty vector control. In EPC D1 celia promoter activity was dependent on cellular density. Dissection of the promoter activity by deletion constructs revealed that a 5 pnme silencer region as well as core promoter region regulated hTERT promoter activity in cancer cells. Whereas the silencer region was important for promoter reguLation in EPC-hTERT ceils as well, only part of the core promoter containing the classical myc-binding site determined the promoter activity. In EPC-D1 cells the silencer region did not pLay a role in promoter regulation, and a more 3 pnme region of the core promoter containing multiple Spl sites was repressed. In summary the human TERT promoter is differentially regulated in esophageal carcinogenesis depending on the transformation/immortalization status of esophageal epithelial cells. Furthermore, the respective pathway inducing cellular immortalization seems to play an important role in the regulation.
M986 Frequent Loss of RUNX3 Gene Expression in Human Bile Duct and Pancreatic Cancer Cell Lines Manabu Wada, Shojiro Yazumi, Shigeo Takaishi, Kazunori Hasegawa, Mitsutaka Sawada, Hiroshi Ida, Chohei Sakakura, Kosei lto, Yoshiaki Ira, Tsutomu Chiba Introduction: RUNX3, a Runt domain transcription factor involved in TGF-6 signaling, is a candidate tumor suppressor gene localized in lp36, a region commonly deleted in a wide variety of human tumors, including those of the stomach, bile duct, and pancreas. Recently, it has been demonstrated that frequent inactivation of RUNX3 is found in human gastric carcinomas, and that RUNX3-null mice develop hyperplasia of the gastric mucosa through activation of cellular proliferation and suppression of apoptosis in epithelial cells. Multiple tumor suppressor pathways are known to be abrogated in bile duct and pancreatic caminomas, and disreguLation of the TGF-6/Smad signahng pathway apparently plays an important role in the pathogenesis of both pancreatic and bile duct carcinomas. In this study, we examined involvement of RUNX3 abnormalities in tumorigenesis of bile duct as well as pancreatic cancers. Material & Methods: We investigated not only expression but also methylation state of RUNX3 in human 10 bile duct and 12 pancreatic cancer cell lines. Result: Seven out of 10 (70%) of the bile duct and 9 of 12 (75%) of the pancreatic cancer cell lines exhibited no expression of RUNX3 by both Nonhem blot analysis and RT-PCR. All of the 16 cell lines that did not express RUNX3 showed methylation of the promoter CpG island of the gene, whereas the 6 cell lines with RUNX3 gene expression were not methylated or only partially methylated in the RUNX3 promoter region. Moreover, treatment with the methylation inhibitor, 5'-aza-2'-deoxycitidine, induced RUNX3 mRNA expression in all of 16 cancer cell lines that originally lacked RUNX3 expression. Finally, hemizygous deletion of RUNX3, as detected by fluorescence in situ hybridization, was found in 15 of the 16 cancer cell lines that lacked RUNX3 expression. Conclusion: We have demonstrated for the first time that RUNX3expression is frequently lost in human bile duct and pancreatic cancer celllines and that methylation of the promoter region and hemizygons deletion are responsible for inactivating the gene. Thus, inactivation of RUNX3 appears to play an important role in tumorigenesis of bile duct and pancreatic carcinomas.
M989 Minichromosome Maintenance Protein 2 Plays a Critical Role in Cellular Proliferation and Senescence Hideki Harada, Hiroshi Nakagawa, Oliver G. Opitz lmroduction: DNA replication is regulated in a carefully orchestrated fashion during discrete phases of the cell cycle. Critical to this process are the minichromosome maintenance proteins (MCM), which are licensing factors that ensure DNA replication occurs only once per cell cycle. In addition, MCM2 protein is deregulated in a variety of cancers and thought to be a useful marker in that context. We have examined the functional propenies of MCM2 protein during senescence, proliferation and transformation in esophageal cells. Methods: MCM2 RNA and protein expression was examined by RT-PCR, Northern blot and Western blot in primary human esophageal epithelial cells (EPC1 and EPC2) and esophageal cancer cell lines (TEl and 3). The chromatin-bindmg assay was employed to detect MCM2 protein bound to chromatin. [3H]Thymidine incorporation assay was done to evaluate DNA synthesis and cell proliferation. Results: The expression of MCM2 (120 kDa) was downregnlated in EPC1 and EPC2 after serial passages and absent in senescent cells, suggesting that it associates with cell proliferation. By contrast, we found that a novel lower molecular mass (60 kDa) isoform of MCM2 was upreguhted after serial passages and abundant in senescent EPC1 and EPC2. Interestingly, this 60 kDa MCM2 isoform was not expressed in TEl and TE3. We also found that there is a reciprocal relationship between the two isoforms in terms of cell confluence and call proliferation in EPC2 as analyzed by the [3H] thymidine incorporation assay. Northern blot demonstrated single transcript of MCM2 in EPC2, indicating that the alternative isoform might be induced through translational or posttranslational modifications. The chromatin-binding assay revealed that the 60 kDa isoform was bound to chromatin together with the 120 kDa MCM2 isoform, suggesting the coordinated regulation of DNA
M987 A Functional Approach for the Identification of a Pancreatic Cancer Tumor Suppressor Gene at 12q21-23 Young-Mi Park, Kay L. Pogue-Geile Background: Loss of heterozygosity studies of pancreatic cancer suggests that many unknown pancreatic cancer tumor suppressor genes are involved in pancreatic cancer. The identification of these genes is critical to the understanding and successful treatment of pancreatic cancer.
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Abstracts