Epigenetic DNA modification by aberrant methylation of cytosine residues is thought to be an important mechanism contributing to tumorigenesis. Methylation of cytosines normally occurs at distinct sites of the genome containing stretches of repeated CpG (CpG islands) often found within promoter areas of transcribed genes. The cytosine methylation pattern is established very early in development by a continuous process of demethylation and de novo methylation (for review see refs. 1,2). Normally, methylation patterns are faithfully maintained through all subsequent cell divisions and are dependent on DNA methyltransferase activity (3). It has been observed, however, that tumour cells often show extensive upregulation of DNA methyltransferase and at the same time hypomethylation of CpG sites (4). The mechanism of this apparent deregulation in cancer cells is not clear but is generally thought that de novo methylation of otherwise nonmethylated genes is the active component of functional disturbance in cancer (4-6). Methylated islands will recruit special methyl-binding proteins and in conjunction with histone deacetylases are then thought to form repressive chromatin states around the promoter regions, leading to transcriptional loss of genes residing downstream (7). If important genes reside within this region loss of functional control in cell proliferation will ensue. It is therefore not surprising that de novo methylation found in cancer includes many tumor-suppressor genes known to date, thus forming an alternative to gene silencing by inactivating deletions (8). KeywordsAberrant MethylationBisulfite ConversionPromoter AreaSubsequent Cell DivisionUnmethylated AlleleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Introduction and Aims Serous and mucinous cystic pancreatic tumors have different clinical behavior. We evaluated whether they also have genotypic differences by analyses of the tumor suppressor genes p16INK4a, p53, and DPC4. Methodology Seven serous cystadenomas (SCA) and seven malignant mucinous cystadenocarcinomas (MCC) were analyzed for alterations in the tumor suppressor genes p16INK4a, p53, and DPC4 by single-strand conformational variant analysis, direct sequencing, and immunohistochemical analysis. Methylation-specific polymerase chain reaction analysis was performed to identify p16INK4a promoter hypermethylation. Clinical data were compared with genetic data. Results None of the seven patients with SCAs but five of the seven patients with MCCs died of the tumor after a median follow-up of 44.5 months (range, 4–169 months). All seven MCCs had alterations in at least one tumor suppressor gene compared with none of the seven SCAs. Of the seven MCCs, three had inactivating p16INK4a promoter hypermethylation, five had p53 alterations, and three had DPC4 mutations. Conclusions The tumor suppressor genes p16INK4a, p53, and DPC4 appear to play an important role in the tumorigenesis of MCCs but not SCAs. These molecular data underscore the clinical and histologic differences of serous and mucinous cystic pancreatic tumors.

Objective To identify the prognostic relevance of the G1/S cell cycle regulator genes p16INK4a, p53, MDM2, and Rb in patients with resected ductal pancreatic cancer (PC). Summary Background Data The tumor suppressor genes p16INK4a, p53, and Rb are altered in PC in 27% to 95%, 40% to 70%, and 5%, respectively. The role of MDM2 is not clearly defined in PC. The prognostic value of these cell cycle regulators has not been clarified. Methods Sixty-two patients with PC with complete follow-up who underwent potentially curative resections were included in the study. An extreme group analysis was performed including the 20 patients with the shortest survival and the 20 patients with the longest survival. Protein expression of p16, p53, MDM2, and Rb was investigated, and mutation analysis of p16INK4a and p53 was performed. p16INK4a promoter hypermethylation was examined by methylation-specific polymerase chain reaction. Results Significantly more tumors in the shortest-surviving patients had p16INK4a alterations compared with tumors of the longest-surviving patients. In contrast, the frequency of p53 alterations was not significantly higher in the shortest-surviving versus the longest-surviving groups. Stabilization of MDM2 and loss of Rb expression were identified in a minority of tumors, independent of survival length. Conclusions The presence of p16INK4a alterations in resected tumors of patients with PC is connected with a worse prognosis, indicating patients that might benefit from adjuvant therapy regimens. p53 alterations, MDM2 overexpression, and loss of Rb expression could not be identified as prognostic markers from this study, but a larger study with greater statistical power might show a different result with regard to p53.

Tumor suppressors that are known to be altered in ductal pancreatic adenocarcinoma (p16 INK4a , TP53 and DPC4) are also identified to be altered in malignant cystic pancreatic tumors in contrast to benign tumors by the present study. Thus alterations of tumor suppressors in cystic pancreatic tumors could be identified as strong indicators of malignancy in the tumor panel of the study.

Functional inactivation of the Rb and p53 pathways appears to be a rite of passage for all cancerous cells. However, p53 and Rb alterations are rare events in neuroendocrine gastroenteropancreatic (GEP) tumors. The CDKN2 locus on chromosome 9p21 sits at the nexus of both pathways harboring tumor suppressor genes, which restrain cell growth by affecting the function of pRb and p53. Therefore, we analyzed the implication of their inactivation in 37 primary neuroendocrine GEP tumors and two cell culture models. RT-PCR analysis revealed loss of expression of at least one of the tumor suppressor genes CDKN2A/p16, CDKN2B/p15, and CDKN2D/p14 with distinct genetic profiles, most frequently in nonfunctional pancreatic tumors (57%) and small intestinal carcinoids (44%), and less commonly in insulinomas (30%) and gastrinomas (22%). DNA analysis and methylation-specific PCR attributed loss of expression to either homozygous deletion or 5'CpG island hypermethylation. 5-Aza-2-deoxycytidine treatment reversed CDKN2A/p16 and CDKN2B/p15 silencing with concurrent growth restraint. Thus, tumor suppressor genes localized in the 9p21 gene cluster are specific targets of inactivation in neuroendocrine GEP tumors, and demethylating agents might hold promise for selective therapy.

Background.p16INK4a alterations are considered to be an early event in pancreatic tumorigenesis and have been described in duct lesions adjacent to pancreatic cancers. This study evaluates whether duct lesions in chronic pancreatitis tissues of patients without pancreatic cancer also harbor genetic alterations in the p16INK4a tumor-suppressor gene, and thus represent high-risk precursors for pancreatic cancer. Methods. Tissues were obtained from 20 pancreatic specimens taken from patients operated on for histologically verified chronic pancreatitis. Pancreatic intraductal neoplasias (PanIN) were identified in hematoxylin-and-eosin-stained slides. p16 protein expression was investigated immunohistochemically in all specimens. DNA from PanIN and non-PanIN tissue was analyzed genetically for p16INK4a mutations by single-strand conformation variation analysis and direct sequencing of the encoding region. Additionally, p16INK4a promoter methylation was analyzed by a methylation specific polymerase test. Results. PanIN-1a lesions were identified in 10 of the 20 chronic pancreatitis specimens. Four of these 10 PanIN specimens (40%), but none of the 20 non-PanIN tissues, revealed a loss of p16 expression in immunohistochemistry. The mutational analysis of the p16INK4a gene showed 1 known polymorphism (c.442G > A; A148T) but no mutations. Two of the 10 specimens with PanIN revealed an inactivating hypermethylation of the p16INK4a promoter. Conclusions. This study shows for the first time that p16INK4a alterations can be observed in a considerable number of PanIN1 in chronic pancreatitis tissues not associated with pancreatic cancer. Therefore, p16INK4a alterations, especially promoter methylation, might indicate high-risk precursors in chronic pancreatitis that might progress to cancer. (Surgery 2001;129:490-7.)

Multiple primary tumors in pancreatic cancer patients might indicate a genetic predisposition to the development of malignancies. In this study we evaluated whether the mutation rate of the TP53 and p16INK4a genes of pancreatic cancers differs in pancreatic cancer patients with and without multiple primaries. Furthermore, we investigated whether pancreatic cancer patients with multiple primaries carry germline mutations in either p16INK4a, TP53, or BRCA2 tumor suppressor genes to detect a genetic alteration that predisposes to the development of different primaries. Fourteen (23%) of 60 pancreatic cancer patients developed histologically verified additional primaries during their lifetimes. Normal constitutional and tumor DNA of the 14 patients with a positive cancer history, but negative family history, were analyzed for p16INK4a, TP53, and BRCA2 mutations by single-strand conformational variant (SSCV) analysis and direct sequencing. Hypermethylation of the p16INK4a promoter region in pancreatic cancers was identified by methylation-specific polymerase chain reaction (PCR; MSP). Four of 14 pancreatic carcinomas carried somatic intragenic p16INK4a mutations, and another four tumors revealed hypermethylation of the p16INK4a promoter region. Somatic intragenic TP53 mutations were identified in six of 14 tumors. None of the pancreatic cancer patients carried TP53 or BRCA2 germline mutations. In contrast, one of 14 pancreatic cancer patients with multiple primaries carried the p16INK4a mutation A68V in his germline. This mutation was localized in the conserved second ankyrin repeat of p16INK4a and did not occur in 100 control patients. The frequency of somatic TP53 and p16INK4a mutations in pancreatic cancer is similar in patients with and without multiple primaries. TP53 and BRCA2 germline mutations seem not to be significantly associated with the occurrence of multiple primaries in pancreatic cancer patients. However, p16INK4a germline mutations might be causative for tumor development in some pancreatic cancer patients with multiple primaries. The genetic investigation of patients with accumulation of different cancers even without a positive family history may be a new approach for the understanding of the relation of different cancers.

Purpose: The aim of this study was to investigate the frequency of three (epi)genetic alterations (p53 and K-ras mutations and p16 INK4a promoter hypermethylation) in symptomatic chronic smokers compared with patients with lung cancer and to evaluate the use of exfoliative material for such analyses. Patients and Methods: Fifty-one patients with histologically confirmed lung cancer and 25 chronic smokers (> 20 pack-years) were investigated for mutations in the K-ras (codon 12) and p53 (codons 248, 249, and 273) genes and for allelic hypermethylation of the p16 INK4a gene. DNA was isolated from sputum and bilateral bronchial lavage, and brushings were taken at bronchoscopy. Results: Forty-one genetic lesions were detected within exfoliative material from the group of 51 patients with lung cancer and 10 lesions in the chronic smoker group. K-ras mutations occurred exclusively in the lung cancer group, whereas p53 mutations and p16 INK4a promoter hypermethylation were also found in chronic smokers. Three of eight chronic smokers who harbored an (epi)genetic alteration were subsequently diagnosed with lung cancer. Analysis of sputum yielded information equivalent to that of samples obtained during bronchoscopy. Conclusion: p16 INK4a promoter hypermethylation and p53 mutations can occur in chronic smokers before any clinical evidence of neoplasia and may be indicative of an increased risk of developing lung cancer or of early disease. K-ras mutations occur exclusively in the presence of clinically detectable neoplastic transformation. Molecular analysis of sputum for such markers may provide an effective means of screening chronic smokers to enable earlier detection and therapeutic intervention of lung cancer. J Clin Oncol 18:3221-3229. © 2000 by American Society of Clinical Oncology.

Background Interleukin S (IL-S), a member of the CXC chemokine family, is a multifunctional cytokine shown to induce angiogenesis, haptotactic migration, and proliferation of certain cancer cells.IL-S is presumed to exert its action after binding to its specific receptors (IL-SRA and IL-SRB).Previously, we have reported that matrix metalloproteinase-9 (MMP-9) was increased by treatment with IL-S in gastric cancer cells.The aims of this study was to examine whether IL-S and IL-S receptors were expressed in human pancreatic cancer and to evaluate the role of IL-S on metastatic potential in pancreatic cancer.Methods The expression of IL-S, IL-SRA and IL-SRB in human pancreatic cancer cells (PANC-I, MIA PaCa-2, Capan-2) were examined by northern blot analysis and western blot analysis.The expressions of IL-S and its receptors were also investigated by immunohistochemistry in human pancreatic cancer tissues using specific antibody.Cytokine induced secretions of IL-S in supernatants of cultured medium from cells were investigated with ELISA (R&D).After treatment with various concentrations of recombinant IL-S (Sigma), the cell proliferations were assayed using Cell Counting Kit™ (Dojindo Labs., Japan).IL-S induced production and gelatinolytic activity of MMP-2 and MMP-9 were analyzed by western blot analysis and gelatin zymogram using conditioned mediums from each cell as samples.Results The expression of IL-S, IL-SRA and IL-SRB were detected in all investigated pancreatic cancer cells.Moreover, the secretion of IL-S was increased with cytokine stimuli.In addition, immunoreactive signals of IL-8 and its receptors were observed in cancer tissues.•Exogenous adding of IL-S did not induce proliferation of human pancreatic cancer cell lines.Surprisingly, the production and gelatinolytic activity ofMMP-2 and MMP-9 were increased by the treatment of recombinant IL-S in a dose-dependent manner.Conclusion These results suggest that IL-S regulate MMP activity and may play an important role in the invasiveness of human pancreatic cancer.

Background/Aims: The molecular mechanisms contributing to the tumorigenesis of insulinomas are poorly understood. Disruption of the cell cycle due to inactivation of the p16INK4a tumor-suppressor gene was identified in a variety of human tumors, including gastrinomas and nonfunctioning endocrine pancreatic carcinomas. In this study the role of p16INK4a in the tumorigenesis of insulinomas was evaluated. Methods: Seventeen insulinomas (14 benign, 3 malignant) were analyzed for genetic alterations in the p16INK4a tumor-suppressor gene by SSCP, PCR-based deletion and methylation-specific assays. p16 expression was determined by immunohistochemistry. Results: One malignant insulinoma showed a homozygous deletion of p16INK4a and another two benign insulinomas revealed aberrant methylation of the p16INK4a promoter region. All three tumors lacked p16 expression according to immunohistochemistry. None of the insulinomas carried intragenic p16INK4a mutations. In total, 17% of insulinomas had p16INK4a alterations. Conclusions: The p16INK4a tumor-suppressor gene contributes to tumorigenesis in only a small subset of insulinomas.

Endokrine Pankreastumoren sind selten und machen etwa 1–5% a11er Pankreastumoren aus. 90% der endokrinen Pankreastumoren, vor allem Insulinome und Gastrinome, sind durch eine extensive Hormonsekretion charakterisiert. Bei 10 % der endokrinen Pankreastumoren handelt es sich um nicht-funktionelle Tumoren ohne Hormonsekretion [1]. Etwa 10–20% der endokrinen Pankreastumoren sind mit der autosomal dominant vererbten Multiplen Endokrinen Neoplasie Typ 1 assoziiert [2]. Das Wissen um genetische Veränderungen, die mit der Initiierung and Progression der Inselzelltumoren einhergehen, ist sehr beschränkt und die vorliegenden Daten basieren auf sehr kleinen Fallzahlen. Das p16 INK4a Tumorsuppressorgen auf Chromosom 9p21 ist bei einer Vielzahl humaner Tumoren, einschließlich denen des exokrinen Pankreas, durch Mutation, homozygote Deletion oder aberrante Promotorhypermethylierung inaktiviert [3, 4]. Aufgrund der engen anatomischen Beziehung zwischen endokrinem und exokrinem Pankreas haben wir 10 nicht-funktionelle Inselzellkarzinome und 8 Gastrinome auf Alterationen im p16 INK4a Tumorsuppressorgen untersucht.

Background/Aim: Molecular mechanisms contributing to the tumorigenesis of neuroendocrine pancreatic tumors are still poorly understood. Therfore we evaluated the role of the p16(INK4) tumor suppressor gene for the genesis of these tumors. Methods: Ten non-functioning islet cell carcinomas and 8 gastrinomas were analysed for alterations in the p16(INK4a) tumor suppressor gene by SSCP, PCR based deletion assay and a methylation-specific PCR. Results: Seven (70%) non-functioning islet cell carcinomas and 2 malignant gastrinomas (25%) showed aberrant hypermethylation or homozygous deletion of the p16(INK4a) gene. Conclusions: The p16(INK4a) tumor suppressor gene plays an important role in the tumorigenesis of non-functioning islet cell carcinomas and malignant gastrinomas.

therapy 175 Bile lipids 515 -secretion 515 Binary complexes 305 Bionormalizer 538 Bisacodyl 69 Bosentan 484 Bullfrog duodenum 324 Caco-2 cells 238 Caerulein 56 Calcitonin gene-related peptide 338 cAMP 324 Cancer 216 -, gastrointestinal tract

The present study investigates nitrosourea-induced rat (Rattus norvegicus) glioma cell lines for the functional status of the p16/Cdkn2a/Ink4a gene, which encodes the p16 cdk4 inhibitor and the alternative reading frame protein, p19ARF. We detected homozygous deletions of the p16/Cdkn2a/Ink4a gene locus in 4 of 5 glioma cell lines (C6, F98, RG2, and RGL.3), but not in the 9L gliosarcoma cell line or in a rat primary fibroblast cell line. RT-PCR demonstrated expression of the p16 and p19ARF mRNAs only in 9L cells and in rat fibroblasts. Comparative genomic in situ hybridization showed that the copy number of rat chromosome RNO5 was not altered in any of the glioma cell lines investigated, indicating that the deletions result from a discrete loss in the region of the p16/Cdkn2a/Ink4a locus. This is the first report of p16/Cdkn2a/Ink4a deletions present in nitrosourea-induced rat glioma cell lines. Since this genetic alteration is also commonly observed in human malignant glial tumors, our results validate the use of chemically induced rat glioma cell lines as an experimental model in the development of gene therapy strategies.

BACKGROUND/AIM:Mutations of the adenomatous polyposis coli (APC) tumor suppressor gene have been described in a subset of pancreatic carcinomas. The APC gene modulates the beta-catenin-Tcf pathway. The major player in this pathway is the beta-catenin protein encoded by the beta-catenin gene. A variety of different tumors, including colon, prostate, endometrial, and hepatocellular carcinomas, carry mutations in exon 3 of the beta-catenin gene. The aim of this study was to determine the role of the beta-catenin gene in the genesis of exocrine and endocrine tumors of the pancreas. METHODS:78 ductal pancreatic adenocarcinomas, 14 ductal pancreatic cancer cell lines, and 33 endocrine pancreatic tumors were evaluated for mutations in exon 3 of the beta-catenin gene by single-strand conformation polymorphism analysis and direct DNA sequencing. In addition, 40 ductal pancreatic adenocarcinomas were analyzed for intracellular beta-catenin accumulation by immunohistochemistry, indicating alterations of the beta-catenin gene. RESULTS:Neither the 111 exocrine and endocrine pancreatic tumors nor the 14 pancreatic cancer cell lines carried mutations in exon 3 of the beta-catenin gene. Intracellular beta-catenin accumulation was not identified in any of the 40 pancreatic adenocarcinomas. CONCLUSION:These data suggest that the beta-catenin gene as the major player of the beta-catenin-Tcf pathway does not play an important role in the genesis of pancreatic tumors.