Response effort and resurgence.

This study provides an initial translational examination of response effort and resurgence. Eleven typically developing adults and five adolescents with autism served as participants across two experiments. Participants received points for touching moving stimuli on a computer screen. The resurgence evaluation consisted of three phases: establishment wherein R1 was reinforced, elimination wherein R1 was placed on extinction while R2 was reinforced, and extinction wherein R1 and R2 no longer resulted in reinforcement. Rate of R1 during extinction was compared across three conditions: intermediate, easy, and difficult. Disparity in effort was created by manipulations of the size and speed of objects that moved about on a computer screen. In Experiment 2, control stimuli were added to the experimental arrangement. Across the two experiments, the magnitude of resurgence was greater when R1 was easy. In Experiment 2, both R1 and control responding were greater in the extinction phase than in the elimination phase in all conditions with all participants. The present study supports the hypothesis that response effort affects resurgence and that less effortful responses are likely to recur with greater magnitude under conditions that produce resurgence than are their more effortful counterparts.

Palladin is overexpressed in the non-neoplastic stroma of infiltrating ductal adenocarcinomas of the pancreas, but is only rarely overexpressed in neoplastic cells.

BACKGROUND: It has recently been suggested that overexpression of palladin in sporadic pancreatic cancer may contribute to pancreatic cancer's invasive and migratory abilities. This hypothesis was based on reverse transcriptase-polymerase chain reaction analyses of bulk pancreatic tissue, yet pancreatic cancer is a complex admixture of neoplastic epithelial cells and desmoplastic stroma. DESIGN: Immunohistochemical labeling of tissue microarrays was used to define the patterns of palladin protein expression in 177 ductal adenocarcinomas of the pancreas. Western blot analysis was used to determine the epitope(s) of palladin recognized by the antibody as well as the relative levels of palladin expression in short-term cultures of stromal fibroblasts, non-neoplastic ductal cells and pancreatic cancer cell lines. RESULTS: Immunolabeling revealed that the palladin protein was strongly overexpressed in non-neoplastic stromal cells in 171 (96.6%) of the 177 evaluable pancreatic cancers. By contrast, the overexpression of palladin protein by the neoplastic epithelial cells relative to normal pancreatic epithelium was observed in only 22 (12.4%) of the 177 cancers. Western blot analysis confirmed that the antibody recognizes the -90 kDa isoform of palladin, and demonstrated that fibroblast cell lines had higher expression of palladin than pancreatic cancer cell lines. CONCLUSIONS: The overexpression of palladin relative to normal pancreas in the majority of pancreatic cancers is limited to non-neoplastic stromal cells. This observation highlights the limitations of relying on bulk tissues when analyzing gene expression. Since palladin is not overexpressed in most pancreatic cancer cells, the overexpression of palladin is not likely to be responsible for pancreatic cancer cells invasive and migratory abilities.

Diagnosing pancreatic cancer using methylation specific PCR analysis of pancreatic juice.

The aim of this study was to determine the utility of detecting methylated ppENK and pi6 in pancreatic juice by methylation specific PCR as a marker of pancreatic adeno-carcinoma. Pancreatic juice samples were collected either intraoperatively, from 92 patients undergoing pancreaticoduodenectomy for benign (n=20) and malignant periampullary disease (n = 72) or endoscopically (by duodenal aspiration after secretin infusion), from 13 patients undergoing investigation for pancreatic disease. Methylated ppENK was detected in the pancreatic juice of 30 (66.7%) of 45 patients with pancreatic ductal adenocarcinoma, in 4 (44.4%) of 9 patients with intraductal papillary-mucinous adenocarcinoma, and in 7 (41.2%) of 17 patients with other periampullary carcinomas, using methylation specific PCR. Methylated pi6 was detected in a lower percentage of these patients (11.1%, 11.1% and 23.5%, respectively). In contrast, methylated ppENK and pi6 were not detected in 20 patients with non-malignant periampullary disease including 12 patients with chronic pancreatitis. Methylated ppENK was detected in 30 of 33 (90.9%) primary pancreatic adenocarcinoma and methylated pi6 was in 6/33 (1 8.2%). Despite the absence of ppENKand pi6 methylation in normal pancreas, methylated ppENK and pi6 was present in the duodenum of 90.5% and 28.6%, respectively of patients without cancer. Further, methylated ppENK and pi6 was seen in 88.9% and 11.1%, respectively of pancreatic juice samples obtained by duodenal aspiration from patients without cancer. We conclude that since ppENK and pi6 are not normally methylated in pancreatic secretions, detection of methylated ppENK and pi6 in pure pancreatic juice obtained by direct cannulation of the pancreatic duct to avoid duodenal secretions may suggest the presence of pancreatic adenocarcinoma

Methylation of cyclin D2 is observed frequently in pancreatic cancer but is also an age-related phenomenon in gastrointestinal tissues.

PURPOSE: Hypermethylation of CpG islands in the promoters of selected genes is a common feature of neoplasia. Aberrant methylation of cyclin D2 has been observed in several cancers. We investigated the methylation of cyclin D2 in aging and pancreatic neoplastic development, and the utility of cyclin D2 methylation as a marker of pancreatic adenocarcinoma. EXPERIMENTAL DESIGN: Methylation-specific PCR was performed on DNA from 165 resected pancreatic exocrine neoplasms [109 adenocarcinomas, 46 intraductal papillary-mucinous neoplasms (IPMNs), and 10 mucinous cystic neoplasms], 14 pancreatic intraepithelial neoplasms, 13 microdissected-normal pancreatic ductal epithelia, 25 normal pancreatic parenchyma, 51 specimens of pancreatic juice obtained perioperatively, 15 pancreatic cancer xenografts, 22 pancreatic cancer cell lines, 59 specimens of normal duodenum, and 49 gallbladders affected by cholecystitis. Cyclin D2 RNA expression was determined in pancreatic cancer cell lines, before and after 5-AZA-2'-deoxycytidine treatment, by reverse transcription-PCR. RESULTS: Methylation of cyclin D2 was identified in 65.1% (71 of 109) of primary pancreatic adenocarcinomas, in 50% (23 of 46) of IPMNs, and in 70% (7 of 10) of mucinous cystic neoplasms, but was detected infrequently in microdissected samples of normal pancreatic epithelia [7.7% (1 of 13)] and in pancreatic intraepithelial neoplasms [14.3% (2 of 14)]. Cyclin D2 methylation was also recognized in 10 of 15 (66.7%) pancreatic cancer xenografts and in 19 of 22 (86.4%) pancreatic cancer cell lines. All of 10 pancreatic cancer cell lines completely methylated at cyclin D2 showed no expression by reverse transcription-PCR. Four of these 10 cell lines were treated with 5-AZA-2'-deoxycytidine, and all 4 showed increased RNA expression of cyclin D2 after treatment. In pancreatic juice, cyclin D2 methylation was detected in 9 of 22 (40.9%) samples from patients with pancreatic cancer and in 6 of 9 (66.7%) patients with IPMNs, but in none of 20 non-neoplastic controls, respectively (P = 0.0013 and P < 0.0001, respectively). Methylation of cyclin D2 was also observed more in non-neoplastic tissues and with increasing age (P = 0.041 in the pancreas, P = 0.047 in the duodenum, and P = 0.0008 in the gallbladder). CONCLUSIONS: The promoter region of cyclin D2 undergoes age-related methylation in multiple tissues, but aberrant methylation is more often detected in tissues and juice samples of pancreatic cancer than in normal tissues. The detection of cyclin D2 methylation in pancreatic juice may aid in the diagnosis of pancreatic adenocarcinoma.

Aberrant methylation of preproenkephalin and p16 genes in pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma.

Pancreatic intraductal neoplasia (PanIN) is thought to be the precursor to infiltrating pancreatic ductal adenocarcinoma. We have previously shown that the preproenkephalin (ppENK) and p16 genes are aberrantly methylated in pancreatic adenocarcinoma. In this study we define the methylation status of the ppENK and p16 genes in various grades of PanINs. One hundred seventy-four samples (28 nonneoplastic pancreatic epithelia, 7 reactive epithelia, 29 PanIN-1A, 48 PanIN-1B, 27 PanIN-2, 14 PanIN-3, 15 invasive ductal adenocarcinomas, and 6 miscellaneous pancreatic neoplasms) were microdissected from 29 formalin-fixed paraffin-embedded surgically resected pancreata, and were analyzed by methylation-specific polymerase chain reaction. Fourteen of 15 (93.3%) invasive pancreatic ductal adenocarcinomas showed methylation of the ppENK gene and 4 of 15 (26.7%) showed methylation of the p16 gene. Nonneoplastic pancreatic epithelia did not harbor methylation of either gene. The prevalence of methylation of the ppENK gene increased significantly with increasing PanIN grade. A similar nonsignificant trend was noted for p16 methylation. Aberrant methylation of the ppENK gene was found in 7.7% of PanIN-1A, 7.3% of PanIN-1B, 22.7% of PanIN-2, and 46.2% of PanIN-3. Aberrant methylation of the p16 gene was found in 12% of PanIN-1A, 2.6% of PanIN-1B, 4.5% of PanIN-2, and 21.4% of PanIN-3. All but one of the PanINs from the 14 pancreata without pancreatic carcinoma was unmethylated with respect to either the p16 or ppENK gene. Our results suggest that methylation-related inactivation of the ppENK and p16 genes is an intermediate or late event during pancreatic carcinogenesis. Because aberrant methylation of ppENK or p16 was more often detected in similar grade PanINs from patients with pancreatic carcinoma than in those with other pancreatic diseases, it may be a useful indicator of the potential malignancy of epithelial cells of the pancreas.

Aberrant CpG island methylation in cancer cell lines arises in the primary cancers from which they were derived.

A higher prevalence of epigenetic inactivation of tumor suppressor genes has been reported in cancer cell line populations compared to primary cancer populations. Cancer-related genes are commonly methylated in cancer cell lines but it is not known the extent to which tumor suppressor genes may be artificially methylated in vitro. We therefore examined 10 pancreatic cancer cell lines and corresponding primary tumors for aberrant DNA methylation of promoter CpG islands of eight genes and seven CpG islands. Using methylation-specific PCR (MSP), methylation was not detected at any of the 15 CpG islands in 15 normal pancreata or in an immortalized normal pancreatic duct epithelial (HPDE) cell line. Of 150 loci examined, 49 loci were methylated in both primary carcinomas and their corresponding cell lines, 95 loci were not methylated in either cell lines or their corresponding primary carcinomas. There were four loci methylated only in cell lines while another two loci were methylated only in primary carcinomas. Overall, the methylation status of primary carcinomas and their cell lines were concordant in 96% of cases (144 of 150) (J statistic; J=0.92, P<0.0001). We conclude that most of the DNA methylation of tumor suppressor genes observed in cancer cell lines is present in the primary carcinomas from which they were derived.