The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo [a]pyrene and 2, 3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murineAhr gene by homologous recombination. Ahr-l mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. TheAhr-1-phenotype is most severe between 0-3 weeks ofage and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis.The Ah receptor (AHR) is a ligand-activated transcription factor that regulates a biphasic pleiotropic response to a variety of structurally related environmental contaminants (1, 2). Upon binding polycyclic aromatic hydrocarbons (PAHs), such as benzo [a]pyrene, the AHR increases the expression of xenobiotic metabolizing enzymes, including the cytochrome P450IA1, P450IA2 and P450IB1-dependent monooxygenases, the glutathione S-transferase Ya subunit, quinone oxidoreductase, and UDP-glucuronosyltransferase (3-8). In response to more potent halogenated aromatic agonists like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the AHR induces xenobiotic metabolism and also mediates a spectrum of toxic responses, including thymic involution, teratogenesis, tumor promotion, wasting, and epithelial hyperplasia and metaplasia (9-11).The AHR is a member of a family of transcription factors containing basic/helix-loop-helix and PAS homology domains (bHLH-PAS) (1). In response to agonist binding within the PAS domain, the cytosolic AHR undergoes a conformational change, translocates to the nucleus, dissociates from the 90-kDa heat shock protein, and dimerizes with a second bHLH-PAS protein known as the Ah receptor nuclear translocator (ARNT) (12-16). This heterodimer interacts with dioxin-responsive enhancer elements upstream of target genes and activates their transcription. Despite our mechanistic understanding of the role of the AHR in regulating xenobiotic metabolism, we still have little understanding of how the AHR mediates the toxic responses of halogenated agonists and why such responses are produced only by high affinity, poorly metabolized ligands such as TCDD.To examine the importance of the AHR in normal vertebrate biology, we have used gene targeting to create mice with a mutation at theAhr locus. We anticipated that such a mutant could provide insights into additional physiological roles of the AHR and would represent a powerful model to understand the toxicology of halogenated aromatic pollutants like TCDD.

Purpose: mTOR and P70 S6 kinase (S6K) play a key role in regulating protein translation. The role of mTOR and S6K in hepatocellular carcinoma has not been investigated, but this pathway is of particular interest because an effective inhibitor, rapamycin, is available. This study was undertaken to determine the prevalence and clinicopathological correlates of mTOR pathway activation in hepatocellular carcinoma and to determine whether rapamycin inhibits the pathway in cell culture.Experimental Design: Total and phosphorylated mTOR and S6K protein expression were studied by immunohistochemistry in hepatocellular carcinomas (n ‫؍‬ 73), fibrolamellar carcinomas (n ‫؍‬ 13), and hepatic adenomas (n ‫؍‬ 15). Results were correlated with tumor growth pattern as defined by the WHO (trabecular, pseudoglandular/acinar, compact, and scirrhous), tumor size, Ki-67 proliferation index, and the modified Edmondson nuclear grade, which has a scale of 1 to 4. HepG2 and Hep3B cell lines were treated with rapamycin to see the effect on proliferation and S6K phosphorylation.Results: Increased expression of total mTOR was seen in 5% of hepatocellular carcinoma, whereas overexpression of phospho-mTOR was evident in 15% of hepatocellular carcinoma. Phospho-mTOR positivity correlated with increased expression of total S6K, which was found in 45% of cases. Total S6K overexpression was positively correlated with tumor nuclear grade, inversely with tumor size, and was unassociated with the proliferation index or WHO growth pattern. Rapamycin treatment of HepG2 and Hep3B cell lines markedly inhibited cell proliferation and reduced S6K phosphorylation in both cell lines.Conclusions: The mTOR pathway is activated in a subset of hepatocellular carcinoma. Rapamycin can inhibit proliferation of neoplastic hepatocytes in cell culture.

DPC4 (MADH4, SMAD4) is a tumor suppressor gene inactivated by allelic loss in approximately 55% of pancreatic adenocarcinomas. Unfortunately, it can be technically very difficult to detect the inactivation of DPC4 at the genetic level because genetic analyses require the microdissection of relatively pure samples of neoplastic and normal tissues. This is especially true for pancreatic adenocarcinomas, which elicit vigorous, non-neoplastic, stromal responses. Immunohistochemical labeling can overcome this hurdle because it preserves morphological information. We therefore studied the expression of the DPC4 gene product in 46 cancers, including 5 cancer cell lines by Western blot analysis and 41 primary periampullary adenocarcinomas by immunohistochemistry. The status of exons 1-11 of the DPC4 gene in all 46 of the cancers had been previously characterized at the molecular level, allowing us to correlate Dpc4 expression directly with gene status. Three cell lines had wild-type DPC4 genes, and Dpc4 expression was detected in all three by Western blot. The two cell lines with homozygously deleted DPC4 genes did not show Dpc4 protein by Western blot analysis. Immunohistochemical labeling revealed that 17 (94%) of the 18 primary adenocarcinomas with wild-type DPC4 genes expressed the DPC4 gene product, whereas 21 (91%) of 23 primary adenocarcinomas with inactivated DPC4 genes did not. Cases in which there was discordance between the immunohistochemical labeling and the genetic analyses were reanalyzed genetically, and we identified a deletion in exon 0 of DPC4 in one of these cases. This is the first report of a mutation in exon 0 of DPC4 in a pancreatic cancer. The contrast between the strong expression of Dpc4 by normal tissues and the loss of expression in the carcinomas was highlighted in several cases in which an infiltrating cancer was identified growing into a benign duct. These observations suggest that immunohistochemical labeling for the DPC4 gene product is an extremely sensitive and specific marker for DPC4 gene alterations in pancreatic carcinomas. The sensitivity and specificity of immunohistochemical labeling for Dpc4 in other periampullary carcinomas has yet to be determined.

Peutz-Jeghers syndrome (PJS) is an autosomal-dominant disorder characterized by hamartomatous polyps in the gastrointestinal tract and by pigmented macules of the lips, buccal mucosa, and digits. Less appreciated is the fact that PJS also predisposes patients to an increased risk of gastrointestinal cancer, and pancreatic cancer has been reported in many PJS patients. It was recently shown that germline mutations of the STK11/LKB1 gene are responsible for PJS. We investigated the role of STK11/LKB1 in the development of pancreatic and biliary cancer in patients with and without the PJS. In a PJS patient having a germline splice site mutation in the STK11/LKB1 gene, sequencing analysis of an intestinal polyp and pancreatic cancer from this patient revealed loss of the wild-type allele of the STK11/LKB1 gene in the cancer. Inactivation of STK11/LKB1, by homozygous deletions or somatic sequence mutations coupled with loss of heterozygosity, was also demonstrated in 4-6% of 127 sporadic pancreatic and biliary adenocarcinomas. Our results demonstrate that germline and somatic genetic alterations of the STK11/LKB1 gene may play a causal role in carcinogenesis and that the same gene contributes to the development of both sporadic and familial forms of cancer.