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Tumors of the exocrine pancreas are a major cause of cancer death and have among the poorest prognosis of any malignancy. Following the "cancer stem cell hypothesis," where tumors are believed to originate in tissue specific stem cells, we screened primary ductal pancreatic carcinomas and cell lines for the expression of possible stem cell factors. We find 32/46 (70%) of primary tumors and 9/10 (90%) of cell lines express PAX6. PAX6 is a transcription factor expressed throughout the pancreatic bud during embryogenesis but not in the mature exocrine pancreas. PAX proteins have also been implicated in maintaining stem cells in a committed but undifferentiated state but a role for PAX proteins in putative pancreas stem cells is not known. We induced a pancreatic carcinoma cell line, Panc-1, to differentiate by transfecting wild-type p53 and treating the cells with differentiation agents gastrin or butyrate. This treatment induces cells to terminally differentiate into a growth-arrested cell with neurite-like processes, express the terminal differentiation marker somatostatin and downregulate PAX6. This phenotype can be replicated by directly inhibiting PAX6 expression. These data support a model where PAX proteins are aberrantly expressed in tumors and downregulation leads to differentiation.

Brain injury from inorganic Pb(2+) is considered the most important environmental childhood health hazard worldwide. The microvasculature of the developing brain is uniquely susceptible to high level Pb(2+) toxicity (ie, Pb(2+) encephalopathy) characterized by cerebellar hemorrhage, increased blood-brain barrier permeability, and vasogenic edema. However, the specific molecular mediators of Pb(2+) encephalopathy have been elusive. We found that Pb(2+) induces vascular endothelial growth factor/vascular permeability factor (VEGF) in cultured astrocytes (J Biol Chem, 2000;275:27874-27882). The study presented here asks if VEGF dysregulation contributes mechanistically to Pb(2+) encephalopathy. Neonatal rats exposed to 4% Pb-carbonate develop the histopathological features of Pb(2+) encephalopathy seen in children. Cerebellar VEGF expression increased approximately twofold (p < 0.01) concurrent with the development of cerebellar microvascular hemorrhage, enhanced vascular permeability to serum albumin, and vasogenic cerebellar edema (p < 0.01). No change in VEGF expression occurred in cerebral cortex that does not develop these histopathological complications of acute Pb(2+) intoxication. Pb(2+) exposure increased phosphorylation of cerebellar Flk-1 VEGF receptors and the Flk-1 inhibitor CEP-3967 completely blocked cerebellar edema formation without affecting microhemorrhage formation or blood-brain barrier permeability. This establishes that Pb(2+)-induced vasogenic edema formation develops via a Flk-1-dependent mechanism and suggests that the vascular permeability caused by Pb(2+) is Flk-1 independent.

BACKGROUND Cigarette smoking is among the few unequivocal risk factors for the development of pancreatic ductal adenocarcinoma (PDAC). Activating mutations in codon 12 of the K-ras protooncogene is a frequent and early molecular event in the pathogenesis of PDAC and a variety of nonmalignant ductal pancreatic lesions. The molecular epidemiologic relation between heavy cigarette smoking and mutational activation of K-ras in PDAC has been examined to a limited extent. The authors have examined the mutational status of K-ras in nonneoplastic pancreata in relation to cigarette smoking status. METHODS Archival formalin fixed paraffin embedded specimens of nonneoplastic pancreata (n = 39) were obtained from the American Cancer Society and evaluated histopathologically. Specimens from age- and gender-matched individuals were stratified into three groups: 1) those who never smoked cigarettes (n = 16), 2) those who smoked 1–2 packs/day for more than 20 years (n = 10 cases), and 3) those who smoked more than 2 packs/day for 20 or more years (n = 13). Cases were preselected from 77 specimens based on the quality, suitability, and cellularity of the archival tissues for analyses. Furthermore, none of the patients died of primary PDAC or had evidence of pancreatic metastases from an extrapancreatic primary tumor. Tissue sections were microdissected and deparaffinized, and genomic DNA was purified by standard proteinase K-phenol-chloroform extraction techniques. Genomic DNA was analyzed for mutations in codon 12 of the K-ras protooncogene by two mutant-allele-enriched polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assays and by multiplex PCR-based ligase chain reaction (LCR) analyses. RESULTS Analyses of multiple microdissected pancreata specimens from 39 cases revealed wild-type K-ras codon 12 sequences in both nonsmoking individuals and those who smoked 1–2 packs/day for 20 or more years. K-ras codon 12 mutations were confirmed by PCR-RFLP and PCR-LCR assays in 5 of 13 pancreata cases (39%) obtained from individuals who smoked more than 2 packs of cigarettes/day for 20 years or more (P < 0.005). The K-ras mutation spectra revealed two GT transversions, one GC transversion and two GA transitions. There was no clear relation between the incidence or spectra of mutations and pancreatic histopathology, as overtly normal pancreata as well as pancreata with squamous metaplasia, periductal fibrosis, and ductal atypia revealed reproducible K-ras alterations. Similarly, among those 34 cases in which a wild-type K-ras sequence was revealed by both approaches, a similar histopathologic profile was evident. CONCLUSIONS Mutational activation of codon 12 of the K-ras protooncogene was confirmed reproducibly by mutant allele-enriched PCR-RFLP and multiplex PCR-LCR analyses in 39% (5 of 13) of archival nonneoplastic pancreata from age- and gender-matched individuals who smoked more than 2 packs of cigarettes/day for 20 or more years. The presence of a mutated or wild-type or K-ras was independent of the histopathologic profile of the 39 cases examined. The data provide further suggestive molecular epidemiologic evidence of an association between a major and unequivocal risk factor for PDAC (heavy cigarette smoking) and mutations in a molecular target (K-ras), the activation of which is an important and early event Cancer 1999;85:326–32. © 1999 American Cancer Society.

Colorectal cancer is the second leading cause of cancer deaths in the United States with an estimated 150,000 diagnosed cases and over 56,000 fatalities annually (Jemal et al., 2006). Approximately one-third to one-half of cases are localized to the colon and rectum and have a favorable prognosis, while one-third to one-half present with regional lymph node metastases at diagnosis and generally are refractory to various chemotherapeutic regimens. Treatment options (surgery, radiation, and chemotherapy) are limited and the disease carries a grave prognosis for many patients. An orthotopic model of colon carcinoma in mice provides a way to evaluate the pathogenesis of tumor growth and metastasis as an aid in developing effective therapies and to better understand the underlying biology of colon tumor growth and metastasis. The protocol described in this unit details the development and characterization of an orthotopic model of murine colon carcinoma in BALB/c mice with diffuse lymphatic and hepatic metastatic spread, closely mimicking the course of the human disease. Curr. Protoc. Pharmacol. 38:14.5.1-14.5.13. © 2007 by John Wiley & Sons, Inc. Keywords: colon carcinoma; athymic nude; BALB/c mice; orthotopic; metastasis

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related deaths in the United States, with a 5-year survival rate of only 2% to 10%. This tumor is aggressive, often metastasizing to distant sites (liver, lung, and adjacent intestines) by the time of diagnosis. Treatment options are limited, and the disease carries a grave prognosis for most patients. An orthotopic model of human PDAC in nude mice provides an excellent way to evaluate the pathogenesis of tumor growth and metastasis in order to develop therapies, to better define the underlying biology of tumor growth and metastasis, and to identify new molecular targets. This unit describes an orthotopic model of human PDAC in athymic nude mice that closely mimics the human condition. It is characterized by diffuse peritoneal, lymphatic, and hepatic metastatic spread and manifestations of a cancer cachexic phenotype. Keywords: Human pancreatic ductal adenocarcinoma; Athymic nude mice; Orthotopic; Metastasis; cachexia