Organization and expression of the human zo-2 gene (tjp-2) in normal and neoplastic tissues.

One of the tight junction components, zonula occludens protein 2 (ZO-2), is expressed as two isoforms, ZO-2A and ZO-2C, in normal epithelia. In pancreatic adenocarcinoma of the ductal type ZO-2A is absent, but none of the common mechanisms of gene inactivation is responsible for lack of ZO-2A expression. In the current study, we report the complete organization of the human zo-2 gene (tjp-2), its alternative splicing, and its expression in normal and neoplastic tissues of several organ sites. In addition to pancreatic adenocarcinoma, ZO-2 was found to be de-regulated in breast adenocarcinoma, but not in colon or prostate adenocarcinoma. The latter are considered to be of acinar rather than ductal type. Thus, our data indicate the importance of zo-2 (tjp-2) gene regulation in ductal cancer development and should help to understand the defects of intercellular interactions, critical for suppressing the malignant phenotype.

zo-2 gene alternative promoters in normal and neoplastic human pancreatic duct cells.

We have observed that 2 forms of zonula occludens 2 (ZO-2) protein, ZO-2A and ZO-2C, are expressed in normal human pancreatic duct cells, but only ZO-2C in pancreatic duct adenocarcinoma. We report here partial organization of the zo-2 gene. Transcription of 2 forms of ZO-2 mRNA is driven by alternative promoters P(A) and P(C). Lack of expression of ZO-2A in neoplastic cells is caused by inactivation of the downstream promoter P(A). Analysis of the promoter P(A) sequence and function in normal and neoplastic cells demonstrated that neither structural changes (mutations) nor a change in the pool of transcription factors is responsible for its inactivation. Although hypermethylation was found in a large number of cancer clones, treatment with 5-aza-2'-deoxycytidine did not fully cause the promoter function to recover. We conclude that the initial down-regulation of zo-2 promoter P(A) activity in pancreatic duct carcinomas is due to the structural or functional alteration(s) in the regulatory elements, localized outside the analyzed promoter region. Methylation of P(A) is responsible for the inactivation of the suppressed promoter at the late stages of tumor development.

Tight junction protein ZO-2 is differentially expressed in normal pancreatic ducts compared to human pancreatic adenocarcinoma.

Differential display of hamster mRNA identified a fragment present in normal pancreatic duct cells that is not expressed in pancreatic duct carcinoma cells. Sequence analysis showed an 88% and 82% identity, respectively, to the cDNA of the canine and human tight junction zo-2 gene. Semi-quantitative RT-PCR analysis of human ZO-2 revealed a striking difference in the expression of various regions of the ZO-2 transcript in normal and neoplastic cells and the presence of an abnormality at the 5'-end of mRNA. RACE analysis identified 2 human ZO-2 mRNAs that encode proteins of different lengths, designated as ZO-2A and ZO-2C. The difference between the 2 forms of ZO-2 is the absence of 23 amino acid residues at the N terminus of ZO-2C compared with ZO-2A. Although ZO-2C was expressed in normal pancreatic cells and a majority of neoplastic tissues analyzed, ZO-2A was undetectable except in one case in all of the pancreatic adenocarcinomas analyzed. This suggests the presence of a yet to be identified motif important for cell-growth regulation within the 23-amino acid residue N-terminal peptide of ZO-2A, MPVRGDRGFPPRRELSGWLRAPG.

Multiple genetic alterations in hamster pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinomas induced in the Syrian golden hamster (SGH) by N-nitrosobis(2-oxopropyl)amine share many similarities with the human disease, including mutations of the K-ras oncogene. In vitro carcinogenesis studies with immortal SGH pancreatic duct cells indicate that neoplastic transformation in this system can occur without mutational inactivation of p53 suppressor gene. In this study we extend the genetic analysis of the in vivo SGH model to increase the number of cases analyzed for the status of K-ras and to determine further the spectrum of alterations involved; we have studied the status of the p53, DCC, and Rb-1 suppressor genes and the status of the mdm2 oncogene, which can involve p53 indirectly. The partial SGH-coding sequence of mdm2 and DCC was determined. K-ras mutation in the second position of codon 12 was present in 17 of 19 (90%) of tumors. Immunohistochemistry and single strand conformation polymorphism analysis showed no evidence of p53 mutation in 21 tumors. RNase protection assays showed overexpression of mdm2 in 5 of 19 (26%) tumors. Semiquantitative reverse transcription-PCR analysis showed a complete or partial loss of DCC expression in 10 of 19 (53%) neoplasms and of Rb-1 (42%) expression in 8 of 19 tumors when compared to matched controls. Deregulation of these genes appears to be significant in SGH pancreatic carcinogenesis as indicated by their frequencies. However, the fact that 6 tumors showed either only a K-ras mutation or the absence of alterations of the 5 genes analyzed indicates that additional as yet unstudied or unknown genes are also involved in SGH pancreatic duct carcinogenesis.

In vitro carcinogenesis of hamster pancreatic duct cells: cellular and molecular alterations.

Neoplastic transformation of Syrian golden hamster (SGH) pancreatic duct cells was induced by in vitro treatment with the direct-acting carcinogens N-methylnitrosourea (MNU) and N-(2-hydroxypropyl)nitrosourea (HPNU), with subsequent selection by sustained culture in serum- and epidermal growth factor (EGF)-deprived medium. The present study examines the efficacy of serum and EGF deprivation as a selection pressure and the effect of the carcinogen dose, frequency and interval of exposure on tumorigenesis and K-ras mutation. Selection of carcinogen-initiated duct cells by serum and EGF deprivation is highly reproducible and effective, increasing the incidence of tumors from 26 to 93% for MNU or from 0 to 100% for HPNU. SGH pancreatic duct cells exposed to 0.5 mM MNU for 13 weeks (long-treatment schedule) produced K-ras mutations at codon 12 in six of six tumors. However, when cells were exposed to 0.125, 0.25 or 0.5 mM MNU daily for 5 days (short-treatment schedule), mutations of K-ras at codon 13 were identified in four of 16 tumors, the remaining 12 showing no mutations. Duct cells exposed to 0.5 mM HPNU by the short-treatment schedule produced K-ras mutations in codon 13 in six of six tumors, as contrasted to 12 tumors that developed from cells exposed to 0.125 or 0.25 mM HPNU, which all contained K-ras codon 12 mutations. The current experiments demonstrate that K-ras mutation in pancreatic carcinogenesis in vitro by MNU or HPNU can be modified by the nature and dose of the carcinogen as well as the frequency and duration of exposure.

Genomic p53 mutation in a chemically induced hamster pancreatic ductal adenocarcinoma.

This study investigates possible alterations in exons 5 through 8 of the p53 gene and altered p53 protein expression in the Syrian golden hamster model of pancreatic ductal adenocarcinoma. In the Syrian hamster p53 sequence, 1469 base pairs are presented for the genomic regions surrounding exons 5 through 8, along with the primer sequences specific for the enzymatic amplification of the individual exons. Single-stranded conformation polymorphism was analyzed on the products obtained by enzymatic amplification of hamster genomic DNA extracted from 2 transplantable pancreatic ductal adenocarcinomas, 6 groups of N-methylnitrosourea (MNU)-treated pancreatic duct cells, and 17 MNU-induced pancreatic ductal adenocarcinomas. The two transplantable adenocarcinomas were a well-differentiated ductal adenocarcinoma established from a N-nitrosobis(2-oxopropyl)amine-induced tumor and a poorly differentiated ductal adenocarcinoma established from a spontaneous tumor. An altered mobility indicated a conformational change in the first part of exon 5 in the solid form of the well-differentiated ductal adenocarcinoma. Direct sequencing of the amplified product revealed an A-->C transversion in codon 135, which corresponds to codon 132 in the human p53 gene. A conformational change in exon 7 was observed in 1 of 6 MNU-treated cell samples, and none of the 17 resultant tumors. Direct sequencing confirmed a deletion of one C of the three in codon 263, which generates a frameshift mutation. No conformational change was observed in any other products. Positive staining with PAb240 or DO7 antibodies against human p53 or with an antibody generated in our laboratory against the hamster p53 fusion protein was observed only in the solid form of well-differentiated ductal adenocarcinoma and in rare cells scattered in 4 of 28 MNU-induced tumors analyzed. This study provides a system to analyze p53 gene alterations in the hamster and is the initial report of a specific p53 mutation in a hamster pancreatic ductal adenocarcinoma.

Exploring pathogenetic mechanisms using transgenic animals.

Molecular technologies for the permanent germ-line transformation of animals are now well established and routine. These new strains of animals, called transgenic, offer an unprecedented opportunity to gain a basic understanding of human genetic disorders. In this brief review we discuss the role of transgenic animals in the creation of new models of human disease and their experimental use in biomedical research. Models are now available for the study of the genetic processes involved in the pathogenesis of neoplasia, diabetes, atherosclerosis, and developmental abnormalities. Many others are available and new ones are being produced at a great rate. Principles of gene replacement therapy are amenable to analysis with transgenic animals and the information gained will be important for the development of rational therapy.

K-ras mutation is an early event in pancreatic duct carcinogenesis in the Syrian golden hamster.

K-ras oncogene mutation has been shown to be a frequent event in pancreatic ductal adenocarcinomas induced by the carcinogen N-nitroso-bis(2-oxopropyl)amine in the hamster. The present study examines the mutational status of the K-ras oncogene in lesions that precede the appearance of invasive ductal adenocarcinomas. Syrian golden hamsters (80-100 g) received 12 weekly doses of 15 mg/kg N-nitroso-bis(2-oxopropyl)amine and were serially sacrificed at 8, 12, 14, 16, or 24 weeks following the initiation of treatment. Ten microns-thick sections of formalin-fixed paraffin-embedded pancreas were examined for hyperplasia, papillary hyperplasia, carcinoma in situ, and invasive and metastatic ductal carcinoma. Marked lesions of interest were scraped from the slide, subjected to polymerase chain reaction-mediated amplification of the first exon of the K-ras gene, and probed by oligonucleotide-specific hybridization for mutations at codon either 12 or 13. Of 186 samples assayed, K-ras codon 12 mutations were detected in 26% of hyperplasias, 46% of papillary hyperplasias, 76% of carcinoma in situ, 80% of adenocarcinomas, and 43% of lymph node metastases. Codon 12 mutations were exclusively G to A changes at the second position. Codon 13 mutations were only detected in 9 of 168 samples. These results suggest that K-ras activation is an early event in N-nitroso-bis(2-oxopropyl)amine-induced pancreatic carcinogenesis in the hamster.

Stimulation of DNA synthesis in pancreatic duct cells by gastrointestinal hormones: interaction with other growth factors.

Pancreatic duct cells of the Syrian hamster were grown as monolayers on thin layers of type I collagen coated onto microporous membranes. The effects of a number of potential trophic factors were tested by their ability to increase [3H]thymidine incorporation into cellular DNA. To measure the effect of growth factors, cells were subjected to a period of growth factor depletion to induce a state of partial quiescence in DNA synthesis. Cells responded with a significant increase in thymidine incorporation after the addition of epidermal growth factor (EGF) alone or a growth factor mixture containing EGF plus insulin, transferrin, selenium, linoleic acid, bovine pituitary extract, triiodothyronine, and dexamethasone. When the serum substitute, Nu Serum IV (5%, vol/vol), was added to this mixture, addition of several gastrointestinal (GI) hormones including secretin, vasoactive intestinal polypeptide (VIP), bombesin, and gastrin caused significant increases in thymidine incorporation at concentrations of 0.01-1 microM. At 1 microM, these hormones stimulated DNA synthesis relative to their respective control in the order secretin (178%) greater than bombesin (153%) greater than VIP (138%) greater than gastrin (126%). Cholecystokinin octapeptide, a known trophic factor for pancreatic acinar cells, did not cause significant increases in thymidine incorporation in cultured duct cells. These results suggest that pancreatic duct cells possess receptors for a number of GI hormones and respond to the trophic effects of hormones known to stimulate pancreatic growth in vivo.

Prognostic implications of proliferative activity and DNA aneuploidy in Astler-Coller Dukes stage C colonic adenocarcinomas.

Paraffin-embedded surgical specimens from 69 patients who underwent resections of otherwise untreated Dukes stage C adenocarcinoma of the colon were examined for proliferative activity, DNA aneuploidy, DNA index, and proportion of aneuploid cells by flow cytometry. Results were correlated to clinical characteristics of the patients and to overall survival times. DNA aneuploid tumors were identified in 60 cases (87%), diploid tumors in 9 cases (13%). The mean S-phase fraction for all cases was 17.6%, with a standard deviation (SD) of 7.8. In univariate statistical analysis, younger patient age, lower tumor proliferative activity, DNA index less than or equal to 1.2, and presence of only 1-4 lymph nodes with tumor involvement were found to be significant predictors of improved patient survival. In multivariate Cox regression analysis, low tumor proliferative activity, younger patient age, and location of the tumor in the right or transverse colon were found to be significant independent predictors of increased patient survival. When tumor proliferative activity was stratified into statistically defined subgroups, patients with tumors of low proliferative activity (S-phase less than mean - 0.5 SD) had significantly longer survival than patients with tumors of moderate proliferative activity (S-phase value greater than mean - 0.5 SD and less than mean +0.5 SD) or high proliferative activity (S-phase greater than mean +0.5 SD). These results suggest that tumor proliferative activity in Dukes C colon carcinoma may be an important biological factor in determining patient prognosis.

Enhancement of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine-induced tumorigenesis in Sprague-Dawley rats by orotic acid.

The effect of orotic acid (OA) on the carcinogenicity of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) in rats was evaluated. A group of 5 week old Sprague-Dawley male rats were placed on a synthetic 20% protein diet containing 1% OA. A second group was placed on a regular, OA-free diet of similar composition. Approximately 2 weeks later, animals from both groups grown to 100 g were treated with 400 mg/kg HPOP delivered continuously for 14 days via 2002 Alzet osmotic pumps implanted s.c. Rats fed the OA diet were kept under this diet for 13 weeks following initiation of HPOP treatment and subsequently were placed on the regular diet for another 12 weeks, at which time they were killed. In the absence of OA, HPOP-treated rats developed adenomas in the kidney and lungs at incidences of 5 and 33% respectively, while pancreas and liver were unaffected. On the other hand, rats fed the OA diet and treated with HPOP developed renal mesenchymal tumors and pulmonary adenomas at incidences of 70 and 65% respectively. In addition, HPOP induced cystic lesions in the pancreas of animals fed the OA diet. The enhancement of the tumorigenic effectiveness of HPOP was at least partly ascribed to the effect of OA treatment on the rate by which carcinogen-induced alkylation of DNA was repaired in various tissues. Accumulation of N7-methylguanine in kidney, lung and pancreas of rats fed the OA diet was 1.6, 1.9 and 2.4 times higher than in respective organs of animals fed the regular diet. Similarly, concentrations of the premutagenic O6-methylguanine (O6-MeG) were 3.0, 3.1 and 2 times greater in the kidney, lung and pancreas of rats fed the OA diet than in the respective organs of those fed the regular diet. Feeding an OA diet to HPOP-treated rats did not have an effect on either the resistance of the liver to this carcinogen or on the level of O6-MeG accumulation in the DNA of this tissue.

Activation of K-ras in transplantable pancreatic ductal adenocarcinomas of Syrian golden hamsters.

High mol. wt genomic DNA was prepared from normal hamster pancreas and the solid and ascites variants of two different hamster transplantable carcinomas, one induced by N-nitrosobis(2-oxopropyl)amine and the other spontaneously occurring. This DNA was transfected into NIH/3T3 cells and resulted in cells that were capable of forming tumors when injected into nude mice. Analysis of the nude mouse tumors by Southern blotting revealed the presence of a band specific for hamster K-ras. Polymerase chain reaction (PCR)-mediated amplification of the K-ras codon 12-13 region of genomic DNA prepared from the transplantable tumors produced a 117 bp fragment which was analyzed by both allele-specific oligonucleotide hybridization and direct DNA sequencing. Oligonucleotide hybridization with probes specific for changes in the first or second position of codons 12 or 13 detected a G to A transversion in the second position of codon 12 in the chemically induced transplantable tumor, and a G to A change in the second position of codon 13 in the spontaneously occurring transplantable carcinomas. The result obtained for the chemically induced tumor was confirmed by direct dideoxy sequencing of the PCR-amplified product. These findings are the first to show a specific oncogene activation in an experimental pancreatic tumor model and also parallel the results recently reported for K-ras mutations in human pancreatic carcinoma.

Characterization of differentiated Syrian golden hamster pancreatic duct cells maintained in extended monolayer culture.

Epithelial cells isolated from fragments of hamster pancreas interlobular ducts were freed of fibroblast contamination by plating them on air-dried collagen, maintaining them in serum-free Dulbecco's modified Eagle's (DME):F12 medium supplemented with growth factors, and selecting fibroblast-free aggregates of duct cells with cloning cylinders. Duct epithelial cells plated on rat type I collagen gel and maintained in DME:F12 supplemented with Nu Serum IV, bovine pituitary extract, epidermal growth factor, 3,3',5-triiodothyronine, dexamethasone, and insulin, transferrin, selenium, and linoleic acid conjugated to bovine serum albumin (ITS+), showed optimal growth as monolayers with a doubling time of about 20 h and were propagated for as long as 26 wk. Early passage cells consisted of cuboidal cells with microvilli on their apical surface, complex basolateral membranes, numerous elongated mitochondria, and both free and membrane-bound ribosomes. Cells grown as monolayers for 3 mo. were more flattened and contained fewer apical microvilli, mitochondria, and profiles of rough surfaced endoplasmic reticulum; in addition, there were numerous autophagic vacuoles. Functional characteristics of differentiated pancreatic duct cells which were maintained during extended monolayer culture included intracellular levels of carbonic anhydrase and their capacity to generate cyclic AMP (cAMP) after stimulation by 1 X 10(-6) M secretin. From 5 to 7 wk in culture, levels of carbonic anhydrase remained stable but after 25 to 26 wk decreased by 1.9-fold. At 5 to 7 wk of culture, cyclic AMP increased 8.7-fold over basal levels after secretin stimulation. Although pancreatic duct cells cultured for 25 to 26 wk showed lower basal levels of cAMP, they were still capable of generating significant levels of cAMP after exposure to secretin with a 7.0-fold increase, indicating that secretin receptors and the adenyl cyclase system were both present and functional. These experiments document that pancreatic duct monolayer cultures can be maintained in a differentiated state for up to 6 mo. and suggest that this culture system may be useful for in vitro physiologic and pathologic studies.

Transdifferentiation of ductular cells into hepatocytes in regenerating hamster pancreas.

The transdifferentiation of regenerating pancreatic cells into hepatocytes in the Syrian golden hamster and Fischer rat is an example of the surprising plasticity of cells in the adult animal. While earlier experiments suggested that these might be derived from acinar cells, unequivocal evidence of this in both models has not been forthcoming. In this paper, we document that pancreatic ductular epithelium in the hamster is the cell of origin and that presumptive hepatocytes can be identified morphologically as early as the 3rd day after the induction of regeneration. The patterns of development of various organelles as characterized by their acquisition of liver-specific protein markers parallels those that have been reported by others during the differentiation of embryonic and postnatal hepatocytes. Colloidal gold-labeled antibody staining of carbamoyl phosphate synthetase, a liver-specific mitochondrial enzyme, and catalase, a peroxisomal enzyme, first appeared 60 hours and 4.5 days postregeneration, respectively. At these times, the morphologic features of the hepatocyte phenotype in the pancreas were ambiguous. Morphometric analysis showed that for both enzymes, the number of gold particles/organelle increased to a maximum by the 9th week. In contrast, urate oxidase, a liver-specific peroxisomal enzyme, was not identified by ultrastructural immunochemistry until the 14th day after regeneration and remained at low levels through the 9th week.

Metabolism and activation of the pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine by isolated hepatocytes and pancreatic cells of the Syrian hamster.

The metabolism of the pancreatic carcinogen N-nitroso-bis(2-oxopropyl)amine (BOP) was studied using primary hepatocytes and acinar and duct cells isolated from Syrian hamsters. Metabolic activation of BOP was verified by detecting its conversion to CO2 covalently bound metabolites and soluble products containing the alpha-carbon of the nitrosamine. At concentrations below 0.2 mM, BOP was completely activated by hepatocytes within 60 min. At high substrate concentration (1 mM) or high cell density (5 x 10(6) cells/ml), reduction of BOP to N-nitroso(2-hydroxypropyl) (2-oxopropyl)amine and N-nitrosobis(2-hydroxypropyl)amine contributed significantly to the metabolic profile. The conditions which favored metabolic activation of BOP were used to compare metabolism by hepatocytes and pancreatic cells. Under such conditions, the ratio of activation products formed by hepatocytes versus those formed by acinar cells was 14.5:1; the corresponding ratio for covalently bound metabolites was 19:1. Hepatocytes activated BOP 106 times more rapidly than duct cells as determined from yields of activation products or 152 times more rapidly as determined from labeling of cellular macromolecules. Acinar cells showed a higher capacity for metabolic activation than duct cells. The ratio for the yield of activation products from acinar versus duct cells was 4.3:1; the corresponding ratio for covalent binding was 5.8:1. The relatively low capacity of pancreatic cells for activation of BOP compared to hepatocytes is in agreement with the low levels of DNA binding in the pancreas compared to other organs after administration of BOP to the hamster in vivo. The observation that the ratio for total covalent binding of BOP in hepatocytes versus acinar cells was higher than that seen previously for the liver versus the pancreas in vivo is consistent with the hypothesis that alkylating agents derived from BOP reach the pancreas after formation in other organs. The liver would be the prime source for such alkylating agents.

Coexpression of glutamine synthetase and carbamoylphosphate synthase I genes in pancreatic hepatocytes of rat.

In the mammalian liver the distribution of ammonia-detoxifying enzymes, glutamine synthetase (GS) and carbamoylphosphate synthase I (ammonia) (CPS-I), is mutually exclusive in that these enzymes are expressed in two distinct populations of hepatocytes that are zonally demarcated in the liver acinus. In the present study we examined the distribution of GS and CPS-I in pancreatic hepatocytes to ascertain if the expression of these two genes in these hepatocytes is also mutually exclusive. Multiple foci of hepatocytes showing no clear acinar organization develop in the adult rat pancreas as a result of a change in the differentiation commitment after dietary copper deficiency. Unlike liver, GS and CPS-I are detected by immunofluorescence in all pancreatic hepatocytes. In situ hybridization revealed that all pancreatic hepatocytes contain GS and CPS-I mRNAs. The sizes of these two mRNAs in pancreas with hepatocytes are similar to those of the liver. The concomitant expression of GS and CPS-I genes in pancreatic hepatocytes may be attributed, in part, to the absence of portal blood supply to the pancreas vis-à-vis the lack of hormonal/metabolic gradients as well as to possible matrix homogeneity in the pancreas.

Toxicology of the pancreas.

The pancreas of mammals is capable of biotransforming drugs and other chemicals and subject to toxic injury by the resultant reactive metabolites. In the case of experimental animals, especially rodents, numerous chemical toxicants have been identified for the exocrine pancreas and, to a lesser degree, the endocrine component. These include alcohol, alloxan, azaserine, dimethylbenzo[a]anthracene, ethionine, 4-hydroxyaminoquinoline-1-oxide, beta-oxidized derivatives of dipropylnitrosamine, oleic acid, and streptozotocin. Depending on the toxicant, the cellular effects span the gamut from acute injury and death, to hyperplasia, metaplasia, and malignant transformation. In contrast to the well-established toxic effects of chronic alcohol abuse on the human pancreas, its susceptibility to toxic injury by therapeutic drugs has become clinically apparent more recently. A drug should be considered as toxic only when it induces acute pancreatitis documented by clinical findings and laboratory tests; the condition improves when the drug is discontinued and worsens when the patient is challenged by readministration of the drug. At present, only 8 therapeutic drugs have fulfilled these criteria and are classified as toxic for pancreas. These are, azathioprine, estrogens, furosemide, methyldopa, pentamidine, procainamide, sulfonamides, and thiazide diuretics. Controlled exposure to cultured component cells of pancreas may be of potential value in identifying such drugs, documenting their toxicity, and allowing for biochemical and toxicologic investigation of the mechanisms involved.