Dante G. Scarpelli

Magnetic Microspheres: A Model System for Site Specific Drug Delivery in Vivo

Summary A novel carrier system for the delivery of chemotherapeutic agents by magnetic means to desired sites has been developed. Results indicate that the carrier, albumin microspheres with entrapped Fe3O4, and adriamycin HCl, can be concentrated at a predetermined site in vivo by a magnetic field. Carrier delivery of adriamycin is supported by the presence of a significant concentration of the drug at the site of carrier localization. This delivery system allows for the accumulation of local adriamycin which is comparable to that achieved by administration of a 100-fold higher dose of the free drug.

Almost total conversion of pancreas to liver in the adult rat: A reliable model to study transdifferentiation

Study of transdifferentiation provides an excellent opportunity to investigate various factors and mechanisms involved in repression of activated genes and derepression of inactivated genes. Here we describe a highly reproducible in vivo model, in which hepatocytes are induced in the pancreas of adult rats that were maintained on copper-deficient diet containing a relatively non-toxic copper-chelating agent, triethylenetetramine tetrahydrochloride (0.6% w/w) for 7-9 weeks and then returned to normal rat chow. This dietary manipulation resulted in almost complete loss of pancreatic acinar cells at the end of copper-depletion regimen, and in the development of multiple foci of hepatocytes during recovery phase. In some animals, liver cells occupied more than 60% of pancreatic volume within 6-8 weeks of recovery. Northern blot analysis of total RNA obtained from the pancreas of these rats revealed the expression of albumin mRNA. Albumin was demonstrated in these pancreatic hepatocytes by immunofluorescence. The advantages of this model over the previously described models are: a) low mortality (10%), b) depletion of acinar cells, and c) development of multiple foci of hepatocytes in 100% of rats.

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.

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.

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. Int. J. Cancer 82:137–144, 1999.

Chemical Carcinogens Without Mutagenic Activity: Peroxisome Proliferators As A Prototype*

Chemical carcinogens can be classified into two categories (i.e. mutagenic and non-mutagenic) on the basis of positive or negative evidence of DNA damage, mutagenicity or chromosomal aberrations in short-term test systems. Evidence indicates that carcinogenic peroxisome proliferators are negative in short-term test systems. This paper outlines approaches which may be useful in identifying a chemical carcinogen without mutagenic activity. It is conceivable that an alteration in DNA, if essential for initiation of neoplasia, may be mediated indirectly by the biological effects of nonmutagenic carcinogens.

Transdifferentiated hepatocytes in rat pancreas

Publisher Summary A specialized exocrine pancreatic cell can transform into a different phenotypic cell—the hepatocyte. Although in an adult animal the pancreatic and liver cells are functionally and morphologically different, both organs arise from gut endoderm, thus, sharing a common ancestry. Under proper conditions, repressed liver-specific genes in pancreatic cells are probably derepressed. The way in which such dormant genes are activated is not clear. Although some carcinogens, such as N -methyl- N -nitroso- guanidine, were shown to induce transdifferentiation, the possibility that 4-HAQO-induced genetic alterations may lead to hepatocyte conversion, when subjected to the added stress of copper depletion and repletion, can be excluded in view of the recent observation that a simple copper depletion-repletion regimen can lead to the development of pancreatic hepatocytes. The stability of a differentiated cell could be due to various molecular mechanisms such as DNA methylation, chromatin structure, DNA protein interactions, and DNA rearrangements. Modification of any of these could initiate transdifferentiation. By utilizing a variety of molecular techniques, it is possible to analyze the control mechanisms of tissue-specific gene regulation in pancreatic hepatocytes. Studies with transdifferentiated hepatocytes may be expected to yield considerable new information about the mechanism(s) of cell differentiation and the attendant transcriptional controls.

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 PA and PC. Lack of expression of ZO‐2A in neoplastic cells is caused by inactivation of the downstream promoter PA. Analysis of the promoter PA 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 PA 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 PA is responsible for the inactivation of the suppressed promoter at the late stages of tumor development. Int. J. Cancer 83:349–358, 1999.

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

SummaryEpithelial 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 Dulbeccos modified Eagles (DME):F12 medium suppleneted 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-triodothyronine, 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. Cell 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 1×10−6M 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 serretin 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.

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 mono- layers 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 p,M. At 1 p.M. these hormones stimulated DNA synthesis relative to their respective control in the order secretin (178%) > bombesin (153%) > VIP (138%) > 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.