The anticancer drug imatinib induces cellular autophagy.

The tyrosine kinase inhibitor imatinib (Gleevec, Novartis Pharmaceuticals Corporation; Basel, Switzerland) is a powerful drug for treatment of chronic myelogenous leukemia (CML) and other malignancies. It selectively targets various tyrosine kinases, thereby leading to growth arrest of respective cancer cells. Given its wide application, it is of high importance to know all related underlying molecular mechanisms. We had previously found that imatinib increases the cellular clearance of intracellular protein aggregates by targeting the abl pathway and thereby upregulating lysosomal activity. Here, we describe that imatinib dose dependently activates the cellular autophagy machinery in mammalian cells, independently of tissue type, species origin or immortalization status of cells. Autophagy is an archetypical cellular degradation mechanism implicated in many physiological and pathophysiological conditions. Our data link for the first time the process of autophagy with the mode of action of imatinib. Induction of autophagy might represent an additional mechanism of imatinib to induce growth arrest, promote apoptosis in cancer cells and eventually even promote tumour regression.

E6 and E7 expression from the HPV 18 LCR: development of genital hyperplasia and neoplasia in transgenic mice.

Human papillomavirus type 18 infection is highly associated with malignant tumors of the genital tract. To investigate the tissue specificity of the HPV long control region (LCR) and the transforming ability of the E6-E7 oncoproteins, an HPV-18 transgene containing the viral LCR and E6 and E7 genes was introduced into mice. Three founder males exhibited enlarged seminal vesicles and preputial glands by 50 weeks of age. A line of transgenic mice was established by in vitro fertilization, and subsequent generations of transgenic males and females were monitored for lesions. Approximately 80% of hemizygous transgenic males exhibited enlarged seminal vesicles and preputial glands as early as 12 weeks of age. Histological examination indicated that this enlargement was due to distension by fluid, along with polyploid hyperplasia of the lining secretory epithelium. E6 and E7 transcripts were limited to affected organs and kidney. Approximately 41% of transgenic females developed cervical neoplasms between 1-2 years of age. Histologically, tumors were mesenchymal rather than epithelial in origin. E6 and E7 transcripts were restricted to cervical tumor tissue and kidney. These findings suggest that the HPV-18 LCR has an element(s) which directs expression specifically to the urogenital tract in transgenic mice.

Zinc finger protein GFI-1 has low oncogenic potential but cooperates strongly with pim and myc genes in T-cell lymphomagenesis.

The gfi-1 gene encodes a zinc finger containing protein that is specifically expressed in T-lymphocytes and is a frequent target of proviral insertion in T-cell lymphoma provoked by infection with MoMuLV--a non acute transforming retrovirus. Expression of a gfi-1 transgene targeted to T-cells by the lck proximal promoter provokes a reduction of peripheral CD4 and CD8 positive T-cells but nevertheless weakly predisposes transgenic animals for the development of T-cell lymphoma. Forced coexpression of the serine/threonine kinase Pim-1 can partially restore normal T-cell numbers in double pim-1/gfi-1 transgenic mice. Moreover, the combinatorial expression of Pim-1 and Gfi-1 leads to accelerated development of T-cell lymphoma with a mean latency period of 114 days. A similar accelerated rate of lymphoma development was observed when lck-gfi-1 mice were crossed with mice that carry a L-myc gene targeted to be expressed at high levels in T-cells. The results show that gfi-1 can act with low activity as a dominant oncogene when overexpressed but also demonstrate that it is most efficient only in the presence of a cooperative partner protein as for example Pim-1 or L-Myc. In addition, the results suggest that Pim-1 and Gfi-1 are acting synergistically in both T-cell lymphomagenesis and T-cell development.

Differential expression of the hyaluronan receptors CD44 and RHAMM in human pancreatic cancer cells.

To explore the putative role of hyaluronan (HA) in tumor invasion in pancreatic cancer, we investigated the expression of the HA receptors CD44s and RHAMM in a panel of human pancreatic cancer cell lines. Expression of CD44s has been found in only 1 of 10 cell lines included in this study. This cell line exhibits a highly differentiated phenotype without any metastatic potential when injected into nude mice. Since it has previously been shown that normal pancreatic duct cells express a high level of CD44s, our results indicate that pancreatic cancer may be accompanied by an almost complete loss of CD44s expression. As demonstrated by PCR amplification, this loss of CD44s expression is due to alternative splicing of CD44 pre-RNA. Although most of the pancreatic cancer cell lines express a complex but identical pattern of variant CD44 gene transcripts, only one higher molecular weight CD44 isoform can be detected in a subset of pancreatic cancer cell lines in Western blot analysis. This variant CD44 molecule represents the epithelial CD44 isoform (CD44v8-v10). When cells are cultured on Matrigel, the expression of additional CD44 variants is induced, suggesting that the extracellular matrix can influence the expression of CD44 isoforms and thereby may facilitate tumor invasion. This induction could be due to a regulatory process in the translation of the CD44 variant mRNAs expressed in pancreatic tumor cells. Molecular cloning of a cDNA encoding human RHAMM reveals that both HA receptors are structurally unrelated. In addition, they share an inverse expression pattern. RHAMM mRNA is overexpressed in pancreatic cancer cell lines exhibiting a poorly differentiated phenotype and a high metastatic potential when injected into nude mice. These results indicate that CD44 and RHAMM differentially contribute to invasion of pancreatic adenocarcinoma; however, these functions still remain to be determined.

Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles.

We report the use of the autofluorescent compound monodansylcadaverine (MDC) for in vivo labeling of autophagic vacuoles. When applied to various cell types (PaTu 8902, MDCK I, PC12, AR4-2J, WI-38) in culture, spherical structures were observed by fluorescence microscopy, predominantly located in the perinuclear region. Only PC12 and WI-38 cells had some of these labeled structures in their filopodiae. Dose-response experiments with PaTu 8902 showed that the optimal concentration for in vivo labeling was 0.05 to 0.1 mM, while cells detached and disintegrated, when MDC concentration exceeded 0.1 mM. After incubation with MDC and subcellular fractionations of PaTu 8902 cells on sucrose density gradients, a narrow fluorescence peak at 20 to 26% sucrose concentration equal to densities of about 1.081 to 1.108 g/cm3 was observed. Ultrastructural analysis of these fractions revealed autophagic vacuoles in different stages of their development. To investigate whether endosomal compartments were also labeled by MDC, we coincubated PaTu 8902 cells with MDC and the fluid-phase markers, RITC-dextran and ferritin, respectively. Fluorescence measurements after subcellular fractionations as well as fine structural analysis indicated that MDC-labeled autophagic vacuoles did not contain fluid-phase markers and were spatially separated from endosomal compartments. We further could demonstrate, after subcellular fractionation procedures, that MDC-labeled organelles contained the lysosomal enzymes acid phosphatase and the mature form of cathepsin D. Membrane markers of rough endoplasmic reticulum (TRAM and sec61 beta), and for smooth endoplasmic reticulum (cytochrome P450) were not detected in the same fractions. These results indicate that MDC accumulates as a selective fluorescent marker for autophagic vacuoles under in vivo conditions and is not present in the early and late endosome.

Hyaluronan is a secretory product of human pancreatic adenocarcinoma cells.

A high molecular weight secretion product of cell lines established from duct cell-derived human pancreatic adenocarcinomas was investigated in this study. After metabolic labeling and molecular sieve chromatography of culture medium, a product appeared in the void volume of a Superose 6 column that could be labeled with [3H]glucosamine, but not with [35S]sulfate. After further purification by anion exchange chromatography it was analyzed and demonstrated to be hyaluronan (HA). CsCl density gradient centrifugation revealed a density of 1.45 g/cm3 in a 4 M guanidinium hydrochloride solution. [3H]Glucosamine-labeled material could be degraded by digestion with hyaluronidase from two sources, but not with heparitinase I or chondroitinase AC. Sugar analysis revealed glucuronic acid and glucosamine at a molar ratio of 1:1. When the amount of HA synthesized by different pancreatic adenocarcinoma cell lines was compared, the values of the cell lines PaTu 8902 and PaTu II were about five- to tenfold higher than those of the lines PaTu 8988s, PaTu 8988t or HPAF, but an order of magnitude lower than in murine 3T3 fibroblasts. HA synthesis per cell decreased with increasing cell density. In serum-free cultures of cell lines with high HA synthesis it was 3 to 5 times higher compared to cultures that were supplemented with serum. We conclude that pancreatic adenocarcinoma cells secrete hyaluronan and thus contribute to the extracellular matrix of the tumor tissue. In pancreatic carcinoma cells, regulation of HA biosynthesis seems not to be positively correlated to proliferation as has been demonstrated for fibroblasts.

Characterization of a transglutaminase expressed in human pancreatic adenocarcinoma cells.

A soluble tissue type transglutaminase (TGases; R-glutaminylpeptide:amine gamma-glutamyltransferase, E.C.2.3.2.13) belonging to a group of widely distributed enzymes which catalyze the reaction between a gamma-carboxyamide group of a protein-bound glutamine residue and various amino groups was characterized in cell lines derived from human pancreatic carcinoma. The enzyme activity was measured by incorporation of [3H]putrescine into N,N-dimethylcasein. It showed a strong dependency on Ca2+, which could not be replaced by Mg2+ but was 80% inhibited by 0.7 mM Mg2+ in the presence of optimal Ca2+ concentration (7 mM). The Km-value in regard to putrescine was 2.6 mM. After centrifugation of cell homogenates at 105,000g 95% of the enzyme activity was found in the supernatant indicating that the TGase in pancreatic tumor cells is soluble. This was further substantiated by immunohistochemistry showing a homogeneous cytoplasmic distribution of the TGase in pancreatic tumor cells. Molecular sieve chromatography and Western blot analysis using an antibody against TGase II from human erythrocytes revealed a molecular mass of 80 kDa. In Northern blots with a cDNA of TGase II from mouse macrophages a single transcript approximately 3.4 kbp in size was detected. Polymerase chain reaction analysis using primers for the coding and 3'-non-coding regions showed in each case a single product with the size expected from the human cDNA of TGase II. Taken these data together, we conclude that human pancreatic adenocarcinoma cells express the soluble tissue type TGase II.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies against 5'-nucleotidase from a human pancreatic tumor cell line: their characterization and inhibitory capacity on tumor cell adhesion to fibronectin substratum.

Four mouse monoclonal antibodies (PTN63, PTN108, PTN124, PTN514) against the ecto-5'-nucleotidase purified from a human pancreatic adenocarcinoma cell line (PaTu II) have been raised and characterized. All four monoclonal antibodies recognize the protein moiety of the glycosylated ecto-5'-nucleotidase. In competition assays it was demonstrated that three of the antibodies (PTN63, PTN108, PTN514) recognize different epitopes within the protein moiety. Furthermore, PTN108, PTN124, and PTN514 reduced the 5'-nucleotidase AMPase activity in contrast to PTN63 having no inhibitory effect. The antibodies show no cross-reactivity with ecto-5'-nucleotidases from rat liver, bull seminal plasma, chicken gizzard and human peripheral blood cells. When assayed by indirect immunofluorescence the antibodies react with the plasma membrane of human pancreatic tumor cells with varying staining intensity. Immunocytochemistry on paraffin sections of normal human pancreas revealed a prominent staining of the pancreatic duct cells. No staining of the acinar and islet cells could be detected. Thus, 5'-nucleotidase is a marker enzyme for pancreatic duct cells and can be used to determine the origin of pancreatic tumor cells. PTN63 reduced the attachment to fibronectin substratum of a human pancreatic adenocarcinoma tumor cell line possessing a high amount of plasma membrane bound ecto-5'-nucleotidase, but had no effect on a cell line lacking the membrane bound AMPase. In contrast, PTN108 and PTN514, which inhibit the AMPase activity, exhibited no influence on the adhesion of human pancreatic tumor cells to fibronectin substratum.

Phenotypical changes of a human pancreatic adenocarcinoma cell line after selection on laminin-1/nidogen (LM/Ng) substratum.

A cell line (PaTu 8902LM) exhibiting an altered phenotypic appearance was selected from a highly dedifferentiated established human pancreatic tumour cell line (PaTu 8902) by repetitive exposure to laminin-1/nidogen substratum and subsequent selection for adherent cells. Polymerase chain reaction analysis for repetitive DNA indicated that both cell lines are genetically very closely related. The original PaTu 8902 line consisted of flat cells growing in monolayers. In contrast, the obtained PaTu 8902LM cells exhibited a spherical morphology and tended to form clusters. Immunofluorescence analysis using antibodies against apical and basolateral marker enzymes indicated that the PaTu 8902LM cells were polarized, arranging their apical surfaces around central lumenal structures when growing in clusters. In addition, the selected PaTu 8902LM cell line exhibited altered levels of a number of differentiation marker enzymes like 5'-nucleotidase, transglutaminase and plasminogen activators. The different morphological characteristics of both cell lines were maintained even after injection into nude mice. In xenografts, PaTu 8902LM cells were grouped around lumenal, duct-like structures, whereas the original PaTu 8902 cell line formed solid tumours composed of undifferentiated cells. Evidence is presented that the PaTu 8902LM cells are not merely selected from preexisting cells, but that the exposure of PaTu 8902 cells to laminin-1/nidogen had induced a stable transdifferentiation towards the phenotype of the epithelial cells lining the pancreatic secretory ducts. Thus the PaTu 8902LM cells resemble more closely those cells from which tumours of the pancreas originate in vivo and therefore might be a useful cell system in future analyses of the biology of pancreatic tumours which are of increasing incidence and clinical importance.

Preparation of a low molecular weight polyethylenimine for efficient cell transfection.

Polyethylenimines (PEIs) of a molecular weight between 25 and about 800 kDa have successfully been used for in vitro and in vivo gene delivery approaches. Recent publications indicated that PEI molecules of lower molecular weight and a small molecular weight range are also efficient transfection reagents with a much lower cytotoxicity compared to high molecular weight PEIs. Here, we describe the application of a molecular sieve chromatography to fractionate a commercially available 25-kDa PEI. We generated three pools of PEIs with molecular weight ranges of 70-360 (I), 10-70 (II), and 0.5-10 kDa (III), respectively. We show that, in comparison with the 25-kDa PEI, pool III increased the expression of luciferase up to 100-fold and the number of transfected cells 2-3 fold. In addition, the kinetics of reporter gene expression was also much faster in pool III, compared with the 25-kDa PEI or with pools I or II. Finally, pool III showed the lowest cytotoxicity in comparison with the other PEI preparations. Thus, we provide a one-step processing of a 25-kDa PEI, resulting in a more effective and also less cytotoxic transfection reagent.

The lysosomotropic agent monodansylcadaverine also acts as a solvent polarity probe.

The autofluorescent substance monodansylcadaverine has recently been reported as a specific in vivo marker for autophagic vacuoles. However, the mechanism for this specific labeling remained unclear. Our results reveal that the common model of ion trapping in acidic compartments cannot completely account for the observed autophagic vacuole staining. Because autophagic vacuoles are characterized by myelin-like membrane inclusions, we tested whether this lipid-rich environment is responsible for the staining properties of monodansylcadaverine. In in vitro experiments using either liposomes or solvents of different polarity, monodansylcadaverine showed an increased relative fluorescence intensity in a hydrophobic environment as well as a Stokes shift dependent on the solvent polarity. To test the effect of autophagic vacuoles or autophagic vacuole lipids on monodansylcadaverine fluorescence, we isolated autophagic vacuoles and purified autophagic vacuole lipids depleted of proteins. Entire autophagic vacuoles and autophagic vacuole lipids had the same effect on monodansylcadaverine fluorescence properties, suggesting lipids as the responsible component. Our results suggest that the in vivo fluorescence properties of monodansylcadaverine do not depend exclusively on accumulation in acidic compartments by ion trapping but also on an effective interaction of this molecule with autophagic vacuole membrane lipids. (J Histochem Cytochem 48:251-258, 2000)

Fluorescence properties and staining behavior of monodansylpentane, a structural homologue of the lysosomotropic agent monodansylcadaverine.

We have recently shown that monodansylcadaverine labels autophagic vacuoles. Analysis of the mechanism underlying the labeling revealed that monodansylcadaverine acts as a lysosomotropic agent, being concentrated into acidic compartments by an ion-trapping mechanism, and as a solvent polarity probe, increasing its relative fluorescence intensity by interacting with membrane lipids that are highly concentrated in the autophagic vacuoles. In this study, we synthesized three structurally related derivatives of monodansylcadaverine, replacing the primary amino group of monodansylcadaverine with a neutral (dansylamylamine; MDH), a polar (dansylaminopentanol; MDOH), or an acidic group (dansylaminovaleric acid; MDA), to replace the lysosomotropic character of the marker. Whereas MDH showed a specific staining of autophagic vacuoles, the polar and acidic derivatives did not show any staining. We further demonstrate that the MDH staining of autophagic vacuoles is independent on the acidic pH and thus on an ion-trapping mechanism, but it still shows the same preferences for autophagic membrane lipids as monodansylcadaverine. We propose that MDH can specifically interact with lamellar bodies of the autophagic type as a solvent polarity probe. Therefore, dansylated aminopentane can be used as a specific marker for autophagic vacuoles in vivo and in fixed cells.(J Histochem Cytochem 49:177-185, 2001)

Receptor mediated antiproliferative effects of the cytotoxic LHRH agonist AN-152 in human ovarian and endometrial cancer cell lines.

Eighty percent of human ovarian and endometrial cancers express receptors for luteinizing hormone-releasing hormone (LHRH). These receptors might be used for targeted chemotherapy with cytotoxic LHRH analogs such as AN-152, in which doxorubicin is linked to agonist carrier [D-Lys6]LHRH. The antiproliferative effects of doxorubicin and AN-152 were assessed in LHRH receptor-positive ovarian (EFO-21, EFO-27) and endometrial (HEC-1A, Ishikawa) cancer cell lines as well as in LHRH receptor negative ovarian SKOV-3 and endometrial MFE-296 lines. The mechanism of action of AN-152 was investigated by a blockage of receptors using an excess of the LHRH agonist [D-Trp6]LHRH. In some cases, confocal laser-scanning microscopy was used to visualize the accumulation of AN-152 or doxorubicin within the cells. In 3 of 4 LHRH receptor-positive cell lines (EFO-21, HEC-1A, Ishikawa) AN-152 was more effective than doxorubicin in inhibiting cell proliferation. The effect of AN-152 was shown to be receptor-mediated because it could be reduced by competitive blockade of the LHRH receptors with [D-Trp6]LHRH. In contrast, AN-152 was less active than doxorubicin in LHRH receptor-negative lines. Confocal laser-scanning microscopy showed an intranuclear accumulation of AN-152 and competitive inhibition thereof by [D-Trp6]LHRH in LHRH receptor-positive cell lines, but no intracellular accumulation of AN-152 could be detected in the receptor-negative SKOV-3 line. These results suggest a selective receptor-mediated action of AN-152 in receptor-positive cell lines.

Effects of monophosphoryllipid-A on the immunization of mice with keyhole limpet hemocyanin- and muramyldipeptide-ganglioside Gfpt1 conjugates.

Since it was considered that an active immunization against ganglioside Gfpt1 (IV2Fuc-, II3NeuAc-Gg4Cer) expressed by human small cell lung cancer cells may be beneficial in the treatment of this neoplasm in humans, an optimal mode of vaccination in model mice was investigated. A novel Gfpt1-muramyldipeptide conjugate (Gfpt1-MDP) was synthesized. Its ganglioside carbohydrate-directed immunogenicity in mice as measured by serum antibody titers was comparable to that of the previously described Gfpt1-keyhole limpet hemocyanin conjugate (Gfpt1-KLH). Similar immunogenicity was displayed by free Gfpt1 in muramyldipeptide-phosphoethanolamine-containing phosphatidyl-choline, -serine (PC,PS) liposomes. Immunization with Gfpt1-vaccines in the presence of monophosphoryllipid A (MPL), in general, raised titers of anti-Gfpt1 antibodies effectively. Immunization with PC, PS-liposomes containing unconjugated Gfpt1 and MPL stimulated the highest titers observed, thereby effectively preventing tumor growth in Balbc nu/nu-mice challenged with human small cell lung cancer cells. However, there was a strong crossreaction of these and most other sera with the structurally related and widely distributed ganglioside Gtet1 (II3NeuAc-Gg4Cer). Only immunization with Gfpt1-KLH conjugate in the presence of MPL stimulated selectively high anti-Gfpt1 antibody titers showing comparably low crossreactivity to ganglioside Gtet1.

Localisation of hyaluronate (HA) in primary tumors and nude mouse xenografts of human pancreatic carcinomas using a biotinylated HA-binding protein.

A biotinylated hyaluronate (HA)-binding protein isolated from bovine cartilage was used to analyze the distribution of HA in nude mouse xenografts derived from human pancreatic adenocarcinoma cell lines as well as in primary human pancreatic adenocarcinomas. The most reproducible results for the localisation of HA were obtained using cryostat sections. When the biotinylated HA-binding protein was applied to histological sections of nude mouse xenografts, the specific staining found could be inhibited by preincubating the HA-binding protein with an excess of HA or by hyaluronidase treatment of the tissue before staining. The highest HA concentration was found at the tumor boundaries, while in the central part of the tumor staining was slight or absent. In cryostat sections of primary tumors HA was found predominantly in the connective tissue immediately around tumor cells or at the border between the tumor and normal pancreatic tissue.

Time course and cellular source of pancreatic regeneration following acute pancreatitis in the rat.

The regenerative capacity of the different cell types in the rat exocrine pancreas has been studied in a model of hormone-induced acute pancreatitis in which pancreatic edema, inflammation, and acinar cell destruction were induced within 12 h of infusion of supramaximal concentrations of cerulein (5 micrograms/kg/h). A sequential biochemical and structural analysis of the pancreas in daily intervals was combined with the autoradiographic quantitation of labeling indices of five cell populations following 3H-thymidine injection at days 1-7 after induction of pancreatitis. Desquamation of acinar cell apical cytoplasm and release of cytoplasmic segments into the acinar lumen on the first day following induction of pancreatitis led to formation of duct-like tubular complexes. Enzyme content in the pancreas decreased progressively following the formation of the edema to levels 15-20% of controls and remained reduced during the initial 5 days. Thymidine incorporation into total DNA showed a biphasic pattern with a distinct peak at day 1 and a second broader peak between days 4 and 7. Autoradiographic quantitation of labeling indices demonstrated the exclusive incorporation into intercalated duct cells and interstitial cells during the initial 24 h, while the second peak was predominantly due to labeling of acinar cells. Larger interlobular ducts and islets did not show changes in labeling index. In vivo labeling with 3H-thymidine during the first day and analysis of labeling indices 14 days later showed the persistence of label in intercalated duct cells and interstitial cells and argued against the stem cell hypothesis and against transformation of duct cells into acinar cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of nucleolar organizer regions with secretory and regenerative process in experimental cerulein-induced pancreatitis in the rat.

Silver staining of nucleolar organizer regions (AgNORs) demonstrates loops of DNA that transcribe ribosomal RNA. Their number and size have been attributed to rDNA transcription activity involved in protein synthesis and thus associated with proliferation. The exact relationship among proliferation, protein synthesis, and expression of AgNORs is, however, not yet well established. We therefore investigated AgNORs in an experimental model of cerulein-induced rat pancreatitis. During secretory stimulation with maximal doses of cerulein (0.25 micrograms/kg/h) for 12 h, AgNOR number and size per nucleus as well as 3H-thymidine label index did not change, although there was a marked increase in pancreatic volume flow, up to 150%, and of protein synthesis rate, up to 180% of the control levels. In contrast, after infusion of supramaximal doses of cerulein (5.0 micrograms/kg/h), AgNOR and 3H-thymidine label values rose significantly, with a distinct peak at day 3 after induction of pancreatitis. Most interestingly, AgNOR number and size were elevated 12 h before DNA replication started as determined by 3H-thymidine incorporation. At the same time intracellular protein synthesis proved to be decreased approximately 30-50% compared to controls. Our data confirm that AgNOR is a marker of proliferation that reflects regulatory events in the cell cycle earlier than 3H-thymidine incorporation. Here we demonstrate for the first time that this phenomenon is independent of the total intracellular protein synthesis rate.

Fibroblast structure and function during regeneration from hormone-induced acute pancreatitis in the rat.

The regeneration of the rat exocrine pancreas from a hormone-induced pancreatitis was investigated. In a previous study it was shown that the [3H]thymidine labeling index of interstitial cells increases 20- to 30-fold on day 1.5 after the induction of pancreatitis. Here we show by electron microscopic autoradiography that 80% of the labeled interstitial cells are fibroblasts. Their replication, fine structure, and collagen biosynthesis was further investigated: By day 2.5 numerous mitotic figures were found, indicating an enhanced proliferative activity of fibroblasts at the early stage of pancreatic regeneration. The ultrastructural analysis revealed that many fibroblasts contain abundant cytoplasm with a well-developed rough endoplasmic reticulum, prominent Golgi complexes, and secretory granules filled with fibrillar material. In contrast, the pancreatic fibroblasts of saline-infused control animals were shown to be spindle-shaped and to contain only very little cytoplasmic organelles. The collagen biosynthesis was quantified by in vivo labeling with [3H]proline and quantification of [3H]hydroxyproline in pancreatic protein hydrolysates. The collagen biosynthesis of experimental pancreata was measured to be 15 times that of controls on days 1.5 and 2.5 after the induction of pancreatitis and to remain fourfold elevated on days 3.5 through 10.5. In pulse-chase experiments using [3H]proline as the labeled precursor for collagen, the newly synthesized collagen was shown to be degraded with a half-life of 35 h. We conclude that replication of pancreatic fibroblasts and collagen biosynthesis as well as collagen degradation play important roles in the early phase of pancreatic regeneration.

Tubular complexes in cerulein- and oleic acid-induced pancreatitis in rats: glycoconjugate pattern, immunocytochemical, and ultrastructural findings.

Ductlike tubular complexes in cerulein-induced pancreatitis and oleic acid-induced pancreatic insufficiency were studied to analyze further their origin and development. Immunocytochemistry for pancreatic enzymes, lectin-binding studies, and ultrastructural investigations were combined with autoradiographic quantitation of labeling indices of ductlike cells in tubular complexes. In one group of rats, pancreatitis was induced by infusion of cerulein (10 micrograms kg-1 h-1). In a second group, pancreatic insufficiency was induced by intraductal injection of oleic acid (50 microliters). The investigations were carried out at distinct intervals following induction of pancreatic injury. In both groups of animals, after 3 days, a significant widening of acinar lumina was paralleled by a decreasing height of acinar cells, which showed pronounced retrogressive changes. At this time, acinar cells bound all of the lectins used and retained their immunoreactivity for amylase, trypsinogen, chymotrypsinogen, and lipase. At further intervals, acinar structures formed typical ductlike complexes, with a progressive loss of immunoreactivity for pancreatic enzymes and a reduced lectin-binding for L-fucose and N-acetylgalactosamine. Autoradiographic quantitation demonstrated no significant labeling of acinar cells undergoing tubular dedifferentiation. In both models, tubular complexes were removed by macrophages. It is concluded that lining cells in tubular complexes represent degenerating acinar cells that have no regenerative potency and have lost their secretory and membrane characteristics.

Repetitive cerulein-induced pancreatitis and pancreatic fibrosis in the rat.

The effect of repetitive inductions of pancreatitis by supramaximal doses of cerulein on pancreatic morphology and collagen content was studied in the rat. Pancreatitis was induced nine times at intervals of about 20 days; 3 days after the last injection of cerulein, pancreatitis was still observed, as indicated by pancreatic weight loss, increase of protein-bound hydroxyproline content, acinar-cell destruction, cellular infiltration, and deposition of collagen fibers. However, 6 weeks later, no differences in the parameters mentioned above were observed between control and cerulein-treated animals. Thus, repetitive induction of pancreatitis in the rat, according to the experimental protocol we used, did not result in pancreatic fibrosis.