Polysialylated-neural cell adhesion molecule expression in rat pituitary transplantable tumors (spontaneous mammotropic transplantable tumor in Wistar-Furth rats) is related to growth rate and malignancy.

Pituitary adenomas are usually benign neuroendocrine tumors. However, some of those that are histopathologically undistinguishable behave aggressively and metastasize. The polysialylated neural cell adhesion molecule (PSA-NCAM), which is highly expressed during the development of the brain and pituitary, is detected in some neuroendocrine tumors and might be relevant as a prognostic marker in pituitary tumors. In the present study, we have searched for PSA-NCAM expression in four lineages of rat pituitary transplantable tumors (SMtTW). Each lineage, maintained by serial tumor grafts under the kidney capsule and skin, differed in its GH/Prl secretion, growth rate, and malignant behavior. PSA-NCAM expression, detected by immunohistochemistry and Western blotting and quantified by ELISA, varied according to the SMtTW lineage. The benign tumors, SMtTW2, with a low growth rate never expressed PSA-NCAM. Another benign lineage, SMtTW3, with a high growth rate expressed a low amount of PSA-NCAM. The highest PSA-NCAM expression was seen in tumors that grew beneath the skin, invaded the kidney, and metastasized (SMtTW4). Tumors of the SMtTW10 lineage, which behaved as either benign or malignant tumors, were heterogeneous in terms of PSA-NCAM expression. In this rat transplantable pituitary tumor model, PSA-NCAM expression correlated in decreasing order with: (a) invasiveness (P < 0.0001), (b) metastases (P = 0.004), (c) ability to grow under the skin (P = 0.006), and (d) growth rate under the kidney capsule (P < 0.01), but not with hormone secretion (r = 0.207). This model, which is very similar to the human pathology, suggests that PSA-NCAM evaluation is of interest in the diagnosis of malignancy and the prognosis of human pituitary tumors. In addition, the SMtTW tumors could be instrumental in evaluating the effects of new therapeutic agents modulating PSA-NCAM expression.

High lysosomal activities in cystic fibrosis tracheal gland cells corrected by adenovirus-mediated CFTR gene transfer.

Human tracheal gland serous (HTGS) cells are now believed to be a major target of cystic fibrosis (CF) gene therapy. To evaluate the efficiency of adenovirus-mediated gene transfer in these cells we tested the adenovirus construction containing beta-galactosidase cDNA. We observed that the endogenous beta-galactosidase activity in cultured CF-HTGS cells was too strong to allow us to detect any exogenous beta-galactosidase activity. Immunohistological study on sections of human tracheal tissue confirmed the presence of beta-galactosidase in the serous component of the submucosal glands. We then looked for other lysosomal activities in normal and CF-HTGS cells. We showed that normal cells already have elevated enzyme values and that CF-HTGS cells contained 2-4-fold more beta-galactosidase, alpha-fucosidase, alpha-mannosidase and beta-glucuronidase activities than normal cells. An analysis of their kinetic constants has shown that this difference could be attributed to a lower K(m) of CF lysosomal enzymes. More importantly, these differences are eliminated after adenovirus-mediated CFTR gene transfer and not after beta-galactosidase gene transfer.

Pancreatic regenerating gene overexpression in the nonobese diabetic mouse during active diabetogenesis.

The reg gene has previously been shown to be associated with regeneration of pancreatic islets. Strategies for influencing the replication and the growth of the beta-cell mass may be important for prevention and/or treatment of type I diabetes. In this study, we have examined the level of reg gene expression at various degrees of diabetogenesis in the pancreas of the NOD mouse (male, female, and cyclophosphamide-treated male) using both human reg cDNA as the probe and dot blot analysis. The expression of the reg gene was found to be significantly increased in female mice compared with male mice, and in both cases, the expression level was not influenced by age. Nondiabetic female mice have a significantly higher expression of the gene than diabetic female mice, and there was a positive correlation between the age of diabetes onset and the reg mRNA level. In addition, overexpression of the reg gene was found in male mice treated by cyclophosphamide, an agent known to be a potent inducer of diabetes in male NOD mice. None of these results were found in the diabetes-resistant control OF1 mice, in which pancreatic reg gene expression did not differ between female and male mice treated or untreated with cyclophosphamide. All of these data suggest that there is a strong correlation between reg gene expression in the pancreas of the NOD mouse and the likelihood of developing diabetes.

Expression of REG protein during cell growth and differentiation of two human colon carcinoma cell lines.

We localized REG protein in Paneth cells and nonmature columnar cells of the human small intestinal crypts and speculated that this protein was associated with growth and/or differentiation. The aim of this study was to determine whether REG protein is present in two human colon cancer cell lines that exhibit enterocytic differentiation after confluence and to investigate changes in the level of its expression during growth and differentiation. Results were compared to those obtained on cells that remain undifferentiated. Western immunoblotting and immunofluorescence demonstrated the presence of REG protein in the three cell lines. With the antisera against human REG protein, the staining was diffusely spread throughout the cytoplasm at Day 2, and after Days 3-4 it appeared to have migrated to cell boundaries. After confluence, we observed only a punctate staining array along cell boundaries, which disappeared at Day 15. REG mRNA expression was demonstrated by RTPCR and REG mRNA hybridization until Day 13, but not after, in the three cell types. REG protein may be involved in cellular junctions. Its presence appears to be associated with the cell growth period and the protein must be downregulated when growth is achieved and differentiation is induced.

cDNA sequence and deduced amino acid sequence of human preprocolipase.

Complementary DNA clones for human pancreatic colipase were identified in human pancreatic cDNA libraries by hybridization with a pool of synthetic oligonucleotides containing all possible coding sequences for amino acids 75 to 80 of the partial human colipase protein sequence (Sternby, et al. Biochim Biophys Acta 1984;784:75). Alignment of overlapping cDNA clones yielded an mRNA sequence of 504 nucleotides [not including the poly(A) tail] encoding a polypeptide of 112 amino acids. The prepeptide comprised 17 amino acids, with an amino-terminal cluster of charged residues followed by a hydrophobic core of 12 residues typical of leader sequences. The deduced human procolipase sequence comprised 95 residues, including a propeptide of 5 residues. It was in complete agreement with the partial sequence previously obtained by protein sequencing. Northern blot analysis revealed that the polyadenylated preprocolipase transcript had a length of approximately 680 nucleotides.

Increased coexpression of CFTR and S100 calcium binding proteins MRP8 and MRP14 mRNAs in cystic fibrosis human tracheal gland cells.

We have demonstrated for the first time, by Northern analysis, the presence of the S-100 calcium binding proteins MRP8 (also called "cystic fibrosis protein") and MRP14 mRNAs in cultured human tracheal gland cells, obtained from normal and cystic fibrosis (CF) patients. A significant increase of these mRNAs in cells of CF origin (as well as that of CFTR mRNA) is shown. These results allow us to assume a potential pretranslational regulation of MRP8 and MRP14 gene expression related to the presence of a mutated CFTR gene.

Regulation of concentrations of mRNA for amylase, trypsinogen I and chymotrypsinogen B in rat pancreas by secretagogues.

Regulation of pancreatic gene expression by the secretory stimulants cholecystokinin-pancreozymin, caerulein and pilocarpine was studied in the rat, by using cloned cDNA probes to quantify concentrations of specific mRNAs (amylase, trypsinogen I and chymotrypsinogen B). It is concluded that long-term pancreatic stimulation results in pre-translational regulation of secretory-protein gene expression, with a preferential accumulation of RNA transcripts encoding serine proteinases, compared with that for amylase.

Regulation of proteolytic enzyme activities and mRNA concentrations in rat pancreas by food content.

Regulation by food content of the expression of genes encoding pancreatic proteases was studied in rats fed diets containing 15%, 25% or 70% protein (w/w) (diet I, II and III). Trypsin, chymotrypsin and elastase activities in pancreas were 1.4, 2.8 and 2 times higher in diet III than in diet I whereas carboxypeptidase A level was unchanged. As compared to diet I, the pancreatic concentration of mRNAs encoding trypsinogen I and chymotrypsinogen B, measured by filter hybridization to specific cDNA probes, were found respectively 3.6 and 3.9 times higher in diet III, and 1.9 and 2.6 times higher in diet II. Elastase I mRNA concentration was 1.8 times higher in diet III, but unchanged in diet II. Procarboxypeptidase A mRNA concentration was not affected. It is concluded to a coordinate pre-translational regulation of serine protease genes expression by the protein content of diet, differing however in amplitude and sensitivity among the three species of enzymes studied.

Trypsinogen expression by two human pancreatic cell lines CFPAC-1 and CAPAN-1. Modulation during spontaneous and induced cell growth.

We previously demonstrated that two human pancreatic adenocarcinoma cell lines, CFPAC-1 (established from a patient with cystic fibrosis) and CAPAN-1, were able to secrete trypsinogens 1 and 2 specifically. In order to analyze the relation of trypsin secretion to differentiation and cell growth, we undertook a comparative study of immunoreactive trypsin 1 (IRT) secretion by the two cell lines during cell growth in the presence and in the absence of various differentiating agents: sodium butyrate (NaBut), dimethylsulfoxide (DMSO), and dexamethasone (DX). In the presence of NaBut, IRT levels in the supernatants of both cell lines were slightly increased, whereas the cellular growth of both cell lines decreased significantly. In the presence of DX, IRT levels in cell culture conditioned media immediately and dramatically decreased, but the cell growth of neither cell line was affected by DX. An important increase in IRT levels was observed when CFPAC-1 cells and CAPAN-1 cells were grown in the presence of DMSO, but for both cell lines the cellular growth decreased in the presence of DMSO. Our data show that neither the IRT secretion level nor the differentiation state of these cell lines correlates with cellular growth, and suggests that the expression of pancreatic proteases by these two tumor cell lines could be either related to a common stem cell with this potential or to a possible acinar origin of pancreatic cancer, as recently proposed by others.

A transformed human tracheal gland cell line, MM-39, that retains serous secretory functions.

Infection with the wild type SV40 virus was used to transform primary cultures of human tracheal gland serous (HTGS) cells. Over 80 different cell lines were obtained, but the majority had lost some of their epithelial and secretory features. However, one of these cell lines, MM-39, was shown to have conserved the physiologic characteristics of the genuine HTGS cells-i.e., the presence of cytokeratin, expression of cystic fibrosis transmembrane conductance regulator mRNA, a level of secretory leukocyte proteinase inhibitor secretion comparable to that of the native cells (25 +/- 3 ng/10(6) cells/h), and the responsiveness to pharmacological agonists: carbachol (+260 +/- 40%), isoproterenol (+260 +/- 40%), and adenosine 5'-triphosphate (+280 +/- 30%). These characteristics describe a transformed cell line of human tracheal gland cells which has retained the features of the native serous cells. As a result, this cell line appears to be a useful tool for large-scale physiologic and pharmacologic studies of bronchial secretion at the cellular level.

Developmental gene expression of trypsinogen and lipase in human fetal pancreas.

BACKGROUND: Very few studies have been reported on the expression of human pancreatic genes during fetal development. We have shown very low lipase immunoreactivity compared with elevated trypsinogen immunoreactivity in a previous immunohistological study of human fetal pancreas during development. METHODS: The expression of these two selectively expressed genes of the exocrine pancreas, trypsinogen and lipase were investigated. The developmental profiles of the corresponding mRNA's were determined from the 13th gestational week. RESULTS: For the two genes, fetal mRNA levels throughout gestation remained significantly lower than the corresponding adult levels. No correlation was found between trypsinogen and lipase gene expression in the fetal pancreas, whereas such a correlation was present in adult pancreas. This may be explained by differences in maturity of the pancreas.

Absence of correlation between reg and insulin gene expression in pancreas during fetal development.

The reg gene characterized in the exocrine pancreas has been found to be expressed in regenerating islets of 90% depancreatized rats and not in normal islets. In humans, it was identified only in the exocrine pancreas. Because the reg protein has been found to be related to islet cell replication and/or beta cell regeneration, we compared the expression of the reg gene with that of chymotrypsinogen of exocrine origin and insulin of endocrine origin. We investigated the expression of the three pancreatic genes in the fetal pancreas during human development using dot-blot analysis. The levels of expression of the corresponding mRNAs did not appear to undergo great changes between the 17th and the 29th wk of gestation. Nevertheless, the fetal mRNA levels for reg and chymotrypsinogen were below that of the adult, with very low levels of reg gene expression in more than half of the studied pancreases. In contrast, the insulin mRNA levels were significantly higher in fetal than in adult pancreases, suggesting that insulin may function as a growth factor during fetal development. Our results indicate that no correlation between reg and insulin gene expression exists in the fetal pancreas during the developmental period studied but, on the contrary, such a correlation was present in the adult pancreas.

Presence of MRP8 and MRP14 in pancreatic cell lines: differential expression and localization in CFPAC-1 cells.

A complex of two calcium binding proteins, MRP8 [also called cystic fibrosis (CF) antigen] and MRP14, proteins known to be expressed in cells of myeloid origin, has been shown to be present in higher amounts in the serum of CF patients and heterozygotes compared with normal subjects. We demonstrated here for the first time, by dot-blot analysis and immunocytochemistry, the expression and the presence of these S100 calcium binding proteins in the pancreatic cell lines CAPAN-1 and CFPAC-1, the latter provided from a patient with CF. Moreover, using immunocytochemical methods, we showed that the localization of MRP8 and MRP14 on the plasma membrane seems to be restricted to the cells expressing a cystic fibrosis transmembrane conductance regulator (CFTR) wild-type protein such as CAPAN-1 cells and CFPAC-1 cells transfected with a plasmid containing the nonmutated CFTR gene (CFPAC-PLJ-CFTR-6 cells). In CFPAC-1 cells, immunoreactivity remains in the cytoplasm throughout the stationary phase. We also showed an increased level of the mRNAs of the two proteins in the CFPAC-1 cells compared with those transfected with the nonmutated CFTR. The demonstration of a difference in the cellular localization of these two proteins and in their mRNA levels in the cell line of CF origin leads us to assume the existence of a possible correlation in the expression of the MRPs with that of the CFTR protein.

Expression of PECAM-1/CD31 isoforms in human brain gliomas.

Microvascular proliferation is a histopathological hallmark of glioblastomas and anaplastic oligodendrogliomas. Platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31) is involved in angiogenesis. PECAM-1 mediates homophilic and heterophilic interactions (with glycosaminoglycans and alphaVbeta3), but deletion of exon 14 results in a loss of heterophilic adhesion. Expression of various PECAM-1 isoforms was searched for in brain gliomas, showing microvascular proliferation (glioblastomas and anaplastic oligodendrogliomas) or not (oligodendrogliomas). In addition, expression of alphaVbeta3 in some tumors was studied by immunohistochemistry. Various tissues and the HUVEC primary cell line were used as controls. Immunohistochemistry showed that PECAM-1 was expressed by all endothelial cells in all tissues and by some tumor cells in glioblastomas and anaplastic oligodendrogliomas. Microvascular proliferation always expressed alphaVbeta3. In addition, some tumor cells in anaplastic oligodendroglioma and glioblastomas expressed it. In all samples examined, PECAM-1 exists under at least two transcriptional isoforms: the whole length molecule and an isoform made by the splicing of exon 14. Western blot analysis revealed in all cases 130 and 110 kDa bands corresponding to the mature form and its precursor respectively. These results suggest that splicing of exon 14 occurs in vivo in various normal and tumoral tissues and may modulate PECAM-1 adhesion according to the presence or not of other PECAM-1 ligands such as alphaVbeta3. Expression of PECAM-1 by tumor cells in glioblastomas and anaplastic oligodendrogliomas may favour angiogenesis by specific PECAM-1 interactions between glial and endothelial cells.

p16INK4a and p19INK4d mRNA expression in neuroglial tumours: correlation with Ki67 proliferation index.

The INK4a-ARF locus encodes two unrelated proteins that both function in tumour suppression: p16INK4a and p19INK4d. Although p19INK4d expression has not been studied in central nervous system (CNS) tumours, it has been reported that p16INK4a inactivation is involved in the growth of glioblastomas. This observation has not been reported in relation to other CNS tumours. To understand further the role of p16INK4a and p19INK4d in neuroglial tumour growth, expression of both p16INK4a and p19INK4d mRNAs was studied by reverse transcription polymerase chain reaction RT-PCR in 59 neuroglial tumours, in which Ki67 labelling indices (LI) were also determined. P16INK4a mRNA was found in all pilocytic astrocytomas (7/7), in all grade II and III astrocytomas (7/7 and 4/4, respectively), in 4/12 glioblastomas, 8/8 oligodendrogliomas, 10/11 anaplastic oligodendrogliomas, 4/7 ependymomas and 3/3 anaplastic ependymomas but not in normal brain. In contrast, p19INK4d mRNA was detected in all tumours and control tissues. p16INK4a expression was associated with a low Ki67 LI in glioblastomas but not in other tumours. P16INK4a expression was not related to anaplasia in oligodendrogliomas and ependymomas. In tumours expressing p16INK4a, in situ hybridization showed a widespread expression of p16INK4a mRNA in tumour cells and in foci of microvascular proliferation. These results strongly support the concept that p16INK4a is involved in the regulation of proliferation in glioblastomas. Other cell cycle regulators which are yet unknown may also play a role in the control of oligodendrogliomas or ependymomas outgrowth. Further studies are required to evaluate the role of p19INK4d in neuroglial tumours.

The PEN5 epitope identifies an oligodendrocyte precursor cell population and pilocytic astrocytomas.

PEN5 is a sulfated polylactosamine carbohydrate epitope first described in a subpopulation of mature natural killer cells. Here we report that it is also expressed in a developmentally regulated fashion in human and rat central nervous systems and that its protein carrier is P-selectin glycoprotein ligand-1 (PSGL-1), a ligand for selectins. In rat neural primary cultures, PEN5 is transiently and selectively expressed by oligodendrocyte precursor cells and marks the transition from proliferative to postmitotic stages. In concordance, in human central nervous system tumors, PEN5 is observed in a subset of oligodendrogliomas and in all pilocytic astrocytomas, a class of tumor of uncertain histogenesis. These data suggest that PEN5-PSGL-1 plays a role in the differentiation of oligodendrocytes and that pilocytic astrocytomas are likely to result from a dysregulation occurring in oligodendrocyte precursor cells at the crucial stage of exit from the cell cycle.

Pancreatic trypsinogen I expression during cell growth and differentiation of two human colon carcinoma cells.

Pancreatic trypsin has been found to induce tight junction or dome formation in some colon cancer cell lines (HT-29, Caco-2), and a tumor-associated trypsinogen, trypsinogen type II, has been isolated from another colon cancer cell line (COLO 205). We have tried to determine if trypsinogen is present and how its expression varies during cell culture in HT-29 Glc+/- and Caco-2 cells, which exhibit enterocytic differentiation, and in HT-29 Glc+ cells, which never differentiate. Trypsinogen mRNA presence and expression were demonstrated in these cells by mRNA hybridization, RT-PCR, cytoimmunofluorescence, Western immunoblot analysis, and gel filtration. Trypsinogen was found to be trypsinogen type I and was mainly in zymogen form in culture media. Differentiating cells exhibited variations in trypsinogen I expression, but cells that remained undifferentiated did not. In the differentiated cells, a high and transient peak in trypsinogen I expression was observed during the first steps of differentiation.