Mechanisms of equinatoxin II-induced transport through the membrane of a giant phospholipid vesicle.

Protein equinatoxin II from sea anemone Actinia equina L. was used to form pores in phospholipid membranes. We studied the effect of these pores on the net transmembrane transport of sucrose and glucose by observing single giant (cell-size) vesicles under the phase contrast microscope. Sugar composition in the vesicle was determined by measuring the width of the halo, which appears around the vesicle in the phase contrast image. The transport of sugars was induced when a vesicle, filled with the sucrose solution, was transferred into the isomolar environment of a glucose solution with added equinatoxin II. Typically, a vesicle grew to a critical size, then the membrane broke by bursting and the vesicle shrank, started to grow again, and the whole process was repeated. The consecutive membrane breaks occurred in the same spot. The observed behavior was interpreted by the diffusion flow of the glucose molecules through the equinatoxin II-induced pores and the consequent increase of the vesicle water content. The burst relaxed the critically strained membrane, which then apparently resealed. A mathematical model of the described behavior was developed and was used to obtain the equinatoxin II-induced membrane permeability for the glucose molecules. Its dependence on the equinatoxin II concentration is in agreement with the previous reports.

Stepwise genetic changes associated with progression of nontumorigenic HPV-18 immortalized human prostate cancer-derived cell line to a malignant phenotype.

Cytogenetics, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) were used to identify genes that are involved in the development and progression of prostate cancer. For that purpose, we chose a cell line established in vitro from a prostatic adenocarcinoma which was nontumorigenic in nude mice and followed its progression to a tumorigenic cell line. Stepwise changes were observed in the cell line as it became tumorigenic. The composite karyotype at the nontumorigenic stage (CA-HPV-10) was 68 approximately 77,XXY,-(1, 9, 13, 14, 19, 22),+(4, 5, 11, 18, 20, 21),+(del(1) (q23q31)=M1 (two copies), +der(9)t(1;9)(q24 approximately q31;p23)=M5(two copies), der(14)t(14;?)(q10;?)=M17 in the majority of metaphases. These two derivative chromosomes were also observed a previous study. Our CGH analysis clearly showed that this deleted region in M1 is, in fact, translocated with derivative M5 and, in reality, is amplified. The cell line established from nodule (SCID 5019 p11), showed a number of new changes, as described; however, the most significant change was amplification of the 8q23 approximately qter region, harboring c-myc. This region was translocated with chromosomes 2, 4, and 16 as der(2)t(2;8)(q33;q23)=M12, der(4)t(4;8)(q34;q23)=M11, and der(16)t(8;16)(q24;q21)=M9. We deduce from our study that amplification of c-myc and other genes in the 8q23 approximately qter region were important in progression but did not lead to tumorigenicity. The population that became tumorigenic (SCID 5019 II) showed almost all of the same changes in the karyotype as observed in the nodular cell line; the only significant change was the appearance of der(11)t(4;11)(q32;q22)=M7 and the addition of another copy of t(3q;7p)=M2. These new changes lead to loss of chromosomes 3p, 4pter approximately q34, 6, 7q21 approximately qter, 11q22 approximately qter, and 18q, and gain of 3q, 7p, 8q23 approximately qter, and 11pter approximately q22, before the cell line became tumorigenic. The clonal selection of the population is proven by the presence of a number of the same derivative chromosomes in both the nodular and tumorigenic cell line. As it progressed to tumorigenicity, some of the same changes observed in the original study re-appear at different stages of malignancy, although it was absent in the nontumorigenic cell line. These are: der(16)t(8;16)(q24;q21)=M9 in the nodular cell line and der(11)t(4;11)(q32;q22)=M7 in the tumorigenic cell line. In our system, amplification of c-myc and other genes in der(2)t(2;8)(q33;q23)=M12,der(4) t(4;8)(q34;q23)=M11 together with the presence of der(16)t(8;16)(q24;q21)=M9 and der(11)t(4;11)(q32;q22)=M5 makes the cell line tumorigenic. It is either nontumorigenic, with the presence of a marker equivalent to der(16)=M9 and der(11)=M7 observed in the original study, and only nodular (SCID 5019 p11, present study), with the presence of number of markers with c-myc amplification (M9, M11, and M12). There is accumulation of all the above-mentioned changes in the same cell before it becomes tumorigenic.

PDX-1 and cell-cell contact act in synergy to promote delta-cell development in a human pancreatic endocrine precursor cell line.

Cell lines from the fetal and adult pancreas that were developed by retroviral transfer of the SV40T and ras(val12) oncogenes lose insulin expression but retain extremely low levels of somatostatin and glucagon mRNA. In contrast to expanded populations of primary human islet cells, none of them express the homeodomain transcription factor PDX-1. When that factor was expressed in the cell lines by retroviral-mediated gene transfer, one of the cell lines, TRM-6, derived from human fetal islets, exhibited a 10- to 100-fold increase in somatostatin gene expression. This is the first report of induction of the endogenous somatostatin gene by PDX-1. Promotion of cell-cell contact by aggregation of TRM-6/PDX-1 into islet-like clusters produced a further 10- to 100-fold increase in somatostatin mRNA, to a level similar to that of freshly isolated islets, which resulted in production of somatostatin protein. Thus, we demonstrate here that signals induced by cell-cell contact act in synergy with PDX-1 to up-regulate the endogenous somatostatin promoter in an immortalized cell line from human fetal islets. This system provides a powerful model for studying human islet cell development and, particularly, the role of cell-cell contact in the differentiation process.

Sustained proliferation of PDX-1+ cells derived from human islets.

Ex vivo expansion of human beta-cells is an important step toward the development of cell-based insulin delivery systems in type 1 diabetes. Here, we report that human pancreatic endocrine cells can be expanded through 15 cell doublings in vitro for an estimated total 30,000-fold increase in cell number. We believe that the cells resulting from these cultures are of beta-cell origin, since they uniformly express the transcription factor PDX-1 (STF-1, IDX-1, IPF-1), which is initially seen only in cells positive for insulin and negative for the ductal cell marker cytokeratin (CK)-19. To rule out the possibility that PDX-1 expression might be induced by the culture conditions used here, cells from isolated human pancreatic ducts were cultured under the same conditions as the islet cells. Cells in these cultures expressed CK-19 but not PDX-1. Although the expanded beta-cells continued to express PDX-1, insulin expression was lost over time. Whether reexpression of islet-specific genes in vitro is essential for successful cell transplantation remains to be determined.

KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: morphoregulatory roles in pancreatic islet development.

Cell adhesion molecules (CAMs) are important mediators of cell-cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell-cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.

Phosphatidylinositol 3-kinase is a negative regulator of cellular differentiation.

Phosphatidylinositol 3-kinase (PI3K) has been shown to be an important mediator of intracellular signal transduction in mammalian cells. We show here, for the first time, that the blockade of PI3K activity in human fetal undifferentiated cells induced morphological and functional endocrine differentiation. This was associated with an increase in mRNA levels of insulin, glucagon, and somatostatin, as well as an increase in the insulin protein content and secretion in response to secretagogues. Blockade of PI3K also increased the proportion of pluripotent precursor cells coexpressing multiple hormones and the total number of terminally differentiated cells originating from these precursor cells. We examined whether any of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in fetal islet cells. The activity of PI3K was inversely correlated with the hepatocyte growth factor/scatter factor-induced downregulation or nicotinamideinduced upregulation of islet-specific gene expression, giving support to the role of PI3K, as a negative regulator of endocrine differentiation. In conclusion, our results provide a mechanism for the regulation of hormone-specific gene expression during human fetal neogenesis. They also suggest a novel function for PI3K, as a negative regulator of cellular differentiation.

Isolation and characterization of a cell line from the epithelial cells of the human fetal pancreas.

Pancreatic cell lines are useful for basic studies of pancreatic biology and for possible application to cell transplantation therapies for diabetes. A retroviral vector expressing simian virus 40 (SV40) T antigen and H-rasval12 was used to infect a monolayer culture of epithelial cells from an 18-wk human fetal pancreas. Infected cells gave rise to a clonal epithelial cell line, designated TRM-1. This cell line expresses epithelial markers as well as gult2 and small amounts of insulin and glucagon. TRM-1 is the first cell line to be generated from the human fetal pancreas and also the first cell line derived directly from the fetal pancreas of any species. The approach that we have used to develop TRM-1 should be applicable to isolating cell lines from other stages of human pancreatic development.

Regulation of proliferation and differentiation of human fetal pancreatic islet cells by extracellular matrix, hepatocyte growth factor, and cell-cell contact.

Ex vivo expansion of human fetal pancreatic endocrine cells is important for biological studies and as a potential tissue source for transplantation in insulin-deficient states. In tissue culture experiments involving the use of hepatocyte growth factor/scatter factor and selected extracellular matrices, we obtained a 30-fold increase in cell number of human fetal pancreatic epithelial cells. This proliferation in monolayer culture was associated with marked downregulation of insulin and glucagon gene expression. However, gene expression increased when the cells were combined into three-dimensional aggregates, suggesting that cell-cell contact mediated mechanisms regulate the transcription of islet-specific genes, a process enhanced by nicotinamide (NIC). After transplantation into nude mice, either as cell suspensions or aggregates, only the cell aggregates treated with NIC developed into mature functional islet-like structures. These are the first experiments to describe the interactions of specific matrices and growth factors in the ex vivo expansion of human fetal pancreatic cells, and they also show the importance of cell aggregates in the context of cellular and molecular events that might positively influence islet cell transplantation.

A role for hepatocyte growth factor/scatter factor in fetal mesenchyme-induced pancreatic beta-cell growth.

We have investigated the role of hepatocyte growth factor/scatter factor (HGF/SF) in the growth and/or differentiation of pancreatic islet beta-cells. We found that in the human fetal pancreas immunoreactive HGF/SF receptor (c-met proto-oncogene product) is preferentially associated with the developing beta-cells. In the adult pancreas, c-met messenger RNA is highly enriched in the islets and the immunoreactive protein is also restricted to the islet beta-cells. HGF/SF messenger RNA content of fetal pancreas-derived fibroblasts is more than 10-fold higher than that of adult fibroblasts. Culture of human fetal pancreatic epithelial cells in conditioned medium from the fetal pancreatic fibroblasts caused a 2.4-fold stimulation of the formation of islet-like cell clusters that was due to both mitogenic and morphogenic effects. Beta-cell proliferation in the cell clusters was stimulated 3.5-fold by the conditioned medium, and this was associated with a marked decrease in insulin content. All of the effects of the conditioned medium were blocked by anti-HGF/SF antibody. Specificity was confirmed by overriding the blocking effect of the antibody with excess recombinant HGF/SF. Conditioned medium from adult pancreatic fibroblasts stimulated islet-like cell cluster formation only slightly, and did not affect beta-cell replication. These results suggest that HGF/SF secreted by fetal fibroblasts is mitogenic to beta-cells. Taken together, our findings indicate an important role for HGF/SF in fetal mesenchyme-induced pancreatic beta-cell growth.

ICA69 is expressed equally in the human endocrine and exocrine pancreas.

Islet cell autoantigen 69 kDa (ICA69) has been reported as a polypeptide antigen expressed in pancreatic beta cells, and autoimmunity against this antigen has been associated with insulin-dependent diabetes mellitus. We have studied the cell type specificity and ontogeny of ICA69 gene expression in man. The ICA69 gene was expressed in all adult human tissues. The level of expression was three-to five-times higher in the pancreas than in the brain, liver, intestine, kidney, spleen, lung or adrenal glands. Pancreatic ICA69 expression increased with age, adult levels being five times higher than the levels present at 13 weeks of gestation. Total RNA from four separate preparations of isolated human islets revealed levels of ICA69 mRNA similar to those found in the pancreas as a whole, although another islet antigen, glutamic acid decarboxylase 65, was highly enriched in the islets. In situ hybridization and immunohistochemical staining of sections of the fetal and adult pancreas revealed expression of the ICA69 gene and protein throughout the acinar, ductal, and islet tissue, but not in the mesenchyme. Analysis of ICA69 mRNA levels in human cell lines indicated expression in neural, endothelial and epithelial cells, but not in fibroblasts. In conclusion, ICA69, although highest in the pancreas, is widely distributed in other human tissues, excluding connective tissue. Within the human pancreas, ICA69 is not enriched in the islets or in the beta cells.

Ontogeny and tissue distribution of human GAD expression.

One of the major beta-cell autoantigens associated with IDDM is GAD. Although GAD expression has been detected in adult islets, transcriptional expression of the GAD genes has not been reported during human pancreatic ontogeny. We therefore analyzed patterns of GAD gene transcription by quantitating the mRNAs encoding both the 65- and 67-kDa isoforms (GAD65 and GAD67, respectively) in human fetal, postnatal, and adult pancreases, as well as in isolated adult islets, and examined their tissue-specific expression. Significant levels of pancreatic GAD65 transcripts were already detected at 13 weeks of gestation and were expressed at higher levels in the fetal and infantile pancreas than in the adult pancreas. Isolated adult pancreatic islets were highly enriched in GAD65 mRNA. In contrast, GAD67 transcripts were not detectable in fetal and postnatal pancreases. In addition to the pancreas, marked GAD expression was detected in the brain, whereas other tissues examined contained either low or undetectable GAD transcripts. Triple immunofluorescent staining of fetal and adult pancreases revealed colocalization of GAD65 with alpha- and beta-cells. In the fetal pancreas, strong immunoreactivity for GAD65 was also evident in epithelial cells, which lacked expression of insulin or glucagon, some of which were present in the ductal epithelium, suggesting that GAD65 expression might correlate with endocrine determination. In summary, 1) this is the first demonstration of GAD65 expression in the human fetal pancreas, implicating a potential role during islet development, and 2) GAD65 may be a useful marker for the identification of primitive islet cells.

Developmental gene expression in the human fetal pancreas.

Differential developmental regulation of pancreas-specific genes has not been reported for the human fetal pancreas. We have therefore undertaken a systematic, quantitative analysis of the transcriptional levels of various genes in the human pancreas at different stages of fetal and postnatal development. Using sensitive ribonuclease protection assays, in situ hybridization, and the polymerase chain reaction, our results indicate the following: 1) Transcriptional levels of insulin and amylin remain lower in the fetal than in the adult pancreas, whereas glucagon and somatostatin mRNA levels are consistently greater after 14 wk gestation than postnatally. These results are in agreement with previous immunohistochemical studies of these gene products. 2) The reg gene exhibits a 20-fold increase in mRNA levels after 16 wk gestation. The gene is expressed exclusively in the acinar cells and does not colocalize with insulin. This restricted exocrine expression does not indicate a direct role for the reg gene in islet development. 3) Glucose transporter 2 and glucokinase mRNA are detectable as early as 13 wk gestation and remain low throughout development. Glucose transporter 1 reaches adult transcriptional levels by 18 wk gestation. The early detection of glucose transporter 2 and glucokinase implies that lack of expression of these "glucose sensor" genes does not account for the known insensitivity of the fetal beta-cells to glucose.

Opposite effects of beta-cell differentiation and growth on reg expression in human fetal pancreatic cells.

Reg is a gene associated with regeneration of pancreatic islets. We have previously shown that nicotinamide induces differentiation of human fetal beta-cells in tissue culture and that hepatocyte growth factor/scatter factor (HGF/SF) is mitogenic for the fetal beta-cells. We now tested whether these conditions, supporting either differentiation or growth, are associated with changes in reg gene expression in human fetal pancreatic cells. Culture for 7 days with 10 mM nicotinamide led to a fourfold decrease in reg mRNA levels (23 +/- 12% of control, n = 5, P < 0.001). In contrast, HGF/SF increased reg expression threefold (302 +/- 68% of control, n = 4, P < 0.05). Nicotinamide, which does not alter the differentiation level of adult beta-cells, did not significantly affect reg expression in adult human islets (84 +/- 4% of control, n = 2, NS). Thus, a higher level of endocrine differentiation is associated with a lower level of reg expression, and a higher rate of beta-cell replication results in increased reg transcription. These results provide the first evidence of a molecular marker, the reg gene, to distinguish between proliferation and differentiation of human beta-cells.

Acid beta-galactosidase: a developmentally regulated marker of endocrine cell precursors in the human fetal pancreas.

Isolation of endocrine cell precursors from the human fetal pancreas will be important to the study of islet cytodifferentiation and eventually for islet transplantation in insulin-dependent diabetes. These precursor cells, from which all four islet endocrine cell types arise, are present within fetal pancreatic ductal epithelium. After enzymatic digestion and culture of the fetal pancreas, we obtained cell clusters resembling islets, but with a high content of undifferentiated cells. Histochemical staining revealed very high acid beta-galactosidase activity in over 70% of cells within the clusters. After transplantation into athymic nude mice, the islet-like cell clusters gave rise to tissue rich in differentiated endocrine cells, but low in beta-galactosidase activity. The histochemical finding of high acid beta-galactosidase activity in endocrine precursor cells was confirmed by direct measurement of lysosomal enzyme activities. In addition, we found that the expression of acid beta-galactosidase was developmentally regulated, peaking at 18-24 weeks gestation and declining to low levels in adult islets. Using a fluorogenic beta-galactosidase substrate, we were able to isolate a subpopulation of cells high in acid beta-galactosidase activity using fluorescence-activated flow cytometry. Evidence identifying these cells as potential islet cell precursors includes, besides the transplantation experiments, the colocalization in vitro of tyrosine hydroxylase, a marker of embryonic islet cells. Thus, our results indicate that high acid beta-galactosidase activity serves as a marker for a population of fetal pancreatic cells with the potential to differentiate and grow into mature pancreatic endocrine cells.

Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells.

The effects of nicotinamide (NIC) on human fetal and adult endocrine pancreatic cells were studied in tissue culture. Treatment of the fetal cells with 10 mM NIC resulted in a twofold increase in DNA content and a threefold increase in insulin content. This was associated with the development of beta cell outgrowths from undifferentiated epithelial cell clusters and an increase in the expression of the insulin, glucagon, and somatostatin genes. DNA synthesis was stimulated only in the undifferentiated cells. Half-maximal doses for the insulinotropic and mitogenic effects of NIC were 5-10 and 1-2 mM, respectively. Islet-like cell clusters cultured with NIC responded to glucose stimulation with a biphasic increase in insulin release (fourfold peak), whereas control cells were unresponsive to glucose. Both control and NIC-treated cells developed into functional islet tissue after transplantation into athymic nude mice. As compared with adult islets, the insulinotropic action of NIC could only be demonstrated in the fetal cells. Our results indicate that NIC induces differentiation and maturation of human fetal pancreatic islet cells. This model should be useful for the study of molecular mechanisms involved in beta cell development.

Phorbol ester synergizes with Ca2+ ionophore in activation of protein kinase C (PKC)alpha and PKC beta isoenzymes in human T cells and in induction of related cellular functions.

Studies described herein were designed to examine the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA), and a Ca2+ ionophore (ionomycin), singly or in combination, on the activation and expression of the Ca(2+)-dependent protein kinase C (PKC) isoenzymes (alpha, beta and gamma) at the protein and messenger RNA (mRNA) levels in T cells. These two agents induce the activation and proliferation of T lymphocytes by mimicking the action of inositol phospholipid-derived second messengers normally generated by triggering of the antigen-specific T-cell receptor (TcR)/CD3 complex. TPA-induced T-cell proliferation, expression of interleukin-2 receptor-alpha subunit (IL-2R alpha) and transferrin receptor, CD3 down-regulation and, lastly, the cytosol-to-membrane PKC translocation (determined by an enzymatic assay or by immunoblotting with a cross-reactive anti-PKC peptide antibody) were all facilitated by ionomycin. Immunoblots with isoenzyme-specific anti-PKC monoclonal antibodies demonstrated expression of immunoreactive PKC alpha, PKC beta and PKC gamma proteins that were translocated to the membrane upon TPA plus ionomycin stimulation. Resting T cells expressed abundant levels of mRNA for PKC alpha and PKC beta, but very low levels (relative to brain) of PKC gamma. TPA increased by two- to threefold the expression of PKC beta, but not of PKC alpha or PKC gamma, mRNA within 12 hr of stimulation. Ionomycin synergized with TPA in increasing the expression of PKC alpha and PKC beta mRNA. The two agents also synergized in inducing expression of additional activation/growth-associated genes, namely the c-myc protooncogene, ornithine decarboxylase (ODC) and IL-2R alpha. Ionomycin alone was inactive (or marginally active) in all of these assays. The translocation of distinct Ca(2+)-dependent PKC isoenzymes to the membrane and the up-regulation of PKC alpha and beta mRNA suggest that at least these two isoenzymes are involved in discrete steps of the pathway leading to T-cell activation and proliferation. Moreover, the combined effects of TPA and ionomycin on T-cell function and cell-surface antigen expression appear to be due, at least in part, to their synergistic activation of distinct PKC isoenzyme(s).

Mitogen-induced human T cell proliferation is associated with increased expression of selected PKC genes.

The induction of T cell proliferation and differentiation into mature effector cells is dependent on two principal exogenous signals that are provided by the antigen or mitogen and IL2. The enzyme protein kinase C (PKC) has a major role in the antigen-receptor signalling pathway in T cells, but appears not to be involved in signalling via the IL2-receptor (IL2-R). Since both pathways trigger a series of sequentially coordinated transcriptional events in which numerous genes are activated, we tested whether a T cell mitogen acting via the TCR/CD3 complex, and IL2, affect the expression of the conventional, Ca(2+)-dependent, PKC genes (alpha, beta and gamma) in T cells. Stimulation of human peripheral blood lymphocytes or an enriched population of human T cells with phytohemagglutinin resulted in augmented mRNA levels of PKC alpha and PKC beta, but not PKC gamma-gene. The response peaked at 24-48 hr when a 3-5-fold increase was observed. Stimulation of IL2-R alpha-expressing T cells with human recombinant IL2 induced cell proliferation and transcription of the IL2-R alpha gene (greater than 100-fold), but did not change mRNA levels of PKC alpha or PKC beta genes. The results suggest that stimulation of human T cells with mitogens acting via the TCR/CD3 complex, that involve activation of PKC, is accompanied also by a late activation of selected PKC genes. By contrast, agonists such as IL2, that operate via a different signalling pathway, do not modify the expression of any of the known conventional PKC genes.