Establishment and immunocharacterization of an immortalized pancreatic cell line derived from the H-2Kb-tsA58 transgenic mouse

Summary This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2K^b-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products α-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.     

EPN: a novel epithelial cell line derived from human prostate tissue

This work reports the isolation and characterization of a line of human, nontransformed and differentiated prostate epithelial cells (EPN) in continuous culture. Primary cultures of epithelial prostate cells were set up using normal tissue isolated from a prostate sample collected after radical prostatectomy for cancer. After 70 passages, EPN cells did not undergo "Hayflike crisis" and were free of fibroblast contamination and were thus subcloned and characterized. EPN cells in culture, as prostate epithelial cells in vivo, express high-molecular weight cytokeratin and Pyk2, whereas they do not express desmin. EPN cells are nontransformed because they do not form colonies in semisolid medium and do not form tumors once injected into nude mice. EPN cells express the functional androgen receptor, which can mediate the mitogenic activity of testosterone. Finally, clonal production of the prostate-specific antigen could be detected in EPN cells. The availability of a line of epithelial nontransformed prostate cell in culture will be useful in investigating the complex process regulating normal prostate physiology as well as the development and progression of prostate tumors.     

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.     

Establishment and characterization of a noradrenergic adrenal chromaffin cell line, tsAM5NE, immortalized with the temperature-sensitive SV40 T-antigen

We established a clonal adrenal medullary cell line, named tsAM5NE, from transgenic mice harbouring the temperature-sensitive Simian virus 40 large T-antigen gene, under the control of the tyrosine hydroxylase promoter. tsAM5NE cells conditionally grew at a permissive temperature of 33°C and exhibited the noradrenergic chromaffin cell phenotype. To understand the characteristics of tsAM5NE cells, we first examined the responsiveness of the cells to ligands of the GDNF (glial cell line-derived neurotrophic factor) family. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not artemin or persephin. At the non-permissive temperature of 39°C, GDNF or neurturin caused tsAM5NE cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, LIF (leukaemia inhibitory factor) did not affect the GDNF-mediated cell proliferation at 33°C, but promoted the survival and differentiation of GDNF-treated cells at 39°C. In the presence of GDNF plus LIF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal markers, indicating that the cells had indeed undergone neuronal differentiation. Thus, we demonstrated that tsAM5NE cells had the capacity to terminally differentiate into neuron-like cells in response to GDNF plus LIF when the oncogene was inactivated by the temperature shift. Thus, this cell line provides a useful model system for studying the mechanisms regulating neuronal differentiation.     

Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.     

Establishment and characterization of a spontaneously immortalized mouse ameloblast-lineage cell line

Tooth development was cooperatively regulated by the epithelial ameloblasts and mesenchymal odontoblasts. Ameloblasts secrete enamel matrix, critical for enamel formation. While there are several reports about establishment of immortalized ameloblast-like cells by introducing viral oncogene, we tried to establish a spontaneously immortalized ameloblast-lineage cell line, maintaining the cell type specific character, including the ability to induce in vitro bio-mineralization. The established cell line (ameloblast-lineage cell; ALC) maintained the expression of several ameloblast specific genes (Amelogenin, Tuftelin, and Enamelin) in long-term culture. They formed calcified nodules after the induction by medium switching from SMEM to DMEM, having high-level alkaline-phosphatase activity. The size and number of calcified nodule formation were enhanced by TGF-beta treatment. Six weeks after sub-cutaneous implantation of ALC to athymic nude mice, we ectopically observed enamel epithelium like structure formation, chondrogenesis, and calcification. These data indicate that ALC is a useful experimental tool to analyze ameloblast character.     

Presence of side-population cells in an immortalized nontumorigenic human liver epithelial cell line

Side-population (SP) cells have been shown to be highly enriched stem cells. We investigated whether an immortalized, nontumorigenic human liver cell line, THLE-5b, contains SP cells. Flow cytometry analysis after Hoechst 33342 staining demonstrated that the THLE-5b line contained a small component of SP cells. These SP cells were essentially eliminated by treatment with verapamil and expressed higher levels of ABCG2 mRNA than non-SP cells. In addition, the level of these SP cells detected by Hoechst 33342 staining was affected by the experimental conditions including the incubation medium. This is the first report of the presence of SP cells in the immortalized, nontumorigenic human liver cell line.     

Establishment and characterization of an epithelial intestinal cell line from rat fetus

An epithelial intestinal cell line has been established from explants of fetal rat small intestine. After the 9th passage (approx. 25 population doublings) epithelial-like cells acquired the properties of a permanent cell line. The epithelial nature of this cell line, and of clone IRD 98 subsequently isolated, is supported by morphological and ultrastructural criteria, and also by the presence of enzymes characteristic of enterocytes, such as aminopeptidase, alkaline phosphatase, gamma-glutamyl transferase, lactase and maltase. The occurrence of the triglyceride pathway enzyme monoacylglycerol acyltransferase and of apoproteins (Apo A1 and Apo E) can also be demonstrated. Taken together, the results presented here provide evidence that clone IRD 98 is an epithelial cell line, most likely originating from the relatively differentiated cell layer of fetal rat small intestine.     

A novel human astrocyte cell line (A735) with astrocyte-specific neurotransmitter function

Summary Studies of brain cell function and physiology are hampered by the limited availability of imortal human brain-derived cell lines, as a result of the technical difficulties encountered in establishing immortal human cells in culture. In this study, we demonstrate the application of recombinant DNA vectors expressing SV40 T antigen for the development of immortal human cell cultures, with morphological, growth, and functional properties of astrocytes. Primary human astrocytes were transfected with the SV40 T antigen expression vectors, pSV3neo or p735.6, and cultures were established with an extended lifespan. One of these cultures gave rise to an immortal cell line, designated A735. All the human SV40-derived lines retained morphological features and growth properties of type 1 astrocytes. Immunohistochemical studies and Western blot analysis of the intermediate filament proteins and glutamine synthetase demonstrated a differentiated but immature astrocyte phenotype. Transport of γ-amino butyric acid and glutamate were examined and found to be by a glial-specific mechanism, consistent with the cell lines’ retaining aspects of normal glial function. We conclude that methods based on the use of SV40 T antigen can successfully immortalize human astrocytes, retaining key astrocyte functions, but T antigen-induced proliferation appeared to interfere with expression of glial fibrillary acidic protein. We believe A735 is the first documented nontumor-derived human glial cell line which is immortal.     

Establishment of a rat Sertoli cell line that displays the morphological and some of the functional characteristics of the native cell

Primary rat Sertoli cells are widely used as a model for mechanistic and toxicological studies, since they are often the target of toxicants in vivo. However, their isolation from testicular homogenates is tedious and requires the regular use of numerous immature animals. It is therefore of great interest to have available established cell lines that are usable in vitro for unlimited periods and closely similar to native cells. To this end, we have established a line of Wistar rat Sertoli cells (SerW3) by immortalization of fresh primary cells with the T antigens of the Simian virus (SV40). When plated on Matrigel, this cell line presents many of the functional characteristics of Sertoli cells in vivo. In addition, they are sensitive to cisplatin and secrete transferrin, although they do not show a clear response to follicle-stimulating hormone. They also present many morphological similarities, including the presence of tight junctions which mimic the natural epithelial barrier. Like Sertoli cells in vivo, they show extensive phagocytic activity. Finally, they display all the characteristics of immortalized, but not transformed, cells, i.e., topo-inhibition and apoptosis at confluence or under serum deprivation.     

Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line

Background  

The Retinal Pigmented Epithelium (RPE) is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa (RP), Fundus Flavimaculatus (FFM), Best's disease and Age-related Macular Degeneration (AMD). We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases.     

Establishment and characterization of a tamoxifen-mediated reversible immortalized mouse dental papilla cell line

Odontoblasts are a type of non-proliferating and terminally differentiated cells that play an important role in the pulpo–dentinal complex. Mouse dental papilla cells (mDPCs), which can differentiate into odontoblast-like cells in vitro, have a limited life span. We combined the traditional strategy of “Cre/LoxP-based reversible immortalization” with a tamoxifen-regulated Cre recombination system to generate a tamoxifen-mediated reversibly immortalized mouse dental papilla cell line, mDPCET. mDPCs were sequentially transduced with a floxed SV40 T antigen-TK (SV40Tag-TK) and an ^ERT2Cre^ERT2-expressing plasmid. Clonal-isolated SV40Tag- and Cre-positive cells showed modified growth characteristics and a significantly extended life span. When mDPCET cells were treated with 4-hydroxytamoxifen, ^ERT2Cre^ERT2 translocated from the cytoplasm to the nucleus and caused the excision of SV40Tag-TK, which led to the reversion of mDPCETs. After the immortalization was reversed, the cells underwent replicative senescence and transitioned into a more differentiated state. Tamoxifen-mediated reversible immortalization, therefore, allows for the expansion of primary mDPCs, leads to the production of odontoblast-like cells that retain most odontoblast-specific properties, and can represent a safe and ready-to-use method due to its simple manipulation.     

Establishment and characterization of an epithelial cell line, SGE1, from isolated rat renal glomeruli

An epithelial cell line, designated as SGE1, has been established from isolated rat renal glomeruli. SGE1 cells are able to grow continuously in a serum-free medium at similar growth rates to those in the medium containing serum. Quantitative studies of the cells in the serum-free condition demonstrated that SGE1 cells required a collagen type I or IV, fibronectin, or laminin-coated substratum for adhesion and growth, and among them, collagen type I and IV were most effective. Essential medium supplements for the adhesion and growth were epidermal growth factor and transferrin, respectively, and both effects were noticeably enhanced with linoleic acid. Morphological observation found that in a monolayer culture, SGE1 cells formed domes, and in a collagen embedding culture, they formed cystic spheres having features of a simple cuboidal epithelium, polarized formation of microvilli and tight junctions as well as a lateral cell membrane with cytoplasmic projections. In addition, SGE1 cells expressed Fx1A antigens, which are nephritogenic antigens on their microvilli.     

Creation and characterization of an immortalized canine myoblast cell line: Myok9

Mammalian Genome - The availability of an in vitro canine cell line would reduce the need for dogs for primary in vitro cell culture and reduce overall cost in pre-clinical studies. An immortalized...     

Differentiation and growth potential of human ovarian surface epithelial cells expressing temperature-sensitive SV40 T antigen

The epithelial ovarian carcinomas arise in the ovarian surface epithelium (OSE) which is the mesothelial covering of the ovary. Studies of human USE have been hampered by the small amounts and limited lifespan of this epithelium in culture. OSE cells expressing SV40 large T antigen (Tag) or the HPV genes E6 and E7 have increased growth potentials but lack some of the normal characteristics of OSE. In this study, we used conditional SV40 Tag expression to produce OSE cells with increased proliferative potentials but relatively normal phenotypes. Primary OSE cultures from three women, one of whom had a BRCA1 mutation, were infected with a temperature-sensitive Tag construct (tsTag), and from these, 28 monoclonal and four polyclonal lines were isolated. The effects of temperature changes were examined in two monoclonal and two polyclonal lines. At the permissive temperature (34 degrees C), these cell lines underwent 52-71 population doublings (PD) compared to 15-20 PD for normal OSE. Nuclear SV40-Tag and p53 expression, demonstrated by immunofluorescence, showed that tsTag was uniformly present and biologically active in all lines. At 34 degrees C, culture morphologies ranged from epithelial to mesenchymal. The mean percentage of cells expressing the epithelial differentiation marker, keratin. varied between lines from 20 to 97%. Collagen type III, a mesenchymal marker expressed by OSE in response to explantation into culture, was present in 24-43% of cells. At 39 degrees C, tsTag was inactivated by 2 d while nuclear p53 staining diminished to control levels over 2 wk. Over 3 d. the cells assumed more epithelial morphologies, keratin expression reached 85-100% in all lines and collagen expression increased significantly in two lines. The cultures with the BRCA1 mutation expressed the most keratin and the least collage n III at both temperatures. As indicated by beta-galactosidase staining at pH 6.0, changes leading to senescence were initiated at 39 degrees C by 6 h and were present in all cells after 24 h. However, the cells underwent 1-3 population doublings over up to 1 wk before growth arrest and widespread cell death, thus providing an experimental system where large numbers of OSE cells with different genetic backgrounds and growth potentials can be studied without the concurrent influence of Tag.     

Establishment and characterization of an abrin-resistant cell line

An abrin-resistant cell line, CHOR 3-4, was isolated from CHOK1 cells which were resistant to a high concentration of abrin (160 ng/ml), and had a 1000-fold higher resistance to abrin that that of CHOK1 cells. CHOR 3-4 cells were about 25-fold more resistant than CHOK1 cells to the N-glycosidase activity of abrin, which was measured by hydrolyzing the N-glycosidic bond of adenine 4324 nucleotide from 3′ end of mammalian 28S rRNA. However, the isolated polysomes of CHOR 3-4 cells had the same sensitivity to abrin as those of CHOK1 cells. On measuring the binding of 125I-abrin to CHOR 3-4 cells, it was decreased to about 20% that of CHOK1 cells. This indicates that the mechanism of the resistance of CHOR 3-4 to abrin is due to the alteration of glycoproteins or glycolipids of cell membrane.     

Quantification of elastase-like activity in 13 human cancer cell lines and in an immortalized human epithelial cell line by RP-HPLC

A sensitive and specific RP-HPLC assay was developed to measure the levels of polymorphonuclear elastase (PMN-E) activity in growing cell cultures. By combining a pre-incubation of the cells with a relatively non-toxic, PMN-E-specific inhibitor, MeOSuc-Ala-Ala-Pro-Val-chloromethylketone (MAAPVCK), the p-nitroaniline formed by the hydrolysis of the substrate MeOSuc-Ala-Ala-Pro-Val-p-NA by PMN-E is quantified. Elastase-like activity was measured in 14 human cells lines: 13 cancer cell lines (HL-60, U-937, A-427, LCLC-103H, YAPC, DAN-G, PA-TU-8902, KYSE-70, -510, -520, 5637, SISO and MCF-7) and one immortalized epithelial cell line (hTert-RPE1). Activity was detected in all lines; the lowest was found in hTert-RPE1 cells while the highest was detected in a pancreas adenocarcinoma line (PA-TU-8902). When the results were normalized according to cell volume instead of cell number, the leukemia line HL-60 had the highest activity and PA-TU-8902 ranked second. A 1 h pre-incubation with 9.0 microM of the irreversible PMN-E inhibitor MAAPVCK led to varying degrees of enzyme inhibition depending on the cell line; the strongest inhibition was observed with the PA-TU-8902 pancreatic cancer cell line (90% inhibition) while the weakest was seen with the A-427 lung cancer cell line (52%). These results indicate that PA-TU-8902 is a suitable in vitro model for testing the efficacy of PMN-E-activated prodrugs of antitumor agents.