Development and characterization of conditionally immortalized gastric epithelial cell lines from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene

Conditionally immortalized gastric epithelial cell lines were established from transgenic rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene. Gastric mucosal cells and epithelial tissues isolated from the stomach of the transgenic rats were cultured at permissive temperature (33 degrees C), and proliferative cells were cloned by colony formation. Six cell lines (designated as RGE1-01, RGE1-02, RGE1-03, RGE1-21, RGE1-22 and RGE2-01) showing epithelial-like morphology have been established. All cells grew at 33 degrees C, but did not at nonpermissive temperature (39 degrees C). High expression level of large T-antigen in the nuclei was observed at 33 degrees C, whereas the expression level was gradually decreased in a time-dependent manner at 39 degrees C. These results suggest that the temperature-sensitive growth characteristics arise as a result of a function of the tsSV40 large T-antigen. None of the cell lines were transformed as judged by anchorage-independent growth assay. Immunocytochemical findings indicated that all cells expressed epithelial cell markers including cytoskeletal (cytokeratin and actin), basement membrane (laminin and collagen type IV) and junctional complex (ZO-1 and desmoplakin I+II) proteins at 33 degrees C. All cells expressed mRNA of cathepsin E, a pit cell marker. Moreover, transepithelial resistance was observed between apical and basolateral sides in the cells. RGE1-22 cells produced prostaglandin E(2). Levels of mRNA for cathepsin E, transepithelial resistance and prostaglandin E(2) were influenced by the nonpermissive temperature. Thus, these conditionally immortalized gastric cell lines which preserve some epithelial cell characteristics will provide a useful in vitro model of gastric epithelium.     

Identification of genes responsive to sodium butyrate in colonic epithelial cells

We identified genes responsive to sodium butyrate (SB) in colonic epithelial cells using cDNA microarrays. Treatment with 2 mM SB of colonic epithelial cells (MCE301), which was derived from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen, arrested cell growth and showed a differentiated phenotype accompanying an increase in alkaline phosphatase activity. Of the approximately 900 genes analyzed, SB down-regulated 25 genes and up-regulated 88 genes by a factor of 2.0 or greater. Northern blot or TaqMan and Western blot analyses confirmed that the mRNA and protein levels of cyclin D1 and the level of proliferating cell nuclear antigen decreased, whereas the levels of integrin beta1 and osteopontin increased. The present results regarding the changes in gene expression, arrived at using microarrays, will provide a basis for a further understanding of the molecular mechanisms of cell growth arrest and differentiation in response to SB in colonic epithelial cells.     

Functional characterization of a conditionally immortalized mouse epididymis caput epithelial cell line MEPC5 using temperature-sensitive simian virus 40 large T-antigen

A conditionally immortalized epididymis caput cell line, MEPC5, was established by infecting primary cultured mouse epididymis caput cells with a temperature-sensitive simian virus 40 large T-antigen. At a permissive temperature of 33 degrees C, the large T-antigen was expressed and the cells grew continuously. However, the downregulation of T-antigen at a nonpermissive temperature of 39 degrees C and the upregulation of cell density at 33 degrees C were associated with growth arrest and the increased protein expression of p21(waf1), a cell cycle inhibitor. The cells expressed epididymal caput-expressed genes such as phosphatidylethanolamine binding protein, polyoma enhancer activator 3, ME1, sulfated glycoprotein-2 (SGP-2), androgen receptor, and retinoic acid receptor alpha. Interestingly, the expression levels of ME1 and SGP-2 were significantly elevated under the cell growth-restricted conditions. The established mouse epididymis caput epithelial cell line MEPC5 retains some characteristics of differentiated epididymis epithelial cells, and should prove an excellent model for studies of gene expression and the physiological functions of epididymis caput epithelial cells.     

Development of a conditionally immortalized testicular Sertoli cell line RTS3-3 from adult transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene

Transgenic mice and rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene are useful for establishing cell lines from tissues. We succeeded in establishing a conditionally immortalized testicular Sertoli cell line, RT3-3, from adult transgenic rats harboring the oncogene. The cells grew at permissive (33 degrees C) and intermediate (37 degrees C) temperatures but not at nonpermissive temperature (39 degrees C). Large T-antigen was expressed at 33 and 37 degrees C, whereas the expression level was gradually decreased at 39 degrees C, suggesting that the temperature-sensitive growth characteristics arise as a result of the function of tsSV40 large T-antigen. The cells showed biochemical features associate with normal Sertoli cells including expressions of mRNAs of sulfated glycoprotein-2 (SGP-2), transferrin (TF) and steel factor. Quantitative polymerase chain reaction revealed that nonpermissive temperature induced increase in the level of SGP-2. Moreover, levels of SGP-2 and/or TF were significantly elevated in the cells treatment with sodium butyrate and retinoic acid, inducers of cellular differentiation. To our knowledge, this is the first report of the establishment of a testicular Sertoli cell line from the transgenic rats. Thus, the conditionally immortalized cell line RTS3-3 with unique characteristics may serve as good experimental in vitro models for basic and applied biology of testicular Sertoli cells.     

Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling

Hepatocyte growth factor (HGF) can promote the regeneration of injured organs, including HGF gene therapy by electroporation (EP) for liver injury. In this study, we investigated the effect of HGF on dextran sulfate sodium-induced colitis and tried to clarify the regenerative mechanisms of colonic epithelial cells and the signaling pathway involved. Colitis was induced by dextran sulfate sodium in mice, together with HGF gene transfer by EP. On day 10, the colitis was evaluated histologically and by Western blot analysis. The colonic epithelial cell line MCE301 was exposed to HGF protein, and its proliferation and activated signaling pathway were analyzed. In vivo, the histological score improved and the number of Ki-67-positive epithelial cells increased in the HGF-treated mice compared with the controls. Western blot analysis showed enhanced expression of phospho-Akt in the HGF-treated mice compared with the controls. In vitro, HGF stimulated the proliferation of MCE301 cells. There was enhanced phospho-Akt expression for more than 48 h after HGF stimulation, although phospho-ERK1/2 was enhanced for only 10 min. LY-294002 or Akt small interfering RNA suppressed cell proliferation induced by HGF. Thus HGF induces the proliferation of colonic epithelial cells via the phosphatidylinositol 3-kinase/Akt signaling pathway. HGF gene therapy can attenuate acute colitis via epithelial cell proliferation through the PI3K/Akt pathway. These data suggested that HGF gene therapy by EP may be effective for the regeneration and repair of injured epithelial cells in inflammatory bowel disease.     

Genetic networks responsive to sodium butyrate in colonic epithelial cells

We performed microarray and computational gene network analyses to identify the detailed mechanisms by which sodium butyrate (SB) induces cell growth arrest and the differentiation of mouse colonic epithelial MCE301 cells. Two thousand six hundred four differentially expressed probe sets were identified in the cells treated with 2mM SB and were classified into four groups. Of these, the gradually increased group and the gradually and remarkably decreased group contained the genetic networks for cellular development and cell cycles or canonical pathways for fatty acid biosynthesis and pyrimidine metabolism, respectively. The present results provide a basis for understanding the detailed molecular mechanisms of action of SB in colonic epithelial cells.     

Establishment and functional characterization of a tracheal epithelial cell line RTEC11 from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen

A tracheal epithelial cell line RTEC11 was established from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen. The cells grew continuously at a permissive temperature of 33 degrees C but not at a non-permissive temperature of 39 degrees C. Morphological and functional investigations demonstrated that the cells were polarized epithelial cells maintaining a regulated permeability barrier function. Interestingly, the expression levels of Muc1 (mucin 1) and Scgb1a1 (uteroglobin), non-ciliated secretory cell markers, and Tubb4 (tubulin beta 4), a ciliated cell marker, were significantly increased under the cell growth-restricted condition. Global gene expression and computational network analyses demonstrated a significant genetic network associated with cellular development and differentiation in cells cultured at the non-permissive temperature. The tracheal epithelial cell line RTEC11 with unique characteristics should be useful as an in vitro model for studies of the physiological functions and gene expression of tracheal epithelial cells.     

Identification of genetic networks involved in the cell growth arrest and differentiation of a rat astrocyte cell line RCG-12

The purpose of the present study is to establish and characterize a conditionally immortalized astrocyte cell line and to clarify the genetic networks responsible for the cell growth arrest and differentiation. A conditionally immortalized astrocyte cell line, RCG-12, was established by infecting primary cultured rat cortical glia cells with a temperature-sensitive simian virus 40 large T-antigen. At a permissive temperature of 33 degrees C, the large T-antigen was expressed and cells grew continuously. On the other hand, the down-regulation of T-antigen at a non-permissive temperature of 39 degrees C led to growth arrest and differentiation. The cells expressed astrocyte-expressed genes such as glial fibrillary acidic protein. Interestingly, the differentiated condition induced by the non-permissive temperature significantly elevated the expression levels of several astrocyte-expressed genes. To identify the detailed mechanisms by which non-permissive temperature-induced cell growth arrest and differentiation, we performed high-density oligonucleotide microarray analysis and found that 556 out of 15,923 probe sets were differentially expressed 2.0-fold. A computational gene network analysis revealed that a genetic network containing up-regulated genes such as RB, NOTCH1, and CDKN1A was associated with the cellular growth and proliferation, and that a genetic network containing down-regulated genes such as MYC, CCNB1, and IGF1 was associated with the cell cycle. The established cell line RCG-12 retains some characteristics of astrocytes and should provide an excellent model for studies of astrocyte biology. The present results will also provide a basis for understanding the detailed molecular mechanisms of the growth arrest and differentiation of astrocytes.     

Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas.

Colon carcinomas appear to arise from the cumulative effect of mutations to several genes (APC, DCC, p53, ras, hMLH1, and hMSH2). By using novel colonic epithelial cell lines derived from the Immorto mouse, named the YAMC (young adult mouse colon) cell line, and an Immorto-Min mouse hybrid, named the IMCE (Immorto-Min colonic epithelial) cell line, carrying the Apc min mutation, we investigated the effect of an activated v-Ha-ras gene on tumor progression. The YAMC and IMCE cell lines are normal colonic epithelial cell lines which are conditionally immortalized by virtue of expression of a temperature-sensitive simian virus 40 (SV40) large T antigen. Under conditions which permit expression of a functional SV40 large T antigen (33 degrees C plus gamma interferon), neither the YAMC nor the IMCE cell line grows in soft agar or is tumorigenic in nude mice. In vitro, when the SV40 large T antigen is inactivated (39 degrees C without gamma interferon), the cells stop proliferating and die. By infecting the YAMC and IMCE cell lines with a replication-defective psi2-v-Ha-ras virus, we derived cell lines which overexpress the v-Ha-ras gene (YAMC-Ras and IMCE-Ras). In contrast to the parental cell lines, under conditions in which the SV40 large T antigen is inactive, both the YAMC-Ras and IMCE-Ras cell lines continue to proliferate. Initally YAMC-Ras cells do not form tumors; however, tumors are visible after 90 days of incubation. IMCE-Ras cells form colonies in soft agar under both permissive and nonpermissive culture conditions. Furthermore, IMCE-Ras cells form tumors in nude mice within 3 weeks. The phenotype of the IMCE-Ras cell line thus clearly demonstrates that a defective Apc allele and an activated ras gene are sufficient to transform normal colonic epithelial cells and render them tumorigenic.     

A Lactobacillus rhamnosus GG-derived Soluble Protein,p40, Stimulates Ligand Release from Intestinal Epithelial Cells to Transactivate Epidermal Growth Factor Receptor

p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis, and preserves barrier function by transactivation of the EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study is to determine the mechanisms by which p40 transactivates the EGFR in intestinal epithelial cells. Here we show that p40-conditioned medium activates EGFR in young adult mouse colon epithelial cells and human colonic epithelial cell line, T84 cells. p40 up-regulates a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) catalytic activity, and broad spectrum metalloproteinase inhibitors block EGFR transactivation by p40 in these two cell lines. In ADAM17-deficient mouse colonic epithelial (ADAM17^−/− MCE) cells, p40 transactivation of EGFR is blocked, but can be rescued by re-expression with WT ADAM17. Furthermore, p40 stimulates release of heparin binding (HB)-EGF, but not transforming growth factor (TGF)α or amphiregulin, in young adult mouse colon cells and ADAM17^−/− MCE cells overexpressing WT ADAM17. Knockdown of HB-EGF expression by siRNA suppresses p40 effects on transactivating EGFR and Akt, preventing apoptosis, and preserving tight junction function. The effects of p40 on HB-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGFR activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion. Thus, these data define a mechanism of a probiotic-derived soluble protein in modulating intestinal epithelial cell homeostasis through ADAM17-mediated HB-EGF release, leading to transactivation of EGFR.     

HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line

The regulation of intercellular adhesion by hepatocyte growth factor (HGF) was examined on a novel nontumorigenic gastric epithelial cell line (IMGE-5) derived from H-2Kb-tsA58 transgenic mice. IMGE-5 cells constitutively expressed cytokeratin 18 and HGF receptors. Under permissive conditions (33 degrees C + interferon-gamma), IMGE-5 cells proliferated rapidly but did not display membrane expression of adherens and tight junction proteins. Under nonpermissive conditions, their proliferation was decreased and they displayed a strong, localized membrane expression of E-cadherin/beta-catenin and occludin/ZO-1. HGF treatment largely prevented the targeting of ZO-1 to the tight junction and induced a significant decrease of the transepithelial resistance measured across a confluent IMGE-5 cell monolayer. HGF rapidly increased the tyrosine phosphorylation of ZO-1 and decreased its association with occludin in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. PI 3-kinase was also involved in HGF-induced migration of IMGE-5 cells. Our results demonstrate that 1) HGF prevents the appearance of ZO-1 in the membrane during epithelial cell differentiation; 2) HGF causes partial relocalization of ZO-1 to the cytoplasm and nucleus and concomitantly stimulates cell dissociation and migration; and 3) IMGE-5 cells offer a useful model for the study of gastric epithelial cell differentiation.     

Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells

Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.     

Comparison of the function of the tight junctions of endothelial cells and epithelial cells in regulating the movement of electrolytes and macromolecules across the cell monolayer

In cell culture, both endothelial and epithelial cell monolayers have been found to generate structurally similar tight junctional complexes, as assessed by thin complexes of the two cell types are, at least in part, responsible for the very different permeability characteristics of native endothelial and epithelial cell monolayers. The purpose of this work was to compare cultured endothelial and epithelial cells with respect to the function of their tight junctional complexes in regulating the movement of macromolecules and ions across the cell monolayers, and define functional parameters to characterize the tight junctional complexes. Bovine aorta endothelial cells and T84 colonic carcinoma epithelial cells were cultured on a microporous membrane support. The permeability coefficients of inulin, albumin, and insulin were determined with the cell monolayers and compared with the permeability coefficients obtained with 3T3-C2 fibroblasts, a cell line that does not generate tight junctions. Electrical resistance measurements across the monolayer-filter systems were also compared. The permeability coefficient of albumin across the endothelial cell monolayer compared favorably with other reported values. Likewise, the electrical resistance across the T84 cell monolayer was in good agreement with published values. Utilizing permeability coefficients for macromolecules as an index of tight junction function, we found that a distinction between a lack of tight junctions (fibroblasts), the presence of endothelial tight junctions, and the presence of epithelial tight junctions was readily made. However, when utilizing electrical resistance as an index of tight junction function, identical measurements were obtained with fibroblasts and endothelial cells. This indicates that more than one index of tight junction function is necessary to characterize the junctional complexes. Although structurally similar, epithelial cell and endothelial cell tight junctions perform very different functions, and, from our data, we conclude that the demonstration of tight junctional structures by electron microscopy is not relevant to the functional nature of the junction: structure does not imply function. A minimal assessment of tight junction function should rely on both the determination of the electrical resistance across the cell monolayer, and the determination of the permeability coefficients of selected macromolecules.     

The response of adult rat sertoli cells,immortalized by a temperature-sensitive mutant of SV40, to 1,2-dinitrobenzene, 1,3-dinitrobenzene, 2,4-dinitrotoluene, 3,4-dinitrotoluene,and cadmium

In this study we test the hypothesis that immortalized adult rat Sertoli cells respond to known testicular toxins in a similar manner to Sertoli cells tested in vivo and in primary culture. This cell line was developed by immortalizing adult rat Sertoli cells with the temperature-sensitive mutant of SV40, ts255, such that the cells proliferate at the permissive temperature of 33 degrees C but express differentiated characteristics at the nonpermissive temperature of 40 degrees C. Confluent monolayers, grown at 33 degrees C or 40 degrees C, were exposed to a range of concentrations of dinitrobenzene (DNB) or dinitrotoluene (DNT) isomers or to cadmium chloride. Cellular response was assessed by neutral-red cell viability assay and ultrastructural changes. Cells grown at 40 degrees C were sensitive to lower concentrations of each toxicant than were cells grown at 33 degrees C. 1,2-DNB was more toxic than 1,3-DNB, and 3,4-DNT was more toxic than 2,4-DNT, as judged by the neutral-red cell viability assay. Ultrastructurally, cells treated with 1,2-DNB or 2,4-DNT showed increased numbers of autophagic vesicles compared to controls. Intercellular penetration of ruthenium red demonstrated breached tight junctions in 1,2-DNB and cadmium-treated cells. From these observations, we conclude that this cell line can serve as a model for studying toxic mechanisms in adult Sertoli cells.     

Establishment and characterization of conditionally immortalized organ of corti cell lines

A culture of cells was isolated from the organ of Corti of 2-week-old H-2Kb-tsA58 (Immortomouse) transgenic mice. All cells of these mice harbor a mutant of the simian virus 40 A-gene, encoding a thermolabile large T-antigen (Tag) protein. At 33 degrees C the Tag protein is functional and induces cell proliferation, but at 39 degrees C it is rapidly denatured and inactivated. Isolated organ of Corti cells growing at 33 degrees C were predominantly small, rounded or fusiform and proliferated rapidly. When moved to 39 degrees C, the cells reduced their rate of proliferation and differentiated into specific morphological phenotypes. Four cell lines were cloned by limiting dilution and characterized by immunofluorescence microscopy and Western blot. The cell lines, named OC-k1, OC-k2, OC-k3 and OC-k4, have been passaged at least 50 times with retention of a stable phenotype. These cell lines were all positive for the neuroepithelial precursor cell marker nestin and for the inner ear cell marker OCP2. In addition, the cells showed reactivity to epithelial and neuronal cell markers, but with a pattern of protein expression different for each clone and different between cells of the same clone growing at 33 degrees C or 39 degrees C. Some of the clones exhibited asymmetric cell division which is a characteristic commonly ascribed to stem cells. These cell lines can be used advantageously to study mechanisms and signals involved in the control of cell differentiation and morphogenesis of the mammalian inner ear and to isolate inner ear specific proteins.     

Conditionally immortalized adrenocortical cell lines at undifferentiated states exhibit inducible expression of glucocorticoid-synthesizing genes.

To facilitate studies on differentiation of adrenocortical cells and regulation of steroidogenic genes, we established cell lines from adrenals of adult transgenic mice harboring a temperature-sensitive large T-antigen gene of simian virus 40. Adrenal glands of the mice exhibited normal cortical zonation including a functionally undifferentiated cell-layer between the aldosterone-synthesizing zona glomerulosa cells and the corticosterone-synthesizing zona fasciculata cells. At a permissive temperature (33 degrees C), established cell lines AcA201, AcE60 and AcA101 expressed steroidogenic genes encoding steroidogenic factor-1, cholesterol side-chain cleavage P450scc, and steroidogenic acute regulatory protein, which are expressed throughout adrenal cortices and gonads. Genes encoding 3 beta-hydroxysteroid dehydrogenase and steroid 21-hydroxylase P450c21, which catalyze the intermediate steps for syntheses of both aldosterone and corticosterone, were inducible in the three cell lines in temperature- and/or dibutyryl cAMP-dependent manners. Notably, these cell lines displayed distinct expression patterns of the steroid 11 beta-hydroxylase P45011 beta gene responsible for the zone-specific synthesis of corticosterone. AcA201 cells expressed the P45011 beta gene at 33 degrees C, showing the property of the zona fasciculata cells, while AcE60 cells expressed it upon a shift to a nonpermissive temperature (39 degrees C). On the other hand, AcA101 expressed the P45011 beta gene at 39 degrees C synergistically with exposure to dibutyryl cAMP. None of these clones express the zona glomerulosa-specific aldosterone synthase P450aldo gene under the conditions we tested. These results show that AcE60 and AcA101 cells display a pattern of the steroidogenic gene expression similar to that of the undifferentiated cell-layer and are capable of differentiating into the zona fasciculata-like cells in vitro.     

Genes involved in nonpermissive temperature-induced cell differentiation in Sertoli TTE3 cells bearing temperature-sensitive simian virus 40 large T-antigen

Sertoli TTE3 cells, derived from transgenic mice bearing temperature-sensitive simian virus 40 large T (tsSV40LT)-antigen, proliferated continuously at a permissive temperature (33 degrees C) whereas inactivation of the large T-antigen by a nonpermissive temperature (39 degrees C) led to differentiation as judged by elevation of transferrin. To clarify the detailed mechanisms of differentiation, we investigated the time course of changes in gene expression using cDNA microarrays. Of the 865 genes analyzed, 14 genes showed increased levels of expression. Real-time quantitative PCR revealed that the mRNA levels of p21(waf1), milk fat globule membrane protein E8, heat-responsive protein 12, and selenoprotein P were markedly elevated. Moreover, the differentiated condition induced by the nonpermissive temperature significantly increased mRNA levels of these four genes in several cell lines from the transgenic mice bearing the oncogene. The present results regarding changes in gene expression will provide a basis for a further understanding of molecular mechanisms of differentiation in both Sertoli cells and cell lines transformed by tsSV40LT-antigen.     

Cell-to-cell communication competence in simian virus 40-transfected rat ovarian cells is reduced following tumor selection

Summary A pSV3neo-transfected rat ovarian cell line (SV-GC) was developed from a primary granulosa culture (GC) to study gap junctional intercellular communication (GJIC) during Simian virus 40 (SV40) transformation. SV-GC expressed SV40 large T-antigen (T-ag), grew indefinitely in culture without luteinization, was anchorage independent, and formed tumors in nude mice. Ultrastructural analysis identified abundant gap junctional membrane and suggested that SV-GC was arrested at an early stage of differentiation. Functional GJIC, measured by a dye transfer technique (gap FRAP), was comparable to that observed in normal granulosa cells, suggesting that the expression of T-ag alone was insufficient to reduce GJIC. However, there was approximately a 50% loss in the rate of GJIC in the nude mouse SV-GC-tumor derived and G418 selected cell line (T-SV-GC). SV-GC→T-SV-GC also resulted in a transition from migration of cells as an epithelial sheet to the dissociation of individual fibroblastoid cells. Tumor cell detachment was also seen in migrating malignant human (A2780 and 547) and rat (DC3) ovarian cell lines. Co-culture combinations of normal (GC)→transformed (SV-GC) → tumor-derived (T-SV-GC) cells indicated that the rate of heterologous GJIC was characteristic of the least communicating partner. Taken together, these data suggested that SV-GC → T-SV-GC represented progression toward metastasis with concomitant reduction of GJIC and adhesiveness. These sequentially derived cell lines may be a useful in vitro model system for studies focusing on the mechanisms involved in the detachment of cells during the progression of ovarian cancer.     

Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression.

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat beta-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.     

Hepatocyte cell lines established from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

To establish cell lines exhibiting differentiation phenotypes, the immortalized cell lines were rapidly established from the primary culture of different tissues of transgenic mice harboring SV40 temperature-sensitive large T-antigen gene. The established cell lines grew at permissive temperature (33 degrees C), but not at nonpermissive temperature (39 degrees C). Several different cell types could be rapidly immortalized and cloned from the adult transgenic mice tissues. Among those cell lines, the established hepatocyte cell lines (TLR cell lines) exhibited liver-specific morphological and biochemical properties, but their properties were not coupled with the growth condition modified by temperature. The hepatocyte cell lines showed an inducibility of P450IA1 by 3-methylcholanthrene as observed in rat livers and this liver-specific function was stable even after 6 months of culture by continuous passages.