Gene expression in clonally derived cell lines produced by in vitro transformation of rat fetal hepatocytes: Isolation of cell lines which retain liver-specific markers (original) (raw)
Related papers
Phenotypic Modulation during Tumorigenesis by Clones of Transformed Rat Liver Epithelial Cells
Cancer Research, 1987
From nine clonal subpopulations (strains) of chemically transformed cultured rat hepatic epithelial cells which were tumorigenic when im planted into 1-day-old isogeneic rats, a cell line was reestablished from each tumor and the cellular properties of the tumor-derived cell lines were compared to those of the corresponding progenitor cells that were implanted to produce the tumors. In seven of eight instances, the cellular DNA content of the tumor-derived cells was virtually identical to the DNA content of the respective progenitor cells, but in one case the tumor cells had twice as much DNA as did their progenitor cells. During the development of tumors in viva, other cellular phenotypic properties often underwent considerable, but variable changes. These changes included the activity of •v-glutamyl transpeptidase, the growth properties on plastic surfaces, and the expression of LDH isozymes. Although there was a relative enhancement in the ability of most of the tumor-derived cells to proliferate or to form colonies in calcium-poor medium, several tumorderived cell lines had very low colony-forming efficiencies in media containing either normal or low levels of calcium. The most consistent association between phenotypes expressed in vitro and tumorigenicity was the ability of cells to form colonies in soft agar; all tumor-derived lines expressed this phenotype, and with some of them this phenotype was acquired only during the process of tumor formation in vivo. These results demonstrate that further phenotypic and genotypic alterations may occur in vivo during tumor formation by chemically transformed cultured cells following their implantation into isogeneic animals; and some of the alterations that occur in vivo may be necessary for the complete expression of tumorigenicity. Although anchorage-independent growth capacity cannot be used to predict the tumorigenicity of clones of rat liver epithelial cells chemically transformed in vitro, this growth property appears to be invariably induced prior to or during the formation of tumors in vivo by these cells.
Cancer Research
Clonal subpopulations of a chemically induced tumorigenic rat liver epithelial cell line were analyzed for their cellular, biochemi cal, and in vitro growth properties and their tumorigenicity after injection into day-old newborn isogeneic rats. The phenotypic properties studied included DMA content; growth rate in culture; activities of 7-glutamyl transpeptidase, NADH diaphorase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase; ability to grow in calcium-poor medium; and ability to form colonies in soft agar. The results show that none of these phenotypes cosegregates with tumorigenicity and there fore is not reliable as a "marker" phenotype for neoplastic trans formation in cultured rat liver epithelial cells. The poor correla tions, either qualitatively or quantitatively, between paratumorigenic phenotypes and tumorigenicity suggest that neoplastic transformation in these cells involves a specific transforming gene locus or loci and that in vitro paratumorigenic phenotypes are merely epiphenomena of neoplastic transformation and pro gression. This study further reveals that the efficiency of the tumorigenicity assay of cultured rat liver epithelial cells in isoge neic newborn rats can be considerably improved by incubating the cells in medium containing only trace amounts of serum prior to transplantation into the host animals.
Cancer Research, 1987
Two epithelial cell lines designated LE/2 and LE/6 were established from cells isolated by centrifugal elutriation from the livers of carcinogentreated rats. Both cell lines exhibit some characteristics of fetal liver cells, such as the expression of the 2.3-kilobase a-fetoprotein mRNA, aldolase A, and láclate dehydrogenases 4 and 5. Primary cultures contain 7-glutamyl Iransferase-positive cells which do not proliferate in vitro. After the first passage, the LE/2 and LE/6 cell lines are uniformly 7glutamyl transferase negative. Neither cell line is transformed as assayed by morphology, anchorage-independent growth, or tumor formation in nude mice. By the 50th passage, LE/6 cells form numerous colonies in soft agar in the presence of epidermal growth factor, while no colonies grow in medium lacking this growth factor. Clonal cell populations derived from five epidermal growth factor-induced soft agar colonies were not tumorigenic in nude mice. This indicates that, although epidermal growth factor-responsive late passage cells had acquired some of the phenotypic properties commonly associated with tumor cells, these cells were not fully transformed. Transformation of LE/6 cells was accom plished by transfection of the ras" oncogene (EJ). Subcutaneous inocu lation of ras" (EJMransfected LE/6 cells produced tumors at the site of injection with histológica! features of moderate to well-differentiated trabecular hepatocellular carcinomas. Tumor cell lines derived from the nude mouse tumors are 7-glutamyl transferase positive and express afetoprotein mRNA. One clonal cell line expresses both a-fetoprotein and albumin mRNA. These results show that nonparenchymal liver epithelial cells transfected with an activated oncogene can give rise to differentiated hepatocellular tumors similar to those induced in livers of rats fed a carcinogenic diet.
Maturation-dependent gene expression in a conditionally transformed liver progenitor cell line
in Vitro Cellular & Developmental Biology-animal, 1998
We have isolated a conditionally transformed liver progenitor cell line with phenotypic similarities to both hepatoblasts (bipotent embryonic liver cells that give rise to hepatocytes and intrahepatic biliary epithelial cells) and liver epithelial cells (primitive hepatic cells isolated from adult livers capable of generating both hepatocytic and biliary lineages). Cell line L2039 was derived from E14 fetal mouse liver after transformation with temperature-sensitive SV-40 large T antigen. At 33° C, these cells have an epithelial morphology with a high nucleocytoplasmic ratio and express both hepatocytic and biliary genes, including albumin, α-fetoprotein, glutamine synthetase, insulinlike growth factor II receptor, fibronectin and laminin, and cytokeratins 8 and 19, a set of markers characteristic for hepatoblasts. The presence of cytokeratin 14, vimentin, and several oval-cell antigens link cell line L2039 to nonparenchymal liver epithelial cell populations though to contain progenitor cells. Serum-free, hormonally defined media conditions and extracellular matrix requirements were determined for growth and differentiation of this cell line. During culture on type IV collagen at 39° C, L2039 cells cease dividing and demonstrate hepatocytic differentiation with the assumption of a hepatocytelike morphology and glucocorticoid-dependent regulation of liver-specific genes, including albumin, α-fetoprotein, phosphoenolpyruvate carboxykinase, and liver-enriched transcription factors. The number of albumin-positive cells increases during culture at 39° C, indicating that L2039 cells convert from a prehepatocytic to a hepatocytic phenotype. Under conditions specific for hepatocytic differentiation, C/EBPs were expressed and differentially regulated, with C/EBPβ and C/EBPδ upregulated early and C/EBPα only slightly expressed after 7 d, indicating that C/EBPα may not be a crucial factor in commitment to the hepatocytic phenotype.
Cell transformation: the role of oncogenes and factors
Mutagenesis, 1986
An attempt is made to draw together diverse areas of biological research which have recently converged and opened up new experimental approaches to understanding the nature of cancer. In particular, the powerful techniques of molecular biology have been brought to bear on tissue culture systems. The case is made for the continued use of cell transformation in vitro as a real and useful model for cancer development. The hallmark of all cancer cells is loss of control over the cell cycle and the cellular elements involved, growth factors, growth factor receptors and signal transducers have been identified and in some instances shown to be encoded in cellular oncogenes. Moreover, as the molecular mechanisms underlying cell growth control are unravelled, those aspects involved in neoplastic change will be identified and this will lead to the development of definitive short-term tests for the detection of chemical carcinogens.
Toxicologic Pathology, 1986
A single exposure to a low concentration (lO-'O moYL) of several tumor promoters, namely 12-0-tetradecanoylphorbol-13-acetate (TPA), phenobarbital (PB), nafenopin, saccharin, teleocidin, benzoyl peroxide, butylated hydroxytoluene (BHT), dichlorodiphenyltrichloroethane (DDT), lindane, clofibrate, and melittin significantly stimulated DNA synthesis of neonatal rat hepatocytes in 4-day-old primary cultures. Thcse cultures wcre kept in low-calcium (0.01 mmoVL) HiWoBa2000 synthetic medium, thereby evoking a neoplastic phenotype in othenvise normal (i.e., non-initiated) cells. The simultaneous addition of a single dose ofalpha-tocopherol (lO-'moVL) or selenous acid (lO-JmoVL), just as that ofexogenous supcroxide dismutasc (SOD) (4), together with each of the above agents fully suppressed the stimulation of hepatocytic DNA synthesis by the xenobiotics. Hence, these findings strengthen the view that superoxide anions (or some other oxidizing compounds) act as the common mediators of the mitogenic effects of various tumor promoters in hepatocytes. Inhibition kinetics studies, in which TPA in a single dose (lO-'OmoVL) was used as the paradigmatic compound together with several kinds of inhibitors of its activity showed that the early mitogenic erects of TPA, i.c., the commitment of quiescent (GO) hepatocytes and the reentry into active cycling of hepatocytes spontaneously poised at the G 1/S boundary, required oxidizing compounds, arachidonate metabolism derivatives, and plasmalemmal calcium-binding sites and transmembrane calcium fluxes. Instead, a later TPAs effect, the flow into DNA synthesis of hepatocytes previously committed to cycle, was shown to be controlled by retinoid-modulable activities, by some product(s) of the lipoxygenase pathway, and again by plasmalemmal calcium-binding sites and transmembrane calcium fluxes. Such results reveal that in the neonatal rat hepatocyte the ability to answer to a single mitogenic stimulus and the metabolic pathways by which this answer is enacted depend upon the mitotic cycle setting of the hepatocytes at the moment of the experimental treatment.
Cancer Research, 1988
The secretion of transforming growth factors (TGFs) a and ßby normal, chemically transformed, and malignant rat liver epithelial cell lines was investigated. The WB-F344 normal cultured rat liver epithelial cell line does not secrete an epidermal growth factor-like (putative!) TGF-a) activity, but several clonal cell strains derived from WB-F344 cells which had been treated with JV-methyl-/V'-nitro-AT-nitrosoguanidine, especially those that expressed high levels of •y-glutamyl transpeptidase, secreted TGF-a-like activity into their conditioned media. Cell lines obtained from tumors which were produced by these cell strains varied in their abilities to secrete TGF-a, even though they all expressed high levels of •Y-glutamyl transpeptidase activity. When two of the non-TGFa-secreting tumor cell lines were transplanted into isogeneic rats, the tumors that formed contained high levels of TGF-a-like activity. Al though epidermal growth-factor (hence, TGF-a also) inhibited the pro liferation of several of these tumor cell lines in monolayer cultures, this growth factor often paradoxically stimulated the anchorage-independent growth of the same cell lines. In contrast to TGF-a-like activity, all cell lines/strains released TGF-/9 activity into their conditioned media. How ever, while both normal or chemically transformed cell strains typically produced the inactive form of TGF-/9, the tumor cell lines tended to produce activated TGF-0 de novo. Anchorage-independent growth of cell lines that produced active TGF-0 was either stimulated, inhibited, or unaffected by TGF-/3. Cell lines that were inhibited by TGF-/3 concur rently produced TGF-a which was usually able to overcome the negative "automne" effect of TGF-/3. We conclude that both TGF-a and TGF-0, singly or in combination, are variously involved in the growth of trans formed rat liver epithelial cells. TGF-a has a predominantly positive autocrine action on the growth of rat liver epithelial tumor cell lines. The "paracrine" effect of TGF-/2 may be at least as important as its autocrine effect in the growth of these transformed epithelial cell lines.