Gradation of carcinogen-induced capacity for anchorage-independent growth in cultured rat liver epithelial cells (original) (raw)
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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.
Cancer Research
The ability of eukaryotic cells in culture to proliferate in calciumpoor medium has been found to characterize populations of transformed cells, but the relationship between this phenotypic property and tumorigenicity at the cellular level is unclear. Thus, we have isolated 14 clonal subpopulations, based on their ability to colonize in calcium-poor medium, from a parental tumorigenic rat hepatic epithelial cell line which was transformed by multiple exposures to A/-methyl-A/'-nitro-A/-nitrosoguanidine. These clonal subpopulations of cells were tested for their ability to grow in soft agar, to express -y-glutamyl transpeptidase activity, and to form tumors upon back-transplantation into isogeneic newborn rats. The results indicated that clonal subpopulations of cells selected by their ability to grow in calcium-poor medium were phenotypically heterogeneous for 7-glutamyl transpeptidase ac tivity and anchorage-independent growth, and, more importantly, they were not more tumorigenic than the phenotypically hetero geneous parental cell line. This observation suggests that the capability of cultured hepatic epithelial cells to grow in calciumpoor medium is not tightly coupled to the tumorigenic phenotype.
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.
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1995
The tumorigenic phenotype in rat liver epithelial cells overexpressing c-myc may depend on a transforming growth factor (TGF)-alpha/epidermal growth factor receptor autocrine loop (L. W. Lee et al., Cancer Res., 51: 5238-5244, 1991). In the present study, we have used constitutive sense and antisense TGF-alpha expression vectors to modify TGF-alpha production in carcinogen-transformed clonal derivatives of a rat liver epithelial cell line, WB-F344, that variably express c-myc, endogenous TGF-alpha, and tumorigenicity. Transgene-mediated TGF-alpha protein production was elevated 2- to 9-fold in derivatives of a low c-myc-expressing transformed cell line, GN4, and 35-fold in a derivative of a high c-myc-expressing cell line, GN6. Although the GN4- and GN6-derived cell lines expressed functional EGF receptor and steady-state c-myc mRNA levels that were comparable to their respective parental cell lines, increased TGF-alpha expression did not increase the tumorigenicity of the derivativ...
Cancer research, 1991
Tumorigenicity was correlated with levels of expression of the genes for transforming growth factor a (TGF-a), epidermal growth factor receptor, c-myc, c-H-ras,, and c-K-ras in a series of 16 clonally derived transformed liver epithelial cell lines. The clonal lines, which varied in tumorigenicity from 0 to 97%, were established from a phenotypically heterogeneous population produced by repeated exposure of diploid WB-F344 (WB) cells to A'-methyl-AT-nitro-A'-nitrosoguanidine. Segregation of gene expression with tumorigenicity among clonal lines was determined by correlating rank orders of gene expression by clones relative to expression by wild-type \VB cells. Only the expression of the c-myc gene correlated with tumorigenicity among all transformed clones. TGF-a gene expression was not correlated with tumorigenicity among all clones, but it was highly correlated with tumorigenicity among clones that expressed the c-myc gene above the median level for all clones (>5-fold the level of expression by WB cells). Even high levels of expression of the TGF-a gene (up to 60-fold the level of expression by WB cells) were not correlated with tumorigenicity among the clones expressing the c-myc gene at levels <5-fold the level of expression by WB cells. Clones which simultaneously overexpressed both c-myc and TGF-a genes at levels above the median levels for all clones were significantly more tumorigenic than were clones which expressed either or both genes at lower than median levels. These results suggest that overexpressed c-myc and TGFii genes cooperate in their association with tumorigenicity. Most of the highly tumorigenic clones that overexpressed c-myc and TGF-a also overexpressed the r-I l-rav and/or the c-K-ras genes; clones that overex pressed neither of the c-ras genes nor the genes for c-myc and TGF-a were not very tumorigenic, while clones that expressed one or both c-ras genes (but not both r-niyr and TGF-a) were variably tumorigenic over an intermediate range.
The Journal of Cell Biology, 1996
Mature adult parenchymal hepatocytes, typically of restricted capacity to proliferate in culture, can now enter into clonal growth under the influence of hepatocyte growth factor (scatter factor) (HGF/SF), epidermal growth factor (EGF), and transforming growth factor et (TGFo 0 in the presence of a new chemically defined medium (HGM). The expanding populations of hepatocytes lose expression of hepatocyte specific genes (albumin, cytochrome P450 IIB1), acquire expression of markers expressed by bile duct epithelium (cytokeratin 19), produce TGFot and acidic FGF and assume a very simplified morphologic phenotype by electron microscopy. A major change associated with this transition is the decrease in ratio between transcription factors C/EBPo~ and C/EBP[3, as well as the emergence in the proliferating hepatocytes of transcription factors AP1, NFKB. The liver associated transcription factors HNF1, HNF3, and HNF4 are preserved throughout this process.