Expression of proto-oncogenes in normal and papovavirus-transformed or -infected rat fibroblasts

Winberry, L; Priehs, Claudette; Friderici, Karen; Thompson, Michael D.; Fluck, Michele M.

doi:10.1016/0042-6822(85)90235-1

Cited by 10 publications

(7 citation statements)

References 54 publications

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“…This finding contrasts with a published report showing that c-Ha-ras RNA is expressed equally in normal and SV40-transformed cells (55). It is of interest that we detected two c-Ha-ras RNA species in the SV40-hepatocyte cell lines.…”

Section: Discussioncontrasting

confidence: 56%

Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.

Woodworth¹,

Kreider²,

Mengel³

et al. 1988

Mol. Cell. Biol.

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Five simian virus 40 (SV40)-hepatocyte cell lines were examined for tumorigenicity and the effect of in vitro passage on the expression of four liver-specific genes (albumin, transferrin, al-antitrypsin, and phosphoenolpyruvate carboxykinase), two oncogenes (c-Ha-ras and c-raJ), and two genes associated with hepatocarcinogenesis (a-fetoprotein and placental-type glutathione-S-transferase). At immortalization and induced expression of c-raf and glutathione-S-transferase-P were observed. Induction of a-fetoprotein was detected with in vitro and in vivo passage only in the CWSV14 cell line and was paralleled by diminished albumin expression. In conclusion, we developed a model system with five SV40-hepatocyte cell lines, tumors induced by them, and tumor cell lines to examine changes in gene expression that accompany the progression from a normal cell to a hepatocellular carcinoma. Because the SV40-hepatocyte cell lines and tumor cell lines remain highly differentiated and vary in the magnitude of expression of specific genes, they can be used to study the molecular mechanisms regulating gene expression, in particular those regulating specific genes associated with differentiation.Because most human neoplasms are carcinomas that develop from differentiated adult epithelium, in vitro model systems for studying transformation of differentiated epithelial cells are particularly important. In many in vitro transformation studies, a primary cell type is used that has the ability to replicate for a limited number of passages in culture, is of fibroblastic origin, and produces an undifferentiated sarcoma in a test animal. Although studies to examine in vitro transformation of primary epithelial cells with defined genetic information have been performed (8-10, 32, 34, 37, 58, 61), the emphasis has been on determining what genes are required for transformation. Our interest was to determine (i) whether transformation of a nonreplicating differentiated epithelial cell could be accomplished, (ii) whether the transformed cells would produce a well-differentiated carcinoma, and (iii) how transformation altered expression of differentiated functions by the epithelial cells in culture and in the tumor.Primary rat hepatocytes are particularly appropriate epithelial cells for studying transformation of differentiated * Corresponding author. cells because they have a limited capacity to replicate and express a variety of well-characterized differentiated functions. We have demonstrated previously that primary hepatocytes can be immortalized by simian virus 40 (SV40) genetic information to yield colonies of replicating epithelial cells that retain the capacity to produce high levels of albumin (24,59). Eleven albumin-producing hepatocyte cell lines have been derived from these albumin-producing colonies (57). Characterization of these cell lines has shown that they express at least some functions associated with differentiation in hepatocytes in normal adult liver. Several of the cell lines produce levels of albumin RNA similar to...

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Section: Discussioncontrasting

confidence: 56%

Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.

Woodworth¹,

Kreider²,

Mengel³

et al. 1988

Mol. Cell. Biol.

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“…Recently, Winberry et al reported that the mRNAs of 10 proto-oncogenes but not of the mos gene were equally expressed in SV40-transformed and untransformed rat F-111 cells (21). Several points can be made about this discrepancy.…”

mentioning

confidence: 93%

“…Third, the original level of c-Ha-ras mRNA in their F-111 cells might have been higher than that in our 3Y1 cells. Actually, we could detect only a trace amount of sis and erbB mRNAs in 3Y1 cells, in contrast to their F-111 cells (21).…”

mentioning

confidence: 94%

Induction of c-Ha-ras transcription in rat cells by simian virus 40 large T antigen.

Segawa¹,

Yamaguchi²

1987

Mol. Cell. Biol.

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Rat 3Y1 cells expressing simian virus 40 large T antigen under the control of the mouse mammary tumor virus long terminal repeat were established. The amount of c-Ha-ras mRNA in those cells was elevated by about 20 times in parallel with large T antigen after exposure to dexamethasone for 48 h. In chloramphenicol acetyltransferase assays with a plasmid containing the c-Ha-ras-1 promoter the increase in c-Ha-ras mRNA was shown to occur at the transcriptional level.Transformation of cells by simian virus 40 (SV40) involves many complex alterations in biological and biochemical properties (19). These changes in cellular growth control and behavior are presumably mediated by perturbations in normal patterns of metabolism and gene expression. A number of individuals have analyzed the changes between transformed and untransformed parental cells (reviewed in reference 19). However, it was not clear whether these changes were the main cause of transformation by SV40 or secondary events induced after the transformation. If one could regulate the amount of large tumor (T) antigen (Ag) in cell clones and analyze the changes induced in parallel with the amounts of large T Ag, these cell clones could be a good tool for examining the phenomena closely related to the transforming function of SV40 large T Ag. For this purpose, we constructed a plasmid (pMMWT-LN) which can express large T Ag in a hormone-dependent manner (Fig. 1). By using rat cells carrying plasmid pMMWT-LN, we examined cellular oncogene expression as a first step in analyzing the changes induced by SV40 large T Ag.Rat 3Y1 cells (8) were transfected with the constructed plasmid pMMWT-LN, and G418-resistant colonies were picked 14 days after transfection. For isolation of cell lines expressing large T Ag in response to dexamethasone (Dx), dot blot analysis of RNA from the above-mentioned colonies was carried out as described previously (10). Some colonies expressed the SV40 large-T-Ag gene irrespective of the presence of Dx (data not shown). Clones which could express the large-T-Ag gene in a Dx-dependent manner were named MM3Y cells. MM3Y cells grew to slightly higher saturation densities in monolayer cultures than did parental 3Y1 cells. However, they could not form colonies in the agarose medium (Fig. 2C). Semiconfluent MM3Y cultures were incubated with or without 1 ,uM Dx for 48 h, and poly(A)+ RNAs were prepared. Northern blot analyses with SV40 DNAs as probes are shown in Fig. 2A. The main bands were doublets of about 2.8 and 2.5 kilobases (kb), corresponding to the sizes of mRNAs for small and large T Ags started in the mouse mammary tumor virus long terminal repeat (LTR), respectively. times greater, respectively, than those in the control culture in the absence of Dx, as measured by counting the radioactivities of the corresponding bands. For immunoprecipitation of large T Ag (14a), semiconfluent cells were cultivated with or without 1 puM Dx for 48 h and then labeled with [35S]methionine for 6 h. The expression of SV40 large T Ag in the presence of Dx ...

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“…The Pst I fragment (0.5 kb) was obtained from isolated plasmids through restriction analysis, agarose gel electrophoresis, and electroeluting. 32P nick-translation labeling was carried out (22) by using [a-32P]dCTP from New England Nuclear, and the resulting labeled DNA was used for dot hybridization according to the method of Winberry et al (23). The total RNA was isolated from individual livers by the method of Chirgwin et al (24).…”

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confidence: 99%

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes increases in expression of c-erb-A and levels of protein-tyrosine kinases in selected tissues of responsive mouse strains.

Bombick

Jankun²,

Tullis³

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT2,3,7,8,-Tetrachlorodibenzo-p-dioxin (TCDD) administered in vivo causes drastic reduction in the weight of the mouse thymus at low doses (e.g., 30 pg/kg single i.p. i jection), the reduction becoming statistically significant after 2 days. To understand the cause for such thymic involution TCDD-evoked changes in various biochemical parameters in this tissue were examined. The most noticeable change was observed in the increased activity of specific protein-tyrosine kinases and protein kinase C and an increased level of p21-associated binding of [3H]GTP. Since no significant change was observed with cAMP-stimulated protein kinases and cAMP levels, the above changes appear to be a selective effect on these special classes of proteins. As a result of a time sequence study it has become apparent that the rise in proteintyrosine kinase activities becomes significant within 24 hr, whereas the rise in protein kinase C does not become sicant until 48 hr. Among protein-tyrosine kinases, pp60c-and probably pps6lskT were found to be significantly elevated by TCDD treatment. In view of similarities between TCDD and thyroid hormones in causing thymic involution, the levels of c-erb-A expression were assessed in the liver by using avian 32P-labeled v-erb-A probe and RNA transfer blot hybridization technique. The results clearly indicate that TCDD has the property to elevate levels of mRNA bearing homology to v-erb-A. Such changes in c-erb-A expression and proteintyrosine kinase occurred only in TCDD-susceptible (responsive) strains but not in tolerant (nonresponsive) strains of mice at the dose tested. Based on such observations a hypothesis has been proposed that TCDD owes its potency to its ability to stimulate the expression of one of a family of DNAs bearing homology to v-erb-A and that one of the major consequences of such an action is stimulation of various tyrosine kinases.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a very toxic, teratogenic, and carcinogenic chemical (1-4). In addition to these deleterious effects, TCDD is known to cause atrophy of thymus (5, 6) in most experimental animals. Since this lesion occurs at sublethal doses of TCDD at an early stage of poisoning, it has been used by several scientists (e.g., refs. 2, 7, 8) as a sensitive indicator of TCDD poisoning.As for the possible cause for such a thymic lesion, Neal et al. (9) have shown that an increase in serum glucocorticoid levels is unlikely. Poland and Glover (7) have found that involution of thymus occurs at low doses of TCDD (30 ,ug/ kg) in sensitive (responsive) strains of mice but only at high doses in tolerant (nonresponsive) strains of mice. These workers used such differential responses among mouse strains as a biochemical indicator for the involvement of specific cytosolic TCDD binding proteins (i.e., TCDD receptor) in the final expression of thymic involution.Our research group has been studying the effects of TCDD on the hepatic plasma membrane receptors. To date, the plasma membrane-bound epidermal growth factor (EGF) r...

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Expression of proto-oncogenes in normal and papovavirus-transformed or -infected rat fibroblasts

Cited by 10 publications

References 54 publications

Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.

Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.

Induction of c-Ha-ras transcription in rat cells by simian virus 40 large T antigen.

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes increases in expression of c-erb-A and levels of protein-tyrosine kinases in selected tissues of responsive mouse strains.

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