Transformation of NIH/3T3 to Anchorage Independence by H-Ras Is Accompanied by Loss of Suppressor Activity

Despite their familiar sensitivity to transformation by dominant-acting ras oncogenes, NIH/3T3 cells carry a ras suppressor. When tested by cell fusion they were able to suppress the anchorage-independent phenotype of both mouse and human cells transformed by activated H-ras or N-ras. This suppression occurred without a decrease in expression of the activated ras oncogene. Ras-transformed NIH/3T3 clones cured of their oncogene by benzamide treatment reverted to a non-transformed phenotype, but had lost the ability to suppress other ras transformants, indicating that their initial transformation was accompanied by suppressor loss. In hamster cells an active ras oncogene increased the rate of chromosome segregation by >100-fold. These results suggest that in vitro transformation of NIH/3T3 cells by ras may be more similar to multistep in vivo tumor development than previously suspected, involving not only expression of an active oncogene but also loss of a suppressor activity, perhaps induced by the clastogenic oncogene.     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of EJ-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

Phospholipid dependence of membrane-bound phospholipase A2 in ras-transformed NIH 3T3 fibroblasts

Although the activation of phospholipase A₂ (PLA₂) in ras-transformed cells has been well documented, the mechanisms underlying this activation are poorly understood. In this study we tried to elucidate whether the membrane phospholipid composition and physical state influence the activity of membrane-associated PLA₂ in ras-transformed fibroblasts. For this purpose membranes from non-transfected and ras-transfected NIH 3T3 fibroblasts were enriched with different phospholipids by the aid of partially purified lipid transfer protein. The results showed that of all tested phospholipids only phosphatidylcholine (PC) increased PLA₂ activity in the control cells, whereas in their transformed counterparts both PC and phosphatidic acid (PA) induced such effect. Further we investigated whether the activatory effect was due only to the polar head of these phospholipids, or if it was also related to their acyl chain composition. The results demonstrated that the arachidonic acid-containing PC and PA molecules induced a more pronounced increase of membrane-associated PLA₂ activity in ras-transformed cells compared to the corresponding palmitatestearate- or oleate- containing molecular species. However, we did not observe any specific effect of the phospholipid fatty acid composition in non-transformed NIH 3T3 fibroblasts. In ras-transformed cells incubated with increasing concentrations of arachidonic acid, PLA₂ activity was altered in parallel with the changes of the cellular content of this fatty acid. The role of phosphatidic and arachidonic acids as specific activators of PLA₂ in ras-transformed cells is discussed with respect to their possible role in the signal transduction pathways as well as in the processes of malignant transformation of cells.     

CD44 protein levels and its biological activity are regulated in Balb/c 3T3 fibroblasts by serum factors and by transformation with the ras but not with the sis Oncogene

CD44s (standard isoform) levels and hyaluronan-binding activity were investigated in Balb/c 3T3 cells and their derivatives transformed with ras or sis oncogenes as a function of serum concentration in the medium. 3T3 cells contained low levels of CD44 and did not bind hyaluronan when grown in medium containing 0.5 or 10% serum. In 5% serum, however, the cells had much higher levels of CD44 and were able to bind hyaluronan. CD44 levels also increased in 3T3 cells restimulated with either 5 or 10% serum after prior maintenance in low serum. In cells restimulated with 5% serum, high levels of CD44 were sustained for at least 72 hr. In cells restimulated with 10% serum, however, the increase in CD44 levels reverted by 48 hr. Transformation of 3T3 cells with ras (but not with sis) oncogene rendered CD44 levels insensitive to serum modulation: ras-transformed cells contained high levels of CD44 and bound hyaluronan at all serum concentrations and at all time points tested. Sis-transformed cells behaved like 3T3 cells in these modulatory changes. Platelet-derived growth factor (PDGF), when supplementing 0.5% serum, mimicked the effects of serum on the levels and hyaluronan-binding capacity of CD44 in 3T3 cells and the CD44-upregulating activity of serum was neutralized by incubation with anti-PDGF antibodies. These data demonstrate that serum factors, specifically PDGF, mediate regulation of CD44 levels in Balb/c 3T3 cells and that transformation of 3T3 cells by ras renders CD44 expression insensitive to the modulating effects of serum in vitro. These results correlate with the metastatic capacity of these cells in vivo. © 1996 Wiley-Liss, Inc.     

Transformation of mouse mammary epithelial cells with the Ha-ras but not with the neu oncogene results in a gene dosage-dependent increase in transforming growth factor-α production

An enhanced expression of transforming growth factor-α (TGFα) was demonstrated in two clones of NOG-8 mouse mammary epithelial cells, NOG-8 SR1 and NOG-8 SR2, that have been transformed by a v-Ha-ras oncogene. The amount of TGFα production in NOG-8 SR1 and NOG-8 SR2 cells was dependent on the level of p21^ras expression in these clones, which directly correlated with their cloning efficiency in soft agar. There was also a decrease in the number of epidermal growth factor (EGF) receptors on the NOG-8 SR1 and NOG-8 SR2 cells that is proportional to the amount of TGFα secreted. These effects were specific for ras because neu-transformed NOG-8 cells grew in soft agar at a comparable level to NOG-8 SR2 cells yet did not show any increase in TGFα production or change in EGF receptor expression.     

In vivo tumorigenicity and in vitro sensitivity to tumor-necrosis-factorα mediated killing of c-Ha-ras-transformed cells

Summary Cellular subclones of high and low tumorigenicity obtained from a mouse c-Ha-ras-transformed clone, were examined for their sensitivity to tumornecrosis-factor (TNF)-mediated cytotoxicity. Cells of the highly tumorigenic subclones showed a significantly enhanced resistance to the cytotoxic effect of TNF plus cyclohexamide (CHI) as compared to cells of the lowtumorigenic subclones. The enhanced resistance to TNF+CHI was not due to a lower expression of TNF receptors on the cells. The c-Ha-ras-transfected cells were transformed and maintained in culture only (C cells). In vivo passage of cells of the initially low-tumorigenic c-Ha-ras subclones through the mouse significantly enhanced the tumorigenic potential of these CTC cells (culture/tumor/culture). In correlation with their enhanced tumorigenicity, the CTC cells were highly resistant to TNF-mediated cytotoxicity as compared to C cells of the same subclone. Furthermore, the involvement of TNF in determining the tumorigenic phenotype of the c-Ha-ras-transformed cells was demonstrated in a more direct manner. Cells of a c-Ha-ras-transformed low-tumorigenic, highly TNF-sensitive subclone were selected by repeated cycles of in vitro exposure to TNF. As a result, a stable, highly TNF-resistant population of cells emerged. These TNF-resistant cells caused more tumors in mice as compared to their original TNF-sensitive cells. These results show that the resistance to the cytotoxic effect of TNF plus cyclohexamide may be involved, at least partially, in the tumorigenic potential of c-Ha-ras-transformed cells and suggest a possible role for TNF in the enhancement of the tumorigenic potential of these cells in mice.     

Sequential changes in MHC antigen expression induced by the v-Ki-ras oncogene

A series of early-passage cell lines were transformed with the v-Ki-ras oncogene with the aim of examining the effect of an activatedras gene on the ability of these cells to express major histocompatibility complex (MHC) antigens. These cell lines were found to undergo multiple phenotypic changes upon transformation and subsequent proliferation. At early passage, the predominant effect ofras was an increased ability to express class II antigens when induced with interferon (IFN). For class I antigens, maximum levels of expression induced with IFN were largely unaffected, however, decreased sensitivity to induction with this lymphokine was noted. With subsequent in vitro or in vivo passage, both class I and class II antigen inducibility was attenuated. The latter phenotypic change was found to be transferable by coculture, implicating a soluble IFN antagonist. Conditioned media fromras-transformed cells treated to activate their latent transforming growth factor (TGF) content mediated similar changes in MHC antigen inducibility, suggesting that TGF\ may be involved in modulating MHC antigen expression inras-transformed cells.     

Rapid hyaluronan uptake is associated with enhanced motility: implications for an intracellular mode of action

Texas red-labeled hyaluronan (TR-HA) is rapidly taken up in a CD44 independent manner into ras-transformed 10T1/2 fibroblasts, where it accumulates in both cell ruffles/lamellae, the perinuclear area, and the nucleus. HA does not accumulate in the cell ruffles/lamellae of parental 10T1/2 cells. Addition of HA to ras-transformed cells promotes their random motility but has no effect on 10T1/2 cell motility. 10T1/2 cells can be modified to take up HA into cell ruffles by exposure to phorbol ester or direct microinjection of HA into cells. Both treatments significantly stimulate 10T1/2 cell motility.     

Basal levels of max are sufficient for the cotransformation of C3H10T1/2 cells by ras and myc

The ras and myc oncoproteins cooperate to transform the established murine fibroblast cell line C3H10T1/2. To determine the impact of overexpression of the myc oncoprotein on the phenotype of C3H10T1/2 cells, two C3H10Tl/2-myc clonal cell lines, SVc-myc 11A and myc neo 13A, were isolated and characterized. Although both C3H10Tl/2-myc cell lines are morphologically indistinguishable from wild-type C3H10T1/2 cells and possess growth properties similar to those of C3H10T1/2 cells, each displays a predisposition to transformation following transfection with the activated form of the human H-ras gene. In C3H10T1/2 cells overexpressing the v-myc or H-ras oncogenes, the levels of mRNA encoding max, the recently identified oligomerization partner of myc, remain unchanged, suggesting that the endogenous level of max in C3H10T1/2 cells is sufficient for a high frequency of transformation by ras and myc. Based on these studies, the C3H10Tl/2-myc clonal cell lines we describe are suitable model systems for examining the molecular role of the myc protein in transformation and for characterizing additional factors that synergize with myc in multistep transformation.     

Dose effects of transfected c-Ha-ras oncogene in transformed cell clones

We have examined the expression of the transformed phenotype in a series of clonal lines of NIH/3T3 cells transfected with the human c-Ha-ras^{Val 12} oncogene and the neomycin phosphotransferase gene. Cells from individual transformed foci were cloned and subjected to detailed analyses of the ras sequences. Three clones were found that expressed approximately one, 2–4, or 4–8 copies of the human c-ras oncogene, respectively. A fourth clone had multiple copies of the transfected sequences, and expressed abundant c-Ha-ras RNA. Analysis of the tranformed phenotype of various clones indicated that cells expressing low levels of mutant c-Ha-ras had lost some of their extracellular fibronectin network, and were barely altered in their cytoskeleton. In contrast, cells expressing abundant c-Ha-ras had lost both their actin and fibronectin networks and showed an increase in plasminogen activator activity. Cells with amplified c-Ha-ras^{Val 12} grew better in low serum, formed large colonies in soft agar and showed enhanced activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis. These results show that the dosage level of the mutant oncogene makes a significant contribution to the transformed phenotype of c-Ha-ras oncogene-transformed cells.     

Malignant transformation of NIH-3T3 and CV-1 cells by a helical mycoplasma,Spiroplasma mirum,strain SMCA

Summary A helical mycoplasma,Spiroplasma mirum strain SMCA, produced malignant transformation in mouse NIH 3T3 cells and monkey kidney CV-1 cells. The transformed cells exhibited morphological changes consistent with the transformed phenotype, grew in soft agar and produced tumors in athymic and BALB/c mice. Transmission electron microscopy revealed structures morphologically similar to mycoplasmas present in the cytoplasm of transformed but not untransformed 3T cells. The time of inoculation ofS. mirum SMCA to 3T3 cells and the passage level of 3T3 cells affected transformation. Editor's statement This paper describes the possible role of a mycoplasma organism, which induces cataracts and brain pathology similar to Creutzfeld Jacob and other degenerative diseases, in malignant transformation of mammalian cells. In addition to this surprising and novel finding is the observation that the mycoplasma resides in an intracellular position. These findings may have important implications for understanding malignant transformation and the nature of the diseases produced by this organism.     

Galectin-3 gene (LGALS3) expression in experimental atherosclerosis and cultured smooth muscle cells

The galectin-3 gene (LGALS3) encodes a β-galactose binding lectin. LGALS3 expression is associated with neoplastic transformation and with differentiation of monocytes to macrophages. Factors involved in migration, proliferation, adhesion and differentiation of vascular smooth muscle cells (SMC) play a major role during atherosclerosis development. Expression of the galectin-3 gene was not detected in quiescent SMC but was activated in aortas of hypercholesterolemic rabbits, in aortas of rats after balloon injury and in cultured SMC. These results suggest that galectin-3 production is involved in the developmental process of atherogenesis.     

A p53 mutation is required for stable transformation of REF52 cells by themyc andras oncogenes

It is known that neoplastic transformation of rodent primary embryonic fibroblasts culturedin vitro requires coexpression at least of two cooperating oncogenes. In the case of transduction into cells of oncogenesras andmyc, the cell transformation is poorly effective. To study some additional factors necessary for such transformation, c-myc and N-ras ^Asp12 were consecutively introduced into REF52 cells by retroviral infection, and the cell cultures obtained were analyzed. Expression ofmyc broke the regulation of the cell cycle, in particular, canceled the G1 phase arrest for cells with damaged DNA, despite the normal function of protein p53 and induction of the p53-responsive genep21 ^Waf1 in these cells. The subsequent transduction ofras led to morphological transformation of cells and an increase of p53 level. However, reversion of the transformed phenotype to normal morphology took place after less than five passages. On this background, rare clones generated the stable transformed cell lines characterized by accelerated proliferation and having a mutation in thep53 gene. Attempts to obtain stable transformed cell lines by transduction ofras into REF52 cells not expressing exogenousmyc were unsuccessful. Analysis of the stable transformed clones revealed a mutation at codon 271 of thep53 gene, a hot spot of mutations, which led to the replacement of arginine by cysteine. In these clones, p53 is accumulated owing to the increased life time, and has a flexible conformation, being able to interact with monoclonal PAb1620 and PAb240 antibodies recognizing alternative protein conformations. The results obtained suggest that p53 participates in negative regulation of the cell cycle under conditions of oncogenic stimulation, and its inactivation is necessary for full transformation of cells by cooperating oncogenesmyc andras.     

The specific anti-cancer activity of green tea (−)-epigallocatechin-3-gallate (EGCG)

Summary. The effect of the green tea polyphenol-(−)epigallocatechin-3-gallate (EGCG) was tested in cultures of normal and transformed NIH-pATMras fibroblasts. In this system transformation can be induced at will by the addition of dexamethasone, which induces the expression of H-ras by activating the mammary tumor virus long terminal repeat (MMTV-LTR) promoter. This facilitates a reliable comparison of the susceptibility of normal and transformed cells to EGCG. It has been shown that EGCG inhibited the growth of transformed but not of the normal fibroblasts. In an attempt to elucidate the mode of the preferential inhibitory activity of EGCG, its effect on growth promoting factors has been examined. The level of ornithine decarboxylase (ODC, EC 4.1.1.17), which is a signal for cellular proliferation, was reduced by EGCG in the transformed but not in the normal cells. EGCG also showed strong inhibition of tyrosine kinase and mitogen-activated protein kinase (MAPK) activities, without affecting the kinases in the normal cells. Similarly, EGCG also preferentially decreased the levels of the oncogenes Ras and Jun in transformed cell. EGCG preferentially induced apoptosis in the transformed fibroblasts. In vitro chemosensitivity tests demonstrated that EGCG inhibited the proliferation of leukemic cells. These findings suggest that EGCG has a therapeutic potential in the combat against cancer. Received July 27, 2001 Accepted September 8, 2001     

DNA methylation is not involved in the structural alterations of Ornithine Decarboxylase or Total Chromatin of c-Ha-rasVal 12 Oncogene-Transformed NIH-3T3 Fibroblasts

The ornithine decarboxylase (odc) gene is an early response gene, whose increased expression and relaxed chromatin structure is closely coupled to neoplastic growth. In various tumour cells, the odc gene displays hypomethylation at the sequences CCGG. Hypomethylation of genes is believed to correlate with chromatin decondensation and gene expression. Since a given pattern of DNA methylation may not be preserved in neoplastic cells, we studied the methylation status of odc gene at the CCGG sequences in c-Ha-ras^{Val 12} oncogene-transformed NIH-3T3 fibroblasts during the growth cycle and relative to their normal counterparts. We found that the methylation state of the odc gene and its promoter and mid-coding and 3' regions remain unaltered during the cell cycle. We also found that in ras oncogene-transformed cells, which display a more decondensed nucleosomal organization of chromatin than the normal cells, the CCGG sequences in bulk DNA and at the odc gene were methylated to the same extent as in the nontransformed cells. These data suggest that DNA hypomethylation at the CCGG sequences is not a prerequisite for chromatin decondensation and cell transformation by the c-Ha-ras^{Val 12} oncogene.     

Phosphoinositide hydrolysis in Ki-ras-transformed fibroblasts stimulated by platelet-derived growth factor and bradykinin

• 1.1. Signal transduction in response to platelet-derived growth factor (PDGF)-BB and bradykinin (BK) have been examined by measuring inositol polyphosphate formation in NIH3T3 fibrobalst and v-Ki-ras -transformed NIH3T3 fibroblast (DT).
• 2.2. The PDGF-induced inositol polyphosphate formation in NIH3T3 was greater than that in DT cells, in which autophosphorylation of PDGF receptor and tyrosine phosphorylation of phospholipase C (PLC)-γ 1 were suppressed when examined by immunoblotting with anti-phosphotyrosine antibody.
• 3.3. On the other hand, BK-stimulation produced a much higher level of inositol polyphosphate in DT cells which have a greater number of BK receptors.
• 4.4. These results indicate that in Ki-ras transformed cells the decrease (caused by PDGF) and the increase (caused by BK) in phosphoinositide hydrolysis are due to the defective autophosphorylation of PDGF receptors leading to a reduction in PLC-γ 1 tyrosine phosphorylation and the overexpression of BK receptors, respectively.