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PubMed2005

Requirement for the SnoN oncoprotein in transforming growth factor beta-induced oncogenic transformation of fibroblast cells.

Transforming growth factor beta (TGF-beta) was originally identified by virtue of its ability to induce transformation of the AKR-2B and NRK fibroblasts but was later found to be a potent inhibitor of the growth of epithelial, endothelial, and lymphoid cells. Although the growth-inhibitory pathway of TGF-beta mediated by the Smad proteins is well studied, the signaling pathway leading to the transforming activity of TGF-beta in fibroblasts is not well understood. Here we show that SnoN, a member of the Ski family of oncoproteins, is required for TGF-beta-induced proliferation and transformation of AKR-2B and NRK fibroblasts. TGF-beta induces upregulation of snoN expression in both epithelial cells and fibroblasts through a common Smad-dependent mechanism. However, a strong and prolonged activation of snoN transcription that lasts for 8 to 24 h is detected only in these two fibroblast lines. This prolonged induction is mediated by Smad2 and appears to play an important role in the transformation of both AKR-2B and NRK cells. Reduction of snoN expression by small interfering RNA or shortening of the duration of snoN induction by a pharmacological inhibitor impaired TGF-beta-induced anchorage-independent growth of AKR-2B cells. Interestingly, Smad2 and Smad3 play opposite roles in regulating snoN expression in both fibroblasts and epithelial cells. The Smad2/Smad4 complex activates snoN transcription by direct binding to the TGF-beta-responsive element in the snoN promoter, while the Smad3/Smad4 complex inhibits it through a novel Smad inhibitory site. Mutations of Smad4 that render it defective in heterodimerization with Smad3, which are found in many human cancers, convert the activity of Smad3 on the snoN promoter from inhibitory to stimulatory, resulting in increased snoN expression in cancer cells. Thus, we demonstrate a novel role of SnoN in the transforming activity of TGF-beta in fibroblasts and also uncovered a mechanism for the elevated SnoN expression in some human cancer cells.

Animals↗
PubMed1983

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.

Animals↗
PubMed1985

Increased secretion of type beta transforming growth factor accompanies viral transformation of cells.

Cells transformed by Harvey or Moloney sarcoma virus secrete at least 40 times as much type beta transforming growth factor as their respective untransformed control cells. The transformed cells bind only 20 to 50% as much type beta transforming growth factor as the control cells, suggesting that transformation causes down-regulation of the type beta transforming growth factor receptor.

Biological Assay↗
PubMed1985

The level of expression of adenovirus type 2 transforming genes governs sensitivity to nonspecific immune cytolysis and other phenotypic properties of adenovirus 2-simian virus 40-transformed cell hybrids.

Syrian hamster embryo cells transformed by adenovirus type 2 (Ad2) or simian virus 40 (SV40) differ markedly in morphology, tumorigenicity, and susceptibility to in vitro lysis by nonspecific cytotoxic cells. Hybrid cells formed by fusing Ad2- and SV40-transformed Syrian hamster embryo cells may express only SV40 T antigens or both SV40 and Ad2 T antigens. Hybrids that express only SV40 T antigens are indistinguishable from the nonhybrid SV40-transformed phenotype, whereas hybrid cells that express T antigens from both viruses closely resemble the nonhybrid parental Ad2-transformed phenotype. Because these hybrid cells have been useful in the study of neoplastic transformation, we determined the amount of viral antigens that they accumulate in an attempt to correlate the level of expression of the transforming viral genes with some of their phenotypic properties. Hybrid cells that expressed proteins from both viruses showed reduced levels of SV40 T antigens compared with those of hybrid cells that did not express Ad2 T antigens. We also found that the production of several cellular proteins that influence cytomorphology was inhibited in hybrid and nonhybrid cells that expressed Ad2 T antigens, and the repression of these cellular proteins correlated with a change in cytomorphology from fibroblastic to spherical. Finally, we showed that the susceptibility of our hybrid cells to in vitro lysis by natural killer cells and activated macrophages, two putative host-effector cells involved in defense against neoplasia, correlated closely with the level of expression of a 58,000-dalton Ad2 protein. The results reported here, together with the results of previous studies, indicate that the oncogenic potential of hybrid cells that express both Ad2 and SV40 antigens is extremely sensitive to Ad2 expression, whereas other phenotypic properties depend on Ad2 expression in a dose-dependent manner.

Actins↗
PubMed1994

Expression and secretion of transforming growth factor-beta in transformed and nontransformed retinal pigment epithelial cells.

The expression and secretion of different isoforms of transforming growth factor-beta (TGF beta) were examined in cultured transformed and nontransformed human retinal pigment epithelial (RPE) cells. Transformed RPE cells were found to express high levels of TGF beta 1 mRNA, low levels of TGF beta 3 mRNA but no detectable TGF beta 2 mRNA. If the cells were grown under serum-free conditions the expression of TGF beta increased. The mRNA expression was accompanied by secretion of TGF beta 1 (but not TGF beta 2) protein into the culture media. By comparison, nontransformed RPE cells were found to secrete similar amounts of TGF beta as transformed cells but predominantly secreted TGF beta 2. The secretion of TGF beta from both transformed and nontransformed RPE cells increased if the cells were grown without serum. In conclusion, the results show that TGF beta is expressed and secreted by transformed and nontransformed human RPE cells and that this expression and secretion are regulated by the presence or absence of exogenous factors.

Blotting, Northern↗
PubMed1995

Antisense GLUT1 RNA suppresses the transforming phenotypes of NIH 3T3 cells transformed by N-Ras.

An antisense approach was attempted to investigate the role of antisense GLUT1 RNA in suppressing tumor cell phenotypes using N-ras-transformed NIH 3T3 cells. The established cell line transformed by ras showed typical biological characteristics of cancer cells, such as increased glucose transport, GLUT1 mRNA contents, and the ability to form colonies on the soft agar. In this system, the plasmids (pMAM-GLUT1(rev)) which can transcribe the antisense GLUT1 RNA were transfected and the accompanying changes in the phenotypes of the ras-transformed cells were observed. The expression of antisense GLUT1 RNA by induction with dexamethasone reduced the glucose transport by 30% (1.97 +/- 0.13 nmoles) after 4 min incubation when compared to the non-induction group of transformed cell (2.85 +/- 0.19 nmoles). Also, the number of colonies sized over 50 microns on the soft agar was reduced significantly in the antisense RNA expressing group compared to non-induction group. These results suggest that the expression of antisense GLUT1 RNA reduced the glucose transport and transforming potential in soft agar possibly by hybridization with GLUT1 mRNA in N-ras-transformed NIH 3T3 cells.

3T3 Cells↗
PubMed1991

Effects of transforming growth factor-beta, transforming growth factor-alpha, and other growth factors on renal proximal tubule cells.

Transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) addition to quiescent, confluent monolayers of rabbit renal proximal tubule cells in primary culture stimulated [3H]thymidine incorporation. TGF-alpha and EGF promoted a 14-fold rise in thymidine incorporation over control levels with half-maximal responses at 2 x 10(-9) M. IGF-1 only promoted a 4-fold rise in thymidine incorporation compared with control values with a half-maximal response of 10(-8) M. Platelet-derived growth factor alone did not stimulate [3H]thymidine incorporation and did not potentiate the effects of EGF or IGF-1 on DNA synthesis, suggesting that platelet-derived growth factor is neither a competence nor a progression growth factor for renal proximal tubule cells. TGF-beta inhibited both baseline and EGF-stimulated [3H]thymidine incorporation after 48 hours of exposure but enhanced EGF-stimulated DNA synthesis at 24 hours. Morphologic evaluation with phase contrast microscopy, scanning, and transmission electron microscopy demonstrated that TGF-beta promoted a dramatic phenotypic transformation of the epithelial monolayer with migration and adhesion of the cells to form solid clusters of adherent cells. Quantitative morphometry demonstrated that this transformation developed 24 hours after TGF-beta exposure, was nearing completion after 48 hours of TGF-beta treatment, and correlated to TGF-beta related inhibition of EGF-induced DNA synthesis (r = -0.82, p less than 0.01). These results demonstrate that EGF and TGF-alpha are the most potent growth promoters for renal proximal tubule cells. IGF-1 is only a modest growth promoter, whereas platelet-derived growth factor has no effect either as a competence or progressive growth factor. TGF-beta inhibited EGF-induced DNA synthesis but only after observable phenotypic transformation of the cells. The degree of TGF-beta promoted transformation on renal tubule cells was highly correlative to th e antiproliferative effect of TGF-beta, suggesting that similar molecular components which promote this phenotypic transformation may also be critical in the antiproliferative effect of TGF-beta.

Animals↗
PubMed1985

[Genetic transformation of somatic cells. VIII. The effect of the DNA methylation inhibitor 5-azacytidine on the stability of thymidine kinase-negative and -positive phenotypes of transformant clone cells].

A study was made of the effect of an DNA methylation inhibitor 5-azacytidine (azaC) on the frequency of reversion to a thymidine kinase-positive (TK+) phenotype in 5-bromodeoxy-uridine (BrdU)-resistant subclones obtained from clones of Chinese hamster cells transformed by thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1). It is shown that in 8 of 15 BrdU-resistant subclones azaC increases 2-1000-fold the frequency of reversion to TK+ phenotype. Variations in the inducibility of reversions to TK+ phenotype indicate that the DNA methylation associated with TK- phenotype affects but differently tk gene of HSV1. Cultivation of TK+ cells of transformant clones in the presence of azaC may lead to stabilization (or decrease in the rate of the loss) of TK+ phenotype, or may not influence the stability of transformant phenotype. The reaction of TK+ cells of transformant clones depends both on genetically determined rate of the loss of TK+ phenotype, and on the structure of transforming DNA introduced to cells. A conclusion is drawn that the TK- phenotype of transformant clone cells arises due to processes which are not associated with methylation of tk gene of HSV1 in spite of the fact that such a methylation may later stabilize significantly the TK- phenotype.

Animals↗
PubMed1985

[Genetic transformation of somatic cells. II. An analysis of the status of the plasmid nucleotide sequences in chromosomal DNA and the thymidine kinase activity in transformant clone cells].

Chinese hamster A238 TK- -cells were transformed with plasmids (derivatives of pBR325) containing thymidine kinase (TK) gene of Herpes simplex virus type 1 (HSV1). The results of dot- and blot-hybridization indicate the presence of pBR325 sequences in the chromosomal fractions of DNA in the transformant clones. These sequences are probably tandemly arranged, and each cluster contains 25--50 copies. SV40 sequences cloned in pBR325 were introduced into the Chinese hamster cells by co-transformation with TK-gene of HSV1-containing plasmid DNA, and all the co-transformant clones selected for TK+-phenotype were shown by hydridization to contain 3V40 DNA fragments. Isoelectrofocusing in polyacrylamide gel shows that thymidine kinase from TK+-transformant clones is of viral type (isoelectric point 7), in contrast to the cellular enzyme (coded by chromosomal gene) having alkaline isoelectric point (pH 9). The results suggest that the true TK+-transformant cells are selected by the procedure used in this study.

Animals↗
PubMed1985

[Genetic transformation of somatic cells. V. Inheritability of the rate of loss of the trait and the stabilization of the transformant phenotype].

Subclones were isolated both on selective and nonselective medium from the Chinese hamster cells transformed by thymidine kinase gene (TK-gene) of Herpes simplex virus (HSV-1) and varying in the rate of the loss of transformant phenotype. The study of the stability of thymidine kinase-positive (TK+) phenotype in cell populations the subclones shows that the nonstability and the rate of the loss of transformant phenotype are the characters that are inherited in the cell generations. Durable cultivation on a HAT-selective medium may lead to a complete or partial (expressed as a reduced rate of the loss of the character) stabilization of TK+-phenotype of the cells of transformant clones. The rate of stabilization of TK+-phenotype may differ depending on the structure of transforming DNA introduced into cells of transformant clones.

Animals↗
PubMed1986

Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor.

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.

Animals↗
PubMed1993

Functional analysis of the carboxy-terminal transforming region of v-Myc: binding to Max is necessary, but not sufficient, for cellular transformation.

To understand the mechanism by which the Myc protein contributes to cell growth and development, our laboratory is studying functions of the avian myelocytomatosis virus 29 (MC29) Gag-Myc protein (v-Myc) in the mouse fibroblast cell line C3H10T1/2. Previously, we identified two specific regions in v-Myc which are required for co-transformation with activated H-ras. One maps to the amino-terminal portion of v-Myc (amino acids 1-137) and has the potential to activate transcription of a basal promoter. The second region spans the carboxy-terminal region of v-Myc (amino acids 244-410), contains a basic/helix-loop-helix/leucine zipper motif and specifies the nuclear location of the protein. In this study, we have generated a series of deletion mutations within the MC29 gag-myc gene to define precisely the carboxy-terminal transforming region using the co-transformation of C3H10T1/2 cells as an assay. v-Myc proteins encoded by selected deletion mutations were also examined for their intracellular location, the ability to interact with the Max protein and the potential to bind specifically to DNA. Our results demonstrate that integrity of both the basic/helix-loop-helix and the leucine zipper motifs of v-Myc is required for co-transforming activity, but that the major nuclear localization signal sequence of v-Myc can be deleted without compromising the ability of v-Myc to cooperate with activated H-Ras p21 to transform C3H10T1/2 cells. In addition, while the binding of v-Myc to Max is necessary for ras/myc co-transformation, it is not sufficient, and also requires the integrity of Myc sequences specifying site-specific DNA binding.

Amino Acid Sequence↗
PubMed1999

Overexpression of transforming growth factor beta-type II receptor reduces tumorigenicity and metastastic potential of K-ras-transformed thyroid cells.

Expression of type II receptor of transforming growth factor beta (TbetaRII) is necessary for this factor to inhibit the growth of thyroid epithelial cells. In rat thyroid transformed cells, the resistance to transforming growth factor beta (TGFbeta) is associated with a decreased expression of TbetaRII mRNA and protein. Reduced TbetaRII expression has also been found in human thyroid differentiated and undifferentiated carcinomas. To investigate the role of TbetaRII in modulating the tumorigenic potential of k-ras-transformed thyroid cells, we transfected these cells with an expression vector carrying the human TbetaRII gene, regulated by an inducible promoter. Isolated clones, overexpressing TbetaRII, showed a reduction in the anchorage-dependent and -independent cell growth, compared with control k-ras-transformed cells. When transplanted in athymic nude mice, the transfected clones presented a decrease in tumorigenicity with respect to the highly malignant parental cells. Moreover, the diminished tumorigenic ability of the clones studied was accompanied by a statistically significant reduction in spontaneous and lung artificial metastases. Taken together, our data demonstrate that TbetaRII acts as a potent tumor suppressor gene when overexpressed in malignant thyroid cells.

Animals↗
PubMed1999

Defects in transforming growth factor-beta signaling cooperate with a Ras oncogene to cause rapid aneuploidy and malignant transformation of mouse keratinocytes.

Genetic inactivation of the transforming growth factor-beta (TGF-beta) signaling pathway can accelerate tumor progression in the mouse epidermal model of multistage carcinogenesis. By using an in vitro model of keratinocyte transformation that parallels in vivo malignant conversion to squamous cell carcinoma, we show that v-ras(Ha) transduced primary TGF-beta1-/- keratinocytes and keratinocytes expressing a TGF-beta type II dominant-negative receptor transgene have significantly higher frequencies of spontaneous transformation than control genotypes. Malignant transformation in the TGF-beta1-/- keratinocytes is preceded by aneuploidy and accumulation of chromosomal aberrations. Similarly, transient inactivation of TGF-beta signaling with a type II dominant-negative receptor adenovirus causes rapid changes in ploidy. Exogenous TGF-beta1 can suppress aneuploidy, chromosome breaks, and malignant transformation of the TGF-beta1-/- keratinocytes at concentrations that do not significantly arrest cell proliferation. These results point to genomic instability as a mechanism by which defects in TGF-beta signaling could accelerate tumor progression in mouse multistage carcinogenesis.

Aneuploidy↗
PubMed2000

Transforming growth factor beta1 selectively regulates ferritin gene expression in malignant H-ras-transformed fibrosarcoma cell lines.

Transforming growth factor beta1 is an important growth regulator in many cell types, usually exerting a negative effect on cellular growth. Inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation, and in some cases, tumor cell proliferation is actually stimulated by TGF-beta1. The present study demonstrates a novel link between alterations in TGF-beta1 regulation during malignant conversion, and the expression of ferritin, an important activity involved in a number of biological functions including iron homeostasis and cell-growth control. A series of H-ras-transformed mouse 10 T 1/2 cell lines, exhibiting increasing malignant potential, was investigated for possible TGF-beta1-mediated changes in ferritin gene expression. Selective induction of gene expression was observed, since only H-ras-transformed cells with malignant potential exhibited marked elevations in ferritin gene expression, in particular, alterations in H-ferritin gene expression. The regulation of H-ferritin gene expression in response to TGF-beta1 did not involve alterations in transcription, but occurred through mechanisms of post-transcriptional stabilization of the H-ferritin mRNA. Additionally, evidence was obtained for a cycloheximide-sensitive regulator of H-ferritin gene expression, since the presence of this protein synthesis inhibitor increased H-ferritin message levels, and in combination with TGF-beta1, cooperated in an additive manner to augment H-ferritin gene expression. These results show for the first time that TGF-beta1 can regulate ferritin gene expression in malignant H-ras transformed cells, and suggest a mechanism for growth factor stimulation of malignant cells, in which early alterations in the control of H-ferritin gene expression are important.

Animals↗
PubMed1999

Bone morphogenetic protein-2 acts synergistically with transforming growth factor-beta3 during endothelial-mesenchymal transformation in the developing chick heart.

In the early embryonic heart, endothelial cells in atrioventricular (AV) and outflow tract (OT) regions are transformed into the invasive mesenchymal cells that form endocardial cushion tissue (endothelial-mesenchymal transformation). It has been reported that bone morphogenetic proteins (BMPs) are transcribed in the AV and OT regions of the embryonic mouse heart. We previously reported that transforming growth factor beta 3 (TGFbeta3) triggers the initial phenotypic changes seen in endothelial-mesenchymal transformation. We cloned BMP2 from embryonic chick hearts and examined its functional role during endocardial cushion tissue formation. In situ hybridization showed BMP2 transcripts in the myocardium of the AV and OT regions, but not in endothelial/mesenchymal cells. Antisense oligodeoxynucleotides to BMP2 inhibited mesenchyme formation in AV endocardium cocultured with associated myocardium. This inhibitory effect was reversed by the addition of recombinant BMP2. In cultured AV endothelial monolayers, recombinant BMP2 did not induce any cellular phenotypic changes characteristic of endothelial-mesenchymal transformation. However, BMP2 enhanced the TGFbeta-induced initial phenotypic changes associated with endothelial-mesenchymal transformation. These results suggest that BMP2 1) plays an important role in the formation of endocardial cushion tissue and 2) acts synergistically with TGFbeta3 in the regulation of this developmental event.

Animals↗
PubMed1976

Transformation-enhancing factor(s) produced by virus-transformed and established cells.

Chick embryo fibroblasts (CEF) and hamster BHK21 cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) release into the culture medium a factor or factors which enhance 2- to 7-fold the formation of transformed foci by chich embryo fibroblasts infected with the Bryan strain of RSV (B-RSV). The factor(s) also increase the number of foci failing to revert to normal phenotype at restrictive temperature (41 degrees C) in cultures infected with a temperature-sensitive mutant (FU-19) of SR-RSV which is defective for transformation. The factor(s) is produced also by BHK21 cells transformed by other tumor viruses and by BHK21 cells passaged for a long time, but not by normal CEF, CEF transformed by B-RSV, CEF infected by FU-19 at 41 degrees C, normal hamster embryo fibroblasts, established but density-inhibited mouse fibroblasts, or BHK21 cells of early passages. The relative enhancement of the number of B-RSV foci can be more than 100-fold when the medium contains fetal calf serum which suppresses focus formation in controls. The focus-enhacing factor(s) appears to act after infection and has been termed, operationally, transformation-enhancing factor(s) or TEF. The factor produced by RS2/3 cells which enhances the formation of B-RSV foci is non-dialyzable and thermolabile, and is presumably a protein. Its molecular weight is between 10(5) and 2 X 10(5) daltons.

Animals↗
PubMed1991

Re-transformation of non-transformed hybrids between c-myc-activating mouse plasmacytoma cells and normal fibroblasts by transfection with activated c-Ha-ras but not c-myc.

In a mouse plasmacytoma S194, c-myc oncogene is rearranged with Ig gene by chromosomal translocation and is consequently activated. We previously reported that transformation of phenotype and expression of rearranged c-myc were repressed in independently isolated hybrid clones, I-1 and IV-10, between S194 and normal fibroblasts. In order to investigate the relationship between transformation of phenotype and oncogene expression, transcriptionally enhanced c-myc or activated c-Ha-ras was transfected into I-1 or IV-10I, a subclone of IV-10. Transfectants expressing high levels of c-myc were found to retain the non-transformed phenotypes. On the other hand, transfectants expressing activated c-Ha-ras showed the transformed phenotypes. These results suggest that enhanced expression of c-myc is not sufficient for re-transformation of the non-transformed hybrid clones between c-myc-activating plasmacytoma cells and normal fibroblasts, but expression of activated c-Ha-ras could diminish or overcome the tumor-suppressive activity of normal fibroblasts.

Animals↗