Rat Embryo Fibroblast Cell Line Research Articles

Calcium and S100B regulation of p53-dependent cell growth arrest and apoptosis.

In glial C6 cells constitutively expressing wild-type p53, synthesis of the calcium-binding protein S100B is associated with cell density-dependent inhibition of growth and apoptosis in response to UV irradiation. A functional interaction between S100B and p53 was first demonstrated in p53-negative mouse embryo fibroblasts (MEF cells) by sequential transfection with the S100B and the temperature-sensitive p53Val135 genes. We show that in MEF cells expressing a low level of p53Val135, S100B cooperates with p53Val135 in triggering calcium-dependent cell growth arrest and cell death in response to UV irradiation at the nonpermissive temperature (37.5 degreesC). Calcium-dependent growth arrest of MEF cells expressing S100B correlates with specific nuclear accumulation of the wild-type p53Val135 conformational species. S100B modulation of wild-type p53Val135 nuclear translocation and functions was confirmed with the rat embryo fibroblast (REF) cell line clone 6, which is transformed by oncogenic Ha-ras and overexpression of p53Val135. Ectopic expression of S100B in clone 6 cells restores contact inhibition of growth at 37.5 degreesC, which also correlates with nuclear accumulation of the wild-type p53Val135 conformational species. Moreover, a calcium ionophore mediates a reversible G1 arrest in S100B-expressing REF (S100B-REF) cells at 37.5 degreesC that is phenotypically indistinguishable from p53-mediated G1 arrest at the permissive temperature (32 degreesC). S100B-REF cells proceeding from G1 underwent apoptosis in response to UV irradiation. Our data support a model in which calcium signaling and S100B cooperate with the p53 pathways of cell growth inhibition and apoptosis.

Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation.

Recent data suggest that aberrant function of the wild type p53 protein (WTp53) may alter cellular survival following DNA damage through cellular pathways involving apoptosis and cell-cycle checkpoints, but little is known concerning it's possible role in DNA repair. In the present study, the ionizing radiation sensitivity was determined for a series of rat embryo fibroblast (REF) cell lines transfected with an activated form of the H-ras oncogene alone, or in combination with a variety of missense-mutant p53 (MTp53) alleles. Transformed REF clones which expressed exogenous MTp53 and p21ras proteins (CLASS II clones) were generally radioresistant in culture as determined by higher values for the surviving fraction after 2 Gy (SF2 value) and the radiation dose required to reduce survival to a fraction of 0.1 (D10 value), compared either to transformed REF clones expressing p21ras protein alone (CLASS I clones), or to non-transfected REF control cell lines expressing baseline endogenous levels of p21ras and WTp53 protein. The increased radioresistance observed in the CLASS II clones (following both HDR- and LDR-irradiation), was significantly correlated with increased expression of MTp53 protein, and a decreased radiation-induced G1 arrest response. The variability observed in clonogenic radiosensitivity among REF clones was not explained by differential radiation-induced apoptosis. Using the Comet assay performed after continuous low dose-rate (LDR)-irradiation, MTp53-expressing REF clones were also found to be more proficient at the rejoining of DNA double-strand breaks (DNA-dsb), compared to WTp53-expressing REF clones. These results suggest that an enhanced DNA and cellular repair capacity may, in part, explain the increased radiation survival observed in some MTp53-expressing transformed fibroblasts and tumours.

Cloning and Identification of Genes That Associate with Mammalian Replicative Senescence

Cellular senescence and limited proliferative capacity of normal diploid cells has a dominant phenotype over immortality of cancerous cells, suggesting its regulation by the expression of a set of genes. In order to isolate the genes that associate with senescence, we have employed a clonal system of conditional SV40 T antigen rat embryo fibroblast cell lines which undergo senescence upon T antigen inactivation. Construction of cDNA libraries from two conditional cell lines and application of differential screening and subtractive hybridization techniques have resulted in the cloning of eight senescence-induced genes (SGP-2/Apo J, α1-procollagen, osteonectin, fibronectin, SM22, cytochrome C oxidase, GTP-α, and a novel gene) and a senescence-repressed gene (FRS-2). Three of these genes encode for extracellular matrix proteins, others are involved in the calcium-dependent signal transduction pathways, while the SGP-2/Apo J gene may have a cellular protective function. RNA analysis has shown that the senescence-associated genes are overexpressed in both normal rat embryonic fibroblasts and human osteoblasts cell cultures undergoing agingin vitro.In comparison, the expression of these genes in a rat fibroblast immortalized cell line (208F cells) was down-regulated after both its partial and its full transformation byrasoncogenes. Thus, cloning of senescence-associated genes opens up new ways to elucidate and/or to modulate aging and cancer.

Signal transduction in fibroblasts stably transformed by [Val12]Ras--the activities of extracellular-signal-regulated kinase and Jun N-terminal kinase are only moderately increased, and the activity of the delta-inhibitor of c-Jun is not alleviated.

Ras-transformed cells often show high levels of expression of activating protein-1 and Ets and of genes regulated by these transcription factors. In analogy with the effects of transient stimulation of Ras, it is assumed that the increase in transcription-factor transactivation in stably transformed cells is due to Ras-induced constitutive activation of mitogen-activated protein kinases. However, this has not been extensively studied. Using specific substrate peptides, we have examined here the activities of two types of mitogen-activated protein kinase, extracellular-signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK), in [Val12]Ras-transformed rat embryo fibroblast cell lines. These activities were elevated 2-3-fold in Ras-transformed cells compared with non-transformed cells with a similar growth rate. Increased ERK activity was not necessarily accompanied by a similar increase in JNK activity. In transformed cells, ERK and JNK activities could be stimulated fourfold and ninefold by phorbol ester and ultraviolet-light treatment, respectively, indicating that only a fraction of these enzymes were constitutively activated in these cells. It has been suggested that inactive JNK downregulates c-Jun transcriptional activity by binding to the c-Jun delta-domain. No decrease in delta-inhibitor activity could be demonstrated in Ras-transformed cells compared with control cells, consistent with the presence of mainly inactive JNK in transformed cells. Treatment of transformed cells wih benzodiazepine 5B, an inhibitor of Ras farnesylation, decreased ERK and JNK activities, and concomitantly caused morphological reversion, reduced growth rate, and normalization of transformation-related gene expression. We conclude that in stably Ras-transformed cells the moderately increased ERK/JNK activities are not coregulated, and that ERK rather than JNK activity correlated with transformation-related gene expression.

Codeletion of the JUN proto‐oncogene and the CDKN2A tumor‐suppressor gene in HRAS‐transformed rat embryo fibroblast cell lines

The cyclin kinase inhibitor p16, encoded by the CDKN2A gene, suppresses the transformation of mouse embryonic fibroblasts by oncogenic RAS.In contrast, the c-JUN transcription factor (a major component of AP-1) has been suggested to be required for RAS transformation of rodent fibroblasts. The CDKN2A gene and the JUNproto-oncogene have both been mapped to rat chromosome band 5q31-33. We here show that both copies of the CDKN2A gene are deleted in four of eight transformed cell lines derived from the transfection of rat embryo fibroblasts (REF) with HRASVAL12. In two cell lines, the homozygous deletions involved a larger area on 5q31—33, which included the JUNproto-oncogene. JUN-defective cells showed high AP-1 binding activity. Both AP-1 binding activity and stromelysin (transin) mRNA expression were found to be RAS-dependent in one of the JUN-defective cell lines. The finding of deletions of the CDKN2A gene in RAS-transformed REF cell lines is consistent with the concept that CDKN2A suppresses transformation by RAS. The occasional concomitant loss of the adjacent JUNproto-oncogene does not prevent establishment of transformed and tumorigenic cell lines. Genes Chromosom. Cancer 20:82–89, 1997. © 1997 Wiley-Liss, Inc.

Codeletion of theJUN proto-oncogene and theCDKN2A tumor-suppressor gene inHRAS-transformed rat embryo fibroblast cell lines

The cyclin kinase inhibitor p16, encoded by the CDKN2A gene, suppresses the transformation of mouse embryonic fibroblasts by oncogenic RAS. In contrast, the c-JUN transcription factor (a major component of AP-1) has been suggested to be required for RAS transformation of rodent fibroblasts. The CDKN2A gene and the JUN proto-oncogene have both been mapped to rat chromosome band 5q31-33. We here show that both copies of the CDKN2A gene are deleted in four of eight transformed cell lines derived from the transfection of rat embryo fibroblasts (REF) with HRASVAL12. In two cell lines, the homozygous deletions involved a larger area on 5q31-33, which included the JUN proto-oncogene. JUN-defective cells showed high AP-1 binding activity. Both AP-1 binding activity and stromelysin (transin) mRNA expression were found to be RAS-dependent in one of the JUN-defective cell lines. The finding of deletions of the CDKN2A gene in RAS-transformed REF cell lines is consistent with the concept that CDKN2A suppresses transformation by RAS. The occasional concomitant loss of the adjacent JUN proto-oncogene does not prevent establishment of transformed and tumorigenic cell lines.

Mutant adenovirus type 9 E4 ORF1 genes define three protein regions required for transformation of CREF cells

Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors in rats, and an essential oncogenic determinant for this virus is Ad9 E4 open reading frame 1 (9ORF1), which encodes a 125-residue cytoplasmic protein with cellular growth-transforming activity in vitro. In this study, we engineered 48 different mutant 9ORF1 genes in an attempt to identify regions of this viral protein essential for transformation of the established rat embryo fibroblast cell line CREF. In initial assays with CREF cells, 17 of the 48 mutant 9ORF1 genes proved to be severely defective for generating transformed foci but only 7 of these defective genes expressed detectable amounts of protein. To further examine the defects of the seven mutant proteins, we selected individual cell pools of stable CREF transformants for the wild-type and mutant 9ORF1 genes. Compared to cell pools expressing the wild-type 9ORF1 protein, most cell pools expressing mutant proteins displayed decreased growth in soft agar, and all generated significantly smaller tumors in syngeneic animals. The altered amino acid residues of the seven mutant 9ORF1 polypeptides clustered within three separate regions referred to as region I (residues 34 to 41), region II (residues 89 to 91), and C-terminal region III (residues 122 to 125). By using indirect immunofluorescence, we also assessed whether the mutant proteins localized properly to the cytoplasm of cells. The region I and region II mutants displayed approximately wild-type subcellular localizations, whereas most region III mutants aberrantly accumulated within the nucleus of cells. In summary, we have identified three 9ORF1 protein regions necessary for cellular transformation and have demonstrated that C-terminal region III sequences significantly influence the proper localization of the 9ORF1 polypeptide in cells.

Human adenovirus early region 4 open reading frame 1 genes encode growth-transforming proteins that may be distantly related to dUTP pyrophosphatase enzymes.

An essential oncogenic determinant of subgroup D human adenovirus type 9 (Ad9), which uniquely elicits estrogen-dependent mammary tumors in rats, is encoded by early region 4 open reading frame 1 (E4 ORF1). Whereas Ad9 E4 ORF1 efficiently induces transformed foci on the established rat embryo fibroblast cell line CREF, the related subgroup A Ad12 and subgroup C Ad5 E4 ORF1s do not (R. T. Javier, J. Virol. 68:3917-3924, 1994). In this study, we found that the lack of transforming activity associated with non-subgroup D adenovirus E4 ORF1s in CREF cells correlated with significantly reduced protein levels compared to Ad9 E4 ORF1 in these cells. In the human cell line TE85, however, the non-subgroup D adenovirus E4 ORF1s produced protein levels higher than those seen in CREF cells as well as transforming activities similar to that of Ad9 E4 ORF1, suggesting that all adenovirus E4 ORF1 polypeptides possess comparable cellular growth-transforming activities. In addition, searches for known proteins related to these novel viral transforming proteins revealed that the E4 ORF1 proteins had weak sequence similarity, over the entire length of the E4 ORF1 polypeptides, with a variety of organismal and viral dUTP pyrophosphatase (dUTPase) enzymes. Even though adenovirus E4 ORF1 proteins lacked conserved protein motifs of dUTPase enzymes or detectable enzymatic activity, E4 ORF1 and dUTPase proteins were predicted to possess strikingly similar secondary structure arrangements. It was also established that an avian adenovirus protein, encoded within a genomic location analogous to that of the human adenovirus E4 ORF1s, was a genuine dUTPase enzyme. Although no functional similarity was found for the E4 ORF1 and dUTPase proteins, we propose that human adenovirus E4 ORF1 genes have evolved from an ancestral adenovirus dUTPase and, from this structural framework, developed novel transforming properties.

A 3 milliTesla 60 Hz magnetic field is neither mutagenic nor Co-Mutagenic in the presence of menadione and MNU in a transgenic rat cell line

The mechanisms by which an electromagnetic field (EMF) influences biological material are poorly understood. One potentially important model suggests that a magnetic field can stabilize free radicals in such a way as to permit their dispersement rather than their return to the ground state ( Okazaki et al., 1988; Scaiano, 1995). We have tested this hypothesis by examining mutagenesis in the E. coli lacI gene target carried in the Big Blue ® rat embryo fibroblast cell line, R2λLIZ. Mutant frequencies were determined in cells exposed to a magnetic field, cells pretreated with the mutagens N-methylnitrosourea (MNU) or 2-methyl-1,4-naphthoquinone (menadione), prior to being held in a 60 Hz 3 milliTesla (mT) magnetic field and cells concurrently exposed to the mutagens and the magnetic field. Menadione was selected because its mutagenic mechanism involves the formation of free radicals, while MNU is an alkylating agent not thought to act through radical formation. According to the radical stabilization hypothesis the application of a magnetic field to menadione treated cells would accentuate the mutagenic effects. Our results failed to indicate that the magnetic field affects mutagenesis by the oxygen-radical mediated mutagen, menadione.

Pituitary-specific chromatin structure of the rat prolactin distal enhancer element.

The location of target DNA sequences within chromatin may affect the ability of trans-acting factors to bind cis-elements and regulate gene transcription. To examine the effect of chromatin structure on the ability of the estrogen-estrogen receptor complex (E2R) to bind its respective DNA binding element within the rat prolactin (rPRL) gene and modulate rPRL gene expression, we have developed cell lines derived from the rPRL-expressing (rPRL+) rat pituitary cell line GH3 and the rPRL-non- expressing (rPRL-) rat embryo fibroblast cell line Rat1. These cell lines contain mini-chromosomes composed of the 5' upstream regulatory region of the rPRL gene driving expression of a reporter gene, Tn5, within a bovine papillomavirus (BPV) vector. The rPRL-Tn5 gene retains the characteristics of cell-specific expression and estrogen inducibility of transcription displayed by the endogenous rPRL gene. The distal enhancer region, which contains an estrogen response element, was found to exist in a nucleosome-free region in pituitary-derived cells even in the absence of estrogen. In contrast, the rPRL distal enhancer in fibroblast cells was found to be randomly packaged into nucleosomes. These results indicate that DNA sequence is not sufficient to position nucleosomes in the rPRL gene. Rather, it suggests that cell-specific factors are present in pituitary cells that modify the chromatin structure of the distal enhancer which allow E2R to bind to its response element.

Interaction of hydro- or lipophilic phthalocyanines with cells of different metastatic potential.

A highly metastatic (4R) and a nonmetastatic (RE4) transformed rat embryo fibroblast cell line were incubated with lipid-soluble Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) and compared for phthalocyanine uptake. The hydrophobic liposome-delivered ZnPc showed a significantly greater uptake by both cell lines than did ZnPcTS. Moreover, the two phthalocyanines appear to interact with cells according to different pathways, as suggested by the different temperature-dependence of the binding process and the different inhibitory action exerted by selected serum proteins, such as lipoproteins and heavy proteins. Under all experimental conditions, the two cell lines exhibited similar interactions with ZnPc and ZnPcTS, suggesting that heterogeneity of the tumor cell population has a minor influence on the accumulation of photosensitizers.

Human adenovirus type 9 E4 open reading frame 1 encodes a cytoplasmic transforming protein capable of increasing the oncogenicity of CREF cells

The induction of estrogen-dependent rat mammary tumors by human adenovirus type 9 (Ad9) requires the Ad9 E4 open reading frame 1 (9ORF1) protein, which alone can transform that rat embryo fibroblast cell line CREF in vitro. In the present study, independent pools of both 9ORF1-expressing and control CREF cells were generated by selection with G418 and compared with respect to transformed properties. Indirect immunofluorescence analyses revealed that more than 99% of the cells that made up the 9ORF1-transfected pools expressed 9ORF1 protein and, together with confocal laser scanning microscopy, indicated that this E4 protein was located predominantly within the cytoplasm of cells. With regard to transformation, cells of the 9ORF1-expressing pools differed from those of control pools by forming foci, displaying morphological alterations, growing more efficiently in soft agar, and reaching higher saturation densities. Following injection into immunocompetent syngeneic rats, the 9ORF1-expressing pool cells exhibited greatly enhanced oncogenicity compared with control pool cells. These results show that 9ORF1 protein (i) localizes predominantly within the cytoplasm, (ii) confers multiple general transformed characteristics to CREF cells in vitro, and (iii) increases the tumorigenic properties of these cells in vivo.

Two Closely Related Isoforms of Protein Kinase C Produce Reciprocal Effects on the Growth of Rat Fibroblasts: POSSIBLE MOLECULAR MECHANISMS

We have previously reported that two closely related protein kinase C (PKC) isoforms, PKC alpha and PKC beta I, had divergent effects on the growth and transformation of the same parental R6 rat embryo fibroblast cell line (Housey, G. M., Johnson, M. D., Hsiao, W.-L. W. O'Brian, C. A., Murphey, J. P., Kirschmeier, P., and Weinstein, I. B. (1988) Cell 52, 343-354; Borner, C., Filipuzzi, I., Weinstein, I. B., and Imber, R. (1991) Nature 353, 78-80). Whereas cells that overexpress PKC beta I lost anchorage dependence, grew to higher saturation densities, and generated small tumors when injected into nude mice, none of these properties were seen with cells that overexpress PKC alpha. In fact, the latter cells grew even slower and to lower saturation densities as compared to control cells. Here we investigate possible molecular mechanisms underlying the reciprocal effects of PKC alpha and PKC beta I. Overexpression of both isoforms enhanced 12-O-tetradecanoyl phorbol-13 acetate-induced expression of the growth regulatory genes c-jun, c-myc, and collagenase and enhanced feedback inhibition of epidermal growth factor receptor binding and cellular levels of diacylglycerol. However, the cells overexpressing PKC beta I differed from those overexpressing PKC alpha by displaying a decreased requirement for growth factors and by the production of a mitogenic factor. Thus, the basis for enhanced growth and transformation of cells overexpressing PKC beta I may be the establishment of an autocrine growth factor loop. These findings may be relevant to the roles of specific isoforms of PKC in carcinogenesis and tumor growth.

The protein kinase C activator, phorbol ester, cooperates with the wild-type p53 species of Ras-transformed embryo fibroblasts growth arrest.

Clone 112 cells, a rat embryo fibroblast cell line cotransfected by an activated ras gene and a temperature-sensitive mutant p53 gene (p53val135) grow well at 37 degrees C but cease DNA synthesis and cell division when shifted to 32 degrees C (Michalovitz, D., Halevy, O., and Oren, M. (1990) Cell 62, 671-680). Characterization of the p53 protein in exponentially growing clone 112 cells at 37 degrees C revealed that both wild-type (reactive with the monoclonal antibody PAb 246) and mutant (reactive with PAb 240) p53 conformational forms are co-expressed. These results indicate that in clone 112 cells the growth suppressor activity of the wild-type p53 species is inactivated at 37 degrees C. We show that clone 112 cells grown at 37 degrees C elicits specific growth inhibition response to stimulation by the tumor promoter phorbol ester, phorbol 12-myristate 13-acetate (PMA). At 37 degrees C, PMA induced nuclear accumulation of the p53 protein, a behavior that is also observed in growth-arrested cells at 32 degrees C. Furthermore, when cells are growth arrested at 32 degrees C, PMA prevented the cells from re-entering the cell cycle when they are shifted back to 37 degrees C. All these observations suggest that PMA can cooperate with the wild-type p53 in cell growth arrest. At 37 and 32 degrees C, PMA stimulation of clone 112 cells resulted in specific enhancement of phosphorylation of the wild-type p53 species but not of the mutant form. We also demonstrate that the growth arrest of clone 112 cells at 37 degrees C is correlated with stimulation of the nuclear wild-type p53-DNA binding activities. The PMA-mediated increase in p53 DNA binding activity coincides with the loss of the PAb 421 epitope on the p53.DNA complex. PAb 421 non-reactivity with p53 has been shown by others to occur in growth-arrested cells and upon phosphorylation of p53 by protein kinase C. We also provide evidence that, in vitro, the protein kinase C mode of phosphorylation stimulates DNA binding activities of purified recombinant wild-type p53 and that in a mutant conformation p53 is not a substrate for protein kinase C. We propose that wild-type p53 and protein kinase C, the cellular receptor of phorbol ester, could participate in the negative feedback controls associated with the phosphoinositide-derived signals common to a number of mitogenic stimulations.

Adenovirus type 9 E4 open reading frame 1 encodes a transforming protein required for the production of mammary tumors in rats.

The E4 region of human adenovirus type 9 (Ad9) transforms established rat embryo fibroblasts and encodes an essential determinant for the production of estrogen-dependent mammary tumors in rats. Testing of the seven Ad9 E4 open reading frames (ORFs) individually for transformation of the established rat embryo fibroblast cell line CREF indicated that only Ad9 E4 ORF1 possessed a significant ability to generate transformed foci on these cells. In contrast, the E4 ORF1 sequences from human Ad5 and Ad12 lacked the transforming potential exhibited by Ad9 E4 ORF1. Cell lines derived from Ad9 E4 ORF1-transformed foci expressed the 14-kDa Ad9 E4 ORF1 protein and formed colonies in soft agar. In addition, the Ad9 E4 ORF1 protein was required for initiation of mammary oncogenesis in vivo, as E4 ORF1 mutant viruses failed while E4 ORF2 and ORF3 mutant viruses succeeded in eliciting mammary tumors in animals. A role for Ad9 E4 ORF1 in tumor maintenance was suggested by the fact that 100% of virus-induced mammary tumors expressed the E4 ORF1 protein. Taken together, the facts that the Ad9 E4 ORF1 protein exhibits transforming potential in culture and is required by Ad9 to produce mammary tumors in animals suggest that Ad9 E4 ORF1 is a new viral oncoprotein.

Oncogene-associated growth behavior and oxygenation of multicellular spheroids from rat embryo fibroblasts.

The basis of the present investigation was the establishment of an oncogene-dependent, genetically determined two-stage carcinogenesis in vitro model as multicellular spheroids. Spheroid formation was achieved with four rat embryo fibroblast cell lines, two of which represent the first step of malignant transformation, known as stage of immortalization. The ras-transfected counterparts of these two parental cell clones represent fully transformed phenotypes. The data obtained show that spheroid volume growth and cellular viability reflect the degree of tumorigenicity in vivo of the different fibroblast types investigated. In addition, ras-transfection alters not only the growth kinetics but also the cellular oxygen metabolism. Furthermore, the results demonstrate very clearly that different fibroblast clones at the same stage of malignant transformation may be characterized by an entirely different growth behavior, morphology and metabolic activity in spheroid culture. This is true, although these cells originate from the same primary cells, differ only in the step of immortalization, and were cultured as spheroids under identical environmental conditions.

Altered ganglioside composition in virally transformed rat embryo fibroblasts

The composition of gangliosides was examined in a normal rat embryo fibroblast cell line (REF52) and in two viral transformants: a polyoma transformant (REF52-PyMLV) and a simian viral 40 transformant (REF52-SV40). The distribution of gangliosides in the cell lines was determined using gas-liquid chromatography and high-performance thin-layer chromatography. N-acetylneuraminic acid was the predominant sialic acid species detected in the three cell lines. The total ganglioside concentration (μg/100 mg dry weight of cells) in the normal, PyMLV, and SV40 lines was 144.7 ± 10.4, 153.8 ± 9.2, and 86.1 ± 6.8, respectively. Gangliosides GM3, GM2, GM1, and GD1a were the major species in the normal and transformed lines. The distribution of these gangliosides, however, differed markedly between the normal and the transformed lines and also between the transformed lines themselves. The transformed cells also differed from the normal cells in growth rate, morphology, and social behavior. The cell line with highest GM3 content (PyMLV) formed islands, whereas the normal and SV40 cell lines, which had lower GM3 levels, grew as monolayers. The findings suggest that PyMLV and SV40 transformation can have multiple and different effects on cellular ganglioside distribution and growth behavior.

Transformation of a continuous rat embryo fibroblast cell line requires three separate domains of simian virus 40 large T antigen.

Mouse C3H 10T1/2 cells and the established rat embryo fibroblast cell line REF-52 are two cell lines widely used in studies of viral transformation. Studies have shown that transformation of 10T1/2 cells requires only the amino-terminal 121 amino acids of simian virus 40 (SV40) large T antigen, while transformation of REF-52 cells requires considerably more of large T antigen, extending from near the N terminus to beyond residue 600. The ability of a large set of linker insertion, small deletion, and point mutants of SV40 T antigen to transform these two cell lines and to bind p105Rb was determined. Transformation of 10T1/2 cells was greatly reduced by mutations within the first exon of the gene for large T antigen but was only modestly affected by mutations affecting the p105Rb binding site or the p53 binding region. All mutants defective for transformation of 10T1/2 cells were also defective for transformation of REF-52 cells. In addition, mutants whose T antigens had alterations in the Rb binding site showed a substantial reduction in transformation of REF-52 cells, and the degree of this reduction could be correlated with the ability of the mutant T antigens to bind p105Rb. There was a tight correlation between the ability of mutants to transform REF-52 cells and the ability of their T antigens to bind p53. These results demonstrate that multiple regions of large T antigen are required for full transformation by SV40.

Transformed rat tracheal epithelial cells exhibit alterations in transforming growth factor-beta secretion and responsiveness.

The purpose of our studies was to define abnormalities in the transforming growth factor-beta (TGF-beta) system of transformed rat tracheal epithelial (RTE) cells that might cause their abnormal growth behavior. We found that many, but not all, of the transformed cell lines were hyporesponsive or unresponsive to the growth inhibitory effects of TGF-beta 1. Scatchard and receptor cross-linking analyses indicated that loss of TGF-beta 1 responsiveness of transformed cells was probably not due to changes in receptor number or affinity, or to changes in expression of the three TGF-beta-binding protein subtypes. Transformed cells were found to secrete far less TGF-beta-like activity (less than 1/10) than primary cells. Cultured normal and transformed RTE cells expressed three TGF-beta 1 transcripts of 2.5, 1.9, and 1.4 kb. In contrast, rat kidney tissue, a rat embryo fibroblast cell line, and a rat liver cell line expressed only the typical 2.5-kb mRNA transcript commonly reported in the literature. In spite of the marked differences in TGF-beta secretion between normal and transformed cells, their levels of TGF-beta 1 mRNA expression were similar. This suggests a change in the posttranscriptional regulation of TGF-beta 1 expression. TGF-beta 2 message was not detected in either normal or transformed RTE cells in culture. These findings are consistent with the hypothesis that the abnormal growth behavior of transformed RTE cells is at least in part due to disturbances of the TGF-beta system.

Low‐level β1 protein kinase C expression in cloned rat embryo fibroblast cells enhances transformation induced by the adenovirus type 5 E1A gene

Expression of the E1A gene of adenovirus type 5 (Ad5) in a cloned rat embryo fibroblast (CREF) cell line results in morphological transformation. The efficiency of E1A-mediated transformation of CREF cells is increased if a wild-type Ad5 E1A gene is cotransfected with a rat beta 1 protein kinase C (beta 1 PKC) gene. A direct demonstration of complementation between a functional-transforming Ad5 E1A gene and beta 1 PK in inducing transformation was demonstrated using Ad5 E1A cold-sensitive mutant (E1Acs) genes. The E1Acs gene enhanced transformation only at the transformation-permissive temperature of 37 degrees C and not at the nonpermissive transforming temperature of 32 degrees C. CREF cells constitutively expressing low levels of beta 1 PKC mRNA were transformed at a higher frequency than parental CREF cells after transfection with an Ad5 E1A gene or infection with wild-type Ad5 or the Ad5 host-range cold-sensitive mutant H5hr1. There was no enhancement of transformation in low-level beta 1 PKC-expressing CREF cells when cultures were grown continuously in the presence of the PKC-inhibitor 1-(5-isoquinolynsulfonyl)-2-methylpiperazine dihydrochloride. Transfected CREF cells expressing low levels of beta 1 PKC mRNA displayed CREF-like morphology and did not form colonies when grown in agar. In contrast, retroviral vector-transformed CREF cells expressing high levels of beta 1 PKC mRNA and beta 1 PKC enzyme activity were morphologically transformed and grew efficiently in agar. These findings indicate that the beta 1 PKC gene, when expressed at low levels, can cooperate with the Ad5 E1A gene in the initiation of viral oncogene-mediated transformation.