Adenovirus E1b-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells

The adenovirus E1b-58kd tumor antigen has been detected in a physical association with a 54 kilodalton cellular protein in adenovirus-transformed mouse cells. Antibody specific for the E1b-58kd protein coimmunoprecipitates a 54 kd protein from transformed, but not from productively infected, cells. Monoclonal antibody specific for the cellular 54 kd protein coimmunoprecipitates the adenovirus E1b-58kd protein from transformed cell extracts. The same or closely related cellular 54 kd protein, associated with the adenovirus E1b-58kd protein, was present in the SV40 large T antigen-54 kd complex previously detected in SV40-transformed mouse cells. The identity of the 54 kd protein is based on the immunological specificities of the anti-54 kd monoclonal antibodies and partial peptide maps of the 54 kd protein associated with the adenovirus and SV40 tumor antigens. The adenovirus E1b-58kd-54 kd complex, like the SV40 large T antigen-54 kd complex, is heterogeneous in size or mass. While all of the cellular 54 kd protein in the adenovirus-transformed cell extract is found in a complex with the E1b-58kd protein, some of the viral 58 kd antigen is detected in a form not associated with the 54 kd protein. The fact that the adenovirus and Sv40 tumor antigens, both required for transformation, can be found in physical association with the same cellular protein in a transformed cell is a good indication that these two diverse viral proteins share some common mechanisms or functions.     

Changes in gene expression and protein phosphorylation in murine cells, transformed or abortively infected with wild type and mutant simian virus 40

The mechanism of SV40-induced cellular transformation was investigated by two-dimensional gel analysis of 35S- and 32P-labeled proteins of various cells. These included rat and mouse cells, either transformed or abortively infected by SV40 wild type, small t deletion mutants, and a large T temperature-sensitive mutant. Synthesis, turnover, or (de)phosphorylation of multiple protein spots was found to be reproducibly and quantitatively influenced by the transformed and/or infected status. Several of these alterations were attributable to the biological activity of either large T or small t antigen. Most changes in 35S-labeled proteins corresponded to a decreased intensity of the gel spots in transformed cells, while hyperphosphorylated proteins were more common than hypophosphorylated ones. About half of the polypeptide alterations in 35S-and 32P-labeled SV40-transformed rat cells, including a set of 35S-labeled small t-dependent changes were shared by Rous sarcoma virus-transformed cells. In contrast, small t-dependent (de)phosphorylation was rarely detected. Phosphoamino acid analysis of selected phosphoprotein spots of rat cells and alkaline hydrolysis of whole two-dimensional gels did not reveal any evidence for increased tyrosine-specific phosphorylation after SV40-induced transformation. Abortively infected mouse cells showed many protein alterations, also observed in stably transformed cells. However, the latter cells contained additional changes, also affecting several phosphoproteins and possibly related to the establishment of transformation. These findings are discussed in relation to the biological functions, known or presumed, for SV40 large T and small t antigens during transformation.     

Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells

Using a newly developed method for microfilament isolation (Matsumura, F., Yamashiro-Matsumura, S. and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644), we have analyzed protein composition of microfilaments in "normal" and transformed rat tissue culture cells. They include REF-52 (an established rat embryo cell line) cells, REF-52 transformed by DNA viruses (SV40 or adenovirus type 5), normal rat kidney cells, and normal rat kidney cells transformed by RNA viruses (Kirsten or Rous sarcoma virus). Microfilaments from normal rat culture cells contain three major tropomyosins (apparent Mr = 40,000, 36,500, and 32,400) and two relatively minor tropomyosins (apparent Mr = 35,000 and 32,000). In transformed cells the levels of one or two of the major tropomyosins (Mr = 40,000 and 36,500) are decreased and the levels of one or both of the minor tropomyosins (Mr = 35,000 and 32,000) are increased. These changes in tropomyosin patterns were also observed in temperature shift experiments with rat-1 cells transformed with a Rous sarcoma virus mutant, temperature-sensitive for transformation. Cell-free translation of whole cell mRNA generated similar tropomyosin patterns on two-dimensional gels, suggesting that changes in the pattern of tropomyosin expression were largely effected at the level of RNA rather than by post-translational modification. Such changes in the tropomyosin composition of microfilaments were consistently found to accompany the various morphological alterations associated with transformation. We suggest that alterations in the pattern of tropomyosin expression are involved in, or cause, rearrangement of stress fibers and that this may be responsible (in part) for morphological transformation.     

Cellular mutation mediates T-antigen-positive revertant cells resistant to simian virus 40 transformation but not to retransformation by polyomavirus and adenovirus type 2.

T-antigen-positive transformation revertant cell lines were isolated from fully simian virus 40 (SV40)-transformed Fisher rat embryo fibroblast cells (REF 52 cells) by methionine starvation. Reversion of the transformed cells (SV-52 cells) was caused by a mutation within the cellular genome. To demonstrate this, we isolated SV40 DNA from the host genome, inserted it into plasmid pSPT18 DNA, cloned it in Escherichia coli, and microinjected it into the nuclei of the REF 52 cells. Fully transformed cells were obtained with the same efficiency (20 to 25%) as after microinjection of wild-type SV40 DNA I. Furthermore, the revertant cells were resistant to retransformation by SV40. Following microinjection of wild-type SV40 DNA I, 42 independent cell lines were isolated. Cells of all analyzed lines acquired additional SV40 DNA copies, but changes in the cell morphology or growth characteristic were not demonstrable. However, the revertants were retransformable with a high efficiency after polyomavirus and adenovirus type 2 infections or microinjection. Also, fusion of the revertant cells with the grandparental REF 52 cells led to restoration of the transformed state.     

Sodium-n-butyrate induces secretion and substrate accumulation of p52 in Kirsten sarcoma virus-transformed rat kidney fibroblasts

1. A group of five transformation-responsive secreted proteins, ranging in molecular mass from 31 to 70 kDa, were identified in cultured normal rat kidney (NRK) fibroblasts. 2. One such protein (p52) was found to be a major secreted and substrate-attached component of NRK cells. 3. Kirsten sarcoma virus-transformed NRK cells failed to accumulate p52 in either the secreted or substrate-associated protein compartments; this protein was inducible, however, in transformed cells by culture in 2 mM sodium-n-butyrate. 4. Kinetics of p52 induction in transformed NRK cells, relative to the time course of increased cell spreading, and its enrichment in the substrate-associated protein fraction suggest that p52 might function in cell-substrate attachment.     

Biochemical localization of the transformation-sensitive 52 kDa (p52) protein to the substratum contact regions of cultured rat fibroblasts. Butyrate induction, characterization, and quantification of p52 in v-ras transformed cells.

A 52 kDa protein (p52) was identified, using differential extraction and electrophoretic criteria, as a major extracellular and substrate-associated component of normal rat kidney (NRK) fibroblasts. Cells transformed with Kirsten murine sarcoma virus (KNRK cells) did not express p52 constitutively, but were inducible for both p52 production and its substrate association during culture in sodium butyrate (NaB)-supplemented growth medium. Comparative analysis of the relative molecular mass, subcellular distribution, and isoelectric complexity (five variants ranging in pI from 5.4 to 6.2) of the 52 kDa species constitutively and inducibly expressed by NRK and KNRK/NaB cells respectively, indicated that they were, indeed, the same protein. p52 selectively localized to cellular fractions enriched in substrate focal contact sites and associated ventral undersurface components. NaB induction of p52 in KNRK cells occurred before cell spreading; other polar compounds, such as dimethyl sulphoxide, which did not induce KNRK cell spreading, similarly failed to elicit p52 production. p52 accumulated more rapidly in (and was quickly released from) the focal-contact-enriched protein fraction of NRK cells compared with its time course of appearance in the medium. These data collectively suggest that p52 is one of a relatively small number of proteins the synthesis of which is either involved in determination of cell shape or regulated as a consequence of cell-shape changes.     

Transformed cells overexpress a cytosolic nucleic acid binding protein

A protein blotting technique was used to identify a 57,000 dalton cytosolic nucleic-acid-binding protein found in neoplastically transformed cell lines. Specifically, greater amounts of this protein were found in Kirsten Murine Sarcoma Virus-, Simian Virus 40-, and methylcholanthrene-transformed Balb 3T3 cells than in comparable untransformed cells. An analogous protein was identified in other transformed mammalian cells. Increased levels of the DNA binding protein in sarcoma virus transformants were shown to be dependent on the continued maintenance of the transformed phenotype. The properties of this protein are compared to those of other previously reported nucleic acid binding proteins.     

Levels of synthesis of primate-specific nuclear proteins differ between growth-arrested and proliferating cells

A monoclonal antibody that reacts specifically with the proliferation-sensitive nuclear proteins, isoelectric focusing (IEF) 8Z30 and 8Z31 (molecular weight (MW), 76,000 charge variants, HeLa protein catalogue number) has been characterized. As determined by indirect immunofluorescence, the antibody stains the nucleolus and nucleoplasm of interphase-cultured cells of primate origin, but does not react with cells of other species. Proteins having similar MWs and isoelectric points as the human or monkey (primates) proteins were not observed in cultured cells of the following species: aves, bat, dog, dolphin, goat, hamster, mink, mouse, pisces, potoroo, rabbit and rat. Quantitative two-dimensional (2D) gel electrophoretic analysis of [35S]methionine-labeled proteins synthesized by normal (quiescent, proliferating) and SV40-transformed human MRC-5 fibroblasts revealed significant differences in the levels of synthesis of both IEF 8Z30 and 8Z31. In quiescent cells the main labelled product corresponded to IEF 8Z31 (ratio IEF 8Z31/8Z30, 2.3), while in the transformed cells the major product was IEF 8Z30 (ratio, 0.62). Normal proliferating fibroblasts exhibited similar levels of both proteins (ratio, 1.21). Combined levels of synthesis of both proteins were 1.50 and 1.20 times as high in the transformed cells as in the quiescent and proliferating cells, respectively. Similar results were observed in other pairs of normal and transformed human cells, such as WI38/WI38 SV40 and amnion/AMA. Modulation of the levels of synthesis of these proteins may play a role in cell proliferation.     

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 (microgram/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.     

Growth factor requirements of normal and polyomavirus middle T gene transformed REF52 cells in serum-free medium: indications of a reduced vasopressin requirement and its relationship to the control of phosphatidylinositol metabolism

The growth factor requirement of normal and polyomavirus middle T gene transformed REF52 cells was studied in serum-free medium in an attempt to elucidate the possible linkage between an altered growth factor requirement and one or more altered physiological properties of the transformed cells. For optimal growth, REF52 cells required vasopressin, epidermal growth factor (EGF), high-density lipoprotein (HDL), hydrocortisone, insulin, transferrin, and fibronectin. Deletion of vasopressin or hydrocortisone from the medium resulted in a 50 to 60% reduction in cell growth and the deletion of HDL, transferrin, or the combination of EGF and vasopressin led to an 80 to 90% growth retardation. The same medium supported the growth of the transformed variant (PyMLV-REF52) at a rate comparable to that of 10% serum, and deletion of hydrocortisone, vasopressin, or the combination of EGF and vasopressin had virtually no effect on PyMLV-REF52 cell growth. In vasopressin-deleted medium, vasopressin elicited a rapid increase of intracellular inositol phosphate levels in REF52 cells and the control of phosphoinositide turnover was strictly regulated. In contrast, both cell proliferation and intracellular inositol phosphate levels of PyMLV-REF52 cells were not affected by vasopressin treatment under identical culture conditions, and control of phosphoinositide metabolism was lost. Thus, a correlation may exist between the trigger of a mitogenic signal and the stimulation of the phosphoinositol pathway by vasopressin in REF52 cells and this relationship was disrupted in PyMLV-REF52 cells.     

Characterization of p51/52, a cell-growth regulated protein of WI-38 cells.

A number of proteins have been identified whose expression or activity is regulated by cell growth. We have produced a monoclonal antibody against a new cell-growth regulated protein found in normal human fibroblasts. We have shown that this antibody recognizes a 51/52-kDa doublet (p51/52) found mainly in normal cells. This doublet is sensitive to degradation by the calcium-activated protease, calpain, breaking down to a 37/38-kDa doublet. The relative amount of the two members of the 51/52-kDa doublet changes when serum-starved cells reenter the cell cycle. Quiescent cells express mainly the 51-kDa form; the 52-kDa form becomes more abundant upon refeeding serum-starved cells. Transformed cells express either very small amounts of this doublet, and then predominantly the 52-kDa form, or no detectable amount of either form. These characteristics distinguish this molecule from several other known growth-regulated proteins such as statin and the anti-oncogene p53.     

The expression of heat shock protein hsp27 and a complexed 22-kilodalton protein is inversely correlated with oncogenicity of adenovirus-transformed cells.

We isolated a monoclonal antibody that immunoprecipitated two proteins of 22 and 27 kilodaltons (kDa) from nononcogenic adenovirus type 5 early region 1 (E1)-transformed rat cells but not from oncogenic adenovirus type 12 E1-transformed rat cells. In a variety of adenovirus-transformed cells including cells transformed by E1A and the c-H-ras oncogene, we found a perfect, inverse correlation between the presence of these two proteins and the oncogenicity of these cells in syngeneic immunocompetent rats. Characterization of the two proteins revealed that they occur in a large (700-kDa) complex and that the 27-kDa protein is identical to the already known 27-kDa (28-kDa) heat shock protein hsp27. The suppression of the hsp27 protein in oncogenic cells is further demonstrated by the fact that its mRNA is absent even after heat-shock induction.     

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 QUEST system for quantitative analysis of two-dimensional gels

The strategies and methods used by the QUEST system for two-dimensional gel analysis are described, and the performance of the system is evaluated. Radiolabeled proteins, resolved on two-dimensional gels and detected using calibrated exposures to film, are quantified in units of disintegrations per minute or as a fraction of the total protein radioactivity applied to the gel. Spot quantitation and resolution of overlapping spots is performed by two-dimensional gaussian fitting. Pattern matching is carried out for groups of gels called matchsets, and within each matchset every gel is matched to every other gel. During the matching process, spots are automatically added to each pattern at positions where unmatched spots were detected in other patterns. This results in enhanced accuracy for both spot detection and for matching. The spot fitting procedure is repeated after matching. Tests show that up to 97% of spots in each pattern can be matched and that fewer than 1% of the spots are matched inconsistently. Approximately 2000 proteins are detected from typical gels. Of these 1600 are high quality spots. Tests to measure the coefficient of variation of spot quantitation versus spot quality show that the average coefficient of variation for high quality spots is 21%. The intensities of the detected proteins range from 4 to 20,000 ppm of total protein synthesis. The QUEST analysis system has been used to build a quantitative database for the proteins of normal and transformed REF52 cells, as presented in the accompanying reports (Garrels, J., and Franza, B. R., Jr. (1989) J. Biol. Chem. 264, 5283-5298, 5299-5312).     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

Altered low density lipoprotein receptor regulation is associated with cholesteryl ester accumulation in simian virus 40 transformed rodent fibroblast cell lines

Summary Several SV40 transformed REF52 cell lines were found to accumulate 5 to 10 times more cholesteryl esters compared to their parent line REF52 when cultured in 10% serum. Under this culture condition, the cholesteryl ester to phospholipid ratio was 0.4∶1 and 2.0∶1 to 3.8∶1 for normal and SV40 tranformed cells, respectively. The mechanism underlying cholesteryl ester accumulation in SV40 transformed lines was investigated. We found that 1) the rate of thede novo cholesterol and cholesteryl ester synthesis was roughly equal in normal and transformed derivatives; 2) the accumulation of cholesteryl esters in the transformants would not occur when cultured in lipoprotein-deficient medium and reappeared upon their return to low density lipoprotein-containing medium; 3) the transformants expressed twice as many low density lipoprotein receptors and were less sensitive to LDL-induced receptor down regulation compared to their nontransformed counterparts. The results indicate that SV40 transformed lines exhibited an accelerated lipid uptake from the culture medium due to an altered regulation of low density lipoprotein receptor activity. Supported by NCI grant CA 38016 Editor's Statement This article reports alteration of cholesterol metabolism in rat cells by viral transformation and provides an explanation for the phenomenon at a molecular level. The results may be widely applicable to other instances of viral transformation or virus infection.