The effect of SV40 transformation on the chromosomal proteins of 3T3 mouse embryo fibroblasts.

The composition and metabolism of chromosomal proteins-histones and nonhistones chromosomal proteins-were examined in normal and SV40 transformed 3T3 mouse cells. Variations were observed, many of which were similar to those previously reported for normal and SV40 transformed W138 human diploid fibroblasts. The possible implications of these viral induced changes in the protein component of the genome for the phenotypic modifications which occur in transformed cells are discussed.     

Cells transformed by a wide variety of agents express higher abundance levels of some cellular RNA species

A cDNA-cloned library was prepared from mRNA synthesized by SV40-transformed mouse cells. Eleven cDNA clones were selected based on their ability to hybridize higher levels of mRNA in SV40-transformed 3T3 cells than in 3T3 cells. These cDNA clones were employed to screen the steady-state levels of cytoplasmic RNAs in a wide variety of viral (SV40, polyoma, adenovirus, and Rous sarcoma virus) and nonviral (methylcholanthrene, embryonal carcinoma) transformed cell lines. Two of the cDNA clones—A17 and 104—detected greater than 40–100-fold higher levels of mRNA in all the transformed cell lines tested when compared to nontransformed cells (3T3, C3HEF). The levels of mRNA complementary to these two cDNAs were regulated in a temperature-sensitive fashion (87–100-fold) in both SV40tsA- and RSV ts-src-transformed murine cell lines. These two cDNA clones detected greater than 100-fold, higher levels of complementary RNA derived from SV40 tumor tissue than in normal mouse liver. RNA species complementary to cDNA clones A17 or 104 were not detected in either actively growing nontransformed cells or in serum-stimulated 3T3 cells. The abundance levels of mRNAs detected by these two cDNA clones appear to be regulated 100-fold or greater by the transformed state, independent of the transforming agent. The higher levels of these RNA species detected in transformed mouse cells appear not to be solely regulated by the state of growth of nontransformed cells.     

The extensive homology between mRNA sequences of normal and SV40-transformed human fibroblasts.

The poly(A)-containing messenger RNA of normal diploid fibroblast and SV40-transformed progeny cells are compared by cross-hybridizing cDNA. We find a high degree of homology between the mRNA from normal and transformed cells. Despite imperfections in the procedure, the technique permits the conclusion that, at most, 3% of the mRNA in the transformed cell has sequences not present in the normal parental cell. Furthermore, much of the difference appears to occur in low and intermediate complexity classes of mRNA molecules. Extension homology in the mRNA sequences of disparate cell lines may be a general phenomenon, and even HeLa cell mRNA is nearly identical to that of diploid human fibroblasts.     

Manganese superoxide dismutase in normal and transformed human embryonic lung fibroblasts

The manganese superoxide dismutase (MnSOD) activity of W138 human embryonic lung fibroblasts and SV40-transformed WI38 cells was measured. Under various growth conditions, the transformed cells always had lower MnSOD activity than their normal cell counterparts. The activity of MnSOD changes greatly with the growth conditions in the WI38 cells, while the MnSOD activity in the tumor cells remained more constant. The amount of immunoreactive MnSOD was measured by Western blotting. In all cases studied, the amount of immunoreactive MnSOD was lower in the transformed cells than in the normal cells.     

SV40 large tumor antigen can regulate some cellular transcripts in a positive fashion

Eleven cDNA clones identified from a cDNA library prepared from the mRNA fraction of SV40 transformed cells detected, by hybridization, higher levels of cellular mRNA in SV40-transformed cells than in nontransformed cells. Three of these cDNA clones detected levels of cellular mRNA that were more than 100-fold greater in SV40tsA transformed cell lines grown at the permissive temperature than in those grown at the nonpermissive temperature. Northern blot hybridizations confirmed these results and in some cases detected RNA species of multiple sizes that were regulated in a temperature-dependent fashion in SV40tsA transformed cell lines. Infection of 3T3 cells with SV40 stimulated the levels of RNAs complementary to these cDNA clones. The results demonstrate that the SV40 large T antigen can regulate the steady state levels of some cellular RNA species.     

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts. II. Metabolism and binding of histones.

Composition, metabolism and extractability of histone fractions from WI38 human diploid fibroblasts and SV40 transformed WI38 fibroblasts are compared. Two alternate procedures were used for isolation of nuclei which allow for either optimal recovery of arginine-rich histones F3 (III) and F2a1 (IV) or for optimal retention of lysine-rich F1 (I) and slightly lysine rich F2b (II b2). While the relative amount of each histone fraction was found to be similar in normal and SV40 transformed cells, substantial increases in the levels of F 3 acetylation and F1 and F2a2 phosphorylation are reported for the histones of SV40 transformed cells. Differences in extractability of arginine-rich histones with 0.25 M HCl are also reported. While F 3 is extracted more rapidly than F 2a1 from nuclei of normal WI38 fibroblasts, the reverse is true in SV40 transformed WI38 cells. These differences are discussed in relation to modification reactions, binding of histones to DNA and SV40-induced alterations in gene readout.     

The relative amounts of the cytoplasmic RNA species in normal, transformed and senescent cultured cell lines.

We have examined the relative quantities of 18S and 28S rRNA, 4S RNA and poly (A) + mRNA in the following cultured cells: the mouse fibroblast lines 3T3 and 3T6 in the resting (contact inhibited) and growing (sparse) states, 3T3 clones transformed with SV40 (SV3T3) and with both SV40 and polyoma SV-Py 3T3), hamster lung fibrobalsts (v79), human cervical carcinoma cells (HeLa), and human diploid fibroblasts at early and late passage. The relative quantities of the RNA species were determined by labeling the cells to equilibrium with 32PO4 and measuring the amount of label in each RNA species. The ratio of mRNA to rRNA varied form 1.1% to 2.7% in the different cell lines, the more rapidly growing cell lines usually giving a higher ratio. In cells experiencing growth limitation either by contact inhibition or due to senescence, the ratio of mRNA to rRNA was about 30% lower than in the corresponding cells in the growing state. In most cell lines the ratio of 4S RNA to 18S rRNA was between 0.8 and 1.2, but in seescent fibroblasts, this ratio increased to greater than 1.7. Senescent fibroblasts also contained much more total RNA per unit of DNA than the same cells at early passage or than 3T6 or 3T3 cells.     

Down-regulation of T-STAR, a growth inhibitory protein, after SV40-mediated immortalization.

Normal human cells can undergo a limited number of divisions, whereas transformed cells may have an extended life span and can give rise to immortal cells. To isolate genes involved in the immortalization process, gene expression in SV40-transformed preimmortal human fibroblasts was compared with expression in SV40-transformed immortalized fibroblasts using an mRNA differential display. We found that the growth-inhibitory protein testis-signal transduction and activation of RNA (T-STAR) a homologue of cell-cycle regulator Sam68, is strongly down-regulated in immortalized cells. Overexpression of T-STAR in the SV40-transformed immortalized cells resulted in a strong reduction of colony formation, whereas deletion of the RNA-binding domain of T-STAR abrogated this effect. Down-regulation of testis-signal transduction and activation of RNA (T-STAR) expression is found only in immortal cells isolated after a proliferative crisis accompanied with massive cell death. The strict correlation of down-regulation of T-STAR expression only in those immortal cells that arose after a clear proliferative crisis suggests that the loss of T-STAR might be necessary to bypass crisis.     

Lowered level of translatable messenger RNAs for manganese superoxide dismutase in human fibroblasts transformed by SV 40

The activity of manganese superoxide dismutase (MnSOD) revealed by specific staining after gel electrophoresis of cell extracts, is decreased in human fibroblasts transformed by SV40. The decrease in enzyme activity is attributable to decreased amount of enzyme protein as determined by radial immunodiffusion. Total fibroblast RNAs were translated in the presence of (35S) methionine in a cell-free translation system and the neo synthesized proteins submitted to immunoprecipitation with an anti MnSOD antiserum. Gel electrophoresis of the immunoprecipitated material followed by fluorography shows that MnSOD is translated as a peptide which is 2000 daltons larger than the mature enzyme subunit. This precursor (pre-MnSOD) is processed in vitro to mature MnSOD by the action of an isolated mitochondrial preparation. Levels of translatable MnSOD mRNA in normal and SV 40 transformed cells were compared in terms of the radioactivities incorporated into pre MnSOD bands. The results indicate that the decreased amount of MnSOD in SV 40 transformed fibroblasts is due to a decreased level of translatable mRNA for MnSOD.     

The cooperative role of the transformation-sensitive glycoproteins, GP140 and fibronectin, in cell attachment and spreading

The detergent-insoluble matrix of cultured human fibroblasts contains cytoskeletal and nuclear components, as well as two major, noncollagenous glycoproteins, fibronectin and GP140. These glycoproteins are stabilized by extensive intermolecular disulfide bonding. GP140, in contrast to fibronectin, is resistant to digestion with trypsin and is not cross-reactive with antisera prepared against fibronectin (Carter, W. G., and Hakomori, S. (1981) J. Biol. Chem. 256, 6953-6960). GP140 was partially purified, under nonreducing conditions, by differential extraction of trypsinized cells with sodium trichloroacetate. Alternatively, a higher yield of GP140 could be obtained under reducing conditions by extraction with urea-dithiothreitol followed by molecular sieve chromatography in the presence of sodium dodecyl sulfate and dithiothreitol. The purified GP140 contained mannose, galactose, and N-acetylglucosamine residues, totaling 2.7% of the molecule. In addition, mild periodate oxidation of GP140 followed by reduction with NaB3H4 under conditions designed to label sialic acid also labeled the peptide portion of the molecule. Amino acid analysis of GP140 detected periodate-sensitive hydroxylysine residues, as well as hydroxylproline, accounting for the periodate/NaB3H4-induced label in the peptide. These hydroxylated amino acids are major components of collagens and collagen-like proteins. The GP140 isolated under nonreducing conditions was found to induce stable cell attachment and cell spreading when coated on plastic surfaces. The cell attachment could not be inhibited with affinity purified anti-fibronectin antibodies. However, trypsinization of cells under conditions that removed surface fibronectin reduced the ability of the cells to bind to the GP140-coated surface. Metabolic labeling of cells with radioactive glucosamine during 1-h cell attachment experiments incorporated label into both fibronectin and GP140, as well as four other carbohydrate-containing components as part of a stable detergent-insoluble matrix, indicating that the cells rapidly glycosylate both fibronectin and GP140 and incorporate them into the matrix. Long term labeling and chase experiments indicated that fibronectin and GP140 in the matrix are subject to very slow turnover. Neither fibronectin nor GP140 were detectable in the detergent-insoluble matrix of SV40-transformed human fibroblasts by either metabolic or cell surface labeling. These results are consistent with the conclusion that fibronectin and GP140 may function in a cooperative manner in cell adhesion and spreading.     

Comparative study of the expression of Rb and p53 genes in human colorectal cancers,colon carcinoma cell lines and synchronized human fibroblasts

We have compared the expression of the retinoblastoma (Rb) and p53 genes in normal human fibroblasts, colon carcinoma cell lines, matched pairs of colorectal tumor tissues and adjacent normal mucosa and in synchronized human diploid fibroblast cell line W138. The increased expression of Rb and p53 RNA was observed in a majority of colorectal cancers in comparison to adjacent normal mucosa and is accompanied by proportional increase in the expression of histone H3 gene. The Rb and p53 RNA levels varied significantly between the various colon carcinoma cell lines. However, we found that the expression of Rb and p53 RNA is regulated differently in cell cycle synchronized normal human fibroblasts. The Rb mRNA level did not change with the position in the cell cycle and did not differ significantly whether the cells were serum deprived or in 10% serum. But p53 mRNA expression follows the same pattern as histone H3 mRNA.     

Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line

A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori-, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori--transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori--transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori- in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells.     

Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene

Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed Wl38 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and Wl38 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and Wl38 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed Wl38 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylat ed RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.     

High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts.

Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells.     

An immortalized xeroderma pigmentosum, group C, cell line which replicates SV40 shuttle vectors

We have established and characterized an immortalized xeroderma pigmentosum (XP), group C, cell line. Transformation of the human fibroblasts was carried out with a recombinant plasmid, pLAS-wt, containing SV40 DNA encompassing the entire early region with a defective origin of DNA replication. The transformed XP cell line, XP4PA-SVwt, and the normal transformed fibroblasts AS3-SVwt, both express SV40 T antigen together with enhanced levels of the transformation-associated cellular protein, p53. XP4PA-SVwt retains the XP UV-repair defective phenotype as demonstrated by low levels of unscheduled DNA synthesis and by the reduced survival of irradiated SV40 virus. Analysis of cellular DNA shows a single major, stable, integration site of pLAS-wt in the XP4PA-SVwt cells. The T antigen in these cells supports efficiently the replication of SV40 based shuttle vectors and should prove suitable for the introduction, expression and selection of genes related to DNA repair and to the study of mutagenesis using defined molecular probes.