Pre-crisis mouse cells show strain-specific covariation in the amount of 54-kilodalton phosphoprotein and in susceptibility to transformation by simian virus 40.

We have used several inbred mouse strains to examine the role of the 54-kilodalton (kDa) cellular phosphoprotein in transformation by the papovavirus simian virus 40. We have measured the endogenous 54-kDa phosphoprotein in cells obtained from these inbred mouse strains. To study the effect of passage, cell cultures were measured for amount of the 54-kDa phosphoprotein at the 2nd and 12th passages. In the absence of any transforming agent, the amount of endogenous 54-kDa phosphoprotein in early pre-crisis mouse cells varied in a strain-specific way. Transformation frequency varied coordinately with endogenous 54-kDa expression. Mouse strains whose cells produced a high level of endogenous 54-kDa phosphoprotein on passage did not further increase its expression after simian virus 40 transformation.     

Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.

To facilitate understanding of the mechanisms underlying pulmonary diseases, including lung cancer and cystic fibrosis, we have transformed and characterized cultures of human tracheal epithelial cells. Cells were transfected by calcium phosphate precipitation with a plasmid containing a replication-defective simian virus 40 (SV40) genome. Colonies of cells with enhanced growth potential were isolated and analyzed for transformation- and epithelial-specific characteristics. Precrisis cells were observed to express the SV40 large tumor antigen, produce cytokeratins, have microvilli, and form tight junctions. After crisis, cells continued to express the SV40 large tumor antigen as well as epithelial-specific cytokeratins and to display the apical membrane microvilli. Apical membrane Cl channels were opened in postcrisis cells exposed to 50 microM forskolin. These channels showed electrical properties similar to those observed in primary cultures. The postcrisis cells have been in culture for greater than 250 generations and are potentially "immortal." In addition to providing a useful in vitro model for the study of ion transport by human airway epithelial cells, the cells can be used to examine stages of neoplastic progression.     

Establishment and characterization of rat pancreatic beta cell lines transformed by simian virus 40

Rat pancreatic beta cells were transformed by simian virus 40 (SV40). The established beta cell lines expressed tumor antigen specific to SV40 and retained the ability to produce immunoreactive insulin (IRI) even at a high passage level. One of 12 transformed beta cell lines, SV-PB1205, was further investigated in vitro and in vivo. Firstly, effects of drugs on replication of the pancreatic beta cells were examined in in vitro experiments. Various drugs increased the uptake of 3H-thymidine into the cells. Those included D-glucose, tetragastrin and secretin. However, such effect was not observed for glucagon and growth hormone. Secondly, the SV40-transformed pancreatic beta cells were transplanted into diabetic rats. This produced such improvement of plasma glucose level at least for 2 weeks. The Significance of those experiments was discussed.     

Establishment and characterization of rat pancreatic beta cell lines transformed by simian virus 40

Rat pancreatic beta cells were transformed by simian virus 40 (SV40). The established beta cell lines expressed tumor antigen specific to SV40 and retained the ability to produce immunoreactive insulin (IRI) even at a high passage level. One of 12 transformed beta cell lines, SV-PB1205, was further investigatedin vitro andin vivo. Firstly, effects of drugs on replication of the pancreatic beta cells were examined inin vitro experiments. Various drugs increased the uptake of³H-thymidine into the cells. Those included D-glucose, tetragastrin and secretin. However, such effect was not observed for glucagon and growth hormone. Secondly, the SV40-transformed pancreatic beta cells were transplanted into diabetic rats. This produced such improvement of plasma glucose level at least for 2 weeks. The Significance of those experiments was discussed.     

Instability of integrated viral DNA in mouse cells transformed by simian virus 40

The state and organization of simian virus 40 (SV40) DNA in tsA mutant-transformed mouse clones were examined early after agar selection in an attempt to elucidate the mechanisms that actively generate the diverse integration patterns found in transformed cells. Although recently selected as a cloned population from agar, A21 cells displayed extremely heterogeneous SV40 DNA patterns when analyzed by agarose gel electrophoresis and Southern blot hybridization. Reselection of clones in agar from A21 at 33 degrees C or 39.5 degrees C and DNA analysis by hybridization demonstrated (i) simplification of the number of integration sites in the new clones; (ii) new sites of integrated SV40 DNA in high molecular weight cell DNA fragments generated by digestion with restriction endonuclease Bgl II; (iii) relatedness between clones with respect to integrated viral sequence arrangement; and (iv) persistence of free viral DNA forms. The majority of free viral DNA appeared to be full length, nondefective SV40 DNA, although a subpopulation of defective viral molecules was also detected. No detectable free SV40 DNA could be observed in A21 clonal derivatives isolated by growth in agar at 39.5 degrees C, indicating that the persistence of free viral forms was regulated by the A gene. These results suggest that the heterogeneity in viral sequences in the A21 cells was generated within a cloned population from which new clones can be derived with different transformed phenotypes and integration patterns.     

Resting state in normal and simian virus 40 transformed Chinese hamster lung cells.

Normal cell deprived of amino acids or serum factors enter a resting state, whereas cells transformed by wild-type simian virus 40 do not. The ability to enter a resting state is temperature-sensitive (ts) in cells transformed by a tsA mutant of simian virus 40. We shown further: (i) that when complete medium is added to resting cells, the length of time until the onset of DNA synthesis often exceeds the length of G1 in growing cells; (ii) that the length of this interval depends upon the conditions used to arrest cell growth; but (iii) that transferring cultures from medium depleted for one factor to medium depleted in a second factor never leads to a round of DNA synthesis; and (iv) that DNA synthesis does not resume rapidly when a resting culture of cells transformed by the tsA mutant is transferred to the permissive temperature in suboptimal medium. A model proposing that in suboptimal conditions cells leave the cell cycle and traverse a branch pathway to enter the resting state is consistent with these findings.     

Alterations in surface glycoproteins and level of sialyltransferase of cells transformed by a temperature-sensitive mutant of simian virus 40.

Mouse cells transformed by a temperature-sensitive mutant of simian virus 40 belonging to complementation group A lost their ability to regulate cell growth when grown at the permissive temperature (35 degrees) but showed the low saturation density of cell growth at the restrictive temperature (39.5 degrees) that is characteristic of normal cells in vitro. Biochemical analysis of the membranes of cells grown under the restrictive and the permissive conditions demonstrated no qualitative temperature-dependent differences either in neutral glycolipids or in acidic glycolipids of the cells. Plasma membrane glycoproteins labeled with radioactive glucosamine showed significantly different patterns on both polyacrylamide gel electrophoresis and electrofocusing. When the levels of glycoprotein glycosyltransferases of the cells were examined, the level of sialyltransferase (CMP-N-acetylneuraminytransferase,EC 2.4.99.1) of the cells grown at the restrictive temperature was low compared with that of cells grown at the permissive temperature. Our results indicate that the level of sialyltransferase is under the control of the gene A function of simian virus 40 and consequently is related to alterations in the cell surface glycoproteins.     

Thermolabile T (tumor) antigen from cells transformed by a temperature-sensitive mutant of simian virus 40.

Partially purified tumor (T) antigen from a strain of Chinese hamster lung cells transformed by wild-type simian virus 40 (SV 40) and either of two temperature-sensitive SV 40 mutants has been studied as a DNA binding protein. The DNA binding activity present in the T-antigen-containing fractions is inhibited by purified hamster anti-T IgG but not by equivalent amounts of nonimmune hamster IgG. T from either wild-type- or tsC219-transformed cells is relatively stable during heating at 44 degrees compared to T prepared from tsA239-transformed cells. These results strongly suggest that T is a product of the SV 40 A gene.     

Failure of human cells transformed by simian virus 40 to form tumors in athymic nude mice.

Four individual lines and one subline of human cells, permanently established in tissue culture after infection with simian virus 40, failed to form tumors when inoculated into athymic nude mice. Under identical conditions, three established human cell lines of neoplastic origin and a spontaneously established human lymphocyte line formed tumors. Nude mice that failed to grow tumors from inocula of simian virus 40-transformed human cells, grew tumors from subsequent injections of authentic human cancer cells. Further efforts to demonstrate an immunologic basis for the growth suppression of human simian virus 40 transformants were also negative. The data suggest that the changes in morphology and in vitro growth behavior induced by the viral information are not sufficient for, or are only coincidentally related to, the neoplastic state.     

Monomer molecular weight of T antigen from simian virus 40-infected and transformed cells.

T-antigens from simian virus 40 (SV 40)-transformed and lytically infected cells have been isolated by immunoprecipitation and their molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T-antigen from SV40-transformed mouse and hamster cells has an apparent molecular weight of 94,000 whereas that from several lines of SV40-infected monkey cells is 84,000. In a wheat germ cell-free system, mRNA from either transformed or productively infected cells is translated into a 94,000 species. Experiments with the protease inhibitors L-l-(tosylamide-2-phenyl)ethylchloromethyl ketone HCl and N-alpha-p-tosyl-L-lysylchloromethyl ketone HCl suggest that the 84,000 species of T-antigen found in infected cells is derived from the larger species by proteolytic cleavage. Further, the cleavage pathway probably involves a two-step reaction with an 89,000 intermediate. The biological significance of the two molecular weight forms of T-antigen is unknown, but the possibility that they have different physiological activities is discussed.     

Antigenic distinctions of glycoproteins in plasma and mitochondrial membranes of lymphoid cells neoplastically transformed by simian virus 40.

Highly purified plasma membranes from hamster lymphocytes transformed by simian virus 40 (GD 248) were compared with the membranes of normal cells by crossed immune electrophoresis, crossed-line immune electrophoresis, and bidimensional isoelectric focusing-immune electrophoresis. Antiserum raised by inoculation of guinea pigs with GD 248 membranes was used as serologic reagent, either directly or after absorption with membranes from normal cells. Bidimensional immune electrophoresis reveals the presence in the plasma membranes of GD 248 cells of at least three antigens not detectable in the membranes from the normal cell population. At least two of these are also present in the mitochondrial membranes of GD 248 cells, but none could be detected in membranes of embryonic fibroblasts. Bidimensional isoelectric focusing-immune electrophoresis indicates that the distinctive antigens of the GD 248 membranes are glycoproteins.     

Purification of simian virus 40 tumor antigen from a line of simian virus 40-transformed human cells.

Simian virus 40 tumor antigen can be isolated in a highly purified state from the nuclei ofSV80 cells, a continuous line of simian virus 40-transformed human fibroblasts. A five-step purification method was used. Its apparent molecular weight (in sodium dodecyl sulfate/polyacrylamide gels) is approximately 90,000-94,000. It contains a detectable amino-terminal residue.     

Expression of simian virus 40 early genes in transformed rat cells is correlated with maintenance of the transformed phenotype.

Early viral polypeptides synthesized in simian virus 40 rat transformants were identified by immunoprecipitation using anti-T (tumor) antigen immune serum. Four polypeptide classes could be identified, which were not detectable in extracts of nontransformed cells and were not precipitated from transformed cell extracts by nonimmune serum. Their apparent M(r) were 92,000, 63,000, 56,000, and 19,000. A similar pattern was observed in extracts from lytically infected cells, but the relative rate of radioactive labeling of the M(r) 63,000 and 56,000 species was in this case significantly lower than in transformed cells. In tsA30 transformants of type A, which maintain the transformed phenotype at high temperature, only minor quantitative variations of this pattern were observed when the cultures were shifted from 33 degrees to 40.5 degrees . In contrast, the rate of labeling of the four virus-specific polypeptides was decreased by 90% or more at high temperature in the temperature-sensitive N transformants. In all cases, a coordinated variation of the radioactivity associated with the different polypeptide classes was observed. These results suggest that the synthesis or processing, or both, of the viral early proteins may be controlled by different mechanisms in various types of simian virus 40 transformants and, furthermore, that it may be under the positive control of a virus-coded protein in transformed cells of type N.     

Assignment of the integration site for simian virus 40 to chromosome 17 in GM54VA, a human cell line transformed by simian virus 40.

GM54VA human cells transformed by simian virus 40 (SV40) were fused with peritoneal macrophages obtained from three different mouse strains. All 27 hybrid clones studied were positive for SV40 tumor antigen in 100% of their cells and contained human chromosome 17. Human chromosome 17 was the only human chromosome present in five of the hybrid clones. Fusion of GM54VA cells and either thymidine kinase (EC 2.7.1.75)-deficient mouse or Chinese hamster fibroblasts resulted in the growth in hypoxanthine-aminopterin-thymidine medium of hybrid clones positive and negative for SV40 tumor antigen. Counterselection of the hybrid clones positive for tumor antigen in medium containing 5-bromodeoxyuridine resulted in the growth of hybrid cells that were negative for tumor antigen. These experiments indicate that negative for tumor antigen. These experiments indicate that SV40 is integrated in only one of the two parental human chromosomes 17. Because the genome of SV40 has been assigned to human chromosome 7 in two other SV40-transformed human cell lines, at least two different integration sites for SV40 would seem to be present in human cells: one located in human chromosome 7 and the other located in human chromosome 17.     

Fragments of the simian virus 40 transforming gene facilitate transformation of rat embryo cells.

Segments of the simian virus 40 (SV40) genome that encode only fragments of large tumor antigen can facilitate immortalization of secondary rat embryo cells. The phenotypes of the immortalized cells range from nearly "normal" to fully transformed. All of the cell lines contain SV40 sequences and express unstable NH2-terminal fragments of large tumor antigen. SV40 small tumor antigen does not appear to be essential for either immortalization or transformation.     

Carcinogen-mediated amplification of viral DNA sequences in simian virus 40-transformed Chinese hamster embryo cells.

Exposure of simian virus 40 (SV40)-transformed Chinese hamster embryo cells to various chemical and physical carcinogens induced SV40 DNA synthesis. Although the carcinogen-mediated amplification of SV40 DNA is regulated by the viral A gene, the induction of viral DNA synthesis does not result in the rescue of infectious virus or the formation of complete viral DNA molecules. Instead, a heterogeneous collection of DNA molecules containing SV40 sequences was generated by treatment with 7,12-dimethylbenz[a]anthracene. Restriction enzyme analysis of the amplified DNA molecules in the Hirt supernatant showed that not all sequences in the integrated SV40 inserts are present. The possibility that amplification of SV40 sequences is a reflection of a general-gene-amplification phenomenon mediated by carcinogens is discussed.     

Appearance of smaller mannosyl-glycopeptides on the surface of a human cell transformed by simian virus 40.

When fucosyl surface glycopeptides from growing normal human cells (WI 38) were compared with those derived from nongrowing cells the former were enriched in high-molecular-weight species. However, a line of human cells (WI 18Va) transformed by simian virus 40 appeared to have fucosyl-glycopeptides similar in size distribution to those from rapidly growing non-transformed cells (WI 38). I propose that the enrichment in high molecular weight species in these cells might be growth- rather than transformation-dependent. Using radioactive mannose to label surface glycopeptides, I observed that those derived from transformed cells (WI 18Va) were smaller than those from rapidly growing normal cells. Thus differences in size distribution may not be adequate criteria to evaluate the growth-dependent alterations in cell surface glycopeptides.