Phenotypic transformation of the host cell enhances polyoma pseudovirion formation.

Phenotypic transformation of the host cell affected the formation of polyoma pseuodovirions. Polyoma virus infection of various transformed derivatives of mouse 3T3 cells resulted in the formation of predominantly pseudovirions, whereas infection of mouse 3T3 cells produced mainly polyoma virus. The effect that transformation of the host cell had on polyoma pseudovirus formation was further demonstrated by using phenotypic revertants isolated from some of the transformed cell lines. The revertants were characterized by their morphology, saturation densities, and colony-forming ability in methylcellulose suspension. By these criteria they were distinct from their transformed parents and similar to 3T3 cells. After infection, the revertants produced predominantly polyoma virus and few pseudovirus. Thus, for the cell lines used in this study, phenotypic transformation enhanced the formationof polyoma pseudovirions.     

Sulfated mucopolysaccharides from normal and virus transformed rodent fibroblasts.

The sulfated mucopolysaccharide composition of normal and virus transformed Balb 3T3 and BHK21 cell lines is reported. It is shown that normal 3T3 cells contain mainly chondroitin sulfate B and heparitin sulfate. Relatively higher amounts of chondroitin sulface AC were observed in polyoma virus transformed 3T3 cells, besides an absolute increase of all the three sulfated mucopolysaccharides in the polyoma and SV 40 transformed cells. It is shown also that the three sulfated mucopolysaccharides are at least in part at the cell surface. Similar differences in sulfated mucopolysaccharide composition of normal and virus transformed BHK cell lines were also observed.     

A cloned polyoma DNA fragment representing the 5'' half of the early gene region is oncogenic.

The two polyoma DNA fragments generated by cleavage with BamHI and EcoRI were cloned in pBR322, and their oncogenic potential was tested in vivo and in vitro. Only recombinant plasmid DNA containing a polyoma DNA fragment which extends clockwise from 58 to 0 map units and include approximately the 5'-proximal half of the early gene region produced tumors in newborn hamsters and transformed rat embryo cells in tissue culture. Southern blotting analysis indicated that the entire 2.2-kilobase polyoma BamHI-EcoRI fragment was intact in both a tumor cell line and a cell line transformed in culture which we examined. The presence of polyoma middle and small T antigen in these lines was demonstrated by immunoprecipitation and tryptic peptide mapping. DNA from a recombinant plasmid containing a polyoma genome deleted between 90 and 4 map units failed to induce tumors or transform cells.     

Cooperation of p53 and polyoma virus middle T antigen in the transformation of primary rat embryo fibroblasts.

Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation.     

Alkaline phosphatase activity and the regulation of growth in transformed mammalian cells

The relationship between alkaline phosphatase activity and cell growth has been studied in hamster cells transformed by different carcinogens. About 90% of normal hamster embryo cells were constitutively positive for alkaline phosphatase activity (AP⁺). However, there were no AP⁺ cells in cell lines transformed after treatment with the chemical carcinogens dimethylnitrosamine or 4-nitro-quinoline-N-oxide and 0.02% and 4% AP⁺ cells in cell lines transformed by polyoma virus or Simian virus 40. The glucocorticoid hormone, prednisolone, induced alkaline phosphatase activity in 12% and 44% of the enzyme-negative (AP^?) cells in cell lines transformed by polyoma or Simian virus 40, but this hormone did not induce alkaline phosphatase activity in AP^? cells from cell lines transformed after treatment with the chemical carcinogens. Treatment of polyoma transformed AP^? cells with the mutagen N-methyl-N′-nitro-N-nitro-soguanidine produced AP⁺ cells, whereas no AP⁺ cells were found after mutagen treatment of AP^? cells from the chemically transformed cell lines. Studies on spontaneous segregation in the polyoma transformed cell line has shown that AP⁺ cells segregated AP^? cells both in vitro and in vivo, although no spontaneous segregation was observed from AP^? to AP⁺ cells. AP⁺ cells, compared to AP^? cells, showed a decrease in DNA synthesis, cell multiplication, the ability to form colonies in soft agar and tumorogenicity in animals. AP^? cells induced for alkaline phosphatase activity by prednisolone, showed the same growth properties in vitro as uninduced AP^? cells. The decreased cell growth found in AP⁺ cells which were constitutive for alkaline phosphatase activity was therefore not found in the hormone induced AP^? cells. The results indicate that constitutive alkaline phosphatase activity appears to be related to the regulation of cell growth and that AP^? cells have a selective advantage over AP⁺ cells.     

State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintergrated viral DNA molecules.

The interaction of polyoma virus with a continuous line of rat cells was studied. Infection of these cells with polyoma did not cause virus multiplication but induced transformation. Transformed cells did not produce infectious virus, but in all clones tested virus was rescuable upon fusion with permissive mouse cells. Transformed rat cells contained, in addition to integrated viral genomes, 20 to 50 copies of nonintegrated viral DNA equivalents per cell (average). "Free" viral DNA molecules were also found in cells transformed by the ts-a and ts-8 polyoma mutants and kept at 33 C. This was not due to a virus carrier state, since the number of nonintegrated viral DNA molecules was found to be unchanged when cells were grown in the presence of antipolyoma serum. Recloning of the transformed cell lines produced subclones, which also contained free viral DNA. Most of these molecules were supercoiled and were found in the muclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specifici infectivity is, however, about 100-fold lower than that of polyoma DNA extracted from productively infected cells, suggesting that these molecules contain a large proportion of defectives.     

Stability of polyoma DNA sequences and virus-coded proteins during tumor formation

To determine the stability of polyoma viral DNA in transformed rat cells during their growth in vivo, we compared the state and arrangement of polyoma virus DNA sequences in virus-transformed rat cell lines before and after their passage in vivo. In cell lines from 12 independent tumors induced by the inoculation of animals with three different transformed cell lines, we could detect no significant changes in the arrangement of viral DNA sequences associated with the in vivo passage of these cell lines. In 13 of 14 tumor cell lines examined, the pattern of polyoma virus tumor antigens, characterized by the presence of the polyoma virus large, middle, and small tumor antigens, was unchanged.     

Similar cell surface antigens on hamster cells transformed by different papovaviruses.

Transformed baby hamster kidney (BHK) cells were tested for surface antigens by an immunocytoadhesion method. The cells were sensitized with rabbit antisera to cell clones transformed by polyoma or by BK virus and then rosetted with erythrocytes coated with antibody to rabbit immunoglobulin. These antisera detected common antigens on BHK cells transformed by either of three papovaviruses, polyoma, BK, or SV40, but apparently not on normal BHK cells.     

Polyoma virus-human cell interactions: persistence of T-antigen in two cell lines with and without transformation.

The interaction of polyoma virus and human cells was investigated. Abortive infection as evidenced by the synthesis of T-antigen was observed in normal fibroblast and abnormal (transformed) cells but not in normal epithelial cells. A high percentage of simian virus 40-transformed WI-18 Va2 and spontaneously transformed BE skin cells produced T-antigen after high-multiplicity infection, but most of the cells rapidly lost antigen-producing capacity upon cell passage, and the cultures became negative by passage 3. All fibroblast cells displayed varying degrees of susceptibility to infection, but most of the cell lines became negative for T-antigen except for two. In one, T-antigen persisted in a small percentage of the cells throughout the lifetime of the culture, without cellular transformation occurring. In the other, the entire culture became morphologically transformed and eventually consisted of 100% T-antigen-positive cells. This is the first time that normal diploid human fibroblast cells have been transformed by polyoma virus.     

Reversion in Polyoma-transformed Cells: Retransformation, Induced Antigens and Tumorigenicity

We studied the properties of two morphologically reverted cell clones isolated as chromosomal segregants from a "hybrid" clone of BHK 21/13 hamster fibroblasts, transformed with polyoma virus. Both clones were less tumorigenic than control transformed cells. They contained no detectable polyoma-specific complement-fixing antigen. Induced transplantation antigen also appeared to be lost or reduced. Both clones could be retransformed with polyoma virus, suggesting that their reversion is due to the loss of viral genes from the transformed cell.     

Distinct transformation phenotypes induced by polyoma virus and simian virus 40 in rat fibroblasts and their control by an early viral gene function.

Several transformed cell lines established from Fisher rat cells (FR 3T3) infected with wild-type polyoma virus or simian virus 40 or early temperature-sensitive mutants (polyoma tsa and simian virus 40 tsA30) were studied for their transformation phenotypes. The distinct patterns which were obtained for polyoma and simian virus 40 transformants led to the conclusion that these two viruses express different transforming abilities in rat cells. The results obtained with temperature-sensitive mutant-derived transformants indicate that all of the transformation characteristics studied so far may be under the control of a viral function in polyoma tsa-transformed cells.     

The dissociation of the surface architecture described by enhanced lectin agglutinability and the transformed phenotype expressed as anchorage independence.

Using a series of cold-sensitive variants of chemically transformed BHK-21 cells, revertants to the normal phenotype derived from a dimethyl-nitrosamine transformed clone of BHK-21 as well as revertants to the normal phenotype derived from polyoma transformed BHK-21 cells we have demonstrated that the surface phenotype described by enhanced agglutinability with Con A and WGA can be dissociated from the transformed phenotype described by anchorage independence (growth in semisolid medium). Specifically we have demonstrated that the surface characteristic of enhanced agglutinability may be found in a variety of cell lines which fail to display to grow in agar. Our work clearly shows that the two phenotypes described are not concomitantly controlled and tends to suggest that the phenotype of enhanced lectin agglutinability may be dissociated from the transformed phenotype.     

Decreased adenylate cyclase responsiveness of transformed cells correlates with the presence of a viral transforming protein

The adenylate cyclase responsiveness of transformed fibroblastic and epithelial cell lines to forskolin, fluoride, guanine nucleotides and cholera toxin was reduced compared to their parental counterparts. This phenomenon was observed in lines transformed by either RNA or DNA tumor viruses, and in the case of polyoma virus, coincided with the expression of middle T antigen. The data suggest that decreased responsiveness of adenylate cyclase to non-hormone activators is a general consequence of viral transformation and may be related to viral regulation of protein kinase activity.     

Aberrant intracellular IGF-1R β-subunit makes receptor knockout cells (IGF1R) susceptible to oncogenic transformation

Insulin-like growth factor 1 receptor (IGF-1R) is important for transformation of cells with cellular and viral oncogenes. This knowledge is mainly based on experiments on IGF-1R knockout mouse fibroblasts, which mostly are unable to transform after introduction of various oncogenes. Recently, we observed two variants of R- cells, one of which (R-s) surprisingly expresses the β-subunit of IGF-1R whereas the other one (R-r) does not. Here we show that the β-subunit is localized intracellularly and forms perinuclear aggregates. It expresses tyrosine kinase activity and appears to be crucial for cell survival since knockdown of it kills the R-s cells. H-RasV12 and/or polyoma middle T-antigen fail to transform R-r, whereas R- cells expressing the β-subunit were transformed as assessed by formation of colonies in soft agar. The oncogenic transformation of R-s cells was, however, abrogated when the aberrant β-subunit was knockdown by siRNA. The occurrence of intracellular IGF-1R, especially in tumor cells, has been widely reported but its function has not been understood. Our study provides evidence that it may be important for cell survival and transformation.     

Enzymatic basis for the structural changes of asparagine-linked sugar chains of membrane glycoproteins of baby hamster kidney cells induced by polyoma transformation

Previous studies indicated that enrichment of the GlcNAc beta 1----6Man alpha 1---- group with concomitant decrease of the GlcNAc beta 1----4Man alpha 1---- group occurs in the complex-type asparagine-linked sugar chains of the membrane glycoproteins of baby hamster kidney cells transformed by polyoma virus. The enzymatic basis of the chemical change is reported in this paper. By using oligosaccharides isolated from the urine of patients with a variety of exoglycosidase deficiencies, beta-N-acetylglucosaminyltransferases in the cell homogenate were successfully assayed separately. Both baby hamster kidney cells and their polyoma transformants contain beta-N-acetylglucosaminyltransferases I, II, IV, V, and VI, but not beta-N-acetylglucosaminyltransferase III. The beta-N-acetylglucosamine residue added by each beta-N-acetylglucosaminyltransferase (GnT) is shown below. (formula see text) Comparative studies of the specific activities of the five beta-N-acetylglucosaminyltransferases in the two cell lines revealed that the value of beta-N-acetylglucosaminyltransferase V in the polyoma transformant was twice of that in the normal cells, while those of the other four transferases in the two cell lines were not significantly different. Therefore the increase in beta-N-acetylglucosaminyltransferase V may be the direct cause of the changes found in the sugar chains of surface glycoproteins in baby hamster kidney cells transformed by polyoma virus.     

Uptake patterns and transport enhancements in cultures of hamster cells deprived of carbohydrates.

Dense cell cultures of the hamster lines, NIL, and polyoma transformed NIL were exposed to culture media containing various sugars (or no sugar). Various responses to these culture conditions were observed as changes in the uptake of galactose and its subsequent metabolism. Cells deprived of sugar have higher uptake rates for galactose and markedly different accumulation products from identical cells treated with sugar. A persistent increase in the transport of the amino acid, cycloleucine, was also observed as a response to culture conditions devoid of sugar     

Rescue of silent integrated polyoma genomes suggests homologous recombination between resident and transfected DNA fragments

Two defective polyoma virus genomes, deleted in the nucleotide sequences coding the N-termini of the tumor antigens, were introduced into Fisher 3T3 rat cells by DNA-mediated gene transfer (transfection). The resulting integrated genomes were incapable of conferring a transformed phenotype to the cells. However, after transfection of these lines with small polyoma fragments overlapping the deleted sequences, transformed clones were isolated. These clones were analyzed by Southern genomic blot hybridization and by isolation in E. coli of plasmids containing viral sequences excised following fusion with mouse polyoma growth-permissive cells. In all cases at least one intact copy of the early region of the polyoma genome was found. Furthermore, restriction sites adjacent to the initial inactive insertion remained unchanged in many of the transformed lines. These results show that functional restoration of the defective polyoma early region involves homologous recombination between the deleted viral genomes integrated in the cellular DNA and the transfecting viral fragments.     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.     

Secondary structure and methylation of DNA in reinoculated tumors and cultures of embryonic cells transformed by oncogenic viruses]

Using the kinetic formaldehyde method the concentration of secondary structure defects (SSD) in the DNAs of ascite leukosis L 1210 cells and cultures of hamster embryonic cells transformed by virus SV-40 and polyoma was evaluated. It was found that this concentration was considerably higher than in the DNAs from normal liver and primary culture of mouse embryonic cells. The occurrence of the defects in malignant cell DNAs is not due to enzymatic degradation of DNA. Using thin-layer chromatography the content of m5C in the DNAs from 17 sources (transformed cell cultures, experimental tumours and liver cells of mouses with Ehrlich ascite carcinoma) were determined. The methylation level for all these DNAs was higher than for normal mouse and rat liver DNAs. No correlation between the SSD concentration and m5C content in the DNAs studied was observed.     

The uptake of actinomycin D by normal and virus transformed BHK21 hamster cells

The uptake of actinomycin-D (AMD) in the hamster cell line BHK21 clone 13, and its polyoma virus-transformed derivative, were compared. In the transformed cell the internal AMD concentration at equilibrium was lower and was reached more quickly. The AMD-binding capacities of nuclei from normal and transformed cells were similar, suggesting that some control of AMD uptake occurs at the plasma membrane. This may be a control on the efflux of AMD since this process has a higher rate constant in transformed cells.