Resistance of simian virus 40-transformed hamster cells to the cytolytic effect of activated macrophages: a possible factor in species-specific viral oncogenicity.

Simian virus 40 (SV40)-transformed hamster cells were relatively resistant to the lytic effect of activated macrophages from animals with chronic intracellular infections. Conversely, SV40-transformed mouse and rat cells and adenovirus 2-transformed hamster cells were highly susceptible to destruction by tumoricidal activated macrophages. The pattern of resistance or susceptibility of SV40-transformed rodent cells was the same whether activated macrophage effectors were obtained from mice, random-bred hamsters, or the inbred LSH hamsters from which some of the SV40-transformed hamster lines were derived. The results suggest that resistance of transformed cells to macrophage-mediated cytolysis may explain in part the species-specific oncogenicity of this DNA virus.     

Adenovirus 2 early gene expression promotes susceptibility to effector cell lysis of hybrids formed between hamster cells transformed by adenovirus 2 and simian virus 40.

Weakly oncogenic adenovirus 2 (Ad2)-transformed LSH hamster cells are sensitive to lysis by spontaneously cytolytic lymphoid cells and activated macrophages, whereas highly oncogenic simian virus 40 (SV40)-transformed LSH cells are relatively resistant to these nonspecific effector cells. Somatic cell hybrids formed between Ad2- and SV40-transformed hamster cells, which expressed Ad2 tumor (T) antigens, exhibited an increased cytolytic susceptibility compared to Ad2 T antigen-negative cell hybrids or nonhybrid SV40-transformed cells. No correlation was found between the expression of SV40 T antigen in hybrid cells and cytolytic susceptibility. The results suggest the existence of a novel function for early Ad2 genome-encoded polypeptides (T antigens) expressed in transformed hamster cells--the induction of susceptibility to destruction mediated by immunologically nonspecific effector cells.     

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.     

Division of BALB/c mouse 3T3 and simian virus 40-transformed 3T3 cells in cellular aggregates

BALB/c mouse 3T3 cells and 3T3 cells transformed by simian virus 40 (SV40) were cultured as aggregates in agitated liquid medium. When maintained with daily medium changes, 3T3 cells incorporated [(3)H]thymidine into acid-insoluble material at a rate (1/4) that of 3T3 cells in logarithmic-phase flat cultures but 16 times that of the same cells in stationary-phase flat cultures. Similarly, SV40-transformed 3T3 cells in aggregates incorporated [(3)H]thymidine at (1/3) the rate of SV40-3T3 cells in logarithimic-phase flat culture and 14 times that of these cells in stationary-phase flat culture. Autoradiographs of aggregates of 3T3 and SV40-3T3 cells incubated for 2 hr in the presence of [(3)H]uridine and [(3)H]thymidine indicated that penetration of the nucleosides into aggregates during this period was limited to the outer four to six cell layers. Aggregates were also incubated in the presence of radiolabeled thymidine and uridine continuously for 4 days. Under these conditions, where nucleoside penetration was not limiting, 100% of the SV40-3T3 cells and 56% of the 3T3 cells incorporated [(3)H]thymidine into acid-insoluble material.The rates of cell division, cell loss, and net cell accumulation in aggregates of 3T3 and SV40-3T3 cells were measured by techniques not influenced by possible alterations in transport, pool size, or penetration. SV40-3T3 cells divided with a doubling time for the total population of 26.0 hr. The total cell number increased more slowly (doubling time of 48.3 hr) because of cell loss, which occurred with a half-time (time for 50% of the cells to be lost) of 53.3 hr. 3T3 cells in aggregates began to divide only after 3 days, then did so with a doubling time for the total population of 76.4 hr. Total cell number decreased (half-time of 26.3 hr) because this rate of cell division was exceeded by the rate of cell loss, which was constant with a half-time of 22.9 hr. The results suggest that 3T3 and SV40-3T3 cells display growth properties in aggregates consistent with their previously reported behavior in conventional flat culture: SV40-transformed 3T3 cells can proliferate under conditions of high cell density to a much greater extent than 3T3 cells can. Both cell lines, however, display an increased capacity to divide in aggregates relative to confluent flat culture, despite conditions of high cell density and absence of anchorage to an artificial solid substrate.     

Serum factor requirements of normal and simian virus 40-transformed 3T3 mouse fibroplasts

Evidence is presented to show the presence in normal rat serum of four different serum factors essential for growth of 3T3 or SV40-transformed 3T3 mouse fibroblasts: a factor that specifically promotes growth of normal 3T3 cells; two factors that specifically promote growth of transformed 3T3 cells; and a factor that sustains viability of both normal and transformed 3T3 cells in serum-free medium, probably without inducing growth of the cells. These factors are separated and partially purified.     

Biological activity of purified simian virus 40 T antigen proteins.

Proteins related to simian virus 40 (SV40) T antigen uere isolated from cells infected with adenovirus 2/SV40 hybrids Ad2+D2 and Ad2+ND1 dp2 as well as from a line of human cells (SV80) transformed by SV40. The 96,000- and 107,000-dalton proteins of SV80 and Ad2+D2, after injection into the cytoplasm of cultured cells, rapidly accumulate in the nuclei, where they remain antigenically reactive for at least 20 hr and trigger DNA synthesis in quiescent cells. By contrast, the 23,000-dalton protein coded by Ad2+ND1 dp2 does not stimulate cellular DNA synthesis. However, all three purified proteins are able to provide helper function for the growth of adenovirus 2 in monkey cells. Thus, purified SV40 T antigen and proteins that share sequences with it retain the ability to carry out at least two functions associated with the product of the A gene of SV40.     

Serum Factor Requirements of Normal and Simian Virus 40-Transformed 3T3 Mouse Fibroblasts

Evidence is presented to show the presence in normal rat serum of four different serum factors essential for growth of 3T3 or SV40-transformed 3T3 mouse fibroblasts: a factor that specifically promotes growth of normal 3T3 cells; two factors that specifically promote growth of transformed 3T3 cells; and a factor that sustains viability of both normal and transformed 3T3 cells in serum-free medium, probably without inducing growth of the cells. These factors are separated and partially purified.     

Membrane Fatty Acid Replacements and Their Effect on Growth and Lectin-Induced Agglutinability

The growth of 3T3 and SV101 3T3 cells in a lipid-depleted medium is enhanced by the addition of biotin or some fatty acids. The extent of enhancement depends on the fatty acid(s) supplied. The presence of linoleate is unique, since it induces a morphological alteration in 3T3 cells resulting in a cell similar to an SV101-transformed 3T3 cell. Analyses of the fatty acids from the membrane phosphatides show that the exogenously supplied fatty acids are incorporated and alter the fatty acid composition. This is most clearly evident with heptadecanoate-grown cells, in which this fatty acid and its derivatives comprise over 45% of the fatty acids in the phospholipids.The fatty acid replacements have a striking effect on the temperature dependence of agglutination by wheat germ agglutinin and concanavalin A, implying that fluidity is involved in agglutination. These temperature dependencies and the effect of fatty acid replacements on them were different for the two lectins, but similar for both transformed and untransformed cells. These observations are interpreted as suggesting that the lipid phase is heterogeneous, and that transformed and untransformed cell membranes have regions of similar fluidity.     

Transformation by simian virus 40 induces virus-specific, related antigens in the surface membrane and nuclear envelope

Nucleus- and mitochondrion-free membranes from hamster lymphocytes transformed by simian virus 40 (SV40), GD248 cells, cause guinea pigs to produce immune sera that reveal the presence in GD248 plasma membranes and mitochondria of two types of glycoprotein that are not detected in membranes of normal lymphocytes [Schmidt-Ullrich, R., Thompson, W. S. & Wallach, D. F. H. (1977) Proc. Natl. Acad. Sci. USA 74, 643-647]. Indirect immune fluorescence of living, SV40-transformed T19 hamster reticulum cells, Balb/c 3T3 mouse fibroblasts, and W18 VA2 human fibroblasts, using the antisera against GD248 membrane, at 4 degrees produced a distinct cell surface fluorescence; however, above 20 degrees , staining at the nuclear perimeter, the SV40 U-antigen reaction, becomes equally prominent. In SV40-transformed cells that had been fixed in cold acetone, as well as in purified GD248 nuclei, thermostable U-antigen staining is dramatic, but there is no reaction for nuclear T-antigen. Rabbit antisera against T19 cells gave immunofluorescence reactions equivalent to those obtained with the antisera against GD248 cells. Normal guinea pig or rabbit sera and cells that had not been transformed by SV40 gave no reaction. Our sera from tumor-bearing hamsters gave only nuclear T-antigen fluorescence. The results indicate the presence of related, SV40-specific antigens in the surface membranes, nuclear envelope, and possibly other intracellular organelles of SV40-transformed cells.     

A Factor from a Transformed Cell Line That Affects Cell Migration

When a monolayer culture of normal Balb/c3T3 cells is wounded by scraping away part of the cell sheet, the cells do not migrate into the cleared area unless there is serum in the culture medium. By contrast, SV40-transformed Balb/c3T3 cells do migrate into the wound area without serum. A quantitative assay for the migration of Balb/c3T3 cells into wounds is described. This assay is used in the partial purification of a migration factor released into serum-free medium by SV28 cells. SV28 is a line of BHK21/13 hamster cells transformed by SV40 chosen for its malignancy. The most purified fractions have about 1500 times the specific activity of whole calf serum. These fractions have an activity that promotes overgrowth of Balb/c3T3 cells to high density and an activity that prolongs cell survival without serum. The SV28 migration factor is not extractable from the medium of untransformed BHK21/13 cells or from serum. This migration factor might contribute to the malignancy of SV28 cells.     

Antigenic Study of Unfertilized Mouse Eggs: Cross Reactivity with SV40-Induced Antigens

Serum obtained from guinea pigs immunized with unfertilized C57BL/6 mouse eggs was found to be cytotoxic in the presence of complement for eggs obtained from syngeneic and allogeneic mice. The anti-egg serum was not cytotoxic for rat eggs, lymph node cells, methylcholanthrene-induced tumor cells, or 3T3 cells obtained either from syngeneic or allogeneic mice. The anti-egg serum was, however, cytotoxic for SV40-transformed 3T3 cells and C57BL/6 cells. After absorption with SV40-transformed cells, anti-egg serum lost its cytotoxicity for mouse eggs.     

Cytoplasmic transfer of DNA containing simian virus 40 sequences into mouse 3T3 cells.

This paper describes the rare cytoplasmic transmission of defective simian virus 40 (SV40) viral DNA from enucleated cells (i.e., cytoplasts) of the SV40-transformed mouse cell line SVT2 (chloramphenicol-resistant) into cybrid cells formed by fusion of these cytoplasts with BALB/c 3T3 cells (thymidine kinase-deficient). The cybrids were selected in medium containing 1% serum, bromodeoxyuridine, and chloramphenicol. They were identified by their 3T3 chromosome content, by the instability of tumor (T)-antigen expression, by their transformed phenotype, and by their drug resistance. The yield of rare cybrids was about 5 x 10(-7) 0.1% of the yield on medium with 10% serum. The presence of the SV40 genome was detected by the expression of SV40-specific T antigen and confirmed (unpublished data) by hybridization of viral DNA probes with restriction enzyme fragments of nuclear DNAs from cybrid clones. Restriction site mapping (unpublished data) showed that at least 1 kilobase of host flanking DNA on each side of the SV40 DNA was included in the transferred segment. The transforming DNA was not stably integrated initially, as judged by cellular heterogeneity in T-antigen expression. Stable T-antigen-positive and negative subclones were recovered in 10% serum; instability could be retained for at least 30 doublings during growth in 1% serum. The instability is interpreted as evidence of non-integration or unstable integration of the transferred DNA into the host genome. The cytoplasmic transfer is interpreted as evidence that chromosomal fragments or intact chromosomes can be transferred rarely through the cytoplasm in cybrid crosses.     

Tumorigenicity of simian virus 40-transformed human cells and mouse--human hybrids in nude mice.

Four different human cell lines transformed by simian virus 40 (SV40) were tested for their tumorigenicity in athymic nude mice. Two of these lines, W18Va2 and GM52VA, were found to be tumorigenic when inoculated at a concentration of 1 x 10(7) cells per mouse. The other two cell lines, LN-SV and GM54VA, were found to induce very small tumors only after the injection of approximately 1 x 10(8) cells per mouse. Somatic cell hybrids between either LN-SV or GM54VA SV40-transformed human cells and normal mouse peritoneal macrophages, which have retained the human chromosomes carrying the SV40 genome, were found to be much more tumorigenic than the SV40-transformed human cell parents. These experiments suggest that the genetic background in which the human chromosomes carrying the SV40 genome are present plays a role in the modulation of the expiration of malignancy.     

Quantitation and characterization of a species-specific and embryo stage-dependent 55-kilodalton phosphoprotein also present in cells transformed by simian virus 40.

A 55-kilodalton (kDal) protein was detected recently in primary cultures of day 12 mouse embryos by immunoprecipitation with serum from simian virus 40 (SV40) tumor-bearing hamsters (T serum), Preliminary evidence suggested that this protein was similar to a cellular 55-kDal protein induced after SV40 transformation of mouse cells. We now show that specific approximately 55-kDal [35S]methionine-labeled proteins precipitate from primary cultures of midgestation mouse, rat, and hamster embryos on addition of T serum or monoclonal antiserum prepared against the SV40-induced mouse 55-kDal proteins. The two-dimensional maps of the [35S]methionine-labeled tryptic peptides of the mouse, hamster, and rat embryo proteins are similar to the maps of the corresponding proteins from SV40-transformed cells. Primary cells from midgestation mouse, hamster, or rat embryos contain one-third to one-half as much 55-kDal protein as a SV40-transformed mouse fibroblast cell and nearly the same amount as F9 mouse embryonal carcinoma cells. The amount of 55-kDal protein is greatly reduced on replating the mouse, rat, or hamster embryo primary cells. The amount of this protein in mouse embryos is dependent on the stage of the embryo. The embryo proteins are phosphoproteins.     

Assignment of the T-antigen gene of simian virus 40 to human chromosome C-7

Hybrid cell clones between mouse cells deficient in thymidine kinase (EC 2.7.1.21) and two different human cell lines transformed by simian virus 40 (SV40) and deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) were examined for SV40 tumor (T) antigen(s). Concordant segregation of the gene(s) for SV40 T antigen and human chromosome C-7 was observed in these hybrids. The human chromosome C-7 which contains the gene(s) for SV40 T antigen is preferentially retained by the majority of the hybrid clones tested. When hybrid clones positive and negative for SV40 T antigen, derived from the fusion of SV40-transformed Lesch-Nyhan fibroblasts with mouse cells, were fused with CV-1 permissive cells, SV40-specific V antigen was observed only in the cultures derived from fusion of the hybrid clones positive for T antigen. This result indicates a linkage relationship between human chromosome C-7, SV40 T-antigen gene(s), and SV40 genome(s) integrated in the human transformed cells.     

Inverse relationship between anti-SV40 TASA and anti-H-2 cytotoxic responses

The in vitro cytotoxic response against H-2d and H-2b SV40-transformed fibroblasts was studied in a 40-h 3H-proline assay. A very low response against SV40 TASA is associated with the H-2d antigens on target cells: however, SV40-transformed H-2d cells are as immunogenic as SV40-transformed H-2b cells and prime against H-2b target cells. The data concerning in vitro amplification of the anti-SV40 TASA response and the involvement of cyclophosphamide-sensitive suppressor populations confirm the comparable immunogenicity of SV40-transformed H-2d AND H-2b cells and cannot account for the haplotype-related behavior observed with SV40-transformed target cells. The study of the response against allogeneic SV40-transformed cells shows the reverse situation: the lower cytotoxic response is now associated with the H-2b antigens on SV40-transformed cells. As suggested by the data presented here, an interaction between SV40 TASA and H-2 antigens might be postulated.