Biochemical properties of media conditioned by simian virus 40-induced hamster tumor cells: correlation with distinct cell phenotypes but not with oncogenicity.

Hamster cells, transformed in vitro by SV40, have been reported to secrete an unidentified factor(s) that inhibits thymidine uptake (TU) by various normal cell types, including activated lymphocytes. It has been postulated that this apparent antiproliferative effect may play an in vivo role in the high tumorigenic capacity of SV40-transformed hamster cells. In keeping with this hypothesis, Adenovirus type 2-transformed hamster cells, which are only weakly tumorigenic, do not inhibit TU by indicator cells in vitro. To study the biological relevance of this phenomenon, we assayed 11 cell lines derived from different fibrosarcomas, induced in Syrian hamsters by SV40, for their ability to inhibit TU by normal rabbit kidney indicator cells. In contrast to cells transformed in vitro by SV40, media conditioned by 6 of 11 tumor-derived cell lines did not inhibit TU. Our results do not support the hypothesis that an antiproliferative factor secreted by SV40-transformed cells promotes the tumor-inducing capacity of these cells. Furthermore, inhibition of TU does not appear to be due to the production of a specific antimitotic peptide, but rather to other biochemical properties of the media conditioned by transformed cells. Finally, these biochemical properties do appear to correlate with specific morphological and growth characteristics of the tumor cells, but probably as an effect and not a cause.

Mitogenic and antimitogenic transforming growth factors secreted by adenovirus 2- and simian virus 40-transformed hamster cells: possible roles in promoting tumorigenesis.

Adenovirus 2 (Ad2)- and simian virus 40 (SV40)-transformed hamster embryo cells differ markedly in a number of phenotypic properties including their potential for inducing tumors in hamsters. Both Ad2- and SV40-transformed cells are immortalized and readily induce tumors in immunoincompetent newborn syngeneic hamsters, but only SV40-transformed cells are highly oncogenic in both adult syngeneic and allogeneic immunocompetent hamsters. The reasons for the difference in the oncogenic potential of the Ad2- and SV40-transformed phenotypes remain elusive. However, recent studies with transforming growth factors (TGFs) indicate that these factors play an important role in determining many phenotypic characteristics of transformed cells. To determine whether TGFs secreted by Ad2- and SV40-transformed hamster embryo cells differ, we have examined the ability of media conditioned by these two transformed cell phenotypes to modulate thymidine uptake in quiescent, untransformed cells. We found that both transformed phenotypes secrete very similar TGF alpha-like mitogenic factors which inhibit binding of 125I-labeled epidermal growth factor to its receptor. Our results also show that SV40-transformed cells, but not Ad2-transformed cells, secrete a powerful mitogenic inhibitor (MI). The MI secreted by SV40-transformed cells is inhibitory for several transformed and untransformed cell types and exerts a cytostatic, not cytolytic, action on untransformed primary hamster embryo cells. MI elutes from size exclusion high-performance liquid chromatography columns with a molecular weight of 24,000. Although MI has about the same molecular weight as TGF beta, it differs from TGF beta in two important respects: it is heat labile and it has a different target specificity for antimitogenic activity. The MI secreted by SV40-transformed cells also inhibits thymidine uptake by concanavalin A-stimulated spleen lymphocytes. This finding suggests that MI might contribute to the extreme oncogenicity of SV40-transformed cells by inhibiting mobilization of immune effector cells at the site of tumor cell proliferation.

The level of expression of adenovirus type 2 transforming genes governs sensitivity to nonspecific immune cytolysis and other phenotypic properties of adenovirus 2-simian virus 40-transformed cell hybrids.

Syrian hamster embryo cells transformed by adenovirus type 2 (Ad2) or simian virus 40 (SV40) differ markedly in morphology, tumorigenicity, and susceptibility to in vitro lysis by nonspecific cytotoxic cells. Hybrid cells formed by fusing Ad2- and SV40-transformed Syrian hamster embryo cells may express only SV40 T antigens or both SV40 and Ad2 T antigens. Hybrids that express only SV40 T antigens are indistinguishable from the nonhybrid SV40-transformed phenotype, whereas hybrid cells that express T antigens from both viruses closely resemble the nonhybrid parental Ad2-transformed phenotype. Because these hybrid cells have been useful in the study of neoplastic transformation, we determined the amount of viral antigens that they accumulate in an attempt to correlate the level of expression of the transforming viral genes with some of their phenotypic properties. Hybrid cells that expressed proteins from both viruses showed reduced levels of SV40 T antigens compared with those of hybrid cells that did not express Ad2 T antigens. We also found that the production of several cellular proteins that influence cytomorphology was inhibited in hybrid and nonhybrid cells that expressed Ad2 T antigens, and the repression of these cellular proteins correlated with a change in cytomorphology from fibroblastic to spherical. Finally, we showed that the susceptibility of our hybrid cells to in vitro lysis by natural killer cells and activated macrophages, two putative host-effector cells involved in defense against neoplasia, correlated closely with the level of expression of a 58,000-dalton Ad2 protein. The results reported here, together with the results of previous studies, indicate that the oncogenic potential of hybrid cells that express both Ad2 and SV40 antigens is extremely sensitive to Ad2 expression, whereas other phenotypic properties depend on Ad2 expression in a dose-dependent manner.

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.

Suppression of the simian virus 40 tumorigenic phenotype in hybrid cells formed from simian virus 40- and adenovirus 2-transformed hamster embryo cells.

Hamster cells transformed by adenovirus 2 (Ad2) or simian virus 40 (SV40) have different tumorigenic phenotypes. In the present study, somatic cell hybrids formed from Ad2- and SV40-transformed hamster cells were used to determine whether possible interactions between the integrated viral genomes would influence the tumorigenic phenotype of hybrid transformed cells. These somatic cell hybrids were of two types, one expressing both Ad2 and SV40 T-antigens and the other expressing only SV40 T-antigens. Tumor induction by hybrid cells that expressed both Ad2 and SV40 T-antigens was reduced in adult syngeneic hamsters and abrogated in adult allogeneic hamsters. These results indicate that the tumorigenic phenotype of transformed somatic cell hybrids that contain both the Ad2 and SV40 genome is governed by the genetic expression of Ad2. This expression may alter the ability of SV40-transformed hamster cells to resist the immunologically nonspecific defenses of the host.

Kinetics of inhibition of papovavirus DNA synthesis by superinfection with adenovirus 2 and non-defective adenovirus 2-simian virus 40 hybrid viruses.

Simian Virus 40 (SV40) DNA synthesis is inhibited in monkey cells by superinfection with adenovirus 2 (Ad2) and various non-defective Ad2-SV40 hybrid viruses. Similarly, BKV (a human papovavirus) DNA synthesis is inhibited in human cells by superinfection with Ad2. Kinetic studies indicate that inhibition begins during the early phase of the Ad2 lytic cycle. Superinfection with Ad2 does not significantly alter the formation of SV40 T antigen. Superinfection with Ad2 late in SV40 lytic cycle is less efficient in the inhibition of SV40 DNA synthesis, and the onset of Ad2 DNA synthesis is delayed, compared to superinfection early in the SV40 lytic cycle. These findings suggest that the Ad2 and SV40 genomes may compete to bind an early AD2 protein which is essential for Ad2 replication, but which blocks SV40 replication.

Regulation of viral transcription in cells infected with iododeoxyuridine-substituted simian virus 40 as a model for the activation by iododeoxyuridine of latent viral genomes.

5-Iododeoxyuridine (IdUrd) induces the expression of viruses in a variety of cell lines that harbor latent viral genomes. Moreover, IdUrd stimulates cellular as well as viral RNA synthesis in certain cells. In order to understand better the action of IdUrd on RNA metabolism, we have examined viral RNA synthesis in monkeys cell infected with IdUrd-substituted simian virus 40 (SV40). Extensively substituted SV40, in which 18 to 35% of the thymidine residues were substituted by IdUrd, was 100-fold less viable (by plaque analysis) than was unsubstituted SV40, although the substituted virus induced 30 to 50% as much viral-specific RNA as did the unsubstituted virus. In contrast, SV40, containing only 10 to 15% IdUrd, substitution was almost as viable as unsubstituted virus, and the substituted SV40 induced 5-fold more viral-specific RNA, as well as longer viral messenger RNA transcripts, than did the unsubstituted virus. These results suggest that the lightly substituted, mutagenized SV40 genome may produce defective proteins which fall to regulate their own transcription. Cellular DNA into which halogenated pyrimidines have been incorporated may also induce the synthesis of defective regulatory proteins, including cellular repressors of transcription which normally maintain the latent state of integrated viral genomes.

Method for determining the extent and copy number of overlapping and nonoverlapping segments of integrated viral genomes.

We analyzed the method of exhaustive hybridization of single-stranded DNA and derived a general relationship between the fraction of the probe DNA hybridized and the sizes and copy numbers of the segments of the viral genome integrated in cellular DNA. The equations employed can be used to analyze integrated DNA comprised of overlapping and nonoverlapping segments of the viral genome. Using these equations, we analyzd the adenovirus type 2 DNA content of a series of hamster cell lines transformed by adenovirus type 2 and several adenovirus type 2-simian virus 40 hybrid viruses. We found no eividence that the integrated viral DNA is comprised of overlapsping segments. However, the number of copies of the integrated segments varies between lines cloned from the same transformed isolate, and copy numbers change during in vivo passage of transformed cells.

Content and expression of integrated viral DNA in hamster cells transformed by nondefective adenovirus type 2- simian virus 40 hybrid viruses.

Hamster cells transformed by adenovirus 2 (Ad2) and five nondefective Ad2-simian virus 40 (SV40) hybrid viruses are all of the Ad2-transformed phenotype. All lines accumulate Ad2 RNA and Ad2 T antigen; two hybrid-transformed lines accumulate SV40 RNA, but only one contains detectable amounts of SV40 antigens. We examines selected lines from this group of transformed hamster cells for Ad2 DNA content and viral RNA expression by using hydroxyapatite chromatography and separated strands of labeled viral DNA as probes. All lines studies contain 1 to 2 Ad2 genome equivalents per haploid equivalent of cell DNA. The expression of Ad2 RNA exhibits two distinct patterns. In one pattern, transcription of the heavy strand of Ad2 DNA is less than that of the light strand, whereas in the second pattern of Ad2 RNA expression the extent of heavy-strand transcription is greatly increased. In the two lines containing SV40 RNA, the entire SV40 (-) strand is transcribed; the (+) strand is not expressed in these cells. These patterns of viral RNA expression suggest the possibility that host promoters may play a role in regulating transcription of integrated viral DNA.

Simian virus 40 DNA replication, transcription, and antigen induction during infection with two adenovirus 2-SV40 hybrids that contain the entire SV40 genome.

The Ad2++hey hybrid virus population produces simian virus 40 (SV40) efficiently during lytic infection, whereas Ad2++ley does not, although both hybrids contain a complete SV40 genome. In this report, we demonstrate the synthesis of nonhydrid SV40 DNA in Ad2++HEY-infected Vero cells, but only early SV40 RNA is transcribed efficiently in Ad2++LEY-infected cells. Ad2++HEY induces SV40 U, T, and V antigens during lytic infection of African green monkey kidney cells, whereas Ad2++LEY induces only SV40 U and T antigens. These variations in the behavior of Ad2++HEY and Ad2++LEY regarding expression of SV40 functions probably reflect differences in the rate of SV40 excision from the hybrid genomes.

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. IX. Template topography in the early region of simian virus 40.

The DNAs of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses contain overlapping segments of the early region of wild-type SV40 DNA. The complementary DNA strands of these five viruses have been separated with synthetic polyribonucleotides in isopycnic cesium chloride gradients. The relative amounts of early and late SV40 template in the DNA of each virus were determined by RNA-DNA hybridization with late lytic SV40 RNA, which contains sequences complementary to both templates. From the distribution of early and late templates in the five overlapping SV40 segments, we conclude that either the entire early region of SV40 is symmetrically transcribed in vivo, or, more probably, that the early SV40 templates are not contiguous.

Evidence for a transcription-control region of Simian virus 40 in the adenovirus 2--Simian virus 40 hybrid, Ad2+ND 1 .

The complementary DNA strands of the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), were separated by isopycnic banding in a CsCl density gradient in the presence of synthetic polyribonucleotides. Separated strands were used in DNA-RNA hybridization reactions with RNA from cells productively infected by Ad2 or SV40, and with complementary SV40 RNA transcribed asymmetrically in vitro. About five times as much Ad2 RNA hybridized to the light stand of Ad2(+)ND(1) as to the heavy strand. Complementary RNA and early SV40 RNA (RNA synthesized before viral DNA replication) had significant homology only with the light strand. Only half as much of a preparation of RNA synthesized before and after viral DNA replication (early-plus-late SV40 RNA) hybridized to the light strand as to the heavy strand. These results indicate that templates for both late and early SV40 RNA are present in Ad2(+)ND(1). Therefore, the small SV40 segment within this virus (10-18% of the SV40 genome) must contain a transcription-control region. Ad2(+)ND(1) should thus be useful in the selective study of transcription as it occurs in cells infected by the oncogenic virus SV40.

Comparison of the biochemistry and rates of synthesis of mesosomal and peripheral membranes in Bacillus subtilis.

The membrane vesicle (beaded chain) portion of the mesosomes and peripheral (ghost) membrane of Bacillus subtilis were obtained by protoplast lysis and separated by differential and sucrose gradient centrifugation. Electron microscopy revealed that both fractions were satisfactorily homogeneous. Comparison of the two membrane preparations showed that they were similar with respect to total protein, total phosphorus, and lipid-soluble phosphorus content. Their protein patterns on acrylamide gel electrophreograms did not differ significantly. A possible point of distinction was revealed by a difference spectrum analysis of their cytochromes. The two preparations showed clear quantitative differences in all five of the enzyme activities assayed. Acrylamide gel electrophreograms of peripheral membrane stained for malate dehydrogenase showed four weak isozyme bands, whereas electrophreograms of mesosome membranes exhibited a single strong peak. (A survey of published data on enzymes in mesosome fractions shows a marked lack of correspondence between different species of bacteria.) Comparison of (3)H-acetate incorporation into the two membrane fractions showed that both were labeled at the same rate. Similarly, (35)SO(4) was taken up by both fractions at a comparable rate and was chased from both comparably. Lipid and protein labeling thus indicates that mesosome vesicle membrane is not a precursor or special growing point of peripheral membrane.