Reversible growth arrest in simian virus 40-transformed human fibroblasts.

A reversible growth arrest of simian virus 40-transformed human fibroblasts has been produced by replacement of methionine in the growth medium by its immediate metabolic precursor, homocysteine, Although these arrested cells exhibit a greatly reduced cloning efficiency when plated in methionine-supplemented medium, they resume rapid proliferation without a lag when subconfluent cells are refed with methionine-supplemented medium. This growth arrest is accompanied by a reduction in the percentage of mitotic cells in the cell population. Furthermore, data obtained using fluorescence-activated cell sorting techniques indicate that the cells are arrested i the S and G2 phases of the cell cycle. This is in contrast to a G1-phase accumulation of cells, which occurs only in methionine-supplemented medium at very high densities and which is similar to the G1 block seen in cultures of normal fibroblasts at high density. The apparent relationship between specific events in the DNA-synthetic and premitotic phase of the cell cycle and methionine dependence in these transformed cultures is discussed.     

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.     

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.     

Signal-mediated nuclear transport in simian virus 40-transformed cells is regulated by large tumor antigen.

Transformation of cultured cells with simian virus 40 (SV40), or transfection with the early region of the SV40 genome, causes a significant increase in both the rate of signal-mediated nuclear transport and the functional size of the transport channels (located in the pore complexes). By microinjecting purified large tumor (T) antigen into the cytoplasm of murine BALB/c 3T3 cells, we have demonstrated that this protein alone can account for the increase in transport capacity. The T antigen-dependent changes can be partially inhibited by cycloheximide and require a functional nuclear localization sequence. Although necessary, the nuclear localization sequence by itself cannot produce the observed variations in nuclear permeability and presumably function in a "helper" capacity, in association with another, as yet unidentified domain.     

Simian virus 40-specific proteins in the membranes of simian virus 40-transformed hamster and mouse cells.

Membranes of simian virus 40-transformed hamster lymphocytes and phagocytes, as well as of transformed mouse fibroblasts, contain two classes of antigenic virus-specific protein. The isoelectric points of these proteins, as defined by isoelectric focusing/immune electrophoresis are at pH 4.5 and 4.7. The molecular weights of the pI 4.5 and pI 4.7 components, determined by isoelectric focusing/dodecyl sulfate polyacrylamide electrophoresis, lie near 58,000 and 90,000-110,000, respectively. The pI 4.5 and pI 4.7 proteins are tentatively identified with the surface (transplantation) and U antigens, respectively.     

Host nuclear proteins expressed in simian virus 40-transformed and -infected cells.

Two new families of host proteins (Mr, 48,000 and 55,000), in additional to the viral large (T) and small tumor antigens, are precipitable, with anti-T antiserum, from cells transformed or infected by the DNA tumor virus simian virus 40 (SV40). Rabbit anti-mouse 48,000 protein antiserum reacts specifically with SV40-infected or -transformed mouse cells to give nuclear staining indistinguishable from T-antigen staining but does not react with SV40-transformed human cells which nevertheless have structurally analogous 48,000 proteins, nor does it give nuclear fluorescence with untransformed mouse cells. Comparison of the partial proteolytic digests of the 48,000 proteins from cultured cells of various mammalian species shows that they are structurally related but not related to the 55,000 or large T-antigen proteins. The 55,000 proteins from the various mammalian species were also structurally related.     

Immunochemical delineation of an oncofetal antigen on normal and simian virus 40-transformed human fetal melanocytes.

Human melanoma cells of uveal origin shed 94,000- and 240,000-dalton glycoproteins in common with most melanoma cell lines of dermal origin. Normal human melanocytes derived from fetal uvea shed a 90,000-dalton glycoprotein that was found to be immunologically identical with the 94,000-dalton glycoprotein of melanoma cells. Expression of this 90,000-dalton molecule was confined to fetal cells of ectodermal origin. After simian virus 40 (SV40) transformation of human fetal melanocytes, there was an apparent increase in molecular size of this component to 94,000 daltons. In contrast, the 240,000-dalton glycoprotein was not synthesized or shed by uninfected or SV40-transformed fetal melanocytes. These data suggest that the 94,000-dalton glycoprotein is an oncofetal antigen of ectodermal origin.     

Enhanced phosphorylation of many endogenous protein substrates in human fibroblasts transformed by simian virus 40

Protein phosphorylation in normal and in simian virus 40-transformed human skin fibroblasts was assessed by two different methods: incubation of whole-cell homogenates with [gamma-(32)P]ATP or labeling of intact cells with Na(2)H(32)PO(4). Phosphorylated proteins were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. With both methods, the Coomassie-blue-stained protein patterns of the three transformed cell lines studied were similar to the patterns of the nontransformed normal human cells. However, although the phosphoprotein autoradiograms of the three transformed cell lines were nearly identical, their patterns were strikingly different from those of the nontransformed cells. Each of the three transformed lines tested showed approximately 25-30 phosphoprotein bands that were significantly enhanced when compared to the patterns of the nontransformed cells. Quantitation of 12 of the enhanced phosphoprotein bands in one of the transformed cell lines showed an average of 4.4 times as much phosphorylation as in the normal cells. The enhanced phosphorylation observed in the transformed cell lines was not dependent on the growth rate of the cells or on cyclic AMP. Furthermore, when homogenates of transformed and nontransformed cells were mixed prior to incubation with [gamma-(32)P]ATP, the resultant phosphoprotein patterns resembed those obtained with transformed cells alone. In addition, an evaluation of the time course of protein phosphorylation revealed that the initial reaction rate was greater in the transformed than in the normal cells, although in both cell types the reaction was complete after 1 min. The results suggest that the simian virus 40-transformed human fibroblasts possess an increased ability to phosphorylate proteins rather than that the normal cells possess a diffusible inhibitor. There appear to be many endogenous cellular substrates for this increased activity.     

Suppression of tumorigenicity in simian virus 40-transformed 3T3 cells transfected with alpha-actinin cDNA.

Human cytoskeletal alpha-actinin cDNA was transfected into highly malignant simian virus 40-transformed BALB/c 3T3 (SVT2) cells that express 6-fold lower levels of alpha-actinin than nontransformed BALB/c 3T3 cells. SVT2 clones expressing various levels of alpha-actinin were isolated and their structure and tumorigenic properties were determined. Transfected SVT2 clones expressing alpha-actinin at levels found in nontumorigenic 3T3 cells displayed a flatter phenotype, a decreased ability to grow in suspension culture in soft agar, and a marked reduction in their ability to form tumors in syngeneic BALB/c mice and in athymic nude mice. Clones overexpressing alpha-actinin at the highest level (about 2-fold higher than 3T3 cells) were completely suppressed in their ability to form tumors in syngeneic BALB/c mice. The results suggest that alpha-actinin, an actin-crosslinking protein that is also localized in cell junctions, may have an effective suppressive ability on the transformed phenotype.     

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.     

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.     

Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40.

Rodent embryo cells immortalized with temperature-sensitive mutants of simian virus 40 large tumor (T) antigen have a proliferative potential that depends on temperature. At the restrictive temperature, heat-inactivation of large T antigen causes p53 release, growth arrest, and cell death. Morphological and molecular analysis indicate that the induced cell death corresponds to apoptosis. Flow cytometric analysis using a combination of forward light scatter and side scatter allows a discrimination of cells committed to apoptosis within the whole population. These cells display a reduction in cell size and a higher cellular density, confirming the apoptotic nature of the cell death. When cells exhibiting the morphological features of apoptosis were stained with a fluorescent probe of the mitochondrial membrane potential, a decreased accumulation of the dye was recorded. Measures of cellular respiration, performed with whole-cell populations, showed that the lower mitochondrial membrane potential (delta psi m) correlates, as expected, with an uncoupling of electron transport from ATP production and is linked to the induction of apoptosis. We also show that this decrease in delta psi m is associated with a decrease in the rate of mitochondrial translation. These events are detected at early stages of the apoptotic process, when most of the cells are not irreversibly committed to death, suggesting that mitochondria could be a primary target during apoptosis.     

Expression of the transformation-sensitive protein "cyclin" in normal human epidermal basal cells and simian virus 40-transformed keratinocytes.

A cell population highly enriched in human epidermal basal cells has been obtained and characterized by using antibodies specific for various cell types in the epidermis. Quantitative two-dimensional gel electrophoretic analysis (isoelectric focusing) of [35S]methionine-labeled polypeptides from basal cells and simian virus 40-transformed keratinocytes showed that the basal cells synthesize very low amounts (less than 0.02% of the total protein) of the nuclear, transformation-sensitive protein cyclin as compared to the transformed cells, which synthesize this protein constitutively (0.15% of the total protein). Very low levels of cyclin were observed in total human epidermis, and preliminary studies of two basaliomas have shown a significant synthesis of this protein in these tumors. Immunofluorescence studies using antibodies to proliferating cell nuclear antigen that immunoprecipitate cyclin confirmed the above observations at least in the case of the cultured cells. Taken together, these results support the notion that cyclin may be a central component of the pathway(s) that controls cell proliferation.     

Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells.

A clonal hamster beta cell line (HIT) was established by simian virus 40 transformation of Syrian hamster pancreatic islet cells. Cytoplasmic insulin was detected in all cells by indirect fluorescent antibody staining, and membrane-bound secretory granules were observed ultrastructurally. Acidified-ethanol extracts of HIT cell cultures contained hamster insulin as determined by radioimmunoassay, radioreceptor assay, and bioassay. One subclone at passage 39 contained 2.6 micrograms of insulin per mg of cell protein. [3H]Leucine-labeled HIT insulin and proinsulin were identical to islet-derived proteins when compared by NaDodSO4/polyacrylamide gel electrophoresis of immunoprecipitates. HIT cell insulin secretion was stimulated by glucose, glucagon, and 3-isobutyl-1-methylxanthine. Insulin secretion at optimal glucose concentration (7.5 mM) was 2.4 milliunits per 10(6) cells per hr. Somatostatin and dexamethasone markedly inhibited HIT insulin secretion. The HIT cell line represents a unique in vitro system for studying beta cell metabolism and insulin biosynthesis.     

Presence of simian virus 40 DNA sequences in human lymphomas

Simian virus 40 (SV40)--a potent oncogenic virus--has been associated previously with some types of human tumours, but not with lymphomas. We examined human tumours for the presence of specific SV40 DNA sequences by PCR and Southern blotting. Viral sequences were present in 29 (43%) of 68 non-Hodgkin lymphomas, and in three (9%) of 31 of Hodgkin's lymphomas. Viral sequences were detected at low frequencies (about 5%) in 235 epithelial tumours of adult and paediatric origin, and were absent in 40 control tissues. Our data suggest that SV40 might be a cofactor in the pathogenesis of non-Hodgkin lymphomas.     

Human mesothelial cells are unusually susceptible to simian virus 40-mediated transformation and asbestos cocarcinogenicity

Mesothelioma, a malignancy associated with asbestos, has been recently linked to simian virus 40 (SV40). We found that infection of human mesothelial cells by SV40 is very different from the semipermissive infection thought to be characteristic of human cells. Mesothelial cells are uniformly infected but not lysed by SV40, a mechanism related to p53, and undergo cell transformation at an extremely high rate. Exposure of mesothelial cells to asbestos complemented SV40 mutants in transformation. Our data provide a mechanistic explanation for the ability of SV40 to transform mesothelial cells preferentially and indicate that asbestos and SV40 may be cocarcinogens.     

Presence of allograft-rejection resistance in simian virus 40-transformed hamster cells and its possible role in tumor development.

LSH Syrian hamster cells transformed in vitro by simian virus 40, which is oncogenic for hamsters, are resistant to rejection by adult allogeneic CB hamsters. In contrast, simian virus 40-transformed cells from other species are usually not oncogenic in immunocompetent autologous or isologous hosts. The ability of simian virus 40 to convey resistance to an allograft-type host response to transformed hamster cells may be important in determining the tumor-inducing capacity of these cells and could, in part, explain the species-specific oncogenicity of this virus.