Simian Virus 40-transformed Cell Line Research Articles

Differentiation-dependent expression of E1^E4 proteins in cell lines maintaining episomes of human papillomavirus type 31b

The life cycle of human papillomaviruses (HPVs) is dependent on epithelial differentiation. Among the viral proteinsexpressed in differentiated epithelial cells are the viral capsid proteins, L1 and L2, as well as the E1^E4 fusion proteins. In this study, the expression and intracellular localization of the E1^E4 proteins of HPV type 31b were examined in both monolayer and raft cultures of the CIN-612 cell line which maintains episomal copies of HPV-31b. In this cell line, a high level of E1^E4 protein expression was observed in the cytoplasm of a small percentage of cells in monolayer culture. A large increase in E1^E4 protein levels was observed upon stratification of the CIN-612 cell line in raft cultures, with E1^E4 protein expression limited to the uppermost layers of the epithelium. A diffuse, slightly grainy cytoplasmic localization of E1^E4 protein was observed in both monolayer and raft culture systems. Although virion synthesis is entirely dependent upon phorbol ester or synthetic diacylglycerol treatment of raft cultures, E1^E4 expression was observed in both treated and untreated monolayer and raft cultures of the CIN-612 cell line. In monolayer cultures of two simian virus 40-transformed cell lines, cos-7 and MK-6, transiently transfected with an E1^E4 expression vector, the distribution of E1^E4 protein was found to differ substantially from that in the CIN-612 cells. In these cell lines E1^E4 protein was found to exhibit a total collapse into either cytoplasmic inclusion granules in the cos-7 cells or a perinuclear halo-like structure in the MK-6 cell line. The host cell, its differentiation state, and the amount of expression can therefore significantly affect the distribution of the E1^E4 proteins.

SV-40 transformation: effect on GM2 ganglioside in cultured cell lines

In previous work, we observed the presence of substantially elevated levels of GM2 after Simian Virus 40 (SV-40) transformation of human fetal brain cells. This elevated level of GM2 contrasted with the reports of many other investigators who had often observed decreased levels of GM2 and a simplification of ganglioside pattern in various non-neural rodent cell lines. In order to determine if the increase in GM2 in the transformed human brain cells would also be found in transformed rodent brain cells, we analyzed ganglioside changes after transformation in mouse brain cell lines and observed the increase in GM3 and low levels or lack of GM2 usually noted in rodent SV-40 transformed cell lines. In addition, we analyzed changes after SV-40 transformation in three human fibroblast lines and found that all three lines contained substantially elevated levels of GM2 after SV-40 transformation. As a result of this study, our earlier work on SV-40 transformed human brain cells, and occasional other reports of high levels of GM2 in human SV-40 transformed cell lines, elevated levels of GM2 may be considered a marker for SV-40 transformed human cells of both fibroblastic and neural origin.

Genetic analysis of indefinite division in human cells: identification of four complementation groups.

Hybrids obtained following fusion of normal human diploid fibroblasts with different immortal human cell lines exhibited limited division potential. This led to the conclusion that the phenotype of cellular senescence is dominant and that immortal cells arise as a result of recessive changes in the growth control mechanisms of the normal cell. We have exploited the fact that immortality is recessive and, by fusing immortal human cell lines with each other, assigned 21 cell lines to at least four complementation groups for indefinite division. A wide variety of cell lines was included in the study to determine what parameters, if any, would affect complementation group assignment. The results indicate that cell type, embryonal layer of origin, and type of tumor do not affect group assignment. There does not appear to be any correlation between expression of an activated oncogene and group assignment. However, all of the immortal simian virus 40-transformed cell lines studied (with the exception of one xerodermapigmentosum fibroblast-derived line) assign to the same group, indicating that this virus immortalizes various human cells by the same processes. The assignment of immortal human cells to distinct groups provides the basis for a focused approach to determine the genes important in normal growth regulation that have been modified in immortal cells.

Hsv‐ and chemical carcinogen‐induced amplification of sv40 dna sequences in transformed cells is cell‐line‐dependent

Eleven simian virus 40-transformed cell lines from 5 different species were tested for their ability to amplify integrated simian virus 40 DNA upon infection with herpes simplex virus type I or treatment with various chemical carcinogens. Four cell lines were positive only for virus-induced gene amplification and two lines were positive for both carcinogen- and virus-induced gene amplification. Individual cell lines were assayed for the presence of an intact SV40 origin of replication, the expression of a functional SV40 T-antigen, and permissivity to herpes simplex virus replication. These parameters were found to be positive in all 6 amplification-competent cell lines. The ability of herpes simplex virus to amplify SV40 DNA sequences in transformed cells is greater than that of chemical carcinogens and can be suppressed by specific inhibitors of the herpes virus-encoded DNA polymerase.

Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.

The steady-state levels of p53 protein and p53 mRNA in transformed and nontransformed cells were examined to elucidate the mechanisms controlling expression of p53. mRNA levels were determined by Northern blot hybridization analysis, employing a p53-specific cDNA clone (M. Oren and A.J. Levine, Proc. Natl. Acad. Sci. U.S.A. 80:56-59, 1983), and protein levels were determined by the Western blotting technique. Analysis of p53 mRNA revealed a single polyadenylated mRNA species migrating at ca. 18S. Levels of p53 mRNA in simian virus 40-transformed cell line (SVT2) and in an homologous nontransformed cell line (3T3) were equivalent, although the steady-state levels of p53 protein were 25- to 100-fold higher in the SVT2 cells than in the 3T3 cells. A study with a non-virus-transformed cell system revealed a different result. Embryonal carcinoma cells (F9) were found to have nearly 20-fold higher levels of p53 mRNA in comparison with differentiated benign progeny cells. In this system the difference in p53 mRNA levels corresponded to the difference in p53 protein levels. Pulse-chase experiments were performed to study the half-life of p53 protein in these four types of cells. The turnover of p53 protein occurred with biphasic kinetics. In addition, it was found that protein synthesis inhibitors placed in the medium during the chase period prevented the turnover of p53 protein in transformed cells, but not in nontransformed (3T3) cells. These results provide evidence that the regulation of p53 expression in cells can occur at the level of p53 mRNA abundancy or p53 protein stability depending upon the experimental system under study, and that a regulated degradation process controls the turnover of p53 protein.

Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.

The steady-state levels of p53 protein and p53 mRNA in transformed and nontransformed cells were examined to elucidate the mechanisms controlling expression of p53. mRNA levels were determined by Northern blot hybridization analysis, employing a p53-specific cDNA clone (M. Oren and A.J. Levine, Proc. Natl. Acad. Sci. U.S.A. 80:56-59, 1983), and protein levels were determined by the Western blotting technique. Analysis of p53 mRNA revealed a single polyadenylated mRNA species migrating at ca. 18S. Levels of p53 mRNA in simian virus 40-transformed cell line (SVT2) and in an homologous nontransformed cell line (3T3) were equivalent, although the steady-state levels of p53 protein were 25- to 100-fold higher in the SVT2 cells than in the 3T3 cells. A study with a non-virus-transformed cell system revealed a different result. Embryonal carcinoma cells (F9) were found to have nearly 20-fold higher levels of p53 mRNA in comparison with differentiated benign progeny cells. In this system the difference in p53 mRNA levels corresponded to the difference in p53 protein levels. Pulse-chase experiments were performed to study the half-life of p53 protein in these four types of cells. The turnover of p53 protein occurred with biphasic kinetics. In addition, it was found that protein synthesis inhibitors placed in the medium during the chase period prevented the turnover of p53 protein in transformed cells, but not in nontransformed (3T3) cells. These results provide evidence that the regulation of p53 expression in cells can occur at the level of p53 mRNA abundancy or p53 protein stability depending upon the experimental system under study, and that a regulated degradation process controls the turnover of p53 protein.

Evidence of transcription from the late region of the integrated simian virus 40 genome in transformed cells: location of the 5' ends of late transcripts in cells abortively infected and in cells transformed by simian virus 40.

By means of S1 mapping, we observed that spliced 16S and 19S viral late mRNAs--in addition to early mRNAs--were present in cytoplasmic polyadenylated RNA preparations from simian virus 40-transformed cell lines of rat or mouse origin containing no detectable amount of free viral DNA. The amounts of early and late virus-specific mRNAs in these lines were quantified by hybridization of radioactive cytoplasmic polyadenylated RNA with cloned region-specific restriction fragments. The relative amount of late viral mRNA produced in these transformed cells was found to be of the same order as that produced in simian virus 40-infected, nonpermissive baby mouse kidney cells. Moreover, by using the S1 nuclease protection method, we compared the 5' ends of late mRNAs produced (i) in transformed cells, (ii) in abortively infected mouse cells, and (iii) in the late phase of the lytic cycle. The 5' ends of late mRNAs both in abortively infected and in transformed cells were less heterogeneous than the 5' ends of late mRNAs produced during the lytic cycle; however, they were a subset of the 5' ends of late transcripts produced in the lytic cycle.

The complex between simian virus 40 T antigen and a specific host protein.

The small DNA tumour virus simian virus 40 (SV40) expresses only two proteins in the cells that it has transformed, large-T and small-t. Large-T has an apparent molecular mass of 94 000 and genetic evidence indicates that it is necessary both for the induction and maintenance of the transformed state. In the lytic cycle of the virus the large-T protein acts to initiate viral DNA replication and appears to regulatea its own transcription. These properties imply that the protein must interact with existing host cell mechanisms in a specific way and that an understanding of these interactions may be important in discovering how the protein acts in 'transforming' non-permissive cells. To identify these interaction structures in the host cell an immunochemical approach has been adopted, and has shown that a fraction of the large-T protein in transformed cells exists in a specific complex with a host-coded phosphoprotein. The host protein has an apparent molecular mass of 53 000. The physiological role of this host protein-T antigen complex has been examined by studying the nature of the complex in a wide variety of different SV40 transformed cell lines. Although in SV40-transformed cells of rodent origin all the host protein present in the cell is complexed to large-T, it was found that in human cells transformed by SV40 only a fraction of the host protein is present in a complex and these cells contain large-T and the host protein in both the free and the complexed state. In SV40-transformed mouse cells that have been selected for loss of the transformed phenotype the complex was still present and in apparently normal amounts. This would indicate that no simple causal relation exists between the presence of the complex and the phenotype of the cell containing it.

Relationship Among Tau Antigens Isolated from Various Lines of Simian Virus 40-Transformed Cells

In addition to the virus-specified tumor antigens, simian virus 40-transformed cells contain at least one other protein which can be immunoprecipitated with serum from animals bearing simian virus 40-induced tumors. This protein, which is designated Tau antigen, has an apparent molecular weight of 56,000 as determined by electrophoresis on acrylamide gels. The relationship among Tau antigens isolated from different lines of simian virus 40-transformed cells was examined by comparing the methionine-labeled tryptic peptides of these proteins by two-dimensional fingerprinting on thin-layer cellulose plates. In this fashion, we initially determined that the Tau antigens isolated from three different lines of transformed mouse cells were very similar. Second, we found that Tau antigen isolated from a line of rat transformants was closely related, but not identical, to the mouse cell Tau antigens. Approximately 70% of their methionine peptides comigrated in two dimensions. Finally, we showed that Tau antigen isolated from a line of transformed human cells was only partially related to the mouse and rat proteins. About 40% of the methionine peptides of the human protein were also contained in the Tau antigens from the other two species. These results strongly indicate that the Tau antigens isolated from these various simian virus 40-transformed cell lines contain common amino acid sequences.

Activation of the viral genome in Simian Virus 40-transformed nonpermissive cells by permissive cell extracts.

Simian virus 40-transformed nonpermissive cells in which neither infectious viral DNA nor virions had previously been detected reproducibly yield low levels of infectious simian virus-40 DNA after treatment with extracts of normal permissive cells. Virions were obtained in one out of 37 experiments. Activation of the viral genome is detected only with normal permissive cell extracts and only in simian virus 40-transformed cell lines that are rescuable by heterokaryon formation or by treatment with chemicals. The activating factor(s) is insensitive to deoxyribonuclease and ribonuclease, but is sensitive to heat at 56 degrees and to proteolytic enzymes.

Response of simian virus 40-transformed cell lines and cell hybrids to superinfection with simian virus 40 and its deoxyribonucleic acid.

Whereas normal human and monkey cells were susceptible both to intact simian virus 40 (SV40) and to SV40 deoxyribonucleic acid (DNA), human and monkey cells transformed by SV40 were incapable of producing infectious virus after exposure to SV40, but displayed susceptibility to SV40 DNA. On the other hand, mouse and hamster cells, either normal or SV40-transformed, were resistant both to the virus and to SV40 DNA. Hybrids between permissive and nonpermissive parental cells revealed a complex response: whereas most hybrids tested were resistant, three of them produced a small amount of infectious virus upon challenge with SV40 DNA. All were resistant to whole virus challenge. The persistence of infectious SV40 DNA in permissive and nonpermissive cells up to 96 hr after infection was ascertained by cell fusion. The decay kinetics proved to be quite different in permissive and nonpermissive cells. Adsorption of SV40 varied widely among the different cell lines. Very low adsorption of SV40 was detected in nonsusceptible cells with the exception of the mKS-BU100 cell line. A strong increase in SV40 adsorption was produced by pretreating cells with polyoma virus. In spite of this increased adsorption, the resistance displayed by SV40-transformed cells to superinfection with the virus was maintained.