Increased susceptibility to SV40 transformation with development and in vitro aging

The incidence of most cancers increases with aging. To examine whether this increased risk might be related to a higher susceptibility of older cells to neoplastic transformation, we transfected rat fibroblasts aged in vivo and in vitro with origin-defective SV40 DNA and measured the number of transformed foci. Substantial increases in the number of transformed foci were observed in cells from adult rats when compared with those of cells from embryos or weanlings. Much higher numbers of foci were also obtained at late passage, when 68% or more of the in vitro lifespan had been completed, while no foci were produced from cells at early or middle passage. To control for changes with aging in uptake, integration, or expression of exogenous DNA, parallel cultures were transfected with a G418 resistance gene. The number of G418-resistant colonies did not increase with aging and, in fact, decreased in late passage embryonic cell cultures. Therefore, increased susceptibility to SV40 transformation appears to be a feature of development and in vitro aging in rat Cells.     

v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures

To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage- independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage- independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.     

A recombinant murine retrovirus for simian virus 40 large T cDNA transforms mouse fibroblasts to anchorage-independent growth.

A recombinant murine retrovirus containing the intact cDNA sequence for the simian virus 40 (SV40) large T antigen (T) was constructed by using the pZIPNeo SV(X)1 vector. Psi 2 packaging cells were then transfected, and G418-resistant clones were used to generate helper-free viral stocks. NIH 3T3 mouse fibroblasts infected by the recombinant T cDNA retrovirus were selected fro G418 resistance. Such cultures synthesized authentic SV40 T and were transformed to anchorage-independent growth at high efficiency. Therefore, this vector has allowed the study of the transformation properties of T under conditions of neutral drug selection and in the absence of SV40 small t antigen.     

Extension of lifespan of human T lymphocytes by transfection with SV40 large T antigen.

Lymphocytes have a finite and predictable proliferative life span in culture similar to that observed in fibroblasts. In general, the senescence of human fibroblasts is inevitable and irreversible, but their proliferative life span can be extended by certain DNA tumor virus oncogenes, such as the large T antigen of the SV40 virus. Here, we show that human T lymphocytes (HTL) can be stably transfected with SV40 large T and that expression of T antigen extended the life span of T cell cultures. PHA-stimulated HTL were transfected with pSV3neo, an expression vector containing the SV40 early region and the neomycin resistance gene. Transfectants were selected for neomycin (G418) resistance. Control HTL, either mock transfected or transfected with pSV2neo (containing the neomycin resistance gene only), ceased proliferation after about 17 population doublings. In contrast, HTL transfected with pSV3neo underwent more than 170 doublings. pSV3neo-transfected cells expressed SV40 large T RNA, detectable by in situ hybridization, and SV40 T antigen, detectable by immunofluorescence. Greater than 95% of the transfected cells were CD4 positive. These results clearly show that SV40 large T enables HTL to escape senescence. Transfection with SV40 large T may be a valuable method for obtaining long term human T cell lines for studies of both aging and immunology.     

Transformation of human epidermal cells by transfection with plasmid containing simian virus 40 DNA linked to a neomycin gene in a defined medium

A human epidermal cell culture was transformed by transfection with a recombinant plasmid containing simian virus 40 DNA with a deletion at the origin and an antibiotic (neomycin or G418) marker. A calcium phosphate-mediated DNA transfection method was optimized for introducing exogenous DNA into cells maintained in a fully defined medium. The transformed cells were propagated for more than 200 population doublings and did not appear to go through a "crisis" period. The growth characteristics of the transformed cells were similar to those of untransformed cells. Major keratins synthesized in the transformed cells were similar to those found in normal epidermal cells. Transformed cells initially transfected with the recombinant plasmid could be propagated for more than 30 passages. Actively growing cells could then be repeatedly selected from cell populations based upon their neomycin (G418)-resistant phenotype for at least another 30 passages. Simian virus 40 T-antigen and extrachromosomal DNA containing plasmid- and SV40-specific DNA sequences were detected in the transformed cells. Because of their nononcogenic phenotype and defined growth requirements, the transformed cells provide a model for examining structural changes during cell proliferation and differentiation, and for exploring the multistage carcinogenesis of human epithelial cells.     

Reactivation of thyroglobulin gene expression in transformed thyroid cells by 5-azacytidine

Transformed rat thyroid cells fail to express thyroglobulin. Cells transformed with a Kirsten murine sarcoma virus carrying a temperature-sensitive ras allele lose their transformation phenotype when shifted to the nonpermissive (39 degrees C) temperature. The thyroglobulin promoter, however, remains inactive. Similarly, transfection of these cells with a thyroglobulin promoter fused to a neomycin resistance reporter gene does not produce clones resistant to G418. Treatment of the transfected cells with the DNA demethylating agent 5-azacytidine reactivates the thyroglobulin promoter and yields stable G418-resistant clones. We show that thyroglobulin promoter activity is correlated with the presence of a thyroid-specific nuclear factor, TgTF1. TgTF1 cannot be detected in transformed cells but reappears after treatment with 5-azacytidine at 39 degrees C. Restoration of Ras activity at 33 degrees C leads to the rapid loss of TgTF1 and G418 resistance.     

Responsiveness of normal and transformed rat embryo cell cultures to poly I-poly C and interferon

A number of normal rat cell cultures as well as cultures transformed spontaneously, by chemicals, and/or by oncogenic viruses were tested for responsiveness to the interferon inducer polyinosinic·polycytidylic acid, or to exogenous interferon. Responsiveness, or lack thereof, had no correlation with subculture passage number, infection with RNA leukemia virus, morphological transformation by oncogenic RNA or DNA viruses, chemical treatment, or the ability of these cells to produce tumors in isologous host animals. The data indicate that lack of response to interferon or to the inducer is neither a necessary prerequisite nor an absolute result of cellular transformation of rat cells.     

Putative role for EPC-1/PEDF in the G0 growth arrest of human diploid fibroblasts

EPC-1/PEDF expression is closely associated with reversible growth arrest in normal human diploid fibroblast-like (HDF) cells and is diminished with proliferative senescence in vitro. EPC-1 expression in HDF cells is induced under conditions of density-dependent contact inhibition and growth factor deprivation. Antiserum generated against EPC-1 recognizes a secreted protein of approximately 50 kDa from medium conditioned by early passage HDF cells, but not from senescent cells. The addition of EPC-1 antiserum to early population doubling level (PDL) cultures near the plateau phase of growth significantly increases the number of cells entering DNA synthesis. Affinity purified EPC-1 antibodies alone enhance the ability of near plateau-phase early PDL WI-38 cells to synthesize DNA by as much as threefold. Further, the addition of recombinant EPC-1 (rEPC-1) to logarithmically growing cells resulted in a marked decrease in the ability of these cells to enter DNA synthesis. We also demonstrate the loss of EPC-1 expression in WI-38 and IMR-90 HDF cell lines with both senescence and simian virus 40 (SV40) transformation. The loss of EPC-1 expression with SV40 transformation occurs at the level of steady-state mRNA and protein accumulation with genomic EPC-1 sequences grossly intact. Taken together, these results suggest that EPC-1 may play a role in the entry of early passage fibroblasts into a G(0) state or the maintenance of such a state once reached.     

Transformation of primary rat kidney cells by fragments of simian virus 40 DNA.

Linear simian virus 40 (SV40) DNA molecules of genome length and DNA fragments smaller than genome length when prepared with restriction endonucleases and tested for transforming activity on primary cultures of baby rat kidney cells. The linear molecules of genome length (prepared with endonucleases R-EcoRI, R-BamHI, and R-HpaII or R-HapII), a 74% fragment (EcoRI/HpaII or HapII-A), and a 59% fragment (BamHI/HapII-A) could all transform rat kidney cells with the same efficiency as circular SV40 DNA. All transformed lines tested contained the SV40-specific T-antigen in 90 to 100% of the cells, which was taken as evidence that the transformation was SV40 specific. The DNA fragments with transforming activity contained the entire early region of SV40 DNA. Endo R-HpaI, which introduced one break in the early region, apparently inactivated the transforming capacity of SV40 DNA, since no transformation was observed with any of the three HpaI fragments tested. Attempts were made to rescue infectious virus from some of the transformed lines by fusion with permissive BSC-1 cells. Infectious virus was only recovered from the cells transformed by circular form I DNA. No infectious virus could be isolated from any of the other types of transformed cells.     

Primary rat cells expressing a hybrid polyomavirus-simian virus 40 large T antigen have altered growth properties.

Expression of simian virus 40 (SV40) large T antigen efficiently immortalizes and transforms primary cells. We previously reported that a hybrid polyomavirus-SV40 large T antigen, PyT1-521-SVT336-708, binds to both p53 and pRb but does not transform an established rat cell line (J. J. Manfredi and C. Prives, J. Virol. 64:5250-5259, 1990). Here we show that this hybrid large T antigen is capable of immortalizing primary rat cells. Plasmids that express resistance to G418 sulfate and either SV40 large T antigen or PyT1-521-SVT336-708 were transfected into primary rat embryo fibroblasts, and cell lines were established. The cell lines that expressed PyT1-521-SVT336-708 were not fully transformed but did exhibit altered growth properties. Although these PyT1-521-SVT336-708-expressing lines did not form foci, they did grow in low serum and grew to a high saturation density; these cell lines also formed colonies in soft agar, but their colonies were much smaller than those seen with an SV40 large-T-antigen-expressing line. PyT1-521-SVT336-708 also demonstrated the ability to cooperate with activated Ha-ras to form foci on primary rat embryo fibroblasts. Surprisingly, two types of morphologies in such lines were observed: refractile and spindle shaped. Although there was no correlation between T-antigen level and morphology, all lines that displayed refractile morphology expressed high levels of p21ras. Since the p53 binding activity of PyT1-521-SVT336-708 appears to be intact, these results suggest that there are functions residing in the amino end of SV40 large T antigen which are necessary for full transformation that are missing from the amino end of polyomavirus large T antigen. Conversely, conferring the ability to bind to p53 on an amino-terminal fragment of polyomavirus large T antigen, although not enough to allow full transformation function, does increase its oncogenic activity in saturation density and soft agar growth assays.     

Cellular senescence involves stochastic processes causing loss of expression of differentiated function genes: transfection with SV40 as a means for dissociating effects of senescence on growth and on differentiated function gene expression

In the accompanying work we demonstrated that the decline in expression of steroid 17 alpha-hydroxylase in mass cultures and clones of adrenocortical cells is the result of a stochastic switching process which yields mixtures of expressing and nonexpressing cells. There is an apparent positive correlation between the replicative potential of adrenocortical cell cultures and the number of cells in the culture that can express 17 alpha-hydroxylase. We investigated this by extending the cells' replicative potential by transfecting them with cloned SV40 virus. Cells from a senescent subclone, with very limited remaining replicative potential, were transfected. The cell population showed a progressive increase in growth rate and gave rise to a line of cells that expressed T antigen and which was apparently immortalized. Induction of mRNA for 17 alpha-hydroxylase by cyclic AMP was absent in this line of cells, as it was in the senescent cells prior to transfection. The cells remained responsive to gene induction by cyclic AMP as evidenced by increases in mRNA and activity for cholesterol side-chain cleavage. The absence of 17 alpha-hydroxylase expression in this line was not the result of interference by SV40 T antigen. When early passage cells were transfected with pSV3neo, which contains the early region of SV40 and neo, and were selected with G418, SV40 T antigen-expressing lines were derived which showed high levels of expression of 17 alpha-hydroxylase after induction with cyclic AMP. These cells maintained high levels of expression of 17 alpha-hydroxylase through four successive recloning events, over a period of replication much longer than that achievable by nontransfected cells. Thus, transfection by SV40 can be used to dissociate effects of senescence on growth and differentiated gene expression. T antigen expression selectively affects growth, but preserves the state of expression of a differentiated function gene as it was prior to transfection.     

Plasmidial maintenance in rodent fibroblasts of a BPV1-pBR322 shuttle vector without immediately apparent oncogenic transformation of the recipient cells.

A recombinant plasmid was constructed (pV69) which comprises a subgenomic fragment of bovine papilloma virus type 1 (BPV1) DNA, part of plasmid pBR322 DNA and a drug resistance gene expressed in both mammalian fibroblasts and Escherichia coli. This gene (vv2) is a modified form of the bacterial neomycin resistance gene (neo) linked to the herpes simplex virus thymidine kinase (tk) promoter (plasmid pAG60), to which the original bacterial neo promoter from transposon Tn5 was added back, upstream of the eukaryotic promoter. It induced kanamycin resistance in E. coli, as well as resistance to the drug G418 in rat and mouse fibroblasts. Its expression in FR3T3 rat cells was enhanced as compared with the original tk-neo construction. After transfer of plasmid pV69 into C127 mouse cells or FR3T3 rat cells, the number of resistant colonies selected in medium containing G418 was one to two orders of magnitude higher than that of transformed foci in normal medium. In eight independent cell lines selected by drug resistance, pV69 DNA was found to be maintained in a plasmidial state, without any detectable rearrangement or deletion and could be transferred back in E. coli. In contrast, cell lines selected by focus formation in normal medium maintained deleted forms of the original plasmid DNA, and only part of them were resistant to G418. Most of the drug-resistant clones had kept the morphology and growth control of the normal fibroblasts. However, with further passages in culture, these cells spontaneously produced transformed foci with increasing frequencies.     

Abrogation of simian virus 40 DNA-mediated transformation of primary C57BL/6 mouse embryo fibroblasts by exposure to a simian virus 40-specific cytotoxic T-lymphocyte clone.

Primary mouse embryo fibroblasts of C57BL/6 origin (B6/MEF) were transformed in vitro by transfection with simian virus 40 (SV40) DNA. The transformation frequency was markedly reduced if the SV40 DNA-transfected cultures were briefly exposed to K11 cells, an SV40-specific clone of cytotoxic T lymphocytes. This abrogation of SV40 transformation in vitro by cytotoxic T-lymphocyte clone K11 was specific, since transformation of B6/MEF cells by adenovirus type 5 DNA was not affected. The approach described here should serve as an ideal model of dissecting immunological events during in vivo tumorigenesis.     

Human papillomavirus type 6b DNA required for initiation but not maintenance of transformation of C127 mouse cells.

We describe the transformation of C127 mouse fibroblasts with human papillomavirus type 6b (HPV-6b) DNA, which is associated primarily with benign tumors of the human genital tract. The major transformed phenotype of the HPV-6b-transfected cells lines, which had been G418 selected, pooled, and maintained without subsequent selection, was tumorigenicity in nude mice. We found that, unlike that reported for other HPVs or papovaviruses, the transformed phenotype was expressed after a delay, in which the cells had undergone extensive culture passages (about 20 passages or 100 generations). Interestingly, the HPV-6b DNA had become reduced or nondetectable in copy number in the cells by the time the transformed phenotype was expressed and in most of the tumors induced by the cells in nude mice, indicating that high levels of HPV-6b DNA were not required for maintenance of the transformed phenotype. Clonal cell lines gave similar results. When continued G418 selection was used to maintain high-copy-number HPV-6b DNA, the cells were tumorigenic, indicating that high levels of HPV-6b DNA did not suppress tumorigenesis. These studies suggest that HPV-6b DNA initiates transformation of C127 cells but is dispensable for expression or maintenance of the transformed phenotype. Transformation by HPV-6b DNA in vitro may provide insights into the HPV type-specific association with benign versus malignant lesions in vivo and may elucidate some of the oncogenic processes involved in tumor progression.     

Correlation of DNA content, p53, T antigen, and V antigen in simian virus 40-infected human diploid cells

Human diploid fibroblasts (HDF) have a finite life span in cell culture which can be extended when transformed with simian virus 40 (SV40). Flow cytometric analysis of SV40-HDF transformation allowed DNA content changes to be correlated with the appearance, quantity, and distribution of T antigen, p53, and V antigen, three proteins associated with this process. These studies demonstrated a shift in the DNA content to tetraploidy, which was correlated with the age of the SV40-HDF but not the time of infection. A significant increase of the epitope recognized by PAb122 to host p53 and the epitope PAb101 to SV40 T antigen occurred at the same time the tetraploid population appeared. However, an antigen reactive with SV40 V antibody was present at high levels in most of the population early after infection, but the levels declined with time. The percentage of PAb101-T antigen-positive cells increased more rapidly in cells infected at a late passage, and this was concomitant with the shift in DNA content to tetraploid. Analysis of the mean fluorescence of total, gated populations (G1, G2, and greater than G2) demonstrated that a threshold level of p53 and T antigen was reached in each compartment of the cell cycle. As the transformed phenotype appeared, a population of cells was continually released into the supernatant, and although these cells had a DNA pattern similar to the monolayer cells, the T antigen and p53 levels were 3-5 times higher in the tetraploid G2 cells. These studies correlated the expression of proteins associated with viral transformation in HDF which vary with time and shift in DNA content.     

Quantitative Characteristics of the Transformation of Hamster Cells by PARA (Defective Simian Virus 40)-Adenovirus 7

An in vitro method for the quantitative measurement of transformation in hamster embryo fibroblasts by the PARA [defective simian virus 40 (SV40)]-adenovirus 7 hybrid has been developed. Transformation by PARA particles followed one-hit kinetics with a ratio of 1 focus-forming unit per 250 plaque-forming units. The method of viral adsorption had a direct effect upon the total number of foci which developed but not on the quantitative aspects of this assay. A fluorescent-focus assay was developed which provided a direct correlation of the observed morphological transformation and the presence of the PARA genome. This fluorescent-focus assay utilized detection of the SV40 tumor antigen, which was present in all foci transformed by PARA. Single foci induced by PARA were isolated and grown into cell lines. Two types of foci were observed and isolated; the first contained cells having a cuboidal or SV40-type morphology, and the second consisted of epithelial or adenovirus-type transformed cells. Both types contained the SV40 tumor and SV40 surface antigens as determined by the indirect fluorescence technique; however, only the epithelial cells contained the adenovirus 7 tumor antigen. All five cell lines which were injected into weanling Syrian hamsters were found to be oncogenic. These cell lines induced antibodies to both SV40 and adenovirus 7 tumor antigens in tumor-bearing animals.     

Regulated replication of an episomal simian virus 40 origin plasmid in COS7 cells.

The replication of a simian virus 40 (SV40) origin-containing plasmid, pSLneo, stably transfected COS7 cells has been studied. pSLneo contains the SV40 origin of replication and encodes the positive selectable marker for G418 resistance. In transient replication assays, pSLneo replicates to a high copy number in COS7 cells. Uncontrolled SV40 plasmid replication has been reported to be lethal to such transfected cells. Thus, it was anticipated that extensive plasmid replication would preclude isolation of permanent cell lines containing pSLneo. However, significant number of G418-resistant colonies arose after transfection of COS7 cells with pSLneo. Cell lines established from these drug-resistant colonies contained between 100 and 1,000 extrachromosomal pSLneo copies per cell. Episomal plasmid DNA in pSLneo/COS7 lines was stably maintained after 2 months of continuous culture in selective medium. Bromodeoxyuridine labeling and density shift experiments demonstrated that replication of pSLneo closely paralleled that of cellular DNA. On average, plasmid DNA did not replicate more than once during a single cell generation period. Regulation of pSLneo replication appeared to be negatively controlled by a cis-acting mechanism. Endogenous copies of episomal pSLneo remained at a stable low copy number during the simultaneous, high-level replication of a newly transfected plasmid encoding SV40 large T antigen in the same cells. These results indicate that regulated replication of an SV40 origin plasmid can be acquired in a cell and does not require the presence of additional genetic elements. The molecular mechanism by which cells enforce this regulation on extrachromosomal SV40 plasmids remains to be defined.     

A diploid rat liver cell culture

Summary Chronic exposure of a cloned rat hepatocyte culture (RL-PR-C) to a subtoxic, sublethal dose of aflatoxin B₁ resulted in malignant transformation. Continuous exposure to aflatoxin B₁ caused increasing tumorigenic potential as tested by back injection into isogenic animals. Control cultures exhibited spontaneous transformation, although approximately 20 passages beyond the chemically induced event. Neither aflatoxin-treated nor control cultures exhibited cytopathological morphology, formation of cell foci, growth in soft agar, or irregular fibroblast-like growth patterns that could be specifically related to the onset of tumorigenic potential. In general, those parameters commonly used to monitor fibroblast cultures for transformation in vitro were not applicable for assessing the tumorigenic potential of these epithelial cells. Karyotypic analyses revealed no specific chromosomal aberrations associated with aflatoxin treatment; however, chromosomal instability was a property of the tumorigenic cell populations. Injection of both aflatoxin-treated and control cultures at passage 56 resulted in tumors indicative of both carcinoma and sarcoma indicating to us the multipotency of these epithelial cells transformed in vitro.     

Transformation of human cultured fibroblasts with plasmids carrying dominant selection markers and immortalizing potential

The disadvantages of using human cultured cells for biochemical and genetic studies are their limited lifespan in vitro and their lack of chemical selection markers. These problems are now overcome by transfecting human cultured fibroblasts with the pSV3-gpt and pSV3-neo plasmid DNA which carry genes coding for the immortalizing SV40 large T-antigen and dominant selection markers. Transformed human fibroblasts were obtained at a frequency of about 10(-5) with both selection systems. These transformed cells showed a twofold increase in growth rate and three to tenfold increase in cell number at confluence. The improved growth characteristics were associated with the expression of the SV40 T-antigen detected with immunoprecipitation. These cell lines also changed from their usual spindle shapes to an epithelioid morphology characteristic of transformed cells. From 60 to 100% of the cells transfected with pSV3 plasmid DNA demonstrated numerical and structural abnormalities in their karyotypes. Cells transfected with DNA from a similar plasmid, pSV2-neo, which differed from the pSV3-neo plasmid only by missing the sequence encoding the complete early region of SV40, neither expressed T-antigen nor showed any change in morphology, improvement in growth characteristics or abnormalities in karyotype. However, they were still selectable with the aminoglycoside G-418. Therefore, by appropriate choice of vector plasmids, dominant selection markers and improved growth characteristics can be imparted separately or simultaneously to human fibroblasts. The morphological, biochemical and chromosomal changes resulting from such transformations must be recognized in using this approach for biochemical and genetic studies.