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.     

High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures

Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures. Transient and stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48 h after bombardment, was abundant, and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed cells by gradually increasing the concentrations of the antibiotic Geneticin (G418). Southern analysis indicated a minimum transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures less than 3 months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with abnormal morphology and a high degree of sterility. Received: 20 January 1999 / Revision received: 1 October 1999 / Accepted: 11 October 1999     

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.     

Efficient transformation and regeneration of carnation cultivars usingAgrobacterium

We have developed an efficient method for transformation and regeneration of plants from carnation,Dianthus caryophyllus L. Whole leaves fromin vitro shoot cultures were mixed withAgrobacterium, cocultivated for 5 days and then plated on 2 µg/l chlorsulfuron (CS). Regenerated shoots and shoot clusters were divided into smaller sections and plated on 3 µg/l CS for selection to produce fully transformed shoots. Geneticin (G418) and kanamycin used were not as effective selective agents as CS. All regenerated shoots were vitrified. These were normalized, rooted and transferred to the greenhouse. 100% of regenerated plants were transformed based on rooting assay, GUS assay, PCR and Southern analysis.     

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells.

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.     

The loss of contact inhibition and anchorage-dependent growth are key steps in the acquisition of Listeria monocytogenes susceptibility phenotype by non-phagocytic cells

Summary— We have previously demonstrated that intestinal and kidney finite cell lines were resistant to L monocytogenes invasion (ie allowed low bacterial entry and no intracellular multiplication) in contrast to the continuous cell lines which were susceptible to Listeria invasion (ie allowed high bacterial entry and intracellular multiplication) (Velge et al (1994a) Med Microbial Immunol 183, 145). The aim of this study was to discover whether epigenetic or genetic cellular modifications could convert L monocytogenes resistant cells into a susceptible phenotype and to determine the cellular steps involved in Listeria susceptibility. Among the 5-azacytidine treated finite cell lines, the untransformed immortal cell lines established remained resistant to L monocytogenes invasion whereas the weakly transformed continuous cell lines established were converted into a susceptible phenotype. Transfection of resistant cells by SV40 large T antigen induced only highly transformed continuous cell lines displaying a susceptible phenotype. Taken together these data show that cell transformation enhanced Listeria invasion. This conclusion was supported by the observation that L monocytogenes was able to induce cell foci within murine finite cell monolayers. This morphological cell transformation was completely reversible and required live bacteria inside cells. In conclusion, we may speculate that the L monocytogenes intracellular multiplication observed within cell foci could be explained by the loss of contact inhibition of the finite cell monolayer. Indeed, the loss of both contact inhibition and anchorage-dependent growth are the key steps involved in the L monocytogenes susceptibility phenotype.     

Immortalization of mutant p53-transfected human fibroblasts by treatment with either 4-nitroquinoline 1-oxide or x-rays

Summary The study of in vitro cell transformation is valuable for understanding the multistep carcinogenesis of human cells. The difficulty in inducing neoplastic transformation of human cells by treatment with chemical or physical agents alone is due to the difficulty in immortalizing normal human cells. Thus, the immortalization step is critical for in vitro neoplastic transformation of human cells. We transfected a mutant p53 gene (mp53: codon 273^Arg-His) into normal human fibroblasts and obtained two G418-resistant mp53-containing clones. These clones showed an extended life span but ultimately senesced. However, when they were treated with either 4-nitroquinoline 1-oxide or X rays, they were immortalized. The immortalized cells showed both numerical and structural chromosome abnormalities, but they were not tumorigenic. The expression of mutant but not wild type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in increased cdk2 and cdc2 kinase activity. However, there was no significant difference between the normal and immortalized human cells in expression of another tumor suppressor gene, p16. These findings indicate that the p53-p21 cascade may play an important role in the immortalization of human cells.     

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.     

X chromosome inactivaton and SV40 transformation of mammalian cells.

Five embryonic mouse cultures and one human fibroblast culture were transformed with SV40. The cultures were studied cytologically to see if the normal pattern of sex chromosome replication was maintained in SV40 transformed cells. Characteristic late replication patterns were observed for both the X and Y chromosomes, and there was no evidence for loss of the inactive X chromosome, even in cells with 4 or more X chromosomes. The human line was heterozygous at two X-linked loci and a clonal analysis showed that the expression of X-linked genes was not affected by SV40 transformation.     

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.     

Spontaneously immortalized mouse mesothelial cells display characteristics of malignant transformation

Abstract. Objectives: Mesotheliomas occur in occult serous cavities after chronic exposure of mesothelial cells to asbestos fibres. Molecular events that contribute to the development of this cancer are therefore not readily accessible for study. We have used in vitro culture systems to study and compare induced and spontaneous transformation events in primary mouse mesothelial cells. Materials and methods: Mouse mesothelial cells were cultivated until small populations of proliferating cells emerged from senescing cultures. Spontaneously transformed cultures of cells were characterized and compared to malignantly transformed cells. Results: Human mesothelial cells had a finite lifespan of 10–15 population doublings when cultured in vitro; mouse mesothelial cells typically exhibit this same pattern. Here, we show that mouse mesothelial cells can be cultured for extended periods and that these cells can transform spontaneously. Lines of spontaneously transformed cells generated in this study are immortal and growth factor‐independent. They display the salient characteristic features of transformation, including increased proliferation rate, lack of contact inhibition, aneuploidy and ability to grow in anchorage‐independent conditions. A subset of these cell lines developed into tumours in syngeneic mice. Comparative gene expression analysis demonstrated that spontaneously transformed cell lines were more closely related to neoplastic cells than to primary cells. Conclusion: These findings have implications for interpretation of in vitro transformation studies, demonstrating broad similarity between spontaneous and induced genetic changes.     

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.     

Reprogramming of cell junction modules during stepwise epithelial to mesenchymal transition and accumulation of malignant features in vitro in a prostate cell model

Epithelial to mesenchymal transition (EMT) is pivotal in tumor metastasis. Our previous work reported an EMT model based on primary prostate epithelial cells (EP156T) which gave rise to cells with mesenchymal phenotype (EPT1) without malignant transformation. To promote prostate cell transformation, cells were maintained in saturation density cultures to select for cells overriding quiescence. Foci formed repeatedly following around 8 weeks in confluent EPT1 monolayers. Only later passage EPT1, but not EP156T cells of any passage, could form foci. Cells isolated from the foci were named EPT2 and formed robust colonies in soft agar, a malignant feature present neither in EP156T nor in EPT1 cells. EPT2 cells showed additional malignant traits in vitro, including higher ability to proliferate following confluence, higher resistance to apoptosis and lower dependence on exogenous growth factors than EP156T and EPT1 cells. Microarray profiling identified gene sets, many of which belong to cell junction modules, that changed expression from EP156T to EPT1 cells and continued to change from EPT1 to EPT2 cells. Our findings provide a novel stepwise cell culture model in which EMT emerges independently of transformation and is associated with subsequent accumulation of malignant features in prostate cells. Reprogramming of cell junction modules is involved in both steps.     

Characterization of the cell cycle of cultured human diploid cells: Effects of aging and hydrocortisone

Age-related changes in the cytokinetics of human diploid cells in vitro have been compared in normal cultures and in cultures in which lifespan has been prolonged by the addition of hydrocortisone to the medium. For both cultures, with advancing age the fraction of cells in the actively proliferating pool decreased and the intercellular variation in cell cycle times increased. The average cell cycle time was prolonged during aging due almost entirely to changes in the duration of G₁. The duration of S remained constant, while a small delay in G₂ was observed in late passage cells near the end of their lifespan. Although the same pattern of change in proliferative parameters occurred in both control and hydrocortisone-treated cultures, the changes were somewhat delayed in the presence of the steroid. The results are interpreted in terms of several cell cycle models and suggest that the events controlling cell proliferation are sensitive to hydrocortisone modulation during the G₁ and possibly the G₂ periods.     

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.     

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.     

THE 'TRANSITION PROBABILITY MODEL' AND THE REGULATION OF PROLIFERATION OF HUMAN DIPLOID CELL CULTURES DURING AGING

Genealogies derived from time-lapse cinemicrophotographic studies of aging human diploid cell cultures were analysed in terms of the ‘transition probability’ model. It was found that the distribution of intermitotic times obtained from middle passage cells deviated only slightly from that predicted by the model. In contrast, the plot for late passage cultures did not fit the predicted pattern and appeared to be composed of multiple curves. These changes are discussed in reference to cellular senescence as expressed by normal human diploid cells in vitro.