SV40 transfection of human kidney epithelial cells and stability of chromosome 11

Simian virus 40 (SV40) transformation has been used in a variety of mammalian cells and has been shown to extend their life span. We therefore decided to apply these results to normal kidney and tumoral cells derived from Wilms' patients. Wilms' tumour, a nephroblastoma which presents in early childhood, has been linked to deletions and rearrangements in chromosome 11. Analysis of gene structure in the 11p13 locus involved in the development of the tumour has been restricted by the very short life-span of the tumoral cells in vitro. We transfected normal kidney WT/NK, tumoral WT/T cells and human foetal kidney HFK cells with 2 SV40-derived plasmids SV3neo (pBR322-SV40-containing neomycin bacterial gene) and SVori- (pMK-origin mutated SV40). We isolated a high number of SV40-transfected cell lines. The efficiency of transfection appeared to be extremely low in WT/T cells compared with HFK and even WT/NK. The life span of the cell lines was increased in relation to their untransfected homologues. However, in all of the cell lines except 3, senescence occurred, after crisis step or not. We looked at different markers associated with SV40 transformation of mammalian cells and specifically with the presence of SV40 T antigen in the cells and its consequences: AIG, tumorigenicity, expression and insertion in genomic DNA of SV40 T antigen. Genetic studies involving karyotypic and restriction fragment length polymorphism (RFLP) analysis demonstrated that, despite a frequent pseudo-diploidy, the cell lines derived have conserved the 11p13 locus.     

Parafibromin tumor suppressor enhances cell growth in the cells expressing SV40 large T antigen

Parafibromin (PF) is a 531-amino acid protein encoded by HRPT2, a putative tumor suppressor gene recently implicated in the autosomal-dominant hyperparathyroidism-jaw tumor familial cancer syndrome and sporadic parathyroid carcinoma. To investigate effects of PF's overexpression on cell proliferation, we performed assays in four different cell lines. The transient overexpression of PF inhibited cell growth in HEK293 and NIH3T3 cells, but enhanced cell growth in the SV40 large T antigen-expressing cell lines such as 293FT and COS7 cells. In 293FT cells, PF was found to interact with SV40 large T antigen and its overexpression promoted entry into the S phase, implying that the interaction enhanced progression through the cell cycle. The tumor suppressor protein PF acts as a positive regulator of cell growth similar to an oncoprotein in the presence of SV40 large T antigen.     

Sensitization of transformed rat cells to parvovirus MVMp is restricted to specific oncogenes

The rat cell line FR3T3 was transformed with the retroviral oncogenes v-myc or v-src, with the DNA tumor viruses SV40 or bovine papilloma virus strain 1 (BPV-1) or with the 69% transforming region of BPV-1. The transformants were compared with the uncloned parental line for their susceptibility to the lytic effect and to the replication of MVMp, an autonomous parvovirus. Expression of v-myc and v-src proteins and of SV40 large T antigen correlated with a greater cell susceptibility to MVMp-induced killing. Thus, the expression of both cytoplasmic and nuclear oncogene products may sensitize rat fibroblasts to MVMp. In contrast, cell lines transformed by BPV-1, including highly tumorigenic and tumor-derived clones, were on the average as resistant as the parental cell line to MVMp infection. A similar resistance to MVMp-induced killing was displayed by BPV-1-transformed NIH3T3 cells. However, supertransformation of one of the BPV-1-transformants by the human EJ-Harvey ras-1 oncogene, known to sensitize FR3T3 and NIH3T3 cells, correlated with an increase in susceptibility to MVMp. Therefore, the failure of BPV-1 transformation to sensitize murine cells to parvoviral attack may be ascribed to the tumor virus rather than to the cells undergoing transformation. Hence, cell sensitization to MVMp appears to be oncogene-specific and cannot be taken as an absolute correlative with neoplastic transformation.     

Design of a selectable reporter for the detection of mutations in mammalian simple repeat sequences

To study the mutator phenotype characteristic of tumors showingwidespread replication errors at simple DNA repeat sequences(RER+), we designed a selectable reporter system for the detectionof such mutations in mammalian cells. A hygromycin B phosphotransferasegene was rendered out-of-frame by the insertion of a (CA)₁₃dinucleotide repeat tract immediately following the ATG startcodon, and subcloned into a retroviral expression vector containinga G418 (neo) selectable marker. Following transduction of thisconstruct into cultured cells, clonal neo+ cell lines were establishedand then tested for their ability to form colonies in hygromycinB-containing medium. Using this system, we found that the HCT116,LS174T and LS180 human colon carcinoma cell lines acquire hygromycinresistance (hyg^r) at a 100-fold higher frequency than the HT29,SW480, DLD-1 and HCT15 human colon carcinoma and NIH3T3 fibroblastcell lines, and at a 25-fold higher rate than the Rat 6 embyrofibroblast cell line. DNA sequence analysis indicated that frameshiftmutations had occurred within the CA dinucleotide repeat tractin HCT116 cells that became hyg^r. Thus, the mutation rates atsimple repeated sequences in mammalian cell lines can be readilydetermined and studied using this system.     

Evidence against a role for SV40 in human mesothelioma

SV40 has been implicated in the etiology of 40% to 60% of human mesotheliomas. These studies could have important medical implications concerning possible sources of human infection and potential therapies if human tumors are induced by this agent. We did PCR-based analysis to detect SV40 large T antigen DNA in human mesotheliomas. None of 69 tumors in which a single copy gene was readily amplified contained detectable SV40 large T antigen sequences. Under these conditions, it was possible to detect one copy of integrated SV40 DNA per cell in a mixture containing a 5,000-fold excess of normal cells using formalin-fixed preparations. Kidney, a known reservoir of SV40 in monkeys, from some of these individuals were also negative for SV40 large T antigen sequences. A subset of mesotheliomas was analyzed for SV40 large T antigen expression by immunostaining with a highly specific SV40 antibody. These tumors as well as several human mesothelioma cell lines previously reported to contain SV40 large T antigen were negative for detection of the virally encoded oncoprotein. Moreover, mesothelioma cell lines with wild-type p53 showed normal p53 function in response to genotoxic stress, findings inconsistent with p53 inactivation by the putative presence of SV40 large T antigen. Taken together, these findings strongly argue against a role of SV40 by any known transformation mechanism in the etiology of the majority of human malignant mesotheliomas.     

Establishment of rat endometrial cell lines by retroviral mediated transfer of immortalizing and transforming oncogenes.

To study hormone responsive genes in differentiated epithelial cells and as a model for endometrial carcinoma, lines were established from primary rat endometrial cells by infection with replication-defective retroviruses carrying oncogenes and the selectable gene neo. The initial step involved immortalization through the large T antigen of SV40 to generate a line we designate RENT4, or with the E1a gene of adenovirus to generate lines referred to as RENE1 and RENE2. Additionally, lines generated by large T antigen of SV40 were superinfected with a replication-defective retrovirus harboring the v-Ha-ras oncogene and selected by the ability to form colonies in soft agar. The latter cell lines appeared fully transformed and were designated RENTR01 and RENTR03. Five established lines were characterized for steroid hormone receptors, alkaline phosphatase activity and their complement of the intermediate filaments vimentin and cytokeratin. With the exception of the RENE1 cells all other lines have normal levels of glucocorticoid receptor, whereas only RENE1, RENE2 and RENT4 were positive for the progesterone receptor. RENTR01, RENTR03 and, to a lesser extent, RENE1 exhibited differential expression of cytokeratins dependent upon whether the cells were grown on a substrata of NIH3T3 cells. When grown on formalin-fixed NIH3T3 cells, RENTR01 and RENTR03 cells appeared to differentiate or rearrange themselves in culture. Individual islands of cells showed a heterogeneous pattern of intermediate filaments with vimentin-positive cells localized to the outer portion of the islands whereas cytokeratin-positive cells are seen on the insides of these structures.     

Transformation of late passage insulin-like growth factor-I receptor null mouse embryo fibroblasts by SV40 T antigen

There is evidence that the insulin-like growth factor-I (IGF-I) receptor is required for transformation by a variety of viral and cellular oncogenes in a mouse embryo fibroblast model. To further investigate the IGF-I receptor signaling pathways that are required for the permissive effect of the receptor on transformation by SV40 T antigen, we established three independent fibroblast cell lines each from wild-type and IGF-I receptor null embryos (R-). We transfected the wild-type and R- cell lines with an SV40 T antigen plasmid and selected three clones from each cell line that expressed T antigen. As in previous reports, none of the cloned R- cell lines expressing T antigen were transformed as measured by the ability to form large colonies in soft agar. However, with further passage, all three T antigen-expressing clones from one of the R- cell lines (R(-)3) formed large colonies in soft agar and the transformation of these T antigen-expressing clones was confirmed by tumorigenesis experiments in immunodeficient mice. DNA microarray analysis comparing gene expression between early passage and late passage R(-)3/T antigen clones showed, among other changes, an increase in the expression of ErbB-3 mRNA in the late passage clones. Also, the expression of ErbB-3 protein was dramatically increased in the late passage R(-)3/T antigen clones. We conclude that late passage IGF-I receptor null mouse embryo fibroblasts can be transformed by SV40 T antigen, and that ErbB-3 may play a role in permitting transformation by T antigen.     

Single-steep transformation of human breast epithelial cells by SV40 large T oncogene.

Normal human mammary epithelial cell (HMEC) cultures originating from 2 mammoplasty reduction surgical samples were transfected with replication-defective SV 40 DNA. Two independent cell lines designated as S2T2 and S1T3, selected for their increased proliferation potential and lifespan, were propagated for greater than 22 months in culture. They maintained a near-diploid karyotype with few chromosomal markers such as trisomy 1q (S1T3) and trisomy 8q (S2T2), which are most common in breast cancer in vivo. Immortalized S1T3 cells were not tumorigenic, whereas S2T2 cells produced slowly growing tumors in nude mice. One tumor was propagated in vitro and the transformed NS2T2 cell line subsequently raised 100% large tumors in the nude mouse. Rearrangement of the SV40 genome was observed in NS2T2 cells, which was not associated with increased expression of large T antigen. S1T3, S2T2 and transformed NS2T2 cell lines expressed cytokeratins CK18, CK19, the mammary-specific antigen DF3, and functional EGF receptors. Single-step immortalization and malignant transformation of human breast epithelial cells can thus occur upon transfection with SV40 large T oncogene. The chromosomal abnormalities observed in these cell lines suggest that they could offer a model for the study of breast-tumor progression in vitro.     

Genomic DNA is captured and amplified during double-strand break (DSB) repair in human cells

Genomic stability is maintained by the surveillance and repair of DNA damage. Here, we describe a mechanism whereby repair of extrachromosomal DNA double-strand breaks (DSBs) in human cells can be accompanied by capture of genomic DNA fragments. The availability of the human genome sequence enabled us to characterize these inserts in cells from a normal individual and from a patient with ataxia telangiectasia (AT), deficient for the damage response kinase ATM and prone to genomic instability. We find AT cells exhibit insertions of human chromosomal DNA fragments in excess of 17 kb during DSB repair, whereas we detected no such genomic inserts in normal cells. However, the presence of simian virus 40 (SV40), used to transform these cell lines, resulted in capture of genomic DNA associated with sites of viral integration in both cell types. The genomic instability at sites of SV40 integration was exported to other sites of DNA damage, and acquisition of the viral origin of replication resulted in gene amplification through autonomous replication of the plasmid harbouring the repaired extrachromosomal DSB. Should this same phenomenon apply to the repair of chromosomal DSBs, genome rearrangements made possible via this DSB insertional repair pose risks to genomic integrity, and may contribute to tumorigenic progression.     

SV40 oncoproteins enhance asbestos-induced DNA double-strand breaks and abrogate senescence in murine mesothelial cells

SV40 virus has emerged as a potential cofactor with asbestos in the development of diffuse malignant mesothelioma, but its precise role in the pathogenesis of this tumor is unclear. SV40 large T antigen is known to inactivate cellular proteins involved in DNA damage and senescence, including p53 and pRb. We hypothesize that SV40 oncoproteins will sensitize mesothelial cells to DNA damage induced by asbestos or chemotherapeutic agents. SV40 oncoprotein expression in murine mesothelial cell lines enhanced spontaneous and asbestos-induced double-strand breaks, indicated by gamma-H2AX foci, and potentiated micronucleus formation. Mesothelial cells exposed to asbestos or bleomycin for 96 h acquired senescent-like morphology and displayed elevated senescence-associated beta-galactosidase activity, reduced bromodeoxyuridine (BrdUrd) incorporation, and reduced colony formation. SV40 oncoprotein expression abrogated the senescent phenotype, and transfected cell lines showed an increase in both BrdUrd incorporation and colony formation after prolonged DNA damage. Murine mesothelial cell lines lacking wild-type p53 due to a point mutation or gene rearrangement also failed to senesce in response to asbestos or chemotherapeutic agents. In addition, stress-induced senescence in human mesothelial cell lines was impaired by SV40 oncoprotein expression (MeT-5A), p53 small interfering RNA, or spontaneous p53 mutation (REN). These studies suggest that exposure to DNA-damaging agents can induce senescence in both murine and human mesothelioma cell lines and suggest a major, although not exclusive, role for p53 in this response. SV40 virus may contribute to mesothelioma progression by impairing stress-induced senescence, in part through p53 inactivation, thereby favoring survival and proliferation of mesothelial cells that have sustained DNA damage.     

Establishment of human retinal pigment epithelial cell lines by oncogenes

The primary human retinal pigment epithelial cells were transfected with oncogenic sequences derived from viruses and cellular homologues of retroviral oncogenes 'protooncogenes' linked to simian virus 40 (SV-40) and retroviral promoters. Foci of cells were noted between 2 to 4 weeks after transfection. Individual colonies of cells were expanded from cultures transfected with SV-40 virion DNA, SV-40 large T antigen gene, Ha-ras oncogene, human and mouse c-myc and adenovirus E1A gene. Established cell lines tested were positive for the specific oncogene sequences by Southern hybridization and also expressed the protein as assayed by immunofluorescence and immunoblot analysis. Cell lines established with SV-40 large T antigen, and SV-40 virion DNA, exhibited epithelioid morphology up to the 25th passage and later became more rounded. However, all cell lines established with other oncogenes continued to retain epithelial morphology. Functional analysis of the cell lines demonstrated the presence of polarity and the ability to phagocytize rod outer segments, characteristics of retinal pigment epithelial cells. The use of oncogenes with immortalization/transformation potential may allow the establishment of cell lines from ocular tissues for analysing the biochemical basis of a disease like retinitis pigmentosa.     

Infrequent existence of simian virus 40 large T antigen DNA in malignant mesothelioma in Japan

Malignant mesothelioma is the most common primary pleural neoplasm. Association of simian virus 40 (SV40) with malignant mesothelioma has been reported, suggesting that SV40 plays an important role in the origin of a subset of these tumors. However, significant geographic variation is present as to how often this association occurs. As no study concerning SV40 in malignant mesothelioma has been reported from Japan, we examined the frequency of SV40 infection in Japanese malignant mesothelioma cases. In pleural malignant mesothelioma tissue from 35 patients in Japan, we sought the presence of SV40 large T antigen DNA using real-time polymerase chain reaction (PCR), as well as expression of the viral protein using immunohistological methods. Real-time PCR demonstrated that two of 35 mesotheliomas contained DNA sequences encoding portions of SV40 large T antigen. None of the 35 malignant mesothelioma specimens showed immunoreactivity for SV40 large T antigen. SV40 infection does not appear to have a major role in the development of malignant mesothelioma in Japan.     

Detection and quantification of SV40 large T-antigen DNA in mesothelioma tissues and cell lines

SV40 viral DNA sequences, particularly large T-antigen (T-ag) DNA, have been reported in malignant pleural mesothelioma (MPM) and suggested to play a role in the tumorigenesis of this cancer. These results remain somewhat controversial owing to variability among a number of laboratories in reported SV40 DNA and protein detection in MPM tissues. This could be explained in part if SV40 DNA is present in relatively low abundance in many MPM tissues determined to contain viral sequences and is therefore difficult to detect. To this end, we investigated the efficacy of real time quantitative PCR in detecting low copy number SV40 DNA sequences, then we quantified SV40 copy number in MPM tissues and cell lines at our institution. Quantitative PCR demonstrated consistent precision and accuracy in detecting SV40 DNA sequences over a large logarithmic range of viral copy number. In addition, SV40 sequences were found in 2/35 tissues and 3/7 cell lines in relatively low abundance. We conclude that SV40 is not a contributing factor in the pathobiology of the majority of MPM tumors from patients at our institution.     

SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation

DNA tumor viruses such as simian virus 40 (SV40) express dominant acting oncoproteins that exert their effects by associating with key cellular targets and altering the signaling pathways they govern. Thus, tumor viruses have proved to be invaluable aids in identifying proteins that participate in tumorigenesis, and in understanding the molecular basis for the transformed phenotype. The roles played by the SV40-encoded 708 amino-acid large T antigen (T antigen), and 174 amino acid small T antigen (t antigen), in transformation have been examined extensively. These studies have firmly established that large T antigen's inhibition of the p53 and Rb-family of tumor suppressors and small T antigen's action on the pp2A phosphatase, are important for SV40-induced transformation. It is not yet clear if the Rb, p53 and pp2A proteins are the only targets through which SV40 transforms cells, or whether additional targets await discovery. Finally, expression of SV40 oncoproteins in transgenic mice results in effects ranging from hyperplasia to invasive carcinoma accompanied by metastasis, depending on the tissue in which they are expressed. Thus, the consequences of SV40 action on these targets depend on the cell type being studied. The identification of additional cellular targets important for transformation, and understanding the molecular basis for the cell type-specific action of the viral T antigens are two important areas through which SV40 will continue to contribute to our understanding of cancer.     

Detection of tumor-specific cell surface antigen of simian virus 40-induced tumors by the isotopic antiglobulin technique

Simian virus 40 (SV40) tumor-specific cell surface antigen (TSSA) was assayed by the isotopic antiglobulin technique. Antisera were produced by immunization of A/LN mice with syngeneic SV40-induced tumor designated SV-A/LN. Positive reactions were only obtained with SV40-transformed mouse or hamster cells which possessed both SV40 TSTA (tumor-specific transplantation antigen) and T antigen. Negative reactions were obtained with untransformed cells or non-SV40 tumors. The T antigen-negative cells (“T- S+” by Tevethia et al., 1968), which lack TSTA and viral RNA or viral DNA, also gave negative reactions. Absorption experiments further confirmed the specificity. These results showed that the SV40 TSSA detected by the isotopic antiglobulin technique was virus-specific, and was correlated with the expression of TSTA.     

Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences.

The normal genomic stability of human cells is reversed during neoplastic transformation. The SV40 large T antigen alters the DNA content in human endometrial stromal cells in a manner that relates to neoplastic progression. Human endometrial stromal cells were transfected with a plasmid containing the A209 temperature-sensitive mutant of SV40 (tsSV40), which is also defective in the viral origin of replication. Ninety-seven clonal transfectants from seven different primary cell strains were isolated. Initial analysis revealed that 20% of the clonal populations (19/97) had an apparent diploid DNA content, 35% (34/97) had an apparent tetraploid DNA content, and the remainder were mixed populations of diploid and tetraploid cells. No aneuploid populations were observed. Diploid tsSV40 transformed cells always give rise to a population of cells with a tetraploid DNA content when continuously cultured at the permissive temperature. The doubling of DNA content can be vastly accelerated by the sudden reintroduction of large T antigen activity following a shift from non-permissive to permissive temperature. Tetraploid tsSV40 transfected cells have a lower capacity for anchorage-independent growth and earlier entry into 'crisis' than diploid cells. These results indicate that during the pre-crisis, extended lifespan phase of growth, the SV40 large T antigen causes a doubling of DNA content. This apparent doubling of DNA content does not confer growth advantage during the extended lifespan that precedes 'crisis'.     

Efficient replication of plasmids containing the SV40 origin in N-myc overexpressing human neuroblastoma cells.

Using test plasmids containing the SV40 origin, we found a wide spectrum of permissiveness to their replication in different human cell lines. N-myc overexpressing neuroblastoma cells were highly permissive. LA-N-1 neuroblastoma cells were the most permissive of all the cell lines that we tested including the homologous CV-1 or COS-1 monkey kidney cells. Other human cell lines expressing various amounts of c-myc, and the 293 cell line expressing adenovirus E1A and E1B exhibited intermediate levels of permissiveness. T24 and EJ bladder carcinoma cells, which do not express the myc genes, were nonpermissive. Transient expression of c-myc or N-myc from plasmid vectors resulted in a modest stimulation of replication. Replication of test plasmids containing different configurations of the SV40 origin region was activated by the myc proteins. The high efficiency of replication in LA-N-1 cells is due to a combination of reasons including the overproduction of N-myc, high efficiency of expression of the SV40 replication initiator protein large T antigen from a cotransfected expression plasmid (containing the T antigen gene under the RSV LTR control), and other unknown host cell replication stimulatory factors. Replication of test plasmids was not detected in N-myc or c-myc overexpressing cells when the T antigen expression plasmid was not provided, showing that the myc proteins cannot substitute for T antigen in SV40 DNA replication.     

Reversion of middle T antigen-transformed Rat-2 cells by Krev-1: implications for the role of p21c-ras in polyomavirus-mediated transformation.

Polyomavirus middle T antigen mediates transformation of cells, at least in part, by its association with and activation of the intrinsic protein tyrosine kinase activity of pp60c-src. pp60c-src, by analogy with pp60v-src, elicits cell proliferation through a signal transduction pathway that includes p21c-ras. Therefore, we tested the possibility that middle T antigen acts upstream of and in the same proliferative signaling pathway as p21c-ras. Co-transfection of Rat-2 cells with plasmids expressing human Krev-1, a dominant suppressor of Ki-ras transformation, and mT antigen resulted in a dose-dependent reduction of mT antigen-induced foci. Krev-1 did not affect the transforming activity of SV40 large T antigen, demonstrating that the transformation-suppressing activity of Krev-1 is specific. To determine the effect of Krev-1 on stably transformed cell lines, Krev-1 DNA was introduced into middle T antigen-transformed Rat-2 cells along with a G418 resistance marker. Of the G418-resistant colonies examined, 1% were morphologically untransformed. Characterization of several morphological revertants revealed that, with the exception of one cell line, all of the cell lines expressed middle T antigen, which was associated with pp60c-src, whose tyrosine kinase activity was similar to that found in the parental transformed cell lines. To determine whether other phenotypic traits associated with transformation were altered in these cell lines, their growth rates and ability to form colonies in agar suspension were examined. The majority of the revertants had longer doubling times, and grew less efficiently in agar suspension compared with their transformed parents. A direct correlation was observed between Krev-1 RNA and protein expression and the efficiency with which the revertants formed colonies in suspension. These results suggest that p21c-ras lies downstream of middle T antigen and pp60c-src in the same proliferative signal transduction pathway.     

Biologic properties of viable deletion mutants of simian virus 40 (SV40) rescued from the cells of an SV40-induced hamster lymphocytic leukemia

A lymphocytic leukemia induced by the oncogenic DNA simian virus 40 (SV40) in an inbred LSH/SsLak Syrian golden hamster was evoked to produce infectious SV40 by fusion of the leukemia cells with grivet monkey kidney (GMK) cells and by exposure of the leukemia cells to the chemical inducers mitomycin C and cycloheximide. Plaque-purified viable substrains of the rescued SV40 when studied by restriction endonuclease digestion of viral DNA were found to contain small deletions within the Hind III restriction fragment C. These deletions lay near the viral origin of DNA replication. Ten plaque-purified substrains of the rescued virus identified by immunofluorescence as being SV40 were found, when compared to the wild-type SV40, to replicate slowly and to form small plaques. Although these substrains transformed NIH/3T3 cells as efficiently as the wild-type SV40 in tissue culture, they were generally less oncogenic in vivo--7 of the 10 failed to induce tumors. The 3 oncogenic SV40-rescued substrains were not found to exhibit "lymphocytotropism," i.e., the capacity to infect and neoplastically transform preferentially hamster lymphocytes. Thus the hamster lymphocytic leukemia originally induced by the wild-type SV40 was most likely a chance-stochastic event rather than the result of tropism-determinism mediated by the virus, as is usually the case with leukemogenic RNA viruses.