SV40-transformed Human Cell Line Research Articles

Abstract 1323: Liver-specific knockout mice and liver-derived cell lines provide insights into potential roles for the GP73/GOLM1 HCC serum biomarker - association with sustained cell proliferation

Abstract Golgi Protein-73kDa (GP73/GOLM1) is a resident cis Golgi Type II membrane protein that is nearly undetectable in normal liver, but whose expression is upregulated in hepatocellular carcinoma (HCC) and whose circulating levels increase in the plasma of patients with chronic liver disease and HCC. As such, it is the subject of ongoing clinical trials investigating its use as a serum biomarker for HCC. GP73/GOLM1 is cleaved by a proprotein convertase and secreted by proliferating cells, an activity possibly elevated in cancer, yet its biological role in the liver and in the pathogenesis of HCC remains unclear. In order to investigate possible biological roles for GP73/GOLM1, liver-specific knockout mice (C57BL/6) were generated using the Cre-loxP system, characterized by a “floxed” GOLM1 gene and Cre recombinase driven by the albumin promoter. GP73/GOLM1 Cre+/−genotypes were confirmed by PCR analysis. GP73/GOLM1Fl/Fl/Cre(+) animals showed no obvious biological phenotype compared to their Cre(-) littermates, and exhibited normal growth, behavior, and successful mating, suggesting that hepatic GP73/GOLM1 is not vital for normal physiological function, as might be hypothesized from its normally low expression in the liver. Previous studies indicated that GP73/GOLM1 expression was not upregulated in post-hepatectomy liver regeneration, suggesting that its activated expression in the liver is linked to inflammatory or carcinogenic processes rather than transiently increased mitosis. Examination of GP73/GOLM1 expression in 14 human HCC cell lines revealed similar expression levels in epithelial and mesenchymal cell lines, and in an SV40-transformed nontumorigenic human liver epithelial cell line. Rodent in vitro models of hepatocellular transformation revealed equal GP73/GOLM1 expression levels in nontumorigenic parent cell lines and their transformed tumorigenic derivatives. Collectively, the data from the in vivo and in vitro models thus far do not support a direct association of GP73/GOLM1 expression with liver mitogenesis or differentiation, but rather suggest that its expression may be linked to a sustained commitment to cell proliferation - such as occurs in cell lines, liver disease and cancerous transformation, promoted by an inflammatory microenvironment. Possible mechanistic roles for GP73/GOLM1 in HCC development and progression are currently being tested in carcinogenesis models with liver-specific knockout mice. A website on GP73/GOLM1 has also been established as a resource for the research community interested in the study of this protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1323. doi:1538-7445.AM2012-1323

Release of Soluble Tumor Necrosis Factor Receptor 1 from Corneal Epithelium by TNF-α–Converting Enzyme-Dependent Ectodomain Shedding

An involvement of tumor necrosis factor-alpha-converting enzyme (TACE)-dependent ectodomain shedding in the release of soluble tumor necrosis factor receptor 1 (sTNFR1) from corneal epithelium was evaluated. In vitro experiments were performed using the human SV40-transformed human corneal epithelial cell (HCEC) line. Ectodomain shedding was stimulated by phorbol myristate acetate (PMA, 3 microM) or peptidoglycan (PGN, 100 microg/mL), with or without TACE inhibition, using TNF-alpha processing inhibitor-1 (TAPI-1, 250 microg/mL) or tissue inhibitor of metalloproteinase-3 (TIMP-3, 2 microg/mL) by addition to the HCEC culture medium. The concentrations of sTNFR1 in culture medium were analyzed by enzyme-linked immunosorbent assay. To induce an inflammatory response in the ocular surface, corneal alkali burn of BALB/c mice was made from a filter paper dipped in 1 N NaOH solution. TNFR1 expression in corneal and conjunctival epithelia was evaluated by immunohistochemistry 28 days after wounding. In HCEC culture medium, sTNFR1 release was significantly increased by the addition of PMA (t-test, P < 0.01) or PGN (P < 0.01). The increased release of sTNFR1 was significantly inhibited by the addition of TAPI-1 or TIMP-3, indicating the possibility of TACE-dependent ectodomain shedding of TNFR1. In the corneal alkali burn model, TNFR1 was expressed in corneal and conjunctival epithelia. TACE-dependent ectodomain shedding of sTNFR1 was recognized in corneal epithelium. In the inflamed ocular surface, TNFR1 was expressed in the corneal and conjunctival epithelia after alkali burn treatment. sTNFR1, released from the ocular surface, may play an anti-inflammatory role in the inflammatory condition.

A Novel Mechanism of Late Gene Silencing Drives SV40 Transformation of Human Mesothelial Cells

Suppression of the late gene expression, usually by integration of the viral DNA into the host genome, is a critical step in DNA tumor virus carcinogenesis. SV40 induces high rates of transformation in infected primary human mesothelial cells in tissue culture, leading to the formation of immortal cell lines (SV40-transformed human mesothelial cell lines, S-HML). The studies described here were designed to elucidate the unusual susceptibility of primary human mesothelial cells to SV40 carcinogenesis. We found that S-HML contained wild-type, mostly episomal SV40 DNA. In these cells, the early genes that code for the viral oncogenes are expressed; at the same time, the synthesis of the late genes, capsid proteins, is suppressed and S-HML are not lysed. Late gene suppression is achieved through the production of antisense RNA molecules. These antisense RNA molecules originate in the early region of the SV40 circular chromosome and proceed in antisense orientation into the late gene region, leading to the formation of highly unstable double-strand RNA, which is rapidly degraded. Our results reveal a novel biological mechanism responsible for the suppression of late viral gene products, an important step in viral carcinogenesis in humans.

Human Corneal Organotypic Cultures

Purpose. In order to test irritancy levels of commercial products, the Draize animal test has been the universally accepted choice. However, since this test uses live animals, many researchers have been trying to develop an alternative organotypic culture. These organotypic cultures, however, utilize animal cells, and it is our belief that a model using human cells would be more predictive for determining human irritancy levels. Methods. Primary human corneal epithelial cells and fibroblasts were separately isolated from a limbal rim and grown in culture. SV40-transformed mouse corneal endothelial cells were seeded onto a membrane and grown to confluence. A homogenous fibroblast/collagen mixture was then added and allowed to gel. After about a week, the epithelial cells were seeded on top of the gel and the constructs were kept submerged in culture for 3-7 days. The constructs were then airlifted to allow for the stratification of the epithelial cells. Following this, the constructs were either fixed and processed for methacrylate sectioning to study morphology, or they were frozen and sectioned for indirect immunofluorescence. Indirect immunofluorescence was performed with ZO-1, a marker of tight junctions; keratins 3 and 12, markers for differentiation; and laminin, a marker of basement membrane components. Initially, a SV40-transformed human endothelial cell line was to be used; however, it did not grow well in this culture system. Results. In these experiments, we tested two methods of isolating epithelial cells. One method, the explant technique-a method we previously used to isolate rabbit epithelial cells-could not be maintained beyond two passages, and when added to the construct only stratified to 2-3 layers of flattened cells. However, with the second method, the dispase technique, the epithelial cells obtained grew rapidly and could be maintained beyond two passages. These cells, when added to the construct, stratified to 5-7 layers and exhibited a more epithelium-like morphology. Staining with ZO-1 indicated that tight junctions in the superficial cells were formed. Staining with keratins 3 and 12 indicated that the epithelial cells were differentiating, and staining with laminin indicated that basement membrane components were being synthesized. Conclusion. Constructing an organotypic culture with human corneal cells is possible, and the morphology of the construct appears to be equivalent to a construct already created with bovine cells that has been used for irritancy studies.

DDB Accumulates at DNA Damage Sites Immediately after UV Irradiation and Directly Stimulates Nucleotide Excision Repair

Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC.HR23B, TFIIH, XPF.ERCC1 and XPG, up to 17-fold for CPDs and approximately 2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors.

Production of Inflammatory Mediators and Cytokine Responsiveness of an SV40-Transformed Human Proximal Tubular Epithelial Cell Line

Proximal tubular epithelial cells (PTEC) play a central role in the physiology of the renal tubulointerstitium. To be able to study the relationship between tubular cells and inflammatory renal diseases the availability of cultured cells is of importance. This study describes an immortalized proximal tubular epithelial cell line which was generated using SV40 DNA. To determine whether the transformation altered the cell line, the transformed cell line was characterized phenotypically using different monoclonal antibodies directed against peptidases, which are characteristic of PTEC, such as adenosine deaminase binding protein (CD26), leucine amino peptidase and carboxy peptidase M by immunofluorescent staining and FACS analysis. All peptidases were clearly present on the parental cell line and the transformed cell line. However, the level of expression of the peptidases was lower on the transformed cell line as compared to the parental nontransfected cells. The morphology of the transformed cell line, determined using a transwell culture system and electron microscopy, showed a polarized morphology of the tubular cells, tight junctions and microvilli. The transformed cell line was compared with the parental proximal tubular epithelial cells in its ability to respond to inflammatory cytokines such as IL-1α, TNF-α, IFN-γ. Stimulation with these cytokines resulted in enhanced production of complement components C2, C3, C4 and factor H, IL-6 and the chemokines IL-8 and MCP-1. The transformed cell line responded in a similar fashion as the parental cell line, although the amount of the different proteins produced was significantly higher in the transformed cell line. Overall, the transformed tubular cell line seems to be a suitable model to study different effects on tubular cells in relation to inflammatory kidney diseases.

Low Catalase Activity in Xeroderma Pigmentosum Fibroblasts and SV40-Transformed Human Cell Lines is Directly Related to Decreased Intracellular Levels of the Cofactor, NADPH

We have previously shown that fibroblasts from ultra-violet (UV) hypersensitive xeroderma pigmentosum patients (XP) are markedly deficient in catalase activity resulting in high intracellular levels of hydrogen peroxide (H 2O 2) following UV irradiation. No direct correlation between catalase activity and repair ability was found since XP variant cells which are proficient in nucleotide excision repair (NER) showed activities as low as those found in NER deficient classical XP groups A and D. However, in contrast to the skin cancer prone XP patients, another NER deficient syndrome, trichothiodystrophy (TTD), which does not exhibit any cancer predisposition, was found to present normal catalase activity. Moreover, it was found that a variety of SV40 transformed human cell lines also showed decreased catalase activities. Our previous data [6]showed that a molecular analysis of the normal, XP, TTD or transformed human fibroblast cell lines did not reveal any differences in levels of catalase transcription or amount of catalase protein subunits. These results incited us to examine the structure/function relationship of the tetrameric active enzyme form of catalase (which is the only one able to carry out H 2O 2 dismutation) with its cofactor NADPH. In the present study, we have measured the effects on catalase activity after adding NADPH either to acellular extracts or during cell culture of the different cell types. The NADPH levels were also quantified directly in intact cells using flow cytometry. Our results show a clear relationship between low catalase activity and striking decrease in intracellular NADPH levels.

Stromelysin-3 Is Induced in Tumor/Stroma Cocultures and Inactivated via a Tumor-specific and Basic Fibroblast Growth Factor-dependent Mechanism

Stromelysin-3 (STR-3) is a recently characterized matrix metalloproteinase (MMP) with a unique pattern of expression and substrate specificity. Unlike other MMPs, STR-3 is consistently and dramatically overexpressed by multiple epithelial malignancies, including carcinomas of the breast, lung, colon, head and neck, and skin. Recent studies suggest that STR-3 promotes the local establishment of epithelial malignancies, contributing to tumor cell survival and implantation in host tissues; however, STR-3's mechanism of action remains undefined. STR-3 is a stromal cell product, prompting speculation that infiltrating stromal cells secrete STR-3 in response to tumor-derived factors. To explore this possibility, we developed a tumor/"stroma" coculture assay in which non-small cell lung cancer (NSCLC) cell lines were grown on confluent monolayers of normal pulmonary fibroblasts. In these tumor/stroma cocultures, NSCLCs stimulate normal pulmonary fibroblasts to secrete STR-3 and release extracellular basic fibroblast growth factor. Thereafter, STR-3 is processed at a unique internal sequence via a basic fibroblast growth factor- and MMP-dependent mechanism to a previously unidentified 35-kDa protein that lacks enzymatic activity. 35-kDa STR-3 is the most abundant STR-3 protein in tumor/stroma cocultures and is only detected when normal pulmonary fibroblasts are cultured with malignant bronchial epithelial cells. Therefore, the tumor-specific processing of STR-3 to the 35-kDa protein is likely to be an important regulatory mechanism.

Improved production of recombinant AAV by transient transfection of NB324K cells using electroporation

Adeno-associated virus (AAV) is useful as an integrating vector for gene transfer. AAV recombinants are generally produced by transient co-transfection methods since it has proven difficult to generate stable packaging cell lines. Acceptable titers of transducing recombinants should be obtainable by optimizing conditions for transient co-transfection. Here, using a luciferase reporter derivative of the AAV infectious plasmid p sub201, we show that substantially higher yields of transducing virus can be obtained using electroporation, rather than calcium phosphate transfection. Furthermore, we observed that electroporation of NB324K cells (an SV40-transformed human cell line) with the helper plasmid, pAAV/Ad, with concomitant adenovirus dl309 infection, gave yields of luciferase transducing recombinant AAV equal or superior to those obtained from the more commonly employed 293 cells. NB324K cells are easier to manipulate and show increased cell-association of the recombinant virus (facilitating its concentration and purification). We also adapted an in situ infected cell hybridization procedure, using a digoxigenin labeled probe, as a general method for determining infectious titer. Titers thus estimated were similar for luciferase-transducing and for alkaline phosphatase-transducing AAV vectors; the estimated titer of the latter agreed with that determined by in situ expression of alkaline phosphatase. We also describe a multiple cloning site derivative of p sub201 which should facilitate generation of further AAV recombinants.

Development and characterization of a human marrow stromal cell line that enhances osteoclast-like cell formation.

We established a human bone marrow stromal cell line (Saka) by infecting marrow adherent cells from semisolid marrow cultures with a recombinant simian virus-40 (SV40) virus. The cells expressed SV40 large tumor antigen, had a fibroblast-like shape, and expressed fibronectin and vimentin. They did not contain detectable alkaline phosphatase activity; express myeloid, lymphoid, or factor VIII-associated antigens; or develop adipocyte-like characteristics with dexamethasone treatment. Polymerase chain reaction analysis of Saka cell RNA detected expression of messenger RNAs for interleukin-6 (IL-6), IL-1 beta, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, and the 1,25-dihydroxyvitamin D3 receptor. Coculture of Saka cells with human marrow mononuclear cells enhanced formation of osteoclast-like multinucleated cells (MNC) in long term human bone marrow cultures. These MNC expressed calcitonin receptors and formed resorption lacunae on dentine. In contrast, coculture of marrow mononuclear cells with other SV40-transformed human marrow stromal cell lines did not increase MNC formation. Conditioned medium from Saka cells or coculture of bone marrow and Saka cells separated by a Millipore membrane did not enhance MNC formation. Addition of a neutralizing antibody to IL-6 or IL-1 beta blocked the effects of Saka cells on MNC formation. These results suggest that marrow stromal cells enhance osteoclast formation in part through direct cell to cell contact and production of IL-6 and/or IL-1 beta.

Distribution of apoptosis-mediating Fas antigen in human skin and effects of anti-Fas monoclonal antibody on human epidermal keratinocyte and squamous cell carcinoma cell lines.

Fast antigen is a cell surface protein that mediates apoptosis. Using immunohistological, flow cytometry and electron microscopic analyses, we investigated the expression of Fas antigen on various skin tissues, and on cultured SV40-transformed human epidermal keratinocyte cell line KJD and human skin squamous cell carcinoma cell line HSC. The Fas antigen was widely distributed in skin components such as the keratinocytes in the lower portion of the epidermis, epidermal dendritic cells, endothelial cells, fibroblasts, apocrine glands, eccrine sweat glands, sebaceous glands, some normal melanocytes and infiltrating lymphoid cells. It was also strongly expressed on the keratinocytes of lichenoid eruptions seen in lupus erythematosus and lichen planus, and on the spongiotic or acanthotic epidermis seen in chronic eczema, adult T-cell leukaemia/lymphoma (ATLL) and atopic dermatitis. Its expression was closely correlated with lymphoid infiltrating cells and it was strongly expressed in lymphoid neoplastic cells, particularly ATLL cells, and fibroblasts seen in dermatofibroma. However, the antigen was not detected on basal cell epithelioma cells, some malignant melanomas or any junctional naevi. The cell lines KJD and HSC strongly expressed the Fas antigen, and crosslinking of the Fas antigen by an anti-Fas monoclonal antibody induced apoptosis of these cell lines. These results indicate that the apoptosis-mediating Fas antigen may play an important role in normal skin turnover and cell differentiation, in immune regulation of skin tumours, and in the pathogenesis of various skin diseases.

The expression of basic fibroblast growth factor and its receptor in cell lines derived from normal human mammary gland and a benign mammary lesion

mRNA for basic Fibroblast Growth Factor (bFGF) was expressed in a series of SV40-transformed human mammary cell lines as molecules of 7.1, 3.6, 2.0 and 1.2 kb. This expression was much weaker in those lines of epithelial morphology than in myoepithelial-like cell lines derived from them. It was confirmed, using northern hybridization to single-stranded RNA probes, that the multiple mRNAs were transcribed from the coding strand for bFGF. bFGF activity was detected in extracts of the cells and the relative amounts of activity corresponded in general to the amounts of mRNA found. Similar results were obtained from spontaneously transformed cell lines derived from a human benign breast lesion. The presence of bFGF protein in the extracts was confirmed by western blotting, which showed a band of 18-19 kDa, migrating in the same position as authentic bFGF; in addition, the myoepithelial-like cells showed prominent bands of bFGF at 24 and 26 kDa. No FGF receptor was detectable by the binding of 125I-bFGF to the SV40-transformed cell lines or to the epithelial cell lines from the benign breast lesion, but both high- and low-affinity receptors were found on myoepithelial-like cells derived from the latter. The results indicate that differentiation to the human myoepithelial-like phenotype in culture is associated with the enhanced expression of bFGF, and it is suggested that bFGF, immunocytochemically detected in the basement membrane of the human breast, may arise, at least in part, from the myoepithelial cells of the mammary parenchyma.

Down-regulation of scatter factor in MRC 5 fibroblasts by epithelial-derived cells. A model for scatter factor modulation.

Scatter factor/hepatocyte growth factor (SF/HGF) is a multifunctional cytokine produced by embryonic fibroblasts and other mesenchymal cells that affects the growth and/or the movement of certain epithelia. Here we report that expression of scatter factor activity by MRC 5 cells, a strain of normal human embryonic lung fibroblasts, is greatly reduced as a result of co-culture of these cells with SVK14, an SV40-transformed human keratinocyte cell line. Using a cDNA probe to the beta chain of human HGF, we have found that the fall in SF activity in MRC 5/SVK14 co-cultures is accompanied by the loss of SF/HGF transcripts. As the inhibition of SF activity coincides with the disappearance of SF/HGF transcript, we conclude that inhibition of the SF activity expressed by MRC 5 cells by co-culture with SVK14 involves transcriptional regulation.

Aberrant splicing of Gs alpha transcript in transformed human astroglial and glioblastoma cell lines.

Alpha subunits of G proteins, which play a vital role in signal transduction, display considerable structural and functional diversity. Point mutations in two forms of alpha subunits, Gs alpha and Gi2 alpha, impairing their GTPase activity, have been detected in endocrine disorders. We report here the presence of truncated Gs alpha transcripts in a human glioblastoma cell line, HS683, and in an SV40-transformed human astroglial cell line, SVG. These transcripts were detected by polymerase chain reaction (PCR) amplification of cDNAs from the cell lines. The truncated Gs alpha transcripts, with deletions in the central region of the molecule, seem to have originated due to aberrant splicing within exonic sequences, which did not conform to the consensus GT/AG splice signals. The presence of a smaller size protein of mol.wt. around 25,000 kd in the SVG and HS683 cell lines, detected by antibodies specific for the C-terminal region of the Gs alpha subunit, seems to be consistent with the presence of truncated Gs alpha transcripts in these cell lines. These aberrantly spliced transcripts, if translated, could synthesize potentially oncogenic Gs alpha subunits deficient in GTPase activity. Whether such molecules, with sometimes relatively large deletions, retain some aspects of their function and are biologically significant remains to be seen.

Detection of translocations of 10p by non-radioactive in situ hybridization of VIM gene in SV40-transformed human cell lines

SV40-transformed human fibroblasts exhibit characteristic chromosome imbalances, fairly well correlated with the activity of enzymes encoded by genes located on chromosome segments either in deficiency or in excess. However, a major discrepancy existed for the expression of vimentin gene (VIM), which was high, even though the map location of the gene (10p) was missing in many cell lines. An in situ hybridization technique using a biotinylated probe for the human VIM was applied to detect eventual cryptic translocations, as chromosome 10p is difficult to identify. In two cell lines (WI 98 and HEL1 HBLT) in which a loss of copy number of 10p was assumed after karyotyping, a signal for VIM was detected in unidentified short arms of derivative chromosomes. This exemplifies that in situ hybridization is a powerful complement to classical cytogenetics to detect rearrangements in highly rearranged karyotypes from transformed or cancerous cells. These results also strengthen the interpretation of the correlation between karyotypic and metabolic imbalances in transformed cells.

Investigations on etiology of Crohn's disease

Many investigators have tried to prove a relationship between Crohn's disease and Mycobacteria. Recent evidence suggests that some autoimmune diseases may be initiated through "molecular mimicry" between mycobacterial stress protein antigens and their human homologs. We investigated whether antibody to stress proteins was more frequent in patients with Crohn's disease than controls. We used ATP binding to separate stress proteins (heat-shock-induced, de novo-synthesized, and constitutively expressed ATP-binding proteins) from crude extracts obtained from Mycobacteria and from an SV40-transformed human epithelial cell line that expresses a heat-shock protein, hsp73, as a complex with SV40 T antigen. We used immunoblots to compare sera from 34 patients with Crohn's disease, 14 with ulcerative colitis, and 14 with duodenal or gastric ulcers (noninflammatory bowel disease control patients). We found no statistically significant pattern or frequency of antibodies against single proteins or a combination of mycobacterial or human stress proteins. These observations do not support the hypothesis that a humoral immune response to stress proteins of Mycobacteria is important in the pathogenesis of Crohn's disease.

Adenovirus infection induces amplification of persistent viral DNA sequences (simian virus 40, hepatitis B virus, bovine papillomavirus) in human and rodent cells

Adenoviruses, types 2 and 12 induce amplification of SV40 DNA sequences in cells of the SV40-transformed human newborn kidney cell line, NB-E. Similarly, integrated hepatitis B virus DNA sequences in the human hepatoma cell line, PLC/*PRF/5, and bovine papillomavirus (BPV) DNA sequences in BPV-transformed mouse cells (ID13) are amplified by adenovirus infection. Thus, similar to herpesgroup or vaccinia viruses or DNA damaging agents, adenoviruses are able to mediate selective DNA amplification in addition to their reported mutagenic and chromosome damaging effects. The role of amplification of integrated viral DNA sequences in development and progression of specific tumors (e.g. hepatocellular carcinoma) remains to be determined.

SV40-mediated tumor selection and chromosome transfer to enrich for cystic fibrosis region

The somatic cell hybrid C121, with chromosome 7 as its sole human component, arose when mouse macrophages SV40 genomes are integrated at 7q31-7q35. We show that hybrids with a reduced chromosome 7 component, but which retain markers linked to the cystic fibrosis locus, can be generated by direct in vivo tumor selection or following chromosome-mediated gene transfer and SV40-mediated cellular transformation. Our methods for chromosome fragmentation and fine-structure mapping can now be applied to the substantial number of SV40-transformed human cell lines, with independent chromosomal integration sites, already available. Our results also suggest that expression of human epidermal growth factor receptor augments the tumorigenic potential of the SV40-transformed C121 hybrid.

Defective repair of UV-damaged DNA in human tumor and SV40-transformed human cells but not in adenovirus-transformed human cells

The DNA repair capacities of five human tumor cell lines, one SV40-transformed human cell line and one adenovirus-transformed human cell line were compared with that of normal human fibroblasts using a sensitive host cell reactivation (HCR) technique. Unirradiated and UV-irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens (Vag) in the various cell types using immunofluorescent staining. The survival of Vag formation for UV-irradiated Ad 2 was significantly reduced in all the human tumor cell lines and the SV40-transformed human line compared to the normal human fibroblasts, but was apparently normal in the adenovirus-transformed human cells. D0 values for the UV survival of Ad 2 Vag synthesis in the tumor and virally transformed lines expressed as a percentage of that obtained on normal fibroblast strains were used as a measure of DNA repair capacity. Percent HCR values ranged from 26 to 53% in the tumor cells. These results indicate a deficiency in the repair of UV-induced DNA damage associated with human tumorigenesis and the transformation of human cells by SV40 but not the transformation of human cells by adenovirus.

High-affinity SV40 T-antigen binding sites in the human genome

We describe two different approaches to isolate human genomic sequences possessing high-affinity binding sites for the simian virus 40 (SV40) large T antigen. First, SV40 T antigen was added to Sau3A-restricted human DNA; the resulting T-antigen-DNA complexes were collected after repeated passages through nitrocellulose filters. The second approach involves the specific immunoprecipitation of chromatin fragments, generated by Sau3A treatment of nuclear chromatin from SV40-transformed human cells. The DNA fragments obtained were cloned in plasmid vectors for further investigation. Using the filter binding approach we isolated four different fragments with high-affinity binding sites. The binding site in one fragment was related to the strong T-antigen binding site 1 in the SV40 genome. The other three fragments contained multiple recognition pentamers, GA(G)GGC. Only one fragment with a high-affinity binding site was identified among the immunoprecipitable chromatin fragments. This DNA fragment belongs to the L1 family of human repetitive DNA. We present evidence suggesting that a significant fraction of human L1 elements possesses T-antigen binding sites. L1-related sequences appear as extrachromosomal elements in an SV40-transformed human cell line, and the amount of extrachromosomal L1 DNA was found to increase after fusion of transformed cells to permissive monkey cells.