pSV2neo Plasmid Research Articles

Viral and cellular oncogene expression during progressive malignant transformation of SV40 transformed human fibroblasts.

In vitro investigation of the multistep neoplastic progression which occurs during transformation of human cells has been hindered by resistance of human cells to both immortalization and tumorigenicity (Mut. Res. 199; 273, 1988). Previously our laboratory established a cell line, HSF4-T12, by transfection of normal human foreskin fibroblasts with the plasmid pSV3-neo which contains the early genes of simian virus 40 (SV40). A multistep progression in karyotypic alterations and transformed phenotype occurred resulting in a neoplastic cell line that was immortal, transformed, and tumorigenic. We have examined changes in the SV40 proteins, large T (T-antigen) and small t (t-antigen) antigens, and in the cellular protein, p53, during progressive transformation of these cells. Total viral protein expression relative to total cellular protein increased following immortalization of HSF4-T12 as did the ratio of T-antigen to t-antigen. Interestingly, no significant change in DNA content accompanied immortalization. However, during the progressive in vitro transformation of HSF4-T12 which occurred primarily post-immortalization, DNA index increased to 1.6 but only small additional increases in T-antigen expression were seen. No consistent or critical role for t-antigen in development of the tumorigenic phenotype was found in this system.

The role of O6-alkylguanine in cell killing and mutagenesis in Chinese hamster ovary cells

Chinese hamster ovary cells with no detectable (less than 200 molecules/cell) O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) were transfected with human cell DNA and pSV2neo plasmid by electroporation. Two stable transformant clones, GC-1 and GC-2, containing 4 X 10(4) and 4-6 X 10(3) methyltransferase molecules/cell respectively were isolated by successive screening in the presence of G418 and 2-chloroethyl-N-nitrosourea (CNU). Only three or four copies of pSV2neo DNA and no repetitive human DNA sequence were detected in these isolates. Secondary transfection of parent cells with GC-1 DNA yielded several clones containing 2-10 X 10(3) methyltransferase molecules/cell. The rate of removal of O6-methylguanine in GC-1, GC-2 and parent cells in vivo reflected their methyltransferase levels, while the N-methylpurines were removed at similar rates in all three cell lines. The differential sensitivity of these cells to several alkylating agents, namely CNU, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and methyl-methane sulfonate (MMS), known to yield different proportions of O6-alkylguanine among the alkyl adducts in DNA, varied widely. The largest and smallest differences in toxic response were observed with CNU and MMS respectively. These cell lines showed no difference in sensitivity to the DNA cross-linking agent psoralen. These data strongly suggest that alkylating agents produce two classes of lethal lesions, one of which is O6-alkylguanine. Induction of mutations at the hypoxanthine-phosphoribosyltransferase locus in these cells lines suggests that, regardless of its relative yield, O6-methylguanine is the major mutagenic lesion for all alkylating agents.

Establishment of a human fetal cardiac myocyte cell line

Human cardiac myocytes undergo degeneration, cytolysis, and necrosis in a number of clinical disease conditions such as myocarditis, dilated cardiomyopathy, and during episodes of cardiac allograft rejection. The precise cellular, biochemical, and molecular mechanisms that lead to such abnormalities in myocytes have been difficult to investigate because at present it is not possible to obtain and maintain viable cell cultures of human adult cardiac myocytes in vitro. However, human fetal cardiac myocytes are relatively easy to maintain and culture in vitro, but their limited availability and growth, variability from one preparation to another, and varying degrees of contamination with endothelial and epithelial cell types have made it difficult to obtain reliable data on the effect of cardiotropic viruses and cardiotoxic drugs on such myocytes. These thoughts prompted us to attempt to derive a cell line of human cardiac origin. Highly enriched human fetal cardiac myocytes were transfected with the plasmids pSV2Neo and pRSVTAg and gave rise to a cell line (W1) which has been maintained in culture for 1 yr. Morphologic and phenotypic analyses of W1 cells by flow microfluorometry and immunoperoxidase techniques indicate that the W1 cell line shares many properties of human fetal cardiac myocytes, but appears not to react with specific antibodies known to react with markers unique to human endothelial, epithelial, skeletal muscle, and dendritic cells. These preliminary data suggest that the W1 cells may provide a unique source of an established cell line that shares many properties ascribed to human cardiac myocytes.

Homologous recombination of a plasmid containing the SV40 early region in rabbit cells.

The plasmid pSV3-neo that carries the SV40 early genetic region and the neor resistance gene underwent homologous recombination between SV40-duplicated sequences of the small t antigen intron and the polyadenylation site after transfection into rabbit vascular smooth muscle cells. This recombination yielded two free plasmids of 1.7 and 3.0 kb that replicated in transfected rabbit cells, while the vector plasmid did not. Replication of the two recombinant plasmids continued for several months in rabbit cells transformed by pSV3-neo, and their structure remained unchanged. Between 15 and 2,510 copies per cell of the 3.0-kb plasmid and between 370 and 1,565 copies per cell of the 1.7-kb plasmid were found in different pSV3-neo-transformed rabbit clones. Similar recombined plasmids were found 3 days after transfection of pSV3-neo onto SV40-transformed rabbit cells. By comparison, pSV3-neo was replicated in simian cells, but no recombined plasmids were detected by Southern analysis. No replication was detected after transfection of pSV3-neo into mouse cells. These results appear to reflect important differences between rabbit and simian cells in the efficiency of homologous recombination involving specific SV40 sequences and/or in the replication of the resulting recombinants. The role of the host cell in the evolution of SV40 sequences may influence the outcome of the infection with this virus.

Exposure of pSV2-Neo Plasmid DNA to X-Rays Enhances Its Ability to Transform Mouse DBT Cells.

[in Japanese]

Over‐expression of the epidermal growth factor receptor in human breast cancer cells fails to induce an Estrogen‐independent phenotype

An association exists in primary human breast tumors between high epidermal growth factor receptor (EGFR) expression and a reduced number or even absence of estrogen receptors (ER). To determine whether an increase in EGFR expression might alter the estrogen responsiveness of an ER-positive human breast cancer cell line, ZR 75-1 cells were cotransfected with a plasmid containing the full-length cDNA for the human EGFR under the transcriptional control of the Harvey murine sarcoma virus (HaMSV) long terminal repeat (LTR) and with a pSV2neo plasmid. Two of the isolated G418-resistant clones were found to constitutively express EGFR levels 15- to 60-fold higher than those found on nontransfected ZR 75-1 cells. The EGFR in these clones were functionally normal since EGF could increase their autophosphorylation and since EGF could enhance the transphosphorylation of p185erbB-2. No change was seen in either the number or affinity of ER in these clones. In addition, the ability of estrogen to stimulate the anchorage-dependent and anchorage-independent growth of these clones was not significantly modified. These results suggest that an increase in EGFR expression alone is not sufficient to induce a hormone-independent phenotype in vitro in human breast cancer cells.

Establishment of transformed swine fibroblast cell lines using SV40 large T antigen

Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.

Relative frequencies of homologous recombination between plasmids introduced into DNA repair-deficient and other mammalian somatic cell lines

Twelve mammalian somatic cell lines, some of them DNA damage-sensitive mutants paired with their respective wild-type parental lines, were assayed for their ability to catalyze extrachromosomal, intermolecular homologous recombination between pSV2neo plasmid recombination substrates. All of the somatic cell lines analyzed are capable of catalyzing homologous recombination; however, there is a wide range of efficiencies with which they do so. Five human cell lines display a fourfold range of recombination frequencies, and six hamster cell lines vary almost 20-fold. Linearizing one of the recombination substrates stimulates recombination in all but one of the cell lines. Two of the three paired mutant cell lines display a threefold reduction in their ability to catalyze homologous recombination when compared to their respective parental cell lines, indicating that the mutations that render them sensitive to DNA damaging agents might also play a role in homologous recombination.

Cytotoxic effects of expression of human superoxide dismutase in bovine adrenocortical cells

Oxygen radicals and the cellular antioxidant enzymes may play a role in cellular senescence. We studied the feasibility of altering oxygen radical metabolism in a normal differentiated cell that undergoes senescence in culture by transfection of an expression vector containing human CuZn-SOD cDNA. Plasmid pRSV2-cSOD was constructed to contain the cDNA for human CuZn-SOD under the regulation of the Rous sarcoma virus long terminal repeat. Early passage cultures of bovine adrenocortical cells were cotransfected with pRSV2-cSOD and a plasmid (pSV3neo) allowing initial selection and continued growth of transfectants. Three passages after isolation of the polyclonal population, as cells grew to confluence, cultures showed focal cell death that spread outward to affect neighboring cells, so that by 72 h most cells had detached from the culture dish. Long term growth of the polyclonal population of transfectants without extensive cell death was achieved by continuous maintenance of low cell density during growth. Southern blot analysis of DNA from the pooled polyclonal population of transfected cells showed the presence of the expected 625 bp band from human CuZn-SOD. However, the intensity of this band indicated that only a minority of cells in the population had integrated the SOD plasmid, and DNA isolated from cells after 25 passages at low cell density showed plasmid sequences only of an altered form, suggesting that cells containing intact human SOD cDNA had been selectively lost from the population. When early passage low density transfectants were allowed to grow back to high cell density, cell death foci were again observed. Additionally, cell fusion with the formation of giant cells with massive multinucleation was observed by fluorescence microscopy after staining cultures with a DNA binding dye. In later stages of this process, the large nuclear mass in such a giant cell became fragmented as the cell detached from the dish and formed the center of a focus of cell death in the surrounding cells. Because cell death prevented the growth of large numbers of transfected cells, it was not possible to demonstrate the involvement of CuZn-SOD in the cytotoxic effect by direct means, but the control plasmid without the CuZn-SOD cDNA insert had no cytotoxic effect. Thus, the introduction of a vector for human CuZn-SOD in a normal differentiated cell caused a cytotoxic effect involving cell death, cell fusion, and nuclear fragmentation.

Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines

The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors.

Drug resistance to cis-diamminedichloroplatinum(II) in chinese hamster ovary cell lines transfected with glutathione S-transferase pi gene

Establishment of Chinese hamster ovary (CHO) cell lines expressing human glutathione S-transferase-pi(GST-π) was performed after cotransfection of pSV2-neo and human GST-π cDNA-carrying plasmid pβactGPi-2. About 30 G418-resistant clones were tested for their expression of GST-π by Northern blot analysis. Two clones, β2–3 and β2–5, expressed a significant amount of GST-π mRNA; and one clone, β1-1, that did not was also used for further study. Western blot analysis with anti-GST-π antibody showed significant increases of GST-π in β2–3 and β2–5, but not in β1-1. Northern blot analysis with the human GST-π cDNA probe showed that the increase in the expression of GST-π-mRNA in β2–3 and β2–5 was respectively 2- and 4-fold higher than that in β1-1. Southern blotting analysis showed that β1-1, β2–3 and β2–5 contained about one copy of the human GST-π cDNA sequence. β2–3 and β2–5 were resistant to 1.4- and 3.0-fold higher doses of CDDP than CHO, respectively, but β1-1 was not. Increased expression of GST-π might be associated with CDDP-resistance in CHO cells.

NS-1 and NS-2 proteins may act synergistically in the cytopathogenicity of parvovirus MVMp

The interaction of parvovirus minute virus of mice (prototype strain, MVMp) with simian virus 40 (SV40)-transformed human cells (NB-E) was investigated by means of transfection with MVMp molecular clones derived from the infectious recombinant plasmid (pMM984). pMM984 inhibits stable transformation of NB-E cells to geneticin resistance (G418 R) upon cotransfection with the selectable pSV2neo plasmid. We show here that this inhibition is not merely caused by a repression of marker gene expression from the SV40 early region promoter in pSV2neo and rather is likely to reflect the cytotoxic action of the parvovirus. Starting from plasmid pMM984, defined mutations were introduced into the genome of MVMp and more particularly into sequences coding for the NS-1 and/or NS-2 nonstructural proteins. In this way we could show that the NS-1 protein is necessary for the inhibition of transformation to G418 R and that the NS-2 protein acts synergistically to enhance this effect. Moreover, results obtained with different viral mutants indicate that the inhibitory action of NS-1 on stable transformation can be dissociated from the ability of this protein both to transactivate the parvoviral p39 promoter of the capsid protein-encoding region and to drive parvoviral DNA amplification. Altogether these data point to a probable direct toxicity of MVMp nonstructural proteins for permissive host cells.

Variation in the loss of O6-methylguanine-DNA methyltransferase during immortalization of human fibroblasts

We have examined O6-methylguanine-DNA methyltransferase (MT) activity in four human fibroblast cell lines during immortalization. Transfection of primary fibroblasts with the plasmid pSV3gpt or pSV3neo, which encode the SV40 large T antigen, confers a transformed phenotype but not immediate immortality. After a period of growth (pre-crisis) the cells enter a quiescent phase (crisis) from which an immortal clone of cells eventually grows out. From measurements of MT activity in extracts of cells taken at different defined stages of the immortalization process, we conclude that the establishment of a Mex- (MT-deficient) cell population is not specifically associated with cellular transformation or with any particular stage of immortalization. It appears that in different cell populations the change from Mex+ to Mex- may occur at different times during the immortalization process and that the change may be very abrupt.

Generation of Metastatic Variants by Transfection of a Nonmetastatic Rat Mammary Epithelial Cell Line with DNA from a Metastatic Rat Mammary Cell Line

Transfection of rat mammary (Rama) 37 epithelial cells, which yield nonmetastasizing adenomas in syngeneic Wistar-Furth rats, with HindIII-fragmented cellular DNA and the drug-resistance plasmids pSV2gpt or pSV2neo yields drug-resistant transformants with a frequency of 10(-4)-10(-5). Transformant cell lines transfected with the following, pSV2gpt alone, pSV2gpt and Rama 37 DNA, pSV2gpt and DNA from a metastasizing cell line Rama 800 (CT set), pSV2neo and salmon sperm DNA, pSV2neo and Rama 800 DNA (C set), all yield tumors when injected subcutaneously into syngeneic rats. A few transformants obtained by cotransfection with DNA from Rama 800 cells produce metastases in lungs and/or lymph nodes. The incidence of such metastases for two transfectants, termed CT4-41 and C18P, is significant at 20 and 24%, respectively, but only half (48%) that achieved with Rama 800 cells. Reintroduction into rats of cells cultured from a metastatic tumor of CT4-41 and of C18P, and from their lung or lymph node metastases, produces either a similar incidence (20-24%) or a significantly higher (48-52%) incidence of metastasis than that of the original transfectants. Cells cultured from nonmetastatic tumors fail to produce any metastatic lesions. When [32P]-labeled gpt or neo DNAs are hybridized to EcoRI-digested cellular DNA of the CT4-41 or C18P series of cell lines, tumors or metastases, gpt binds to one major fragment of 3,800 basepairs, and neo to two major fragments of 5,700 and 4,200 basepairs. The same cell lines produce hybridizing mRNAs of 1,500 and 1,900 bases for the CT4-41 series and 2,000-2,400 bases for the C18P series. It is suggested that transfection of DNA from the metastatic cells causes the nonmetastatic cells to become metastatic, in a genetically dominant manner, but additional steps are required for this process to become established and expressed at a level equivalent to that of the original, metastasizing donor cells.

The in vivo clearance of Ha-ras transformants by natural killer cells

The experiments in this study were designed to test the hypothesis that natural killer (NK) cells play a role in host surveillance against early neoplastic changes in the malignant process. C3H 10T1/2 mouse fibroblasts were transfected with a pSV2-neo plasmid vector which contains EJ, the mutated c-Ha-ras, regulated by its own promoter. Control cells were transfected with pSV2-neo alone and did not contain the ras gene. Oncogene-transfected cells were compared with control cells for lung colony formation following tail vein injection into C3H mice. Intravenous injection of ras-transfected 10T1/2 cells induced marked lung colony formation in vivo, whereas C3H 10T1/2 parental lines or 10T1/2 cells transfected with pSV2-neo alone induced no lung colonies in C3H mice. The colonising potential of ras transfectants could be decreased by augmentation of NK activity by injection of polyinosinic cytidylic acid and increased by depletion of NK effectors with anti-asialo GM1. Experiments with beige mice demonstrated that the mortality of syngeneic, NK-deficient C3H-bg/bg mice injected with ras tranfectants was significantly greater than similarly treated NK-normal C3H(-)+/bg littermate controls. The results support the view that NK cells are capable in vivo of recognizing early defined stages in the neoplastic process initiated by oncogenes.

Expression of oncomodulin does not lead to the transformation or immortalization of mammalian cells in vitro.

A recombinant plasmid (pMTONCO) containing the coding sequences for rat oncomodulin under the direction of the metallothionein promoter was constructed. pMTONCO was co-transfected with the pSV2-NEO plasmid into primary mouse kidney cells or Rat-1 cells using the calcium phosphate technique and stable transformants were isolated after selection with G418. Transcription from the metallothionein promoter was inducible with heavy metals and produced an oncomodulin-specific mRNA. The presence of oncomodulin protein in stable cell lines was verified by immunoprecipitation with specific antisera. While a plasmid encoding the polyomavirus T-antigens was able to prolong the life-span of primary mouse kidney cells in culture, no equivalent activity was noted when the pMTONCO plasmid was used to transfect primary cells. When expressed in Rat-1 cells, oncomodulin did not affect the growth properties of these cells, nor did it predispose cells to higher frequencies of oncogenic transformation to a viral oncogene. We conclude that oncomodulin is neither an immortalizing nor transforming agent in vitro.

Decreased stable transfection frequencies of six X-ray-sensitive CHO strains, all members of the xrs complementation group

Six X-ray-sensitive strains (xrs) of the Chinese hamster ovary (CHO) cell line, all of which have a defect in double-strand break (dsb) rejoining, have been investigated for their proficiency in DNA transfection assays. All 6 strains and clonal isolates derived from them, show a decreased stable transfection frequency using the plasmids pSV2neo and pSV2gpt after transfection by either the CaPh method or the polybrene method. The magnitude of this effect is DNA concentration dependent and is more marked after transfection with higher DNA concentrations (5–20 μg DNA). A spontaneous X-ray-resistant reactivant (or revertant) of one xrs strain also acquired the elevated transfection frequency of the wild-type strain providing evidence for a causal relationship between the decreased transfection frequency and the xrs phenotype. In contrast, the strains show no defect when transfection is assayed using a transient transfection system. Since the transient transfection assay only depends on the uptake and transcriptional activity of foreign DNA, and does not necessitate DNA integration, this suggests that the xrs strains do not have a defect in the uptake of foreign DNA, but might have a defect in integration or the processing of DNA molecules prior to integration.

Synthesis of l‐3,4‐Bihydroxyphenylalanine by Tyrosine Hydroxylase cDNA‐Transfected C6 Cells: Application for Intracerebral Grafting

In the present study, we obtained genetically manipulated nonneuronal cells which synthesize a catecholamine precursor for future use in intracerebral grafting. Human type 1 tyrosine hydroxylase (TH; EC 1.14.16.2) cDNA was inserted into eukaryotic expression vector pKCRH2 and was co-transfected into C6 cells with plasmid pSV2neo. Expression of the TH minigene was screened by immunohistochemical staining with TH antibody and immunoblotting analysis. Several clones of the C6 transfectants that produce TH molecules were obtained. These cells showed TH activity, and the product, L-3,4-dihydroxyphenylalanine (L-DOPA), was detected intracellularly due to the absence of L-amino acid decarboxylase (EC 4.1.1.28) activity. It was found that a large amount of L-DOPA was released from the cells into the culture medium. These transfectants were transplanted into rat brain, and the expression of TH was examined immunohistochemically. On the 10th day following transplantation, a mass of C6 cells which was heavily stained with TH antibody was observed in the brain. These findings may provide us with an opportunity to investigate the effects of intracerebral transplantation of nonneuronal cells that produce catecholamine or its precursor.

Clonal populations of the mouse mammary cell line, COMMA-D, which retain capability of morphogenesis in vivo

Clonal populations were isolated from the mouse mammary cell line, COMMA-D, by transfection with a dominant-selectable gene, pSV2Neo, which confers resistance to the antibiotic, G418. Seven of twenty-four clones isolated retained the ability of the parental line to repopulate cleared mammary fat pads in vivo as ductal-alveolar hyperplasias. Two sublines designated CDNR2 and CDNR4 retained hyperplastic growth potential after multiple passages in vitro with low incidence of tumor formation. A third subpopulation, CDNR1, contained a single integration site for the pSV2Neo plasmid indicating a bonafide clonal origin for this subline. CDNR1 cells displayed heterogeneous growth phenotypes in vivo including hyperplasia, adenocarcinoma, and bone formation. Functional differentiation of CDNR1 cells organized as alveolarlike structures in vivo or on floating collagen gels in vitro was observed as determined by immunoperoxidase staining for the milk-specific protein, casein. Overall, the results indicate that a subset of cells from the COMMA-D cell line may be functionally analogous to stem cells existing in the mammary gland.

A dual-circular plasmid structure dependent on DNA replication generated in monkey COS7 cells and cell extracts

When monkey COS7 cells are transfected with plasmids pSVod or pSV2-neo, a DNA structure can be detected consisting of two circular forms linked by a duplex bridge. Generation of this structure is enhanced by camptothecin, an inhibitor of DNA topoisomerase I. Generation of dual-circles in vitro , using a DNA replication system with added T-antigen, requires template DNA with an SV40 origin. Heterogeneous dual-circles can be visualized involving two initially independent molecules of different size. Implications for in vitro studies of certain types of recombination are discussed.