African Green Monkey Kidney CV-1 Research Articles

Bisphenol A affects androgen receptor function via multiple mechanisms

Bisphenol A (BPA), is a well-known endocrine disruptor compound (EDC) that affects the normal development and function of the female and male reproductive system, however the mechanisms of action remain unclear. To investigate the molecular mechanisms of how BPA may affect ten different nuclear receptors, stable cell lines containing individual nuclear receptor ligand binding domain (LBD)-linked to the β-Gal reporter were examined by a quantitative high throughput screening (qHTS) format in the Tox21 Screening Program of the NIH. The results showed that two receptors, estrogen receptor alpha (ERα) and androgen receptor (AR), are affected by BPA in opposite direction. To confirm the observed effects of BPA on ERα and AR, we performed transient transfection experiments with full-length receptors and their corresponding response elements linked to luciferase reporters. We also included in this study two BPA analogs, bisphenol AF (BPAF) and bisphenol S (BPS). As seen in African green monkey kidney CV1 cells, the present study confirmed that BPA and BPAF act as ERα agonists (half maximal effective concentration EC50 of 10–100nM) and as AR antagonists (half maximal inhibitory concentration IC50 of 1–2μM). Both BPA and BPAF antagonized AR function via competitive inhibition of the action of synthetic androgen R1881. BPS with lower estrogenic activity (EC50 of 2.2μM), did not compete with R1881 for AR binding, when tested at 30μM. Finally, the effects of BPA were also evaluated in a nuclear translocation assays using EGPF-tagged receptors. Similar to 17β-estradiol (E2) which was used as control, BPA was able to enhance ERα nuclear foci formation but at a 100-fold higher concentration. Although BPA was able to bind AR, the nuclear translocation was reduced. Furthermore, BPA was unable to induce functional foci in the nuclei and is consistent with the transient transfection study that BPA is unable to activate AR.

Endocrine disruption effects of 2,2′,4,4′,6-pentabromodiphenylether (BDE100) in reporter gene assays

Polybrominated diphenyl ethers (PBDEs) constitute an important group of flame retardants. 2,2',4,4',6-Pentabromodiphenylether (BDE100) is a prominent PBDE congener in some human populations. The potential of BDE100 to modulate responses mediated by the estrogen (ER), thyroid hormone (ThR) or androgen receptors (AR) were investigated by use of transactivation reporter gene assays. The African green monkey kidney CV-1 cell transiently transfected with the constructed reporter gene plasmid ERE-TATA-Luc and pUAS-tk-Luc with luciferase (Luc) under control of the estrogen response (ERE), or thyroid hormone response (ThRE) elements were used to evaluate (anti)estrogen and thyroid effects of BDE100. The (anti)androgenic potency of BDE100 was also evaluated by use of MDA-kb2 cells, which were stably transfected with MMTV-luciferase. The assays displayed appropriate responses to known natural estrogen 17β-estradiol (E2), ThR ligand triiodothyronine (T3), and the AR agonist 5α-dihydrotestosterone (DHT). 10 or 50 μM BDE100 significantly up-regulated expression of Luc under control of the ER. Antiestrogenic potency was observed for BDE100 (IC50 = 6.21 μM). Co-exposure to 50 μM BDE100 significantly enhanced expression of Luc caused by 5 nM T3. BDE100 was antiandrogenic at 10 and 50 μM with an IC50 of 28.60 μM BDE100. These results suggest that BDE100 can modulate the endocrine system in multiple ways by interfering with several hormonal signaling pathways simultaneously.

The Suggested Physiologic Aryl Hydrocarbon Receptor Activator and Cytochrome P4501 Substrate 6-Formylindolo[3,2-b]carbazole Is Present in Humans

Dioxins and other polycyclic aromatic compounds formed during the combustion of waste and fossil fuels represent a risk to human health, as well as to the well being of our environment. Compounds of this nature exert carcinogenic and endocrine-disrupting effects in experimental animals by binding to the orphan aryl hydrocarbon receptor (AhR). Understanding the mechanism of action of these pollutants, as well as the physiological role(s) of the AhR, requires identification of the endogenous ligand(s) of this receptor. We reported earlier that activation of AhR by ultraviolet radiation is mediated by the chromophoric amino acid tryptophan (Trp), and we suggested that a new class of compounds derived from Trp, in particular 6-formylindolo[3,2-b]carbazole (FICZ), acts as natural high affinity ligands for this receptor. Here we describe seven new FICZ-derived indolo[3,2-b]carbazole-6-carboxylic acid metabolites and two sulfoconjugates, and we demonstrate the following. (i) FICZ is formed efficiently by photolysis of Trp upon exposure to visible light. (ii) FICZ is an exceptionally good substrate for cytochromes P450 (CYP) 1A1, 1A2, and 1B1, and its hydroxylated metabolites are remarkably good substrates for the sulfotransferases (SULTs) 1A1, 1A2, 1B1, and 1E1. Finally, (iii) sulfoconjugates of phenolic metabolites of FICZ are present in human urine. Our findings indicate that formylindolo[3,2-b]carbazols are the most potent naturally occurring activators of the AhR signaling pathway and may be the key substrates of the CYP1 and SULT1 families of enzymes. These conclusions contradict the widespread view that xenobiotic compounds are the major AhR ligands and CYP1 substrates.

Contamination and Effects of Perfluorochemicals in Baikal Seal (Pusa sibirica). 2. Molecular Characterization, Expression Level, and Transcriptional Activation of Peroxisome Proliferator-Activated Receptor α

To investigate the biological effects of perfluorochemicals (PFCs) and to identify biomarkers of exposure to PFCs, this study focused on the effects mediated by peroxisome proliferator-activated receptor alpha (PPARalpha) in Baikal seals (Pusa sibirica). We cloned a full-length cDNA, encoding PPARalpha from the liver of Baikal seal, which has a deduced open reading frame of 468-amino acid residues with a predicted molecular mass of 52.2 kDa. Comparison of the amino-acid sequence of Baikal seal PPARalpha with that of other mammalian PPARalpha showed considerable similarities with PPARalpha of dog (97%), human (95%), rat (92%), and mouse (91%). The quantitative real-time RT-PCR analyses of tissues from Baikal seals revealed that PPARalpha mRNAs were primarily expressed in the liver, kidney, heart, and muscle. The hepatic expression levels of PPARalpha mRNA showed a positive correlation with the expression levels of immunochemically detected cytochrome P450 (CYP) 4A-like protein, indicating that the PPARalpha-CYP4A signaling pathway in Baikal seal is likely conserved. This study also developed an in vitro PPARalpha reporter gene assay using African green monkey kidney CV-1 cells transiently transfected with Baikal seal PPARalpha cDNA expression vector and a reporter vector containing a peroxisome proliferator-responsive element The in vitro reporter gene assay displayed significant response to clofibrate, which is a known PPARalpha agonist in humans and rodents. Treatmentwith perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), or perfluorooctane sulfonate (PFOS) induced PPARalpha-mediated transcriptional activity in a dose-dependent manner, showing the lowest-observed-effect concentrations of 62.5, 125, 125, 62.5, and 125 microM, respectively. In the livers of wild Baikal seals, expression levels of PPARalpha mRNA showed a significant positive correlation with PFNA levels. Moreover, expression of hepatic CYP4A-like protein was significantly correlated with the hepatic concentrations of PFNA and PFDA. These results suggest modulation of the PPARalpha-CYP4A signaling pathway by PFCs in the wild Baikal seals. Our study demonstrates that the PPARalpha-mediated response may be a useful biomarkerto evaluate potential biological effects of PFCs in wildlife.

Rho Family Guanine Nucleotide Exchange Factor Brx Couples Extracellular Signals to the Glucocorticoid Signaling System

Glucocorticoids regulate many crucial biologic functions through their cytoplasmic/nuclear glucocorticoid receptors (GR). Excess, deficiency, or alteration in tissue sensitivity to glucocorticoids has been associated with major causes of human morbidity and mortality. Brx, a cytoplasmic Rho family guanine nucleotide exchange factor, binds to and influences the activity of several nuclear hormone receptors. We examined the functional and molecular interactions between GR and Brx. The glucocorticoid sensitivity of lymphocytes obtained from mice haplo-insufficient for Brx was significantly decreased. Conversely, GR-mediated transcriptional activity of a glucocorticoid response element (GRE)-mediated glucocorticoid-responsive promoter was enhanced by Brx in a guanine nucleotide exchange factor domain-dependent fashion. Brx interacted with GR, forming a ternary complex with RhoA. In a chromatin immunoprecipitation assay, Brx and RhoA were co-precipitated with GREs only in the presence of ligand-activated GR. Extracellularly administered lysophosphatidic acid, which activates its signaling cascade through a specific membrane GTP-binding protein (G-protein)-coupled receptor in a G-protein alpha(13)-, Brx-, and RhoA-dependent fashion, enhanced GR transcriptional activity, whereas depletion of endogenous Brx attenuated this effect. These findings suggest that glucocorticoid signaling and, hence, the tissue sensitivity to glucocorticoids, may be coupled to extracellular signals via Brx and small G-proteins. Nuclear Brx might act as a local GRE-GR-transcriptosome activator by mediating the effect of small G-proteins on glucocorticoid-regulated genes.

VACCINIA VIRUS MEDIATED p53 GENE THERAPY FOR BLADDER CANCER IN AN ORTHOTOPIC MURINE MODEL

We determined if vaccinia virus (VV) mediated delivery of human tumor suppressor p53 is safe and effective for bladder tumor therapy in an orthotopic murine model. We used recombinant VV (rVV) vectors to express transgenes in murine bladder cancer MB-49 cells in culture and those growing orthotopically in syngeneic mice. Cultured MB-49 cells were infected with rVV expressing reporter genes (rVV-L15) or p53 (rVV-TK-53) to measure virus infection and apoptosis induction. Orthotopic MB-49 tumors in C57/Bl6 mice were treated with intravesical instillation of rVV, and the tumor incidence, survival and transgene expression were determined. Productive virus infection in vitro was observed in MB-49 cells, although at somewhat lower efficiency than in African Green Monkey kidney CV-1 cells (American Type Culture Collection, Manassas, Virginia). Expression of transgenes in vitro correlated with the virus dose. Cells infected with rVV underwent apoptosis with rVV-TK-53 inducing far greater cell death than rVV-L15. The rVV-L15 virus had no effect on tumor incidence but it increased mean survival compared with control. Instillation of rVV-TK-53 decreased the tumor incidence and 33% of mice survived treatment. At necropsy all nonsurviving mice had bladder tumor, whereas 2 survivors in the rVV-TK-53 treated group were tumor-free. Immunohistochemistry of tumors detected expression of the human p53 gene product in tumor cells. To our knowledge we report for the first time that recombinant vaccinia virus expressing human p53 can induce the death of MB-49 tumor cells in vivo, not only through the lytic effect of the virus, but also through expression of the death inducing p53 transgene. Further studies are needed to shed light on the mechanisms of rVV-TK-53 mediated tumor apoptosis and the antitumor immune response.

Norethisterone is bioconverted to oestrogenic compounds that activate both the oestrogen receptor α and oestrogen receptor β in vitro

In the present study, we used [ 3H]norethisterone to explore the bioconversion of this compound to A-ring reduced metabolites in African Green Monkey Kidney CV-1 cells and breast cancer T-47D cells. Additionally, we analyzed the capability of each norethisterone tetrahydro-reduced compound to bind the human oestrogen receptors α and β and transactivate an oestrogen-sensitive reporter gene. The results showed that norethisterone is mainly metabolized to 3α,5α-norethisterone (>85% of total [ 3H]norethisterone added) by CV-1 and T-47D cells, and that both A-ring tetrahydro-reduced metabolites exhibit different capabilities to displace [ 3H]17β-oestradiol from the oestrogen receptor α and β, being 3α,5α-norethisterone the weakest competitor. We also found that 3α,5α-norethisterone and 3β,5α-norethisterone activate both oestrogen receptors at nanomolar concentrations and that the transactivation induced by the oestrogen receptor α was generally higher (1.7- to 4.0-fold) than that provoked by the β receptor isoform. In oestrogen receptor α-transfected CV-1 and T-47 D cells, the oestrogenic-like potency of the 3β,5α-tetrahydro-reduced form was similar to that exhibited by 17β-oestradiol and 2.5- to 4.0-fold higher than that shown by the 3α,5α-reduced compound; conversely, in the oestrogen receptor β system the potency of the natural ligand was higher than that presented by the 3β,5α-tetrahydro-reduced metabolite. In CV-1 cells expressing the oestrogen receptor β, the transactivation potency of 3β,5α-norethisterone was ∼2-fold higher than that exhibited by its 3α,5α-tetrahydro-reduced isomer, whereas in T-47D cells the potency of the 3α,5α-tetrahydro-reduced compound was slightly higher than that shown by the 3β,5α A-ring reduced norethisterone metabolite. These results demonstrate that CV-1 and T-47D cells possess the enzymatic machinery to bioconvert norethisterone into the 5α-reduced, 3α-hydroxylated form and that neither 3α,5α- or 3β,5α-norethisterone exhibit preference or selectivity towards a particular oestrogen receptor isoform to induce a particular oestrogenic effect in these cell lines.

Cell Surface Expression of a Specific Antigenic Site on the Catalytic Subunit of (Na+ + K+)-ATPase

Structural localization of a peptide region, KRQPRNPKTDKLVNE, in the catalytic subunit of (Na+ + K+)-ATPase was investigated using a specific antibody directed against this peptide in cultured African green monkey kidney CV-1 cells. Immunofluorescence staining of frozen cell sections shows that an anti-KRQPRNPKTDKLVNE antibody (SSA95) interacts with its antigenic site and binds to the extracellular side of the cell membrane. Indirect immunofluorescence and flow cytometric analyses confirmed the presence of this epitope on intact cell surfaces. These results suggest that the KRQPRNPKTDKLVNE region of the (Na+ + K+)-ATPase is expressed on the cellular membrane surface.

Transactivation of progestin- and estrogen-responsive promoters by 19-nor progestins in African Green Monkey Kidney CV1 cells.

New and more potent progestins and antiprogestins suitable for reproductive therapy and contraception are currently the target of intensive research. The design of such drugs has been hampered by the complex technology required for screening these compounds at the molecular level. To solve this problem, we developed an in vitro cell system that allows detection of the progestagenic effects of a given compound using a PRE2-TATA-CAT reporter vector transiently introduced in a cell line stably transfected with the rabbit progesterone receptor (PR). The African Green Monkey Kidney CV1 (AGMK-CV1) cell line was chosen because these cells do not express endogenous steroid receptors; the selected clone stably expressing the rabbit PR has been maintained in our laboratory for more than 2 yr without detectable losses in PR content and progestagenic response. The presence and function of the PR were assessed by immunohistochemical and saturation analyses as well as by monitoring transactivation of the PRE2-TATA-CAT reporter gene. In this cell line, the PR is expressed at a concentration of 0.170 fmol/mg of protein, and the receptor is localized within the cell nucleus in either the presence or absence of the potent synthetic progestin R5020. This PR-expressing cell system allowed study of the in vitro progestational activity of several 19-nor progestins. The antiprogestin RU486 inhibited CAT activity induced by R5020; norethisterone (NET), levonorgestrel (LNG), and gestodene (GSD) induced PRE2-TATA-CAT activity at concentrations similar to those of R5020, whereas NET A-ring-reduced metabolites induced CAT activity at an extent lower than (5alpha-NET) or similar (3beta,5alpha-NET) to that of the precursor compound. The PRE2-TATA-CAT induction by 17beta-estradiol was also analyzed and no crossreactivity was detected. However, when the ERE-VitA2-TK-CAT (estrogen-responsive element-vitellogenin A2-thymidine kinase promoter-CAT) reporter vector and the estradiol receptor alpha or beta were cotransfected, CAT activity was induced in the presence of 17beta-estradiol, and NET tetrahydro-reduced derivatives. The results indicate that this AGMK-CV1-PR cell assay system appears to be suitable for measuring the effects of different synthetic progestins at the transcriptional level. In this assay system, NET, LNG, and GSD exhibit potent progestational effects at the transcriptional level. In the particular case of NET, the assay system allowed us to determine that the single or multiple hormonal transcriptional effects of this compound are partially mediated by its A-ring-reduced derivatives.

Biological activity profiles of 1alpha,25-dihydroxyvitamin D2, D3, D4, D7, and 24-epi-1alpha,25-dihydroxyvitamin D2.

We have synthesized several 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D] derivatives and evaluated their biological activity in terms of their binding affinity for the vitamin D receptor (VDR) and vitamin D-binding protein (DBP), antiproliferative or differentiation-inducing effects on human promyelocytic leukemic HL-60 cells, and transcriptional activity on a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter, including two vitamin D-responsive elements (VDREs), and human osteocalcin gene promoter, including a VDRE in transfected human osteosarcoma MG-63 cells. Furthermore, human VDR- or retinoic acid X receptor alpha (RXR alpha)-mediated luciferase activities of the derivatives were also measured by a one-hybrid system in human epitheloid carcinoma, cervix HeLa cells and African green monkey kidney CV-1 cells. Binding affinity for VDR, bone-resorbing activity, antiproliferative and cell-differentiating effects, transactivation potencies on target genes and VDR- or RXR alpha-mediated gene regulations of 1alpha,25(OH)2D2 and 1alpha,25(OH)2D4 were almost comparable to the effects of 1alpha,25(OH)2D3 while 24-epi-1alpha,25(OH)2D2 and 1alpha,25(OH)2D7 were much less active than 1alpha,25(OH)2D3 in these respects. This is the first report concerning biological assessment of 1alpha,25(OH)2D2, 1alpha,25(OH)2D3, 1alpha,25(OH)2D4, 24-epi-1alpha,25(OH)2D2 and 1alpha,25(OH)2D7 at the molecular level, especially with regards to the structural differences at the 24R- or 24S-methyl group and a double bond between carbons 22 and 23 in the side chain of 1alpha,25(OH)2D derivatives.

Constitutive activation of the aromatic hydrocarbon receptor.

The ligand-activated aromatic hydrocarbon receptor (AHR) dimerizes with the AHR nuclear translocator (ARNT) to form a functional complex that transactivates expression of the cytochrome P-450 CYP1A1 gene and other genes in the dioxin-inducible [Ah] gene battery. Previous work from this laboratory has shown that the activity of the CYP1A1 enzyme negatively regulates this process. To study the relationship between CYP1A1 activity and Ah receptor activation we used CYP1A1-deficient mouse hepatoma c37 cells and CYP1A1- and AHR-deficient African green monkey kidney CV-1 cells. Using gel mobility shift and luciferase reporter gene expression assays, we found that c37 cells that had not been exposed to exogenous Ah receptor ligands already contained transcriptionally active AHR-ARNT complexes, a finding that we also observed in wild-type Hepa-1 cells treated with Ellipticine, a CYP1A1 inhibitor. In CV-1 cells, transient expression of AHR and ARNT leads to high levels of AHR-ARNT-dependent luciferase gene expression even in the absence of an agonist. Using a green fluorescent protein-tagged AHR, we showed that elevated reporter gene expression correlates with constitutive nuclear localization of the AHR. Transcriptional activation of the luciferase reporter gene observed in CV-1 cells is significantly decreased by (i) expression of a functional CYP1A1 enzyme, (ii) competition with chimeric or truncated AHR proteins containing the AHR ligand-binding domain, and (iii) treatment with the AHR antagonist alpha-naphthoflavone. These results suggest that a CYP1A1 substrate, which accumulates in cells lacking CYP1A1 enzymatic activity, is an AHR ligand responsible for endogenous activation of the Ah receptor.

Tissue-specific enhancer of the human multidrug-resistance (MDR1) gene.

Identification of cis-regulatory sequences is a first step in analyzing the regulation of the human multidrug-resistant 1 (MDR1) gene which encodes the 170-kilodalton membrane P-glycoprotein in normal tissues and tumor cells. We have studied several overlapping genomic clones containing the 5'-flanking region of the gene. These clones span about 30 kilobases (kb) of contiguous DNA containing 10 kb of the gene and 20 kb of the 5'-flanking sequence. The nucleotide sequence of the first exon and the 2 kb preceding the exon were determined. DNA sequences containing the 5'-flanking regions were linked to the chloramphenicol acetyltransferase (CAT) gene. For transient CAT assay, we have employed six cell lines, including human cancer KB, vincristine-resistant VJ-300 derived from KB, mouse adrenal tumor Y-1, African green monkey kidney CV-1, mouse fibroblast NIH3T3, and human adrenal carcinoma SW-13 cells. Promoter activity was very weak regardless of the length of the promoter region in mouse adrenal tumor Y-1 and monkey kidney CV-1 cells, in which endogenous P-glycoprotein was expressed. Introduction of a 700-base genomic DNA fragment from a site located at 10 kb far upstream of the initiation site increased the transcription of the CAT gene in Y-1, CV-1, and SW-13 cells. However, no significant increase in the CAT activity could be observed in NIH3T3, KB, and VJ-300 cells. This fragment markedly augmented the expression of the CAT gene regardless of orientation or position, and it acted in a cell type-specific manner even with heterogenous promoters. Our present study suggests that the 700-base pair fragment may carry a tissue-specific transcriptional enhancer that is active in at least some adrenal and kidney-derived cell lines.

Primary sequence and heterologous expression of nuclear pore glycoprotein p62.

The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in nuclear transport. We have cloned, sequenced, and expressed the full-length rat p62 cDNA. The rat p62 mRNA is 2,941 nucleotides long and encodes a protein of 525 amino acids containing 30% serine and threonine residues. The amino acid sequence near the amino-terminus contains unique tetrapeptide repeats while the carboxy-terminus consists of a series of predicted alpha-helical regions with hydrophobic heptad repeats. Heterologous expression of rat p62 in African Green Monkey Kidney COS-1 cells and CV-1 cells was detected using a species-specific antipeptide serum. When transiently expressed in COS-1 cells, rat p62 binds wheat germ agglutinin and concentrates at the spindle poles during mitosis. In CV-1 cells cotransfected with rat p62 cDNA and SV40 viral DNA, rat p62 associates with the nuclear membrane without interfering with the nuclear transport of SV40 large T antigen. The ability to express p62 in tissue culture cells will facilitate analysis of the role of this pore protein in nuclear transport.

Tissue culture fixation with diimidoesters—IV. A comparison between adipimidate and aldehydes of their effects on cell size and morphology

The effects of fixation with dimethyladipimidate (DMA) on cell size and morphology were compared with those from aldehydes (glutaraldehyde, formaldehyde) using African green monkey kidney CV1 cells. Fixation with any of the three fixatives makes the cells resistant to any considerable size alteration. Glutaraldehyde has the strongest stabilizing effect, formaldehyde occupies an intermediate position while DMA forms the least cross-links of all. Morphologically (phase contrast microscopy), DMA-fixed and aldehydes-fixed cells stained with haematoxylin/eosin were similar except that with DMA the nuclei exhibit a granular thread-like structure with apparent granular nucleolei. This was also observed in isolated nuclei. Furthermore, in DMA-fixed unstained cells the cytoplasmic filament bundles are very distinct. This feature is not present to the same extent in aldehydes-fixed cells. Data from stained CV1 monolayers, from determinations of free amino groups and from gel electrophoresis show that DMA fixation does not preclude a subsequent reaction with glutaraldehyde or formaldehyde.

Tissue culture fixation with diimidoesters—III. Influence of adipimidate on cell size, thermal and mechanical stability

The effects of fixation with dimethyladipimidate (DMA) on cell size and stability to thermal and mechanical stresses were studied using African green monkey kidney CV1 cells. The variables investigated were fixative and buffer concentrations, type of buffer and rinsing after fixation. Buffer concentration had little effect on cell size, whereas DMA concentration had greater one. Type of buffer seemed to have no effect. Measurements that were carried out without washing the fixative after fixation indicated marked changes on cellular size, while post-fixation washing recovered these changes. The above parameters also influence the morphologic appearance of cells growing on petri dishes, either by showing the cytoskeletal bundles to some extent or by being vacuolated. DMA fixation renders the cells stable to thermal and mechanical stresses without or with small effect on cell diameter.

Structural state of newly replicated closed circular simian virus 40 DNA.

We have studied the early transition of newly replicated, segregated daughter molecules of simian virus 40 (SV40) into their mature, fully supercoiled state. The DNA of SV40 replicating in African green monkey kidney CV1 cells was chronically labeled with [14C]thymidine and pulse-labeled with [3H]thymidine. The cells were lysed and the viral DNA was isolated. Density gradient centrifugation of viral DNA in cesium chloride revealed that the pulse-labeled, newly synthesized, closed circular supercoiled DNA molecules banded at a slightly higher density (delta sigma = 0.0025) than the chronically labeled DNA, suggesting that the newly completed molecules were in a different structural state. Electrophoresis of DNA in agarose gels at appropriate chloroquine concentrations demonstrated that the mobility of the pulse-labeled closed, superhelical DNA was retarded relative to that of the chronically labeled DNA. These observations indicated that the newly completed SV40 DNA molecules existed in a structural state more relaxed than that of mature DNA by one or two linking numbers.

Purification of transiently transfected cells by magnetic affinity cell sorting

A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-anti-body-coated magnetic particles in the presence of a sorting solution designed tominimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained.

Pausing in simian virus 40 DNA replication by a sequence containing (dG-dA)27.(dT-dC)27.

A 200 bp sequence including a stretch of 54 base pairs of alternating guanosine and adenosine nucleotide residues [(dG-dA)27.(dT-dC)27] was cloned in the simian virus 40 (SV40) genome between the KpnI and HpaII sites. This sequence was discovered earlier as part of a region limiting the amplification of sequences adjacent to an integrated polyoma virus in a transformed rat cell line. The newly constructed DNA was transfected into African Green monkey kidney CV1 cells and the variant virus was isolated by plaque-purification. The insertion was stably maintained and the variant virus grew more slowly than the wild type, had lower titers and gave smaller plaques. In mixed infection experiments, the variant was found to be stable, though the wild type replicated more rapidly. Pulse labeling experiments indicated that the unusual inserted sequence acts as a pause site for fork progression during DNA replication, as evidenced by the rate of incorporation of radioactively labeled nucleotides into various regions of the SV40 genome. Statistical fit of the experimental curves with theoretically generated curves suggested the pause of fork progression to be about one minute.

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.

A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells

We have constructed a shuttle vector plasmid for studying mutagenesis in mammalian cells. The plasmid replicates in cell lines permissive for SV40 virus as well as in the bacterium Escherichia coliand carries a bacterial suppressor tRNA gene ( supF) that can serve as a mutagenesis marker. The plasmid replicates as efficiently as SV40 virus in African Green Monkey kidney CV1 cells, indicating that all traces of the inhibitory sequences normally found in pBR322 and its derivatives have been removed. The design of the plasmid and the small size of the mutagenesis target gene decrease the probability of recovering spontaneous deletion mutations that have been shown to occur at high frequency during passage in mammalian cells. The frequency of spontaneous-mutant plasmids recovered after passage in CV1 cells is substantially lower than with other vectors described previously. When the plasmid DNA is treated with UV radiation before passage in CV1 cells, mutants are observed at a frequency about 20-fold above the spontaneous background.