Demonstration of Infectious Deoxyribonucleic Acid in Transformed Cells I. Recovery of Simian Virus 40 from Yielder and Nonyielder Transformed Cells

Deoxyribonucleic acid (DNA) was extracted from virus-free simian virus 40 (SV40)-transformed hamster, mouse, and monkey cells and was inoculated into simian cells in the presence of diethylaminoethyl (DEAE)-dextran; infectious SV40 was recovered by using DNA from cell lines which fail to yield virus by the fusion technique as well as from cell lines which readily yield virus by fusion. The rescued virus was identified as SV40 by three methods: (i) neutralization of plaque formation by specific antiserum; (ii) induction of synthesis of viral-specific antigens detected by immunofluorescence; and (iii) presence of papovavirus particles seen by the electron microscope. Treatment of the transformed cell DNA with deoxyribonuclease or omission of the DEAE-dextran prevented the rescue of virus. Large amounts of transformed cell DNA were required (>10 mug/culture of 10(6) cells) to effect rescue of SV40 by passage through monkey cells. A linear response was obtained between the input of DNA with inocula between 10 and 45 mug of DNA/culture and the yield of SV40 recovered. Biological activity was demonstrable irregularly when the transformed cell DNA was assayed directly in the presence of DEAE-dextran. The DNA induced plaque formation in about 50% of the trials as well as the synthesis of SV40 tumor and viral antigens in rare simian cells. The infectious DNA appeared to be associated with cellular DNA. The infectivity was found in the pellet of precipitated DNA obtained by the Hirt technique and was inactivated by boiling for 15 min. These properties are characteristic of linear cellular DNA and not of free, circular SV40 DNA.     

Rescue of infectious virus from permissive monkey cells containing simian virus 40 DNA fragments.

Permissive TC7 cells were separately transfected with simian virus 40 (SV40) EcoRI/Hap II A (74% genome) DNA fragments and EcoRI/Hap II B (26% genome) DNA fragments in the presence of DEAE-dextran. Fusion of the progeny of recipient cells receiving the A fragment, TC7 (SV40/74) cells, with TC7 (SV40/26) cells, which had received the B fragment, resulted in SV40 rescue. TC7 (SV40/74 + 26) cells, which had simultaneously received both complementary subgenomes, either spontaneously produced SV40 upon subculture or yielded virus upon treatment with iododeoxyuridine. In addition, fusion of rat cells containing the EcoRI/Hap II A fragment with TC7 (SV40/26) cells resulted in SV40 rescue. Cytopathology, V-antigen production, neutralization, and electron microscopy were parameters used to verify that the rescued virus was SV40. No infectious virus was produced when the combinations of cells fused did not total a complete SV40 genome equivalent.     

Efficient gene transfer by sequential treatment of mammalian cells with DEAE-dextran and deoxyribonucleic acid

A variation of the classical DEAE-dextran method of gene transfer was developed for efficient transfection of HeLa cells with plasmid DNA. A brief exposure of the cells to medium containing DEAE-dextran was found to be sufficient for subsequent uptake of pRSVcat and to be superior to cocultivation of the cells with DEAE-dextran plus DNA. This sequential method of gene transfer is nontoxic and yielded up to 60% of HeLa cells positive for a surface protein encoded by the transfected sequence. The implications of this sequential transfection technique regarding the mechanisms of gene transfer are discussed.     

Murine teratocarcinoma: A model for virus-cell interaction in a differentiating cell system

The stem cell of the murine teratocarcinoma is refractory to infection with Simian virus 40 and polyoma. Utilizing various procedures, we attempted to alter this block to infection by modifying the infection procedure. Multiple infections with high-titer SV₄₀ and pretreatment of cells with DEAE-dextran or the carcinogen 4-nitroquinoline l-oxide did not induce embryonal carcinoma cells to produce T- antigen. Co-infection with adenovirus 5, which infects the embryonal carcinoma, and SV₄₀ did not induce the expression of SV₄₀ Tantigen. Therefore, these procedures did not overcome the block to virus infection. The assay for the SV₄₀ T antigen was immunofluorescence; however, the immunoprecipitation technique did not detect T antigen in the infected embryonal carcinoma cells. Finally, the viral DNA present in the embryonal carcinoma was examined for its ability to replicate. These studies showed that viral DNA was not replicating as assayed by the viral DNA's sensitivity to UV irradiation when replicating in the presence of 5-bromodeoxyundine.     

Rescue of SV40 following transfection of TC7 cells with cellular DNAs containing complete and partial SV40 genomes

Summary Infectious SV40 virions could be rescued from permissive TC7 cells within one to three subcultures following cotransfection with two cellular DNAs, each containing a complementary portion of the SV40 genome. SV40 virions could also be rescued by transfection of TC7 cells with cellular DNAs from a variety of SV40 transformed cells containing complete genome equivalents but not from cells containing subgenomes alone or defective genomes. Infectious virus was not rescued if the transfecting DNA species was treated with DNAase or if the DEAE-dextran pretreatment of the recipient cells was omitted.deceased     

Superinfection of Simian Virus 40-Transformed Permissive Cells with Simian Virus 40

Evidence that the resistance of simian virus (SV40)-transformed permissive cells to superinfection with SV40 is due to lack of virus uptake is presented. When virus uptake is enhanced, the events of infection proceed as in normal permissive cells, resulting in production of infectious virus.     

Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures.

Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.     

Binding of polycation DEAE-dextran to Chinese hamster ovary cells induces reversible Ca2+ influx and inhibits capping of concanavalin A acceptor proteins

Binding of the polycation DEAE-dextran to the cell surface of HA-1 CHO cells caused a marked increase in 45Ca2+ exchange influx. The effect was fairly selective for Ca2+, undirectional (efflux was not increased) and was rapidly reversed by treatment with polyanion dextran sulfate. 45Ca2+ influx could not be stimulated by treatment with multivalent lectins or fibronectin. In addition to stimulating 45Ca2+ flux, DEAE-dextran inhibited the capping of concanavalin-A acceptor proteins. Inhibition of capping occurred over the same DEAE-dextran concentration range (20-200 micrograms/ml) which stimulated 45Ca2+ uptake, possibly implicating increased cellular [Ca2+] in the inhibition of concanavalin A acceptor protein capping in this cell type. The profound effect of DEAE-dextran on cellular Ca2+ uptake and the rapid reversal of the effect by dextran sulfate might make the polycation a useful agent for the induction of transient increases in cellular [Ca2+].     

Plaque Formation by 55 Rhinovirus Serotypes

Plaque production by 55 rhinoviruses in several lines of HeLa cells is reported. Forty-nine types produced macroscopic plaques under the conditions described previously employing 30 mM MgCl(2) and 30 mug/ml of DEAE-dextran in overlay and M HeLa cells. Basically three kinds of plaques were observed: clear large and intermediate sized plaques and small turbid plaques. Five rhinoviruses produced plaques only in a sensitive clonal line isolated from the M HeLa line. Rhinovirus type 4 produced plaques after the pretreatment of cells with actinomycin D. Enhancement of plaque formation by MgCl(2) has been demonstrated to date with 17 rhinoviruses. Some rhinoviruses were enhanced either by DEAE-dextran or by dextran sulfate in the overlay. No inhibitors were found in any of 10 bovine sera tested. Rhinovirus type 2 was visualized inside HeLa cells by electron microscopy and there appeared to be a very high ratio of physical particles per infectious unit of virus.     

Gene transfer method for transient gene expression, stable transformation, and cotransformation of suspension cell cultures.

A new method was developed to study transient gene expression, stable transformation, and cotransformation in suspension cells, such as mouse myeloma and erythroleukemia cells. This method involves attachment of cells to a concanavalin A-coated tissue culture dish, treatment of cells with DEAE-dextran to adsorb plasmid DNA to the attached cells, and finally treatment with a 40% solution of polyethylene glycol to facilitate the uptake of DNA by the cells. Plasmids pSV2cat and pSV2neo were used as markers to optimize the conditions for transient gene expression and stable transformation, respectively, of mouse myeloma and erythroleukemia cells. This method was successfully used to obtain cotransformants of mouse myeloma cells.     

Comparison of DNA facilitators in the uptake and intracellular fate of infectious herpes simplex virus type 2 DNA.

The comparative efficiencies of polyornithine, CaCl2 and DEAE-dextran in enhancing the infectivity of exogenous herpes simplex virus (HSV) type 2 DNA were examined. CaCl2 was 12-times more effective in promoting genetic expression of viral DNA than DEAE-dextran, while polyornithine did not mediate any HSV DNA infectivity. A comparison of sedimentation profiles of DNA extracted from cells inoculated with viral DNA in the presence of each facilitator revealed that input 56 S HSV DNA underwent marked alteration with time. There was also extensive processing of a 20 S DNA species. The data indicated that the biological efficiency of each facilitator was related to the amount of 20 S DNA remaining at the final time periods. One possible explanation for the efficiency of CaCl2 as a facilitator was that it allowed for the reutilization of the 20 S DNA species during HSV replication. The persistence of the 20 S peak in cells treated with DEAE-dextran was a measure of the decreased efficacy of this facilitator. Finally, the absence of a 56 S peak and the greatly elevated levels of 20 S DNA at final time points in polyornithine-treated cells accounted for the failure of this compound to promote HSV DNA infectivity.     

Phosphorylation of proteins in cytoplasmic and nuclear fractions of human cells treated with double-helical RNA and human recombinant interferon type I]

The dynamics of protein kinases activity in nuclear and cytoplasmic fractions of human fibroblasts treated by preparations of natural and synthetic dsRNA (ridostin, rifastin, larifan and poly(I).poly(C), DEAE-dextran and dsRNA complexes with DEAE-dextran), as well as by preparations of recombinant alpha-2 and beta-1 interferons was obtained. The early activation of enzymes in treated cells extracts and their presence in dsRNA-activated and nonactivated forms were found. In cytoplasmic cellular fractions treated by interferons the dsRNA dependent protein kinases (nonactivated forms- were prevalent.r In contrast, in dsRNA treated cells or dsRNA complexes with DEAE-dextran treated ones the dsRNA independent protein kinases (activated forms) were found, while dsRNA dependent forms induced by interferons were found at later periods. Nuclear protein kinases are mainly dsRNA independent making possible the supposition of their intracellular activation by incoming dsRNA or interferon-induced formation of ds-structures in cellular nuclei. In phosphorylated proteins spectre the 90, 69, 45-40 and 30-35 kDa polypeptides were found. At early intervals in nuclear fractions was found a nuclease resistant and partially EDTA resistant high molecular phosphorylated complex (120 kDa). The complex is, probably, capable of dissociation to low mol mass components. DEAE-dextran induces strong activation of protein kinases in cytoplasm and nuclei and increases the content of activated forms of enzyme in larifan treated cells.     

Conditions that affect binding of DNA by cultured plant cells and protoplasts: An autoradiographic analysis

Summary The binding of the¹⁴C-labelledSalmonella typhimurium DNA or³H-labelled soybean SB-1 DNA to cultured soybean cells (Glycine max L. Merr.) (SB-1) could be increased at least 100-fold by choosing the proper incubation conditions. The uptake of DNA by cells could completely be inhibited by the addition of an excess of unlabelled thymidine, indicating that the observed uptake of DNA by cells most probably is simply uptake of DNA degradation products. Autoradiograms, prepared from SB-1 protoplasts that were previously incubated with DNA, showed that the DNA was not associated with the protoplasts, but only with aggregates of cell wall material contaminating the protoplast preparation. When protoplasts and DNA were incubated in the presence of DEAE-dextran, the amount of DNAse resistant radioactivity increased 40 times. Again, the autoradiograms showed that most if not all DNAse-resistant material was associated with cell wall materials. Our observation that it is cell wall contaminants in protoplast preparations which account for most of the DNA binding demonstrates the need for caution in interpreting experiments on the binding and uptake of DNA by plant protoplasts.NRCC No. 16353.     

Characterization of saturable binding sites for rabies virus.

A specific, saturable receptor for rabies virus was analyzed on cultured cells of neural or non-neural origin. Viral attachment kinetics were enhanced by DEAE-dextran, an effect which in turn enhanced the apparent infectivity of the virus inoculum. Under optimized conditions, the attachment of metabolically labeled ERA strain rabies virus obeyed the laws of mass action, whereby the amount of virus bound to cells varied proportionally with the concentration of cells or virus. Attachment was sensitive to changes of temperature and pH, did not require divalent cations such as Mg2+ or Ca2+, and occurred despite prior treatment of cells with proteolytic or sialic acid-specific enzymes. Saturation of the cell surface with rabies virus could be accomplished with 3 X 10(3) to 15 X 10(3) attached virions per cell. Competition for the rabies receptor occurred with rabies nonpathogenic variant virus, RV194 -2, and vesicular stomatitis virus. Reovirus type 3, another neurotropic virus, failed to inhibit rabies virus binding, and West Nile virus only slightly inhibited rabies virus binding, suggesting independent cellular receptors were recognized by these viruses. Isolated rabies virus glycoprotein failed to compete in an equivalent manner. However, solubilization of BHK-21 cells with octylglucoside yielded a chloroform-methanol-soluble extract which blocked rabies virus attachment. The binding inhibition activity of this extract was resistant to proteases but could be destroyed by phospholipases and neuraminidase, suggesting a phospholipid or glycolipid component at the receptor site. These data provide evidence for a rhabdovirus-common mechanism for cellular attachment to cells in culture.     

Effects of mixed micellar lipids on carotenoid uptake by human intestinal Caco-2 cells

We reported previously that lysophosphatidylcholine remarkably enhanced β-carotene uptake from bile acid-mixed micelles by human intestinal Caco-2 cells. In the present study, we evaluated how mixed micelle components other than phospholipids, viz., fatty acids, monoolein, and cholesterol, affect carotenoid uptake by Caco-2 cells. Each component influenced the β-carotene uptake in a different way depending on micellar composition. Oleic acid at 200 μM significantly enhanced uptake in the absence of lysophosphatidylcholine. Cholesterol at 40 μM significantly reduced uptake in the presence of lysophosphatidylcholine, while no reduction was found in the presence of 200 μM oleic acid. Facilitated diffusion was suggested partly to mediate uptake in mixed micelles, except for mixed micelles containing 200 μM oleic acid. Uptake mediated by facilitated diffusion was approximately 20% of total uptake. Mixed micellar lipids have the potential to modify intestinal uptake.     

Substrate-dependent differences in growth and biological properties of fibroblasts and epithelial cells grown in microcarrier culture

Normal diploid human fibroblasts and first passage monkey kidney epithelial cells were examined for growth and metabolic activity on microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells cm2 of surface area) on the glass microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells/cm2 of surface area) on the glass microcarriers than they did on the DEAE-dextran microcarriers and morphological differences were observed between the cells growing on the two substrates. On the DEAE-dextran microcarriers, the cells were much more resistant to protease-mediated detachment than were the cells on the glass microcarriers. In these respects, the cells grown on the glass microcarriers were similar to cells grown in conventional monolayer culture. Interestingly, the cells grown on the DEAE-dextran microcarriers expressed higher levels of proteolytic enzyme activity than the cells grown on the glass microcarriers. Substrate-dependent differences in prostaglandin production also occurred--both in unstimulated cells and in cells stimulated with 12-0-tetradecanoyl phorbol acetate. The unstimulated cells on the glass microcarriers produced slightly higher levels of three different prostaglandins than did the cells on the DEAE-dextran microcarriers. However, after stimulation the levels were much higher in the DEAE-dextran microcarrier cultures than in the glass microcarrier cultures. In contrast to these results, there was no significant, substrate-dependent difference in the production of infectious herpes simplex virus. Taken together, these findings suggest that when commercially-useful cells such as normal fibroblasts and epithelial cells are grown in large quantities on microcarriers, the nature of the substrate may have a profound effect on the growth and physiology of the cells. They also suggest that when microcarriers are used, unexpected results based on preliminary work in conventional monolayer culture may be obtained.     

Control of Interferon Synthesis: Effect of Diethyl-aminoethyl-Dextran on Induction by Polyinosinic-Polycytidylic Acid

Interferon production in cultures of rabbit kidney cells (RKC) stimulated with 10 to 250 mug of polyinosinic-polycytidylic acid (poly I.poly C) per ml peaked at 3 to 4 hr after the exposure of cells to inducer and rapidly declined thereafter. On the other hand, RKC stimulated with poly I.poly C (10 or 2 mug/ml) in the presence of diethylaminoethyl (DEAE)-dextran (100 or 20 mug/ml, respectively) produced a protracted interferon response, with the release of interferon continuing for over 24 hr. The kinetics of interferon production in RKC stimulated with lower concentrations of the mixture of poly I.poly C and DEAE-dextran were similar to the response produced by poly I.poly C alone (10 to 250 mug/ml). Only the responses that terminated early were paradoxically enhanced by treatment with low doses of actinomycin D or with cycloheximide. Cells stimulated with 50 mug of poly I.poly C/ml showed hyporesponsiveness to a second interferon induction with poly I.poly C when restimulated 7 hr after primary induction. This hyporesponsiveness could be overcome by restimulating with higher concentrations of the poly I.poly C-DEAE-dextran complex. The results are compatible with the hypothesis that the early termination of interferon production and hyporesponsiveness to repeated induction with poly I.poly C are due to a cellular repressor exerting negative control on interferon synthesis, and that the increased cellular uptake of poly I.poly C in the presence of DEAE-dextran may effectively neutralize the repressor. These results also suggested that the often observed different kinetics and the varied effects of inhibitors of ribonucleic acid or protein synthesis on interferon responses in various cells and in cells stimulated with different inducers (such as with viruses as compared with polynucleotides) need not imply the existence of fundamentally different mechanisms of interferon production.     

Nature of permeability changes in membrane of HeLa cells adsorbing Sendai virus

Adsorption of Sendai virus to HeLa cells induced in them an increased permeability to K+, Na+, Ca++, deoxyglucose, but not to fluorescein. The stimulation of uptake of 42K was temperature-dependent, did not occur below 15 degrees C, and was not inhibited by ouabain. The virus-induced increase in the uptake and release of 42K and of 3H deoxyglucose could not be mimicked by treatment of cells with linoleic acid, a procedure which increased the fluidity of the cellular membranes. The stimulatory effect of 0.5 mM ATP on the release of deoxyglucose was enhanced several fold in the presence of Sendai virus. These results seem to indicate the possible involvement of membranal enzymes such as e.g. protein kinase in the permeability changes induced by Sendai virus.     

Oxygen uptake, acidification of medium and nitrate uptake induced by blue light in nitrate-starved Chlorella cells

Blue light-induced oxygen uptake of the colorless mutant of Chlorella kessleri (No. 9.80) was 30-40% higher in the presence of exogenous glycine than in its absence. None of the other amino acids tested had this effect. Moreover, mutant cells in which glutamine synthetase was inhibited by methionine sulphoximine, accumulated approximately 65% more ammonium ions under blue irradiation in the presence of exogenous glycine than in its absence. The protein kinase C inhibitors, staurosporine or K252a, reduced the enhancement of oxygen uptake by approximately 40%. The present results indicate that blue light-dependent deamination of endogenous glycine might be a prerequisite for enhanced oxygen uptake in Chlorella. This blue light-induced oxygen uptake was not influenced by the inhibitors of protein phosphatase, calyculin A or okadaic acid. On the contrary, calyculin A and okadaic acid had a marked effect on the acidification of the suspension medium and nitrate uptake induced by blue light in Chlorella cells. The different responses to the inhibitors of protein kinase and phosphatase suggest the presence of different pathways among the blue light signal transduction operating on oxygen uptake, acidification of the medium and nitrate uptake in Chlorella.