Contaminants within bacterial plasmid preparations trigger apoptosis in liposome transfected OVCAR3, but not in SKOV3 or AZ224 human ovarian cancer cells

Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.     

Effect of lysogeny on transfection and transfection enhancement in Bacillus subtilis.

Strains of Bacillus subtilis 168 lysogenic for bacteriophage phi105 transfer with deoxyribonucleic acid (DNA) isolated from bacteriophage SPO2 at a higher efficiency than non-lysogenic strains. This enhancement of transfection was not the result of recombination between bacteriophages SPO2 and phi105. Superinfection marker rescue increased transfection with DNA from bacteriophage phi105 occurred simultaneously with the addition of the transfecting DNA. Again, this enhancement of transfection was not the result of recombination but rather a protection of the transfecting DNA by the superinfecting bacteriophage. The ability of the superinfecting bacteriophage to protect the transfecting DNA from inactivation was maximal when the bacteria were just becoming competent. Bacteriophage phi1 cannot replicate after the transfection of competent bacteria lacking a functional DNA replication system, whereas bacteriophage phi1 was able to replicate after infection of competent bacteria grown under comparable conditions. These observations support the hypothesis that GAPase and an inducible repair system play an important role in the development of competence.     

Isolation by polymerase chain reaction of a cDNA whose product partially complements the ultraviolet sensitivity of xeroderma pigmentosum group C cells

A xeroderma pigmentosum (XP) cell line from complementation group C has been complemented to attain ultraviolet (UV) resistance and DNA repair proficiency, by transfection with a human expression cDNA library, followed by selection to UV resistance. We now show that the transfected cDNAs can be rescued from cellular DNA of a secondary transformant by its in vitro amplification using expression-vector-specific oligodeoxyribonucleotides as primers in a polymerase chain reaction. The amplified cDNAs were cloned into a mammalian expression vector. Their transfection into XP cells identified a single cDNA which specifically complemented the UV sensitivity of a group-C-derived cell line to the same partial UV-resistance levels exhibited by the transformant from which the cDNAs were rescued.     

Relationship Between Competence for Transfection and for Transformation

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).     

Inhibition of initiation of simian virus 40 DNA replication during acute response of cells irradiated by ultraviolet light.

To study the mechanism by which ultraviolet (UV) light inhibits DNA replication, we examined the effects of UV 254 nm irradiation on the replication of simian virus 40 (SV40) DNA and SV40-based plasmid in monkey cells. The study was designed to determine the relative contributions made by inhibition of replication initiation and chain elongation to the immediate inhibition of DNA replication following UV irradiation. We used two-dimensional neutral-alkaline electrophoresis to examine the behaviour of replication intermediates unambiguously. Kinetic analysis using this technique showed that initiation of replication started to decline at 15 min post-irradiation. When the pulse label incorporated in SV40 replication intermediates before irradiation was chased for 1 h, most of the label was found in mature Form I and II molecules. This indicated that replication elongation took place on damaged template. We also used a transfection technique to show that heavily irradiated plasmids replicated efficiently in unirradiated transfected cells. By the transfection technique, we observed that UV irradiation of host cells dose-dependently inhibited replication of transfected non-irradiated plasmids, suggesting that the inhibition of DNA replication is due to a global change in cellular physiology induced by UV. This change was also apparent from poor staining of the chromatin by fluorescent-DNA-binding dyes immediately after UV irradiation of intact cells. We conclude that a significant fraction of chain elongation proceeds on damaged templates and DNA replication during the acute response of cells irradiated with UV is mainly controlled by the inhibition of replication initiation.     

Effects of human interferon on cellular response to UV in UV-sensitive human cell strains

Cells of a human RSa cell line, with high sensitivity to UV killing and low capacity for DNA repair, when pretreated with 1-100 units/ml of human interferon (HuIFN) preparations for more than 12 h before irradiation, acquired an enhancement of UV-induced DNA-repair replication synthesis in association with recovery from inhibition of total cellular DNA synthesis and UV survival. Prompt and transient induction of plasminogen activator activities was also found within 5 min after UV irradiation in the cells pretreated with HuIFN but not in the cells non-pretreated with HuIFN. The enhancement and induction effects of HuIFN were observed, irrespective of the kind of HuIFN preparation used (alpha, beta or gamma, and natural or recombinant) and in other UV-sensitive fibroblast cells which were derived from Cockayne syndrome and xeroderma pigmentosum fibroblasts (XP1KY). However, all of the enhancement of DNA-repair synthesis and the induction of plasminogen activator activities by HuIFN was suppressed by treatment with cycloheximide immediately after UV irradiation.     

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.     

Properties of electroporation-mediated DNA transfer in Escherichia coli

Efficient and reproducible DNA-transfection was attained in E. coli, by electroporation. The yield of the transfectants was affected by pretreatment of the recipient cells as well as by the composition of the electroporation medium. Using a single pulse procedure, relationships among the electrical parameters, the transfection efficiency, and the cellular viability were investigated in 10 mM Tris-HCl buffer (pH 7.5) containing 5% sucrose. Certain sodium salts (e.g., citrate, phosphate, and sulfate) were promotive, whereas Mg2+, DEAE-dextran, and polyvinylpyrrolidone were inhibitory to the transfection. Heterologous nucleic acids (native DNA, denatured DNA, and tRNA) exerted only a marginal effect on transfection with a viral replicative-form DNA. The efficiency of DNA transfer was affected by culture conditions, and bacteria grown at a higher temperature were more competent. The electroporation system was more efficient than an improved CaCl2 method, not only in transfection with viral single- and double-stranded DNAs, but also in transformation with plasmid DNAs.     

UV stimulation of DNA-mediated transformation of human cells.

Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells (complementation groups A and F), which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. No stimulation was found after damaging vector DNA by treatment with DNase or gamma rays. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome.     

In vitro mutation of Haemophilus influenzae transforming deoxyribonucleic acid by ultraviolet radiation at -70 degrees C

Previous studies have shown the non-mutability of Haemophilus influenzae either by UV irradiation of the cells or by irradiating the transforming DNA and transformation of competent cells. In the present work, we present evidence of transforming DNA mutation in vitro by UV irradiation at -70 degrees C, which upon transformation of competent cells showed a rise in the mutation frequencies of novobiocin resistance of the order of several hundredfold. Also we performed experiments using the UV-irradiated DNA either sonicated or DNase-treated, which allowed us to propose that such rise in mutation frequency is probably due to the integration of DNA carrying premutagenic photoproducts to the recipient cells' genome. We think that the key point was the low temperature at which the DNA was irradiated in order to obtain the mutagenic effects, since it is likely that at -70 degrees C, the main photoproducts are not the cyclobutane dimers, but are the spore photoproducts, which are probably responsible for the damage that leads to mutagenic effects.     

Inhibition of transformation and transfection in Haemophilus influenzae Rd9 by lysogeny.

Haemophilus influenzae Rd9 lysogenic for temperate bacteriophage N3 was found to be virtually nontransformable and nontransfectable. This inhibition of transformation and transfection was due partly to the decreased capacity of competent lysogenic cells for irreversible binding of deoxyribonucleic acid (DNA) and partly to some events taking place after adsorption of the DNA. The unadsorbed DNA was not degraded by the competent lysogenic cells.     

Mechanism of Transfection with Deoxyribonucleic Acid from the Temperate Bacillus Bacteriophage φ105

Bacteriophage phi105 is a temperate phage for the transformable Bacillus subtilis 168. The infectivity of deoxyribonucleic acid (DNA) extracted from mature phi105 phage particles, from bacteria lysogenic for phi105 (prophage DNA), and from induced lysogenic bacteria (vegetative DNA) was examined in the B. subtilis transformation system. About one infectious center was formed per 10(8) mature DNA molecules added to competent cells, but single markers could be rescued from mature DNA by a superinfecting phage at a 10(3)- to 10(4)-fold higher frequency. Single markers in mature DNA were inactivated at an exponential rate after uptake by a competent cell. Prophage and vegetative DNA gave about one infectious center per 10(3) molecules added to competent cells. Infectious prophage DNA entered competent cells as a single molecule; it gave a majority of lytic responses. Single markers in sheared prophage DNA were inactivated at the same rate as markers in mature DNA. Prophage DNA was dependent on the bacterial rec-1 function for its infectivity, whereas vegetative DNA was not. The mechanism of transfection of B. subtilis with viral DNA is discussed, and a model for transfection with phi105 DNA is proposed.     

Free radical-mediated transgene inactivation of macrophages by endotoxin

Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin. Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers. The ROS scavenger N-t-butyl-alpha-phenylnitrone, the H(2)O(2) scavenger catalase, and the.OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O(2)(-) scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that.OH formed by H(2)O(2)-dependent, metal-catalyzed Fenton reaction play a major role in this process.     

Tripeptide, Arg-Gly-Asp, inhibits the transfection of protein-linked DNA of bacteriophage M2

The effect of tripeptide, Arg-Gly-Asp (RGD), on the transfection activity of Bacillus phage M2 DNA was examined. The transfection activity decreased when M2 DNA was preincubated with RGD before it was added to competent cells.     

Restriction of hemimethylated DNA by the Bacillus subtilis R system

Summary The effects of restriction by the BsuR system on hemimethylated SPP1 DNA were investigated. In vitro, single-stranded nicks were introduced in the nonmodified strand of the hemimethylated DNA at the same sites as recognized in nonmodified homoduplex DNA. Transfection with BsuR-treated hemimethylated DNA was severely reduced.In vivo, transfection with hemimethylated DNA was also severely reduced in competent B. subtilis R cells. In contrast, transfection of protoplasts of the R strain with this DNA was not affected. The apparent restriction by competent cells was attributed to the special mode of processing of transfecting DNA.     

Bacteriophage N3 of Haemophilus influenzae. IV. Intracellular events during infection by Haemophilus influenzae phage and transfection by its DNA

Intracellular events following infection of competent Haemophilus influenzae cells by N3 phage or transfection by DNA from phage were examined. After infection by whole phage three forms of intracellular phage DNA were observed by sedimentation velocity analysis. These forms are probably twisted circles, open circles and linear duplexes. In transfection only about 15% of the phage DNA is efficiently taken up by the competent cells. After entry of phage DNA into wild-type cells in transfection the DNA is degraded at early times, but later some of the fragments are reassembled, resulting in molecules that sediment faster than the monomer length of phage DNA. These presumably concatamer forms are generated by recombination. In strain rec-1 the fast-sedimenting molecules do not appear and degradation of phage DNA is even more pronounced than in the wild-type cells. Since rec-1 is transfected with much lower efficiency than the wild-type our hypothesis is that both fragmentation and generation of fast-sedimenting phage DNA by recombination are required for efficient transfection. These results also show that although phage N3 codes for its own recombination system it cannot operate in the early stages of transfection and succesful transfection is entirely dependent upon the host recombination system.     

Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair

The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination of nonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of (3)H-labeled 35S viral RNA. The copy number of the integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration.     

Frequency of infection and efficiency of transfection of Rhizobium meliloti cells and spheroplasts.

Competent cultures of Rhizobium meliloti cells and spheroplasts obtained by various methods were infected with DNA of phage 1A. The Frequency of infection among the cells and spheroplasts was 2 X 10(-8)-5 X 10(-10). The efficiency of transfection calculated from the ratio of plaque forming units to infective DNA molecule of phage 1A was 5 X 10(-8) to 10(-10). Frequency of infection and efficiency of transfection among the competent cells were by one order of magnitude higher in the case of the spheroplasts. The use of various media did not noticeably alter the efficiency of transfection.     

Two-wavelength fluorescence assay for DNA repair

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP). Both proteins can be excited by visible light with a maximum at 488 nm, but EGFP emits with a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This makes it possible to monitor the expression of the two genes simultaneously by measuring the fluorescence at two wavelengths. HEK293 cells were cotransfected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At different time intervals after transfection the fluorescence of EGFP was determined relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used to calculate the number of UV lesions in DNA and hence the repair capacity of the host cells. It was found that HEK293 cells were able to repair approximately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average.     

Killing of Haemophilus influenzae Cells by Integrated Ultraviolet-Induced Lesions from Transforming Deoxyribonucleic Acid

Highly competent cultures of Haemophilus influenzae are inactivated by exposure to transforming deoxyribonucleic acid (DNA) irradiated with ultraviolet light (UV). As a function of UV dose to the DNA, the killing goes to a maximum and then decreases. The killing of H. influenzae by unirradiated H. parainfluenzae DNA, reported by other workers, is enhanced by low doses of UV, but drops off at high doses. Since there are no such lethal effects in a strain of H. influenzae that takes up DNA normally but does not integrate it, it is concluded that the killing is associated with integrated UV lesions. All the killing of wild-type cells due to irradiated DNA is eliminated by photoreactivation of the DNA. The killing of an excisionless strain of H. influenzae, however, is not eliminated by maximal photoreactivation of the irradiated transforming DNA. The nonphotoreactivable fraction of killing in the excisionless strain increases with increasing dose. The kinetics of the killing-dose curves may be explained only partially in terms of UV-induced loss of integration. It is postulated that the number of pyrimidine dimers relative to other DNA components integrated decreases at higher UV doses.