ABSTRACT
Tumor-directed gene therapy, such as "suicide gene" therapy, requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Current vector strategies have been ineffective in achieving these goals. This report introduces the attenuated (thymidine kinase (TK)-negative) replication-competent vaccinia virus (VV) as a potential vector for tumor-directed gene therapy by studying the biodistribution of VV in animal tumor models. A TK-deleted recombinant VV (Western Reserve strain) expressing luciferase on a synthetic promoter was constructed. Luciferase activity was measured in vitro after transduction of a variety of human and murine tumor cell lines and in vivo after intraperitoneal (i.p.) delivery in C57BL/6 mice with 7-day i.p. tumors (10(6) MC-38 cells). Three other in vivo tumor models were examined for tumor-specific gene expression after intravenous delivery of VV (human melanoma in nude mice, adenocarcinoma liver metastasis in immunocompetent mice, and subcutaneous sarcoma in the rat). In addition, a replication-incompetent vaccinia (1 microg of psoralen and ultraviolet light, 365 nm, 4 minutes) was tested in vitro and in vivo and compared with active virus. Luciferase activity in i.p. tumors at 4 days after i.p. injection of VV was >7000-fold higher than lung, >3000-fold higher than liver, and >250-fold higher than ovary. In addition, intravenous injection of VV resulted in markedly higher tumor luciferase activity compared with any other organ in every model tested (up to 188,000-fold higher than liver and 77,000-fold higher than lung). Inactivation of the virus resulted in negligible gene expression in vivo. In summary, VV has a high transduction efficiency in tumor cells with high levels of gene expression. The results suggest a selective in vivo replication of TK-deleted VV in tumor cells. Replication competent, TK-deleted VV appears to be an ideal vector for testing the in vivo delivery of toxic genes to tumor cells.
Subject(s)
Genetic Therapy , Genetic Vectors/genetics , Neoplasms, Experimental/therapy , Thymidine Kinase/genetics , Vaccinia virus/genetics , Animals , Biomarkers, Tumor , Disease Models, Animal , Ficusin/pharmacology , Gene Expression/drug effects , Gene Expression/radiation effects , HT29 Cells , Humans , Luciferases/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Nude , Mutation , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Photosensitizing Agents/pharmacology , Rats , Rats, Inbred F344 , Thymidine Kinase/metabolism , Transfection/drug effects , Transfection/radiation effects , Tumor Cells, Cultured , Ultraviolet Rays , Virus ReplicationABSTRACT
Several lines of evidence suggest that an IFN-gamma-producing, Th1/Tc1 phenotype may be optimal for tumor rejection. Recent work has indicated that IFN signaling on tumor cells is important for protection against carcinogenesis. However, the potential involvement of IFN signaling among host immune cells has not been carefully examined. To this end, Stat1-deficient mice were employed as tumor recipients. In contrast to wild-type mice, Stat1-/- mice failed to reject immunogenic tumors and did not support regression of poorly immunogenic tumors when treated with an IL-12-based vaccine. T cells from immunized Stat1-/- mice produced 50% of the levels of IFN-gamma and lacked cytolytic activity compared with wild-type mice, and NK lytic activity also was not observed. Lack of cytolytic function correlated with a failure to up-regulate serine esterase activity. Thus, IFN-mediated signaling on host cells is required for the development of antitumor lytic effector cells.
Subject(s)
Cytotoxicity, Immunologic , DNA-Binding Proteins/biosynthesis , Mast-Cell Sarcoma/immunology , Mast-Cell Sarcoma/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Trans-Activators/biosynthesis , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cytotoxicity, Immunologic/genetics , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Graft Rejection/etiology , Graft Rejection/genetics , Graft Rejection/immunology , Interleukin-12/administration & dosage , Interleukin-12/genetics , Interleukin-12/immunology , Mast-Cell Sarcoma/genetics , Mast-Cell Sarcoma/pathology , Mice , Mice, Inbred DBA , Mice, Knockout , STAT1 Transcription Factor , Signal Transduction/genetics , Signal Transduction/immunology , T-Lymphocytes, Cytotoxic/cytology , Trans-Activators/deficiency , Trans-Activators/genetics , Transfection/immunology , Transfection/radiation effects , Tumor Cells, Cultured , VaccinationABSTRACT
Gene therapy is an evolving discipline which today relies primarily on viral systems for gene transfer. The primary reason that plasmid vectors have not been widely used for gene therapy trials is their relatively low rate of stable gene transfer. We show here that both ionizing irradiation and hydrogen peroxide can each increase the gene transfer efficiency of plasmids. Hydrogen peroxide improves gene transfer in a linear dose-dependent manner. At equitoxic doses, hydrogen peroxide improves gene transfer by 20-fold over untreated cells and approximately 5 times above that seen for radiation, and this improvement correlates with both the total amount of DNA damage induced and the amount of residual damage after 4 hr of repair. These data suggest that DNA damaging agents may be useful to improve human gene therapy.
Subject(s)
DNA Damage/drug effects , DNA Damage/radiation effects , Gene Transfer Techniques , Animals , Cell Line , Fibroblasts , Genetic Vectors , Humans , Hydrogen Peroxide/pharmacology , Lung Neoplasms , Mice , Transfection/drug effects , Transfection/radiation effects , Tumor Cells, CulturedABSTRACT
This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy.
Subject(s)
DNA/genetics , Plasmids/genetics , Transfection/genetics , Cell Line , DNA/radiation effects , HeLa Cells , Humans , Radiation Dosage , Transfection/radiation effectsABSTRACT
The vast majority of clinical protocols involving gene therapy today rely on viral vectors for gene transduction. The primary reason that plasmid vectors have not been widely used for gene therapy trials is their relatively low rate of stable gene transfer. We show here that ionizing radiation can improve plasmid transfection efficiency in both normal and neoplastic human and mouse cells. As high as 1,400-fold improvement in transfection efficiency can be seen in primary human fibroblasts treated with 9 Gy. Radiation improves transfection efficiency in a dose-dependent manner of only linearized plasmid DNA in transformed or immortalized cells, but of both linearized and supercoiled plasmid in normal human fibroblasts. The gene transfer dose-response curves are linear for neoplastic cell lines and exponential for primary cell lines. This suggests that radiation can improve gene integration by at least two mechanisms, one that may require free DNA ends and one that does not. The 2-hr delay described here, from the time of irradiation to the beginning of enhanced gene integration, suggests an inducible process that becomes active after the bulk of the radiation damage has been repaired. Our data further suggest that radiation may be useful to target human gene therapy using plasmid vectors.
Subject(s)
Gene Transfer Techniques , Radiation, Ionizing , Transfection/radiation effects , Animals , Cation Exchange Resins , Cell Line , Dose-Response Relationship, Radiation , Fibroblasts , Genetic Vectors/genetics , Humans , Lipids , Mice , Plasmids/genetics , Radiation Tolerance , Recombination, GeneticABSTRACT
In experiments with NIH3T3 mouse fibroblast the authors showed a dose-dependent (gamma- or UV-radiation) increase in the immortalized fibroblast transformation by purified DNA preparations from Ehrlich ascites tumour cells. The transformant yield was the highest when the transfection started within the first few minutes after irradiation, when radiation lesions occurred in the genome and the system of enzymic DNA repair was activated. The proteolytic activity inhibition by treating the exposed cells with phenylmethanesultonyl fluorine reduced the radiation-induced transformation.
Subject(s)
Cell Transformation, Neoplastic/radiation effects , Chromatin/radiation effects , DNA Damage/radiation effects , DNA, Neoplasm/pharmacology , Animals , Carcinoma, Ehrlich Tumor/genetics , Cell Line, Transformed , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/metabolism , Cells, Cultured/drug effects , Cells, Cultured/enzymology , Cells, Cultured/radiation effects , Chromatin/drug effects , Chromatin/metabolism , DNA Damage/drug effects , DNA Damage/physiology , DNA Repair/drug effects , DNA Repair/physiology , DNA Repair/radiation effects , DNA, Neoplasm/isolation & purification , Dose-Response Relationship, Radiation , Fibroblasts/drug effects , Fibroblasts/enzymology , Fibroblasts/radiation effects , Gamma Rays , Mice , Time Factors , Transfection/drug effects , Transfection/physiology , Transfection/radiation effects , Ultraviolet RaysSubject(s)
Cells/radiation effects , Lasers , Optics and Photonics , Animals , Caenorhabditis/embryology , Caenorhabditis/radiation effects , Cell Fusion/radiation effects , Cells/ultrastructure , Genetic Engineering , Mitochondria/radiation effects , Optics and Photonics/instrumentation , Spindle Apparatus/radiation effects , Transfection/radiation effectsABSTRACT
In vitro, the photodynamic compound benzo[a]pyrene (BAP) generates singlet oxygen efficiently when irradiated in organic solvents. It also photogenerates superoxide anion radical in water and can act as a photoreducing agent in the absence of oxygen. In vivo, the hemolysis of human erythrocytes, the inactivation of Escherichia coli cells representing a series of strains differing in excision repair and catalase proficiency, and the inactivation of Haemophilus influenzae transforming DNA activity were used to characterize the phototoxicity of BAP in the presence of near-UV light (290-400 nm). The results are consistent with BAP behaving as a photosensitizer that generates both superoxide and singlet oxygen, and that damages chiefly membranes. DNA does not seem to be a major target in the phototoxic reactions investigated.
Subject(s)
Benzo(a)pyrene/toxicity , Cell Survival/drug effects , DNA, Bacterial/genetics , Haemophilus influenzae/genetics , Hemolysis/drug effects , Transfection , Benzo(a)pyrene/radiation effects , Cell Survival/radiation effects , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/radiation effects , Gene Expression , Hemolysis/radiation effects , Humans , Oxygen/metabolism , Photochemistry , Superoxides/metabolism , Transfection/drug effects , Transfection/radiation effects , Ultraviolet RaysABSTRACT
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 micrograms 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.
Subject(s)
Transfection , Animals , Cell Line , Chloramphenicol O-Acetyltransferase/analysis , Chloramphenicol O-Acetyltransferase/genetics , Clone Cells , Cricetinae , Cricetulus , Female , Genetic Complementation Test , Kinetics , Ovary , Plasmids , Transfection/radiation effects , X-RaysABSTRACT
Following transfection of genomic mouse DNA into an SV40 transformed fibroblast cell line from a patient with Xeroderma pigmentosum (complementation group A, XPA), a single UV resistant cell clone was isolated out of a total of 10(4) independent transfectants. The recipient XPA cell line has as yet not produced spontaneous revertants among 2.2 x 10(8) cells. The isolated cell clone contains 50-70 kb of mouse sequences which are heavily amplified (500-fold), and has acquired both intermediate resistance to UV killing and intermediate unscheduled DNA synthesis (UDS) capacity. By continued passage without selective pressure, cells were generated, which had lost both the dominant marker gene and repetitive mouse sequences. Single colonies of these cells were still intermediately resistant to UV suggesting that either undetected unique mouse DNA had segregated from the bulk of repetitive DNA, or, more likely, that the initially isolated transfectant was a spontaneous revertant. This documents that a persuasive clone isolated can still be a false positive (spontaneous revertant) and that an extremely laborious approach may lead into a dead end.
Subject(s)
DNA/radiation effects , Gene Amplification , Transfection/radiation effects , Ultraviolet Rays , Xeroderma Pigmentosum/genetics , Animals , Base Sequence/radiation effects , Blotting, Southern , Cell Line, Transformed , DNA Mutational Analysis , Humans , Mice , PhenotypeABSTRACT
Trisomy 21 (Down's syndrome, DS) is the most frequent chromosomal aberration. Triplication of a small region of chromosome 21, the fragment 21q22 is sufficient to cause the DS phenotype including immunodeficiency, premature aging, neurodegenerations, mental retardation and an increased risk of leukemia. Chromosomal aberrations caused by X-ray irradiation were observed in DS lymphocytes and DS fibroblasts, but the correlation to cell death or repair deficiency was not clear. We approached this problem and report here on a profound X-ray repair deficiency of DS cells. With a colorimetric viability assay we observed an UV sensitivity of DS fibroblasts at doses beyond 14 Jm-2 but no significant X-ray sensitivity. By the nucleoid sedimentation technique, a deficient restoration of nucleoids in DS cells after X-ray irradiation was demonstrated. The same features apply for cells, which contain an overexpressed Cu/Zn-superoxide dismutase (SOD-1) gene. Radiation sensitivity of DS cells and SOD-1 overexpressing cells resemble those of ataxia telangiectasia (AT) fibroblasts. Additionally, DS and AT cells exert lack of inhibition of DNA synthesis after X-ray irradiation.
Subject(s)
Down Syndrome/pathology , Fibroblasts/radiation effects , Superoxide Dismutase/genetics , Cell Line , Cells, Cultured , Copper/metabolism , Down Syndrome/etiology , Down Syndrome/genetics , Gene Expression Regulation/radiation effects , Humans , Superoxide Dismutase/metabolism , Superoxide Dismutase/radiation effects , Transfection/radiation effects , Ultraviolet Rays/adverse effects , X-Rays/adverse effects , Zinc/metabolismABSTRACT
We have previously demonstrated that murine colon tumor cells transfected with the gene coding for the hemagglutination antigen (HA) of influenza virus acquire an inherent immunogenicity, fail to grow in syngeneic mice, and demonstrate an ability to cross-protect against a challenge with parental nontransfected cells. In the present study the immunogenic potential of HA-transfected cells correlated with cell-surface HA expression, as measured both by a fluorescence-activated cell sorter and by radio-labeled antibody binding. HA-transfected immunogenic cells had a median lethal dose (LD50) that was 10,000-fold greater than that of nontransfected cells. Most importantly this study demonstrated that HA-transfected cells retained their immunogenicity after X-irradiation with 12,000 rad. This characteristic makes their potential usefulness in treating human neoplasia more plausible.
Subject(s)
Antigens, Viral/radiation effects , Colorectal Neoplasms/immunology , Hemagglutinins, Viral/radiation effects , Influenza A virus/radiation effects , Transfection/radiation effects , Animals , Antigens, Viral/immunology , Binding Sites, Antibody , Cell Line , Clone Cells/immunology , Clone Cells/radiation effects , Clone Cells/transplantation , Female , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinins, Viral/genetics , Influenza A virus/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasm TransplantationSubject(s)
DNA Damage , DNA/genetics , Animals , Buffers , Cells, Cultured , Free Radicals , Mutagens , Plasmids , Rats , Simplexvirus/genetics , Thymidine Kinase/genetics , Transfection/radiation effectsABSTRACT
Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.
Subject(s)
Genetic Vectors/radiation effects , Mutation , Sunlight , Transfection/radiation effects , Ultraviolet Rays , Animals , Base Composition , Base Sequence , Cell Line , Dose-Response Relationship, Radiation , Escherichia coli/genetics , Molecular Sequence Data , PlasmidsABSTRACT
Ionizing radiation induces a number of molecular changes in cells, including DNA damage, mutation, genetic recombination, gene amplification, and chromosomal rearrangement. The studies described here make use of the process of DNA-mediated gene transfer to examine the molecular effects of ionizing radiation. Two Chinese hamster ovary cell lines, the wild-type, AA8-4, and a DNA repair-deficient line, EM9-1, that is sensitive to ionizing radiation, were transfected with the recombinant DNA plasmid, pSV2-GPT, either in the absence or presence of high-molecular-weight carrier DNA. Following transfection, cell populations were irradiated with graded doses of 137Cs gamma-rays. Results demonstrate that, on a per viable cell basis, ionizing radiation hinders the transfection of this plasmid when tested in the presence of carrier DNA. A similar dose response was seen for both the wild-type (AA8-4) and mutant (EM9-1) lines. However, in the absence of carrier DNA, 137Cs gamma-rays clearly enhanced the gene transfer process. An enhancement factor of 3-5 was seen for AA8-4 cells and 2-3 for EM9-1 cells. This enhancement occurred at relatively low doses (e.g., 50 cGy) and was not substantially elevated by larger doses.
Subject(s)
DNA Damage , DNA Repair , Transfection/radiation effects , Animals , Cell Line , Cricetinae , Gamma Rays , Gene Expression Regulation/radiation effects , Plasmids , Transformation, Genetic/radiation effectsABSTRACT
When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells.
Subject(s)
Genetic Vectors , Mitomycins/pharmacology , Mutation , Simian virus 40/genetics , Transfection/drug effects , Animals , Base Composition , Base Sequence , Cell Line , Chlorocebus aethiops , Genes , Genetic Vectors/radiation effects , Kidney , Mitomycin , Molecular Sequence Data , RNA, Transfer/drug effects , RNA, Transfer/genetics , Transfection/radiation effects , Ultraviolet RaysABSTRACT
The enhancement effects of ionizing and ultraviolet radiation on the efficiency of DNA-mediated gene transfer were studied. The established cell line, Rat-2, consists of cells that are density-dependent contact-inhibited and produce flat monolayers in vitro. When these cells are infected with SV40 virus, a small fraction of cells becomes morphologically "transformed" due to the stable expression of the viral A-gene. Rat-2 cells are competent for DNA-mediated gene transfer, deficient in thymidine kinase activity (TK-), and will die in HAT selective media. Confluent Rat-2 cells were transfected with purified SV40 viral DNA (via calcium phosphate precipitation), irradiated with either X rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were nonlinear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X rays or 330 MeV/amu argon particles at the Berkeley BEVALAC showed a higher frequency of HAT+ colonies/survivor than unirradiated transfected cells. In both cases the enhancement contained a linear and a higher order component in dose, but the argon ions were at least twice more efficient than X rays in producing enhancement per unit dose. Rat-2 cells transfected with pOT-TK5, X-irradiated, and assayed for either TK transformation or A-gene transformation showed the same dose dependence for radiation enhancement. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK transformation by both X rays and ultraviolet radiation. SV40 A-gene products are not necessary for the radiation enhancement of the efficiency of gene transfer. This in vitro system will be used to study the lesions produced by ionizing radiation on mammalian cell DNA that may act as substrates for integration of exogenously introduced plasmid DNA.
Subject(s)
Simian virus 40/genetics , Transfection/radiation effects , Animals , Cell Line , Plasmids , Radiation Genetics , Radiation, Ionizing , Rats , Thymidine Kinase/genetics , Transformation, Genetic/radiation effects , Ultraviolet RaysABSTRACT
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.
Subject(s)
Pyrimidine Dimers/genetics , Transformation, Genetic/radiation effects , DNA/radiation effects , DNA Repair , Deoxyribodipyrimidine Photo-Lyase , Genes, Dominant , Humans , Recombination, Genetic/radiation effects , Transfection/radiation effects , Ultraviolet Rays , Xeroderma Pigmentosum/geneticsABSTRACT
The DNA of bacteriophage SPO2c12 was treated with methylmethane sulfonate (MMS), beta-propiolactone (BPL), 2-anthramine (AA) or benzo[a]pyrene (BP) and then exposed to 254-nm radiation. Competent Bacillus subtilis host cells were transfected with DNA subjected to the carcinogen-UV treatment or with DNA treated with carcinogen only. Survival curves were obtained for loss of plaque-forming ability as a function of UV dose. The UV sensitivity of DNA treated with MMS, BPL or AA was not significantly different from that of untreated DNA. The results indicate that in competent B. subtilis the pathways for repair of alkylating agent damage and for repair of UV damage are probably different.
Subject(s)
Bacillus subtilis/genetics , Carcinogens/toxicity , DNA Repair , DNA/radiation effects , Bacteriophages/genetics , Benzo(a)pyrene/toxicity , DNA, Viral/genetics , Methyl Methanesulfonate/toxicity , Propiolactone/toxicity , Transfection/drug effects , Transfection/radiation effects , Ultraviolet RaysABSTRACT
Monkey kidney cells, either untreated or pretreated with UV-light at 254 nm or mitomycin C, were transfected 24 hours later with the intact or UV-irradiated DNA from the thermosensitive tsB201 simian virus 40 mutant unable to grow at 41 degrees C. The survival of the viral progeny obtained from the UV-irradiated DNA is increased in pretreated cells compared to the survival of the viral progeny obtained in untreated cells. Irradiation of the viral DNA enhances the reversion frequency of the viral progeny towards a wild type phenotype able to grow at 41 degrees C. Pretreatment of the cells with UV or mitomycin C does not increase the reversion frequency.