RESUMO
UV radiation and other carcinogenic agents induce an increase in DNA-binding activity to the early domain of the simian virus 40 (SV40) minimal origin in both SV40-permissive and SV40-nonpermissive cells. The increase is due to posttranslational modification of a preexisting protein, since it occurs in the presence of cycloheximide or anisomycin. Binding of this factor is an absolute requirement for the UV-induced SV40 DNA amplification in Co631 cells in vivo. A synthetic double-stranded oligonucleotide covering the early domain sequence totally blocked the UV-induced amplification in competition experiments. Point mutants of the sequence and unrelated oligonucleotides which could not bind the factor also did not block SV40 amplification. Inhibitors of protein synthesis caused an immediate increase of both early-domain factor activity (perhaps by prolonging mRNA half-life for the factor or for a modifying enzyme) and DNA amplification. The effects of UV and cycloheximide on SV40 amplification were superaddition.
Assuntos
DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Amplificação de Genes/efeitos da radiação , Vírus 40 dos Símios/genética , Animais , Anisomicina/farmacologia , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Cricetinae , Cicloeximida/farmacologia , Dano ao DNA , DNA Viral/genética , Amplificação de Genes/efeitos dos fármacos , Dados de Sequência Molecular , Mutação , Oligodesoxirribonucleotídeos/metabolismo , Biossíntese de Proteínas , Raios UltravioletaRESUMO
Iodine-125, in the form of 5-[125I]iododeoxyuridine (I-UdR), was incorporated into the DNA of SV40 transformed Chinese hamster embryo cells. Disintegration of the 125I led to increased cell killing with increasing dose as measured by the colony-forming ability of single cells. The D37 (the dose at which 37% of the cells survive) amounts to 95 decays per cell, corresponding to 0.66 Gy. Variations in the copy number of specific DNA sequences was measured by using dispersed cell blotting with sensitive DNA hybridizations. A 13-fold amplification of the viral DNA sequences (SV40) and a twofold amplification of two cellular oncogenes of the ras-family (Ki-ras and Ha-ras) were found. Other cellular genes, like the alpha-actin gene, were not amplified, and no variation in gene copy number was detected after incubation of cells with cold I-UdR. We suggest the observed gene amplifications are induced by the densely ionizing radiation emitted by the decay of the incorporated 125I atoms.
Assuntos
Transformação Celular Viral , Amplificação de Genes/efeitos da radiação , Radioisótopos do Iodo , Oncogenes/efeitos da radiação , Vírus 40 dos Símios , Animais , Sobrevivência Celular/efeitos da radiação , Cricetinae , Técnicas In Vitro , Vírus 40 dos Símios/genéticaRESUMO
Simian Virus 40 wild type (SV40)-transformed Chinese hamster embryo cells (Co631) contain about five viral copies integrated per cell genome. These SV40 sequences were used as endogenous indicator genes to study the response of mammalian cells to radiation at the gene level. An increase in copy number was detected by dispersed cell blotting and Southern analysis in combination with specific DNA hybridization. All types of radiation tested induce a 15- to 25-fold amplification of SV40 sequences without producing intact virus. The amplification is dose dependent and increases with time after irradiation: a maximum effect is observed at Day 3 after alpha particle or uv exposure and at Day 6 after gamma-ray exposure. A RBE of 6 can be calculated for alpha particles if amplification rates at Day 3 are compared. However, when the maximum effect is considered independent of time, no difference between different types of radiation is observed. Southern blots of genomic DNA show that not all integrated SV40 sequences are amplified upon radiation. Amplified sequences are found either in restriction fragments of relatively high molecular weight or in unit size fragments. SV40 amplification is selective in that the amplification of other genes, e.g., of alpha-actin, dhfr (dihydrofolate reductase), and of two oncogenes of the ras family (Kirsten ras and Harvey ras), was below detection level.
Assuntos
Partículas alfa , Raios gama , Amplificação de Genes/efeitos da radiação , Radiação Ionizante , Raios Ultravioleta , Animais , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Cricetinae , Cricetulus , Relação Dose-Resposta à Radiação , Feminino , Genes/efeitos da radiação , Ovário/citologiaRESUMO
Survival and G2 delay of L929 mouse fibroblasts exposed to 3.4-MeV alpha particles depend on the cell age at the time of irradiation. Greatest sensitivity for both endpoints has been found at the G1/S transition: The surviving fraction of G1/S cells is reduced to 0.11 following 1 Gy of alpha particles compared to 0.31 for early G1 cells. The G2 + M transit time rises from 3 hr for control cells to 22 and 30 hr for cells irradiated with 0.3 Gy in G2 or at the G1/S boundary, respectively. Cells irradiated in early G1 do not show increased G2 + M transit times. Growth delay as calculated for the entire population increases linearly with dose by 23 hr/Gy of alpha particles.
Assuntos
Amerício , Sobrevivência Celular/efeitos da radiação , Interfase/efeitos da radiação , Células L/efeitos da radiação , Partículas alfa , Animais , Camundongos , Fatores de TempoRESUMO
A number of carcinogenic and cocarcinogenic agents induce new gene products in mammalian cells including primary human skin fibroblasts. These have been defined by cDNA cloning techniques, by protein resolutions in 2D PAGE and by the detection of new enzymatic functions. The uniform and transient genetic reaction is tentatively called the genetic stress response.
Assuntos
Modelos Genéticos , Estresse Fisiológico , Animais , Cricetinae , Cicloeximida/farmacologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Amplificação de Genes , Humanos , Interleucina-1/farmacologia , Biossíntese de Proteínas , Proteínas/genética , Proteínas/efeitos da radiação , RNA/genética , RNA/metabolismo , Estresse Fisiológico/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transcrição GênicaRESUMO
In comparison with primary cell cultures, SV40-transformed human skin fibroblasts, either from healthy donors or from patients suffering from ataxia-telangiectasia (AT) or xeroderma pigmentosum, are more resistant to the cytotoxic action of low LET 60cobalt gamma-rays as well as to high LET alpha-particles. Resistance factors calculated from D10's lie between 1.4 and 2.0. Northern blot analysis reveals spontaneous overexpression of the oncogenes c-myc, Ki-ras and c-raf and of the tumour suppressor gene p53 as a consequence of SV40 transformation. For c-myc, the increased expression is due to gene amplification and gene rearrangement. An even further increase in the expression of c-myc has been found for AT cells (AT5BI-VA) after moderate doses of 60cobalt gamma-irradiation. A possible correlation between SV40-induced changes in gene expression and cellular radioresistance is discussed.
Assuntos
Transformação Celular Viral/genética , Regulação Viral da Expressão Gênica/efeitos da radiação , Oncogenes , Vírus 40 dos Símios/genética , Partículas alfa , Amerício , Radioisótopos de Cobalto , Transferência de Energia , Raios gama , Amplificação de Genes , Genes myc , Genes p53 , Genes ras , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-raf , PeleRESUMO
SV40-transformed Chinese hamster embryo cells were exposed to 60Co gamma-irradiation and the fate of the integrated SV40 sequences was pursued over a period of 20 days following radiation exposure. As shown by colony hybridization, integrated SV40 sequences were amplified in surviving and non-surviving cells. At later times, however, clonal sublines of surviving cells grown for 20-30 cell generations after irradiation had lost most of their amplified SV40 copies but showed altered restriction fragment patterns indicating reintegration of SV40 sequences at new sites of the hamster genome. This suggests that 60Co gamma-irradiation can generate mutations by inducing over-replication of chromosome segments that are then substrates of enzymatic rearrangements.
Assuntos
Sobrevivência Celular/efeitos da radiação , DNA Viral/genética , Amplificação de Genes , Vírus 40 dos Símios/genética , Animais , Linhagem Celular Transformada , Radioisótopos de Cobalto , Raios gamaRESUMO
To investigate the role of myc-overexpression on radiation-induced amplification of the dihydrofolate reductase gene (DHFR) we compared diploid Chinese hamster ovary cells (CHO-9) to cells of the same line that had been stably transfected with a dexamethasone-inducible c-myc cDNA. The application of flow-cytofluorometry and fluorescent in situ hybridization (FISH) allowed the evaluation of an increase in DHFR gene copy number following radiation treatment without the use of a preceding selection procedure. We show that DHFR gene amplification may occur independently of p53 status in cells overexpressing c-myc.
Assuntos
Amplificação de Genes , Genes myc , Animais , Southern Blotting , Células CHO , Cricetinae , Citometria de Fluxo , Amplificação de Genes/efeitos da radiação , Expressão Gênica/efeitos da radiação , Hibridização in Situ Fluorescente , Tetra-Hidrofolato Desidrogenase/genética , Fatores de Tempo , TransfecçãoRESUMO
Ionizing and UV radiations induce amplification of SV40 DNA sequences integrated in the genome of Chinese hamster cells and increase amplification of the dihydrofolate reductase (DHFR) gene during methotrexate selection in human skin fibroblasts of a patient with ataxia telangiectasia. By cell fusion experiments it could be shown that SV40 gene amplification is mediated by one or several diffusible trans-acting factors induced or activated in a dose dependent manner by all types of radiation. One of these factors binds to a 10 bp sequence within the minimal origin of replication of SV40. In vivo competition with an excess of a synthetic oligonucleotide comprising this sequence blocks radiation-induced amplification.
Assuntos
Amplificação de Genes/efeitos da radiação , Animais , Ataxia Telangiectasia/enzimologia , Ataxia Telangiectasia/genética , Sequência de Bases , Linhagem Celular , Cricetinae , DNA/genética , DNA/efeitos da radiação , Replicação do DNA/genética , Replicação do DNA/efeitos da radiação , Humanos , Dados de Sequência Molecular , Vírus 40 dos Símios/genética , Tetra-Hidrofolato Desidrogenase/genéticaAssuntos
Interfase/efeitos da radiação , Partículas alfa , Amerício , Animais , Cafeína/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , Relação Dose-Resposta à Radiação , Interfase/efeitos dos fármacos , Mitose/efeitos da radiaçãoAssuntos
Ciclo Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Interfase/efeitos da radiação , Animais , Argônio , Carbono , Contagem de Células/efeitos da radiação , Células Cultivadas , Cricetinae , Cricetulus , Relação Dose-Resposta à Radiação , Neônio , Radioterapia de Alta Energia , Eficiência Biológica Relativa , Raios XAssuntos
Mitose/efeitos da radiação , Animais , Células Cultivadas , Cricetinae , Cricetulus , Citometria de Fluxo , InterfaseRESUMO
Two gamma-ray hypersensitive cell lines, human ataxia telangiectasia (AT) and murine severe combined immune deficiency (SCID) cells, proved to be very competent in amplifying their dihydrofolate reductase (DHFR) gene under methotrexate selection stress. Over a period of months, methotrexate-resistant clones were obtained which were able to grow in progressively increasing methotrexate concentrations up to 1 mM. By then methotrexate-resistant AT and SCID cells had amplified their DHFR gene 6- and 30-fold, respectively, and showed very high DHFR mRNA expression. In contrast, related cells with normal radiosensitivity (human GM637 and mouse BALB/c fibroblasts) did not show DHFR gene amplification under comparable conditions. This correlation of the capacity of DHFR gene amplification and gamma-ray hypersensitivity in AT and SCID cells suggests that gene amplification may have a mechanism(s) in common with those involved in repair of gamma-radiation-induced damage. No difference in cell killing could be observed following exposure to densely ionizing alpha particles: AT and SCID cells exhibited comparable survival rates to GM637 and BALB/c cells, respectively.
Assuntos
Partículas alfa , Ataxia Telangiectasia/genética , Amplificação de Genes/efeitos dos fármacos , Metotrexato/farmacologia , Tolerância a Radiação , Tetra-Hidrofolato Desidrogenase/genética , Animais , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Fibroblastos/efeitos da radiação , Raios gama , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCIDRESUMO
During selection for methotrexate (MTX) resistance the metastatic subclone BSp73ASML of a spontaneous rat adenocarcinoma and a metastatic transfectant containing the metastogene META-1 underwent amplification of the dihydrofolate reductase (DHFR) gene at accelerated rates in contrast to non-metastatic but closely related BSp73AS cells. A four log increase in MTX resistance was associated with a 16-fold amplification and increased expression of the DHFR gene. The capacity for gene amplification in metastatic BSp73ASML cells was correlated with a deletion in the p53 gene and enhanced expression of the oncogene c-myc due to a 10-fold amplification of the myc gene. Increased expression of Ki-ras and c-raf in the non-metastatic BSp73AS cells seems to confer tumorigenicity but not permissivity for gene amplification.
Assuntos
Adenocarcinoma/patologia , Metotrexato/farmacologia , Tetra-Hidrofolato Desidrogenase/genética , Adenocarcinoma/genética , Animais , DNA de Neoplasias/genética , Resistência a Medicamentos , Amplificação de Genes/efeitos dos fármacos , Genes myc , Genes p53 , Genes ras , Metástase Neoplásica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-raf , Ratos , Células Tumorais CultivadasRESUMO
Alkali-labile sites produced by gamma-irradiation in dry RF-DNA of bacteriophage phi chi 174 were found to be lethal lesions with respect to the plaque-forming ability.
Assuntos
DNA Viral/efeitos da radiação , Efeitos da Radiação , Álcalis , Centrifugação Zonal , Radioisótopos de Cobalto , Colífagos , Vírus de DNA , DNA Circular/efeitos da radiação , Relação Dose-Resposta à Radiação , Raios gamaRESUMO
Treatment of Chinese hamster embryo cells with alpha irradiation (4 MeV, emitted by 241americium) induces a 15-fold amplification of integrated SV40 sequences. The extent of amplification depends on the dose of irradiation and on the presence of a functional T-antigen encoded by the SV40 A gene. The inducing signal can be transmitted to a non-irradiated nucleus following cell fusion. Amplification is therefore the result of some trans-acting process, and this could explain how SV40 amplification can occur after doses of alpha irradiation that are too low to cause direct DNA damage within the SV40 replicon.
Assuntos
Partículas alfa , DNA Viral/análise , Amplificação de Genes/efeitos da radiação , Vírus 40 dos Símios/genética , Animais , Sequência de Bases , Fusão Celular , Células Cultivadas , Cricetinae , Probabilidade , Vírus 40 dos Símios/efeitos da radiaçãoRESUMO
In an attempt to study the regulatory properties of cells required for gene amplification, the capacity for amplification of the dehydrofolate reductase gene (dhfr) was determined in F9 teratocarcinoma stem cells during differentiation. By stepwise selection of surviving cells exposed to progressively increasing concentrations of methotrexate (MTX) up to 1,000 microM within 4 months, non-differentiated F9 cells reached a more than 40-fold amplification of their dhfr gene, elevated levels of dhfr mRNA and a 5-log increase in MTX resistance. Exposure to 5 Gy of 60Co-gamma-irradiation accelerated the process of amplification. In contrast, F9 cells that had been differentiated to endodermal cells by treatment with retinoic acid (RA) did not grow in MTX concentrations above 0.1 microM, either with or without radiation pretreatment and did not amplify the dhfr gene to any measurable extent over the same period of time. Upon treatment of methotrexate-resistant (F9-MTXr) cells with retinoic acid, loss of the amplified DNA in the absence of selection was significantly retarded. The ability to amplify the dhfr gene was correlated with the occurrence of origin binding activity in vitro (early domain of SV40 minimal origin of replication). These data indicate an increase in genomic stability and rigorous control of origin activity by differentiation.
Assuntos
Células-Tronco/citologia , Teratoma/genética , Tretinoína/farmacologia , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Amplificação de Genes , Metotrexato/farmacologia , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Teratoma/metabolismo , Teratoma/patologia , Células Tumorais CultivadasRESUMO
Exponentially growing Chinese hamster V79 cells were exposed to 4 MeV alpha particles emitted by an americium-241 source. The chromatin of cells arrested in G2 by alpha irradiation is severely damaged, though all cells were still capable to condensate their chromatin after fusion with mitotic cells. In addition to the common types of aberrations (breaks, gaps, dicentrics and exchanges) cells were found possessing one or more chromosomes with long stretches of undercondensed chromatin. Repair of these lesions was indicated by site-specific unscheduled DNA synthesis and by the observation that condensation of these regions improved during G2 arrest.