RESUMEN
The rat vascular smooth muscle cell (VSMC) line A10 (ATCC CRL 1476) was stably transfected with a human c-fos promoter-driven luciferase reporter gene to monitor thrombin receptor activation and subsequent induction of c-fos expression. Selective activation of the endogeneous thrombin receptor by the thrombin receptor activating peptide (TRAP1-6), SFLLRN, is shown here to result in a significant transient increase of intracellular [Ca2+], dose-dependent induction of c-fos promoter-mediated luciferase activity, and stimulation of DNA synthesis. These data demonstrate that A10 cells and reporter line derivatives thereof possess a functional thrombin receptor very similar or identical to that previously described. Results obtained with various signal transduction modulating or inhibiting agents support previous notions showing that thrombin receptor activation by SFLLRN is coupled to events involving p21ras activation, protein tyrosine kinase, and activation of PKC. The A10 reporter line described here proved to be a helpful and reliable tool to study alpha-thrombin and TRAP1-6-mediated intracellular events, since it retained most of the spectrum of biological responses found in primary VSMC cultures.
Asunto(s)
Genes fos , Músculo Liso Vascular/metabolismo , Receptores de Trombina/metabolismo , Animales , Línea Celular , ADN/biosíntesis , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Luciferasas/genética , Fragmentos de Péptidos/farmacología , Ratas , Receptores de Trombina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Trombina/farmacología , TransfecciónRESUMEN
DNA amplification of the helper-dependent parvovirus AAV (adeno-associated virus) can be induced by a variety of genotoxic agents in the absence of coinfecting helper virus. Here we investigated whether the origin of AAV type 2 DNA replication cloned into a plasmid is sufficient to promote replication activity in cells treated by the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A pUC19-based plasmid, designated pA2Y1, which contains the left terminal repeat sequences (TRs) representing the AAV origin of replication and the p5 and p19 promoter but lacks any functional parvoviral genes is shown to confer replication activity and to allow selective DNA amplification in carcinogen-treated cells. Following transfection of plasmid pA2Y1 or plasmid pUC19 as a control, density labeling by a bromodeoxyuridine and DpnI resistance assay suggested a semi-conservative mode of replication of the AAV origin-containing plasmid. Furthermore, the amount of DpnI-resistant full-length pA2Y1 DNA molecules was increased by MNNG treatment of cells in a dose-dependent manner. In addition, DNA synthesis of plasmid pA2Y1 was studied in vitro. Extracts derived from MNNG-treated CHO-9 and L1210 cells displayed greater synthesis of DpnI-resistant full-length pA2Y1 molecules than did nontreated controls. Experiments with specific enzyme inhibitors suggested that the reaction is largely dependent on DNA polymerase alpha, DNA primase, and DNA topoisomerase I. Furthermore, restriction endonuclease mapping analysis of the in vitro reaction products revealed the occurrence of specific initiation at the AAV origin of DNA replication. Though elongation was not very extensive, extracts from carcinogen-treated cells markedly amplified the AAV origin region. Our results, including electron microscopic examination, suggest that the AAV origin/terminal repeat structure is recognized by the cellular DNA replicative machinery induced or modulated by carcinogen treatment in the absence of parvoviral gene products.
Asunto(s)
Adenoviridae/genética , Replicación del ADN , ADN Viral/biosíntesis , Amplificación de Genes/efectos de los fármacos , Metilnitronitrosoguanidina/farmacología , Adenoviridae/efectos de los fármacos , Adenoviridae/crecimiento & desarrollo , Animales , Células Cultivadas , Cricetinae , Replicación del ADN/efectos de los fármacos , ADN Viral/efectos de los fármacos , Desoxirribonucleasas de Localización Especificada Tipo II , Conformación de Ácido Nucleico , Plásmidos , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Transfección , Replicación ViralRESUMEN
We studied the effects of helper-dependent parvovirus AAV [adeno-associated virus] type 2 on carcinogen-inducible resistance to methotrexate (MTX) and adriamycin (ADR) in Chinese hamster ovary cells. Both types of drug resistance were monitored by determination of the number of drug-resistant colonies normalized for the respective value of plating efficiency under non-selective conditions. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) drastically enhanced the frequency of resistance to MTX and ADR. By contrast, infection of cells with AAV-2 prior to treatment with MNNG markedly inhibited carcinogen-induced drug resistance. Infection by AAV alone did not exert any effect. Analysis of the dihydrofolate reductase (dhfr) gene copy numbers of individual MTX-resistant clones derived from MNNG-treated and non-treated cultures revealed similar frequencies (60-80%) and amplitudes of dhfr gene amplification (2- to 8-fold) irrespective of prior AAV treatment. Hence, carcinogen-induced enhancement of MTX-resistance could reflect an increase in the frequency of dhfr gene amplification among the survivors of MNNG treatment. On the other hand, inhibition of carcinogen-inducible drug resistance by AAV suggests an interference of the virus with cellular responses to genotoxic stress, thus leading to enhanced cell killing under altered growth conditions. Possible mechanisms responsible for the inhibitory effect of AAV and its relevance in relation to tumor chemotherapy are discussed.
Asunto(s)
Dependovirus/patogenicidad , Doxorrubicina/antagonistas & inhibidores , Metotrexato/antagonistas & inhibidores , Metilnitronitrosoguanidina/farmacología , Ovario/efectos de los fármacos , Animales , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Ensayo de Unidades Formadoras de Colonias , Cricetinae , Cricetulus , ADN/efectos de los fármacos , ADN/genética , Resistencia a Medicamentos , Femenino , Amplificación de Genes/efectos de los fármacos , Ovario/enzimología , Plásmidos/efectos de los fármacos , Plásmidos/genética , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
Chemical compounds can cause amplification of specific DNA sequences. DNA amplification may result in an enhanced production of gene products which help cells to cope with the chemicals. This may lead to a resistance of the cells toward the agent. Additionally, initiation of transformation or progression of transformed cells to tumorigenicity may also involve DNA amplification. Therefore, it is of interest to study the potential of chemicals to induce DNA amplification. This report focuses on the investigation of a variety of chemicals in 2 systems with which the amplification of viral DNA is measured within cells in culture. One model system comprises the measurement of SV40 DNA content in an SV40-transformed Chinese hamster cell line following chemical treatment. Antitumor agents as well as genotoxic and non-genotoxic compounds were studied in this system as a first step to determine the DNA amplification-inducing potential of a variety of differently acting chemical compounds. Also, a novel assay based on adeno-associated virus infection of cells is described. This system may offer the possibility of studying DNA amplification in a variety of different target cells. For the future, the need is stressed to develop and analyze versatile systems to study amplification of specific target genes in untransformed cells and in tumor cells.
Asunto(s)
Carcinógenos , ADN Viral/efectos de los fármacos , Dependovirus/genética , Amplificación de Genes/efectos de los fármacos , Mutágenos , Animales , Línea Celular Transformada , Cricetinae , Cricetulus , Genes Virales , Virus 40 de los Simios/genéticaRESUMEN
We studied DNA amplification of helper virus-dependent parvoviruses [adeno-associated virus (AAV)] following genotoxic treatment of a number of mammalian cell lines from different species including primary, immortalized, and tumorigenic cells. All cell lines, either infected with AAV or transfected with parvoviral DNA, readily amplified AAV DNA in the absence of helper virus following treatment of cells with a wide variety of genotoxic agents like chemical carcinogens, UV, heat shock, and metabolic inhibitors of DNA replication or protein synthesis. In addition, we show that in the SV40-transformed Chinese hamster cell lines CO60 and CO631 carcinogen-induced AAV DNA amplification may result in a complete AAV replication cycle giving rise to infectious AAV progeny. Our results demonstrate that AAV DNA amplification induced by genotoxic agents is completely independent of the presence of viral helper functions. Because its induction is not restricted to a specific cell type or to a malignant phenotype, AAV DNA amplification may represent a marker for cellular genotoxic stress response.
Asunto(s)
ADN Viral/genética , Dependovirus/genética , Amplificación de Genes , Genes Virales , Metilnitronitrosoguanidina/farmacología , Animales , Línea Celular , Línea Celular Transformada , ADN Viral/efectos de los fármacos , Dependovirus/efectos de los fármacos , Humanos , Plásmidos , Virus 40 de los Simios/genéticaRESUMEN
Inhibitors of DNA polymerase alpha (aphidicolin, phosphonoacetic acid, phosphonoformic acid) efficiently inhibit initiator-induced amplification of SV40 DNA sequences in the SV40-transformed Chinese hamster cell line CO631. Amplification is also inhibited by various protease inhibitors (antipain, leupeptin, aprotinin, alpha-I-antitrypsin, epsilon-amino-caproic acid, soy-bean protease inhibitor), by the non-initiating but DNA-damaging agent caffeine, and by sodium butyrate, which inhibits DNA synthesis by histone modification. In contrast, an inhibitor of topoisomerase II, nalidixic acid, enhances amplification when applied simultaneously with initiating treatment. This latter compound does not induce amplification when applied without initiator. Cycloheximide induces DNA amplification in the same way as chemical and physical carcinogens. This amplification can still be observed when protein synthesis is completely blocked. The data suggest a complex mechanism of selective DNA amplification. The possible involvement of proteases leading to a functional modification of DNA polymerase alpha is discussed.