RESUMEN
We have investigated the genotoxicity of two 3'-derivatives of cytidine, 2,3'-O-cyclocytidine (3'-cycloC) and beta-xylocytidine (xyloC), in human leukemia and solid tumor cell lines. Both derivatives were found to be cytotoxic at micromolar concentrations. For example, in the alveolar tumor cell line A549 which was included in all experiments as a reference, drug concentrations required to induce 50% inhibition of cell growth (D50 values) equalled 55 microM for 3'-cycloC and 80 microM for xyloC. Compared with the response of this reference cell line, none of the solid tumor cell lines tested--representing five different malignancies--displayed significant hypersensitivity to these drugs, while the acute lymphoblastic leukemia cell lines proved to be hypersensitive (range of D50 values, 5-13 microM). To gain insight into the modes of cytotoxic action of xyloC and 3'-cycloC, we compared the effect on DNA metabolism of these compounds with that of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of semi-conservative DNA replication and long-patch excision repair. As seen with araC, the xylo compound strongly inhibited both DNA replicative synthesis and the repair of DNA damage induced by UV light and 60Co gamma-radiation. In gamma-irradiated A549 cells, the extent of repair inhibition by 1 mM xyloC was approximately 40% of that inhibited by araC, and concomitant exposure of the irradiated cultures to xyloC plus araC gave rise to a synergistic response. Since araC was employed at a concentration (0.1 mM) which produced a maximal effect on DNA repair when applied alone, the observed synergistic response implies that the mode of action of xyloC on DNA repair is different from that of araC. In contrast to that observed with xyloC, 3'-cycloC proved to be a very weak inhibitor of DNA replication and repair, strongly suggesting that the genotoxic action of the latter analog may be through a mechanism other than inhibition of DNA synthesis.
Asunto(s)
Ancitabina/toxicidad , Citarabina/toxicidad , Citidina/análogos & derivados , Leucemia/tratamiento farmacológico , Leucemia/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidad , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Niño , Citarabina/administración & dosificación , Citidina/toxicidad , Daño del ADN , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/efectos de la radiación , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Leucemia/patología , Neoplasias/patología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
Methylation in vitro of calf thymus DNA, a supercoiled plasmid, poly(dG).poly(dC), and poly(dGdC).poly(dGdC) by N-nitroso(acetoxy-methyl)methylamine and N-nitroso(acetoxybenzyl)methylamine in the presence of esterase, and by N-nitrosomethylurea was investigated. Although there were differences in the amounts of 7-methylguanine and O6-methylguanine formed in the various DNA substrates, the methylation pattern was the same for each of these methylating agents. The three compounds reacted identically when methylation of a portion of a 345 bp restriction fragment of the plasmid pBR322 was examined at nucleotide resolution by a sequencing assay. They also showed a tendency to react preferentially with particular guanines. These data suggest that the three N-nitroso compounds methylate DNA via a common intermediate such as the methyl diazonium ion, which exhibits some sequence specificity.
Asunto(s)
ADN , Compuestos Nitrosos , Secuencia de Bases , Enzimas de Restricción del ADN , Dimetilnitrosamina , Guanina/análogos & derivados , Metilación , Metilnitrosourea , Nitrosaminas/metabolismo , Plásmidos , PolidesoxirribonucleótidosRESUMEN
alpha-Acetoxy-N-nitrosodimethylamine, an activated derivative of the carcinogen N-nitrosodimethylamine, methylated in vitro a plasmid containing the human c-Ha-ras-1 proto-oncogene, resulting in the generation of a transforming oncogene, assayed by transfection into NIH 3T3 cells. The resulting transformed cells were tumorigenic and metastatic in immune-deprived mice. Further transfection using tumor DNA led to the formation of three secondary NIH 3T3 transformants. DNA from these secondary transformants contained human ras gene sequences. Two of the three secondary transformants contained G----A mutations at guanine 35 in codon 12, and the third secondary transformant retained the wild-type sequence at codons 12, and 61. For the latter, the activating mutation was not determined. These results demonstrate that a simple methylating agent can activate a normal human ras proto-oncogene to a transforming oncogene.
Asunto(s)
Dimetilnitrosamina/análogos & derivados , Regulación de la Expresión Génica , Genes ras/efectos de los fármacos , Animales , Southern Blotting , Pruebas de Carcinogenicidad , Línea Celular , Transformación Celular Neoplásica , ADN/metabolismo , Análisis Mutacional de ADN , ADN Polimerasa Dirigida por ADN , Dimetilnitrosamina/farmacología , Amplificación de Genes , Humanos , Masculino , Metilación , Ratones , Ratones Endogámicos CBA , Sondas de Oligonucleótidos/síntesis química , Sondas de Oligonucleótidos/genética , Plásmidos , Proto-Oncogenes Mas , Polimerasa Taq , TransfecciónRESUMEN
We have previously reported some effects of DNA repair on the transition frequencies produced by an O6-methyl-guanine (MeG) or an O6-n-butyl-guanine (BuG) paired with C at the first position of the third codon in gene G of bacteriophage phi X174 form I' DNA (Chambers et al. 1985). We now report experiments in which the transition is produced from T:MeG or T:BuG, instead of C:MeG or C:BuG, located at this site. The site-modified DNAs were transfected into cells with normal DNA repair as well as into cells with repair defects (uvrA, uvrB, uvrC, recA, uvrArecA). The lysates were screened for phage carrying the expected transition using a characteristic change in phenotype. The data demonstrate that the transition frequency from T:BuG is low (0.3% of total phage progeny) in cells with normal repair (Escherichia coli AB1157) and increases 7-fold in uvrA cells (E. coli AB1886). A similar increase is seen in uvrB and uvrC cells (AB1885, AB1884). These data, like our previous data, indicate BuG is repaired primarily by excision. In contrast to this, the transition frequency from T:MeG is high (5 +/- 2%) in cells with normal repair. After induction of alkyl transfer repair in E. coli AB1157, the transition frequency goes up 5-fold. Compared with cells with normal repair, the transition frequency goes up 2-fold in uvrA, uvrB and uvrC cells; it goes up 1.5-fold in recA cells (E. coli AB2463). The data reinforce our earlier conclusion that MeG is repaired primarily by alkyl transfer, but the ABC excinuclease as well as RecA protein inhibit this repair process.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Bacteriófago phi X 174/metabolismo , Reparación del ADN , Guanina/análogos & derivados , Bacteriófago phi X 174/genética , Composición de Base , Replicación del ADN , ADN Viral/genética , ADN Viral/metabolismo , Guanina/metabolismoRESUMEN
Reaction of N-nitrosomethyl(acetoxymethyl)amine in vitro in the presence of esterase with a plasmid containing the human c-Ha-ras-1 gene has been shown to generate a transforming oncogene when the methylated DNA is transfected into NIH 3T3 cells. Our results show that an activated nitrosamine can mutate a normal cellular proto-oncogene, a reaction that may be the necessary first step in the multistage process of tumour induction.
Asunto(s)
Carcinógenos/farmacología , ADN/efectos de los fármacos , Dimetilnitrosamina/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Proto-Oncogenes , Animales , Línea Celular , Dimetilnitrosamina/farmacología , Humanos , Técnicas In Vitro , Metilación , Ratones , Ratones Endogámicos CBA , Plásmidos , Proto-Oncogenes Mas , TransfecciónRESUMEN
Using site-specific mutagenesis, we have examined the mutagenic activity in vivo of O6-methylguanine or O6-n-butylguanine located at a preselected site in gene G of bacteriophage phi X174. The experiments were designed so that the phage mutant produced by a targeted transition from either of these alkylated derivatives would be recognizable by a simple plaque assay. Spheroplasts derived from normal and repair-deficient cells were transfected, and the lysates were screened for mutant virus. In cells with normal repair, DNA carrying the methylguanine produced the expected transition in 15% of the total phage; DNA carrying the butylguanine produced the same mutation in 0.3% of the phage. In cells deficient in excision repair (uvrA) the transition frequency went up by a factor of 8 for O6-butylguanine and down by a factor of 40 for O6-methylguanine. In cells deficient in recombination (recA), the transition frequency increased 1.5-fold for butylguanine and decreased by a factor of 8 for methylguanine. The data show that both methyl- and butylguanine produce site-directed transitions in phi X174; the transition occurs in recA cells; the frequency of the transition is influenced by both recA and uvrA mutations; the recA and uvrA mutations alter the transition frequency for methylguanine and butylguanine in opposite directions.