RESUMO
The comet assay, one of the most widely used techniques for the evaluation and detection of DNA strand breaks, is frequently employed in vivo. In vitro assays are usually performed with mammalian cell lines, clearly not the best choice for tests on aquatic genotoxicity. Here we evaluated a fish hepatoma cell line (RTH-149) and a primary blood cell culture from the intertidal colonial tunicate Botryllus schlosseri as possible model targets for comet assays using the genotoxic agent H2O2. We found that DNA strand break levels in RTH-149 fitted dose-dependent responses better than the tunicate cells. Moreover, in B. schlosseri controls, 34% of the cells were already ranked as severely damaged. Assays were then performed on water samples from the polluted Kishon river (Israel) on three different dates, using RTH-149 cells (50% dilutions, 2-h exposures). In all cases, high genotoxicity of the river water was revealed by evaluating comet percentages, average tail lengths and DNA damage levels. This assay was found to be fast and sensitive, appropriate to be employed as a part of a monitoring program. The use of B. schlosseri blood cells should be validated in additional work.
Assuntos
Ensaio Cometa/métodos , Dano ao DNA/efeitos dos fármacos , Monitoramento Ambiental/métodos , Mutagênicos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Peixes , Água Doce/análise , Peróxido de Hidrogênio/toxicidade , Israel , UrocordadosRESUMO
A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 'gain of function' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53(R175H) mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53(R175H), was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial-mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53(R175H) mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53(R175H) mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.
Assuntos
Transição Epitelial-Mesenquimal , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Substituição de Aminoácidos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Epigênese Genética , Histonas/metabolismo , Humanos , Masculino , Mutação , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Proteína 1 Relacionada a Twist/genética , Regulação para CimaRESUMO
Estuarine and marine near-shore environments are often subjected to heavy metal pollution. We establish a bioassay using the quantitative evaluation of metallothionein (MT) transcript in the fish hepatoma cell line, RTH-149, as a tool for detecting heavy metal pollution in brackish-marine water containing other pollutants in addition to heavy metals. RT-competitive polymerase chain reaction was used for the quantitative evaluation of the transcript in absolute units. Cadmium was used as a model pollutant to optimize two parameters of the assay: exposure periods (24, 96, 144 h) and salinity (0%, 25%, 50%, 75%, 100% sea water). Results revealed that salinity at or below 25% sea water at an exposure period of 144 h are the preferable conditions for detecting MT mRNA levels for in vitro assays employed on water samples from highly polluted brackish habitats.