RESUMO
OBJECTIVES: To develop a real-time quantitative RT-PCR method for BRCA1 mRNA and then use it for the study of BRCA1 gene expression in human MCF-7 breast cancer cells after their exposure to antineoplastic agents and gamma irradiation. DESIGN AND METHODS: The developed QRT-PCR method is based on the real-time monitoring of a fluorescein-labeled TaqMan probe, specific for BRCA1 mRNA, during PCR in the LightCycler. A BRCA1 PCR amplicon was purified, quantitated and used as a standard of known concentration for the development and analytical evaluation of the assay. The method was applied to study the alteration of BRCA1 gene expression after exposure to taxol, doxorubicin, 5-fluorouracil, etoposide or gamma irradiation in human MCF-7 breast cancer cells. RESULTS: The developed method is quantitative, highly specific for mRNA and highly sensitive (detection limit of 4 BRCA1 copies per mug of total RNA). We observed a reduction of BRCA1 expression for all antineoplastic agents used, while the gamma irradiated MCF-7 cells had an increase of expression with a peak at the 10 Gy dose. CONCLUSIONS: The developed BRCA1 QRT-PCR method is quantitative, highly sensitive and specific. The proposed method is rapid, automated, and cost effective and can be used to study BRCA1 expression in a variety of clinical samples.
Assuntos
Proteína BRCA1/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Aminoácidos , Antineoplásicos , Sequência de Bases , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/metabolismo , Feminino , Raios gama , Humanos , Hipoxantina Fosforribosiltransferase/genética , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
OBJECTIVES: To evaluate the effect of antineoplastic agents on the expression of human telomerase reverse transcriptase (hTERT) splice variants in MCF-7 cells. DESIGN AND METHODS: We have developed a luminometric hybridization assay for hTERT beta plus transcript. MCF-7 cells were isolated before and after treatment with antineoplastic agents. A combination of nested RT-PCR and the developed luminometric hybridization assay was used for the specific detection of hTERT beta plus transcript in treated and untreated MCF-7 cells. Amplification of all hTERT splicing variants by nested PCR in the same samples was also performed. RESULTS: MCF-7 cells treated with taxol and etoposide were found positive for all hTERT splicing variants, while the expression of hTERT beta plus transcript did not differ significantly before and after exposure. MCF-7 cells treated with doxorubicin and 5-fluorouracil did not express any of hTERT splicing variants. In the presence of cisplatin, three splicing variants of hTERT were detected. CONCLUSIONS: The developed hybridization assay is highly sensitive and specific for the detection of hTERT beta plus transcript in clinical samples.