RESUMO
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.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , RNA Neoplásico/metabolismo , Telomerase/genética , Linhagem Celular Tumoral , Primers do DNA/genética , Proteínas de Ligação a DNA , Humanos , Hibridização Genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Telomerase is a general diagnostic and prognostic molecular tumor marker since it is expressed in the majority of human tumors in contrast to most healthy tissues. Alternate splicing of human telomerase reverse transcriptase (hTERT) has been shown to affect telomerase activity. PATIENTS AND METHODS: We have developed a hybridization assay that selectively detects the hTERT beta-plus transcript. Biotinylated PCR products were captured on streptavidin-coated microtiter wells, hybridized with digoxigenin-labeled probes and detected by a highly sensitive luminometric reaction. RESULTS: The method was applied in ten colorectal tumor forceps biopsies and their corresponding normal tissues. Six out of ten tumors were positive for hTERT beta plus transcript whereas none of the corresponding normal tissues were found positive. There was a complete concordance between the hybridization assay and real-time PCR. When the method was applied in peripheral blood of 20 breast cancer patients with metastatic disease and 21 healthy blood donors, 14 patients (70%) were found positive while all 21 healthy blood donors were negative. CONCLUSION: The developed hybridization assay is highly sensitive and specific for the detection of hTERT beta-plus transcript in clinical samples.