The clinical efficacy of camptothecins, a family of drugs specifically targeting topoisomerase I, is under evaluation for the treatment of malignant gliomas, that are among the most strikingly chemotherapy-unresponsive tumours. Therefore, it is particularly important to understand the molecular mechanisms underlying the response to camptothecin (CPT) in this type of cancer. Agilent’s Oligo 60-mer technology was used to identify genes that are transcriptionally regulated by CPT in two glioma cell line U87-MG and DBTRG-05, during a time-course treatment ranging from 2 to 72 hours. These two models were chosen because of different responses to CPT, as shown by the results obtained from the tests of vitality and apoptosis. We adopted an experimental design consisting of six after-treatment time points compared to an untreated zero-time common reference. Each time point was replicated by performing a dye-swap. To assess differentially expressed genes the transformed data were analysed by MAANOVA (MicroArray ANalysis Of VAriance) data analysis package of R programming environments. We focused on the common mechanisms induced by CPT as well as on the pathways that may explain the substantial variation in the response to the drug in U87-MG and DBTRG cell lines. Moreover, several modulated transcripts were also confirmed by RT-PCR. Many of the genes, that were found to be modulated in both target cells, are specifically involved in regulating cell cycle, DNA repair and /or apoptosis in response to DNA damage, such as cyclin-dependent kinase inhibitor 1A (p21), growth arrest and DNA damage-inducible protein 45 (GADD45), and activating transcription factor 3 (ATF3). Nevertheless, a significantly higher number of down-regulated genes, which are involved in the inhibiton of cell cycle, was found in U87-MG cells. The transcription of other genes such as amphiregulin (AREG), epiregulin (EREG) and MYC, possibly involved in survival signalling in response to genotoxic stress, showed a distinctive regulation depending on the cell line, suggesting a different role of the epidermal growth factor receptor pathway in U87-MG and DBTRG cells. A combination of oligo microarray technology with informatics is a good premise to develop specific drug-gene profiles to be used as molecular markers for potential response to chemotherapy.
Silingardi P., Morandi E., Severini C., Quercioli D., Vaccari M., Horn W., et al. (2006). OLIGONUCLEOTIDE MICROARRAY TIME-SERIES ANALYSIS TO EVALUATE CAMPTOTHECIN EFFECTS: A GLIOBLASTOMA MODEL. PHILADELPHIA : American Association for Cancer Research (AACR).
OLIGONUCLEOTIDE MICROARRAY TIME-SERIES ANALYSIS TO EVALUATE CAMPTOTHECIN EFFECTS: A GLIOBLASTOMA MODEL
SILINGARDI, PAOLA;MORANDI, ELENA;QUERCIOLI, DANIELE;VACCARI, MONICA;HORN, WOLFANGO;COLACCI, ANNAMARIA
2006
Abstract
The clinical efficacy of camptothecins, a family of drugs specifically targeting topoisomerase I, is under evaluation for the treatment of malignant gliomas, that are among the most strikingly chemotherapy-unresponsive tumours. Therefore, it is particularly important to understand the molecular mechanisms underlying the response to camptothecin (CPT) in this type of cancer. Agilent’s Oligo 60-mer technology was used to identify genes that are transcriptionally regulated by CPT in two glioma cell line U87-MG and DBTRG-05, during a time-course treatment ranging from 2 to 72 hours. These two models were chosen because of different responses to CPT, as shown by the results obtained from the tests of vitality and apoptosis. We adopted an experimental design consisting of six after-treatment time points compared to an untreated zero-time common reference. Each time point was replicated by performing a dye-swap. To assess differentially expressed genes the transformed data were analysed by MAANOVA (MicroArray ANalysis Of VAriance) data analysis package of R programming environments. We focused on the common mechanisms induced by CPT as well as on the pathways that may explain the substantial variation in the response to the drug in U87-MG and DBTRG cell lines. Moreover, several modulated transcripts were also confirmed by RT-PCR. Many of the genes, that were found to be modulated in both target cells, are specifically involved in regulating cell cycle, DNA repair and /or apoptosis in response to DNA damage, such as cyclin-dependent kinase inhibitor 1A (p21), growth arrest and DNA damage-inducible protein 45 (GADD45), and activating transcription factor 3 (ATF3). Nevertheless, a significantly higher number of down-regulated genes, which are involved in the inhibiton of cell cycle, was found in U87-MG cells. The transcription of other genes such as amphiregulin (AREG), epiregulin (EREG) and MYC, possibly involved in survival signalling in response to genotoxic stress, showed a distinctive regulation depending on the cell line, suggesting a different role of the epidermal growth factor receptor pathway in U87-MG and DBTRG cells. A combination of oligo microarray technology with informatics is a good premise to develop specific drug-gene profiles to be used as molecular markers for potential response to chemotherapy.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
