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Gene expression time-series analysis of camptothecin effects in U87-MG and DBTRG-05 glioblastoma cell lines.

Morandi E, Severini C, Quercioli D, D'Ario G, Perdichizzi S, Capri M, Farruggia G, Mascolo MG, Horn W, Vaccari M, Serra R, Colacci A, Silingardi P - Mol. Cancer (2008)

Bottom Line: To address this issue, we had previously compared the expression profile of human U87-MG glioblastoma cells with that of a CPT-resistant counterpart, giving evidence that the development of a robust inflammatory response was the main transcriptional effect associated with CPT resistance.In U87-MG cells we also found inflammation response and IL1-beta induction, as late transcriptional effects of Topo I treatment but these changes were only partially involved in the senescence development, as shown by IL1-beta gene silencing.Moreover, our results helped in identifying some key genes whose expression seemed to be associated with the execution of senescence or apoptosis in U87-MG and DBTRG-05 cells, respectively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Excellence Environmental Carcinogenesis, Lab, Mater, Environmental Protection and Health Prevention Agency, Emilia-Romagna Region EPA, Bologna County, Italy. emorandi@arpa.emr.it

ABSTRACT

Background: The clinical efficacy of camptothecin (CPT), a drug specifically targeting topoisomerase I (TopoI), is under evaluation for the treatment of malignant gliomas. Due to the high unresponsiveness of these tumours to chemotherapy, it would be very important to study the signalling network that drives camptothecin outcome in this type of cancer cells. To address this issue, we had previously compared the expression profile of human U87-MG glioblastoma cells with that of a CPT-resistant counterpart, giving evidence that the development of a robust inflammatory response was the main transcriptional effect associated with CPT resistance. Here we report time-related changes and cell line specific patterns of gene expression after CPT treatment by using two p53 wild-type glioblastoma cell lines, U87-MG and DBTRG-05, with different sensitivities to TopoI inhibition.

Results: First, we demonstrated that CPT treatment brings the two cell lines to completely different outcomes: accelerated senescence in U87-MG and apoptosis in DBTRG-05 cells. Then, to understand the different susceptibility to CPT, we used oligo-microarray to identify the genes whose expression was regulated during a time-course treatment, ranging from 2 h to 72 h. The statistical analysis of microarray data by MAANOVA (MicroArray ANalysis Of VAriance) showed much less modulated genes in apoptotic DBTRG-05 cells (155) with respect to the senescent U87-MG cells (3168), where the number of down-regulated genes largely exceeded that of the up-regulated ones (80% vs. 20%). Despite this great difference, the two data-sets showed a large overlapping (60% circa) mainly due to the expression of early stress responsive genes. The use of High-Throughput GoMINER and EASE tools, for functional analysis of significantly enriched GO terms, highlighted common cellular processes and showed that U87-MG and DBTRG-05 cells shared many GO terms, which are related to the down-regulation of cell cycle and mitosis and to the up-regulation of cell growth inhibition and DNA damage.Furthermore, the down-regulation of MYC and DP1 genes, which act as key transcription factors in cell growth control, together with the inhibition of BUB1, BUB3 and MAD2 mRNAs, which are known to be involved in the spindle checkpoint pathway, were specifically associated with the execution of senescence in U87-MG cells and addressed as critical factors that could drive the choice between different CPT-inducible effectors programs. In U87-MG cells we also found inflammation response and IL1-beta induction, as late transcriptional effects of Topo I treatment but these changes were only partially involved in the senescence development, as shown by IL1-beta gene silencing.

Conclusion: By comparing the transcription profile of two glioblastoma cell lines treated with camptothecin, we were able to identify the common cellular pathways activated upon Topo I inhibition. Moreover, our results helped in identifying some key genes whose expression seemed to be associated with the execution of senescence or apoptosis in U87-MG and DBTRG-05 cells, respectively.

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Time-dependent expression profile of AREG, GEM, MYC and MIG6. Log2 (expression ratio) was plotted versus time.
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Figure 5: Time-dependent expression profile of AREG, GEM, MYC and MIG6. Log2 (expression ratio) was plotted versus time.

Mentions: The lists of differentially expressed genes were numerically completely different. Applying a p-value threshold of 0.01 and 0.05 for U87-MG and DBTRG-05 cells respectively, the total number of differentially expressed genes was 3168 in the U87- and only 155 in the case of the DBTRG-05. To facilitate the comparison between the two data-sets, we restricted the analysis to those genes that reached a log2 (expression ratio) value above 1.5, at least in one of the six time-series data points. By doing that, we reduced the overall number of differentially expressed genes to 713 for U87-MG cells and to 92 for DBTRG cells (see Additional files 1 and 2 to retrieve the complete lists of genes returned from MAANOVA analysis and the filtering process). Based on the time dependent expression profile, we defined the up- and down-regulated genes within the two data sets. Interestingly, this analysis, that is summarized in the Venn diagrams reported in Figure 4, demonstrated that most of the genes, whose expression was affected by the treatment with CPT, were indeed down-regulated (almost 80%) in U87-MG cells, while a substantial balance between up- and down-regulation was observed in the DBTRG-05. Despite this difference, the two data-sets were largely overlapping, being more than the 60% of the genes found in DBTRG-05 cells also represented in the U87-MG population. In Table 1 and Table 2, we reported the lists of the common up-regulated genes and the down-regulated ones, respectively. Only four genes (AREG, MYC, MIG-6 and GEM) showed a different time-dependent expression profile between the two data-sets (Figure 5). Indeed, CPT treatment induced the expression of these genes in DBTRG-05 cells while repressed them in U87-MG cells. For selected time-points, three of these genes (AREG, MYC, GEM) were chosen to be confirmed through a semi-quantitative RT-PCR analysis, together with other genes (CDKN1A, TXNIP, GADD45A, EREG) known to be CPT transcriptional targets (Figure 6). Taking into account that we used a semi-quantitative approach, we found overlapping results between two techniques except for AREG in U87- and EREG in DBTRG-05 cells whose over-expression was undetectable in microarray.


Gene expression time-series analysis of camptothecin effects in U87-MG and DBTRG-05 glioblastoma cell lines.

Morandi E, Severini C, Quercioli D, D'Ario G, Perdichizzi S, Capri M, Farruggia G, Mascolo MG, Horn W, Vaccari M, Serra R, Colacci A, Silingardi P - Mol. Cancer (2008)

Time-dependent expression profile of AREG, GEM, MYC and MIG6. Log2 (expression ratio) was plotted versus time.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2556695&req=5

Figure 5: Time-dependent expression profile of AREG, GEM, MYC and MIG6. Log2 (expression ratio) was plotted versus time.
Mentions: The lists of differentially expressed genes were numerically completely different. Applying a p-value threshold of 0.01 and 0.05 for U87-MG and DBTRG-05 cells respectively, the total number of differentially expressed genes was 3168 in the U87- and only 155 in the case of the DBTRG-05. To facilitate the comparison between the two data-sets, we restricted the analysis to those genes that reached a log2 (expression ratio) value above 1.5, at least in one of the six time-series data points. By doing that, we reduced the overall number of differentially expressed genes to 713 for U87-MG cells and to 92 for DBTRG cells (see Additional files 1 and 2 to retrieve the complete lists of genes returned from MAANOVA analysis and the filtering process). Based on the time dependent expression profile, we defined the up- and down-regulated genes within the two data sets. Interestingly, this analysis, that is summarized in the Venn diagrams reported in Figure 4, demonstrated that most of the genes, whose expression was affected by the treatment with CPT, were indeed down-regulated (almost 80%) in U87-MG cells, while a substantial balance between up- and down-regulation was observed in the DBTRG-05. Despite this difference, the two data-sets were largely overlapping, being more than the 60% of the genes found in DBTRG-05 cells also represented in the U87-MG population. In Table 1 and Table 2, we reported the lists of the common up-regulated genes and the down-regulated ones, respectively. Only four genes (AREG, MYC, MIG-6 and GEM) showed a different time-dependent expression profile between the two data-sets (Figure 5). Indeed, CPT treatment induced the expression of these genes in DBTRG-05 cells while repressed them in U87-MG cells. For selected time-points, three of these genes (AREG, MYC, GEM) were chosen to be confirmed through a semi-quantitative RT-PCR analysis, together with other genes (CDKN1A, TXNIP, GADD45A, EREG) known to be CPT transcriptional targets (Figure 6). Taking into account that we used a semi-quantitative approach, we found overlapping results between two techniques except for AREG in U87- and EREG in DBTRG-05 cells whose over-expression was undetectable in microarray.

Bottom Line: To address this issue, we had previously compared the expression profile of human U87-MG glioblastoma cells with that of a CPT-resistant counterpart, giving evidence that the development of a robust inflammatory response was the main transcriptional effect associated with CPT resistance.In U87-MG cells we also found inflammation response and IL1-beta induction, as late transcriptional effects of Topo I treatment but these changes were only partially involved in the senescence development, as shown by IL1-beta gene silencing.Moreover, our results helped in identifying some key genes whose expression seemed to be associated with the execution of senescence or apoptosis in U87-MG and DBTRG-05 cells, respectively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Excellence Environmental Carcinogenesis, Lab, Mater, Environmental Protection and Health Prevention Agency, Emilia-Romagna Region EPA, Bologna County, Italy. emorandi@arpa.emr.it

ABSTRACT

Background: The clinical efficacy of camptothecin (CPT), a drug specifically targeting topoisomerase I (TopoI), is under evaluation for the treatment of malignant gliomas. Due to the high unresponsiveness of these tumours to chemotherapy, it would be very important to study the signalling network that drives camptothecin outcome in this type of cancer cells. To address this issue, we had previously compared the expression profile of human U87-MG glioblastoma cells with that of a CPT-resistant counterpart, giving evidence that the development of a robust inflammatory response was the main transcriptional effect associated with CPT resistance. Here we report time-related changes and cell line specific patterns of gene expression after CPT treatment by using two p53 wild-type glioblastoma cell lines, U87-MG and DBTRG-05, with different sensitivities to TopoI inhibition.

Results: First, we demonstrated that CPT treatment brings the two cell lines to completely different outcomes: accelerated senescence in U87-MG and apoptosis in DBTRG-05 cells. Then, to understand the different susceptibility to CPT, we used oligo-microarray to identify the genes whose expression was regulated during a time-course treatment, ranging from 2 h to 72 h. The statistical analysis of microarray data by MAANOVA (MicroArray ANalysis Of VAriance) showed much less modulated genes in apoptotic DBTRG-05 cells (155) with respect to the senescent U87-MG cells (3168), where the number of down-regulated genes largely exceeded that of the up-regulated ones (80% vs. 20%). Despite this great difference, the two data-sets showed a large overlapping (60% circa) mainly due to the expression of early stress responsive genes. The use of High-Throughput GoMINER and EASE tools, for functional analysis of significantly enriched GO terms, highlighted common cellular processes and showed that U87-MG and DBTRG-05 cells shared many GO terms, which are related to the down-regulation of cell cycle and mitosis and to the up-regulation of cell growth inhibition and DNA damage.Furthermore, the down-regulation of MYC and DP1 genes, which act as key transcription factors in cell growth control, together with the inhibition of BUB1, BUB3 and MAD2 mRNAs, which are known to be involved in the spindle checkpoint pathway, were specifically associated with the execution of senescence in U87-MG cells and addressed as critical factors that could drive the choice between different CPT-inducible effectors programs. In U87-MG cells we also found inflammation response and IL1-beta induction, as late transcriptional effects of Topo I treatment but these changes were only partially involved in the senescence development, as shown by IL1-beta gene silencing.

Conclusion: By comparing the transcription profile of two glioblastoma cell lines treated with camptothecin, we were able to identify the common cellular pathways activated upon Topo I inhibition. Moreover, our results helped in identifying some key genes whose expression seemed to be associated with the execution of senescence or apoptosis in U87-MG and DBTRG-05 cells, respectively.

Show MeSH
Related in: MedlinePlus