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

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.

Figure 1

CPT-induced acute citotoxicity and apoptosis. A. Dose-response analysis of CPT effects on U87-MG and DBTRG-05 cells. On Y-axis the percentage of growth, calculated according to the protocol used by the National Cancer Institute, is reported. The GI50 values for both cell lines were calculated by linear regression analysis of the log (dose/effect) curve. B. Annexin-V apoptosis assay was performed after a 24-h treatment with 0.5 and 5 μM CPT. DMSO and TNF-alpha plus actinomycin D were used as negative and positive controls, respectively. Each data point is the average of three independent experiments; bars, standard errors.

Figure 2

Apoptotic response of DBTRG-05 and U87-MG cells exposed to CPT working concentrations. A. Percentages of Annexin-V positive DBTRG-05 cells treated for 24 and 72 hours with 0.2 μM CPT. * p < 0.05, Student's t-test. ** p < 0.01, Student's t-test. B. Percentages of Annexin-V positive U87-MG cells treated for 24 and 72 hours with 1 μM CPT; bars, standard errors. ** p < 0.01, Student's t-test.

Figure 3

BrdU Flow cytometry assay of U87-MG and DBTRG-05 cells exposed to CPT and SA-β Gal staining in treated U87-MG cells. A. Time dependent changes in DNA content following treatment, from 2 to 72 hours, of U87-MG and DBTRG-05 cells with 1 μM and 0.2 μM CPT, respectively. DMSO: solvent control B. SA-β Gal staining of CPT-exposed U87-MG cells (CPT) versus control (NT). For this purpose, a 72 h treatment with 1 μM CPT was performed. Counts were made on three random fields of about 100 cells.

Figure 4

Venn-diagram representation of gene transcripts up- (A) and down-regulated (B) by CPT in either/both U87-MG and DBTRG-05 cell lines.

Figure 5

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

Figure 6

Semi-quantitative RT-PCR post-validation of microarray results. A. Gene expression changes for seven genes (CDKN1A, GADD45, TXNIP, MYC, GEM, AREG, EREG) were measured at selected time points after CPT treatment by semi-quantitative RT-PCR and normalized to β-actin expression of time- and dose- matched controls. The RT-PCR reactions were analyzed with the DNA1000 kit of the Agilent 2100 Bioanalyzer to give a gel-like image. B. At each selected time point after CPT treatment, expression fold change values obtained with semi-quantitative RT-PCR and microarrays were compared.

Figure 7

Effects of IL-1beta gene silencing on CPT-induced senescence in U87-MG cells. A. Real- time analysis of IL-1beta gene silencing. IL-1beta mRNA levels were significantly reduced (** p < 0.01, Student's t-test) with respect to mRNA level of the non silenced CPT-treated cells. IL1-B relative expression was obtained by performing the comparative method [48]. Firstly, data were normalized to GAPDH, then to a calibrator, consisting of mRNA obtained from CPT treated cells. The results were expressed as 2^^-ΔΔCt, where ΔCt of each sample was defined as Ct_{(target gene = IL1-beta)} - Ct_GAPDH, and ΔΔCt = ΔCt_{(sample = cDNA from CPT+iRNAs treated cells)} - ΔCt calibrator_{(cDNA from CPT treated cells)}. ΔΔCt calibrator is always equal to 0 so that 2^^-ΔΔCt is always 1. B. SA-β gal activity, in response to 1 μM CPT, was quantified in U87-MG cells in the presence or in the absence of IL-1beta gene silencing. DMSO, solvent control. Bars, standard error. * p < 0.05, Student's t-test. C. 250 cells from either CPT and solvent exposed cells or IL-1beta-silenced/CPT-treated U87-MG cells were sub-cultured in fresh medium in five replicates. Cells were maintained in culture for 10 days, with bi-weekly medium changes, then were fixed with methanol, stained with 10% aqueous Giemsa and scored for colony formation. Only colonies containing more than 50 cells were counted. Clonal efficiency was calculated as the mean number of colonies per plate with respect to the solvent. Bars, standard error. * p < 0.05, Student's t-test. ** p < 0.01, Student's t-test.