Dynamics of chemosensitivity and chromosomal instability in recurrent glioblastoma

Abstract

Glioblastoma multiforme is characterised by invasive growth and frequent recurrence. Here, we have analysed chromosomal changes in comparison to tumour cell aggressiveness and chemosensitivity of three cell lines established from a primary tumour and consecutive recurrences (BTL1 to BTL3) of a long-term surviving glioblastoma patient together with paraffin-embedded materials of five further cases with recurrent disease. Following surgery, the BTL patient progressed under irradiation/ lomustine but responded to temozolomide after re-operation to temozolomide. The primary tumour -derived BTL1 cells showed chromosomal imbalances typical of highly aggressive glioblastomas. Interestingly, BTL2 cells established from the first recurrence developed under therapy showed signs of enhanced chromosomal instability. In contrast, BTL3 cells from the second recurrence resembled a less aggressive subclone of the primary tumour. Although BTL2 cells exhibited a highly aggressive phenotype, BTL3 cells were characterised by reduced proliferative and migratory potential. Despite persistent methylation of the O6-methylguanine-DNA methyltransferase promoter, BTL3 cells exhibited the highest temozolomide sensitivity. A comparable situation was found in two out of five glioblastoma patients, both characterised by enhanced survival time, who also relapsed after surgery/chemotherapy with less aggressive recurrences. Taken together, our data suggest that pretreated glioblastoma patients may relapse with highly chemosensitive tumours confirming the feasibility of temozolomide treatment even in case of repeated recurrence.

Figures

Figure 1

CGH analysis profiles (for interpretation see Pirker et al, 2003) of (A) primary tumour-derived BTL1, (B) first recurrence-derived BTL2, and (C) second recurrence-derived BTL3 cells. Arrows in (A) indicate typical changes for glioblastoma multiforme. Asterisks in (B) indicate additional chromosomal aberrations in BTL2 as compared to BTL1. In (C) the asterisk indicates an aberration present also in BTL2 but not BTL1. Crosses in (C) represent changes unique to BTL3.

Figure 2

Cytogenetic analysis of the BTL glioblastoma cell model. Representative CGH (A), FISH (B, C), and CDD banding (D–F) analyses of the indicated cell lines are shown. Arrows in (A) indicate typical changes for glioblastoma multiforme. For FISH analysis (B, C) a paint for whole chromosome 7 (red) and a BAC clone for the EGFR locus (green) were used. (D and E) show representative karyograms of the two subclones detected in BTL1.

Figure 3

In vitro growth dynamics of the BTL glioblastoma cell model. Growth curves for the indicated cell lines were established as described in Material and Methods. One of three experiments with comparable results is shown.

Figure 4

Migratory potential of the BTL glioblastoma cell model. Scratch assays were performed as described in Materials and Methods. (A) Representative photomicrographs taken at the indicated time points are shown. (B) To determine the relative closure of the gap, photomicrographs as shown in (A) were taken at different time points and measured using MetaMorph 6.1 software. The gaps’ widths immediately after wounding were arbitrarily set as 1. At least three experiments with comparable results were performed.

Figure 5

Antiproliferative and cytotoxic effects of temozolomide against BTL cells. (A) The indicated BTL cell lines were exposed to increasing temozolomide concentrations for 48 h and the rate of DNA synthesis was determined by 3H-thymidine incorporation. Data are given relatively to the untreated controls set as 1. Means of two experiments in triplicates are shown. (B) BTL cells were exposed for 5 days to the indicated temozolomide concentrations and the percentage of apoptotic/dead cells was determined by Hoechst 33258 and PI staining as described in Materials and Methods. One representative example out of three experiments delivering comparable results is shown.

Figure 6

Expression of chemoresistance-related genes in BTL glioblastoma cells. mRNA (A) and protein (B) levels encoded by the indicated chemoresistance genes were determined by RT–PCR and Western blot, respectively. (C) Methylation of the MGMT promoter was detected by methylation-specific PCR and a representative experiment is shown (m=methylated; u=unmethylated). Protein extracts of the glioblastoma cell lines GL80, GL54, and GL52 were used as positive controls for MGMT, MVP, and MRP1, respectively.