gms | German Medical Science

Objective: At genetic level Glioblastoma multiforme (GBM) are characterized by a high degree of chromosomal instability and aneuploidy. During the cell cycle, the mitotic spindle checkpoint controls fidelity of spindle attachement to chromatids and thereby of chromosome segregation. It has been shown that defects in this checkpoint may lead to the development of aneuploidy and tumorigenesis. Here, we analysed this checkpoint function in GBM cell lines and primary cells. In addition, we investigated GBM cell lines and biopsies from patients with astrocytic tumors for alterations in mRNA and protein expression of the key checkpoint proteins MAD1, MAD3, BUB1, BUBR1, BUB3 and MPS1. Also their mutational status was determined.

Methods: Checkpoint function was investigated by FACS analysis after Nocodazole induced spindle damage in GBM cell lines U87, U138, U251, U343 and U373. These cells also were used for determination of checkpoint protein expression by semiquantitative RT-PCR and Western-blotting, respectively. In addition, a panel of 15 astrocytoma WHO grade 2, 15 GBM and 3 normal brain biopsies were analysed. cDNA-sequencing was used to check for mutations.

Results: Generally, the mitotic checkpoint was functional in GBM cell lines. High Nocodazole concentrations (> 1.5 µM) caused a mitotic cell cycle arrest and cells accumulated in G2/M phase. However, low Nocodazole-concentrations (0.75 µM) allowed cells to slip through the checkpoint, leading to more aneuploid and apoptotic cell populations. On the mRNA level, there was upregulation of BUB1 expression concomitantly with increasing WHO grading of the glioma and MPS1 was overexpressed by tumor tissue in comparison to normal brain samples. On the protein level, these alterations were less obvious, but displayed the same tendency. There were no sequence mutations detectable.

Conclusions: Mitotic checkpoint function is weakened in GBM cells. This may be due to alterations in checkpoint protein expression levels, which may cause an imbalance in the checkpoint signalling network. Thereby, cells become more prone to chromosomal instability and more sensitive to induction of apoptotic cell death. Since cells are most sensitive to gamma-irradiation during the G2/M phase of the cell cycle, alterations in mitotic checkpoint function may have implications for efficacy of GBM standard therapy.