gms | German Medical Science

Objective: Hypoxia and aneuploidy are hallmarks of glioblastoma multiforme (GBM). Fidelity of spindle attachment to chromatids and chromosomal segregation are controlled by the mitotic spindle checkpoint. This checkpoint may be disturbed by protein dysregulation and hypoxic conditions. Low oxygen supply in conjunction with a weakened mitotic checkpoint may therefore accelerate chromosomal instability and participate in increasing malignancy of GBM.

Method: Immunohistochemistry was used to detect mitotic checkpoint proteins in matched samples of 24 astrocytomas WHO grade II (LGA) and 10 GBM (10 primary and 4 secondary GBM). Tumor grade, recurrence of disease, and patient's survival was correlated with protein expression. mRNA expression of the checkpoint proteins MAD1, MAD2, MAD3, BUB1, BUBR1, BUB3, and MPS1 was analyzed by semiquantitative and quantitative RT-PCR in U87, U251, and U343 cells. Mitotic checkpoint function was examined in these cells under different oxygen concentrations (21%, 5%, and 1% O₂) by FACS analysis after Nocodazole (0.075µM, 1µM, and 3µM) induced spindle damage.

Results: MAD2 is a key regulator of the mitotic spindle checkpoint. Its expression was very variable in samples from different patients and there was a tendency for prolonged survival of GBM patients displaying lower MAD2-expression. However, whereas there was high expression of this factor in LGA, its expression was significantly reduced in GBM. In addition, BUB1 was upregulated concomitantly with increasing WHO grading and MPS1 was overexpressed by tumor tissue. Such alterations may weaken the checkpoint function. It has been shown that high-grade gliomas are highly hypoxic tumors. Therefore, we tested the checkpoint function under hypoxic cell culture conditions. Severe hypoxia (1% O₂) revealed a weakened mitotic checkpoint in U87, U251, and U343 cells. In contrast to normoxic conditions, cells continued to proliferate even if spindle damage was induced by Nocodazole. The degree of aneuploidy increased under these conditions.

Conclusions: Alterations of mitotic checkpoint protein expression like MAD2, BUB1, and MPS1 are observed in human malignant gliomas and may participate in a disturbed checkpoint function. Hypoxia escalates the effect of checkpoint malfunction to aneuploidy. Normalization of checkpoint protein expression and O₂-levels could stabilize the chromosomal content, activate the mitotic checkpoint under radiation and chemotherapy, and slow down tumor growth rates.