gms | German Medical Science

Objective: MAD2A is a key regulator of the mitotic spindle checkpoint, which controls fidelity of spindle fiber attachment to sister chromatides. Defects in this checkpoint may contribute to chromosomal instability and may be involved in tumorigenesis. Since human malignant gliomas are highly aneuploid, we examined whether there are alterations in MAD2A expression and investigated how such alterations influence checkpoint function and proliferation rates of glioblastoma (GBM) cells.

Methods: Expression of MAD2A was determined by semiquantitative RT-PCR and Western-blotting, respectively, in a panel of 15 astrocytoma WHO II, 15 glioblastoma WHO IV and 3 normal brain biopsies and in an array of GBM cell lines. U251 and U373 GBM cells were transfected either with an MAD2A expression construct for protein overexpression or with specific siRNA for protein knock-down. Spindle checkpoint function was analysed by FACS analysis in presence or absence of the microtubuli damaging agent Nocodazole. Cell-survival and proliferation rates were monitored in real time for several days using the xCELLigence system (Roche).

Results: MAD2A expression was very diverse in glioma tissue samples from different patients, ranging from no detectable protein to very high expression levels. In comparison to untreated cells MAD2A depleted cells showed broadened peaks in FACS analysis. After Nocodazole treatment, which normally induces the mitotic checkpoint, no cell cycle arrest in G2/M phase could be observed and peaks were flattened and less confined compared to control cells. These results were confirmed by real time proliferation measurements. Untreated and MAD2A overexpressing cells arrested after Nocodazole treatment, knock-down cells continued to proliferate. However, MAD2A overexpression led to reduced proliferation rates (Control: 0.026 cycles/h, Overexpression: 0.019 cycles/h).

Conclusions: MAD2A is a key regulator of the mitotic spindle checkpoint in GBM cells. Recently, it has been shown that MAD2A-induced chromosome instability participates in lung tumor recurrence. Therefore, its loss in some GBM tissues could be of disadvantage for the patient, since the mitotic checkpoint is switched off. MAD2A overexpression reduces cell proliferation and may not only be a prognostic marker, but normalization of its expression levels could also be a therapeutic strategy to stabilize the chromosomal content and to slow down tumor growth rates.