IJUP08 - Universidade do Porto

Analysis of the efficiency of the mitotic checkpoints in
glioblastoma cell lines
A. Nascimento 1,3 , E. Logarinho 2 , O. Martins 2 , R. M. Reis 2 and H. Bousbaa 3
1 Faculty of Science, University of Oporto and ICBAS, University of Oporto
2 Life and Health Sciences Research Institute, School of Health Sciences, University of Minho
3 Instituto Superior de Ciências da Saúde – Norte, Centro de Investigação em Ciências da Saúde
(CICS), Grupo de Biologia Molecular e Celular, CESPU
The mitotic checkpoint modulates the timing of anaphase initiation in response to improper
alignment of chromosomes at the metaphase plate. The BUB and MAD gene families
encode proteins which are part of a large multi-protein complex which are believed to be
key components of the checkpoint regulatory pathway [1][1]. Failure of this surveillance
system can lead to genomic instability and could be responsible for the increased incidence
of aneuploidy and possible driving forces in, tumorigenesis [3]. Glioblastomas are the most
common and malignant form of primary adult brain tumors[4]. These tumors are
characterized by marked chromosomal instability, with gains and/or losses of
chromosomes, referred as aneuploidy [6]. In order to get insight to the mechanism that lead
to aneuploidy in glioblastomas, we assessed the efficiency of the mitotic checkpoints and
possible molecular alterations in two glioblastoma cell lines.
To evaluate mitotic arrest efficiency, mitotic index (percentage of viable cells arrested in
mitosis) was determined, in untreated as well as in cell cultures treated with the
microtubule-disrupting drug nocodazole for 16 hours, by cell-rounding under phase
contrast microscopy. The molecular alterations in checkpoint genes were evaluated at the
protein levels by Western blotting analysis. For that, total protein extracts were prepared
from glioblastoma cells and the protein amounts were determined by quantitative
immunoblotting using specific antisera. The protein expression levels were compared to
those in HeLa cells, using the alpha-tubulin levels as reference standards.
Concerning the efficiency of the mitotic checkpoint, glioblastomas showed a low mitotic
index, a lack of mitotic arrest when incubated with nocodazole together with an alteration
in the expression levels of the mitotic checkpoint proteins examined. These results confirm
our suspicion of a deficient mitotic checkpoint in glioblastoma cells which, through
premature onset of anaphase, lead to chromosome mis-segregation. Our finding might
provide an explanation for the chromosomal defects seen in glioblastoma.
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[2] McGowan, C. H. (2003). Regulation of the eukaryotic cell cycle in Progress in Cell Cycle
Research 5: 1-4.
[3] Cahill, D. P., et al. (1998).Mutations of mitotic checkpoint genes in human cancers in Nature,
392(6673), 300-3.
[4] Isaka, T., et al. (2003). Chromosomal variations within aneuploid cancer lines in Journal of
Histochemistry and Cytochemistry, 51(10): 1343-53.
[5] Kleihues P, and Cavenee WK; The WHO classification of tumors of the nervous system,
International Agency for Research on Cancer, 2000.
[6] Draviam, V.M. et al., (2004) Chromosome segregation and genomic stability in Current
Opinion in Genetics and Development 2004 Apr;14(2), 20-5
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