The Consequence of Centrosome Anomalies after Irradiation

Increased numbers of centrosomes after irradiation can contribute to the formation of multipolar spindles [26, 27]. It is likely that multipolar spindles may lead, in most cases, to loss of cell viability through lethal mitotic events. However, the exact fate of cells containing multipolar spindles after irradiation is uncertain. To address this issue, we treated MIA PaCa-2 pancreatic cancer cells with 10 Gy y-radiation and the mitotic cells were collected after 24 h by the method of mitotic shake-off [27]. Staining of these mitotic cells with an anti-y-tubulin antibody revealed that over 60 % of them displayed multiple centrosomes which were dispersed chaotically throughout the condensed chromosomes (Figure 17.3A). The cells were then re-plated in culture dishes and incubated with complete medium. After 24 h, we found that ~ 80% of the reseeded cells turned into giant cells containing multiple nuclear fragments of various sizes and shapes (Figure 17.3B and C). A prolonged culture of these multinucleated cells for 7 days resulted in cell swelling, membrane blebbing, and detachment from the culture dishes in most cells. We have also observed a positive correlation between the fraction of cells with multiple centrosomes and the fraction with mitotic cell death characterized by the appearance of multi- or micronucleated cells after irradiation. To determine the effect of centrosome aberrations on radiation-induced cell killing we trans-fected p21, a Cdk inhibitor known to block centrosome duplication [55], into cells before irradiation. Forced expression of p21 by adenovirus gene transfer inhibited the induction of multiple centrosomes and partially prevented cells from undergoing cell death after irradiation. Collectively, these findings indicate that radiation-induced centrosome anomalies may give rise to the multinucleated phenotype, which eventually results in cell death.

Alternatively, it is possible that cells containing abnormally high number of cen-trosomes could be eliminated through the apoptotic pathway. Because the centro-some is involved in multiple signal transduction pathways and interacts with a number of regulatory proteins [70], it could play an important role in apoptosis and its dysregulation could trigger apoptotic signaling [71]. Evidence supporting a link between centrosomes and apoptosis comes from studies that report the localization of several proteins involved in the regulation of apoptosis, including p53 and poly(ADP-ribose) polymerase (PARP), to centrosomes [42, 72-74]. Sandal and coworkers have further demonstrated that Irod/Ian5, a protein also localized to centrosomes, functions as an inhibitor of y-radiation- and okadaic acid-induced

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