Abstract
Centrosomal p53 has been described for three decades but its role is still unclear. We previously reported that, in proliferating human cells, p53 transiently moves to centrosomes at each mitosis. Such p53 mitotic centrosome localization (p53-MCL) occurs independently from DNA damage but requires ATM-mediated p53Ser15 phosphorylation (p53Ser15P) on discrete cytoplasmic p53 foci that, through MT dynamics, move to centrosomes during the mitotic spindle formation. Here, we show that inhibition of p53-MCL, obtained by p53 depletion or selective impairment of p53 centrosomal localization, induces centrosome fragmentation in human nontransformed cells. In contrast, tumor cells or mouse cells tolerate p53 depletion, as expected, and p53-MCL inhibition. Such tumor- and species-specific behavior of centrosomal p53 resembles that of the recently identified sensor of centrosome-loss, whose activation triggers the mitotic surveillance pathway in human nontransformed cells but not in tumor cells or mouse cells. The mitotic surveillance pathway prevents the growth of human cells with increased chance of making mitotic errors and accumulating numeral chromosome defects. Thus, we evaluated whether p53-MCL could work as a centrosome-loss sensor and contribute to the activation of the mitotic surveillance pathway. We provide evidence that centrosome-loss triggered by PLK4 inhibition makes p53 orphan of its mitotic dock and promotes accumulation of discrete p53Ser15P foci. These p53 foci are required for the recruitment of 53BP1, a key effector of the mitotic surveillance pathway. Consistently, cells from patients with constitutive impairment of p53-MCL, such as ATM- and PCNT-mutant carriers, accumulate numeral chromosome defects. These findings indicate that, in nontransformed human cells, centrosomal p53 contributes to safeguard genome integrity by working as sensor for the mitotic surveillance pathway.
Highlights
Numeral and structural defects of the centrosomes are prevalent in cancer and are thought to contribute to tumorigenesis by promoting abnormal mitotic spindles and chromosomal instability[1] or increasing tumor cells invasiveness[2]
We show that inhibition of p53 mitotic centrosome localization (p53-MCL) results in centrosome fragmentation and cell death in nontransformed human cells, but not in mouse cells and tumor cells, and that centrosomal p53 works as sensor for the mitotic surveillance pathway
10 μm. ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 but only a mild centrosome amplification (7.5% in CTRi vs. 15% in CEP131-depletion by RNAi (CEP131i)) as previously reported[36]. These results show that inhibition of p53-MCL induces centrosome fragmentation in nontransformed human cells but not in tumor cells (Fig. 3d)
Summary
We previously described p53-MCL in hemopoietic LCLs and PBMCs by double immunofluorescence (IF) with anti-p53 and anti-γ-tubulin antibodies (Abs)[23,29,30]. Acute depletion of p53 induces centrosome fragmentation in nontransformed human cells we attempted to inhibit p53-MCL through different independent strategies and analyzed the effects on centrosome number and structure by double IF for γtubulin and centrin-2 (Fig. 2a). B U2OS cells were transfected with the empty vector (ctr) and the vector expressing the HSPA9p and analyzed as described in (a) In these cells, p53-MCL was even more significantly reduced than in HFs, but no sign of centrosome fragmentation was detected. Comparable results, i.e., accumulation of p53 foci in response to centrinone, were obtained with 53BP1ΔRPE1 cells in which 53BP1 was knocked out by CRISPR/Cas[9] (Supplementary Fig. 4b) These results indicate that centrosome-loss leaving p53 orphan of its mitotic centrosomal localization, promotes the formation of discrete foci of p53Ser15P that, in turn, allow the recruitment of 53BP1 foci that triggers the mitotic surveillance pathway (Fig. 7d). An increase of aneuploid metaphases were observed in those cells in which p53-MCL was defective, including RPE1 cells expressing HSPA9p, further supporting a link between p53-MCL and the mitotic surveillance pathway
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