Abstract
Hepatitis B virus X protein (pX), implicated in hepatocarcinogenesis, induces DNA damage because of re-replication and allows propagation of damaged DNA, resulting in partial polyploidy and oncogenic transformation. The mechanism by which pX allows cells with DNA damage to continue proliferating is unknown. Herein, we show pX activates Polo-like kinase 1 (Plk1) in the G(2) phase, thereby attenuating the DNA damage checkpoint. Specifically, in the G(2) phase of pX-expressing cells, the checkpoint kinase Chk1 was inactive despite DNA damage, and protein levels of claspin, an adaptor of ataxia telangiectasia-mutated and Rad3-related protein-mediated Chk1 phosphorylation, were reduced. Pharmacologic inhibition or knockdown of Plk1 restored claspin protein levels, Chk1 activation, and p53 stabilization. Also, protein levels of DNA repair protein Mre11 were decreased in the G(2) phase of pX-expressing cells but not with Plk1 knockdown. Interestingly, in pX-expressing cells, Mre11 co-immunoprecipitated with transfected Plk1 Polo-box domain, and inhibition of Plk1 increased Mre11 stability in cycloheximide-treated cells. These results suggest that pX-activated Plk1 by down-regulating Mre11 attenuates DNA repair. Importantly, concurrent inhibition of Plk1, p53, and Mre11 increased the number of pX-expressing cells with DNA damage entering mitosis, relative to Plk1 inhibition alone. By contrast, inhibition or knockdown of Plk1 reduced pX-induced polyploidy while increasing apoptosis. We conclude Plk1, activated by pX, allows propagation of DNA damage by concurrently attenuating the DNA damage checkpoint and DNA repair, resulting in polyploidy. We propose this novel Plk1 mechanism initiates pX-mediated hepatocyte transformation.
Highlights
Polo-like kinase 1 (Plk1) Is Activated in G2 Phase of Immortalized pX-expressing Hepatocytes—Elevated protein levels of Plk1 occur in many human cancers [33, 34, 60], including HBV-mediated liver cancer [35]
The degradation of claspin and phosphorylation of Chk1 were restored following treatment with MG132 (Fig. 2C). These results demonstrate that in the G2 phase of pX-expressing cells, claspin is targeted for degradation by Plk1 despite the presence of DNA damage. To verify by another approach that activation of Plk1 by pX attenuates the DNA damage checkpoint, we examined the status of the inhibitory phosphorylation of residue Tyr-15 of mitotic kinase Cdc2 [65]. 10 h after release from double thymidine block (DTB), pXexpressing cells exhibited the following: 1) ␥-H2AX immunostaining indicative of DNA damage; 2) minimal p-H3 immunostaining as a marker of mitotic entry, and 3) phosphorylation of Tyr-15 of Cdc2 (Fig. 3A)
Plk1 Activated by pX Induces Polyploidy in Immortalized Hepatocytes—Because Plk1 activity increases the number of pX-expressing cells with DNA damage entering mitosis (Figs. 7 and 8), we examined whether pX-mediated Plk1 activation results in polyploidy
Summary
Overexpression of Chk or depletion of Plk delayed exit from the DNA damage checkpoint [21], indicating that Plk has a role in checkpoint adaptation in mammalian cells. Upon completion of DNA repair, Plk mediates recovery from the DNA damage checkpoint by inducing proteasomal degradation of claspin [13]. Our recent studies have shown that Topors [29, 30], following phosphorylation by Plk, suppresses p53 sumoylation in the absence of DNA damage, inducing instead ubiquitination and proteasomal degradation of p53 [31]. In HPV-16 E7-expressing cells, claspin is degraded despite DNA damage; these cells display elevated levels of Plk protein [38]. The mechanism by which Plk contributes to pX-mediated oncogenic transformation is not yet understood
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