Hepatitis B Virus X Protein Increases the Cdt1-to-Geminin Ratio Inducing DNA Re-replication and Polyploidy

Abstract

Hepatitis B virus X protein (pX) is implicated in hepatocellular carcinoma pathogenesis by an unknown mechanism. Employing the tetracycline-regulated pX-expressing 4pX-1 cell line, derived from the murine AML12 hepatocyte cell line, we demonstrate that pX induces partial polyploidy (>4N DNA). Depletion of p53 in 4pX-1 cells increases by 5-fold the polyploid cells in response to pX expression, indicating that p53 antagonizes pX-induced polyploidy. Dual-parameter flow cytometric analyses show pX-dependent bromodeoxyuridine (BrdUrd) incorporation in 4pX-1 cells containing 4N and >4N DNA, suggesting pX induces DNA re-replication. Interestingly, pX increases expression of endogenous replication initiation factors Cdc6 and Cdtl while suppressing geminin expression, a negative regulator of rereplication. In comparison to a geminin knockdown 4pX-1 cell line used as DNA re-replication control, the Cdt1/geminin ratio is greater in 4pX-1 cells expressing pX, indicating that pX promotes DNA re-replication. In support of this conclusion, pX-expressing 4pX-1 cells, similar to the geminin knockdown 4pX-1 cells, continue to incorporate BrdUrd in the G 2 phase and exhibit nuclear Cdc6 and MCM5 co-localization and the absence of geminin. In addition, pX expression activates the ATR kinase, the sensor of DNA re-replication, which in turn phosphorylates RAD17 and H2AX. Interestingly, phospho-H2AX-positive and BrdUrd -positive cells progress through mitosis, demonstrating a link between pX-induced DNA re-replication and polyploidy. Our studies highlight a novel function of pX that likely contributes to hepatocellular carcinoma pathogenesis. Chronic hepatitis B virus (HBV)4 infection results in the development of hepatocellular carcinoma (HCC) by the fourth or fifth decade (1) by an unknown mechanism. In HBV-mediated HCC the rate of chromosomal aberrations is significantly increased in comparison to HCC associated with other risk factors (2-4). However, the mechanism by which genomic changes initiate HCC development is not yet understood (5-7). Herein, employing the HBV X protein (pX) as the oncogenic signal, we investigate whether pX expression induces chromosomal abnormalities, resulting in HCC pathogenesis.The link between HBV-mediated HCC and pX is derived both from clinical evidence (8) as well as animal and cell culture transformation studies (9). Specifically, integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion, with most tumors displaying sustained expression of pX (8). Importantly, pX, which is essential for the viral life cycle (10), is a multifunctional protein inducing activation of the cellular mitogenic ras-raf-MAPK, c-Jun NH 2 -terminal kinase, and p38MAPK pathways (11) and transcription of select viral and cellular genes (9). These pX activities deregulate cellular gene expression, resulting either in unscheduled cell cycle progression (12) or apoptosis (13), depending on the growth conditions. Specifically, pX expression sensitizes the less-differentiated 4pX-1 hepatocyte cell ...

HBx Protein of Hepatitis B Virus Promotes Reinitiation of DNA Replication by Regulating Expression and Intracellular Stability of Replication Licensing Factor CDC6

“…This surplus CDC6 can participate in licensing of origins in quiescence and early G 1 phase, which may induce untimely initiation of DNA replication. In agreement with this observation, it has been reported that HBx-expressing murine AML12 hepatocytes overexpress CDC6 and Cdt1 origin licensing factors and exhibit polyploidy (29).…”

“…Despite ever increasing data proving that HBx adversely affects cell cycle regulatory proteins and causes DNA replication defects, there is no direct evidence linking viral HBx with DNA replication. Further, the regulation of DNA replication proteins such as CDC6 and Cdt1 by HBx is also poorly understood (29).…”

Polo-like Kinase 1 Activated by the Hepatitis B Virus X Protein Attenuates Both the DNA Damage Checkpoint and DNA Repair Resulting in Partial Polyploidy

“…In optimal growth factor conditions, pX-expressing cells do not undergo apoptosis but instead exhibit accelerated and unscheduled S phase entry, activation of the DNA damage checkpoint, and eventual progression to mitosis (49). Interestingly, in optimal growth conditions, pX expression promotes DNA re-replication-induced DNA damage by enhancing expression of replication initiation factors Cdt1 and Cdc6, while inhibiting expression of geminin, the negative regulator of re-replication (52). Intriguingly, despite DNA re-replicationinduced DNA damage, these pX-expressing hepatocytes proceed through mitosis, propagate damaged DNA, and generate daughter cells that are partially polyploid (Ͼ4N DNA) (52).…”

“…Interestingly, in optimal growth conditions, pX expression promotes DNA re-replication-induced DNA damage by enhancing expression of replication initiation factors Cdt1 and Cdc6, while inhibiting expression of geminin, the negative regulator of re-replication (52). Intriguingly, despite DNA re-replicationinduced DNA damage, these pX-expressing hepatocytes proceed through mitosis, propagate damaged DNA, and generate daughter cells that are partially polyploid (Ͼ4N DNA) (52). Partial polyploidy induced by pX results in oncogenic transformation (40).…”

DNA replication licensing control and rereplication prevention

“…Perturbation of the balance between Cdt1 and geminin by HBX may be a direct cause of DNA rereplication in this case. Although the authors did not address how HBX deregulates the licensing factors, the significantly altered mRNA levels of Cdt1, Cdc6 and geminin suggest that HBX may exert its effect by regulating transcription of these genes (Rakotomalala et al, 2008). Alternatively, the fact that HBX is able to interact with Ddb1, a common linker protein in the CUL4 ubiquitin complex, and disrupt CRL4…”