Hepatitis B virus X protein increases the Cdt1-to-geminin ratio inducing DNA re-replication and polyploidy.
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 G2 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 high-light a novel function of pX that likely contributes to hepatocellular carcinoma pathogenesis.
Rakotomalala
Lova
L
Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Studach
Leo
L
Wang
Wen-Horng
WH
Gregori
Gerald
G
Hullinger
Ronald L
RL
Andrisani
Ourania
O
D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry*; D002460:Cell Line; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005434:Flow Cytometry / Q000379:methods; D064248:Geminin; D022781:Hepatocytes / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008954:Models, Biological; D015739:Nocodazole / Q000494:pharmacology; D009687:Nuclear Proteins / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011123:Polyploidy; D015534:Trans-Activators / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D064248:Geminin; C577997:Gmnn protein, mouse; D009687:Nuclear Proteins; C498645:Ris2 protein, mouse; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D004247:DNA; D015739:Nocodazole