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

Lova Rakotomalala,Leo Studach,Wen-Horng Wang,Gerald Gregori,Ronald L Hullinger,Ourania Andrisani

doi:10.1074/jbc.m802751200

Lova Rakotomalala, Leo Studach + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m802751200

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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 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.

Highlights

Fifth decade [1] by an unknown mechanism
hepatitis B virus (HBV) pX Induces Polyploidy—Expression of pX in the immortalized mouse hepatocyte 4pX-1 cell line [14] induces accelerated cell cycle progression, unscheduled S phase entry followed by a transient S phase pause, activation of the G2/M checkpoint, and eventual progression through the cell cycle [12]
To understand how pX causes these chromosomal abnormalities, the tetracycline-regulated pX-expressing 4pX-1 cell line was synchronized by growth factor withdrawal as described [14]; to re-enter the cell cycle, 4pX-1 cells were first treated for 10 h with 10% fetal calf serum followed by removal of tetracycline for 10 h to allow pX expression; nocodazole was added for the last 6 h of cell growth (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Cell lines 4pX-1 [14] and 4pX-1-p53kd [15] were used. pX expression was initiated by removal of tetracycline [14]. Flow Cytometry and Live Cell Sorting— 4pX-1 and 4pX-1p53kd cell lines were growth factor-deprived for 18 h as described [14] and subsequently grown for 10 h with 10% fetal calf serum followed by removal of tetracycline for an additional 10 h; nocodazole (250 ng/ml) was added for the last 6 h. Cells were washed 3 times with PBS and incubated in blocking buffer containing PBS and 10% goat serum (Sigma) for 30 min followed by a 2-h incubation with BrdUrd-fluorescein isothiocyanate-conjugated antibody (1:300) and propidium iodide (75 mg/ml). Double Thymidine Block— 4pX-1 and 4pX-1-gemininkd cell lines at 20% confluence were grown for 19 h in growth medium [14] containing 5 ␮g/ml tetracycline and 2 mM thymidine (Sigma) followed by an additional incubation for 9 h in the same growth medium without thymidine.

WCE isolated from synchronized

RESULTS

DISCUSSION