ABSTRACT
UNLABELLED: Chronic hepatitis B virus (HBV) infection is linked to development of hepatocellular carcinoma (HCC). The HBV X protein (pX) is implicated in HCC pathogenesis acting as a weak oncogene or a cofactor in hepatocarcinogenesis. pX induces DNA re-replication, DNA damage, and partial polyploidy in a poorly differentiated, immortalized hepatocyte cell line. In this study we employed sorted, pX-induced polyploid cells to investigate their growth and oncogenic transformation potential over the course of 70 cell doublings. Immediately after live cell-sorting, nearly 40% of pX-induced polyploid cells undergo apoptosis, whereas the surviving cells exhibit proliferation sensitive to p53. After 40 cell generations the pX-expressing polyploid cultures exhibit loss of p53 function and become growth factor- and anchorage-independent, indicative of oncogenic transformation. The pX-induced polyploid cultures in the course of 70 cell generations undergo progressively increasing DNA damage, propagate damaged DNA to daughter cells, and display increased expression of a cluster of proliferation genes shown to be elevated in human HCC, including HBV-HCC. One of these genes is the mitotic kinase Polo-like kinase 1 (Plk1). Oncogenic transformation is suppressed in the absence of pX expression, and significantly, by inhibition of Plk1. These results identify Plk1 as crucial in pX-mediated oncogenic transformation. CONCLUSION: Partial polyploidy induced by pX is not immediately associated with oncogenic transformation. Continued DNA damage for 40 cell generations is reproducibly associated with loss of p53 function, enhanced expression of Plk1, and oncogenic transformation. Because Plk1 expression is also elevated in HBV-HCC tumors, this in vitro cellular model simulates liver cancer progression and pathogenesis in chronic HBV patients. Inhibition of Plk1 activity suppresses pX-mediated oncogenic transformation, identifying Plk1 as a promising therapeutic target for HBV-mediated HCC.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Cell Cycle Proteins/metabolism , Cell Transformation, Neoplastic , Liver Neoplasms/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Animals , Apoptosis , Carcinoma, Hepatocellular/virology , Cell Line , Cell Proliferation , DNA Damage , DNA Replication , Disease Progression , Gene Expression Regulation, Neoplastic , Hepatitis B, Chronic/complications , Liver Neoplasms/virology , Mice , Polyploidy , Tumor Suppressor Protein p53/metabolism , Viral Regulatory and Accessory Proteins , Polo-Like Kinase 1ABSTRACT
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.
