Proteins ZNF198 and SUZ12 are down-regulated in hepatitis B virus (HBV) X protein-mediated hepatocyte transformation and in HBV replication - PubMed (original) (raw)

Proteins ZNF198 and SUZ12 are down-regulated in hepatitis B virus (HBV) X protein-mediated hepatocyte transformation and in HBV replication

Wen-Horng Wang et al. Hepatology. 2011 Apr.

Abstract

Chronic hepatitis B virus (HBV) infection is a major etiologic factor in hepatocellular carcinoma (HCC) pathogenesis, involving effects of chronic liver inflammation and of the weakly oncogenic HBV X protein (pX). pX-mediated hepatocyte transformation requires Polo-like kinase1 (Plk1) activity, but the mechanism is not fully understood. We identified by a genome-wide short hairpin RNA (shRNA) library screen the genes zinc finger, MYM-type 2 (ZNF198) and suppressor of zeste 12 homolog (Drosophila) (SUZ12) whose protein depletion rescues pX-expressing cells from DNA damage-induced apoptosis. ZNF198 and SUZ12 are components of chromatin remodeling complexes and associate with promyelocytic leukemia (PML) nuclear bodies. Knockdown of ZNF198 and SUZ12 by small interfering RNA (siRNA) reduced p53 stability and DNA repair, rescued pX-expressing hepatocytes from DNA damage-induced apoptosis, and increased pX-induced polyploidy and oncogenic transformation, suggesting ZNF198 and SUZ12 have a role in pX-mediated transformation. Interestingly, during pX-mediated transformation the protein but not messenger RNA (mRNA) levels of ZNF198 and SUZ12 progressively decreased, whereas Plk1 levels increased. Inhibition of Plk1 activity restored protein levels of ZNF198 and SUZ12. In addition, transfected Polo-box-domain (PBD) of Plk1 coimmunoprecipitated with ZNF198 and SUZ12, suggesting that these proteins are Plk1 substrates. Elevated Plk1 and reduced protein levels of ZNF198 and SUZ12 were also observed in human liver cancer cell lines derived from HBV-related tumors and in the presence of HBV replication. Importantly, knockdown by siRNA of ZNF198 and SUZ12 enhanced HBV replication.

Conclusion: Reduced protein levels of ZNF198 and SUZ12 and elevated Plk1 occur during pX-mediated hepatocyte transformation in human liver cancer cell lines, as well as during HBV replication, underscoring the significance of these genes both in HBV-mediated HCC pathogenesis and HBV replication. We propose Plk1 activity down-regulates ZNF198 and SUZ12, thereby enhancing both HBV replication and pX-mediated oncogenic transformation.

2011 American Association for the Study of Liver Diseases.

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Figures

Fig. 1

Knockdown of ZNF198 and SUZ12 increases cell survival in conditions of pX-mediated apoptosis. A. Immunoblots of ZNF198, SUZ12, Topors, and PML, using lysates from the respective knockdown cell lines. 4pX-1 cells with stable integration of pGIPZ empty vector were constructed in parallel. B. and C. Lysates from indicated cell lines grown ± pX and doxorubicin (0.5 μg/ml) for 6 h or 2 days, were immunoblotted for p53 in B., and active caspase3 in C. Actin is loading control. D. Quantification of cell survival of indicated cell lines grown ± pX and doxorubicin (0.5 μg/ml) for 6 days.

Fig. 2

Defective DNA repair in ZNF198 and SUZ12 knockdown cells. A. Immunofluorescence microscopy of Mre11 performed as described (45), using the indicated cell lines treated with or without doxorubicin (0.5 μg/ml) for 6 h. 60× magnification. B. EcoRI-linearized pGL2-Luciferase plasmid DNA transfected for 24 h. Luciferase activity quantified per μg of protein.

Fig. 3

Knockdown of ZNF198 and SUZ12 increases pX-induced polyploidy and oncogenic transformation. A. Flow cytometric profiles of indicated cell lines grown ± pX for 16 h; nocodazole (300 nM) was added during the last 6 h as described (13). Arrows indicate chick erythrocytes used for calibration. Quantification of polyploid cells (>4N DNA) from indicated cell lines from three independent flow cytometric experiments is shown in Table I. B. pX-induced polyploid cells were isolated by live cell sorting as described (15) and grown in soft agar for 12 days ± pX. C. Quantification of colonies formed in soft agar from three independent experiments.

Fig. 4

Elevated Plk1 and reduced protein levels of ZNF198 and SUZ12 during pX-mediated transformation and in human liver cancer cell lines. A. Diagram shows the in vitro pX-mediated transformation model previously described in (15). B. and C. Immunoblots of ZNF198, SUZ12 and Plk1, using lysates from B., 4pX-1 cells grown without pX, and P2-P5 cultures grown with pX; and C., indicated human liver cancer cell lines. “Relative intensity” quantified vs. actin. D. Real-time PCR quantification of ZNF198 and SUZ12 mRNA, using RNA isolated from 4pX-1 cells without pX and P1-P5 cultures with pX expression. E. Immunoblots of ZNF198 and SUZ12, using lysates from P4 cultures treated with 500 nM BI 2536 for 0–6 h. F. Immunoblots of ZNF198, SUZ12 and FLAG, using FLAG immunoprecipitates from 4pX-1 cells transfected with PBD-FLAG plasmid DNA ± pX.

Fig. 5

Elevated protein levels of Plk1 and reduced ZNF198 and SUZ12 during HBV replication. A. Quantification of HBV replication by real-time PCR exactly as described (41). (Left) Ct values of serially diluted HBV1.2 ayw plasmid DNA and template DNA from isolated intracellular virions obtained from HepAD38 cells grown ± tetracycline for 5 and 10 days. (Right panel) Quantification of HBV genome equivalents from data shown in left panel. B. Immunoblots of ZNF198, SUZ12, and Plk1 using lysates of HepAD38 grown ± tetracycline for 5, 10, and 20 days. C. PML immunoprecipitates of lysates from B., immunoblotted for ZNF198 and SUZ12. D. and E., PML immunofluorescence microscopy of indicated cell lines (60× magnification).

Fig. 6

siRNA knockdown of ZNF198, SUZ12 and PML enhances HBV replication**. A.** Immunoblots of SUZ12, ZNF198 and PML using HepAD38 cells transfected with indicated siRNAs grown as follows: tetracycline addition for 24 h after siRNA transfection (−HBV replication), followed by tetracycline removal for 1 day (+HBV replication). B. Quantification of HBV genome equivalents using template DNA from isolated intracellular virions from HepAD38 cells transfected with indicated siRNAs, grown ± tetracycline for 1, 5 and 10 days.

Fig. 7

Diagram shows effects of down-regulation of ZNF198 and SUZ12 identified by our studies. We propose Plk1-mediated down-regulation of ZNF198 and SUZ12 links oncogenic transformation and HBV replication.

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Proteins ZNF198 and SUZ12 are down-regulated in hepatitis B virus (HBV) X protein-mediated hepatocyte transformation and in HBV replication - PubMed (original) (raw)