Kinetics of DHBV RNA transcription, DNA replication and cccDNA formation in DSL212 cells.

<p>DSL212 cells were seeded in 6-well plates and cultured in the presence of tetracycline (1 μg/ml) until cell monolayers became confluent. Cells were then cultured in media without tetracycline and harvested at the indicated days since the removal of tetracycline. Total cellular RNA, cytoplasmic core DNA and total cellular Hirt DNA were extracted and analyzed by Northern and Southern blot hybridization, respectively. (A) For viral RNA analysis, each lane was loaded with 5 μg of total RNA. pgRNA, pregenomic RNA; sRNA, mRNAs specifying the two envelope proteins. Ribosomal RNA (28S and 18S) served as loading controls. For DHBV core DNA (B) and Hirt DNA (C) analysis, each lane represents the amount of viral DNA extracted from one half of cells in a well of 6-well plate. RC, relaxed circular DNA; DSL, double stranded linear DNA; SS. single stranded DNA; cccDNA, covalently-closed circular DNA. Unit length of linear DHBV DNA (lane 10) and core or Hirt DNA extracted from dstet5 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043270#pone.0043270-Guo4" target="_blank">[40]</a> cultured in the absence of tetracycline for 8 days (lane 11) served as controls.</p

DHBV DNA replication and cccDNA formation in a panel of cells lines that are defective in NHEJ DNA repair pathway.

<p>Cell lines used in this experiment are CHO-K1 and its derived cell line Xrs-5 harboring defective gene of the p86 subunit of the Ku autoantigen, four human fibroblast cell lines GM16133, GM16135, GM16147 and GM16089 that are defective in XRCC1, the catalytic subunit of DNA-PK, XRCC4 and ligase IV, respectively. The cells are infected with AdDHBV1S at a MOI of 10 and infected cells are harvested on day 3 post infection. DHBV core-associated (upper panel) and Hirt DNA (lower panel) were extracted and analyzed by Southern blot hybridization.</p

Subcellular distribution of DHBV DNA replication intermediates in HepG2 cells.

<p>(A) DSL212 cells were cultured in the absence of tetracycline for 6 days. Cytoplasm and nuclei were fractionated with QIAgen Qproteome Cell Compartment Kit by following the manufacturer's directions. DHBV core-associated DNA and Hirt DNA were extracted from whole cell, cytoplasm and nuclear fractions were analyzed by Southern blot assay. (B) DHBV core-associated DNA and Hirt DNA were extracted from whole cell, cytoplasm and nuclear fractions of HepG3 cells (a HepG2-derived stable cell line containing an integrated DHBV head-to-tail unit-length DNA dimer) were analyzed by Southern blot assay.</p

DHBV DNA replication and cccDNA formation in a panel of human cell lines infected by AdDHBV1S.

<p>The indicated cell lines were seeded onto 6-well plate and infected with AdDHBV1S at a MOI of 10. Five days post infection, DHBV core-associated (A) and Hirt DNA (B) were extracted and analyzed by Southern blot hybridization. Each lane represents the amount of viral DNA extracted from one half of cells in a well of 6-well plate.</p

Role of Ku80 in DHBV cccDNA formation from dslDNA precursors.

<p>(A) CHO-K1 and Xrs-5 cells were transfected with plasmid DHBV-1S or 1Sdsl-3, respectively. On the day five post transfection, the cells were harvested and DHBV core-associated (upper panel) and Hirt DNA (lower panel) were extracted and analyzed by Southern blot hybridization. Unit-length DHBV genomic DNA served as a molecular weight control. (B) Xrs-5 cells were co-transfected with plasmid 1Sdsl-3 and vector plasmid pUC119 (lane 2), plasmid expressing wild-type Ku80 or Ku80-YFP fusion protein, respectively. The cells were harvested on day 5 post transfection. Core (upper panel) and Hirt (middle panel) DNA were detected by Southern blot hybridization. Ku80 and Ku80-YFP expression were detected by Western blot assay (lower panel). β-actin served as a loading control.</p

Schematic representation of cccDNA biosynthesis pathways from rc and dslDNA.

<p>See text for detailed explanation.</p

Cell Permissiveness on DHBV replication, deproteinization and cccDNA formation.

<p>Note: <i>^a</i> each “<b>+</b>” represents one quarter of quantitative signal for each DHBV DNA species from HepG2 cells by DNA hybridization; <i>^b</i> undetectable by DNA hybridization; <i>^c</i> not determined due to cell death after AdDHBV1S infection.</p

Suppression of AKT Anti-Apoptotic Signaling by a Novel Drug Candidate Results in Growth Arrest and Apoptosis of Hepatocellular Carcinoma Cells

<div><p>Hepatocellular carcinoma (HCC) is the third most common cause of cancer fatalities worldwide, with limited treatment options and five year survival rates of between <5 and 15%. To address this medical need, we conducted a screen of a drug-like small molecule library for HCC-selective cytotoxins. We report here the identification of a disubstituted aminothiazole termed HBF-0079, with remarkable selective toxicity for HCC-derived cell lines versus non-HCC liver lines and most other cancer lines. HBF-0079 caused irreversible growth arrest and apoptosis of the HCC lines Huh7, Hep3B, HepaRG as well as the hepatoblastoma line HepG2, with CC₅₀ values from ∼0.7−7.7 µM, while more than 45 µM was needed to achieve CC₅₀ values for the immortalized normal hepatocyte lines THLE-2 and PH5CH. Of the sixty cancer lines from the National Cancer Institute panel, only five exhibited >50% growth inhibition by HBF-0079. In Huh7 cells, HBF-0079 induced cell cycle arrest in G1 and concomitant apoptosis, and its effects were irreversible after removal of the compound. These observations corroborate a loss of AKT phosphorylation at the mTORC2-targeted residue S473, with concurrent loss of phosphorylation of the mTORC1 targets SK6 and 4EBP1 in Huh7 but not PH5CH cells. Finally, growth of Hep3B-derived tumors in a murine xenograft model was significantly repressed by the compound through either systemic or intratumoral administration of formulated HBF-0079. The potential for development of this drug candidate is discussed.</p> </div

HBF-0079 modulates cell-growth and anti-apoptotic signaling through AKT and mTOR.

<p>Log-phase Huh7 were cultured for 3 days in the absence or presence of HBF-0079 at indicated concentrations, or 0.5% DMSO, lysates were analyzed by Western blotting using Abs specific for (A) total Akt, Akt phosphorylated at S473, Akt phosphorylated at T308, or β-Actin; (B) mTOR targets 4E-BP1 phosphorylated at T37/T46, S6K phosphorylated at S371/394, total PKCα, PKCα phosphorylated at S657, and β-Actin; (C) β-catenin and β-Actin; (D) Log-phase PH5CH cells were treated, cultured and analyzed for AKT, 4E-BP1 and S6K as in (A,B). Details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054595#s2" target="_blank">Methods</a>.</p

Cytotoxicity spectrum of HBF-0079 and the analogue IHVR-04042.

<p>Cells were seeded at sub-confluence, as in text, and cultured for 3 days in the absence and presence of the indicated drug, with concentrations ranging from 0.016 to 50.0 mM. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054595#s2" target="_blank">Methods and materials</a> for details.</p>1<p>Cell line name, and tissue of origin.</p>2<p>The concentration that is cytotoxic to 50% of the cells (CC₅₀) is reported in micromolar value, with standard deviation values as determined in 4–7 experiments.</p