Specific uptake mediated by cell-surface DHCR24.

<p>(<i>a</i>) HCC cell lines (HuH-7, Hep3B, and PLC/PRF/5) and HeLa cells were incubated with 2-152a MAb at 4°C (a temperature that inhibits endocytosis) or 37°C (physiological temperature) for 2 h, and then incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. (<i>b</i>) HuH-7 cells were seeded at a density of 5 × 10³ cells/well in 96-well tissue culture plates. After incubation for 24 h, serial dilutions of 2-152a MAb or mouse IgG were added in the presence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was then assessed using the BrdU ELISA assay kit. Average viability was calculated relative to the viability of untreated cells, which was set at 100%. (<i>c</i>) HeLa, Hep3B, and PLC/PRF/5 cells were treated with 2-152a MAb or mouse IgG (10 μg/mL) in the presence or absence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was determined using a BrdU ELISA assay kit. Percent viability was calculated relative to the viability of untreated cells, which was set at 100%. *, <i>p</i> < 0.05.</p

3β-Hydroxysterol Δ24-Reductase on the Surface of Hepatitis C Virus-Related Hepatocellular Carcinoma Cells Can Be a Target for Molecular Targeting Therapy

<div><p>In our previous study, we demonstrated that 3β-hydroxysterol Δ24-reductase (DHCR24) was overexpressed in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), and that its expression was induced by HCV. Using a monoclonal antibody against DHCR24 (2-152a MAb), we found that DHCR24 was specifically expressed on the surface of HCC cell lines. Based on these findings, we aimed to establish a novel targeting strategy using 2-152a MAb to treat HCV-related HCC. In the present study, we examined the antitumor activity of 2-152a MAb. In the presence of complement, HCC-derived HuH-7 cells were killed by treatment with 2-152a MAb, which was mediated by complement-dependent cytotoxicity (CDC). In addition, the antigen recognition domain of 2-152a MAb was responsible for the unique anti-HCV activity. These findings demonstrate the feasibility of using 2-152a MAb for antibody therapy against HCV-related HCC. In addition, surface DHCR24 on HCC cells exhibited a functional property, agonist-induced internalization. We showed that 2-152a MAb-mediated binding of a cytotoxic agent (a saponin-conjugated secondary antibody) to surface DHCR24 led to significant cytotoxicity. This suggests that surface DHCR24 on HCC cells can function as a carrier for internalization. Therefore, surface DHCR24 could be a valuable target for HCV-related HCC therapy, and 2-152a MAb appears to be useful for this targeted therapy.</p></div

152a ChAb can bind to surface DHCR24 and shows anti-HCV activity.

<p>(<i>a</i>) HuH-7 (1 × 10⁶) cells were incubated with the light or heavy chain of the chimeric Ig (152a Ch-L, 152a Ch-H) or 152a ChAb (1 μg/mL, respectively) at 4°C for 2 h, and then with an Alexa Fluor 488-conjugated goat anti-human IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. (<i>b</i>) HuH-7 (1 × 10⁶) cells were incubated with 152a scFv-hIgG1-Fc at 4°C for 2 h, and then incubated with Alexa Fluor 488-conjugated goat anti-human IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. (<i>c</i>) Subgenomic HCV replicon FLR3-1 cells were plated in a 96-well plate at a density of 5 × 10³ cells/well and allowed to adhere overnight. Then, the supernatant was removed, and the cells were treated with the light or heavy chain of the chimeric Ig (152a Ch-L, 152a Ch-H) or 152a ChAb (1 μg/mL, respectively) for 72 h. HCV replication was evaluated by measuring luciferase activity using the Bright-Glo Luciferase Assay System. *, <i>p</i> < 0.05. (<i>d</i>) Simultaneously, the viability of FLR3-1 cells was evaluated by measuring the absorbance (OD at 450 nm) using the WST-8 Cell Counting Kit. Experiments were performed 3 times with triplicate wells.</p

Complement-dependent cytotoxicity (CDC) induced by 2-152a MAb.

<p>HeLa, HepG2, and HuH-7 cells (1 × 10⁶) were incubated with 2-152a MAb or mouse IgG (final concentration, 1 or 10 μg/mL) in the presence of guinea pig complement (the complement:cell ratio was 1:8 for HeLa and HepG2 and 1:20 for HuH-7) for 30 minutes at 30°C. Cell viability was measured using the WST-8 cell counting kit, and cell killing was calculated by comparing death in the experimentally treated cells with that in the untreated cells. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01.</p

Construction of a chimeric antibody and scFv consisting of the 2-152a MAb antigen-binding domain and the human IgG constant domain.

<p>(<i>a</i>) The 2-152a VL and VH cDNAs were isolated from hybridoma #2-152a and cloned into pFUSE2ss-CLIg-hk and pFUSEss-CHIg-hG1, respectively. HEK293 cells were co-transfected with the expression vector harboring the chimeric Ig (152a Ch-L; pFUSE2ss-152aVL-CLIg-hk, 152a Ch-H; pFUSEss-152aVH-CHIg-hG1). The 152a Chimera Ab or chimeric Ig (152a Ch-L, 152a Ch-H) was secreted into the culture medium and then purified from the culture medium by using protein A/G/L sepharose. (<i>b</i>) The 152a scFvfragments (152a VL-VH, 152a VH-VL) were constructed by SOE-PCR and cloned into pFUSE-hIgG1e4-Fc2. HEK293 cells were transfected with the 152a scFv-hIgG1-Fc expression vectors (pFUSE-scFv152a(VLVH)-hIgG1-Fc and pFUSE-scFv152a(VHVL)-hIgG1-Fc). 152a scFv-hIgG1-Fc fusion protein was secreted into the culture medium, and then the protein was purified from the culture medium by using protein A/G/L sepharose. (<i>c</i>) Schematic diagram of scFv152a-hIgG1-Fc. The scFv fragments (152a VL-VH or 152a VH-VL) derived from 2-152a MAb were fused to the Fc portion of human IgG1.</p

Overexpression of DHCR24 on the cell surface of HCC cell lines was decreased by treatment with U18666A and cyclosporin A.

<p>(<i>a</i>) Cell lysates of each cell line (containing 50 μg of protein) were separated by 10% SDS-PAGE and analyzed by western blotting with 2-152a MAb and an anti-actin MAb. Normal hepatic cell lines: NKNT, and TTNT. HB-derived cell line: HepG2. HCC-derived cell lines: HuH-7, Hep3B, and PLC/PRF/5. (<i>b</i>) HuH-7 cells were treated with U18666A (final concentration, 1 μM) for 48 h, and then the surface expression of DHCR24 was analyzed by flow cytometry. (<i>c</i>) HuH-7 cells were treated with cyclosporin A (final concentration, 5 or 10 μM) or solvent (cremophor) for 48 h, and then the surface expression of DHCR24 was analyzed by flow cytometry.</p

Specific uptake mediated by cell-surface DHCR24.

<p>(<i>a</i>) HCC cell lines (HuH-7, Hep3B, and PLC/PRF/5) and HeLa cells were incubated with 2-152a MAb at 4°C (a temperature that inhibits endocytosis) or 37°C (physiological temperature) for 2 h, and then incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. (<i>b</i>) HuH-7 cells were seeded at a density of 5 × 10³ cells/well in 96-well tissue culture plates. After incubation for 24 h, serial dilutions of 2-152a MAb or mouse IgG were added in the presence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was then assessed using the BrdU ELISA assay kit. Average viability was calculated relative to the viability of untreated cells, which was set at 100%. (<i>c</i>) HeLa, Hep3B, and PLC/PRF/5 cells were treated with 2-152a MAb or mouse IgG (10 μg/mL) in the presence or absence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was determined using a BrdU ELISA assay kit. Percent viability was calculated relative to the viability of untreated cells, which was set at 100%. *, <i>p</i> < 0.05.</p

Double immunofluorescence localisation of B220 (Green) and NF-κB p65 (Red) in HCV-Tg mice and the fractionation analysis of mouse tissues.

<p><b>A</b>: Co-localisation of NF-κB p65 immunoreactivity with B220 is indicated by arrows. (a–b) Cells double-positive for B220 and NF-κB in the control mouse (CD19cre). (c–d) Cells double-positive for B220 and NF-κB in the asymptomatic HCV-Tg mouse (RzCD19cre). (e–f) Cells double-positive for B220 and NF-κB in the lymphomatous HCV-Tg mouse (RzCD19cre). <b>B</b>: Quantitative analysis of the ratio of double-positive cells among B220-positive cells in each HCV-Tg mouse. Bar graph indicates the percentage of cells with NF-κB p65 nuclear translocation in B220-positive cells. <b>C</b>: Bar graph shows the ratio of double-positive cells within the B220-positive cells in normal, asymptomatic and lymphomatous HCV-Tg mice. Ho: Hoechst33342 Data are presented as means ± S.E., * P<0.05, ** P<0.01, *** P<0.001. <b>D</b>: Western blot analysis: tissues from the spleen of controls (224–2, 3) or HCV-Tg mice without BCL (217–3, 224–4, 232–3) or with BCL (56–5, 69–5) were fractionated into nuclear and cytoplasmic fractions. NF-κB p50 and p65 were detected by antibodies. Relative ratios of quantitation by imager are indicated. GAPDH was detected as a loading control of the cytoplasmic fraction.</p

The expression of genes involved in oncogenic pathways associated with BCL.

<p><b>A</b>: Highly modified gene signals in B cell lymphoma in RzCD19Cre mice BCL vs. B cells in RzCD19Cre male (Pair 1) or female (Pair 3) mice (left), and the genes modified by HCV expression in B cells in male (Pair 2) or female (Pair 4) (right). Red indicates the relative enhancement of the expression ratio of the processed signal (Test/Control, 532/635), and green indicates the relative reduction of expression. <b>B</b>: Quantification of Fos mRNA in HCV-, HCV+ B cells and HCV-Tg BCL in mice (numbers of individual mice were indicated) by quantitative RT-PCR. Fos mRNA was normalised against 18S rRNA, and the relative ratio was calculated. Vertical bars indicate S.D. <b>C</b>: Quantification of C3 mRNA in HCV-, HCV+ B cells and HCV-Tg BCL in mice. C3 mRNA was normalised against 18S rRNA, and relative ratio was calculated. Vertical bars indicate S.D. <b>D</b>: Quantification of LT βR mRNA in HCV-, HCV+ B cells and HCV-Tg BCL in mice by quantitative RT-PCR. RNA copies per total RNA (μg) were indicated and vertical bars indicate S.D.</p

Mice subjected to microarray analysis.

<p>*BCL: B cell lymphoma; ⁺4EBP(+/−): heterozygous knockout of 4E-BP1 gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091373#pone.0091373-TsukiyamaKohara2" target="_blank">[73]</a>.</p