Figure 1.
Prostate cancer-specific expression driven by the DD3 promoter in the presence of WPRE.
A. The luciferase activity of the DD3 promoter in the presence of WPRE is expressed compared to the pGL3-control (activity = 1000). Cells were seeded in 24-well plates. Forty-eight hours after infection with the reporter plasmids pGL3-control or pGL3-DD3-WPRE, the cells were harvested, and the luciferase activity was measured. The activity of pGL3-DD3-WPRE relative to the pGL3-control is presented as the mean ± standard deviation (SD) of three transfections. B. The expression of E1A by Ad.DD3.D55 (without WPRE) and Ad.DD3.D55 in different cell lines was evaluated. α-Tubulin was evaluated as a loading control. C. Crystal violet assay. Cells on 24-well plates were infected with different viruses at the indicated MOIs. Cytotoxicity was observed by crystal violet staining at five days after infection.
Figure 2.
Characterization of Ad.DD3.D55-PTEN.
A. Schematic diagram of Ad.DD3.D55-PTEN. The endogenous promoter of E1A was replaced by the DD3 promoter, and the E1B-55K gene was deleted. The PTEN expression cassette was inserted into Ad.DD3.D55 to construct Ad.DD3.D55-PTEN. ITR, inverted terminal repeat. B. Characterization of Ad-PTEN, Ad.DD3.D55 and Ad.DD3.D55-PTEN expression by Western blot analysis. The expression of PTEN, E1A and E1B-55K by Ad-PTEN, Ad.DD3.D55 or Ad.DD3.D55-PTEN in CL1 cells was evaluated. Ad.WT was used as a positive control. α-Tubulin was evaluated as a loading control. C. Relative mRNA levels of PTEN and E1A in 22RV1 cells infected with Ad-PTEN, Ad.DD3.D55 and Ad.DD3.D55-PTEN. GAPDH was evaluated as an internal reference. The data are presented as the mean ± SD of three separate experiments. D. Western blot analysis of the total and phosphorylated Akt protein levels in CL1 cells infected with Ad.DD3.D55 and Ad.DD3.D55-PTEN. E. Analysis of tumor sections derived from tumors treated with PBS or one of various adenoviruses by hematoxylin and eosin staining and hexon and PTEN immunostaining. Scale bar, 100 µm.
Figure 3.
Prostate cancer-specific cytotoxicity of Ad.DD3.D55-PTEN.
A. MTT assay. The six cell lines indicated above were seeded on 96-well plates and infected with different viruses at an MOI of 10. Cell viability was measured by MTT assays at 0, 24, 48, 72, 96 and 120 hours post-infection. The results are presented as the mean ± SD of five separate experiments and are expressed as the percentage relative to mock-treated control cells. B. Crystal violet assay. Cells on 24-well plates were infected with different viruses at the indicated MOIs. Cytotoxicity was observed by crystal violet staining at five days after infection.
Figure 4.
Ad.DD3.D55-PTEN induces apoptosis of prostate cancer cells.
A. CL1 and 22RV1 cells were stained with Hoechst 33258 at 48 hours after viral infection. The arrows indicate the apoptotic cells. Scale bar, 10 µm. B. Flow cytometric analysis of propidium iodide-stained cells was performed at 48 hours after infection. The percentages of sub-G1 cells are shown. The results are presented as the mean ± SD of three separate experiments (** indicates P<0.01; * indicates P<0.05; N/S indicates P>0.05).
Figure 5.
Ad.DD3.D55-PTEN activates caspase-mediated apoptosis signaling pathways.
A. Western blot analysis of apoptosis-related proteins in CL1 cells infected with Ad.DD3.D55 or Ad.DD3.D55-PTEN. B. CL1 cells were treated with the indicated agents and cell viability was analyzed by the MTT assay after 120 hours. For the Z-VAD-fmk inhibition assay, cells were pre-treated with Z-VAD-fmk (20 µM) 2 h before virus infection. The results are presented as the mean ± SD of three separate experiments (** indicates P<0.01; * indicates P<0.05; N/S indicates P>0.05). C. Analysis of the mitochondrial membrane potential of CL1 cells by FACS after JC-1 staining. The percentage of cells in the trapeziform region is shown.
Figure 6.
Ad.DD3.D55-PTEN inhibits the growth of prostate cancer cell xenograft tumors.
A. Antitumor effect of infection of CL1 xenograft tumors with different adenoviruses. The tumor volume was measured and is presented as the mean ± SD. B. Ad.DD3.D55-PTEN inhibits the growth of CL1 xenograft tumors. Analysis of the viral kinetics of Ad-PTEN or Ad.DD3.D55-PTEN-treated 22RV1 tumors by real-time PCR. Adenovirus E3 region was amplified to evaluate the viral DNA content. β-actin DNA was used as an internal control. (NS means P>0.05, no statistical significance). C. Analysis of tumor sections derived from tumors treated with PBS or one of various adenoviruses by TUNEL staining. Scale bar, 100 µm.
Table 1.
Nucleotide sequence of oligonucleotide primers used for virus identification, RT-PCR and real-time PCR.