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. 2014 Oct;15(10):1358-66.
doi: 10.4161/cbt.29842. Epub 2014 Jul 14.

Dissecting the roles of E1A and E1B in adenoviral replication and RCAd-enhanced RDAd transduction efficacy on tumor cells

Fang Wei  1 , Huiping Wang  1 , Xiafang Chen  1 , Chuanyuan Li  2 , Qian Huang  3
Affiliations

Dissecting the roles of E1A and E1B in adenoviral replication and RCAd-enhanced RDAd transduction efficacy on tumor cells

Fang Wei et al. Cancer Biol Ther. 2014 Oct.

Abstract

Oncolytic viruses have recently received widespread attention for their potential in innovative cancer therapy. Many telomerase promoter-regulated oncolytic adenoviral vectors retain E1A and E1B. However, the functions of E1A and E1B proteins in the oncolytic role of replication-competent adenovirus (RCAd) and RCAd enhanced transduction of replication defective adenoviruses (RDAd) have not been addressed well. In this study, we constructed viruses expressing E1A alone, E1A plus E1B-19 kDa, and E1A plus E1B-19 kDa/55 kDa. We then tested their roles in oncolysis and replication of RCAd as well as their roles in RCAd enhanced transfection rate and transgene expression of RDAd in various cancer cells in vitro and in xenografted human NCI-H460 tumors in nude mice. We demonstrated that RCAds expressing E1A alone and plus E1B-19 kDa exhibited an obvious ability in replication and oncolytic effects as well as enhanced RDAd replication and transgene expression, with the former showed more effective oncolysis, while the latter exhibited superior viral replication and transgene promotion activity. However, RCAd expressing both E1A and E1B-19 kDa/55 kDa was clearly worst in all these abilities. The effects of E1A and E1B observed through using RCAd were further validated by using plasmids expressing E1A alone, E1A plus E1B-19 kDa, and E1A plus E1B-19 kDa/55 kDa proteins. Our study provided evidence that E1A was essential for inducing replication and oncolytic effects of RCAd as well as RCAd enhanced RDAd transduction, and expression of E1B-19 kDa other than E1B-55 kDa could promote these effects. E1B-55 kDa is not necessary for the oncolytic effects of adenoviruses and somehow inhibits RCAd-mediated RDAd replication and transgene expression.

Keywords: adenoviral vector; gene expression; gene transduction; replication competent adenovirus; replication defective adenovirus; tumor; tumor gene therapy.

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Figures

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Figure 1. RCAd enhanced RDAd-carried GFP expression in cancer cells. The representative pictures of GFP expression. The different type of tumor cells were infected with 25 MOI of Ad-EGFP alone or co-infected with Ad-EGFP (25 MOI) and different RCAd (10 MOI) and the pictures were taken by epi-fluorescence microscopy (A). FACS analysis of GFP expression. The NCI-H460 cells were infected with different MOI of AdGFP (25, 50, and 100 MOI) and co-infected with Ad-EGFP (25 MOI) and different RCAd (2, 5, and 10 MOI). The mean fluorescent intensity of GFP was analyzed by FACS 72 h after infection. Data were average of 3 individual wells for each viral vector infection and at least 3 times of infection (B). *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 2. Virus replication and oncolytic effect of RCAd. The cancer cell lines NCI-H460, A549, Hep3B, NCI-H1299, and U251MG were infected with 10 MOI of different RCAd, wild-type adenovirus dl309 was used as a control. The cell lysates were prepared at 72 h and viral titers were measured by plaque-forming assay. The titer values are shown as mean ± SD from triplicates (A). All cancer cells mentioned above were infected with different RCAd at 10, 25, 50, 100, and 500 MOI respectively. Cell viability was measured using CCK-8 assay 7 d post-infection. Each infection were performed in 5 wells and repeated 3 times. Data are shown as the mean ± SD and compared with AdGFP alone (B). *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 3. RCAd enhanced AdGFP transduction in tumor in vivo. A total of 5 × 106 NCI-H460 cells were injected subcutaneously into both hind limbs of Balb/C nude mice. When the tumor size reached 10 mm in diameter, animals were divided into 3 groups (n = 3). A total of 5 × 107 RCAd plus 1 × 108 AdGFP were injected into the tumor on the right hind limb, while about 1 × 108 AdGFP alone were injected into the tumor on the left hind limb as control. Forty-eight hours later, in vivo imaging was performed by NightOwl LB 981 Molecular Imaging System (Berthold Technologies) (A). Comparison of fluorescence intensity in NCI-H460 tumors, data are shown as ratio of RCAd plus Ad-GFP (right leg) over AdGFP alone (left leg) (B).
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Figure 4. RCAd enhanced AdIL2 transduction inhibited tumor growth. Human xenograft tumors were established by subcutaneous injection of approximately 1 × 107 NCI-H460 tumor cells into the right hind limbs of mice. When tumors reached 5 mm in diameter, mice were randomly divided into 3 groups, with 8 mice in each group. The injection of AdIL2 (5 × 107 pfu) alone or AdIL2 (5 × 107 pfu) plus Adpx21 (1 × 107 pfu) significantly inhibited tumor growth in comparison with saline injected tumors (A). In another set, 72 h after virus injection mice were sacrificed and the tumor tissues were excised and homogenized in PBS (n = 4). IL2 in the supernatant were measured by ELISA. The tumors received Adpx21 plus AdIL2 had increased IL2 expression than that received AdIL2 alone (B). Data are shown as the mean ± SD. *P < 0.05.
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Figure 5. Differential effect of different element of E1 genes on AdGFP replication. SMMC7721, NCI-H460, and A549 cells grown on 6-well plates were transfected with 2 µg of plasmid DNAs using lipofectamine 2000, empty vector pZap was used as control. Twenty-four hours after transfection, cells were infected with 10 MOI of AdGFP. Four hours later AdGFP were removed and cells were washed 3 times using PBS and cultured with normal medium. The representative pictures of GFP expression were taken by epi-fluorescence microscopy 24 h later after infection (A). A summary of FACS analysis for GFP expression in cells mentioned above (B). GFP DNA detected by quantitative PCR (C). GFP mRNA detected by quantitative RT-PCR (D). E1a mRNA detected by quantitative RT-PCR (E).
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Figure 6. A schematic diagram of replication competent adenovirus constructed in this study. E1a-P, E1a promoter; TERT-P, the telomerase (TERT) promoter; E1a and E1b, early E1 genes of the adenovirus; IRES, internal ribosome entry site; E1b19k, gene expresses E1b-19 kDa protein; E1b55k, gene expressing E1b-55 kDa protein (A). Confirmation of E1A, E1B-19 kDa, and E1B-55 kDa protein expression by western blot. NCI-H460 cancer cells were infected with different adenoviruses at a 10 MOI (pfu/cell) for 48 h and then the cell lysates were analyzed by western blot. GAPDH (36 kDa) was used as control (B).
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