Volume 10, Issue 5, Pages 938-949 (November 2004) Oncolytic effects of adenovirus mutant capable of replicating in hypoxic and normoxic regions of solid tumor  Won-Kyung Cho, Young Rim Seong, Yeune Hee Lee, Min Ji Kim, Kyung-Sun Hwang, Jinsang Yoo, Seeyoung Choi, Cho-Rok Jung, Dong-Soo Im  Molecular Therapy  Volume 10, Issue 5, Pages 938-949 (November 2004) DOI: 10.1016/j.ymthe.2004.07.023 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Construction of and analysis of E1A expression from oncolytic adenoviruses. (A) HepG2 cells were transfected with the indicated plasmids harboring luciferase as a reporter gene under the transcriptional control of the E1A promoter or SV40 minimal promoter linked to 5 × HRE from the human VEGF promoter (5 × VEGF-HRE-pSV40 min) and grown for 24 h under normoxia. Cells were further incubated under normoxic (20% O2) or hypoxic (1% O2) conditions for 16 h, before the reporter assay was performed. Data are the means ± SD of three independent experiments in duplicate. (B) Replication-competent or -defective adenoviruses constructed in this study and Ad.Δ55.E1A (dl1520; Onyx-015) are schematically illustrated. ψ, packaging signal; pA, polyadenylation signal. (C) Huh-7 cells were infected with Ad.E1A.Δ55, Ad.Δ55.HRE, or Ad.stuffer.HRE.Δ55 at an m.o.i. of 20 and exposed to normoxia or hypoxia for 20 h before the cell lysates were subjected to Western blot analysis with the indicated antibodies. Molecular Therapy 2004 10, 938-949DOI: (10.1016/j.ymthe.2004.07.023) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Oncolytic effect of Ad.Δ55.HRE on parental Huh-7 and Huh-7 hepatoma cells constitutively expressing HIF-1α. (A) Clear cell lysates of parental Huh-7 and Huh-7 cells constitutively expressing HIF-1α (Huh-7/HIF-1α) were immunoprecipitated (IP) with anti-Flag antibody followed by immunoblotting with anti-Flag antibody. Molecular mass markers in kDa are shown at the left. (B) Huh-7 or Huh-7/HIF-1α cells were infected with Ad.HRE.luc (Fig. 1B) encoding luciferase as a reporter gene under the control of the 5×VEGF-HRE-pSV40 min at an m.o.i. of 1. Luciferase activity in equivalent cell lysates was determined at 20 h posttransduction. Data are the means ± SD of three independent experiments in duplicate. (C) Huh-7 and Huh-7/HIF-1α cells were infected with Ad.Δ55.HRE (lanes 2, 4, 6, and 8) or Ad.E1A.Δ55 (lanes 1, 3, 5, and 7) at the indicated m.o.i. and incubated under normoxia or hypoxia for 4 days. Oncolytic effect of the adenoviruses was evaluated by staining viable cells with crystal violet. The experiment was repeated more than twice and a representative is shown. (D) Huh-7 cells were infected with Ad.Δ55.HRE or Ad.E1A.Δ55 at an m.o.i. of 0.5. Cells were harvested at the indicated times. Adenoviruses from the culture media and the infected cells were titered on a 293 plaque assay. Data are the means ± SD of two independent experiments. Molecular Therapy 2004 10, 938-949DOI: (10.1016/j.ymthe.2004.07.023) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Oncolytic effects of Ad.Δ55.HRE on PC-3 prostate, Panc-1, and AsPC-1 pancreatic tumor cells. (A) PC-3 tumor cells were mock infected or infected with Ad.E1A.Δ55 or Ad.Δ55.HRE at an m.o.i. of 20 and incubated under normoxia or hypoxia for 20 h before Western blot analysis with the indicated antibodies. (B) PC-3 tumor cells were mock infected or infected with Ad.E1A.Δ55 or Ad.Δ55.HRE at the indicated m.o.i. and incubated for 4 days under normoxia, and viable cells were stained with crystal violet. (C) Panc-1 and AsPC-1 tumor cells were infected with Ad.Δ55.HRE (lanes 2 and 4) or Ad.E1A.Δ55 (lanes 1 and 3) at the indicated m.o.i. and incubated for 4 days under normoxia, and survival cells were stained with crystal violet. The experiment was repeated more than twice and a representative is shown. Molecular Therapy 2004 10, 938-949DOI: (10.1016/j.ymthe.2004.07.023) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Oncolytic effects of Ad.Δ55.HRE on HepG2 hepatoma and A549 lung tumor cells. (A) The tumor cells were infected with the indicated adenoviruses at an m.o.i. of 20, incubated for 20 h under normoxic or hypoxic conditions, and lysed for Western blot analysis with the indicated antibodies. (B) HepG2 cells were infected with the indicated adenoviruses at an m.o.i. of 1 and incubated for 4 days under normoxic or hypoxic conditions, before adenoviruses from the culture media and the infected cells were titered on a 293 plaque assay. Data are the means ± SD of three independent experiments. (C) HepG2 or A549 cells were infected with Ad.E1A.Δ55 or Ad.Δ55.HRE at the indicated m.o.i. and incubated under normoxic or hypoxic conditions for 4 days, before cells were stained with crystal violet. The experiment was repeated more than twice and a representative is shown. Molecular Therapy 2004 10, 938-949DOI: (10.1016/j.ymthe.2004.07.023) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Cytotoxic effects of Ad.Δ55.HRE and Ad.E1A.Δ55 on normal fibroblast cells. (A) MRC-5 and IMR-90 fibroblast cells were mock infected (lanes 1, 4, 7, and 10) or infected with Ad.Δ55.HRE (lanes 3, 6, 9, and 12) or Ad.E1A.Δ55 (lanes 2, 5, 8, and 11) at an m.o.i. of 20 and incubated for 20 h under normoxic or hypoxic conditions before cell lysates were immunoblotted with the indicated antibodies. (B) MRC-5 and IMR-90 cells were mock infected or infected with Ad.Δ55.HRE (lanes 2, 4, 6, and 8) or Ad.E1A.Δ55 (lanes 1, 3, 5, and 7) at the indicated m.o.i. and incubated under normoxic or hypoxic conditions for 4 days. Cytotoxic effect of the viruses was evaluated by staining viable cells with crystal violet. The experiment was repeated more than twice and a representative is shown. Molecular Therapy 2004 10, 938-949DOI: (10.1016/j.ymthe.2004.07.023) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 6 In vivo antitumor activity of Ad.Δ55.HRE in PC-3 prostate tumor-bearing nude mice and long-term survival analysis. PC-3 tumor xenografts developed in nude mice were intratumorally injected daily for 5 consecutive days with 2 × 108 pfu of Ad.E1A.Δ55 (n = 4), Ad.Δ55.HRE (n = 5), or PBS (n = 4) as a control. (A) Tumor growth and (B) body weight of the mice were measured at the indicated times. Data are presented as the mean tumor volumes or body weights ± SD and are indicative of one of two such experiments with a similar outcome. (C) In the experiments described for (A), the fraction of animals alive within the different treatment groups was plotted against days after xenografting. Molecular Therapy 2004 10, 938-949DOI: (10.1016/j.ymthe.2004.07.023) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 7 In vivo antitumor activity of Ad.Δ55.HRE in MDA-MB-435 tumor-bearing mice and immunohistochemical analysis of E1A, hexon, and HIF-1α expression in tumor xenografts. (A) MDA-MB-435 xenografts were established subcutaneously in the dorsal flanks of Balb/c nude mice. Once tumors reached about 150–200 mm3 in volume, they were intratumorally injected daily for 5 consecutive days with PBS (n = 6) or 2 × 108 pfu of Ad.E1A.Δ55 (n = 6) or Ad.Δ55.HRE (n = 7). Tumor growth was measured at the indicated times. Data are presented as the mean tumor volumes ± SD. (B) Animals in (A) were sacrificed at 37 days after initial treatment, and tumors were excised and subjected to immunohistochemical analysis using anti-E1A and anti-hexon antibodies as described under Materials and Methods. The numbers of tumor cells containing E1A or hexon proteins, which appeared as brown spots, were counted in three different fields of tumor samples from mice at × 200 magnification and averaged, respectively. Representative photographs with the mean numbers of the immunostained E1A or hexon proteins are shown. (C) Tumors were excised from PC-3 and MDA-MB-435 xenografts developed in nude mice and subjected to immunostaining with anti-HIF-1α antibody. Brown-stained cells are positive for the expression of HIF-1α. Controls are tumor tissues incubated without primary antibody. Representative photographs (original magnification × 200) are shown. Molecular Therapy 2004 10, 938-949DOI: (10.1016/j.ymthe.2004.07.023) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions