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. 2014 Mar 4;22(5):986–998. doi: 10.1038/mt.2014.7

Figure 1.

Figure 1

Adenoviruses and cells used in this study and in vitro characterization. (a) Schematic representation of viruses used in this study. CAV2 is the canine wild-type virus serotype 2. CAV2RGD contains an RGD motif (FiberRGD) in the HI-loop of the CAV2 fiber. ICOCAV15 and ICOCAV17 are canine conditionally replicative adenoviruses, based on CAV2RGD, in which the endogenous E1a promoter have been modified inserting four palindromic E2F-binding sites and one Sp-I-binding site (403p) at the 403 nucleotide position of the virus genome, 21 base pairs of E1a region (E1aΔ21) homologous to Δ24 in human oncolytic adenoviruses have been deleted. ICOCAV17 is armed with the human PH20 hyaluronidase (PH20) under the control of the canine IIIa protein Splicing Acceptor (IIIaSA). MLP, Major late promoter; pA, polyadenylation signal. (b) Western blot of pRb in canine tumor cells and dog epidermal keratinocytes (DEK). Both hypophosphorylated (pRb) and hyperphosphorylated (ppRb) forms of the protein were resolved. Abrams cell line expressed low levels of ppRb. α-Tubulin was used as loading control. (c) Comparison of infectivity between CAV2 and CAV2RGD in four canine tumor cell lines. Abrams, 17CM98, D17, and CML1 cells were infected with an equal number of viral particles per cell of each virus using two different doses. Immune staining against viral hexon protein was performed 24 hours after infection, and the number of infected cells counted. The ratio between CAV2RGD and CAV2 of three independent assay results ± SD is shown. (d) ICOCAV15 total production yields in a cycle-arrested nontumor canine cell line. DEK were seeded in 24-wells plates. After 20 days in confluence, DEK were infected with 8 transducing units (tu)/cell of CAV2RGD or ICOCAV15. Two days postinfection, total virus production was measured. Three independent assay results ± SD are shown. (e) Virus production of ICOCAV15 in tumor cells. Different tumor cell lines were infected with a dose of virus that resulted in >80% transduction (20 tu/cell for D17 and CML1 and 30 tu/cell for Abrams and 17CM98). Two days after infection, total virus production was measured in triplicate. Results ± SD are shown. (f) Hyaluronidase expression in cells infected with ICOCAV17. DK28Cre cells were infected with ICOCAV15 or ICOCAV17 with 20 tu/cell. After 48 hours, supernatants from infected cells were incubated with a solution of Hyaluronan (HA) for 0, 1, 2, 3, or 4 hours and analyzed. Four samples with known concentration of purified hyaluronidase were used as references. HA degradation was measured by absorbance (600 nm). #Statistical significance between CAV2 and CAV2RGD (P < 0.05) by two-tailed unpaired Student's t-test; *Statistical significance between CAV2RGD and ICOCAV15 (P < 0.05) by two-tailed unpaired Student's t-test.