Value toward other neoplastic diseases.ResultsGeneration of the recombinant adenovirusesThe structures of the recombinant adenoviruses constructed for this study PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26080418 are presented in Figure 1A, including Ad-hTERT-E1a-Apoptin and Ad-CMV-E1aApoptin, in which the E1a gene is under the control of the hTERT promoter or CMV promoter, respectively, and Apoptin is controlled by a CMV promoter. We generated two additional control replicating adenoviruses without Apoptin, Ad-hTERT-E1a and Ad-CMV-E1a, in which E1a is expressed by the hTERT or CMV promoters, respectively. The two replication-incompetent adenoviruses (lacking the E1a gene) were Ad-hTERTApoptin and Ad-CMV-Apoptin, in which hTERT promoter or CMV promoter drove Apoptin expression, respectively. An empty adenovirus vector (designated Ad-mock) was used as control.Transgene expression and characterization of recombinant adenovirusesIn the melanoma cell lines (A375 and B16), infection with Ad-hTERT-E1a-Apoptin, Ad-CMV-E1a-Apoptin, Ad-hTERT-Apoptin and Ad-CMV-Apoptin resulted in significant production of Apoptin proteins (15 kDa), whereas no Apoptin protein was detected after AdhTERT-E1a, Ad-CMV-E1a or Ad-mock infection (Figure 1B and 1C). In the HEM cell line, infection with AdCMV-Apoptin, Ad-hTERT-E1a-Apoptin and Ad-CMVE1a-Apoptin, but not Ad-hTERT-Apoptin, Ad-hTERTE1a, Ad-CMV-E1a or Ad-mock, produced Apoptin protein (Figure 1D). A375 or B16 cells infected with AdhTERT-E1a-Apoptin, Ad-CMV-E1a-Apoptin, AdhTERT-E1a and Ad-CMV-E1a produced E1a proteins (36 kDa); however, cells infected with Ad-hTERT-Apoptin, Ad-CMV-Apoptin or Ad-mock did not (Figure 1B and 1C). Infection of HEM with IRC-022493MedChemExpress Setmelanotide Ad-CMV-E1a-Apoptin or Ad-CMV-E1a resulted in E1a production, whereas infection with Ad-hTERT-E1a-Apoptin, Ad-hTERT-E1a, Ad-hTERT-Apoptin, Ad-CMV-Apoptin or Ad-mock resulted in barely detectable E1a expression (Figure 1D). These results demonstrated that Ad-hTERT-E1a-Apoptin had cancer cell-selective replication properties, and the Apoptin transgene was effectively expressed.Xiao et al. Molecular Cancer 2010, 9:10 http://www.molecular-cancer.com/content/9/1/Page 3 ofFigure 1 Schematic of the recombinant adenoviruses and the adenovirus-mediated transgene expression. (A) Schematic diagram depicting the organization elements in the recombinant adenoviruses. Polyadenylation signal sequence is designated as pA. The indicated cells were infected with Ad-mock (control) or indicated recombinant viruses at a MOI of 100 for 48 h. Western blot analysis was performed to detect (B) Apoptin or E1a protein from A375 cells, (C) Apoptin or E1a expression in B16 cells, or (D) Apoptin or E1a expression in HEM cells. Infection of normal HEM human epidermal melanocytes (D) with Ad-CMV-E1a or Ad-CMV-E1a-Apoptin, but not Ad-hTERT-E1a or Ad-hTERT-E1a-Apoptin, resulted in production of E1a proteins, whereas in A375 (B) and B16 (C) melanoma cells, infection with all these replication-competent recombinant adenoviruses generated E1a proteins. In HEM cells (D), infection with Ad-CMV-E1a-Apoptin and Ad-CMV-Apoptin resulted in Apoptin production, whereas infection with Ad-hTERT-Apoptin or Ad-hTERT-E1a-Apoptin resulted in barely detectable of Apoptin production. In A375 (B) and B16 (C) cells, infection with Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-CMV-E1a-Apoptin, or Ad-hTERT-E1a-Apoptin generated significant Apoptin production. 1. Ad-mock; 2. Ad-CMV-Apoptin; 3. Ad-hTERT-Apoptin; 4. Ad-CMV-E1a; 5. Ad-hTERT-E1a; 6. Ad-CMV-E1a-Apoptin; 7. Ad-hTERT-E1a-A.
