br Recombinant adenoviruses Ad Apoptin
Recombinant adenoviruses Ad-Apoptin-hTERTp-E1a (Ad-VT), Ad-hTERTp-E1a (Ad-T), Ad-Apoptin (Ad-vp3),
and Ad-mock were constructed and preserved in our labora-tory (Laboratory of molecular Virology and Immunology, Military Medical Science Academy of the PLA, Chang-chun, China) (Fig. 1) . The constructed shuttle plasmids were cotransfected into HEK-23 cells, and then a recombi-nant adenovirus was produced by homologous recombina-tion. The virus was purified using an Adeno-X Virus Purification kit (BD Bioscience Clontech, San Diego, CA) and the virus titer was detected using an Adeno-X Rapid titer kit (BD Bioscience Clontech, San Diego, CA).
Female BALB/c nude mice aged 4 to 5 weeks were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of China. The animal experimental protocols were approved by the Insti-tutional Animal Care and Use Committee of the Chinese Academy of Military Medical Science, Changchun, China (10ZDGG007). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
2.2. Construction and identification of PC-3-luc cells
(Promega, Madison, WI) and 4 ml of Effectene Trans-fection Reagent (QIAGEN, Beijing, China). After trans-fection for 24 hours, PC-3 A83 01 were digested and adjusted to a density of 100 cells/well, and added to a new 6-well plate. At the same time, a certain concentra-tion of geneticin (G418) was used for screening, and fresh medium was changed every 2 days. G418 was used until a single resistant clone appeared; the above well-grown clones were selected into 96-well plates at 100 ml/well, and the G418 (BD Bioscience Clontech, San Diego, CA) concentration was maintained. When the cell confluence reached 80% or more, the cells were transferred to 24-well plates and culture was continued. Similarly, when the cell confluence reached 80% or more, they were transferred to a 12-well plate and assayed for their luciferase activity.
The preliminary screened cell clones were cultured at 5 £ 103 cells/well in 96-well cell culture plates, and cul-tured at 37˚C and 5% CO2. After 48 hours of culture, the luciferase activity of each cell clone was detected using a ONE-Glo Luciferase Assay System (Promega). Subse-quently, the cell clone with the highest fluorescence value was continuously cultured for 6 to 8 weeks, and its lucifer-ase activity (RLU) was detected every 5 generations using the luciferase assay kit to observe whether the luc gene was stably expressed.
Fig. 1. Schematic diagram of four recombinant adenovirus vectors constructed using shuttle vectors.
2.3. Detecting the biological characteristics of PC-3-luc cells
The characterization of PC-3-luc cells was performed using cell growth curve, cell cycle assays, and immunoblot-ting. The PC-3 and PC-3-luc cells were cultured at 5 £ 103 cells/well in 96-well cell culture plates, and cultured at conditions of 37˚C and 5% CO2 for 24 hours. A 96-well cell culture plate was taken at each of 7 different time points (1, 2, 3, 4, 5, 6, and 7 days), and the culture solution of each well was discarded, and then 110 ml WST-1 solu-tion was added to each well (10 ml WST-1 and 100 ml serum-free and no antiantibody DMEM medium), cultured continued for 1.5 hours in the dark, and the absorbance at 450 nm was then measured.
The PC-3 and PC-3-luc cells were cultured at 2 £ 105 cells/well in 12-well cell culture plates, and cultured at 37˚ C and in 5% CO2 for 24 hours. The PC-3 and PC-3-luc cells were harvested and washed 3 times with phosphate-buff-ered saline (PBS). The PC-3 and PC-3-luc cells were resus-pended in 5 ml 75% ethanol (precooled at 4˚C) and incubated at 4˚C for 18 hours in the dark. Subsequently, the PC-3 and PC-3-luc cells were washed 3 times with PBS and then 500 ml propidium iodide (PI)/RNase solution was added. After 20 minutes of incubation, the sample was transferred to a labeled flow tube and detected using flow cytometry (BD Bioscience Clontech, San Diego, CA).
105 cells/well in 6-well cell culture plates, and cultured at 37˚C and in 5% CO2 for 24 hours. The PC-3 and PC-3-luc cells were collected by centrifugation. Cell lysates were used to resuspend the cells, then they were stored on ice bath for 5 minutes. They were centrifuged at 600 g for 5 minutes and the supernatant was centrifuged obtain both the cytosol and the mitochondria. The precipitants were dis-solved by buffer (10 mmol/L Tris [pH 7.4], 150 mmol/L NaCl, 1% Triton X-100, 5 mmol/L EDTA [pH 8.0]); all samples were analyzed by Western blotting. The following primary antibodies were used: rabbit b-actin (Cat. 4970), rabbit anti-Cyclin D1 (Cat. 2978), rabbit anti-CDK2 (Cat. 2546) (all from Cell signaling technology, Danvers MA). The following secondary antibodies were used: Peroxidase-Conjugated Goat anti-Rabbit IgG (H+L) (Cat. ZB2301, ZSGB-BIO, CN).