Figure 12
AAV2 infection of different breast cancer cells and induction of apoptosis/cell death results in altered expression profiles of different cell cycle tumor suppressors proteins. (A) MCF-7 (B) MDA-MB-468 and (C) MDA-MB-231 monolayer cell cultures were synchronized by trypsinization of 80% confluent cultures and plating at a density of 1 × 106 cells in E medium. The cells were incubated for 10 to 12 h at which point at least two-thirds of the cells are maximally synchronized in the G1 phase followed by infection with AAV2. This time point was designated time zero (t = 0). Cell pellets were collected each day over a 7 day period. Cells were passaged 1:2 on day 2 and day 5. Detection of cell cycle tumor suppressor proteins was performed by Western blotting. Total protein extracts were prepared as described previously [48]. Sixty micrograms of total protein extracts from AAV2 infected and control cells were resolved in SDS-polyacrylamide gel electrophoresis (PAGE) gels. The pRb protein hyperphosphorylated (Hyper-pRb) and hypophosphorylated (hypo-pRb) forms were detected using the rabbit polyclonal antibody (Santa Cruz). The p21WAF1, p27KIP1 and p16INK4 tumor suppressors were detecting using rabbit polyclonal antibodies (Santa Cruz) as described in Materials and Methods. The p53 protein was detected using a mouse monoclonal antibody (Oncogene).