Fluctuations and Ultrastructural Localization of Oncoproteins and Cell Cycle Regulatory Proteins during Growth and Apoptosis of Synchronized AGF Cells 1

AGF cells were synchronized by blocking the cell cycle at the G1]S boundary with high concentrations of thymidine (thymidine block) for 11 h. Prolongation of the thymidine block from 11 h to 20 h resulted in apoptosis. Early changes in cellular and nuclear morphology were monitored by confocal microscopy, transmission electron microscopy, and scanning electron microscopy. The fluctuations in the levels of the proliferation cell nuclear antigen (PCNA), cyclin A, CDC-2, c-myc, and p53 proteins were monitored in synchronized cultures and in cells undergoing apoptosis by immunofluorescence staining, flow cytometry, and Western immunoblotting. When assayed by immunofluorescence staining and flow cytometry, the levels of cydin A and PCNA increased about 2-fold during the S phase, and the level of CDC-2 was fairly constant during S and slightly decreased during late S/G2. The level of c-myc also increased about 2-fold during the S phase, whereas the level of p53 increased only slightly during S. Most importantly, however, the level of staining for c-myc, p53, cyclin A, CDC-2, and PCNA increased 50%-150% during apoptosis compared to the levels observed in cells at G1/S. In contrast, the levels of actin and vimentin, although increased during S, were decreased during apoptosis compared to the levels observed at G~/S. Western blot analysis of the steady state levels of PCNA, cyclin A, and CDC-2 revealed an increase in the levels of all three proteins during S, with higher levels of these proteins observed in apoptotic cells compared to the levels observed in cells at Gt/S. Similarly, the levels of p53 and c-myc proteins increased during S and were also high in apoptotic cells. Interestingly, high levels of these two proteins were observed also in cells arrested at Gt/S. AGF cells undergoing apoptosis were immunostained for c-myc, p53, PCNA, cyclin A, and CDC-2 and were viewed by confocal microscopy. Apoptotic cells exhibited increased staining for c-myc and p53 in the blebbing nuclei. Furthermore, we observed for the first time that CDC-2, cyclin A, and PCNA proteins were associated mostly with the plasma membrane and the cytoplasm of log phase cells. However, in cells undergoing apoptosis, these proteins were found exclusively in the nuclei of apoptotic cells. These results suggest a possible active role for c-myc, p53, and the cell cycle regulatory proteins in the process of nuclear blebbing and apoptosis.