REGULATION OF CELL-GROWTH AND APOPTOSIS IN SYNCHRONIZED AGF CELLS - INVOLVEMENT OF ONCOGENES AND CELL-CYCLE REGULATORY PROTEINS

Cell synchrony was induced in AGF cells by blocking of the cell cycle at GI-S boundary with high concentrations (2 mM) of thymidine for 11 h . Prolonged arrest of cells in GI-S (15 h-20 h) induced progressive an d time dependent apoptosis. Early morphological changes in cellular an d nuclear morphology (blebbing) were monitored by Transmission Electro n Microscopy (TEM), Scanning Electron Microscopy (SEM) and by staining of nuclei with Hoechst and propidium iodide and stained cells viewed by fluorescence and confocal microscopy. The fluctuations in the level s of the proliferation cell nuclear antigen (PCNA), cyclin A, CDC-2, c -myc and p53 proteins were monitored in synchronized cultures and in c ells undergoing apoptosis by immunofluorescence staining and flow cyto metry. As expected, the levels of cyclin A and PCNA increased during t he S phase and the level of CDC-2 decreased during late S/G2. Similarl y, the level of c-myc increased during the S phase, whereas the level of p53 did not change much during S phase. Most importantly, however, the level of staining for c-myc, p53, cyclin A, CDC-2 and PCNA increas ed markedly during apoptosis. In contrast, the level of actin vimentin and tubulin, although increased during S phase, were markedly decreas ed during apoptosis. AGF cells stained for c-myc during apoptosis, and viewed by confocal microscopy, revealed increased staining for c-myc in the blebbing nuclei. These results, taken together, suggest a possi ble active role for c-myc in the process of nuclear blebbing and apopt osis.