APOPTOSIS INVOLVED IN DENSITY-DEPENDENT REGULATION OF RAT FIBROBLASTIC 3Y1 CELL-CULTURE

When rat 3Y1 fibroblastic cells are cultured toward confluency, the ce lls go through maximum cell density (overshoot) before reaching post-c onfluence stationary cell density, After overshooting, a number of flo ating cells are found in the medium. In a long-term culture, a cyclic change in cell number, an increase after each medium refreshment and s ubsequent cell loss within a few days has been observed so that the ce ll populations in the monolayer maintain post-confluence stationary ce ll density at a constant level. The floating cells excluded trypan blu e, but they had no ability to attach to the substrate and to form colo nies after being reseeded in fresh medium. They had condensed and unif ormly electron-dense chromatin with sharply circumscribed edges. Their DNA contained a laddering pattern in harmony with internucleosomal cl eavage. The features were those of apoptosis. When floating cells appe ared, apoptotic bodies were also observed in the monolayer. Most of th em were found within the cytoplasm of intact cells, suggesting that ap optotic bodies were also faded away from the culture by being rapidly engulfed by neighboring intact cells. These suggest that apoptosis and subsequent detachment from the monolayer or engulfment by neighboring intact cells, in addition to inhibition of cell division, are basic m echanisms on the process of density-dependent regulation in monolayer culture of rat 3Y1 cells.