AN ADHERENT CELL MODEL TO STUDY DIFFERENT STAGES OF APOPTOSIS

Apoptosis in the classical thymocyte model occurs very rapidly making it difficult to study the intermediate steps in the process, An alternative adherent cell model is characterized and proposed in this paper, HT29 cells treated with a teniposide were collected at various times for morphological and biochemical assessments, Large DNA breaks (450-500, 350-400, 100-200 kb) were observed in these cells between 6 and 24 h, The larger DNA breaks appeared initially and in progression such that the smaller DNA break of 100-200 kb became apparent by 24 h. These changes in DNA corresponded with an increase in cell diameter and a gradual rounding and detaching of cells from each other but not from the tissue culture plates, The smallest DNA break of 23-50 kb appeared at 48 h and persisted throughout the 96 h of incubation. DNA ladders of 180- to 200-bp oligomers were also observed between 48 and 96 h and these coincided with the presence of small floating cells. Changes in cell adherence after teniposide treatment have permitted the consistent isolation of cells in four distinct morphological and biochemical stages of apoptosis: (1) ''preapoptotic,'' (2) ''swelling,'' (3) ''rounding,'' and (4) ''floating.'' The main advantages of this adherent cell model are: (1) apoptosis occurs very slowly (minimum of 48 h) permitting the observation of progressive changes; (2) cells from four stages of apoptosis can be used to study the sequence of events of other biochemical and genetic factors involved in the process; and (3) extracellular matrix proteins are present in this model so their participation in apoptosis, if any, can be determined. (C) 1995 Academic Press, Inc.