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 altern ative 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 (450-5 00, 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 t hat 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 g radual rounding and detaching of cells from each other but not from th e tissue culture plates, The smallest DNA break of 23-50 kb appeared a t 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 t hese coincided with the presence of small floating cells. Changes in c ell adherence after teniposide treatment have permitted the consistent isolation of cells in four distinct morphological and biochemical sta ges of apoptosis: (1) ''preapoptotic,'' (2) ''swelling,'' (3) ''roundi ng,'' and (4) ''floating.'' The main advantages of this adherent cell model are: (1) apoptosis occurs very slowly (minimum of 48 h) permitti ng the observation of progressive changes; (2) cells from four stages of apoptosis can be used to study the sequence of events of other bioc hemical and genetic factors involved in the process; and (3) extracell ular matrix proteins are present in this model so their participation in apoptosis, if any, can be determined. (C) 1995 Academic Press, Inc.