APOPTOTIC DEATH IN EPITHELIAL-CELLS - CLEAVAGE OF DNA TO 300 AND OR 50 KB FRAGMENTS PRIOR TO OR IN THE ABSENCE OF INTERNUCLEOSOMAL FRAGMENTATION

To date, apoptosis has been characterized biochemically by the product ion of 180 - 200 bp internucleosomal DNA fragments resulting from the activation of an endonuclease(s). The principal morphological feature of apoptosis is the condensation of chromatin and it has been assumed that this may reflect the oligonucleosomal fragmentation pattern. We h ave re-examined this dogma by comparing the biochemical and morphologi cal features of cell death in several epithelial cell types (HT-29-I1 colon adenocarcinoma, CC164 mink lung, DU-145 human prostatic carcinom a and MCF-7 human breast adenocarcinoma) and one mesenchymal cell line (H11ras-R3 ras-transformed rat fibroblasts). Cell death was induced e ither by serum deprivation, TGF-beta1 or etoposide, or by leaving cell s to reach confluence. Cell death was assessed with respect to detachm ent from monolayers, morphological changes and DNA integrity. The DNA- binding fluorophore Hoechst 33258 revealed chromatin condensation patt erns consistent with apoptotic cell death in all cell types except MCF -7 cells. Using field inversion gel electrophoresis in conjunction wit h conventional 2% agarose gel electrophoresis, cleavage of DNA to 50 k bp fragments was observed in atl cases except MCF-7 cells. This preced ed the appearance of oligonucleosomal fragments in HT-29-I1, CC164 and H11ras-R3 cells. Although the DNA of DU-145 cells fragmented into 50 kbp units, and although the cells exhibited classical apoptotic morpho logy, no subsequent internucleosomal cleavage was observed. These resu lts suggest that changes in the integrity of DNA indicative of the rel ease of chromatin loop domains occur before cleavage at internucleosom al sites is initiated and that the tatter is not an essential step in the apoptotic process.