LOSS AND SHEDDING OF SURFACE-MARKERS FROM THE LEUKEMIC MYELOID MONOCYTIC LINE THP-1 INDUCED TO UNDERGO APOPTOSIS

Previous studies have established that a relationship exists between a poptosis and cell surface (ecto-) peptidase activity. Thus dose-depend ent increases were found both in ectopeptidase activities and in the p roportion of cells undergoing apoptosis in HeLa cell monolayers after exposure to UV and other perturbants causing arrest of DNA synthesis ( indirectly or directly as a result of DNA damage). The nature of the c orrelation made no distinction as to whether an increase in peptidase activity was causal of, or consequential to apoptosis, nor whether the increase was a general response by all cells. As a wider approach to understanding the possible role played by ectopeptidases in apoptosis, we report the effect on expression of a known ectopeptidase, aminopep tidase N (CD13), by a myelomonocytic cell line induced to undergo apop tosis. Using THP-1 cultures exposed to low concentrations of ethanol, we used FACS technology to sort for early apoptotic cells that have an increased ability to sequester the vital dye Hoechst 33342 while excl uding nonvital dyes. Apoptosis was verified by light, fluorescence, an d transmission electron microscopy, and the presence of DNA fragmentat ion. These early apoptotic cells showed a significant loss in CD13 lab eling. Another surface marker, CD33, behaved similarly, whereas CD14 w as lost globally, and not just by the apoptotic cells. Peptidase assay s confirmed that an aminopeptidase was shed into the bathing media and that this activity was inhibitable both by bestatin and by a CD13 neu tralizing monoclonal antibody. In treated cells, there was no evidence for an increase in cell surface protease activity directed toward a h ighly aliphatic nonapeptide substrate used as a model for TGF-alpha sc ission from its precursor form. However, other cell surface proteases of different specificity are presumably responsible for the observed s hedding of CD13. (C) 1996 Wiley-Liss, Inc.