CELLULAR EVENTS IN FAS APO-1-MEDIATED APOPTOSIS IN JURKAT T-LYMPHOCYTES/

In the present study we investigated the Fas-mediated cellular events using the human leukemic T cell line, JURKAT. Ligation of the Fas rece ptor with a monoclonal antibody (IgM) resulted in the rapid (within 3 h) induction of apoptosis and was characterized by a sequence of disti nct morphological and biochemical events. Thus, plasma membrane blebbi ng, condensation of the chromatin, and formation of high-molecular-wei ght (HMW) DNA fragments were the earliest events observed (by 45 min). They were followed by cleavage of DNA into oligonucleosomal-length fr agments (laddering pattern) and the formation of apoptotic bodies, and finally, rounding of the apoptotic cells and complete cleavage of DNA into oligonucleosomal-length fragments occurred. The mitochondria rem ained structurally intact up to the stage of oligonucleosomal-length D NA cleavage, and the ability of the cells to exclude trypan blue was n ot compromised throughout the time course of the experiments. In contr ast to many other model systems, apoptosis in JURKAT cells after anti- Fas treatment did not require the presence of extracellular Ca2+ or Mg 2+ and was only partially inhibited by Zn2+. In addition, Fas-mediated apoptosis was unaffected by the presence of free radical scavengers o r inhibitors of protein phosphatases, protein kinases, and nitric oxid e synthesis. However, the serine protease inhibitors, N-tosyl-L-phenyl alanine chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI) p revented anti-Fas-induced apoptosis in JURKAT cells, Low concentration s of these inhibitors blocked oligonucleosomal-length, but not HMW, DN A fragmentation. The latter required a higher concentration of TPCK or DCI to block. In addition, low concentrations of DCI also prevented F as-mediated plasma membrane blebbing. In summary, our results suggest that proteolysis plays a central role in Fas-mediated apoptosis and th at distinct proteolytic enzymes are involved in HMW DNA fragmentation, and oligonucleosomal-length DNA fragmentation, as well as in plasma m embrane blebbing. (C) 1995 Academic Press, Inc.