Apoptosis is induced in cells with cytolytic potential by L-leucyl-L-leucine methyl ester

Previous studies have demonstrated that the selective toxicity of leucyl-leucine methyl ester (LeuLeu-OMe) for cytotoxic lymphocytes and myeloid cells is dependent on intracellular conversion to membranolytic metabolites by the acyl transferase activity of the granule enzyme dipeptidyl peptidase I (DPPI) that is enriched in these cells. The mechanism of cell death remained unclear, however, and was the subject of the experiments reported here. When human U937, HL60, or THP-1 myeloid tumor cell lines or murine CTLL-2 cells were treated with Leu-Leu-OMe, early release of both cytosolic ⁵¹Cr and soluble [³H]TdR labeled DNA fragments was observed, whereas antibody + C treatment of these cells caused only ⁵¹Cr release. Killing of U937 or THP-1 cells by incubation with the lysosomotropic amino acid methyl ester, Phe-OMe also induced only ⁵¹Cr release without evidence of DNA fragmentation. Preincubation with Zn²⁺, a known inhibitor of endonuclease activity prevented Leu-Leu-OMe-in-duced ⁵¹Cr or [³H]TdR release from these cell lines, but had no effect on antibody + C or Phe-OMe-induced ⁵¹Cr release. Zn²⁺ also prevented Leu-Leu-OMe-mediated killing of normal human CD16⁺ NK cells. Zn²⁺ had no inhibitory effect on Leu-Leu-OMe uptake or intracellular conversion to (Leu-Leu)_n-OMe metabolites by these cell lines. Moreover, Zn²⁺ did not inhibit ⁵¹Cr release from nonnucleated E or nucleated U937 targets induced by extracellular production of DPPI-generated metabolites of LeuLeu-OMe. Thus, killing of cytotoxic lymphocytes and myeloid cells by Leu-Leu-OMe appears to be dependent on generation of metabolites with membranolytic properties, but cell death induced by this process does not involve simple lysis of the plasma membrane. Rather, intracellular production of DPPI generated (Leu-Leu)_n-OMe metabolites appears to trigger, an additional Zn²⁺-sensitive process that is associated with induction of apoptosis in cells with cytolytic potential.