THE ROLE OF APOPTOSIS IN ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY AGAINST MONOLAYERS OF HUMAN SQUAMOUS-CELL CARCINOMA OF THE HEAD AND NECK TARGETS

Antibody-dependent cellular cytotoxicity (ADCC) against squamous cell carcinoma of the head and neck (SCCHN) targets in the presence of huma n/mouse chimeric monoclonal antibodies (cMAbs), SF-25 and 323/A3, is m ediated by natural killer (NK) cells. In 4-hr Cr-51-release assays wit h SSCHN targets in suspension, ADCC was always significantly better (P < 0.01) than that measured in parallel with the same target cells in monolayers. No differences were observed in the level of expression of the relevant antigens recognized by cMAbs on these targets. To better explain the difference, ,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetraz olium bromide (MTT) monolayer and [H-3]thymidine-release assays were u sed. Cytostasis and cell. death measured in monolayer MTT assays and D NA fragmentation measured in [H-3]thymidine-release assays were signif icantly higher (P = 0.028) than cytotoxicity determined using Cr-51-la beled SCCHN monolayers. Cell death observed in monolayer MTT assays wa s blocked by pretreating SCCHN targets with cycloheximide or actinomyc in-D or by paraformaldehyde fixation of effector cells. The presence o f apoptotic cells in monolayers co-incubated with effector cells was d emonstrated in situ by labeling fragmented ends of DNA with fluorescei n-conjugated dUTP and terminal deoxynucleotidyl transferase and also b y flow cytometry of target cells obtained from such monolayers. Our re sults indicate that NK cells preferentially utilize membrane lysis (ne crosis) in ADCC with tumor cell targets in single-cell suspensions. Ho wever, necrosis is not efficient in monolayers. In the presence of cMA bs, apoptosis is the primary mechanism of NK cell-mediated killing in monolayers of SCCHN targets, which were found to express receptors for tumor necrosis factor and fas ligand. (C) 1996 Academic Press, Inc.