The mechanism of killing of B-lymphoma cells by 111In-conjugated antibodies.
OBJECTIVE
To determine the mode of killing of B-lymphoma cell lines by 111In-conjugated antibodies (Ab).


METHODS
Cells were treated with a range of radiolabeled Ab concentrations to produce a level of killing from 80 - 99.9%, as determined by a long-term proliferation assay. On days 1, 2, 3 and 4, apoptosis was evaluated in individual cells by two assays: (i) Mitochondrial function by JC-1 staining; and (ii) presence of cleaved PARP (poly-ADP-ribose-polymerase) by immunofluorescence. Five cell lines were tested, including two non-Burkitt lines, RL and FSCCL.


RESULTS
In preliminary experiments, it was realized that the standard JC-1 assay, which is widely used, does not distinguish between apoptotic and necrotic cells. The JC-1 assay becomes useful if it is combined with a stain for lysed cells, Sytox green, and then is able to distinguish between viable, apoptotic and necrotic cells. Of the 5 cell lines tested, only Ramos had a large population of apoptotic cells detected by the modified JC-1 assay, but all four of the other cell lines showed evidence of cleaved PARP, albeit to markedly different degrees. The caspase inhibitor, Z-VAD (N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone), significantly inhibited the killing of Daudi cells.


CONCLUSIONS
Apoptosis was activated in at least four of the five cell lines tested, and participated in the killing of the cells. Staining for cleaved PARP was much more sensitive than the modified JC-1 assay, for most cell lines, probably because it can detect cells that have already lysed, but which died via apoptosis. In addition, the methodological issues discussed may lead to a better understanding of the role of apoptosis in tumor cell cytotoxicity.
Mattes
M Jules
MJ
Garden State Cancer Center at the Center for Molecular Medicine and Immunology, Belleville, New Jersey 07109, USA. mjmattes@gscancer.org
D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004307:Dose-Response Relationship, Radiation; D004583:Electrons; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007205:Indium Radioisotopes / Q000378:metabolism*; D016393:Lymphoma, B-Cell / Q000378:metabolism* / Q000473:pathology; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000378:metabolism; D009336:Necrosis; D009842:Oligopeptides / Q000737:chemistry; D016499:Radioimmunotherapy / Q000379:methods*
D004791:Enzyme Inhibitors; D007205:Indium Radioisotopes; D009842:Oligopeptides; C517629:benzyloxycarbonyl-valyl-alanyl-aspartic acid