Apoptosis and DNA fragmentation precede TNF-induced cytolysis in U937 cells.
The hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF-mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2-3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1-2 h prior to cell death as defined by trypan blue exclusion or 51Cr release. DNA fragmentation was not a non-specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF-resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF-mediated cytotoxicity. Two agents known to inhibit TNF-mediated cytotoxicity, ZnSO4 and 3-aminobenzamide, were shown to inhibit TNF-induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNF lytic pathway in the U937 model system.
Wright
S C
SC
Palo Alto Institute for Molecular Medicine, Mountain View, California 94043.
Kumar
P
P
Tam
A W
AW
Shen
N
N
Varma
M
M
Larrick
J W
JW
D001549:Benzamides / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects*; D002860:Chromium Radioisotopes; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D006801:Humans; D014316:Tritium; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015032:Zinc / Q000494:pharmacology
D001549:Benzamides; D002860:Chromium Radioisotopes; D014409:Tumor Necrosis Factor-alpha; D014316:Tritium; C025160:3-aminobenzamide; D004247:DNA; D015032:Zinc