Detection of tumor necrosis factor induced nuclear damage with p-phenylenediamine.
A simple technique has been used to observe the effects of rTNF alpha on nuclear morphology. Using the intercalating dye p-phenylenediamine to stain nuclei, we detected TNF alpha-induced nuclear alterations which characteristically occur during apoptosis in the TNF alpha sensitive U937 cell line. Nuclear alterations were visible prior to the loss of plasma membrane integrity and subsequent cell death. A subclone of U937 cells was isolated in which TNF alpha failed to alter either cell viability or nuclear morphology. TNF alpha resistant U937 cells, however, retained the ability to bind, internalize and degrade TNF alpha. These results suggest that nuclear damage induced by TNF alpha in sensitive U937 cells occurs early and precedes cell death as measured by dye exclusion assays. Staining cells with p-phenylenediamine and visualization with a 520 nm fluorescence filter provides a rapid and simple method to monitor apoptotic cell death.
Salcedo
T W
TW
Department of Pathology, State University of New York, Stony Brook 11794-8691.
Fleit
H B
HB
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000187:drug effects; D010655:Phenylenediamines; D011994:Recombinant Proteins / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods*; D013936:Thymidine / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D010655:Phenylenediamines; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C029728:4-phenylenediamine; D013936:Thymidine