Analysis of TNF alpha-induced DNA strand breaks at the single cell level

Abstract

Treatment of L929 cells with TNF alpha initiates apoptosis and subsequent cell death. The authors have visualized sites of DNA damage in situ by using DNA polymerase to synthesize new strands from the DNA strands breaks as starting point. Biotin-dUTP was incorporated into the newly synthesized strand and visualized by immunocytochemistry. DNA strand breaks were first observed 3 to 4 hours after contact with TNF alpha and preceded cell death. Limiting doses of TNF alpha caused DNA strand breaks only in a subpopulation of L929 cells. At a low dose, TNF alpha led to DNA damage without any subsequent loss of cell viability. The new assay also detects DNase-induced single strand breaks and thus is able to visualize apoptotic as well as non-apoptotic types of DNA damage.