DNA damage as a basis for 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyranoside) (etoposide) cytotoxicity.

The precise mechanism of action of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16), an important chemotherapeutic agent, has yet to be determined. VP-16 has been shown to cause single-strand breaks (SSBs) in DNA, but their relationship to cytotoxicity has not been determined. We have investigated the action of VP-16 using mouse leukemia L1210 cells in culture. By using the alkaline elution technique, we reaffirmed the occurrence of SSBs in DNA over the drug concentration range 1 to 60 microM. We were able to demonstrate additional types of DNA damage in the form of DNA double-strand breaks and DNA-protein cross-links within the same dose range. The number of double-strand breaks formed per SSB was consistent over this dose range and greater than that found after exposure of L1210 cells to gamma-irradiation. DNA SSBs and double-strand breaks were also shown to occur in isolated nuclei, indicating that cytoplasmic components are not required for this drug action. Colony formation by L1210 cells in soft agar decreased over a drug concentration range similar to that which produced DNA damage. The correlation between the effective dose range in the colony-forming assay and the DNA scission experiments supports the hypothesis that DNA breakage is responsible for drug cytotoxicity. The demonstration of strand scission in isolated nuclei may provide an experimental model for elucidating the exact mechanism of action of VP-16.