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DNA Damage as a Basis for 4'-Demethylepipodophyllotoxin-9-(4,6-O-ethylidene-ß-D-glucopyranoside) (Etoposide) Cytotoxicity¹

Clinical Pharmacology Program, Department of Pharmacology, and Division of Medical Oncology, Department of Medicine, University of Florida, Gainesville, Florida 32610

The precise mechanism of action of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-ß-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 µM. 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 -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.

¹ Supported by Bristol Laboratories, Syracuse, N. Y. Presented in part at the Annual Meeting of the American Association for Cancer Research, St. Louis, Mo., April 1982 (14).

² Supported by American Cancer Society Institutional Grant 1N62-V.

³ Supported by Grant RCDA CA-00537 from the National Cancer Institute. To whom requests for reprints should be addressed.

Received 6/16/82. Accepted 10/11/82.

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