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DNA Strand Breaks Produced by Etoposide (VP-16,213) in Sensitive and Resistant Human Breast Tumor Cells: Implications for the Mechanism of Action

Clinical Pharmacology Branch, Clinical Oncology Program [B. K. S., N. H., L. D.], and Laboratory of Molecular Pharmacology [D. K., Y. P.], National Cancer Institute, NIH, Bethesda, Maryland 20892

Pleotropic resistant human breast cancer cells (MCF-7), selected for resistance to Adriamycin, were used to study the production of DNA strand breaks by etoposide (VP-16) and its relationship to drug cytotoxicity. It was shown that the resistant MCF-7 cell line was cross-resistant to VP-16, and the degree of resistance was found to be 125–200-fold. Alkaline elution studies indicated that the parental cell line was very sensitive to VP-16 which caused extensive DNA strand breakage. In contrast, little DNA strand breakage was detected in the resistant MCF-7 cells, even at very high drug concentrations, indicating a good agreement between strand breaks and cytotoxicity. Further studies indicated that the nuclei isolated from the parental cell line were more resistant to VP-16-induced DNA strand breaks than the intact cells, while the opposite was found in the resistant cell line. In addition, the alkaline elution studies in isolated nuclei showed only a 2-fold reduction of VP-16-induced DNA breaks in nuclei from the resistant cells. In agreement with this result, it was found that nuclear extract from the resistant cells produced 2-3-fold less VP-16-induced DNA breaks than that from the sensitive cells in ³²P-end-labeled SV40 DNA. VP-16 uptake and efflux studies indicated that there was a 2–3-fold decrease in net cellular accumulation of VP-16 in the resistant cells. Although the reduced uptake of VP-16 and decreased drug sensitivity of topoisomerase II appear to contribute to the mechanism of action and the development of resistance to VP-16, they do not completely explain the degree of resistance to VP-16 in this multidrug-resistant MCF-7 cell line indicating that other biochemical factors, such as activation of VP-16, are also involved in drug resistance and suggesting that the resistance is multifactorial.

¹ To whom requests for reprints should be addressed, at Bldg. 10, Room 6N-119, National Cancer Institute, NIH, Bethesda, MD 20892.

² Present address: Department of Radiation and Clinical Oncology, Hadassah University Hospital, Jerusalem, Israel 91120.

Received 10/21/87. Revised 3/ 1/88. Revised 6/ 7/88. Accepted 6/14/88.

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