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Induction of Apoptosis in MCF-7:WS8 Breast Cancer Cells by ß-Lapachone¹

Department of Human Oncology and the University of Wisconsin Comprehensive Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin 53792 [S. M. W., J. J. P., S. M. P., K. L. B., D. A. B.]; and Preparative Synthesis Core Facility, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [W. G. B.]

ß-Lapachone (ß-lap) affects a number of enzymes in vitro, including type I topoisomerase (Topo I); however, its exact intracellular target(s) and mechanism of cell killing remain unknown. We compared the cytotoxic responses of MCF-7:WS8 (MCF-7) human breast cancer cells after 4-h pulses of ß-lap or camptothecin (CPT), a known Topo I poison. A direct correlation between loss of survival and apoptosis was seen after ß-lap treatment (LD₅₀ = 2.5 µM). A concentration-dependent, transient sub-2 N preapoptotic cell population was observed at 4–8 h. Estrogen deprivation-induced synchronization and bromodeoxyuridine-labeling studies revealed an apoptotic exit point near the G₁-S border. Apoptosis activated by ß-lap was closely correlated with cleavage of lamin B but not with increases in p53/p21 or decreases in bcl-2. Loss of hyperphosphory-lated forms of the retinoblastoma protein was observed within 5 h, but cyclins A, B1, and E levels were unaltered for up to 72 h after 5 µM ß-lap. Topo I and Topo II levels decreased at >24 h. Logarithmic-phase MCF-7 cells were not affected by 1 µM ß-lap.

In contrast, dramatic and irreversible G₂-M arrest with no apoptosis was observed in MCF-7 cells treated with 1 µM CPT, monitored for 6–10 days posttreatment. MCF-7 cells treated with supralethal doses of CPT (5 µM) resulted in only 20% apoptosis. No correlation between apoptosis and loss of survival was observed. MCF-7 cells exposed to >5 µM CPT arrested at key cell cycle checkpoints (i.e., G₁, S, and G₂-M), with little or no movement for 6 days. Ten-fold increases in p53/p21 and 2–5-fold decreases in bcl-2, Topo I, Topo II, and cyclins A and B1, with no change in cyclin E, were observed. Temporal decreases in bcl-2 and cleavage of lamin B corresponded to the minimal apoptotic response observed.

ß-Lap activated apoptosis without inducing p53/p21 or cell cycle arrest responses and killed MCF-7 cells solely by apoptosis. In contrast, concentration-dependent increases in nuclear p53/p21 and various cell cycle checkpoint arrests were seen in MCF-7 cells after CPT. Despite dramatic p53/p21 protein induction responses, CPT-treated MCF-7 cells showed low levels of apoptosis, possibly due to protective cell cycle checkpoints or the lack of specific CPT-activated apoptotic pathways in MCF-7 cells.

¹ Funded by the efforts of Sara Hildebrand through the Breast Cancer Inspiration Fund and the Breast Cancer Research Fund. This work was also funded by United States Army Medical Research and Material Command Breast Cancer Initiative Grant BC971431 (to D. A. B.), by a United States Army postdoctoral fellowship (to J. J. P.), and by a University of Wisconsin-Madison Comprehensive Cancer Center core grant.

² To whom requests for reprints should be addressed, at Department of Human Oncology, K4/626 Clinical Science Center, 600 Highland Avenue, University of Wisconsin-Madison, Madison, WI 53792. Phone: (608) 262-4970; Fax: (608) 263-8613; E-mail: boothman@humonc.wisc.edu.

Received 9/26/97. Accepted 3/ 4/98.

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