INDUCTION OF APOPTOSIS IN MCF-7-WS8 BREAST-CANCER CELLS BY BETA-LAPACHONE

beta-Lapachone (beta-lap) affects a number of enzymes in vitro, includ ing type I topoisomerase (Topo I); however, its exact intracellular ta rget(s) and mechanism of cell killing remain unknown. We compared the cytotoxic responses of MCF-7:WS8 (MCF-7) human breast cancer cells aft er 4-h pulses of beta-lap or camptothecin (CPT), a known Topo I poison . A direct correlation between loss of survival and apoptosis was seen after beta-lap treatment (LD50 = 2.5 mu M). A concentration-dependent , transient sub-2 N preapoptotic cell population was observed at 4-8 h . Estrogen deprivation-induced synchronization and bromodeoxyuridine-l abeling studies revealed an apoptotic exit point near the G(1)-S borde r. Apoptosis activated by beta-lap was closely correlated with cleavag e of lamin B but not with increases in p53/p21 or decreases in bcl-2. Loss of hyperphosphorylated forms of the retinoblastoma protein nas ob served within 5 h, but cyclins A, B1, and E levels were unaltered for up to 72 h after 5 mu M beta-lap. Topo I and Topo II alpha levels decr eased at >23 h. Logarithmic-phase MCF-7 cells were not affected by les s than or equal to 1 mu M beta-lap. In contrast, dramatic and irrevers ible G(2)-M arrest with no apoptosis was observed in MCF-7 cells treat ed with 1 mu M CPT, monitored for 6-10 days posttreatment. MCF-7 cells treated with supralethal doses of CPT (5 mu M) resulted in only simil ar to 20% apoptosis. No correlation between apoptosis and loss of surv ival was observed. MCF-7 cells exposed to >5 mu M CPT arrested at key cell cycle checkpoints (i.e., G(1), S, and G(2)-M), with little or no movement for 6 days. Ten-fold increases in p53/p21 and 2-5-fold decrea ses in bcl-2, Topo I, Topo II alpha, and cyclins A and B1, with no cha nge in cyclin E, were observed. Temporal decreases in bcl-2 and cleava ge of lamin B corresponded to the minimal apoptotic response observed. beta-Lap activated apoptosis without inducing p53/p21 or cell cycle a rrest responses and killed MCF-7 cells solely by apoptosis. In contras t, concentration-dependent increases in nuclear p53/p21 and various ce ll cycle checkpoint arrests were seen in MCF-7 cells after CPT. Despit e dramatic p53/p21 protein induction responses, CPT-treated MCF-7 cell s showed low levels of apoptosis, possibly due to protective cell cycl e checkpoints or the lack of specific CPT-activated apoptotic pathways in MCF-7 cells.