Induction of human breast cancer cell apoptosis from G(2)/M preceded by stimulation into the cell cycle by Z-1,1-dichloro-2,3-diphenylcyclopropane

We have shown previously that Z-1,1-dichloro-2,3-diphenylcyclopropane (a.k. a. Analog II, A(II)) inhibits human breast cancer cell proliferation regard less of estrogen receptor status or estrogen sensitivity, and that its cell ular targets include microtubules. In the present study, we investigated th e apoptosis-inducing effects of A(II). MCF-7, MCF-7/LY2, and MDA-MB-231 cel ls all showed nuclear fragmentation in response to 100 mu M A(II) when stai ned with Hoechst 33342 and examined by fluorescence microscopy. Pulsed fiel d gel electrophoretic analysis showed that each of the cell lines also deve loped specific high molecular weight DNA fragments: a low level of 1-2 Mb f ragments appeared after 6 hr, while 30-50 kb fragments accumulated subseque ntly. At 24 hr of drug exposure, the majority of cells became nonadherent, and the 30-50 kb fragments were restricted to detached MCF-7 and MDA-MB-231 cells. Roth adherent and detached MCF-7/LY2 cells exhibited these fragment s. A previous study by single-color (propidium) flow cytometry demonstrated that A(II) blocks MDA-MB-231 cells in G(2)/M of the cell cycle. More refin ed analyses in the present study showed this same result for MDA-MB-231 cel ls, but MCF-7 and MCF-7/LY2 cells did not reveal apparent drug-induced cell cycle block. A(II) demonstrated growth inhibitory, cell cycle-perturbing, and hypodiploidy-inducing activity against other human breast carcinoma lin es, i.e. BT-20, CAMA-1, and SKBR-3, but no such actions in the non-tumorige nic, "normal" human breast epithelial line MCF-10A. Bromodeoxyuridine label ing and two-color flow cytometric analysis, however, suggested that A(II) c aused stimulation into S phase, and that G(2)/M was the phase of the cell c ycle from which cells apoptosed. A(II) caused cell rounding, detachment fro m the growth matrix, and nuclear shrinkage and fragmentation in parallel wi th biochemical changes, Cycloheximide inhibited A(II)-induced cell death, i ndicating that its toxicity requires de novo protein synthesis. (C) 1998 El sevier Science Inc.