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The chemotherapeutic drug paclitaxel induces microtubular stabilization and mitotic arrest associated with increased survivin expression. Survivin is a member of the inhibitor of apoptosis (iap) family which is highly expressed in during G2/M phase where it regulates spindle formation during mitosis. It is also constitutively overexpressed in most cancer cells where it may play a role in chemotherapeutic resistance. MCF-7 breast cancer cells stably overexpressing the sense strand of survivin (MCF-7(survivin-S) cells) were more resistant to paclitaxel than cells depleted of survivin (MCF-7(survivin-AS) despite G2/M arrest in both cell lines. However, survivin overexpression did not protect cells relative to control MCF-7(pcDNA3) cells. Paclitaxel-induced cytotoxicity can be enhanced by retinoic acid and here we show that RA strongly reduces survivin expression in MCF-7 cells and prevents paclitaxel-mediated induction of survivin expression. Mitochondrial release of cytochrome c after paclitaxel alone or in combination with RA was weak, however robust Smac release was observed. While RA/paclitaxel-treated MCF-7 (pcDNA3) cultures contained condensed apoptotic nuclei, MCF-7(survivin-S) nuclei were morphologically distinct with hypercondensed disorganized chromatin and released mitochondrial AIF-1. RA also reduced paclitaxel-associated levels of cyclin B1 expression consistent with mitotic exit. MCF-7(survivin-S) cells displayed a 30% increase in >2N/<4N ploidy while there was no change in this compartment in vector control cells following RA/paclitaxel. We propose that RA sensitizes MCF-7 cells to paclitaxel at least in part through survivin downregulation and the promotion of aberrant mitotic progression resulting in apoptosis. In addition we provide biochemical and morphological data which suggest that RA-treated MCF-7(survivin-S) cells can also undergo catastrophic mitosis when exposed to paclitaxel.

All-trans retinoic acid inhibits growth associated with downregulation of cyclin D1 and can cause low level apoptosis in estrogen receptor positive breast cancer cell lines. The cyclin D1 gene is amplified and/or the protein overexpressed in about one-third of breast cancers. Constitutive expression of cyclin D1 in estrogen receptor positive MCF-7 and ZR-75 breast cancer cells (MCF-7(cycD1) and ZR-75(cycD1)) Increased the fraction of cells in S phase and reduced the G1 accumulation following retinoic acid treatment compared with control cells. However, culture of MCF-7(cycD1) with 1 microM all-trans retinoic acid resulted in about threefold greater growth inhibition compared with vector-transfected cells. Hoechst staining of DNA and in situ DNA end-labeling analysis indicated that MCF-7(cycD1) and ZR-75(cycD1) cultures contained 4-6-fold more retinoic acid-induced apoptotic nuclei as vector-transfected cells. Retinoic acid treatment of vector-transfected clones resulted in Bax protein activation as assessed by exposure of the NH(2)-terminus of Bax but the proportion of cells containing activated Bax was increased in cyclin D-expressing cells treated with retinoic acid. The latter cells also displayed both immunocytochemical and biochemical evidence of translocation of cytochrome c into the cytosol following RA-treatment. Retinoic acid markedly decreased the Bcl-2 levels in MCF-7 and ZR-75 cells. Accordingly, coexpression of Bcl-2 and cyclin D1 rendered the cells resistant to retinoic acid-induced apoptosis. We conclude that constitutive expression of cyclin D1 sensitizes ER-positive breast cancer cells to a retinoic acid-induced mitochondrial death pathway involving Bax activation, cytochrome c release and caspase-9 cleavage.