Ectopic expression of cyclin D1 amplifies a retinoic acid-induced mitochondrial death pathway in breast cancer cells

All-trans retinoic acid inhibits growth associated with downregulation of c yclin D1 and can cause lon level apoptosis in estrogen receptor positive br east 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 c ells (MCF-7(cycD1) and ZR-75(cycD1)) Increased the fraction of cells in S p hase and reduced the G1 accumulation following retinoic acid treatment comp ared with control cells, However, culture of MCF-7(cycD1) with 1 muM all-tr ans retinoic acid resulted in about threefold greater growth inhibition com pared with vector-transfected cells. Hoechst staining of DNA and lit situ D NA end-labeling analysis indicated that MCF-7(cycD1) and ZR-75(cycD1) cultu res contained 4-6-fold more retinoic acid-induced apoptotic nuclei as vecto r-transfected cells. Retinoic acid treatment of vector-transfected clones r esulted in Bar protein activation as assessed by exposure of the NH2-termin us of Bar but the proportion of cells containing activated Bar was increase d in cyclin D-expressing cells treated with retinoic acid. The latter cells also displayed both immunocytochemical and biochemical evidence of translo cation of cytochrome c into the cytosol following RA-treatment, Retinoic ac id markedly decreased the Bcl-2 levels in MCF-7 and ZR-75 cells. Accordingl y, coexpression of Bcl-2 and cyclin D1 rendered the cells resistant to reti noic acid-induced apoptosis, We conclude that constitutive expression of cy clin D1 sensitizes ER-positive breast cancer cells to a retinoic acid-induc ed mitochondrial death pathway involving Bax activation, cytochrome c relea se and caspase-9 cleavage.