Key role of the cyclin-dependent kinase inhibitor p27(kip1) for embryonal carcinoma cell survival and differentiation

Hexamethylen-bisacetamide (HMBA) represents the prototype of a group of hyb rid polar compounds, which induce differentiation in a variety of transform ed cells including human embryonal carcinoma cells, Therefore, HMBA has bee n used in the differentiation therapy of cancer for patients with both hema tological and solid malignancies. Upon HMBA treatment, the embryonal carcin oma cell line NTERA-2 clone D1 (NT2/D1) accumulates in G1 and undergoes ter minal differentiation. Here we demonstrate that growth arrest and different iation of NT2/D1 cells induced by HMBA involve increased expression of the cyclin-dependent kinase inhibitor p27, enhanced association of p27 with cyc lin E/CDK2 complexes and suppression of kinase activity associated to cycli n E/CDK2 (but not to cyclin D3/CDK4), When HMBA differentiation was induced in the presence of p27 antisense oligonucleotides, NT2/D1 cells failed to arrest growth properly and, in parallel with the reduction of the anti-apop totic Bcl-2 gene expression, cells underwent massive programmed cell death. Conversely, constitutive expression of p27 into NT2/D1 cells induced a mar ked reduction in the growth potential of these cells and partially reproduc ed HMBA-induced modification of surface antigen expression (down-regulation of SSEA-3 expression and up-regulation of VINIS-3 expression). Expression of p21 induced growth arrest but not differentiation. Likewise, inhibition of CDK2 by transfection of a dominant negative CDK2 in NT2/D1 cells or trea tment with the kinase inhibitor olomucine induced growth arrest but not dif ferentiation, Therefore, we propose that p27 represents a crucial molecule in HMBA signaling that cannot be replaced by p21, Furthermore, the results obtained with CDK2 inhibitors demonstrate that the block of CDK2; activity is sufficient for growth arrest but not for cell differentiation and sugges t that, at least in these cells, growth arrest and differentiation are regu lated by two overlapping but different pathways.