Key role of the cyclin-dependent kinase inhibitor p27kip1 for embryonal carcinoma cell survival and differentiation.
Hexamethylen-bisacetamide (HMBA) represents the prototype of a group of hybrid polar compounds, which induce differentiation in a variety of transformed cells including human embryonal carcinoma cells. Therefore, HMBA has been used in the differentiation therapy of cancer for patients with both hematological and solid malignancies. Upon HMBA treatment, the embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) accumulates in G1 and undergoes terminal differentiation. Here we demonstrate that growth arrest and differentiation of NT2/D1 cells induced by HMBA involve increased expression of the cyclin-dependent kinase inhibitor p27, enhanced association of p27 with cyclin E/CDK2 complexes and suppression of kinase activity associated to cyclin 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-apoptotic Bcl-2 gene expression, cells underwent massive programmed cell death. Conversely, constitutive expression of p27 into NT2/D1 cells induced a marked reduction in the growth potential of these cells and partially reproduced HMBA-induced modification of surface antigen expression (down-regulation of SSEA-3 expression and up-regulation of VINIS-53 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 treatment with the kinase inhibitor olomucine induced growth arrest but not differentiation. 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 suggest that, at least in these cells, growth arrest and differentiation are regulated by two overlapping but different pathways.
Baldassarre
G
G
Servizio Oncologia Sperimentale E, Istituto Nazionale Tumori, Napoli, Italy.
Barone
M V
MV
Belletti
B
B
Sandomenico
C
C
Bruni
P
P
Spiezia
S
S
Boccia
A
A
Vento
M T
MT
Romano
A
A
Pepe
S
S
Fusco
A
A
Viglietto
G
G
D000081:Acetamides / Q000494:pharmacology; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics; D015295:Antigens, Tumor-Associated, Carbohydrate; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D042846:CDC2-CDC28 Kinases; D018236:Carcinoma, Embryonal / Q000378:metabolism / Q000473:pathology*; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000235:genetics; D016213:Cyclins / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic; D006028:Glycosphingolipids / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007701:Kinetin; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics; D011687:Purines / Q000494:pharmacology; D016160:Retinoblastoma Protein / Q000378:metabolism; D000077546:Roscovitine; D055746:Stage-Specific Embryonic Antigens; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D000081:Acetamides; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D015295:Antigens, Tumor-Associated, Carbohydrate; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D006028:Glycosphingolipids; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D011687:Purines; D016160:Retinoblastoma Protein; D055746:Stage-Specific Embryonic Antigens; D025521:Tumor Suppressor Proteins; C052924:stage-specific embryonic antigen-3; D000077546:Roscovitine; D050760:Cyclin-Dependent Kinase Inhibitor p27; C090046:olomoucine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; C014026:hexamethylene bisacetamide; D007701:Kinetin