p53-mediated growth suppression in response to Nutlin-3 in cyclin D1 transformed cells occurs independently of p21.
Interaction of cyclin D1 with cyclin-dependent kinases (CDK) results in the hyperphosphorylation of the RB family of proteins, thereby inactivating the tumor-suppressive function of RB. Our previous findings suggest that constitutive cyclin D1/CDK activity inhibits p53-mediated gene repression by preventing the appropriate regulation of CDK activity by the CDK inhibitor p21, a transcriptional target of p53. To study the role of cyclin D1 in driving human mammary cell transformation, we expressed a constitutively active cyclin D1-CDK fusion protein (D1/CDK) in immortalized human mammary epithelial cells. D1/CDK-expressing human mammary epithelial cells grew anchorage-independently in the presence of wild-type p53, consistent with the idea that D1/CDK disrupts downstream p53 signaling. Using this transformation model, we examined the sensitivity of the D1/CDK-expressing cells to Nutlin-3, an HDM2 antagonist that activates p53. Surprisingly, treatment of D1/CDK-transformed cells with Nutlin-3 prevented their anchorage-independent growth. The Nutlin-3-induced growth arrest was enforced in D1/CDK-expressing cells despite the presence of hyperphosphorylated RB implicating a p53-dependent, RB-independent mechanism for growth suppression. Further analysis identified that CDC2 and cyclin B1, key cell cycle regulators, were stably down-regulated following p53 stabilization by Nutlin-3, consistent with direct interaction between p53 and the CDC2 and cyclin B1 promoters, leading to the repression of transcription by methylation. In contrast to D1/CDK expression, direct inactivation of p53 resulted in no repression of CDC2 and no cell cycle arrest. We conclude that induction of p53 by Nutlin-3 is a viable therapeutic strategy in cancers with constitutive CDK signaling due to the direct repression of specific p53 target genes.
Kan
Charlene E
CE
Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic and Department of Genetics, Case Western Reserve University, Case Comprehensive Cancer Center, Cleveland, Ohio 44195, USA.
Patton
John T
JT
Stark
George R
GR
Jackson
Mark W
MW
D048708:Cell Growth Processes / Q000187:drug effects / Q000502:physiology; D002471:Cell Transformation, Neoplastic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019938:Cyclin D1 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D045325:HCT116 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D042361:Mammary Glands, Human / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D010879:Piperazines / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D016160:Retinoblastoma Protein / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D007093:Imidazoles; D010879:Piperazines; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D019938:Cyclin D1; C482205:nutlin 3