Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression.
We have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1-S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.
Grillo
Mary
M
Oncology Research, Wyeth Research, Pearl River, NY 10965, USA.
Bott
Matthew J
MJ
Khandke
Neha
N
McGinnis
John P
JP
Miranda
Miriam
M
Meyyappan
Muthupalaniappan
M
Rosfjord
Edward C
EC
Rabindran
Sridhar K
SK
D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051358:Cyclin-Dependent Kinase 4 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005260:Female; D016193:G1 Phase; D020868:Gene Silencing; D006801:Humans; D047468:Immunoprecipitation; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000494:pharmacology*; D016160:Retinoblastoma Protein / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; D019938:Cyclin D1; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D013752:Tetracycline