title: Synchronized prostate cancer cells for studying androgen regulated events in cell cycle progression from G1 into S phase.
abstract: Androgen-ablation is a most commonly prescribed treatment for metastatic prostate cancer but it is not curative. Development of new strategies for treatment of prostate cancer is limited partly by a lack of full understanding of the mechanism by which androgen regulates prostate cancer cell proliferation. This is due, mainly, to the limitations in currently available experimental models to distinguish androgen/androgen receptor (AR)-induced events specific to proliferation from those that are required for cell viability. We have, therefore, developed an experimental model system in which both androgen-sensitive (LNCaP) and androgen-independent (DU145) prostate cancer cells can be reversibly blocked in G(0)/G(1) phase of cell cycle by isoleucine deprivation without affecting their viability. Pulse-labeling studies with (3)H-thymidine indicated that isoleucine-deprivation caused LNCaP and DU145 cells to arrest at a point in G(1) phase which is 12-15 and 6-8 h, respectively, before the start of S phase and that their progression into S phase was dependent on serum factors. Furthermore, LNCaP, but not DU145, cells required AR activity for progression from G(1) into S phase. Western blot analysis of the cell extracts prepared at regular intervals following release from isoleucine-block revealed remarkable differences in the expression of cyclin E, p21(Cip1), p27(Kip1), and Rb at the protein level between LNCaP and DU145 cells during progression from G(1) into S phase. However, in both cell types Cdk-2 activity associated with cyclin E and cyclin A showed an increase only when the cells transited from G(1) into S phase. These observations were further corroborated by studies using exponentially growing cells that were enriched in specific phases of the cell cycle by centrifugal elutriation. These studies demonstrate usefulness of the isoleucine-deprivation method for synchronization of androgen-sensitive and androgen-independent prostate cancer cells, and for examining the role of androgen and AR in progression of androgen-sensitive prostate cancer cells from G(1) into S phase.
authors.affiliation: Vattikuti Urology Institute, Henry Ford Health Sciences Center, Detroit, Michigan 48202, USA.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Cifuentes
authors.forename: Richard
authors.initials: R
authors.lastname: Croxen
authors.forename: Mani
authors.initials: M
authors.lastname: Menon
authors.forename: Evelyn R
authors.initials: ER
authors.lastname: Barrack
authors.forename: G Prem-Veer
authors.initials: GP
authors.lastname: Reddy
mesh_terms: D000728:Androgens / Q000502:physiology; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division; D003470:Culture Media; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016193:G1 Phase; D006801:Humans; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016196:S Phase; D014407:Tumor Cells, Cultured
chemical_list: D000728:Androgens; D018797:Cell Cycle Proteins; D003470:Culture Media; D019925:Cyclin A; D019927:Cyclin E; D007532:Isoleucine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases