Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27Kip1
Adrienne L. Katner, Praveen Gootam, Quoc Bao Ly Hoang, Ewa Jaruga, Qiangwei Ma, and Walter Rayford2
Departments of Urology, Louisiana State University Health Sciences Center, New Orleans, LA, 70112
Submitted to Prostate
ABSTRACT
Background: Adp27Kip1, a recombinant adenovirus was evaluated for expression of p27, a cyclin-dependent kinase inhibitor (CDKI) and tumor suppressor protein, in human prostate carcinoma cells. Effects of p27Kip1 on cell cycle and apoptosis were analyzed.
Methods: We evaluated the effects of over-expression of p27Kip1 in the human prostate carcinoma cell lines LNCaP, DU-145 and PC-3 in vitro and in vivo. Growth curve studies, cell cycle analysis, TUNEL, and annexin V-FITC apoptosis analyses were conducted to determine effects of p27Kip1 on cell cycle. CDKI activity assays and westerns were conducted to determine presence/activities of CDKIs.
Results: Adp27Kip1-induced protein levels increased in a dose-dependent manner; p27Kip1 protein was detected within 6 hours of infection with Adp27Kip1 and remained stable for at least 48 hours. The activities of Cdk2, Cdk4, and Cdc2 kinases were inhibited 24 hours after infection with Adp27Kip1. BrdU incorporation demonstrated a dose-dependent decrease in S-phase cells 24 hours post-infection. TUNEL analysis revealed an induction of apoptosis (10 pfu/cell) within 48 hours of infection in all cell lines. Growth curve analyses demonstrated that Adp27Kip1 infection inhibited proliferation of all cell lines tested and decreased cell numbers for Adp27Kip1-infected LNCaP and PC-3 cells by 96 hours. Cell cycle analysis of DNA content demonstrated an accumulation of cells in G0/G1-phase 24-120 hours after Adp27Kip1-infectio. In vivo studies demonstrated a reduction in LNCaP xenograft tumor growth rates in mice injected with Adp27Kip1.
Conclusions: Exogenous p27Kip1 over-expression results in cell cycle regulation in the human prostate carcinoma cell lines tested, representing the first use of this vector on prostate cancer cell lines in vitro and in vivo. Moreover, p27Kip1 expression is associated with an increase in early apoptosis, which represents a recently discovered function for this protein. It also represents the first time this association has been observed in prostate carcinoma cell lines. This strategy may provide a therapeutic alternative for the treatment of adenocarcinoma of the prostate, as suggested by in-vivo results.