Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27(Kip1).

Abstract

Adp27(Kip1), 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 p27(Kip1) on cell cycle and apoptosis were analyzed. We evaluated the effects of overexpression of p27(Kip1) in the human prostate carcinoma cell lines LNCaP, DU-145, and PC-3 in vitro and in vivo. Growth curve studies, cell cycle analysis, terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL), and annexin V-fluorescein isothiocyanate apoptosis analyses were conducted to determine effects of p27(Kip1) on cell cycle. CDKI activity assays and Western blots were conducted to determine presence/activities of CDKIs. Adp27(Kip1)-induced protein levels increased in a dose-dependent manner; p27(Kip1) protein was detected within 6 hr of infection with Adp27(Kip1) and remained stable for at least 48 hr. The activities of Cdk2, Cdk4, and Cdc2 kinases were inhibited 24 hr after infection with Adp27(Kip1). Bromodeoxyuridine incorporation demonstrated a dose-dependent decrease in S-phase cells 24 hr postinfection. TUNEL analysis revealed an induction of apoptosis (10 pfu/cell) within 48 hr of infection in all cell lines. Growth curve analyses demonstrated that Adp27(Kip1) infection inhibited proliferation of all cell lines tested and decreased cell numbers for Adp27(Kip1)-infected LNCaP and PC-3 cells by 96 hr. Cell cycle analysis of DNA content demonstrated an accumulation of cells in G0/G1-phase 24-120 hr after Adp27(Kip1)-infection. In vivo studies demonstrated a reduction in LNCaP xenograft tumor growth rates in mice injected with Adp27(Kip1). Exogenous p27(Kip1) overexpression 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, p27(Kip1) 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 study provides support for the further development of Adp27(Kip1) as a potential therapeutic vector in the treatment of adenocarcinoma of the prostate.