Androgen stimulated cellular proliferation in the human prostate cancer cell line LNCaP is associated with reduced retinoblastoma protein expression

Abstract

To elucidate the mechanism of androgen-dependent cellular proliferation in prostate cancer, androgen-dependent alterations of individual cell cycle regulatory proteins in the androgen-sensitive prostate cancer cell line LNCaP were evaluated. LNCaP cells were deprived of androgens by culture in steroid-depleted media for 5 days, which resulted in the maximal accumulation of cells in G(0)/G(1) phase of the cell cycle. The mitogenic concentration of the synthetic androgen R1881 was established as 0.1 nM using cell proliferation assay. Protein and mRNA levels of particular cyclins, cyclin-dependent kinases (Cdks), cyclin-dependent kinase inhibitors (Ckis), and the retinoblastoma proteins (Rb) were assessed. Androgen stimulation resulted in a post-transcriptional reduction in Rb protein levels, an increase in Rb phosphorylation at serine 780 and an accumulation of high molecular weight Rb protein species. Androgen stimulation also induced the expression of the Cdk2 and Cdk1 as well as their regulatory partners, cyclin A and cyclin B, resulting in a corresponding increase in cyclin A/Cdk2 activity in vitro. Pulse-chase showed decreased Rb protein stability in androgen-treated LNCaP cells. Collectively, our findings suggest a novel mechanism of androgen-dependent prostate cancer growth in which androgen stimulation results in decreased Rb protein expression in LNCaP cells. The observation of decreased Rb protein stability in the setting of increased phosphorylation supports the concept of phosphorylation mediated protein degradation. We propose that the observed reduction in Rb protein level occurs through Rb degradation via the ubiquitin/proteasome pathway, and is preceded by selective Rb phosphorylation by cyclin A/Cdk2 and cyclin B/Cdk1.

Expression of a hyperactive androgen receptor leads to androgen-independent growth of prostate cancer cells

“…Previous work has suggested that reduced Rb protein expression may be responsible for androgen-induced proliferation of LNCaP cells (Taneja et al 2002). In support of this, we observed that R1881 repressed Rb expression in LNCaP cells (C14 cells; Fig.…”

“…4). Rb protein expression was repressed by androgen in C14 cells, as shown previously (Taneja et al 2002). Interestingly, Rb expression was significantly reduced in V94 cells, but only in the absence of androgen, when compared with C14 cells.…”

A role for cyclin A1 in mediating the autocrine expression of vascular endothelial growth factor in prostate cancer

“…For the R1881 treatment, 0.5 Â 10 6 /ml cells were Vascular endothelial growth factor in prostate cancer B Wegiel et al maintained in medium containing charcoal-stripped fetal bovine serum (cFBS) (HyBond) before the treatment as previously described (Taneja et al, 2001). 0.1, 1, 5, 10, 15 and 20 nM R1881 (Dupont, Boston, MA, USA) dissolved in 70% ethanol were then added into the medium, and cells were collected after 72 h.…”

Therapeutic potential of CDK inhibitor NU2058 in androgen-independent prostate cancer

“…Reduced levels of p27, an inhibitor of CDK2, and increased levels of CDK2 and cyclin E are indicators of poor prognosis and associated with androgen independence (Guo et al, 1997;Macri and Loda, 1998;Gregory et al, 2001;Karan et al, 2002). Androgen withdrawal caused G1 arrest in androgen-dependent LNCaP cells and the reintroduction of androgens resulted in increased expression of cyclins A and B1, and CDK1 and CDK2 with resultant increase in CDK2 activity and pRb phosphorylation, but no change in cyclins D1 or E or CDK4 or -6 or p16, p21 or p27 (Taneja et al, 2001). Antiandrogens block the androgen-androgen receptor (AR) binding that promotes cell cycle progression and inhibits cell cycle arrest (Masiello et al, 2002), therefore one potential tool for the treatment of AIPC is to inhibit cell cycle progression by inhibiting CDK2 directly.…”