Rapid, non-destructive, cell-based screening assays for agents that modulate growth, death, and androgen receptor activation in prostate cancer cells.

BACKGROUND: We developed non-invasive, cell-based screening assays to rapidly and biologically assess factors that modulate prostate cancer growth and affect androgen receptor (AR) activity. METHODS: LNCaP cells, which stably express enhanced green fluorescent protein (EGFP) either constitutively or upon AR activation, were treated with a variety of agents, and then monitored by fluorescence and MTS assays for dose-dependent changes in cell number and AR activity. RESULTS: The assays were validated for rapid, fluorescence-based, quantitative measurement for the presence of growth and AR modulators. Using these assays, we found that osteoblast conditioned media (CM) enhanced prostate cancer cell growth, but not AR activity. After priming with androgen (<1 nM R1881), forskolin or the pesticide dichlorvos enhanced AR activation, whereas interleukin-6 (IL-6) inhibited it. CONCLUSION: These non-destructive, cell-based assays enable rapid systematic monitoring of the effects of drugs or complex mixtures on prostate cancer cell growth and/or AR activity.

Estrogen added intermittently, but not continuously, stimulates differentiation and bone formation in SaOS-2 cells.

Although it is well established that estrogen inhibits bone resorption, its effects on bone formation remain controversial. We studied the effects of intermittent and continuous treatment with estrogen on bone formation in vitro using long term cultures of SaOS-2 cells under conditions that permit mineralization. SaOS-2 cells cultured in dexamethasone, ascorbic acid and beta-glycerophosphate for up to 17 d formed mineralized bone nodules as visualized by von Kossa staining. Electron microscopic analysis of ultrathin sections of representative mineralized nodules showed the presence of mineral deposits, collagen fibrils and osteocytes. Both the mineralized nodule numbers and areas increased exponentially with time of culture after addition of beta-glycerophophate at day 8. Intermittent addition of 17beta-estradiol (E(2)) for 6 h or 24 h of every 48 h starting at day 3 or day 8 to the end of culture period resulted in a specific time- and dose-dependent stimulation of mineralized bone nodule number and area, and alkaline phosphatase activity which were accompanied with increase in cell numbers. On the other hand, continuous treatment with E(2) added every 48 h had no effect. The estrogen receptor alpha (ERalpha) mRNA expression was stimulated after 6 or 24-h (intermittent), but not after 48-h (continuous) treatment with E(2). The stimulatory effect of E(2), when added intermittently, but not continuously, on differentiation and bone formation in human osteoblasts in culture may be relevant to previous reports of stimulatory effects of E(2) on bone formation in vivo.