AR-agonists induce cellular senescence in LNCaP and C4-2 human prostate cancer cell lines in a concentration dependent manner. Androgen dependent growing LNCaP and castration resistant C4-2 PCa cell lines were incubated with DMSO as solvent control and different concentrations of the synthetic and more stable androgen R1881 or the natural androgen DHT for 72 h. Because DHT is metabolized rapidly, DHT was added daily. 1 pM R1881 is defined as low androgen levels (LAL) and 1 nM R1881 as supraphysiological androgen levels (SAL). Cells were fixed and analyzed for SA β-Gal activity using a light microscopy and 3x 200 cells were counted and as means of the triplets in percent diagramed. A. SA β-gal activity of LNCaP cells treated with R1881 (methyltrienolone). B. SA β-gal activity of LNCaP cells treated with DHT. C. SA β-Gal staining in LNCaP cells at 200x magnification by phase microscopy. Upper panel: Arrowheads indicate the blue precipitations in the cytoplasm in senescent cells. Lower panel depicts the formation of senescence associated heterochromatic foci (SAHF) via DAPI staining of the nucleus. For fluorescence microscopy a 1000x magnification was used. Arrowheads mark accumulation of heterochromatic foci. D. SA β-Gal activity of the human castration resistant PCa cells C4-2. E. Microscopy of cells after SA β-Gal activity detection (upper panel) and DAPI staining with SAHF formation (lower panel) after androgen treatment of C4-2 cells.