Repeated exposure of LNCaP cells to senescence-inducing stimuli via cyclic AD induces outgrowth of an androgen-refractory subpopulation.

<p>(<b>A</b>) Colony formation in GFP-labeled LNCaP cells and by crystal violet staining. Colonies indicates clones that exist in a transiently arrested state in the bulk population and resume division when androgen-replete conditions are restored after initial CSS culture. The indicated cells underwent one round of switch back to FBS media (SB1) following senescence in CSS culture. Following 21 days in FBS culture, clonal outgrowths were observed via crystal violet staining (∼0.1%). The insets show light microscopy and FITC images of a representative colony that has begun to proliferate in FBS media. (<b>B</b>) Schematic of the switch back (SB) method for generating androgen-refractory population of cells (designated LNCaP-SB5). (<b>C</b>) Proliferation curves for SB0 and SB5 cells in CSS. Cells were harvested on Day 7 and immunoblotted for cyclin A expression (inset) as a proliferation marker. (<b>D</b>) Morphology differences between LNCaP SB0 and SB5 cell populations. (<b>E</b>) Percentage SA-beta-gal positive cells: 87% LNCaP- SB0, 43% LNCaP-SB5. (<b>F</b>) PI cell cycle analysis (S phase: 0.7% LNCaP-SB0, 6.5% LNCaP-SB5). (<b>G</b>) Immunoblotting of 35 µg protein lysates from indicates samples against the denoted proteins. Note that AR expression remains elevated in SB5 versus SB0 cells. Androgen-refractory LNAI cells are shown as a positive control. GAPDH expression is used to normalize for protein loading. (<b>H</b>) Relative proliferation ability of SB5 in different co-cultures under AD. FL1-A indicates intensity of FITC signal and the right hand gate (V2-R) contains GFP-positive (SB5) cells whereas V2-L contains the co-mixed unlabeled cells. Representative flow profiles with the percentages (in red) of each co-mixed population are shown at the indicated time points. (<b>I</b>) Quantitation of GFP-positive (SB5) cell numbers under different co-culture conditions after 7 days of CSS culture are graphed. Quantitation was accomplished via the cell counting parameters from the CFlow software for counterpart cytometric profiles to those shown in (I).