Figure 2.

Transient exposure of cells cultured at low density to androgens induced a self-sustained but reversible growth arrest in the G0/G1 phase of cell cycle. (A) Scheme of experimental design for transitory R1881 treatments. Cells were cultured for 7 d under LD-hypo conditions in the presence R1881 at the indicated concentration, then washed 3 times and further cultured in the presence of the indicated compounds (LD-hypo + R1881 → LD-hypo ± none or GSK or NSK). GSK: 8 mM GSH, 0.2 µM SB505124 and 0.2 µM K02288; NSK: 8 mM NAC, 0.2 µM SB505124 and 0.2 µM K02288. For cloning efficiency measurement, cell colonies were counted at day 27. RT-qPCR and flow cytometry analysis were performed at day 7 (LD-hypo and LD-hypo + R1881) and day 14 (LD-hypo + R1881 → LD-hypo). (B and C) RT-qPCR analysis of the variations in the expression of the dormancy signature genes according to the indicated cell culture conditions in LNCaP* and VCaP cells. R1881 was used at a 0.5 nM concentration. Values are the mean ± sd of 2 independent experiments. Asterisks indicated statistical significance for the differences in mRNA levels between cells cultured under LD-hypo and LD-hypo + R1881 (red asterisks) or LD-hypo + R1881 → LD-hypo (black asterisks) conditions. (D and E) Inhibition of the cloning of LNCaP* and VCaP cells by R1881 and its subsequent reversal by the GSK or NSK mix. Data are averaged from 2 and 3 independent experiments for LNCap and VCaP cells respectively. Statistical significance of the effect of R1881 compared with non-treated cells (red asterisks) and of the effects of the GSK or NSK mix compared with cells treated with R1881 only (blue asterisks) are indicated. (F and G) Cell cycle analysis of LNCaP* and VCaP cells after a 7-days culture in the presence 0.5 nM R1881 followed by a 7-days culture without R1881 under LD-hypo conditions. Data are from the same experiment as those displayed in Fig. 1E and F.