Figure 6.

Cell cycle effects of methylated pRb. (A) U2OS cells stably expressing inducible wild-type pRb, pRb K810R or pTRE2 (empty vector control) were treated with 1 μg/ml doxycycline (Dox) for 72 h to induce expression. Induced pRb was detected using anti-Flag antibody. (B) Inducible wild-type pRb and pRb K810R U2OS cells were plated onto glass coverslips and treated as in (A). Induced pRb was detected using anti-Flag antibody and DAPI was used to visualize nuclei. (C) U2OS cells stably expressing inducible wild-type pRb, pRb K810R or pTRE2 (empty vector control) were treated with 1 μg/ml doxycycline for 72 h to induce expression. Cells were then prepared for cell cycle analysis by flow cytometry as detailed in Materials and methods. The mean percentage of cells observed in G1 was then derived from triplicate samples for each condition. The data were expressed in graphical format as the relative difference between the mean percentage of G1 cells before and after induction with doxycycline. (D) U2OS cells were transfected with 20 nM non-targeting (N) or Set7/9 (S) siRNA for 72 h. Cells were also treated with 10 μM etoposide for 16 h (Et) where indicated. Cells were then prepared for cell cycle analysis by flow cytometry as detailed in Materials and methods. The mean percentage of cells in each stage of the cell cycle was then derived from triplicate samples. The data were expressed in graphical format as the difference between the mean percentages of cells treated with Set7/9 siRNA or non-targeting siRNA (under the presence and absence of etoposide (Et)) (i). Actual G1 values were as follows: no etoposide treatment, non-targeting siRNA=67.16±1.20%, Set7/9 siRNA=61.38±0.48% (P<0.01); with etoposide treatment, non-targeting siRNA=56.74±0.61%, Set7/9 siRNA=48.46±0.23% (P<0.01). The corresponding immunoblot for this analysis is also shown (ii). (E) U2OS cells were transfected with 20 nM non-targeting (N) or Set7/9 (S) siRNA for 72 h. Cells were also treated with 10 μM etoposide (Et) and 300 μM olomoucine for 16 h where indicated. Cell extracts were prepared and immunoblotted with the indicated antibodies. (F) Model depicting the effect of methylation on Cdk-dependent growth control. Cdk phosphorylation of pRb relieves cell cycle arrest by facilitating the expression of E2F target genes. Under conditions of methylation, pRb remains in a hypophosphorylated growth-arresting state, thereby limiting E2F target gene expression assisting cell cycle arrest.