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Comparative Study
. 2008 May 1;7(9):1262-8.
doi: 10.4161/cc.7.9.5945. Epub 2008 Mar 7.

Distinct roles for p107 and p130 in Rb-independent cellular senescence

Brian D Lehmann  1 , Adam M Brooks, Matthew S Paine, William H Chappell, James A McCubrey, David M Terrian
Affiliations
Comparative Study

Distinct roles for p107 and p130 in Rb-independent cellular senescence

Brian D Lehmann et al. Cell Cycle. .

Abstract

Telomere attrition, DNA damage and constitutive mitogenic signaling can all trigger cellular senescence in normal cells and serve as a defense against tumor progression. Cancer cells may circumvent this cellular defense by acquiring genetic mutations in checkpoint proteins responsible for regulating permanent cell cycle arrest. A small family of tumor suppressor genes encoding the retinoblastoma susceptibility protein family (Rb, p107, p130) exerts a partially redundant control of entry into S phase of DNA replication and cellular proliferation. Here we report that activation of the p53-dependent DNA damage response has been found to accelerate senescence in human prostate cancer cells lacking a functional Rb protein. This novel form of irradiation-induced premature cellular senescence reinforces the notion that other Rb family members may compensate for loss of Rb protein in the DNA damage response pathway. Consistent with this hypothesis, depletion of p107 potently inhibits the irradiation-induced senescence observed in DU145 cells. In contrast, p130 depletion triggers a robust and unexpected form of premature senescence in unirradiated cells. The dominant effect of depleting both p107 and p130, in the absence of Rb, was a complete blockade of irradiation-induced cellular senescence. Onset of the p107-dependent senescence was temporally associated with p53-mediated stabilization of the cyclin-dependent kinase inhibitor p27 and decreases in c-myc and cks1 expression. These results indicate that p107 is required for initiation of accelerated cellular senescence in the absence of Rb and introduces the concept that p130 may be required to prevent the onset of terminal growth arrest in unstimulated prostate cancer cells lacking a functional Rb allele.

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Figures

Figure 1
Figure 1
Irradiation-induced senescence is dependent on functional p53 protein. (A) DU145 and DU/WT p53 cells were either mock (0 Gy) or irradiated (4 Gy) and stained for β-galactosidase activity five days later. (B) The percentage of β-galactosidase positive cells were scored using bright-field microscopy. (C) DU145 and DU/WT p53 cells were stained for β-galactosidase activity and DAPI to reveal SAHF. DAPI fluorescence was converted to grayscale and inverted to demonstrate areas of condensed DNA.
Figure 2
Figure 2
Irradiation-induced senescence is independent of Rb. DU145 and DU/WT p53 cells were monitored for changes in protein expression associated with the onset of senescence. Cells lysates were immunoblotted for p53, c-myc, skp2, cks1, p27, cdk2, cyclin E, cyclin D1, cyclin B, cyclin A, p107 and p130 at 0, 1, 2, 3 and 5 days post-irradiation (4 Gy). β-Actin served as a protein loading control.
Figure 3
Figure 3
Rb-independent senescence in DU/WT p53 cells requires functional p107 protein. (A) Experimental timeline for DU/WT p53 cells treated with either missense siRNA, p107 siRNA, p130 siRNA or p107 and p130 siRNA. Cells were treated with siRNA for 2 d and re-treated at 4 d. Senescence-associated β-galactosidase staining was done 5 d after IR (4Gy). (B) Cell lysates were prepared prior to IR at 2 d post siRNA transfection and at 5 d after IR to evaluate knockdown of p107 and p130. (C) Number of SA-β-gal positive cells were scored as a percentage for the various treatments. (D) Images show representative SA-β-galactosidase staining and SAHF using DAPI of DU/WT p53 cells. Arrows indicate individual cells staining positive for SA-β-gal (black) and SAHF (white). Cell containing condensed chromosomes undergoing mitosis (M) are labeled accordingly.
Figure 4
Figure 4
Rb-independent senescence is mediated by p53-dependent stabilization of p27. Substantial DNA damage activates ATM/ATR, which in turn stabilizes p53. p53 can stabilize p27 directly by repressing the transcription of cks1 or indirectly by repressing c-myc transcription, relieving p27 repression and preventing c-myc dependent transcription of cks1. The stabilized p27 then inhibits both cyclin E and cyclin D, essential for G1-S progression. Unphosphorylated p107 is then able to permanently halt the cell cycle and maintain premature senescence. (------) lines indicate tentative and hypothetical interactions.
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