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. 2009 Sep 25;1(10):845-54.
doi: 10.18632/aging.100091.

Reduced transcriptional activity in the p53 pathway of senescent cells revealed by the MDM2 antagonist nutlin-3

Baoying Huang  1 , Lyubomir T Vassilev
Affiliations

Reduced transcriptional activity in the p53 pathway of senescent cells revealed by the MDM2 antagonist nutlin-3

Baoying Huang et al. Aging (Albany NY). .

Abstract

The p53 tumor suppressor plays a key role in induction and maintenance of cellular senescence but p53-regulated response to stress in senescent cells is poorly understood. Here, we use the small-molecule MDM2 antagonist, nutlin-3a, to selectively activate p53 and probe functionality of the p53 pathway in senescent human fibroblasts, WI-38. Our experiments revealed overall reduction in nutlin-induced transcriptional activity of nine p53 target genes and four p53-regulated microRNAs, indicating that not only p53 protein levels but also its ability to activate transcription are altered during senescence. Addition of nutlin restored doxorubicin-induced p53 protein and transcriptional activity in senescent cells to the levels in early passage cells but only partially restored its apoptotic activity, suggesting that changes in both upstream and downstream p53 signaling during senescence are responsible for attenuated response to genotoxic stress.

Keywords: MDM2; apoptosis; cancer; p53; senescence.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1.
Figure 1.
WI-38 cells cease to proliferate after extensive in vitro passaging and become senescent. (A) Cell cycle analysis after BrdU incorporation in early passage and senescent cells. S phase cells are within the rectangle. (B) Senescent WI-38 cells stain for SA-β-Gal activity. Scale bar is 50 μm. (C) SAHF in senescent WI-38 cells. Early passage and senescent cells were immunostained with anti-HP-1γ antibody (green) and counter stained with DAPI (red). Scale bar is 20 μm.
Figure 2.
Figure 2.
Transcriptional activity of p53 target genes in senescent WI-38 fibroblasts. (A) Basal transcription of p53 target genes is not compromised in senescent cells. Total RNA from early passage and senescent WI-38 cells was isolated and the expression of specific mRNAs was determined by quantitative PCR. Expression levels of each individual mRNA (from early passage and senescent cells) were normalized to 18S rRNA. Expression levels in senescent cells were calculated as fold change from the expression levels in early passage cells. The standard deviation (SD) was calculated from four independent experiments. (B) Basal expression levels of p53-regulated microRNA in senescent cells. Expression levels of individual microRNAs from early passage and senescent cells were determined by quantitative PCR, and normalized to RNU48 as an internal control. Expression levels in senescent cells were calculated and presented as in (A).
Figure 3.
Figure 3.. Transcriptional activity of nutlin-induced p53 is attenuated in senescent WI-38 cells.
(A) Protein level of p53 and two target genes, p21 and MDM2, in early passage and senescent cells. Cells were incubated in the presence of 10 μM nutlin-3a for 24 hours, lysed and subjected to Western blotting as described. (B) Induction of p53 target genes by nutlin-3a is decreased in senescent cells. Cells were treated as in (A), RNA was extracted and subjected to quantitative PCR. Fold induction is calculated as change in post compared to pre-treatment expression levels, both normalized to 18S rRNA. (C) Total expression levels of p53 target genes are reduced in nutlin-3a treated senescent cells. Cells were treated as in (B). Gene expression levels after exposure to nutlin are shown normalized to expression levels in early passage cells (100%). (D) Induction of p53 regulated microRNAs by nutlin-3a is decreased in senescent cells. Cells were treated as in (B). MicroRNA expression is determined by quantitative PCR and normalized to RNU48. Fold induction is calculated as in (B).(E) Total expression levels of p53-regulated microRNA are reduced in nutlin-treated senescent cells. Cells are treated as in (B), microRNA determined as in (D), and data presented as in (C).
Figure 4.
Figure 4.. Doxorubicin-induced apoptosis in senescent cells.
(A) Senescent WI-38 cells are resistant to doxorubicin-induced apoptosis. Early passage and senescent cells were incubated in the presence of 300 nM doxorubicin for 72 hours and the fraction of apoptotic cells was determined by the Annexin V assay. (B) Western blot analysis of early passage and senescent WI-38 cells treated with 300 nM doxorubicin for 24 hours. (C) Transcriptional activity of p53 target genes in doxorubicin-treated senescent WI-38 cells. Early passage and senescent cells were exposed to 300 nM doxorubicin, RNA was extracted for and data analyzed as in Figure 3B. (D) Effect of doxorubicin on the relative expression levels of p53 target genes in senescent cells. Cells were treated as in (C) and data calculated and presented as in Figure 3C. (E) Nutlin raises doxorubicin-induced p53 protein level in senescent cells. Early passage and senescent WI-38 cells were exposed to 300 nM doxorubicin, 10 μM Nutlin-3a, or combination of both for 24 hours prior to collection for Western analysis. (F) Nutlin increases apoptosis induced by doxorubicin in senescent cells. Senescent WI-38 cells were treated with 10 μM nutlin-3a, 300 nM doxorubicin or 10 μM nutlin-3a plus 300 nM doxorubicin for 72 hours and the apoptotic cell fractions were measured by the Annexin-V assay. (G) Nutlin restores transcriptional response to doxorubicin-induced p53 in senescent cells. Senescent cells were treated with 10 μM nutlin-3a, 300 nM doxorubicin or 10 μM nutlin-3a plus 300 nM doxorubicin for 24 hours and expression levels of indicated genes were determined by quantitative PCR, normalized, and calculated as fold change. (H) Nutlin restores the transcription of doxorubicin-induced p53 target genes in senescent cells to early passage levels. Early passage cells were exposed to 300 nM doxorubicin and senescent cells to 300 nM doxorubicin plus 10 μM nutlin-3a. 24 hours after treatment, mRNA levels were determined by quantitative PCR and normalized to expression levels in early passage cells (100%).
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