Results: 5

1.
Figure 2

Figure 2. From: Nutlin-3a Activates p53 to Both Down-regulate Inhibitor of Growth 2 and Up-regulate mir-34a, mir-34b, and mir-34c Expression, and Induce Senescence.

mir-34 expression was increased by nutlin-3a treatment in a p53-dependent manner. A, NHF-hTERT cells were treated with 10 μmol/L nutlin-3a for indicated time points. Total RNA (10 ng) extracted from each of the treated cells was used for real-time reverse transcription–PCR (RT-PCR) analysis of mir-34a, mir-34b, and mir-34c and RNU66 expression. RNU66 transcripts were used as an internal control. Statistical analysis was performed by Student’s t test. Columns, average of three independent experiments; bars, SD. B, the human tumor cell lines shown were left untreated or treated with 10 μmol/L nutlin-3a for 24 h. Total RNA (10 ng) was then extracted and used for real-time RT-PCR analysis of mir-34a and RNU66 expression. RNU66 transcripts were used as an internal control. Statistical analysis was performed by Student’s t test. Columns, average of three independent experiments; bars, SD. C, total RNA (10 ng) was then extracted from the same cell lines as above and used for real-time RT-PCR analysis of mir-34b and RNU66 expression. RNU66 transcripts were used as an internal control. Statistical analysis was performed by Student’s t test. Columns, average of three independent experiments; bars, SD. D, the human tumor cell lines were left untreated or treated with 10 μmol/L nutlin-3a for 24 h. Total RNA (10 ng) was then extracted from the cells and used for real-time RT-PCR analysis of mir-34c and RNU66 expression. RNU66 transcripts were used as an internal control. Statistical analysis was performed by Student’s t test. Columns, average of three independent experiments; bars, SD.

Kensuke Kumamoto, et al. Cancer Res. ;68(9):3193-3203.
2.
Figure 1

Figure 1. From: Nutlin-3a Activates p53 to Both Down-regulate Inhibitor of Growth 2 and Up-regulate mir-34a, mir-34b, and mir-34c Expression, and Induce Senescence.

Nutlin-3a induces primary and immortalized normal human fibroblasts to undergo senescence in a p53-dependent manner. A, normal human fibroblasts, GM08402, and NHF-hTERT were treated with 10 μmol/L nutlin-3a, nutlin-3b, or left untreated (NT) for 7 d. SA-β-gal assay was performed after the treatment. Magnification, 200 ×. B, three primary fibroblast strains, GM08402, IMR-90, and MRC-5, were treated with 10 μmol/L nutlin-3a, nutlin-3b, or left untreated for 7 d. The percentage of cells stained by SA-β-gal was calculated. Columns, average of five random fields per dish of three independent experiments; bar, SD. C, NHF-hTERT, an E6-expressing cell line (NHF-hTERT E6), and a p53 shRNA-expressing cell line (NHF-hTERT p53 shRNA) were treated with 10 μmol/L nutlin-3a, nutlin-3b, or left untreated for 7 d. SA-β-gal was assayed after the treatment. Columns, as above; bars, SD. D, NHF-hTERT, NHF-TERT E6 (E6), and NHF-hTERT p53 shRNA (p53 shRNA) cells were treated with 10 μmol/L nutlin-3a (3a), nutlin-3b (3b), or left untreated for 24 h. As a positive control, each cell line was treated with 1 μmol/L doxorubicin (dox) for 24 h. Cell lysates from these cells were immunoblotted with the indicated antibodies.

Kensuke Kumamoto, et al. Cancer Res. ;68(9):3193-3203.
3.
Figure 5

Figure 5. From: Nutlin-3a Activates p53 to Both Down-regulate Inhibitor of Growth 2 and Up-regulate mir-34a, mir-34b, and mir-34c Expression, and Induce Senescence.

Down-regulation of ING2 induces cellular senescence. A, NHF-hTERT cells were treated with the following siRNA oligonucleotides at 100 nmol/L concentration: a random sequence control (control), an ING2 siRNA which reduces ING2 levels by almost 50% (ING2-1), and an ING-2 siRNA which almost completely inhibits ING2 expression (ING2-2) for 24 h. After the treatment, 10 μmol/L nutlin-3a or nutlin-3b was added to cells for 72 h. Cells were stained for SA-β-gal assay. Magnification, 200 ×. B, after the procedure as above, the percentage of blue-stained cells was calculated. Columns, five random fields were counted per dish in each experiment. Three separate experiments were done in triplicate, and the results were averaged. Bars, SD. C, 100 nmol/L siRNA ING2-2 or control was transfected to 041 p53 −/− cells. 24 h later, cells were treated with 10 μmol/L nutlin-3a or nutlin-3b for 72 h. SA-β-gal assay was performed. The positive stained cells were counted and shown as percentage. Columns, same as above; bars, SD. D, protein lysates were made from cells treated as in B and immunoblotted with the indicated antibodies.

Kensuke Kumamoto, et al. Cancer Res. ;68(9):3193-3203.
4.
Figure 4

Figure 4. From: Nutlin-3a Activates p53 to Both Down-regulate Inhibitor of Growth 2 and Up-regulate mir-34a, mir-34b, and mir-34c Expression, and Induce Senescence.

p53 binds to ING2 regulatory region and suppresses luciferase activity. A, HCT116 p53+/+, HCT116 p53−/−, NHF-hTERT, NHF-hTERT E6, and RKO cells were treated with 10 μmol/L nutlin-3a or the same amount of DMSO for 24 h. After the treatment, ChIP was performed as described in Material and Methods. B, EMSA was carried out using biotinated oligonucleotides generated from the candidates of p53 consensus DNA-binding sequence on the ING2 regulatory regions, ING site 1 and site 2. Recombinant p53 protein and anti-p53 (DO-1) antibody were used for detecting shifted or super-shifted bands. Each unlabeled oligonucleotide (100-fold excess) was used as a competitor. C, four kinds of luciferase constructs using pGL-3 basic vector were designed as A, B, C, and D. Each 1.6 μg of a pGL-3 luciferase reporter and 0.16 μg of Renilla luciferase assay vector pRL were cotransfected into the p53 isogenic pairs of HCT116 and RKO cells. At 24 h later, 10 μmol/L nutlin-3a or the same amount of DMSO was added to cells for 12 h. Variation in transfection efficiency was normalized by Renilla luciferase activity. Columns, average of relative luciferase activity in three independent experiments; bars, SD.

Kensuke Kumamoto, et al. Cancer Res. ;68(9):3193-3203.
5.
Figure 3

Figure 3. From: Nutlin-3a Activates p53 to Both Down-regulate Inhibitor of Growth 2 and Up-regulate mir-34a, mir-34b, and mir-34c Expression, and Induce Senescence.

ING2 expression was decreased by nutlin-3a treatment in a p53-dependent manner. A, NHF-hTERT cells treated with 10 μmol/L nutlin-3a, nutlin-3b, or untreated for 24 h were lysed for Western blot analysis of ING2 proteins. B, p53 wild-type cells (HCT116, RKO, LS174T, A549, MCF-7, and NHF-hTERT), p53 mutant cells (SW620, WiDr, and H157), and p53 null cells (HCT116 p533/3, H1299, and Calu-6) were treated with 10 μmol/L nutlin-3a for 24 h. The same amount of DMSO was added as a control. Total RNA extracted from each of the treated cells was used for real-time RT-PCR analysis of ING2 and GAPDH mRNA expression. GAPDH mRNA transcripts were used as an internal control. Columns, average of three independent experiments; bars, SD. C, top, HCT116 p53+/+ and RKO cells were treated with varying doses of nutlin-3a (0, 2.5, 5, 7.5, and 10 μmol/L) for 24 h. Bottom, HCT116 p53+/+ and RKO cells were treated with 10 μmol/L nutlin-3a or the same amount of DMSO for indicated time points (0, 1, 4, 8, and 24 h). ING2 and p21 mRNA transcripts were measured by real-time RT-PCR analysis using indicated samples. These expressions were normalized by GAPDH mRNA expression. Columns, average of three independent experiments; bars, SD. D, the p53 isogenic pairs of HCT116 and NHF-hTERT were treated with 10 μmol/L nutlin-3a for indicated time points. The expression of ING2 was determined by Western blot analysis using total cell lysates prepared from these cells. β-Actin was probed as an internal control.

Kensuke Kumamoto, et al. Cancer Res. ;68(9):3193-3203.

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