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Figure 2.
Fig. 2. Inhibition of MDM2-p53 binding by Nutlin-1 activates
the p53 pathway in cells with wild-type p53. (A) SW480 (mutant
p53) and HCT116 (wild-type p53) cells were incubated with the
indicated concentrations of Nutlin-1 for 8 hours and p53, MDM2,
and p21 proteins were analyzed in the cell lysates by Western
blotting. (B) Nutlin-1 treatment induces the expression of the
p21 gene but not the p53 gene. Cells with wild-type p53 (HCT116,
RKO, and H460a) were treated with Nutlin-1 for 8 hours, and the
change in the level of transcription was measured by
quantitative PCR and expressed as fold induction compared with
the untreated control. (C) Nutlin-1 arrests cell cycle in the
G[1] and G[2] phases. HCT116 and SJSA-1 cells were incubated
with 4 µM Nutlin-1 or an equivalent amount of solvent for
22 hours and an additional 2 hours with 10 µM BrdU, and
cell cycle distribution was analyzed after propidium
iodide/fluorescein isothiocyanate-antibody to BrdU staining
(30). Cells within the rectangles are in S phase. (D)
Antiproliferative and cytotoxic activity of Nutlin-1.
Exponentially growing cancer cells with wild-type p53 (HCT116,
RKO, and SJSA-1) or mutant p53 (MDA-MB-435 and SW480) were
incubated with a range of concentrations for 5 days and the cell
mass and viability were measured by the MT T assay. (E)
Inhibition of clonogenic cell growth. Cancer cells with
wild-type p53 (HCT116 and RKO) or mutant p53 (MDA-MB-435, SW480,
and PC3) were seeded at a low cell density and their ability to
form colonies was measured after 5 days of incubation with
Nutlin-1. (F) p53 activation by Nutlin-1 does not involve Ser15
phosphorylation on p53. Cancer cells were treated with
doxorubicin (1 µM), etoposide (10 µM), or Nutlin-1
(6 µM) for 20 hours, and the amount of total p53 and p53
phosphorylated on Ser15 was determined by Western blottingin
aliquots of cell lysates normalized for total protein.
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