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PDBsum entry 1rv1
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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In vivo activation of the p53 pathway by small-Molecule antagonists of mdm2.
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Authors
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L.T.Vassilev,
B.T.Vu,
B.Graves,
D.Carvajal,
F.Podlaski,
Z.Filipovic,
N.Kong,
U.Kammlott,
C.Lukacs,
C.Klein,
N.Fotouhi,
E.A.Liu.
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Ref.
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Science, 2004,
303,
844-848.
[DOI no: ]
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PubMed id
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Abstract
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MDM2 binds the p53 tumor suppressor protein with high affinity and negatively
modulates its transcriptional activity and stability. Overexpression of MDM2,
found in many human tumors, effectively impairs p53 function. Inhibition of
MDM2-p53 interaction can stabilize p53 and may offer a novel strategy for cancer
therapy. Here, we identify potent and selective small-molecule antagonists of
MDM2 and confirm their mode of action through the crystal structures of
complexes. These compounds bind MDM2 in the p53-binding pocket and activate the
p53 pathway in cancer cells, leading to cell cycle arrest, apoptosis, and growth
inhibition of human tumor xenografts in nude mice.
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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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Figure 4.
Fig. 4. In vivo antitumor activity of MDM2 inhibitors. Nude
mice (10 animals per dose group) bearing subcutaneous human
cancer xenografts (SJSA-1) with mean volumes of 185 mm3 received
200 mg/kg of an oral dose of Nutlin-3 (racemic) twice daily or
10 mg/kg of intravenous doxorubicin once a week for 3 weeks. The
tumor volumes were measured and recorded periodically duringthe
course of the study. P < 0.001 for Nutlin-3 and doxorubicin
compared with corresponding vehicle controls. Error bars show
SEM.
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The above figures are
reprinted
by permission from the AAAs:
Science
(2004,
303,
844-848)
copyright 2004.
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