Download p53

請改震動模式
Robert A. Weinberg
The Biology of Cancer
First Edition
Chapter 9:
p53 and Apoptosis: Master
Guardian and Executioner
Copyright © Garland Science 2007
Outlines:
-p53 mutations in human cancer
-Facts about p53
-Mutant p53 interferes with normal p53 function
-p53 turnover & p53 stabilization upon DNA damage
-p53: guardian of the genome
-Mdm2 and ARF battle over the fate of p53
-ARF and p53-mediated apoptosis protect against cancer by monitoring
intracellular signaling
-Apoptosis vs. Necrosis
-mitochondria and cytochrome c
-families of Bcl-2, caspases & inhibitors of apoptosis
p53: co-IP by anti-serum against SV40 tumor
(mouse embryonic carcinoma)
IP by
anti-sera
p53
-a tumor suppressor working as a transcription factor
-frequently mutated in human cancer
-germ-line mutation is related to Li-Fraumeni syndrome
(Tag)
(p53)
-p53, a cellular protein, was pulled down by anti-SV40 anti-serum.
-p53 is present in elevated amounts in transformed cells.
Figure 9.1-2 The Biology of Cancer (© Garland Science 2007)
Mutations of p53 in human cancers
-p53 gene is mutated in 30 to 50% of commonly
occurring human cancers.
-75% of the tumor-associated p53 alleles are
missense mutations.
Q: Do tumor cells benefit from the mutant alleles than the null alleles?
How can mutant p53 proteins foster tumor cell formation?
Figure 9.4 The Biology of Cancer (© Garland Science 2007)
Li-Fraumeni syndrome
-early onset
-multiple malignancies, including glioblastoma, leukemias, carcinomas of the breast,
lung and pancreas, wilms tumor, and soft tissue sarcomas.
-clusters of R337H mutation in adrenal cortical carcinoma (under acidic condition, the
tetramerization of R337H mutants is less stable.)
Figure 9.20 The Biology of Cancer (© Garland Science 2007)
p53 as a TSG: Survival of p53-/--mice
-Knock-out mice lacking p53 gene had no apparent defect in
development, but had a shorter life span (about 5 months), dying
most often from lymphomas and sarcomas.
Figure 9.5 The Biology of Cancer (© Garland Science 2007)
Effects of p53 on cell transformation
Transformation of rat embryo fibroblasts (REFs)
-Wild-type p53 suppresses ras-induced cell transformation, whereas the mutant p53
collaborates with ras to induce transformation.
Q: How could the mutant p53 alter the behavior of the wild-type rat embryo fibroblasts?
How could they countervail the action of the wild-type alleles?
(act as dominant-interfering or dominant-negative mutant and through homotetramer
formation)
Figure 9.3 The Biology of Cancer (© Garland Science 2007)
Facts about p53
(A) Domains
(B) Mutations
(C) p53 consensus binding site
(x2)
(Pu-Pu-Pu-C-A/t-T/a-G-Py-Py-Py)2
-p53 contains 5 domains conserved evolutionarily throughout species. In addition to the
transactivation domain and DNA binding domain, it also contains an oligomerization domain
at C-terminus.
-Most of p53 mutations are located within the DNA binding domain.
-p53 works as a transcription factor which binds as a tetramer to the p53 consensus sites
(452 sites identified in human genome by CHIP assay)
Figure 9.6 The Biology of Cancer (© Garland Science 2007)
Mechanism of p53 dominant-negative mutations
Four of the α-helices of the
tetramerization domains
form two pairs assembled
in right angle. Introduction
of an amino acid
substitution destablizes the
tetramer.
In a cell that is heterozygous at the p53 locus, 15/16
of the subunits may lack full normal function.
Mutant embryonic stem cells demonstrate that the
cells with mutant allele present in heterozygous
configuration showed substantial reductions in p53
function, as compared to the ES cells lacking one of
the two p53 gene copies (i.e., carrying a null allele of
p53).
Figure 9.7 The Biology of Cancer (© Garland Science 2007)
p53 response
-p53 is a highly unstable protein, being broken down by proteolysis soon after it is synthesized.
-A variety of cell-physiologic stresses can cause a rapid increase in p53 levels. Basically, DNA
damage and de-regulated growth signals cause p53 stability.
-The accumulated p53 protein induces a number of responses through transactivation of
downstream target genes. Among them, the cytostatic and pro-apoptotic powers of p53 represent a
major threat to incipient cancer cells.
Figure 9.8 The Biology of Cancer (© Garland Science 2007)
Effects of p53: growth arrest and apoptosis
p53-induced growth arrest upon
DNA damage
Exposure of cells to X-rays strongly
increases p53 level, which halts cell
cycle advance by subsequent induction
of p21, a widely acting CDK inhibitor.
Figure 9.910 The Biology of Cancer (© Garland Science 2007)
p53-induced apoptosis upon
DNA damage
Thymocytes from mice of various p53
gene background displayed different
survival rates after X-irradiation. The
loss of viability was attributed to
apoptosis.
Table 9.2 The Biology of Cancer (© Garland Science 2007)
Control of p53 level by a negative-feedback loop
mediated through Mdm2
*
-In normal cells, p53 is rapidly turned-over
by a negative-feedback loop.
-Mdm2, a downstream target gene of p53,
works as an E3 ligase which binds p53 and
facilitates ubiquitin-mediated degradation
of p53.
Mdm2 (mouse double minute 2) was initially
identified as a protein encoded by double-minute
chromosomes present in murine sarcoma cells.
Subsequently, the human homolog Hdm2 was
found to be frequently amplified in sarcomas. It is
also overexpressed in lung cancer. Mdm2, an
oncogene, works by antagonizing p53 and
prevents entrance of a cell into cell cycle arrest or
into apoptotic suicide program.
-Mdm2 and p53 are locked in a death grip.
mdm2-/- : embryonic lethal,
due to runaway p53 activity
-/p53
: developed normally
mdm2-/- p53-/- : developed normally
Figure 9.11 The Biology of Cancer (© Garland Science 2007)
Phosphorylation of p53 protects p53
from Mdm2 binding
yellow space-filling model: p53 residues 18-27, the mdm2
interaction domain
blue mesh: the surface of mdm2 complementary pocket
Phosphorylation of p53 by kinases such as Chk2, ATM and ATR
prevents this interaction, and spares p53 from Mdm2-mediated
ubiquitination and subsequent degradation.
Figure 9.13 The Biology of Cancer (© Garland Science 2007)
Accumulation of p53 in p53-mutant cells
Anti-p53 Ab
(nuclear staining)
Mutant p53 is unable to induce Mdm2 transcription, and escapes
degradation which leads to its accumulation to very high level.
Figure 9.17 The Biology of Cancer (© Garland Science 2007)
p53: Guardian of the Genome
Control of p53 levels by
positive and negative signals affecting Mdm2
-In normal cells, mdm2 keeps p53 level
very low.
-Under stress, DNA damage-sensing
kinases phosphorylate p53, and
prevents the binding of Mdm2.
These kinases also phosphorylate and
inactivate Mdm2, and blocks the
binding of Mdm2 to p53.
-Actions of some oncogene-mediated
signals:
-mitogenic signal acts through AP1
and Ets1 to collaborate with p53 to
greatly increase the transcription of
Mdm2.
-Akt/PKB phosphorylates mdm2 and
facilitates its nuclear translocation.
-ARF binds to Mdm2 and inhibits its
action.
Figure 9.13 The Biology of Cancer (© Garland Science 2007)
*
INK4a/ARF Locus: LOH of chromosome 9p21
-p16INK4A gene encodes the important inhibitor of CDK4/6 that initiate pRb phosphorylation.
-Another gene, p19ARF (in mouse or p14ARF in human) encodes from the same locus, using an
alternative promoter. P19 is transcribed, sharing the exon 2 of the p16, but in an alternative
reading frame.
-Forced expression of ARF causes a strong inhibition in cell proliferation. However, this
inhibition was not observed in cells lacking wt-p53 function. This indicates that the growthinhibitory power of ARF depends absolutely on the presence of functional p53 in cells.
-In wt-cells, ARF expression causes a rapid increase in p53 level.
Figure 9.14 The Biology of Cancer (© Garland Science 2007)
ARF sequesters Mdm2 from antagonizing p53
ARF is transactivated
by E2F1.
-ARF binds to Mdm2 and sequesters Mdm2 in the nucleolus.
Consequently, p53 is expressed at an increased level.
-The p19ARF gene is an extremely important TSG, because
ARF has a central role in increasing p53 level. In many human
cancer cells that retain wt-p53, p53 function was eliminated by
inactivating the ARF gene.
Figure 9.15a The Biology of Cancer (© Garland Science 2007)
Control of apoptosis by ARF
Eμ-myc transgenic mice
Caspases (3, 7, 8, & 9)
Bcl2 family protein (Bim,
Noxa, PUMA), Apaf-1, and p73
apoptosis
(p53-independent)
(p53-dependent)
-Some oncogenic signals favor apoptosis through their ability to induce E2F activity, which
leads to increased ARF expression, suggesting the involvement of this signaling in the
elimination of cells with overly active E2F signaling.
-The ARF+/GFP Eμ-myc mice developed fatal tumors more rapidly then did those carrying only
the Eμ-myc transgene, and the cells in these tumors shed their remaining wt-ARF allele,
suggesting the loss of myc-mediated pro-apoptotic function in the absence of ARF function,
allowing its proliferative effects to dominate and drive tumor formation.
Figure 9.15b The Biology of Cancer (© Garland Science 2007)
E2F1-mediated induction of apoptosis
E2F-induced apoptosis may serve as an anti-cancer mechanism to eliminate
unwanted, pre-neoplastic cells.
Figure 9.16 The Biology of Cancer (© Garland Science 2007)
Apoptosis and normal morphogenesis
Sculpting the Digits in Developing Mouse Paw
Day 1
The paw in the mouse embryo
stained with a dye that
specifically labeled apoptotic
cells.
Day 2
The interdigital cell death
eliminates the tissue
between the developing
digits.
Metamorphosis of a Tadpole into a Frog
(TUNEL assay)
(An increase in thyroid hormone in the blood induces
metamorphosis, including apoptosis in the tail.)
Apoptosis
normal cell
chromatin
condensation
HeLa
Lymphocytes
membrane blebing
Formation of DNA ladders
fused
to form syncytium
Golgi bodies
fragmented
Pyknosis
Phagocytosis of apoptotic bodies
Staining of p-Histone 2B
Figure 9.18 The Biology of Cancer (© Garland Science 2007)
Apoptosis: Programmed Cell Death
Anti-apoptosis of Bcl-2-like proteins
Generation of mice with
myc or Bcl-2 transgenes
Pro-survival effects of Bcl-2-like proteins
increased
mortality,
dying from
lymphoma
died from
plasma cell
tumors
Figure 9.22 The Biology of Cancer (© Garland Science 2007)
Mitochondria and cytochrome c
Network-like mitochondria
a human liver cell
-Cytochrome c resides in the space between the inner
and outer mitochondrial membranes, where it functions
to transfer electrons as part of oxidative phosphorylation.
-When signals trigger the initiation of apoptosis, the outer
mitochondrial membrane become depolarized (i.e., loses
its normal voltage gradient), and cytochrome c is released
into cytosol, cytochrome c associates with other proteins
to trigger a cascade of events yielding apoptotic death.
Figure 9.23 The Biology of Cancer (© Garland Science 2007)
As apoptosis proceeds (from B, C, to
D), cytochrome c staining becomes
increasingly uniform in the
cytoplasm.
Bcl-2 family proteins
-share a common BH3 domain, some with BH1, BH2, TM and BH4
Figure 9.25 The Biology of Cancer (© Garland Science 2007)
Foaming inter- and intra-molecular complexes
of Bcl-2 family proteins
Intermolecular interaction:
Intramolecular interaction:
Upon binding of a BH3-only protein to
Bcl-xl, the α-helix of the BH3 protein
binds to the groove of Bclxl lying
between the BH1 and BH3 domains,
thereby inhibiting Bclxl.
Bax protein tucks its own C-terminal
α-helix tail into the groove between
BH1 and BH3 domain.
Figure 9.25b The Biology of Cancer (© Garland Science 2007)
Balance between Pro- and anti-apoptotic proteins
(A) the well-consolidated kidney tissue in the wild-type mouse
(B) wide-spread apoptosis and kidney disease in the bcl-/- mouse
(C) Deletion of one copy of bim reversed the phenotype observed in the bcl-/- mouse.
Figure 9.26 The Biology of Cancer (© Garland Science 2007)
Bak/Bax complex formation upon apoptosis
-Left panel: Bax is normally found in the cytosol (not visible here), whereas Bak is
attached to the out mitochondrial membrane (black spots).
-Right panel: Upon initiation of apoptosis, Bak coalesce with Bax in punctate foci on the
surface of mitochondria, which participates in mitochondrial fragmentation and
contributes to cytochrome C release.
Figure 9.25c The Biology of Cancer (© Garland Science 2007)
Interplay between the
pro- and anti-apoptotic Bcl-2 family proteins
Figure 9.27c The Biology of Cancer (© Garland Science 2007)
Apoptotic signals acting through Bcl-2-related proteins
Cytoplasmic sequestration of BH3 proteins
Cyt c release
-Various cell-physiologic stresses operate through different pro-apoptotic proteins to antagonize Bcl-2 protein.
-The Bcl-2 protein is thereby prevented from neutralizing Bax, the dominant pro-apoptotic protein that functions
to release cytochrome c from the mitochondrial membrane.
Figure 9.27a The Biology of Cancer (© Garland Science 2007)
The Caspase Cascade Involved in Apoptosis
Initiator caspase
Effector caspase
(executioner caspase)
-Multiple copies of the initiator procaspases are brought together by adaptor proteins. Activation is
achieved by either cleaving each other by the small amount of protease activity of the initiator
procaspase or a conformational change induced when they are brought together in a complex or
aggregates.
The apoptotic caspase cascade
The wheel of death
Figure 9.29 The Biology of Cancer (© Garland Science 2007)
Death receptors and ligands
Homotrimers of death ligand
Attacking of cancer cells (with Fas receptor)
by cytotoxic T lymphocytes (with FasL)
CTL
Cancer cell
Figure 9.31a The Biology of Cancer (© Garland Science 2007)
Conversion of
intrinsic and extrinsic apoptotic pathways
Figure 9.32 The Biology of Cancer (© Garland Science 2007)
Activation of apoptosis by p53
transactivation of Fas, Bax,& IGFBP-3
Figure 9.33 The Biology of Cancer (© Garland Science 2007)
Anti-apoptotic strategies in cancer
Blue: decrease of pro-apoptotic activity
Red: increase of anti-apoptotic activity
Figure 9.34 The Biology of Cancer (© Garland Science 2007)
Table 9.5 The Biology of Cancer (© Garland Science 2007)
The apoptotic circuit board
Figure 9.37 The Biology of Cancer (© Garland Science 2007)