Ch. 9: TP53 and Apoptosis Flashcards
Where was TP53 first discovered?
in SV40 DNA tumor virus
- viral large T protein binds to TP53 and perturbs its function
TP53 Tumor suppressor gene (TSG)
loss of fxn mutation -> cancer
- not typical TSG
- dominant negative allele
Dominant negative
When a mutant allele effects the WT allele in a negative way
Most cancers that have altered TP53, involve a…
missense mutation
Most cancers that have altered TP53, involve a missense mutation, Suggesting…
tumor cells benefit more
from a slightly altered TP53 protein than from complete loss of TP53 protein
TP53 functions as a…
tertramer
Four individual TP53 proteins
come together to make…
functional TP53
If any of these tetramers
contain a TP53 subunit with a
point mutation it will be…
non-functional
In normal cells: TP53 is…
continuously made and
degraded at a high rate = low steady-state levels
Why should a cell invest energy into making a protein if it’s rapidly going to be degraded?
Provides a mechanism to rapidly increase the cellular concentration of TP53 in response to certain physiological signals.
Ex- A cell can double the concentration of TP53 in 20 minutes by
blocking its degradation.
What is responsible for TP53 continuous destruction?
negative feedback loop with MDM2
what is the negative feedback loop that regulates TP53
- TP53 tetramer is a transcription factor.
- It activates the transcription of MDM2.
- MDM2 protein binds to TP53
tetramers and initiates TP53
ubiquitylation and subsequent
proteasomal degradation
What induces TP53 levels in a normal cell?
- X-Rays
- UV radiation
- Chemo. drugs that damage DNA
- Inhibitors of DNA synthesis
- Low oxygen (hypoxia)
- Increase in reactive oxygen species (ROS)
- Introduction of MYC oncogene
- … and many more
TP53 is required for…
cell death in response to DNA damage.
What causes rapid increases in TP53 protein levels?
a variety of cell physiological stresses
TP53 undergoes post-
translational modifications which induces…
a number of different responses
How does DNA damage induce TP53 in a normal cell?
- ssDNA- single stranded DNA
activates ATR kinase - DSBs- double stranded DNA breaks activates ATM kinase
- Both ATR and ATM kinases can phosphorylate TP53 which causes it to be stabilized
- TP53 protein is no longer degraded and accumulates in the cell
How does DNA damage induce TP53 in a
normal cell?
ATR/ATM kinases further increase TP53 protein levels by
phosphorylating MDM2 at specific sites which inactivate MDM2.
* This causes TP53 protein levels to further increase in a cell. TP53 can then activate cell cycle arrest, senescence, or apoptosis.
Is MDM2 a tumor suppressor gene or proto-
oncogene?
- Tumor suppressor gene- loss of MDM2 expression/function would cause transformation.
- Oncogene- gain of MDM2
expression/activation would cause transformation
Active RB protein inhibits…
E2F transcription factors
E2F transcription factors activate…
the transcription of ARF
ARF is a…
negative regulator of MDM2
If cells have lost proper control of RB, and have high levels of E2F activity, what would you expect to happen based on the above pathway?
If RB is not functioning properly, E2F is left uninhibited so the transcription of ARF is constitutively active and MDM2 will not be negatively regulated -> TP53 levels increase -> apoptosis
Anti-cancer mechanism
if cells lose proper growth control, they have a molecular mechanism to sense this and induce programmed cell death via TP52
TP53 functions as a…
transcription factor
TP53 as a transcription factor
Activates transcription of
genes involved in cell cycle
arrest, apoptosis, and DNA
repair.
* TP53 halts cell cycle in
response to DNA damage and
attempts to aid in the repair
process
TP53 halts the ______ by activating the transcription of ____
- cell cycle
- p21
p21 inhibits…
CDK2 and CDK1
TP53 halting the cell cycle
Can halt cell cycle progression at multiple points in the cell cycle.
* At the same time, TP53 activates txn of cellular DNA repair machinery.
* Gives cells a chance to pause and repair DNA. If DNA is repaired cell cycle can resume.
TP53 is considered the…
“guardian of the genome”
TP52 as the guardian of the genome
TP53 gives cells a chance to repair their DNA before cell division occurs.
* If severe irreparable DNA damage has occurred in a cell TP53 also has a mechanism to induce permanent senescence or apoptosis. Allows
organism a way to deal with mutant cells and their damaged genomes.
* Next time, talk about apoptosis and how TP53 initiates apoptosis.
With prolonged DNA damage, TP53 can induce…
permanent senescence
senescence
permanent form of cell cycle arrest
replicative senescence
type of senescence, associated with growth potential of cells in cell culture, associated with short telomeres
Purpose of apoptosis
- Used to maintain appropriate
numbers of cell types in human tissues
Highly controlled process.
How apoptosis is highly controlled
- 1) Plasma membrane: form multiple blebs (small protrusions)
- Cell surface appears to be boiling
2) Nucleus is condensed and fragments, chromosomal DNA is cleaved into small segments
3) Apoptotic cell is broken up into small pieces and are ingested by a specialized
cell, phagocyte. The phagocyte will break the molecules in the apoptotic cell down with enzymes. All traces of
the apoptotic cell are now gone. Apoptotic cell
within a macrophage
Rolls of apoptosis in biology
- Development-
* Way to get rid of excess cells to make well- formed functional tissues and organs.
(Kind of like a chisel)
Ex- During paw formation, cells in- between digits undergo apoptosis to form distinct digits - Adult tissue homeostasis-
* In some tissues new cells are made when needed and they undergo apoptosis when no
longer biologically necessary
* Ex- Adult mammary gland. During pregnancy mammary epithelial cells are made in great numbers. Mammary epithelial cells contribute
to milk formation after birth. When the newborn baby weans (stops nursing, getting
milk from mom), mom’s mammary epithelial cells undergo apoptosis as milk production is not necessary anymore. - Anti-cancer mechanism
* Cells with serious DNA damage are triggered to undergo apoptosis. This helps
get rid of cells with mutant DNA that could contribute to cancer formation.
How does the mitochondria control apoptosis on a
molecular level?
Cytochrome C is a molecule usually present in the inner-mitochondrial space (between the inner and outer
mitochondrial membrane).
* Usually, involved in electron transport chain -> ATP production.
* Apoptosis is first triggered by the release of Cytochrome C into the cytoplasm of the cell
How does Cytochrome C leak out of the mitochondrial inner space?
Under normal conditions:
* BAX/BAK- pro-apoptotic proteins. Have the ability to make a mitochondrial outer membrane pore (MOMP).
* BCL2/and others- anti-apoptotic proteins. Interact with BAX/BAC and inhibit them from making pores.
* BCL2/others > BAX/BAC
* Cytochrome C stays inner
mitochondrial space
Under apoptotic conditions:
* BIM/BAD/NOXA/PUMA- pro-
apoptotic proteins.
* Bind to BCL2/other anti-apoptotic proteins, BCL2 no longer able to bind to
BAX/BAK.
* Bind to BAX/BAK and promote pore formation.
* BAX/BAK can make pores,
cytochrome C leaks into the
cytoplasm.
Many different signals can induce apoptosis through mitochondrial pore formation (Examples)
Example 1:
* TP53 activation promotes the txn of NOXA and PUMA (pro-apoptotic proteins). These promote BAX/BAK pore formation.
Example 2:
* Loss-of-anchorage, causes
upregulation of BIM/BMF (pro-
apoptotic proteins.) These
promote BAX/BAK pore formation.
*Don’t have to know this who figure, just the portion highlighted here.
What happens after Cytochrome C is released into
the cytoplasm?
How does Cytoplasmic Cytochrome C promote
apoptosis?
Apoptosome
a complex composed of 7 Cytochrome C molecules and 7 APAF1 proteins.
* Helps initiate apoptosis.
* Pro-caspase 9 -> Caspase 9
protease
an enzyme that breaks down
proteins and peptides
Activated Caspase 9 is a protease
Caspase 9 cleaves pro-caspase 3.
* This activates caspase 3.
* Caspase 3 can then cleave and activate another caspase.
* Protease Cascade keeps going until “death substrates” are cleaved
death substrates
critical components of
the cell that are degraded as part of apoptosis
initiator caspases
activate the caspase cascade
* Ex- Caspase 9
executioner caspases
destroy critical components of the cell.
* Ex- Caspase 3, 6, 7
* Ex- Cleavage of nuclear lamins is involved in break down of nucleus
* Ex- Cleavage of cytoskeleton such as actin leads to collapse of cytoskeleton and formation of blebs that protrude
from the plasma membrane.
Apoptosis can also be initiated by death receptors.
Ligand binding to death receptors causes activation of initiator pro-caspases.
* These can further activate
executioner caspases.
* These degrade cellular proteins involved in apoptosis.
TP53 can also activate apoptosis via inducing
death receptor expression
Stabilized TP53 tetramers activate transcription of a death receptor, FAS.
* Since death receptors activate initiator caspases, apoptosis is activated
Cancer and apoptosis
Cancer cells come across a lot of physiological stressors that can trigger apoptosis.
* Therefore, most, if not all, cancers have found ways to inactivate mechanisms
that contribute to apoptosis.
* Most important and widely used strategy by most cancer cells to inhibit apoptosis is to inactivate TP53 pathway
Anti-apoptotic strategies used by cancer cells
1) Inactivation of TP53 pathway.
* Examples
* inhibition of ARF
* increase in MDM2
2) Alterations that inhibit
mitochondrial pore formation.
* Examples
* inhibition of BAX (normally makes a pore)
* activation of BCL2 (normally inhibits pore formation)
Blue- Pro-apoptotic proteins are decreased in some cancers
Red- Anti-apoptotic proteins are increased in some cancers
