L8 Flashcards
Describe the discovery of p53
- 53 kDa protein that interacted with
the viral SV40 T-antigen
- 3T3 mouse fibroblast Ccell lysates incubated with N or T and then immunoprecipitated with:
N= normal hamster serum
T= hamster antiserum reactive with SV40-transformed cells - Also found to be present in non-SV40 transformed cells (F9; mouse embryonal
carcinoma cells) and in mouse cells transformed by exposure to a chemical
carcinogen - led to the conclusion that
p53 was of CELLULAR ORIGIN (ENDOGENOUS protein)
Is p53 oncogene? Describe what happens in each scenario
1) ras and p53 deletion mutant
2) ras + p53 val-135 point mutant
3) ras + wild-type p53
1) ras and p53 deletion mutant
- Deletion: supports transformation
2) ras + p53 val-135 point mutant
- 1) Initial observation: Co-transfection with ras results in transformation. Is p53 an oncogene?
3) ras + wild-type p53
- P53 suppresses transformation
Mutant alleles of p53 (TP53) are frequent in human tumors. What % of ovary tumors have a TP53 mutation?
50%
Describe graph on slide 7
Kaplan-Meier survival analysis
- Shows % survival of different p53 genotypes as a function of age (days). Individuals that are homozygous for wildtype p53 have the highest survival, then those who are heterozygous (+/-), and individuals with both mutant copies of p53 have the lowest % survival (development of sarcomas and leukemias)
Majority of mutant p53 alleles are _____ mutations. Most of them affect the area of the DNA fxning in what?
- Missense (a point mutation in which a single nucleotide change results in a codon that codes for a diff AA)
- Most mutations affect DNA CONTACT or PROTEIN CONFORMATION
Majority of mutant APC, ATM, BRCA1 alleles are _____ mutations
frameshift (deletion or insertion in a DNA sequence that shifts the way the sequence is read)
Describe the structure of p53.
Homotetrameric transcription factor
In a cell that is heterozygous for p53, ____ of the subunits may lack fully normal function
15/16
What is the effect of a mutant p53 allele?
The mutant allele has a dominant negative effect (MORE severe than a deletion allele)
- altered gene product acts antagonistically to the wild-type allele
How do somatic vs. germline TP53 mutations differ? How are they similar?
- SOMATIC TP53 mutations contribute to SPORADIC cancer
- GERMLINE TP53 mutations cause a rare cancer predisposition
called Li-Fraumeni Syndrome - BOTH somatic and germline mutations are usually followed by LOH during tumour progression
Many mutant p53 forms lose tumor suppressor activity and acquire _______ activities and they also gain new ______. Example of oncogenic properties?
- Many mutant p53 forms lose tumor suppressor activity and acquire DOMINANT-NEGATIVE activities and they also gain new ONCOGENIC properties e.g. when mutant p53 is transfected into p53 -/- null cells, the result was ENHANCED ABILITY to form TUMOURS
What are the 5 oncogenic properties (tumour initiation and progression) of mutant p53? What are their underlying mechanisms?
- Proliferation and survival
- Differentiation block
- Drug resistance and inhibition of apoptosis
- Genetic instability
- Angiogenesis, invasino, and metastasis
- Inactivation of wild-type p53
- Inactivation of p63 and p73
- Interaction w/TFs (e.g. VDR)
- Structure specific DNA binding
- NF-kb pathway activation
- Inhibition of autophagy
- Cooperation w/Ras in transformation
- ATM pathway inactivation
- Topoisomerase I activation
What are the 3 classes of p53 mutations?
- LOF (loss of fxn)
- Transcriptional regulation of genes that mediate growth-suppression, apoptosis, DNA repair etc. - DN (dominant neg)
- GOF (gain of fxn)
- transcriptional regulation of genes that mediate proliferation, drug-resistance, survival, and metastasis etc.
Draw a diagram connecting the 3 classes of p53 mutations
Slide 13
Which property of p53 is selected for during cancer development?
N/A
What are 6 stress signals cause a rapid increase in p53 protein levels? In response to these signals, what are the possible actions of p53?
p53 receives signals from an array of surveillance systems
- Lack of nucleotides
- UV radiation
- Ionizing radiation
- Oncogene signaling
- Hypoxia
- Blockage of transcription
Lead to increased p53 lvls. p53 can either fxn in
- cell cycle arrest
- > can lead to SENESCENCE (irreversible) or RETURN to PROLIFERATION (reversible) - DNA repair
- block of angiogenesis
- apoptosis
Nickname of p53
Master guardian
Using a diagram show the pathway of diff stress signals on p53 and its action
Slide 14
Describe the control of p53 levels by Mdm2, provide a diagram
Slide 15
Mdm2 = mouse double minutest
- In cases of cell stress, p53 activate downstream transcriptional targets inc Mdm2. When Mdm2 is transcribed and translated, its protein will bind p53. Mdm2 is a ubiquitin ligase, thus acts to ubiquitinate p53 and cause its degradation. This forms a NEG FEEDBACK loop; high lvls of p53 leads to transcription of p53 regulator, which binds p53 and cause its degradation. Thus half-life of p53 is very rapid ~20 mins.
What events lead to p53 STABILIZATION and DEGRADATION
Stabilization of p53:
- DNA damage sensing kinases such as ATM and ATR act via Chk2
and phosphorylate p53 (which prevents Mdm2 binding) and also phosphorylate Mdm2 itself (on N terminus) so it cannot associate with p53.
Degradation of p53:
- Via Mdm2
- Suvival signals act through AP1 and Ets TFs to collaborate with p53 to promote Mdm2 transcription and translation
- these survival signals also activate Akt/PKB kinase which phosphorylates (C terminus) and activates Mdm2; the resulting active Mdm2 then triggers the Ubiquitlyation and proteasomal degradation of p53.
Define Mdm2 and Hdm2.
Mdm2 = mouse double minutes 2 HDM2 = Human homolog
How does HDM2 fxn as oncogene
- oncogene (i.e. gain of function leads to transformation)
- Mechanism of transformation involves increased degradation of p53 i.e. loss of a tumor suppressor
What is the Mdm2/HDM2 gene product?
= E3 ubiquitin ligase that targets proteins for proteasomal degradation; attaches ubiquitin to lysine residues of target (i.e. p53)
Where can Phosphorylation of Mdm2 occur
1) N terminus (region
that interacts with p53
2) central acidic region (docking site for binding partners);
multiple P sites in each region
What is ARF protein and how does it affect regulation
= Alternate Reading Frame product of the CDKN2A
locus accumulates in the nucleolus where it forms stable complexes with Mdm2 and acts as a tumor suppressor by initiating p53
dependent cell cycle arrest and apoptosois;
- ARF inhibits mdm2, thus promotes p53 which promotes p21
activation and inactivates cell cycle genes
- (i.e. ARF is a negative regulator of a negative regulator…)
Elevated ARF causes an ___ in p53 (by inactivating Mdm2)
Increase
What is apoptosis?
- Apoptosis = Greek, “falling off” of petals from flowers, or leaves from trees – for the betterment of the organism as a whole
- Descriptive term for the phenotype of cells undergoing a particular morphological form of PROGRAMMED CELL DEATH
- Characteristic morphology includes cell shrinkage, membrane blebbing, morphological conservation of most organelles, nuclear condensation, and phagocytosis of dying cells
- Important in normal development and homeostasis
- Evolutionarily conserved
How does apoptosis differ from necrosis?
Apoptosis
- Cell shrinkage
- Membrane blebbing
- Nuclear condensation (Pyknosis)
- Phagocytosis my macrophage
- Morphological conservation of most organelles
- Fragmented golgi bodies
Necrosis
- Cell swelling
- PM rupture
- Induction of inflammatory response
Define Pyknosis
condensation of chromatin and COLLAPSE of NUCLEAR STRUCTURE
4 Fxn of apoptosis; dysfxns
- Deleting structures
- Sculpting tissues
- Cell # culling
- Deleting damaged cells
- Developmental abnormalities
- Neurodegenerative disease
- Autoimmune diseases and cancer
How is apoptosis affected in Cancer
Cancer cells actively inhibit apoptosis – expect DECin apoptotic ACTIVATORS, INC in INHIBITORS
First apoptosis gene implicated in cancer?
- Bcl-2 gene = b cell lymphoma/leukemia 2
- an anti-apoptotic gene upregulated in lymphoma; highlighted the importance of homeostatic balance
Describe the 2 pathways of apoptosis
I. Death Receptor “Extrinsic” pathway
1. Death ligands bind to death receptors of the Tumor Necrosis Factor
(TNF) Receptor family containing death domains
2. Activated receptors cluster and recruit adaptor proteins
3. Pro-caspase 8 binds and is activated
4. Downstream effector caspases are activated
5. DNA and protein substrate cleavage
6. Apoptotic phenotype
II. Mitochondrial “Intrinsic” pathway
1. Perturbation of mitochondria; increased permeability of mitochondrial
outer membrane
2. Cytochrome c is released (regulated by Bcl-2 family members)
3. Apoptosome formation: Cyt c +Apaf-1 (apoptotic protease activating
factor-1) + pro-caspase 9
4. Caspase 9 activation and downstream effector caspase activation
5. Apoptotic phenotype
Define caspases
Cysteine aspartyl-specific proteases
Draw a diagram of the 2 main pathways of apoptosis
Slide 35
Define FADD
= Fas-associated Death Domain (FADD) Protein
Define DISC
DISC = Death Inducing Signaling Complex
Caspase 10
- initiator caspase in death receptor signaling
- Processed by initiator caspase 8
What are initiator caspases?
- initiator caspases interact with upstream adaptor molecules through protein-protein interaction domains called CARD (Caspase Activation and Recruitment Domain) and DED (Death Effector Domain)
- Once processed, caspase 10 cleaves and activates executioner caspase 3 and 7
What are the 2 key features of all 3 extrinsic apoptosis signaling cascades
- cell surface receptors
2. caspase-dependency
Describe 3 ways Caspase are activated
- Processing by an upstream caspase (to produce mature enzyme which is a heterotetramer consisting of two p20/p10 hetero- dimers).
- Induced proximity: death receptor complexes recruit several molecules of pro-caspase 8 resulting in high concentration of zymogen;; mutual cleavage and activation by low intrinsic protease activity of the pro- enzyme
- Association with a regulatory subunit: caspase 9 is activated by association with Apaf-1 and cytC.
