L6: p53 Flashcards
(10 cards)
tumour suppressors
Cancer cells release excess go signals- growth factors, gf receptors, other tf in genes involved in cellular proliferation and growth, excess growth signals
Stop signals- tumour suppressor genes: p53, pRB, p21, p16
Carcinogenes effect genes like p53 as they are crucial for maintaining genomic integrity
What are tumour suppressor genes (TSGs)
TSGs encode proteins responsible for regulating cell growth, division, and survival.
Their primary function is to prevent the formation of tumours by maintaining genomic integrity and inhibiting the uncontrolled proliferation of cells.
They act as brakes on the cell cycle to prevent unchecked growth.
They facilitate the correction of damaged DNA to ensure stability during replication.
When cells are severely damaged, TSGs can trigger programmed cell death.
Some TSGs inhibit the invasion and spread of cancerous cells to other tissues.
Types of TSGs
- Gatekeepers: Directly regulate cell cycle progression and apoptosis. Examples: RB (retinoblastoma protein) and p53. Loss of gatekeeper function leads to unregulated cell division.
- Caretakers: Maintain genomic stability by repairing DNA damage and preventing mutations. Examples: BRCA1/BRCA2 (involved in homologous recombination repair) and MLH1 (part of the mismatch repair pathway). Mutations in caretakers allow the accumulation of genetic errors, increasing cancer risk.
- Landscapers: Regulate the extracellular environment to suppress tumor formation. Example: APC (adenomatous polyposis coli), which modulates cell-cell adhesion and signalling.
TSGs regulate cell cycle checkpoints and DNA integrity
p53 and G1
There is cross-talk between p53 and the Rb (retinoblastoma) pathway, especially during the G1 phase of the cell cycle.
When p53 senses DNA damage, it activates the transcription of p21, a cyclin-dependent kinase (CDK) inhibitor.
p21 inhibits cyclin-CDK complexes, which normally phosphorylate (and thus inactivate) Rb.
If Rb is not phosphorylated, it remains active and continues to bind and inhibit E2F, a transcription factor needed for S-phase entry.
This halts the cell cycle, allowing time for DNA repair.
When Rb is phosphorylated (i.e. in the absence of p21 or DNA damage), it releases E2F, allowing the transcription of genes required for DNA synthesis and progression into the S phase.
p53
The p53 gene: the guardian of the genome
The p53 gene is located on chromosome 17 (17p13.1) and encodes a 393-amino acid protein with functional domains for transactivation, DNA binding, and tetramerization.
Normal Functions of p53: p53 plays a pivotal role in cellular responses to stress. Its functions include:
DNA Damage Detection: p53 acts as a sensor for DNA damage caused by radiation, toxins, or replication errors.
Cell Cycle Arrest: When activated, p53 induces the expression of p21, a CDK inhibitor, to halt the cell cycle at G1/S or G2/M checkpoints. This pause allows time for DNA repair before the cell divides.
DNA Repair Activation: p53 promotes the transcription of genes involved in DNA repair pathways, such as GADD45.
Apoptosis: If the damage is irreparable, p53 triggers apoptosis by activating genes like BAX, PUMA, and NOXA, which promote mitochondrial dysfunction and cell death.
Diff cellular stressors like dna damage, metabolic stress, hypoxia, dereg growth activate-(oncogenic stress mutation in oncogene is sensed by cell as stress) which in turn can induce other cellular stresses (dna damage, metabolic stress not hypoxia) p53
P53 transcription does not change
Accumulates - stabilised and accumulates?
It outputs protecting individual cells or tissues
P53- quiescence, senesence, apoptosis
P53 indudces temporary cellular arrest- quiscence
Induce cell death
Its role as a tumur suppressor is ascribed mainly to response to dna damage and/or deregulated growth
evidence for p53 as a tumour suppressor
TP53 gene is the most frequently mutated gene (>50%) in human cancer
(mostly missense mutations that impair DNA binding)
Chromosome 17 short arm deletions (where the TP53 gene resides)
are often accompanied by mutations in the remaining TP53 allele (LOH)
in p53 null/mutant tumours, deletion of one allele of tp53 is often associated with mutation in the other
any tumours lack p53 expression all together
WT p53 actively suppresses oncogene-mediated cellular transformation
p53 binding to dna
Most p53 mutations occur in the region responsible for DNA binding. These mutations prevent p53 from binding to DNA and carrying out its function as a transcription factor.
Loss of p53 function can happen in several ways:
Complete loss of the TP53 gene,
Loss of one allele (heterozygosity),
Deletion of the chromosome arm where p53 is located (17p),
Or point mutations in the DNA-binding domain.
Even heterozygous mutations (where only one of the two alleles is mutated) can lead to loss of function. This is because p53 functions as a tetramer (a complex of four subunits), and the presence of even one mutant subunit in the tetramer can disrupt the entire complex — a phenomenon called dominant-negative effect.
p53 functions as a tetramer — four p53 molecules come together to bind to specific DNA recognition sites and regulate target genes.
One of its key targets is MDM2 (Mouse Double Minute 2 homolog), an E3 ubiquitin ligase.
p53–MDM2 Feedback Loop:
p53 activates the transcription of MDM2, forming a negative feedback loop.
When p53 levels rise (e.g., during DNA damage), it increases MDM2 expression.
MDM2 then binds to p53, ubiquitinates it, and sends it to the cytoplasm for degradation, thereby reducing p53 levels.
This regulation prevents p53 from remaining active when it is no longer needed.
Regulation of MDM2:
Growth and survival signaling pathways (e.g., Akt) can phosphorylate and activate MDM2, enhancing its ability to degrade p53.
Oscillations and p53 Stability:
This feedback loop causes out-of-phase oscillations between p53 and MDM2 protein levels.
In normal cells, p53 has a short half-life (around 6–20 minutes), keeping its protein levels low under non-stressed conditions.
However, p53 is constantly transcribed, so if it becomes stabilized (e.g., during DNA damage), it can accumulate quickly, allowing a rapid stress response — making it a highly effective cellular sensor.
p53 stabilisation by dna damage
DNA Damage Response and p53 Stabilisation
DNA damage triggers signalling pathways that stabilize p53 and inhibit its degradation by MDM2.
Types of DNA Damage:
Single-strand breaks (SSBs):
Generally easier to repair.
Recognized by proteins like Replication Protein A (RPA).
This recruits ATR (Ataxia Telangiectasia and Rad3-related) kinase.
ATR phosphorylates proteins including MDM2, which impairs its ability to degrade p53.
Double-strand breaks (DSBs):
More severe and complex to repair.
Sensed by the MRN complex (MRE11–RAD50–NBS1).
This activates ATM (Ataxia Telangiectasia Mutated) kinase.
ATM phosphorylates both p53 and MDM2.
MDM2 Regulation:
Under normal conditions, MDM2 is activated (phosphorylated at Ser166 and Ser188) by growth signals (e.g., via Akt), allowing it to bind and degrade p53.
In response to DNA damage:
ATR/ATM phosphorylates MDM2 at different sites (e.g., Ser359), which prevents MDM2 from binding to p53.
ATM also directly phosphorylates p53, further stabilizing it.
Outcome:
MDM2 inhibition + p53 phosphorylation = p53 stabilisation.
Stabilized p53 accumulates in the nucleus, binds to DNA, and activates transcription of target genes involved in:
DNA repair
Cell cycle arrest (quiescence)
Senescence
Apoptosis
CDKN2A
CDKN2A Gene and Its Products
The CDKN2A gene produces two distinct tumour suppressor proteins via alternative splicing:
p16^INK4a
p14^ARF (in humans) or p19^ARF (in mice)
Functions:
1. p16^INK4a
Inhibits cyclin D/CDK4 or CDK6 complexes.
This prevents phosphorylation of Rb, so Rb remains active and continues to inhibit E2F (a transcription factor required for S-phase entry).
This action halts cell cycle progression in G1.
Frequently mutated or deleted in cancers.
- ARF (p14 in humans / p19 in mice)
Plays a critical role in p53 stability.
ARF inhibits MDM2, the E3 ubiquitin ligase that normally degrades p53.
This allows p53 to accumulate, promoting:
Cell cycle arrest (via p21)
DNA repair
Apoptosis
Interaction with Rb and E2F:
When Rb is phosphorylated, it releases E2F, allowing E2F to activate genes involved in cell cycle progression — including ARF.
So paradoxically, E2F activity (usually pro-proliferative) can lead to ARF expression, which stabilises p53 and triggers an anti-proliferative response.
The Importance of the p53-p21 Pathway:
Without p53, p21 cannot be induced.
Without p21, cyclin-CDK complexes remain active and phosphorylate Rb, inactivating it.
Inactive Rb means continuous E2F activity, which drives uncontrolled proliferation — a hallmark of cancer.
ARF
ARF Activation by Oncogenic Signals
Oncogene activation (e.g., Ras, Myc) can trigger the transcription of an alternative product from the INK4a locus: ARF (p14^ARF in humans, p19^ARF in mice).
This acts as a tumour-suppressive mechanism to counteract oncogenic stress.
How ARF Stabilizes p53:
Oncogenic signalling increases ARF levels.
ARF binds to MDM2, the E3 ubiquitin ligase that normally targets p53 for degradation.
This binding inhibits MDM2’s function, so it cannot ubiquitinate p53.
As a result, p53 becomes stabilised and accumulates in the cell.
Role of MDM2 Phosphorylation:
Normally, growth and survival pathways (e.g., Akt) phosphorylate MDM2 at Serine 166 and 188, which activates MDM2 and promotes p53 degradation.
However, ARF interferes with MDM2’s ability to bind to p53 — even when MDM2 is in its phosphorylated, active form.
Therefore, in the presence of oncogenic stress, ARF overrides MDM2 activity, ensuring p53 is not degraded.
Summary:
Oncogene → ARF ↑ → MDM2 inhibition → p53 stabilisation
This allows p53 to trigger cell cycle arrest, senescence, or apoptosis in response to potentially cancerous signals.
p53 post translational modifications
p53 is tightly regulated in the cell through multiple post-translational modifications (PTMs). These modifications occur mainly in response to cellular stress and control p53’s stability, localisation, and activity.
Key PTMs of p53:
Phosphorylation
Occurs mainly on serine and threonine residues.
Triggered by stress signals such as DNA damage.
Reduces p53’s interaction with MDM2, preventing degradation.
Promotes stabilisation and activation of p53.
Acetylation
Occurs on lysine residues.
Increases p53’s ability to bind DNA and activate transcription of target genes.
Often carried out by acetyltransferases like p300/CBP.
Stabilises p53 and enhances its tumour suppressor function.
Ubiquitination
Also occurs on lysine residues.
Performed by MDM2, leading to proteasomal degradation of p53.
Balances p53 levels in unstressed cells.
