p53 Part 1

Question 1

What is TP53?

Answer 1

This is the gene most frequently targeted tumour suppressor gene, it is a transcription factor that induces over 100 well characterised proteins and functions in both conditions of low and acute cellular stress

Question 2

What are the functions of basal levels of p53?

Answer 2

These regulate metabolism, detoxify reactive oxygen species and repair DNA
Inducing a copper transporter (SCO2) which is necessary for cytochrome C oxidase assembly
Indirect suppression of the IKK/NFkappaB pathway
Induction of the TP53-inducible glycolysis and apoptosis regulator (TIGAR gene)
Induction of antioxidant enzymes
Promoting of repair of low levels of DNA damage

Question 3

What affect does loss of p53 have on normal cellular metabolism?

Answer 3

The inability to produce cytochrome C oxidase suppresses oxidative phosphorylation and promote glycolysis (the Warburg effect)
It also results in activation of NF-kappaB and the induction of the GLUT3 glucose transporter allowing the cell to sustain high rates of glycolysis and conditions permissive for transformation

Question 4

What are the functions of TIGAR?

Answer 4

This suppresses glycolysis by directing glucose metabolism through the pentose phosphate pathway, increasing NADPH and GSH concentrations, decreasing ROS concentrations and reducing DNA damage
Loss of P53, and therefore loss of TIGAR expression can promote glycolysis, limit the pentose phosphate pathway leading to genome instability

Question 5

What antioxidant enzymes are induced by basal levels of P53?

Answer 5

Gluathione peroxidase 1, mitochondrial superoxide dismutase 2 and sestrins

Question 6

What occurs to basal antioxidant enzyme expression with loss of P53?

Answer 6

There are elevated 8-oxo-guanine concentrations and mutation rates

Question 7

How does basal P53 expression lead to repair of low levels DNA damage?

Answer 7

Induction of NER proteins (XPC and XPE) that recognize DNA damage and activates the BER enzyme 8-oxo-guanine glycosylase

Question 8

What are the functions of p53 under high stress cells?

Answer 8

It induces reversible arrest, senescence or apoptosis this is generally believed to be a part of its tumour suppressor function
P53 mediated responses to DNA damage are responsible for toxicity side effects pf anti-cancer therapy

Question 9

What is the Hayflick Limit?

Answer 9

In somatic non-stem cells, telomeres shorten with each cell division this leads to the Hayflick limit where due to telomere attritions cells undergo a limited number of divisions before becoming senescent
This is due to the telomere attrition activating a DNA damage response

Question 10

How is arrest of the cell cycle by p53 induced?

Answer 10

This is mediated by p21cip1 and microRNAs induced by p53 this includes miR-34a/b/c that suppressed Cdk4, cycle E and HGFR expression

Question 11

What is senescence defined by?

Answer 11

Cell cycle arrest and the expression of markers such as senescence-associated beta-galactosidase
P16INK4a, p15INK4b
Heterochromatin-binding proteins HP-1alpha and 1gamma
Histone H3K9 trimethylation

Question 12

What mediates Apoptosis?

Answer 12

BH3 only regulators especially p53-upregulated modulator of apoptosis (PUMA) Others include Bid, NOXA and Bax
Death receptors such as Fas, DR4 and DR5 may also be upregulated

Question 13

What occurred when p53-transgenic mice were generated in which three lysine residues in the DNA binding domain (acetylated when p53 is activated) were mutated to arginine?

Answer 13

These mice could not induce p21, cell cycle arrest and senescence, PUMA, BAX, DR5 and apoptosis
They do however suppress tumour development suggesting that arrest, senescence and apoptosis are not needed for tumour suppression
They also retained the ability to inhibit expression of GLUT3, Induce TIGAR, GLS2 (a mitochondrial transglutaminase that deanimates glutamine to glutamate), induce the Cu-transporter SCO2 needed for oxidative phosphorylation

Question 14

What is the relationship between Cancer cells and the TCA cycle?

Answer 14

Cancer cells use TCA cycle intermediates for biosynthesis and have high rates of glycolysis to generate ATP
The ability of tumour cells to have this altered metabolism may underlie tumour suppression

Question 15

What are the primary negative regulators of p53?

Answer 15

MDM2 and MDM4/MDMX with genetic deletion of either of these causing embryonic death due to unrestrained p53 activity we can determine that these are specific for p53 as if both they and p53 are deleted then the embryo will survive

Question 16

What is the role of MDM2 and MDM4/MDMX in human cancer?

Answer 16

These are each overexpressed with gene amplification in more than 10% of human cancers and without gene amplification in others. Loss of P53 function occurs after moderate increases in concentration of both MDM2 and MDM4/X or after large increases in one of these

Question 17

What is the normal function MDM2 and MDM4/X?

Answer 17

Under non stressed conditions MDM2 acts as an E3 ubiquitin ligase which causes p53 degradation while MDM4 suppresses P53 transcriptional activity
In the presence of stress MDM2 changes specificity to cause degradation of itself and MDM4 stimulating p53 transactivation function where MDM2 is induced completely degrading MDM4 maximising p53 activity
After the stress MDM2 will return to its p53 specificity

Question 18

How was the Ras oncogene discovered?

Answer 18

It fully transformed immortalised NH3T3 fibroblasts but if it was introduced to non immortalised mouse fibroblasts this induced senescence leading to the discovery of the two step process to transform cells where immortalisation with a gene such as myc and transformation with a gene like Ras

Question 19

What is p19ARF (mouse)/ p14ARF(human)?

Answer 19

These are induced by Ras and are inhibitors of MDM2 which leads to p53 activation and senescence
The myc oncogene promoted both vigorous cell division and sensitivity to apoptosis exertin a selective pressure for the loss of p53 or p19 when one of these tumour suppressor genes was inactivated then the cells could become resistant to senescence or apoptosis were immortalised and could be transformed by Ras

Question 20

How can MEFs be made resistant by transformation by myc+ras?

Answer 20

Deletion of a transcription repressor of the POK class (pokemon)
Conversely infection of MEFs with retroviral vectors expressing pokemon+myc or ras efficiently induced transformation as the expression of pokemon substitutes for the loss of p19ARF or p53 in transforming cells

Question 21

What is pokemon?

Answer 21

This is a transcriptional repressor of p14, over expression of this promotes transformation and is found in many human cnacer

Question 22

What typically occurs in clinical human cancers to the pRb and p53 pathways?

Answer 22

Typically both of these are lost for example retinoblastomas retain normal p53 gnees but have elevated concentrations of MDMX or MDM2 oftn as a consequence of gene amplification
Bladder cancers lose pRb and p53 function but retain p16/14 exression or lose p16/14 function while retaining pRb and p53 expression
Glioblastomas lose pRb and p53 function or co-delete p16 and p14 or co-amplify CDK4 and MDM2