SUMMARY NOTES
Lecture 3: p53 tumour suppresor
Discovery of p53
-p53 is a tumour suppressor gene prevalently disrupted in cancer via multiple
mechanisms. Exhibits pleiotropy, it has many different anti-cancer functions.
-TP53 is located within chromosome 17, arm p, cytogenetic band 17p13.1.
-Papovavirus- simian virus 40
• Tumour causing DNA virus in model systems. Transforms mouse
fibroblasts which gain ability to form tumours when injected back into
mice.
• Harvested antisera after tumour growth, immunoprecipitated proteins.
Antibody bound to large T-antigen from virus and 53kDa protein from
mouse host. Called protein p53, binds to viral oncoprotein.
• Since then, discovered many oncoviruses which express proteins which
bind and inactivate p53- viral oncoproteins.
> Model papilloma virus, human T-cell leukemia virus type I.
p53 mutations
-Introducing wild type tp53 cDNA at same time as injecting transformed
fibroblasts block tumour formation.
-Conversely, introducing loss of function p53 from tumour cell lines enhances
transformation of cells by oncogene (e.g., H-Ras).
-Knockout mice for p53 succumb in a dose dependent manner to many cancers.
-Point mutations
• Generally non-synonymous coding (in exons) point mutations which
produce aberrant full-length proteins. Change function of protein.
• Enhance tumorigenesis when introduced experimentally. Point mutant
p53 alleles are dominant-negative, loss of function of one gene affects
more than 50% of proteins since p53 is a tetramer. Protein encoded by
gene loses ability of normal function but can also interfere with wild type
molecule of protein.
• Haploinsufficient tumour suppressor means loss of one copy is enough for
progression towards tumorigenesis.
• Positive-dominant mutations are subclass that inactivate p53 tumour
suppressor activities and cause it to acquire additional capabilities with
roles in activating invasion and metastasis.
, > Gain of function- can drive invasion for progression and metastasis.
Promotes tumourigenesis more than deletion.
-Rarer mutations
• Deletions and insertions.
• Gross deletions where part of or whole gene is lost
• Intragenic mutations (indels) which lead to frameshift mutations.
-Origin of p53 aberrations
• Spontaneous (somatic)- events occur in single cell, often LOH in tumours.
• Inherited (germline)- rarer, LOH also occurs.
p53 and genomic integrity
-p53 controls cell proliferation and genomic integrity. Intertwined functions.
• p53 prevents DNA damage accumulating (particularly from ss and ds
breaks).
-Acts as a transcription factor, binds to consensus sequence and upregulates
transcription of several genes, many involved in cell cycle and genome
maintenance.
, • p53 mutations are predominantly in the coding region for the domain that
binds DNA, result in loss of DNA binding.
• Via activity as TF, stops. ell cycle and boosts ability to repair DNA.
p53 regulation
-p53 is actively transcribed in cells then activated after stress (e.g., DNA damage)
post-translationally.
• dsDNA break -> p53 protein stabilization + post-translational modification
-> gene transcription of TSGs (p21, DNA repair enzymes)
• PTM code on p53- specific residues modified via phosphorylation,
acetylation, ubiquitylation, methylation, sumolynation. Signal hub for
many responses to stress signals.
• PTMs may also affect stability of p53 independent of Mdm2, directly
affect transcriptional activation potential (target selection), sequester
p53 from nucleus and affect DNA binding of p53.
-Mdm2 major node in regulation of p53. Keeps p53 in off state by causing its
degradation.
• p53 polypeptide attached to chains of ubiquitin by covalent linkage-
polyubiquitylation
> Results in proteasomal mediated degradation of polyubiquitylated p53.
Ubiquitin acts as recognition signal for proteasome.
> Reaction catalyzed by Mdm2, ubiquitin ligase. Keeps low levels of p53
despite continuous translation.
, • Stress signals compromise degradation, inhibit Mdm2. Result in PTM
modifications of p53, enhancing activity.
• For example, ATR and ATM kinases sense DNA damage -> protein kinase
cascade through checkpoint kinases results in p53 phosphorylation ->
Mdm2 does not bind phosphorylated p53.
• Mdm2 is a p53 target gene, to avoid p53 activation in absence of stress
p53 exerts negative feedback by upregulating its levels
> Point mutant p53 can no longer bind Mdm2, resulting in big
accumulations of non-functional p53.
-Mdm2 is thus an oncogene via its inhibitory action on p53 TSG. Its
overexpression along with other oncogenes can drive cancer.
• Murine lymphoma model- transplanted lymphocytes engineered to
overexpress c-myc +/- Mdm2. Mdm2/ myc mice had very low survival
rates compared to individual oncogenes.
• Mdm2 and paralogues (Mdmx) amplified in some cancers (increased
number of gene copies). Even if p53 not mutated, function is suppressed
during tumorigenesis due to these genetic alterations.
Lecture 3: p53 tumour suppresor
Discovery of p53
-p53 is a tumour suppressor gene prevalently disrupted in cancer via multiple
mechanisms. Exhibits pleiotropy, it has many different anti-cancer functions.
-TP53 is located within chromosome 17, arm p, cytogenetic band 17p13.1.
-Papovavirus- simian virus 40
• Tumour causing DNA virus in model systems. Transforms mouse
fibroblasts which gain ability to form tumours when injected back into
mice.
• Harvested antisera after tumour growth, immunoprecipitated proteins.
Antibody bound to large T-antigen from virus and 53kDa protein from
mouse host. Called protein p53, binds to viral oncoprotein.
• Since then, discovered many oncoviruses which express proteins which
bind and inactivate p53- viral oncoproteins.
> Model papilloma virus, human T-cell leukemia virus type I.
p53 mutations
-Introducing wild type tp53 cDNA at same time as injecting transformed
fibroblasts block tumour formation.
-Conversely, introducing loss of function p53 from tumour cell lines enhances
transformation of cells by oncogene (e.g., H-Ras).
-Knockout mice for p53 succumb in a dose dependent manner to many cancers.
-Point mutations
• Generally non-synonymous coding (in exons) point mutations which
produce aberrant full-length proteins. Change function of protein.
• Enhance tumorigenesis when introduced experimentally. Point mutant
p53 alleles are dominant-negative, loss of function of one gene affects
more than 50% of proteins since p53 is a tetramer. Protein encoded by
gene loses ability of normal function but can also interfere with wild type
molecule of protein.
• Haploinsufficient tumour suppressor means loss of one copy is enough for
progression towards tumorigenesis.
• Positive-dominant mutations are subclass that inactivate p53 tumour
suppressor activities and cause it to acquire additional capabilities with
roles in activating invasion and metastasis.
, > Gain of function- can drive invasion for progression and metastasis.
Promotes tumourigenesis more than deletion.
-Rarer mutations
• Deletions and insertions.
• Gross deletions where part of or whole gene is lost
• Intragenic mutations (indels) which lead to frameshift mutations.
-Origin of p53 aberrations
• Spontaneous (somatic)- events occur in single cell, often LOH in tumours.
• Inherited (germline)- rarer, LOH also occurs.
p53 and genomic integrity
-p53 controls cell proliferation and genomic integrity. Intertwined functions.
• p53 prevents DNA damage accumulating (particularly from ss and ds
breaks).
-Acts as a transcription factor, binds to consensus sequence and upregulates
transcription of several genes, many involved in cell cycle and genome
maintenance.
, • p53 mutations are predominantly in the coding region for the domain that
binds DNA, result in loss of DNA binding.
• Via activity as TF, stops. ell cycle and boosts ability to repair DNA.
p53 regulation
-p53 is actively transcribed in cells then activated after stress (e.g., DNA damage)
post-translationally.
• dsDNA break -> p53 protein stabilization + post-translational modification
-> gene transcription of TSGs (p21, DNA repair enzymes)
• PTM code on p53- specific residues modified via phosphorylation,
acetylation, ubiquitylation, methylation, sumolynation. Signal hub for
many responses to stress signals.
• PTMs may also affect stability of p53 independent of Mdm2, directly
affect transcriptional activation potential (target selection), sequester
p53 from nucleus and affect DNA binding of p53.
-Mdm2 major node in regulation of p53. Keeps p53 in off state by causing its
degradation.
• p53 polypeptide attached to chains of ubiquitin by covalent linkage-
polyubiquitylation
> Results in proteasomal mediated degradation of polyubiquitylated p53.
Ubiquitin acts as recognition signal for proteasome.
> Reaction catalyzed by Mdm2, ubiquitin ligase. Keeps low levels of p53
despite continuous translation.
, • Stress signals compromise degradation, inhibit Mdm2. Result in PTM
modifications of p53, enhancing activity.
• For example, ATR and ATM kinases sense DNA damage -> protein kinase
cascade through checkpoint kinases results in p53 phosphorylation ->
Mdm2 does not bind phosphorylated p53.
• Mdm2 is a p53 target gene, to avoid p53 activation in absence of stress
p53 exerts negative feedback by upregulating its levels
> Point mutant p53 can no longer bind Mdm2, resulting in big
accumulations of non-functional p53.
-Mdm2 is thus an oncogene via its inhibitory action on p53 TSG. Its
overexpression along with other oncogenes can drive cancer.
• Murine lymphoma model- transplanted lymphocytes engineered to
overexpress c-myc +/- Mdm2. Mdm2/ myc mice had very low survival
rates compared to individual oncogenes.
• Mdm2 and paralogues (Mdmx) amplified in some cancers (increased
number of gene copies). Even if p53 not mutated, function is suppressed
during tumorigenesis due to these genetic alterations.
