Tumour biology Flashcards
Name the purines and pyrimidines in DNA and who they pair with
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Purines : adenine, guanine
Pyrimidine: cytosine, thymine
A = T (2 hydrogen bonds)
G 三 C (3 hydrogen bonds)
How is DNA packaged?
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DNA is an acid with highly negative charge. Double-helix - 2 anti-parallel strands – polymers of nucleotides
DNA helix wraps around nucleosomes consisting of histones (highly +ve charge) -> forms chromatin fibre -> coiled and packaged into chromosomes
Name the bases in RNA? What is the difference between RNA and DNA?
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- Single stranded, shorter, U instead of T
Purines: adenine, guanine
Pyrimdine: cytosine, uracil
A = U (2 hydrogen bonds)
G 三 C (3 hydrogen bonds)
Name the difference types of RNA and their assoc RNA polymerases?
rRNA (ribosomal)- up to 5kb- structural - made by RNA Pol 1
mRNA (messenger) - 1-10kb- carry messages to encode proteins - made by RNA Pol II
tRNA (transfer)- 76-90bp (very small) - made by RNA pol III
What are exons and introns?
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Exons: coding DNA
introns: non-coding DNA
Define anaplasia?
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Lack of differentiation and loss of morphological characteristics. Cellular and nuclear pleomorphism (different sizes). Hyperchromatic nuclei. Loss of orientation/polarity. Increased nuclear:cytoplasm ratio. Frequent mitoses +/- multipolar spindles. Giant cells
What is dysplasia and CIS?
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Dysplasia is disordered growth, usually in epithelium. Loss of cellular uniformity and architectural orientation, pleomorphism, increased mitotic figures in abnormal location (not just basal layer). Cells retain their polarity (unlike anaplasia). Usual progressive maturation is lost
Carcinoma In situ: full thickness dysplasia with basement membrane intact and no extension to subepithelium
Both can progress to cancer but can normalise
What is metaplasia?
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Reversible change of one cell type to another cell type- eg Barretts (squamous to columnar) or smoker’s bronchial epithelium (columnar to squamous). - may be pre-malignant
What is hyperplasia?
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Increase in number of cells in a tissue eg HRT and endometrium. May be pre-malignant
What are the basic steps of making a protein from DNA? What are post-translational modifications?
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Transcription (DNA transcribed 5’ to 3’ by RNA polymerase binding to promoter and unzipping DNA strand until reaches a terminator sequence. Forms pre-mRNA which gets 5’ cap and introns spliced out and polyA tail (facilitates binding to ribosome) to form mRNA and travels to ribosome from nucleus)
Translation - involves all 3 types of RNA, occurs in a ribosome (made of 2 structural rRNA/protein subunits - ribosome moves to each codon and tRNA brings the amino acids binding to codon by its complementary anticodon sequence- can be free floating in cytoplasm or attached to endoplasmic reticulum) - starts at AUG (methionine/open reading frame) - stops at stop codon (UAG/UGA/UAA) - polypeptide chain formed. Genetic code is redundant – more codons for 1 AA (64 codons = 20 AAs)
Post-translational modifications.
- Chemical modifications that generate heterogeity in proteins, modifying end product of expression and
regulating protein function
- Cleaving – Proteases cut sections leading to activation
- Ubiquitination – tagging with Ubiquitin, leading to degredation by Proteosomes
- Phosphorylation – adding Phosphate groups to change function – activate/deactivate
- Also Acetylation and Methylation – NB: Glycosylation is NOT a post-translational modification
Describe the process of transcription? Where and how does it start?
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Usually started at 5’ end of DNA - contains a nucleotide sequence that make up the promoter region.
TATA box - located near the start of transcription is one of the most important regulatory elements
TBP (tata box-binding protein) is a generic transcription factor crucial for the initiation.
Response elements: short DNA sequence in promoter recognised by specific transcription factor.
Reaches stop codon - 3 codons always indicates stop protein synthesis - UAA, UAG, UGA
What is a somatic vs germ line mutation?
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Somatic mutation- occur in somatic cells and only affect the individual in which the mutation arises
Germ-line mutation- alter gametes and passed on to offspring
Name some types of point mutations?
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Substitutions- transitions/transversions
Deletions
Insertions
Describe the two types of base pair substitutions?
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Transitions: convert a purine to another purine
- 4 types - A↔G, T↔C
- most result in a synonymous substitution (no change in amino acid due to degenerate code)
Transversions: convert a purine to a pyrimidine and vice versa
- 8 types
- more likely to result in non-synonymous mutations
Can result in:
- Nonsynonymous/misense mutation - base pair substitution results in a different amino acid eg sickle cell
- Nonsense mutation: base pair substitution results in stop codon (short protein)
- Neutral non-synonymous mutation: base pair substituion results in substitution of an AA with similar chemical properties (does not affect function)
- synonymous/silent mutation
What type of mutation does an insertion/deletion of a base cause?
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Frameshift mutation: deletions or insertions non divisible by 3 result in translation of incorrect AAs/codons.
What is a misense mutation? What is a nonsense mutation? What is a silent mutation?
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Missense: Change from one AA to another due to a base pair substitution.
Nonsense: Base pair substitution results in a stop codon
Silent: which code for the same amino acid due to degenerate nature
What is an inversion? What is a duplication?
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Duplications – Repeat of 1 or more nucleotides to another DNA sequence
Inversions – inverted sequence of 2 or more nucleotides within a DNA sequence
What is chromothripsis?
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When a chromosome shatters and in an attempt to repair the damage many incorrect junctions occur. Can disrupt tumour suppressor genes and produce oncogenic fusion genes. Shattered DNA fragments may also form extrachromosomal DNA.
CAUSES CANCER
Produces multiple mutations at once. Detected by FISH.
Potential causes include ionising radiation, telomere dysfunction, aborted apoptosis.
Which type of UV radiation causes the most cancer?
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UVB
UVA - reaches most acellular dermis (wavelength og 320-380nm)
UVB- reaches epidermis (wavelength 290-320nm)
UVC- absorbed by ozone, rarely reaches skin
Note need a wavelength of <100nm to cause ionising event - hence UV radiation is not ionising. Does have enough energy to break chemical bonds.
How does UV radiation cause cancer?
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UVB
Conjugated double bonds in the rings of the nitrogenous bases of DNA absorb UV radiation.
- causes CYCLOBUTANE PYRIMIDINE DIMERS- cause a bend in DNA helix so DNA polymerase cannot read DNA template -> it preferentially incorporates an A reside so TC/CC dimers restored to TT dimers -> result in transitions (TC -> TT, CC-> TT). PYRIMIDINE DIMERS UNIQUE TO SKIN CANCER. Induces NER.
- Also get 6,4 photoproducts- abasic site
UVA
- indirectly damages DNA via free-radicals, water is fragmented generating ROS -> cause DNA damage
- G-> T transversions characteristic
DNA-protein crosslinks are also important lesions in cells exposed to UV radiation. Crosslinks are
particularly disruptive, as they occur mostly in the area of the chromosome that is undergoing replication.
What is characteristic of UVB radiation damage?
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Pyramidine dimers - 2 types
- cyclobutane pyrimidine dimers (2/3)
- 6,4 photoproducts (1/3)
What is the carcinogen in coal tar from cigarettes and how does it cause mutations?
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Polycyclic aromatic hydrocarbons : Benzo (a)pyrene (the most well-known carcinogen in tobacco smoke)
PAHs metabolised (by CYP1A1 enzyme) -> forms ultimate carcinogen -> forms adducts with purine bases -> results in G-> T transversions.
What in nitrosamines and nitrosamines causes cancer?
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Found in tobacco, preserved fish and meats during smoking. Principal carcinogenic product is alkylated O6 guanine derivatives.
What are DNA mismatches?
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DNA can base pair incorrectly leading to DNA structure distortion
Tautomeric shifts
Deamination
Loss of bases: depurination, depyrimidination
What is a tautomeric shift?
A tautomer is a structural isomer.
Thymine and guanine usually in keto forms (C=O) -> undergo spontanous isomerisation to enol form (C-OH) - means they can join to keto forms of T and G
Cytosine and adenine usually in amino forms (C-NH2) -> undergoes spontaneous isomerisation to imino form (C=NH) -> means they can join to amino forms of C and A
What is deamination
Type of mismatch: Loss of an amino group (from C, G or A) can happen spontaneously and result in conversion of bases.
Cytosine -> uracil
Adenine-> hypoxanthine
Guanine -> xanthine
Name the different types of DNA repair?
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Direct repair Base exicision repair- most common Nucleotide exicison repair Mismatch repair DSB repair - HR - NHEJ
What is direct DNA repair and name some examples of direct DNA repair?
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Damage is recognised by a protein factor and directly chemically reversed
Bulky alkyl adducts
- recognised by O6- alkylguanine DNA alkyltransferase (AGT/ MGMT), the damaged base is flipped out of the DNA helix- methyl moeity is transfered to AGT protein
- MGMT, known as O6-methylguanine-DNA methyltransferase,
- AGT is a suicide enzyme- only does one round of demethylation and it is not regenerated.
Pyrimidine dimer - NOT in placental mammals
- recognised by photolyase which absorbs blue light and breaks the cyclobutane ring
Describe the process of base excision repair.
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Targets chemically altered bases due to oxidation, deamination and alkylation (eg 8-oxoguanine- failure to remove this results in G->T transversion mutation)
Initiated by specific DNA glycosylases (eg OGG1, MUTYH) that recognise and remove specific damage to leave an AP- apurinic/apyrimidinic site
- Scaffold protein XRCC1
- AP site cleaved by endonuclease
- repair takes place
- DNA polymerase B replaces the nucleotide and ligase 3 and 1 fills the gap
- Poly (ADP-ribose) polymerase- PARP- interacts with single strand breaks and synthesise poly (ADP-ribose) chain that signal to other DNA repair proteins and leads to modification of histones and relaxation of chromatin to increase accessibility
Short patch repair = one nucleotide. Long patch = 2-10 nucleotides.
Single strand breaks recognised by alkaline solution assay.
Important in repairing radiation induced damage.
What is the role of PARP in DNA repair?
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SSB
- Poly (ADP-ribose) polymerase- PARP- interacts with single strand breaks and synthesise poly (ADP-ribose) chain that signal to other DNA repair proteins and leads to modification of histones and relaxation of chromatin to increase accessibility
What types of damage is NER useful for repairing?
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Specific to helix distorting lesions e.g.
UV induced DNA damage pyrimidine dimers
Bulky DNA adducts e.g. polycyclic aromatic hydrocarbons
Cisplatin
Describe NER?
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Helix-distorting lesions
Recognition of damaged site (XPC and XPA) lead to unwinding of DNA by helicases (XPB and XPD) and excision of a short ssDNA segment by ERCC1-XP F nuclease (usually 10-20 bases) containing damage
Lots of other XP proteins involved
DNA polymerase copies the undamaged strand and DNA ligase seals off the ends.
No loss of information. PCNA (proliferating cell nuclear antigen) is associated with DNA polymerase.
Two forms- differ in how they recognise the damage
- GG-NER- global genome repair occurs continuously but takes a long time to spot the abnormality - continuously scanning for helix distortion
- TC-NER- transcription coupled repair- actively transcribed strand of DNA is repaired with greater efficacy - RNA polymerase comes across the DNA backbone kink and stalls - NER recruited and repairs - transcription can continue
When does mismatch repair occur?
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Corrects errors that arise spontaneously during DNA replication. When there are base-base mismatches or insertions/deletions introduced due to slippage during replication of repetitive sequences (e.g. microsatellites).
What disease is caused by NER deficiencies?
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Xeroderma pigmentosum
Defects in 1 of 7 proteins XP A-G
Rare AR disorder
Extremely sensitive to UV light
No radiosensitvity
1000x risk Skin cancer, 20x risk other malignancies, life expectancies 20-30s, keratitis, mental retardation, premature dementia
How does mismatch repair work?
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hMSH2/3 (indels) or 2/6 (mismatch) (also called MutS) recognises distorted structure in DNA and recruits MutL heterodimers (MLH1/PMS1) which have endonuclease activity - this is the MMR complex - allows strand discrimination by putting nicks into the incorrect strand. PCNA is also recruited. Exonuclease cuts out the nucleotides. DNA polymerase/ligase resolves. Gap is filled by polymerase and sealed by ligase.
Which cancer syndrome is caused by mismatch repair deficiencies?
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HNPCC/lynch - hereditary non-polyposis colorectal carcinoma = colorectal/endometrial/ovarian cancer. Autosomal dominant. Less severe than FAP.
Describe the process of NHEJ?
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- DNA ends recognised by Ku 70/80 (XRCC6/5) proteins -> recruit DNA-PK catalytic subunit. The Ku heterodimer has a high affinity for DNA ends and forms a close-fitting asymmetrical ring that
threads onto a free end of DNA - DNA-PKcs associates with Ku70/80 to form the DNA-PK holo-enzyme. DNA-PK holds ends together. DNA-PK phosphorylates H2Ax so opening chromatin.
- recruits artemis protein which trims ends.
- DNA ligase IV forms a tight complex with XRCC4. XRCC4 and ligase 4 rejoin ends
If DNA ends compatible they can be directly ligated together but often processed (resected) then ligated back together. Error-prone, mutagenic, quick, efficient, throughout cell cycle, can lead to chromosome translocations and telomere fusion
Important in repairing radiation induced damage, particularly in late responding tissues.
important in V(D)J recombination - therefore get immunodeficient if mutated - SCID
What are the pros and cons and NHEJ?
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Quick 2-4hrs
LOST DNA, MUTAGENIC
When in the cell cycle does NHEJ occur?
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Can occur at any point.
Very important in G1 PHASE of cell cycle as HR doe not occur.
Non proliferating normal tissues with low mitotic rate eg brain, kidney cord sit in G1 phase so if you were to inhibit NHEJ then would get back late toxicity.
What has overriding control and orchestrates response to DSBs?
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ATM
- phosphorylation of histone H2AX by ATM/DNA-PK causes recruitment of proteins to site of damage. H2AX phosphorylation represents an important step in the formation of nuclear foci of DNA repair
proteins (also phosphorylated by ATR and DNA-PK)
Describe HR
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MRN complex- made up of Rad50/Mre11/Nbs1 complex binds to ends of DNA and uses endonuclease activity to create single strand 3’ end overhangs. (Mre11 also activates ATM)
Rad52 binds to DNA termini
RAD51 is a recombinase and forms a nucleoprotein filament that facilitates strand invasion
for homologous recombination. Rad51 binds to exposed ends to form nucleoprotein - BRCA 1/2 aids in nuclear transport of Rad51 and Rad52 helps with binding of Rad 51 to exposed ends. Needs the BRCA1-PALB2-BRCA2 complex.
Rad51 helps to search for homologous template
When homologue is found the 3’ end of ssDNA serves as a primer to initiate DNA synthesis.
BCM protein (deficient in Bloom’s syndrome) help migrate the junctions towards each other
Resolvases restore the junctions known as Holliday junctions.
Important in repairing radiation induced damage. No loss of information
What does the fanconi anaemia pathway do?
Repair inter- strand cross link which block replication forks - repairs stalled replication forks. Bulky adducts so NER started - causes dsDNA breaks as on both sides so HR then started. Fanconi proteins important in this NER/HR process of interstrand repair. Therefore Fanconi anaemia patients very sensitive to cisplatin.
Assembly of FA complex which recruits nucleases.
Breaks DNA and lesion is bypassed by translesional synthesis and further repaired with NER
Gap in 2nd strand is a double strand break and is repaired by HR
Fanconi anaemia patients get a lot of quadriradials
What is the double hit hypothesis
One copy of a gene is inherited mutated (germ line) in all cells of the body. Later on a second copy gets randomly mutated (somatic) which pushes the cell towards oncogenic transformation
What is synthetic lethality?
Genetic concept that describes buffering effect genes have on each other functions. If one targets a synthetic lethality partner of mutated gene one will achieve selected cell death in only cancer. PARP1 inhibitors in BRCA1/HR deficient cancers is the main example.
Why are BRCA and PARP synthetically lethal?
PARP is a key mediator in BER
BRCA1/2 key mediator in HR
In BRCA mutant cell HR is dysfunctional so cells heavily rely on BER. Because BER is blocked the single strand breaks become DSBs and HR not working to toxic to cells.
What causes genetic instability?
Replication errors- eg mismatch repair
Replication problems- impeded progress
Damage to DNA
Mitotic errors - chromosome segregation defects eg spindle assembly checkpoints.
What is a transcription factor?
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Protein that binds to gene promoters and regulates transcription
3000 transcription factors regulate 20,000 genes
What is the structure of a transcription factor?
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Contain a DNA binding domain, transcriptional activation domain (Recruits the generaly replication complex), dimerisation domain and ligand binding domain (e.g. steroid hormone for steroid receptors).
4 types of DNA binding domains: - helix-turn-helix motif - Leucine- zipper motif - helix- loop-helix motif - Zinc finger motif These domains are characteristic protein formations that enable transcription factor to bind to DNA
How is the activity of a transcription factor regulated?
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Synthesis in particular cell types only
Covalent modifications eg phosphorylation
ligand binding
Dimerisation - exchange of partner proteins
Give some examples of transcription factors?
AP-1 (jun and fos family, dimerise in different ways)
Myc family (Myc, max, mad, mxi)- dimerise in different ways
Steroid hormones
RAR - retinoic acid receptor
p53 - mutated in 50% cancers
What is AP-1 and how does it work?
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Transcription factor.
AP1- binds to TPA response element or to cyclic AMP response element in the promoter of target genes
AP-1 is made up of two components and is produced by dimers from 4 families: Jun, Fos, ATF/CREB and MAF family. 18 possible combinations. Fos and jun contain leucine zipper dimerisation domain. Promote proliferation generally.
Jun B acts as a negative regulator of growth when dimerised with Jun
AP-1 activated by specific signals e.g. growth factors (MEK/ERK pathway), ROS, radiation
AP-1 made from c-Fos and c-Jun can transform a normal cell to malignant cell
Activates cyclin D
How do steroid hormone receptors act as transcription factors?
Superfamily of steroid hormone receptors act as ligand-dependent transcription factors.
Nuclear receptor family (48 members) eg Androgen receptor, oestrogen receptor, glucocorticoid receptor, mineralocorticoid receptor, progresterone, PPAR, retinoic acid, thyroid hormone receptor, vitamin D receptor.
Contain a zinc finger DNA binding domain, ligand binding domain for specific steroid hormones and dimerisation domain.
Each domain specific for that hormone.
Steroid hormones pass through cell membrane and bind to intracellular receptor in cytoplasm or nucleus -> receptors move to nucleus and activate transcription through specific DNA response elements.
What is the retinoic acid receptor (RAR) and how does it work?
Transcription factor
RAR is located in nucleus and is a heterodimer with RXR. This heterodimer acts as a transcriptional repressor in absence of retinoic acid (derived from vit A) by recruiting co-repressors. It binds to RA response element (RARE) in target genes. In vitamin A presence, it binds to co-activators instead.
Aberrant forms of RARs are characteristic in some forms of leukaemia. PML-RAR recruits HDACs and DNMTs to silence differentiation genes - all-trans retinoic acid is used in treatment of APML to change the PML-RARA to an activating rather than silencing transcription factor = differentiation.
What can a somatic mutation upstream of TAL1 gene produce?
TAL1 (T cell Acute Leukaemia 1)- oncogene which codes for basic helix-loop-helix transcription factor -> create a super enhancer which upregulates expression -> loads of transcription factors.
What makes up chromatin, nucleosomes and histones
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Chromatin : thread of DNA 1m long when stretched out (60%), assoc RNA (5%), protein (35%). Structural scaffold tightly wound to form chromosomes.
Nucleosomes formed into fibres then radial loops.
Nucleosome: 147 base pairs of DNA wrapped 1.7 x around core histone proteins. Histone core is an octomer of histones (2 of each of H2A/H2B/H3/H4 and then H1 is associated with the linker DNA between nucleosomes)
Histone: contains domains for histone-histone, histone-DNA interactions and NH2-terminal lysine rich; COOH rich terminal tail domains which can be modified eg methylated/phosphorylated.
- Histones play a role in protecting against ionising radiation induced damage
What is epigenetics?
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Heritable information that is encoded by modifications of genome/chromatin components (not a change in DNA sequence so NOT mutations)
What are the two most common types of epigenetic modification?
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Histone modification
DNA methylation
Both can be acquired or inherited.
What types of histone modification are there?
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Acetylation (euchromatin)
Methylation (H3K4me3 euchromatin; H3K9me, H3K27me3 is heterochromatin)
Phosphorylation
Ubquination
Measured by ChIP-Seq
Describe histone acetylation?
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Alters chromatin structure and effects gene expression. Acts as a docking signal for recruitment/replusion of chromatin Histone acetyltransferases (HATs) - add acetyl group to histone tail lysines - neutralises positive charge on lysine residues and relaxes chromatin folding - transcriptional activators recruit HATs
Histone deacetylases (HDACs) - remove acetyl group.
Name some examples of histone acetylation causing cancer.
EP300 gene codes for p300 protein a HAT
P300 usually acts as a tumour suppressor. Mutated in epithelial cell tumours.
Chromosomal translocation produces PML-RAR (APML)- recruits HDAC to promoter region of RA target genes and represses the expression of genes -> blocks cell differentiation. Give trans-retinoic acid = no longer recruits HDAC - genes activated instead = differentiation.
Oncohistones = mutated histones that cause cancer.
Where does DNA methylation occur on DNA?
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Addition of methyl group to position 5 of cytosine in CG dinucleotide
ONLY occurs at cytosine nucleotides which are situated 5’ to guanine (CpGs)
Where do you find the most CpGs?
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CpG is unequally represented in genome- which may be due to 5-methylcytosine easily deaminating to thymine causing a C-> T transition.
CpG islands- clusters of CpG located in promoter region of genes.
Methylated cytosines are mainly found in repressed genes eg X chromosome, inactivated genes, imprinted genes -> methylation is a heritable signal and assoc with compact chromatin structure and maintains gene silencing
Not clear how it works to silence - perhaps stops binding of transcription factor; recruit chromatin modifying enzymes; induce mutation due to deamination.
What are DNMTs and what types are there?
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DNA methyltransferases - mediate the covalent addision of a methyl group from a methyl carrier SAM (S-adenosyl-methionine).
Three DNMTs:
- DNMT1- during DNA replication this methylates DNA if original strand was methylated- allows inheritance of methylation
- DNMT3a, DNMT 3b- involved in de novo methylation
Overall do cancer cell have more or less methylation of DNA than normal cells?
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20-60% less methylation - causes genetic instability
Global hypomethylation with hypermethylation of specific gene promoters
30% breast cancers ER negative due to hypermethylation of ERalpha.
BRCA1 can be inactivated by methylation
MGMT (glioblastoma), APC (colorectal), MLH1- commonly methylated.
How do you detect DNA methylation?
Sodium bisulfite treatment- converts unmethylated cytosine to uracil by deamination.
Then do methylation specific PCR
Change grading of GBM (MGMT promoter methylation)
What are microRNAs (miRNAs)
Small non-protein coding RNAs (18-25 nucleotides) regulate expression of mRNAs
Able to repress hundreds of gene targets post transcriptionally -> powerful regulators of growth, differentiation and apoptosis.
Repress gene targets binding to 3’UTR of their target mRNA blocks translation
Can be oncogenes or tumour suppressors
What are telomeres?
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Protect ends of the chromosomes from digestion by nuclear enzymes and prevent induction of mechanisms of repair of DNA double strand breaks.
Composed of several thousand TTAGGG repeats bound by a protein complex called shelterin complex on 3’ overhang - forms T loop/G quadruplexes so no dsDNA break detected
Telomeres can be transcribed into a lncRNA that contains telomere repeat-containing RNA (TERRA) - essential in maintaining length of telomeres.
Telomere length and distance to a gene can also affect gene expression: telomere position effect.
What is the end replication problem? What is the Hayflick limit?
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DNA shortens by 100-200 DNA bases with each round of replication due to limits of DNA polymerases. DNA synthesised in 5’ to 3’ direction therefore complementary strand is synthesised in fragments (Okazaki fragments) with a RNA primer to initiate - these primers are lost so therefore the strand shrinks. Enter senescence once threshold length reached. If cells bypass senescence due to mutation, telomeres become critically short»_space; chromosome instability»_space;apoptosis
Hayflick limit: number of times a cell can replicate - limited by number of telomeres.
What is telomerase?
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A ribonucleoprotein containing human telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTR) which maintains the telomere length eg stem cells.
hTERT uses hTR as a template to add new repeats to telomeric DNA
AGAINST CENTRAL DOGMA OF BIOLOGY as synthesising DNA from RNA
- Physiologically observed in stem cells, germ cells and hair follicles – cells that need to be immortal
How does cancer affect telomeres?
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90% cancer upregulates telomerase, likely through reversal of epigenetic changes that occur on differentiation to
somatic cell lineages
Patients with lower TERT levels have better prognosis, promoter mutations in TERT»_space; increase telomerase activity»_space; cancer
- melanoma and other cancers have found mutations in TERT promoter.
- c-myc increases expression of hTERT gene.
Telomere shortening occurs in response to replication, ssDNA breaks and oxidative damage so may act as a tumour suppressor limiting replicative potential
What are the 4 types of protein involved in the transcription of growth factor signal?
Growth factors
Growth factor receptors (many are TKIs)
Intracellular signal transducers
Nuclear transcription factors
Name some types of EGFRs and what is their general function?
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- EGFR/ErbB1/HER1 - ErbB2/HER2 - no ligand binding activity - acts as a heterodimer for others - ErbB3/HER3 - no tyrosine kinase activity ErbB4/HER4
Family of receptor tyrosine kinases - important for transduction of a signal from an EXTRACELLULAR growth factor through the cell where regulates gene expression. Form homodimers or heterodimers after ligand binding.
RTKs can be indirectly activated by cross talk:
o G-protein coupled receptors activate proteases»_space; cleave precursor growth factors»_space; release of
ligands»_space; RTK activation
o ErbB2 receptors trans-activated by other RTKs and integrins
What is the structure of an EGFR
Extracellular ligand binding domain (cetuximab binding)
Single hydrophobic transmembrane domain
Cytoplasmic protein tyrosine kinase domain (except HER2 does not bind a known ligand, just acts as a co-receptor and HER3 only has weak kinase activity) (Osimertinib etc binding)
How does the EGF signal get inside the cell?
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Binding of EGF to receptor
EGFR receptor dimerisation (one EGF bound to each receptor)
Conformational change in the receptor causes autophosphorylation (one half of dimer phosphorylates other half due to kinase activation)
The phosphorylated tyrosine residues create high affinity binding sites for Src homology 2 (SH2) domains. GRB2 binds to SH2 domains on the EGFR and binds SOS via its SH3 domain so translocating it to the cell membrane. SH3 domains interact with SOS which recruits Ras. Causes Ras to release GDP and bind GTP, activating it.
Ras activates RAF/MEK/ERK and PI3K
SH2&3 domains mediate protein-protein interaction in pathways activated by TKs. Proteins that contain SH2/3 domains = Grb2, ABL, SRC and PI3K
Signal can be terminated by further phosphorylation of EGFR, removal of tyrosine phosphorylation, receptor endocytosis and degradation, and binding of receptor inhibitors.
What is Ras and what is its function
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Ras is a GTP binding protein. It is a GTPase. Intracellular transducer. When bound to GDP inactive. SOS releases Ras from GDP and it binds to GTP. (SOS [son of sevenless] is a guanine nucleotide exchange factor)
Binary switch
K-RAS, N-RAS, H-RAS
Ras is loosely bound to inside of cell membrane (also found on subcellular membrane compartments such as endoplasmic reticulum)
Causes activation of Raf (MAPKKK) and PI3K
Mutation results in Ras protein that can’t covert GTP»_space; GDP – therefore remains permanently “on”
- Most commonly mutated oncogene in humans - 30% of human tumours carry Ras mutation – (mainly in
Codons 12, 13, 61) e.g. K-Ras in colorectal cancer, N-Ras in thyroid cancer
- Anti-cancer targets against Ras oncogene include farneslyation transferase inhibitors (Ras requires posttranslational modification by farneslation to become active), up-stream targets (e.g. EGF-receptor -Cetuximab or HER2 – Trastuzumab) and down-stream receptors (e.g. Raf – Sorafenib or MEK – Trametinib)
- Ras hard to target directly (“undruggable”) – binding affinity of GTP to Ras is much higher than that of ATP to kinases and there are no binding domains for small molecule TKIs
What is Raf?
A serine/threonine kinase- MAPKKK
- Raf recruited to cell membrane and binds to RAS-GTP -> activates it -> signal transducer and carries the signal away from the cell membrane.
Describe the Ras-Raf Pathway?
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GF - extracellular
EGFR - dimerisation and autophosphylation -> SH2/3 domains
RAS - GTP (liberated by SOS from GDP)
RAF (MAPKKK)
phosphorylates MEK (MAPKK) (dual tyrosine and serine/threonine kinase)
Phosphorylates MAPK/ERK - serine-threonine kinase
MAPKs -> enter nucleus through facilitated diffusion and phosphorylates Fos to activate AP-1 transcription factors (made up of Jun and Fos family members) and Myc family transcription factors (myc, max, mad, Mxi) dimerise in different ways.
Describe the PI3-k pathway?
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PI3K - a lipid kinase interacts directly with Ras. PI3K phosphorylates PIP2 to PIP3 (in cell membrane).
PIP-3 recruits serine/threonine kinase PDK-1 to cell membrane
Then serine/threonine kinase AKT recruited and phosphorylated and activated by PDK-1.
Activated AKT takes signal from the membrane and is involved in anti-apoptotic signals by phosphorylating distant target proteins
- mTOR (serine/threonine kinase) - downstream target Akt which is involved in promoting anabolic programmes eg lipid/nucleotide synthesis.
Akt can also travel to nucleus to the nucleus where is can phosphorylate transcription factors like FOXO (forkhead box O!!!!!!!!)
INHIBITED BY PTEN via PIP3
What is Src?
Intracellular tyrosine kinase- plays role in proliferation, adhesion, invasion and motility
Src normally phosphorylated which blocks its SH2 and SH3 domain.
Src activated by tyrosine kinase receptors like EGFR - reveals its active domains.
Integrins activate focal adhesion kinase/Src complex. Activated FAK-Src functions to promote cell motility (by inhibiting E cadherin and promoting disassembly of focal adhesions), cell cycle progression and cell survival.
First oncogene discovered - from Rous Sarcoma Virus. v-SRC - lacks negative regulatory domain so constitutively active.
What is an oncogene? What is a proto-oncogene?
Proto-oncogene: normal cellular genes that encode proteins that stimulate proliferation/cell viability/inhibit cell
death (GO). c-GENE NAME
Oncogenes: Mutated genes whose protein product is produced in higher quantities or whose altered product has increased activity and therefore acts in a dominant manner and contributes to carcinogenesis. v-GENE NAME
Discovered in transforming retroviruses in animals (gene “captured” from host organism), v-onc found to be homologous to mammalian genes. Origin of names e.g. K-Ras – Kirsten rat sarcoma virus (K-Ras)
Types
- retroviruses - rous sarcoma virus
- growth factors e.g. PDGF
- growth factor receptor e.g. RET, EGFR, HER2
- Intracellular signal tranducers eg Ras (mutated in 30% tumours), BRaf
- Transcription factors
> 100 oncogenes have been identified.
Give an example of growth factor as an oncogene
Proto-oncogene = c-sis produces PDGF
Oncogene = v-sis causes unregulated growth via activation of PDGF pathway - inappropriately cytoplasmic so constitutively active.
Give an example of a growth factor receptor oncogene
Proto-oncogene- RET - TK receptor
Transduces signal to glial derived neurotrophic factor (GDNF) family ligands when in heterodimer with cofactors
- familial medullary thyroid cancer
MEN2A and MEN2B
Oncogenic activation can lead to constitutive activation by dimerisation or increased kinase activity
v-ERBB = truncated EGFR = no extracellular domain = constitutively active. Also often activated by point mutations infering with growth factor binding and gene amplification.
HER2 over-expression driving cancer cell proliferation – a genetic copy number
variation or epigenetic change»_space; over-expression or ErbB2. ErbB2 homo/heterodimerization»_space; amplified oncogenic signalling
Mutations»_space; inducing activation/inhibiting inactivation E.g. L858R mutation of EGFR – most common EGFR mutation causing cancer, leads to permanently ‘active’ structural conformation, or E.g. EGFR viii deletion mutation – loss of extra-cellular domain, therefore ligand independent and constitutionally ‘active’ – linked to cisplatin/cetuximab resistance in oral
cancers
Geneomic rearrangement/translocation»_space; improper localisation/activation E.g. Fusion of intracellular domain of ALK with dimerization/oligomerization domain of
another protein
Name some intracellular transducers that can become oncogenes
RAS
- 30% human cancers
- loss of GTPase activity- usually required to return active RAS-GTP to RAS-GDP causing constitutive activation
B-RAF
- melanoma (V600E)- causes constitutive kinase activity and insensitivity to feedback
Genes that code for cytoplasmic TKs can become oncogenes:
- SRC- colon cancer Tyr530 on Src cannot form its inactive phosphorylated form so always active
- MAPK
- ABL (nuclear tyrosine kinase - 9:22 translocation forms BCR-ABL which form homooligomeric complexes that autophosphorylate so constitutively active. Also stays in cytoplasm instead of nucleus)
Name some transcription factors that can become oncogenes?
AP-1
- components of AP-1- jun and fos are encoded by protooncogenes c-jun, c-fos.
- truncation at end of v-fos means it produces an mRNA with a longer half life.
- both c-fos and c-jun increased by overexpression and amplification
C-myc
- chromosomal translocation of myc (chromosome 8) to a location that falls within regulation of strong promoter of imumunoglobulin genes (chromsome 14) increases expression of myc gene -> Burkitts lymphoma
What are the mechanisms of oncogenic activation
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- Point mutations and deletions in coding regions e.g. loss of GTPase function in Ras so constituvely activated
- Mutations in gene promoter region
- Chromosomal translocations e.g. BCR-ABL, IMH-myc + insertional mutagenesis
- Gene amplification from the normal 2 copies in diploid genome eg HER2
How long is the cell cycle?
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Average length 16hrs
15hrs interphase (G1 is most variable and longest in length)
1hr mitosis (shortest phase)
Varies depending on cell type
Chromosomes can only be observed in mitosis due to condensation
When is a cell irreversibly committed to the cell cycle?
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On passing G1 restriction point
Describe the order of the cell cycle
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G0: Terminally differentiated – not dividing but expressing proteins required for cellular house-keeping. Most lymphocytes are in G0 and awaiting the appropriate antigen, which will stimulate to re-enter cell cycle at G1. mitogens/growth factors induce cells to re-enter cycle
Gap1: Longest part of cell cycle/most variable. Intense (mRNA) transcription of proteins for DNA synthesis
Check-point at G1 ensures favourable cell division conditions
G1/S checkpoint: Cyclin D + cdk4/6 = Rb hypophosphorylation + cyclin E/CDK2 = Rb hyperphosphorylation = S phase proteins + cyclin E/A/CDK2 = origin of replication complex.
S phase: DNA duplicated into 2 sister chromatids. Replicate two complete sets of all 46 chromosomes (2n to 4n DNA)
G2 phase: Synthesis of protein required for mitosis and cytokinesis. Checkpoint at G2 to confirm DNA replicated correctly and fit to proceed to Mitotic phase
G2/M checkpoint: Cyclin B/A + cdk1 = chromosome condensation/nuclear envelope breakdown/spindle formation
Mitosis: prophase, metaphase, anaphase, telophase
M checkpoint: chromatids lined up on metaphase plate = anaphase promoting complex = anaphase occurs
G2/M (due to mitotic catastrophe) >G1>early S>late S (due to presence of sister chromatid to use for HR) = radiosensitivity
How do cyclin-cdk complexes exert their effect?
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Cyclins: Regulate progression through the cell cycle. Binding of Cyclin to CDK = conformational change = reveals active site of CDK. Concentration in cell rise and fall throughout cell cycle. Cyclins are present only during short periods within the cell cycle and are controlled by their own degradation. Degraded by ubiquitin proteasome.
CDKs: concentration is constant. When activated by cyclins, they phosphorylate target proteins including transcriptional regulators, cytoskeleton proteins, nuclear pore, envelope proteins, histones. Dephosphorylation is important for resetting the cycle.
Where does cyclin D act and how?
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Drives progression through G1
Binds to cdk4/6
ONe of its final targets is EGF signalling pathway
Phosphorylates Rb = releases HDAC = transcription of cyclin E
Where does cyclin E act and how?
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Important for G1-S phase progression. Cyclin E/CDK2.
Hyperphosphorylates Rb = E2F released and drives transcription of S phase proteins.
Where does cyclin A act and how?
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Important for S phase progression. Formation of origin of replication complex with CDK2
Directs G2 and G2->M phase. Drives nuclear envelope breakdown, spindle formation and chromosome condensation.Important for G2/M checkpoint with CDK1:
Where does cyclin B act and how?
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Directs G2 and G2->M phase. Drives nuclear envelope breakdown, spindle formation and chromosome condensation.
Binds to CDK1
What are cyclin dependent kinases?
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Serine/threonine kinases that regulate progression of the cell cycle via phosphorylation.
What are the mechanisms of cdk regulation
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- Association with cyclins - activates cdk by conformational change revealing active site, cyclins degraded by proteasomes after being flagged by ubiquitin
- Association with cdk inhibitors - 2 families p16ink4a (INK) family and p21 (cip/kip) family.
- INK4 proteins (p16, p15, p18, p19) bind cdks 4/6 and interfere with binding to cycle D. p14 ARF inhibits the MDM2-mediated degradation of p53. p16INK4A is a cell cycle inhibitor that prevents phosphorylation of RB by CDK4. p14ARFis an MDM2 inhibitor thereby causing p53 levels to increase, resulting in greater cell cycle inhibition.
- p21 family members (p21, p27, p57) interact with both cyclins and their associated cdks and block ATP binding sites
- phosphorylation causing activation e.g. by CDK activating kinase (CAK)
-phosphorylation causing inactivation e.g. by Wee1 kinase.
- dephosphorylation causing activation (cell cycle division 25 phosphatase cdc25 dephosphorylates Wee1 site). CHK1/2 (serine/threonine kinase) phosphorylate and inhibits cdc25 - activated by DNA damage.
What is a key substrate of cyclin d - cdk4/6 complex?
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Rb protein
How does Rb protein regulate the G1 checkpoint?
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Rb protein is a key substrate of cyclin d-cdk4/6 substrate
Rb regulates activity of E2F transcription factor- crucial for expression of genes needed for S phase.
Hypophosphorylated Rb sequesters HDAC and E2F so transcription repressed.
Cyclin d-cdk4/6 causes partial phosphorylation of Rb and release of HDAC -> repression relieved for some genes like cyclin E
Additional phosphorylation by cyclin E- cdk2 causing release of E2F
-E2F (transcription factor) initiates the transcription of genes required to enter S phase including cyclin A
This occurs in response to growth signals
DNA damage is detected and repaired to ensure accurate transmission of genetic
material – halts cell cycle at G1 until DNA repaired, ensuring faulty DNA is not
replicated in S phase. DNA damaged detected by p53. ↑ p53 protein → ↑p21 level → inhibits cyclinE-cdk2 complexes →inhibits phosphorylation of Rb protein
i.e Rb proteins remain bound to transcription factor E2F→ G1/S arrest for DNA repair
What happens at the G2 checkpoint?
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Blocks entry into M phase in cells that have incurred DNA damage - allowing repair
DNA damage activates either -> ATM or ATR -> phosphorylate and activate Chk 2 and Chk1 respectively
Chk 1/2 prevents cdks from becoming active by phosphorylating and inactivating Cdc25.
Halts cell cycle progression
After successful repair- PLK1 which targets Chk1 for degradation and inhibits Chk2.
Describe the steps in mitosis
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Prophase- appearance of chromosomes as result of condensation, nuclear membrane breakdown, separation of duplicated centrosomes, Mitotic spindle assembled (a set of microtubules extending from the centriole which will
later attach to chromosomes)
Metaphase- align chromosomes on metaphase/equatorial plate and assembly of microtubules to form mitotic spindle
Anaphase- spindle pulling apart and separating chromatids, move towards spindle poles
Telophase- accumulation of chromosomes at their respective poles, reforming nuclear membrane, chromosome decondensation and cytokinesis (cytoplasmic division)
Chromosome refers to the whole package of DNA: pre S phase this contains 2n (double-stranded helix), post S phase contains 4n (two sister chromatids with double-stranded helix). Chromatid is 2n.
Centromere is part of chromosome where spindle attaches
What happens at the spindle assembly checkpoint/mitotic checkpoint?
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Signalling cascade that ensures the correct chromosomal segregation during mitosis and production of two genetically identical nuclei. Anaphase-promoting complex (ubiquitin-ligase) inhibits securin which usually inhibits separase - therefore APC activates separase which separates the chromatids by degrading cohesin so activating anaphase. APC is inhibited until the chromatids are lined up on the metaphase plate.
APC also inactivates cyclins so prevents re-entry into cell cycle.
What does anaphase complex do?
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An ubiquitin–protein ligase that regulates mitosis
During metaphase unattached chromatid pairs recruit proteins to form the mitotic checkpoint complex that inhibits anaphase complex. Once chromatids attached to microtubules they stop inhibiting it.
Anaphase complex targets securin - once degraded protease separase is activated
Separase cleaves cohesin link between sister chromatids allowing them to separate
APC also marks cyclins for degradation
What are aurora kinases?
Aurora kinases A, B, C
Regulate important aspects of mitosis
Serine -threonine kinases that phosphorylate target proteins
Aurora kinase A (STK15): localises to centrosomes during interphase; thought to play role in centromere maturation and spindle formation.
Aurora kinase B (STK12): involved later in mitosis, role in spindle attachment to chromosomal centromeres and the
spindle checkpoint, in chromosomal segregation and cytokinesis
Aurora kinase C (STK13): active during late mitosis. Localises to spindle poles
Aurora kinases frequently overexpressed in cancers
What mutations in cdks are you aware of?
Miscoding mutation in cdk4 stops it binding to INK4 inhibitors in subset melanoma patients
Cdk4 required for development of mammary gland tumours
Overexpression of cdk6 in some leukaemias
Name some tumour suppressor genes?
BRCA1/2
PTEN
Rb
p53
What is a tumour suppressor gene?
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Tumor suppressor genes represent the opposite side of cell growth control, normally acting to inhibit cell proliferation and tumor development. In many tumors, these genes are lost or inactivated, thereby removing negative regulators of cell proliferation and contributing to the abnormal proliferation of tumor cells.
Functions in DNA damage repair, blocking cell division and controlling apoptosis. Are usually recessive – both genes need to be mutated (exceptions are P53 and P27). Exceptions are due to haploinsufficiency – ONE mutated allele can lead to cancer as only half the normal quantity of protein is produced which isn’t enough to stop tumour formation in that specific case
Most familial cancer syndromes are due to mutations in tumour suppressor genes apart from MEN2A/2B which is RET.
What is the role of BRCA1?
Recruitment of Rad51 to DSBs - HR. (Also has broader role in NHEJ and RNA polymerase - ubiquitination. Thought to inhibit oestrogen receptor mediated transcription hence less breast/ovarian.
What is the role of BRCA2?
The localization of RAD51 to the DNA double-strand break requires the formation of the BRCA1-PALB2-BRCA2 complex.
What is PTEN?
Gene encoding a phosphatase with dual specificity. It can act as a protein and lipid phosphatase.
PTEN -> dephosphorylates the membrane lipid PIP3 -> PIP2
so inhibiting the PI3K pathway
How does PTEN mutation cause cancer?
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Loss of inhibitory dephosphorylation activity of PTEN (for PIP3) can result in a consituitively active PI3K pathway.
What syndromes does a germline mutation of PTEN cause?
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Cowden disease aka hamartoma syndrome. Autosomal dominant. BET – breast, endometrial and thyroid cancers.
Describe the differences in the sporadic and familial form of retinoblastoma
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Tumour suppressor gene
Familial (40% cases)
- one germline mutation and second sporadic- often results from somatic mitotic recombination in which normal gene is replaced with the mutant copy - Knudson’s 2 hit hypothesis
- often bilateral
- autosomal dominant
- also associated with small cell lung cancer
Sporadic form (60% cases)
- both mutations occur somatically in the same retinoblast.
- low chance of this occuring > once so usually only affects one eye
What is Rb?
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Transcriptional co-factor that can bind to transcription factors and either inhibit or induce transcription factor activity
Rb has >100 known protein binding partners
Main role is to regulate G1 -> S phase transition
Facilitates activity of E2F and chromatin remodelling enzymes
Cell cycle arrest can be induced by Rb via stabilisation of CDK inhibitor p27.
What are the upstream activators of p53?
DNA damage
Aberrant growth signals
Cell stress- radiation, drug, hypoxia, nucleotide depletion
What are the downstream responses possible when p53 is activated?
Cell cycle arrest or senescence (p21 - inhibits CDKs so inhibits G1/S and G2/M checkpoint)
DNA repair (DNA repair proteins e.g. XPC)
Apoptosis (e.g. PUMA)
Inhibition of angiogenesis (thrombospondin 2)
What is the structure of p53 protein
p53 - phosphoprotein transcription factor containing 4 distinct domains:
- Amino-terminal transactivation domain and MDM2 binding site
- DNA binding domain containing Zn ion
- Tetramerisation domain
- carboxy-terminal regulatory domain
-p53 binds as a tetramer to p53 response element
How is p53 regulated?
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Regulated at level of protein degradation not gene expression
Main regulator = MDM2
p53 present transiently as constantly being degraded.
Other regulators: MDMX and HAUSP (removes Ubiquitin form p53)
What is MDM2?
Main regulator of p53 protein
Ubiquitin ligase (flags protein for proteolysis)
Modifies the carboxy-terminal domain of p53 tagging it for degradation.
It also binds and inhibits p53 transactivation domain and transports protein into cytoplasm away from nucleus
How is MDM2 regulated?
p53 stimulates production of MDM2
Low amounts of p53 will reduce transcription of MDM2
What is the activation pathway (upstream of p53) when a DSB occurs?
DSB -> stimulates ATM -> phosphorylates and activates Chk2 -> ATM + Chk2 phophorylate amino-terminal sites of p53 -> interferes with binding of MDM2
What is the activation pathway (upstream of p53) when cellular stress occurs?
Cell stress -> activates ATR -> casein kinase II -> phosphorylates p53
What is the activation pathway (upstream of p53) for oncogene activation?
Activated oncogenes eg Ras -> activity of protein p14arf -> sequesters MDM2 to nucleolus of nucleus
What are the downstream activators once p53 has been activated?
INHIBITION OF CELL CYCLE
Transcriptional induction of p21 gene -> product p21 inhibits several cyclin-cdk complexes and causes pause in G1->2 transition
APOPTOSIS
- several mediators of apoptosis transcriptionally regulated by p53
- induces pro-apoptotic proteins NOXA, PUMA, p53AIP1
- tips balance regulated by Bcl-2 towards apoptosis
DNA repair and angiogenesis
- Gene XPC is involved in nucleoside excision repair and is regulated by p53
- Thrombospondin an inhibitor of angiogenesis is also regulated by p53
- Induction of miRNAs is important for inhibiting stem call and preventing mets
How does p53 decide which of the downstream outcomes occur?
P53 chooses between apoptosis and cell cycle pausing via – phosphorylation of Ser46 (apoptosis); pulses (pause) vs sustained expression (apoptosis); presence of cofactors e.g. ASPP (apoptosis stimulating proteins of p53) push to apoptosis
What regulates p21?
p53 and MIZ 1 bind to promoter and induce transcription resulting in cell cycle inhibition
However if myc is present it competes with p53 and binds to p21 and inhibits transcription blocking cell cycle inhibition
What is the most common type of mutation in p53? How does this compare to other tumour suppressor genes?
> 75% p53 mutations = missense mutations - most of these are located in the DNA binding domain
Differs from classical tumour suppression genes -> tend to have nonsense or frameshift mutations that lead to inactivated truncated proteins.
What is Li-Fraumeni and how is it inherited?
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Germline mutation of p53
AD disease - haploinsufficiency can be sufficient to cause cancer. p53 important in apoptosis, DNA repair, senescence.
25 x increase risk of developing cancer <50yrs. 50% will have cancer before aged 30.
Sarcomas, HER2+ breast cancer, leukaemia, brain tumours, choroid plexus tumours (specific - refer for testing), adrenocortical tumours (specific - refer for testing. Very sensitive to carcinogens (if smoker will definitely get lung cancer, if have ionising radiation for cancer then will get radiation induced sarcoma)
RADIOSENSITIVE - sarcomas
However, their tumours are actually radioresistant so radiotherapy less effective.
Patients with Li-fraumeni syndrome do not usually lose other p53 gene, remain heterozygous. This does not fit with Knudsons hypothesis. Why might this be?
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Reduced amounts of p53 (haploinsufficiency) can cause transformation, also specific mutations may result in varied amounts of tumour suppressor.
Some p53 do not lead to loss of function but instead form altered protein that interacts with the normal p53 and inactivates it. p53 is tetramer so just one abnomral protein inactivates whole tetramer
What is the continuum model of tumour suppression?
Integrates the two hit hypothesis with broader concept
Subtle dosage effects of tumour suppressor either as changes in level of expression or protein activity
eg p53- one dominant negative mutation can cause tumour suppression
What can viruses do to p53?
Adenovirus E1A, papillomavirus E6 & E7 etc inactivate Rb and p53.
Some do this using uquitin-proteosome system to degrade it.
How does HPV virus affect p53?
p53 is rarely mutated
binding of HPV protein E6 to p53 causes it to be flagged for degradation
Polymorphisms in p53 gene leads to differences in risk for cervical cancer. eg pts with 2 allelles coding for Arg have 7 x increased risk as it is more susceptible to degradation by HPV E6
What is apoptosis?
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Highly regulated process of programmed cell death, active process. ATP dependent.
- Highly regulated, not associated with an inflammatory response
- Reduction of cellular/nuclear volume
- Chromain condesation/DNA ladder formation - multiples of 80bp
- Nuclear fragmentation and plasma blebbing
- Cell contents packaged in membrane bound bodies
- Engulfment by phagocytes - The exposure of phosphatidylserines (phospholipids) on the exterior of the plasma membrane is the signal that initially recruits phagocytes. Ordinarily, phosphatidylserine is sequestered on the inner leaflet of the phospholipid bilayer and is not displayed on the cell’s surface. Macrophages then produce antiinflammatory TGFbeta to ensure no inflammatory response is produced.
- 2 pathways - Intrinsic (mitochondrial driven) and Extrinsic (receptor driven signalling)
- Both mediated by Caspases (Cysteine-rich ASpartate ProteASES):
o first synthesised as inactive Procaspases – activated by cleavage of Aspartate residues, then cause
further cleavage»_space; cascade of amplification»_space; amplification of apoptosis signal
o Initiator Caspases = 2/8/9/10, Executioner Caspases = 3/6/7 (note Caspase 3 serves as a paracrine signal from dying cells to stimulate proliferation of surviving cells) - Can be stimulated by extracellular signals - “death factors” or by intracellular insults – DNA damage/oxidative
stress
What is necrosis?
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Sloppy process whereby cells swell, cell membranes become leaky and cells spill out contents into surrounding tissue and cause inflammation. Uncontrolled, in response to trauma. Swelling of cytoplasmic organelles. Moderate chromatin condensation
Necroptosis: Necroptosis is a programmed form of necrotic cell death. Necrotic cell death has been considered a form of passive cell death. However, the discovery that TNF-alpha mediated necrosis can be inhibited by a specific inhibitor of RIP1 kinase, necrostatin-1, led to the concept of necroptosis. Necroptosis has now been established as a regulated necrotic cell death pathway controlled by RIP1 and RIP3 kinases. Under conditions that are insufficient to trigger apoptosis, TNF-alpha activates TNFR1 and in turn induces the recruitment of RIP1 kinase and other proteins to form complex I. Subsequently, these proteins dissociate from TNFR1 and RIP1 can be found in the cytosol in complex IIb, which includes RIP1 and RIP3. The formation of complex IIb leads to necroptosis
What are caspases?
Specific proteases that act like molecular scissors to cleave intracellular proteins at aspartate residues
he initiator caspases (caspases-2, -8, -9 and -10), which activate the downstream caspases, and the executioner caspases (caspases-3, -6 and -7), which cleave cellular substrates.
Describe the extrinsic pathway of apoptosis?
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Death factor (eg Fas ligand/TNF) -> binds to transmembrane death receptors (Fas/TNF-r). FAS is transmembrane on T cells, TNFa is free.
Receptors form homotrimers undergo conformational change and expose intracellular death domains.
Intracellular adaptor proteins (FADD/TRADD) transduce signal to caspases
Recruit pro-caspase 8 via death effector domains.
Pro-caspase 8 activate when close together by self cleavage
CASPASE 8 IS INITIATOR -> cascade of caspases -> executioner caspases (3,6,7)
Proteolysis of target proteins
(caspases also cleave Rb suggesting it may have a role in inhibiting apoptosis)
A death factor (e.g. FAS ligand) bound to plasma membrane of another cell or a soluble factor (e.g TNF or
TRAIL) is received by a transmembrane death receptor/TNF receptor
Receptor undergoes conformational change (e.g. trimerisation) which exposes the intracellular Death Domain
- This allows intracellular adaptor proteins e.g. FAS associated death domain (FADD) protein or TNF-receptor
associated death domain (TRADD) protein to bind to Death Domain and recruit Procaspase 8 (The ligandreceptor-death domain protein-Procaspase 8) complex is called a Death Inducing Signalling Complex - DISC
- Self cleavage and activation of Procaspase 8»_space; Caspase 8»_space; cascade of Caspase activation»_space; activation of
executioner Caspases 3/6/7
- Inhibited by c-FLIP which blocks Procaspase 8 recruitment and activation
What is the initiator caspase in extrinsic apoptosis?
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Caspase 8
What inhibits extrinisic apoptosis?
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c-Flip
Binds to FADD or caspase 8 and inhibits caspase 8 recruitment and activation
Describe the process of intrinsic apoptosis?
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Stimuli inside the cell eg DNA damage
Induced via Bcl-2 family which act at outer mitochondrial membrane.
One group of Bcl proteins inhibit and one group promote apoptosis.
On activation of BH3 only proteins, Bid and bim activate Bax -> translocates to mitochondrial membrane -> oligomerises into the membrane -> causes membrane to become more permeable and release apoptotic mediators -> cytochrome c joins to Apaf-1 and recRuits pro-caspase 9 to form apoptosome 7 spoked wheel of death -> activates caspase cascade
Smac/DIABLO released from mitochondria inhibit IAPs that normally block caspases.
- Regulated by BCL-2 family which are divided into two groups with opposing functions:
- Pro-apoptotic: Bax, Bak and also BH3-only proteins: Bim, Bid, Bax, Bak, Noxa, Puma
o These stimulate pore formation on Mitochondrial membrane through which Cytochrome C and
Procaspase 9 are relseased
o Apoptotic signal»_space; BIM + BID bind to and activate BAX»_space; conformal change in BAX»_space; BAX monomers
insert into Mitochondrial membrane»_space; form oligomers»_space; pore formation - Pro-survival: BCL-2, BCL-xl, BCL-W, MCL-1
o These bind and sequester Pro-apoptotic factors to inhibit apoptosis
o Pro-survival factors e.g. BCL-xl cause dissociation of BAX oligomers - Cytochrome C release triggers Caspase activation and formation of Apoptosome (Seven-spoked wheel of
death:
o Spokes: APAF-1 (bind to Procaspase 9 via CARD domain)
o Tip of spokes: Cytochrome C
o Dome (Hub): Procaspase 9
o Binding of APAF-1 to Procaspase 9»_space; activation to (initiator) Caspase 9»_space; begins caspase cascade»_space;
activation of (executioner) Caspases 3/6/7 - Inhibitors of apoptosis (IAPs) can bind and inhibit activated caspases
- Smac/DIABLO, also released from mitochondrion inhibit IAPs
- Intrinsic pathway triggered by DNA damage OR damage to plasma membrane – radiation activates
sphingomyelinase on plasma membrane»_space; generates ceramide»_space; second messenger to activate apoptosis via mitochondria
How do the anti-apoptotic proteins work in intrinsic apoptosis?
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Cause dissociation of Bax oligomers from the mitochondrial membrane
What are the pro apoptotic members of the Bcl family?
BH3- only, pro apoptotic
- Bad
- Bik
- Bid
- Bim
- Noxa
- Puma
Pro apoptotic other members
- Bax
- Bok
- Bak
What are the anti- apoptotic members of the Bcl family?
Bcl-2 Bcl-w A1 Boo Mcl-1 Bind to and inhibit pro-apoptotic proteins
What is the process of release of apoptotic mediators form mitochondrial membrane called?
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MOMP
Mitochondrial Outer Membrane Permeabilisation
How is the extrinsic and intrinsic pathway of apoptosis linked?
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Caspase 8 (extrinsic pathway) can cleave and activate Bid -> stimulate intrinsic pathway via Bax.
ATM kinase phosphorylates Bid and is required to cause cell cycle arrest
How is p53 involved in apoptosis?
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p53 functions both in the nucleus and cytoplasm by transcription dependent and independent means.
FUNCTIONS LINKED TO PUMA
- p53 can repress expression of anti-apoptotic factors (Bcl-2, IAPs)
- PUMA (p53 upregulated modulator of apoptosis) - member of Bcl-2 family is essential for p53 induced apoptosis
p53 activates PUMA which acts as an enabler for release of Bcl-x from p53 so p53 can activate Bax
Are cancer cells “closer” to apoptosis?
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Yes. Cancer cells are in a pro-apoptotic state that is inhibited by IAPs, whereas normal cells are in a non-apoptotic state that require caspase activation to initiate apoptosis.
TNF-related apoptosis-inducing ligand (TRAIL/APO-2L) is a member of the TNF family that promotes apoptosis by binding to the transmembrane receptors TRAIL-R1/DR4 and TRAIL-R2/DR5. Its cytotoxic activity is relatively selective to the human tumor cell lines without much effect on the normal cells. Potential target for drugs.
How can cancer alter the extrinsic pathway of apoptosis?
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Sunburn/UV causes clustering of Fas death receptors and activation of caspase cascade (mutation in Fas-r may lead to increased risk of skin cancer)
Loss of caspase 8 in SCLC and neuroblastomas
How can cancer alter the intrinsic pathway of apoptosis? How does this affect treatment?
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More common than extrinsic pathway
Mutations in p53/MDM2/ATM/Chk2
Bcl-2 translocation t(14,18) - B cell lymphoma
Loss of BH3-only proteins e.g. deletion/mutation.
Mutations in bax and bid (Bax >50% mutated in colorectal)
Apaf-1 (co-activator of caspase 9) mutated and repressed in mets melanoma
XIAP induced leukaemia, lung, prostate cancer (supress caspase 9/3/7)
MAKE CANCERS RESISTANT TO CHEMOTHERAPY
eg loss of bax makes resistant to 5FU in colorectal
PI3K:
- Activation of PI3K»_space; activates AKT, AKT blocks pro-apoptotic BIM + BAD
- Therefore PI3K activation inhibits apoptosis»_space; increased risk of proliferation and cancer
What are alternative death pathways that are caspase independent and what do they involve?
Use alternative proteases eg calpains, cathepsins, serine proteases
eg autophagy, mitotic catastrophe
What are cancer stem cells?
DEFINITELY IN CURRICULUM
Rare cells within a tumour that have the ability to self renew and give rise to phenotypically diverse cancer cells with limited replicative potential that make up the rest of the tumour. This self renewal provides an extended window for mutations. Cancer stem cells are heterogeneous but are generally more radioresistant and chemoresistant than the more proliferative tumor cell populations, which can be attributed to high anti-oxidant levels, slow cycling, mdr1 expression, and reprogramming. Cancer stem cells appear to have higher levels of free radical scavengers, abnormal activation of developmental pathways, hyperphosphorylation of checkpoint kinases which collectively tend to drive their superb resistance to radiation.
Cancer stem cells = clonogenic cells that survive treatment (can be selected for by chemo/radiotherapy)
They:
- Are capable of self-renewal
- Have differentiation capacity
- Can continually sustain the tumour
- Are resistant to treatment – e.g. by DNA repair, drug efflux, anti-apoptotic
- Are responsible for recurrence
How is it proposed that the dergulation of self renewal of stem cells occurs in cancer?
Normal stem cells maintain a balance between self renewal and differentiation. Loss of balance can lead to unregulated self renewal.
Alternatively differentiated cells may acquire a mutation which reactivates self renewal programme
What is wnt signalling pathway?
evolutionarily conserved cell-cell communication system that is important for stem cell renewal, cell proliferation and cell differentiation both during embryogenesis and during adult tissue homeostasis.
19 members of Wnt proteins - INTER cellular signalling molecules.
Lipid modficiations of Wnt by protein porcupine play a role in its secretion from cell.
Describe the Wnt pathway when no Wnt ligand present?
In cytoplasm (intracellular)
- Several proteins (APC, Axin, glycogen synthase kinase & casein kinase) aggregate together to form a degradation complex
- complex targets β- catenin (a transcriptional co-activator) for degradation by phosphoylating and ubquintinating it.
- In absence of β-catenin transcription is repressed by transcriptional repressor Groucho
Describe the Wnt pathway when Wnt ligand is present?
Wnt (extracellular) binds to its transmembrane receptor (can be G protein coupled & TKRs) called Frizzled.
Frizzled co receptor LRP undergos change and its cytoplasmic tail is phosphorylated by GSK3 &CKI)
This allows β-catenin to escape from degradation complex and move to nucleus
Acts as a co-activator (with Bcl-9 & pygopus) of T cell factor/LEF family of transcription factors (eg c-myc, cyclin D)
