Tumour biology Flashcards
Name the purines and pyrimidines in DNA and who they pair with
Purines : adenine, guanine
Pyrimidine: cytosine, thymine
A = T (2 hydrogen bonds)
G 三 C (3 hydrogen bonds)
How is DNA packaged?
DNA is an acid with highly negative charge
DNA helix wraps around nucleosomes consisting of histones (highly +ve charge) -> forms chromatin fibre -> coiled and packaged into chromosomes
Name the bases in RNA?
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 (matrix) - 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?
Exons: coding DNA
introns: non-coding DNA
Define anaplasia?
Lack of differentiation and loss of morphological characteristics. Cellular and nuclear pleomorphism (different sizes). Hyperchromatic nuclei. Loss of orientation/polarity.
What is dysplasia and CIS?
Dysplasia is disordered growth. Loss of cellular uniformity and architectural orientation, pleomorphism, increased mitotic figures.
CIS: full thickness dysplasia with BM intact
Both can progress to cancer
What is metaplasia?
Change of one cell type to another cell type- eg Barretts (squamous to columnar) - may be pre-malignant
What is hyperplasia?
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?
Transcription
Translation - involves all 3 types of RNA, occurs in a ribosome
Post-translational modifications
Describe the process of transcription? Where and how does it start?
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 transcription factor crucial for the initiation
Reaches stop codon - 3 codons always indicates stop protein synthesis - UAA, UAG, UGA
What is a somatic vs germ line mutation?
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?
Substitutions- transitions/transversions
Deletions
Insertions
Describe the two types of base pair substitutions?
Transitions: convert a purine to another purine
- 4 types - A↔G, T↔C
- most result in a synonymous substitution
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?
Frameshift mutation: deletions or insertions non divisible by 3 result in translation of incorrect AAs/codons.
What is a misense mutation?
Change from one AA to another due to a base pair substitution.
What is a nonsense mutation?
Base pair substitution results in a stop codon
What is chromothripsis?
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.
CAUSES CANCER
Which type of UV radiation causes the most cancer?
UVB
UVA - reaches most acellular dermis (wavelength og 320-380nm)
UVB- reaches epidermis (wavelength 290-320nm)
UVC- absorbed by ozone, rarely reaches skin
How does UV radiation cause cancer?
UVB
- causes CYCLOBUTANE PYRIMIDINE DIMERS- cause a bend in DNA helix so DNA polymerase cannot read DNA template -> it preferentially incorporates an A reside so TT dimers often restored to TC/CC dimers -> result in transitions (TC -> TT, CC-> TT). PYRIMIDINE DIMERS UNIQUE TO SKIN CANCER
- 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
What is characteristic of UVB radiation damage?
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?
Polyaromatic hydrocarbons : Benzo (a)pyrene 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?
Found in tobacco, preserved fish and meats during smoking
Principal carcinogenic product is alkylated O6 guanine derivatives
What are DNA mismatches?
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 G
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?
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?
Damage is recognised by a protein factor and directly chemically reversed
Bulky alkyl adducts
- recognised by O6- alkylguanine DNA alkyltransferase (AGT/ MDMT), the damaged base is flipped out of the DNA helix- methyl moeity is transfered to AGT protein
- AGT is a suicide enzyme- only does one round of demethylation and it is not regenerated.
Pyramidine dimer - NOT in placental mammals
- recognised by photolyase which absorbs blue light and breaks the cyclobutane ring
Describe the process of base excision repair.
Targets chemically altered bases (eg 8-oxoguanine- failure to remove this results in G->T transversion mutation)
Initiated by DNA gylcosylases (eg OGG1, MUTYH) - recognise and remove damage to leave an AP- apurinic/apyrimidinic site
- Scaffold protein XRCC1
- AP site cleaved by endonuclease
- repair takes place
- DNA polymerase 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
What is the role of PARP in DNA repair?
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?
UV induced DNA damage
Specific to helix distorting lesions eg pyramidine dimers, bulky DNA adducts
Cisplatin
Describe NER?
Recognition of damaged site leaded to 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
Two forms- differ in how they recognise the damage
- GG-NER- global genome
- TC-NER- transcription coupled repair- actively transcribed strand of DNA is repaired with greater efficacy
When does mismatch repair occur?
Corrects errors that arise spontaneously during DNA replication
What disease is caused by NER deficiencies?
Xeroderma pigmentosum Rare AR disorder Extremely sensitive to UV light No radiosensitvity Skin cancer, keratitis, mental retardation, premature dementia
How does mismatch repair work?
hMSH2/3 or 2/6 (also called MutS) recognises distorted structure in DNA and induces formation of a complex which leads to incision in the NEWLY synthesised strand of DNA. Gap is filled by polymerase and sealed by ligase.
Which cancer syndrome is caused by mismatch repair deficiencies?
HNPCC
Describe the process of NHEJ?
- DNA ends recognised by Ku 70/80 (XRCC6/5) proteins -> recruit DNA-PK catalytic subunit
- DNA-PK is whole complex including the Ku proteins which holds ends together
- recruits artemis protein
- XRCC4 and ligase 4 rejoin ends
If DNA ends compatible they can be directly ligated together but often processed (resected) then ligated back together.
What are the pros and cons and NHEJ?
Quick 2-4hrs
LOST DNA, MUTAGENIC
When in the cell cycle does NHEJ occur?
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?
ATM
- phosphorylation of histone H2AX by ATM/DNA-PK causes recruitment of proteins to site of damage
Describe HR
MRN complex- made up of Rad50 and Mre11 complex binds to ends of DNA and uses endonuclease activity to create single strand 3’ ends
Rad52 binds to DNA termini
Rad51 binds to exposed ends- BRCA 1/2 aids in nuclear transport of Rad51 and Rad52 helps with binding of Rad 51 to exposed ends.
Rad51 helps to seach for homologous template
When homologue is found the 3’ end of ssDNA serves as a primer to initiate DNA synthesis.
Resolvases restore the junctions known as Holliday junctions.
What does the fanconi anaemia pathway do?
Repair inter- strand cross link which block replication forms
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
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.
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?
Protein that binds to gene promoters and regulates transcription
3000 transcription factors regulate 20,000 genes
What is the structure of a transcription factor?
Contain a DNA binding domain, transcriptional activation domain, dimerisation domain and ligand binding domain.
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?
Synthesis in particular cell types only
Covalent modifications eg phosphorylation
ligand binding
Dimerisation
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
What is AP-1 and how does it work?
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 Jun & Fos family (Jun, Jun B, JunD, Fos, FRA1 etc) - contain leucine zipper dimerisation domain.
Jun B acts as a negative regulator
AP-1 activated by specific signals eg GF, ROS, radiation
AP-1 can transform a normal cell to malignant cell
How do steroid hormone receptors act as transcription factors?
Superfamily of steroid hormone receptors act as ligand-dependent transcription factors.
Nuclear receptor family (42 members) eg Androgen receptor, oestrogen receptor, glucocorticoid 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 acts as a transcriptional repressor in absence of retinoic acid (derived from vit A)
It binds to RA response element (RARE) in target genes as a heterodimer with RXR.
Aberrant forms of RARs are characteristic in some forms of leukaemia
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
Chromatin : thread of DNA (60%), assoc RNA (5%), protein (35%)
Nucleosome: 147 base pairs of DNA wrapped 1.7 x around core histone proteins. Histone core is an octomer of histones
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.
What is epigenetics?
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?
Histone modification
DNA methylation
Both can be acquired or inherited.
What types of histone modification are there?
Acetylation
Methylation
Phosphorylation
Ubquination
Describe histone acetylation?
Alters chromatin structure and effects gene expression. Acts as a docking signal for recruitment/replusion of chromatin Histone acetyltransferases (HATs) - add acetyl group - 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 coses 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.
Where does DNA methylation occur on DNA?
Addition of methyl group to position 5 of cytosine
ONLY occurs at cytosine nucleotides which are situated 5’ to guanine (CpGs)
Where do you find the most CpGs?
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
What are DNMTs and what types are there?
DNA methyltransferases - mediate the covalent addision of a methyl group from a methyl carrier.
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?
20-60% less methylation
Global hypomethylation with hypermethylation of specific gene promoters
30% breast cancers ER negative due to hypermethylation
BRCA1 can be inactivated by methylation
MGMT, MLH1- commonly methylated.
How do you detect DNA methylation?
Sodium bisulfite treatment- converts unmethylated cytosine to uracil but deamination.
Then do methylation specific PCR
Change grading of GBM
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?
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
What is the end replication problem?
DNA shortens by 100-200 DNA bases with each round of replication due to limits of DNA polymerases
What is telomerase?
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
How does cancer affect telomeres?
90% cancer upregulates telomerase
- melanoma and other cancers have found mutations in TERT promoter.
- c-myc increases expression of hTERT gene.
Telomere shortening occurs in response to DNA 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?
EGFR - ErbB1/HER1 - ErbB2/HER2 - ErbB3/HER3 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
What is the structure of an EGFR
Extracellular ligand binding domain
Single 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)
How does the growth factor signal get inside the cell?
Binding of EGF to receptor
EGFR receptor dimerisation
Conformational change in the receptor causes autophosphorylation (one half of dimer phosphorylates other half due to kinase activation)
The phosphorylated tyrosine resiues create high affinity binding sites for Src homology 2 (SH2) domains.
SH2&3 domains mediate protein-protein interacction in pathways activated by TKs.
Proteins that contain SH2/3 domains = Grb2, ABL, SRC, PI3K
SH3 domains interact with SOS which recruits Ras
What is Ras and what is its function
Ras is a GTP binding protein.
When bound to GDP inactive. SOS releases Ras from GDP and it binds to GTP
Ras is loosely bound to inside of cell membrane
Causes activation of Raf (MAPKKK) and PI3K
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?
GF - extracellular
EGFR - dimerisation and autophosphylation -> SH2/3 domains
RAS - GTP (liberated by SOS from GDP)
RAF (MAPKKK)
phosphorylates MEK (MAPKK)
Phosphorylates MAPK
MAPKs -> enter nucleus targets 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?
PI3K - a lipid kinase interacts directly with Ras.
PIP-3 recuits PDK-1 to cell membrane
Then AKT recuited 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 involed 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
What is an oncogene?
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.
Types
- retroviruses - rous sarcoma virus
- GFs
-GFR
- Intracellular signal tranduscers eg Ras, Raf
- Transcription factors
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
Give an example of a growth factor receptor oncogene
Proto-oncogene- RET - TK receptor
Transduces signal to glial derived neurotrophic factor family ligands
- papillary thyroid cancer
MEN2A and MEN2B
Oncogenic activation can lead to constitutive activation by dimerisation or increased kinase activity
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 constiutive 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 form so always active
- ABL (nuclear kinase)
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.
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
- Point mutations and deletions in coding regions
- Mutations in gene promoter region
- Chromsomal translocations + insertional mutagenesis
- Gene amplification eg erbB2
How long is the cell cycle?
Average length 16hrs 15hrs interphase 1hr mitosis 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?
On passing G1 restriction point
Describe the order of the cell cycle
G0 -> mitogens/GFs induce cells to re-enter cycle G1 - CYclin D + cdk4/6 G1 checkpoint S phase Cyclin E + cdk2 Cyclin A + cdk2 G2 phase Cyclin B/A + cdk1 G2 checkpoint Mitosis M checkpoint
How do cyclin-cdk complexes exert their effect?
Upon binding cyclin induces a conformational change in the catalytic subunit of the cdk partner revealing its active site -> 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?
Drives progression through G1
Binds to cdk4/6
ONe of its final targets is EGF signalling pathway
Plays role in regulation of cyclin E
Where does cyclin E act and how?
Important for G1-S phase progression
Binds to cdk2
Where does cyclin A act and how?
Important for S phase progression
Binds to cdk2
Where does cyclin B/A act and how?
Directs G2 and G2->M phase
Binds to cdk1
What are cdks?
Serine/threonine kinases that regulate progression of the cell cycle via phosphorylation.
What are the mechanisms of cdk regulation
- association with cyclins - activates cdk, cyclins degraded by proteasomes after being flagged by ubquitin
- Association with cdk inhibitors - 2 families p16ink4a (INK) family and p21 (cip/kip) damily.
- INK proteins bind cdks 4/6 and interfere with binding to cycle D
- p21 family members interact with both cyclins and their assoc cdks and block ATP binding sites
- addition of phosphate groups that activate or inactive cdk activity.
What is a key substrate of cyclin d - cdk4/6 complex?
Rb protein
How does Rb protein regulate the G1 checkpoint?
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
This occurs in response to growth signals
What happens at the G2 checkpoint?
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 1 and Chk2
Chk 1 prevents cdks from becoming active
After sucessful repair- PLK1 which targets Chk1 for degradation and inhibits Chk2.
Topoisomerase II - relieves torsional stresses by making dsDNA breaks in order to detangle ready for anaphase.
Describe the steps in mitosis
Prophase- appearance of chromosomes as result of condensation, nuclear membrane breakdown, separation of duplicated centrosomes, assembly of mitotic checkpoint proteins at centromeres.
Metaphase- aliging chromosomes on metaphase plate and assembly of microtubules to form mitotic spindle
Anaphase- spindle pulling apart and separating chromatids
Telophase- accumulation of chromosomes at their respective poles, reforming nuclear membrane, chromosome decondensation and cytokinesis
What happens at the spindle assembly checkpoint/mitotic checkpoint?
Signalling cascade that ensures the correct chromosomal segregation during mitosis and production of two genetically identical nuclei
What does anaphase complex do?
An ubuiquitin–protein ligase that regulates mitosis
During metaphase unattached chromatid pairs recruit proteins that inhibit anaphase complex. Once chromatids attached to microtubules they stop inhibiting it.
Anaphase complex targets securin - once degraded protease separase is activated
Separase cleaves protein link between sister chromatids allowing them to separate
What are aurora kinases?
Aurora kinases A, B, C
Regulate important aspects of mitosis
Serine -threonine kinases that phosphorylate target proteins
Aurora kinase A: localises centrosomes during interphase & thought to play role in centrosome maturation
Aurora kinase B: activity highest later in mitosis- role in spindle attachment and chromosome segragation
Aurora kinaseC - active during late mitosis
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?
Hereditary syndromes that cause cancer predispositions
What is the role of BRCA1?
Recruitment of Rad51 to DSBs - HR
What is the role of BRCA2?
More diverse role in HR and regulation of transcription
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
PIP2 inhibits the PI3K pathway
How does PTEN mutation cause cancer?
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?
Cowden disease
- harmartomas, risk of breast, endometrial and thyroid tumours
Describe the differences in the sporadic and familial form of retinoblastoma
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
- often bilateral
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?
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
DNA repair
Apoptosis
Inhibition of angiogenesis
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?
Regulated at level of protein degradation not gene expression
Main regulator = MDM2
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