Tumour biology Flashcards

Question

What is a tautomeric shift?

Answer 1

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

Answer 2

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

Answer 3

``` Direct repair Base exicision repair- most common Nucleotide exicison repair Mismatch repair DSB repair - HR - NHEJ ```

Answer 4

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

Answer 5

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.

Answer 6

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

Answer 7

Specific to helix distorting lesions e.g. UV induced DNA damage pyrimidine dimers Bulky DNA adducts e.g. polycyclic aromatic hydrocarbons Cisplatin

Answer 8

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

Answer 9

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).

Answer 10

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

Answer 11

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.

Answer 12

HNPCC/lynch - hereditary non-polyposis colorectal carcinoma = colorectal/endometrial/ovarian cancer. Autosomal dominant. Less severe than FAP.

Answer 13

- 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

Answer 14

Quick 2-4hrs LOST DNA, MUTAGENIC

Answer 15

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.

Answer 16

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)

Answer 17

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

Answer 18

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

Answer 19

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

Answer 20

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.

Answer 21

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.

Answer 22

Replication errors- eg mismatch repair Replication problems- impeded progress Damage to DNA Mitotic errors - chromosome segregation defects eg spindle assembly checkpoints.

Answer 23

Protein that binds to gene promoters and regulates transcription 3000 transcription factors regulate 20,000 genes

Answer 24

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 ```

Answer 25

Synthesis in particular cell types only Covalent modifications eg phosphorylation ligand binding Dimerisation - exchange of partner proteins

Answer 26

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

Answer 27

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

Answer 28

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.

Answer 29

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.

Answer 30

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.

Answer 31

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

Answer 32

Heritable information that is encoded by modifications of genome/chromatin components (not a change in DNA sequence so NOT mutations)

Answer 33

Histone modification DNA methylation Both can be acquired or inherited.

Answer 34

Acetylation (euchromatin) Methylation (H3K4me3 euchromatin; H3K9me, H3K27me3 is heterochromatin) Phosphorylation Ubquination Measured by ChIP-Seq

Answer 35

``` 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.

Answer 36

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.

Answer 37

Addition of methyl group to position 5 of cytosine in CG dinucleotide | ONLY occurs at cytosine nucleotides which are situated 5' to guanine (CpGs)

Answer 38

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.

Answer 39

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

Answer 40

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.

Answer 41

Sodium bisulfite treatment- converts unmethylated cytosine to uracil by deamination. Then do methylation specific PCR Change grading of GBM (MGMT promoter methylation)

Answer 42

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

Answer 43

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.

Answer 44

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 >> chromosome instability >>apoptosis Hayflick limit: number of times a cell can replicate - limited by number of telomeres.

Answer 45

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

Answer 46

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 >> increase telomerase activity >> 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

Answer 47

Growth factors Growth factor receptors (many are TKIs) Intracellular signal transducers Nuclear transcription factors

Answer 48

``` - 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 >> cleave precursor growth factors >> release of ligands >> RTK activation o ErbB2 receptors trans-activated by other RTKs and integrins

Answer 49

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)

Answer 50

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.

Answer 51

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 >> 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

Answer 52

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.

Answer 53

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.

Answer 54

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

Answer 55

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.

Answer 56

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.

Answer 57

Proto-oncogene = c-sis produces PDGF Oncogene = v-sis causes unregulated growth via activation of PDGF pathway - inappropriately cytoplasmic so constitutively active.

Answer 58

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 >> over-expression or ErbB2. ErbB2 homo/heterodimerization >> amplified oncogenic signalling Mutations >> 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 >> improper localisation/activation E.g. Fusion of intracellular domain of ALK with dimerization/oligomerization domain of another protein

Answer 59

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)

Answer 60

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

Answer 61

1. Point mutations and deletions in coding regions e.g. loss of GTPase function in Ras so constituvely activated 2. Mutations in gene promoter region 3. Chromosomal translocations e.g. BCR-ABL, IMH-myc + insertional mutagenesis 4. Gene amplification from the normal 2 copies in diploid genome eg HER2

Answer 62

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

Answer 63

On passing G1 restriction point

Answer 64

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

Answer 65

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.

Answer 66

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

Answer 67

Important for G1-S phase progression. Cyclin E/CDK2. Hyperphosphorylates Rb = E2F released and drives transcription of S phase proteins.

Answer 68

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:

Answer 69

Directs G2 and G2->M phase. Drives nuclear envelope breakdown, spindle formation and chromosome condensation. Binds to CDK1

Answer 70

Serine/threonine kinases that regulate progression of the cell cycle via phosphorylation.

Answer 71

- 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.

Answer 72

Rb protein

Answer 73

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

Answer 74

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.

Answer 75

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

Answer 76

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.

Answer 77

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

Answer 78

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

Answer 79

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

Answer 80

BRCA1/2 PTEN Rb p53

Answer 81

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.

Answer 82

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.

Answer 83

The localization of RAD51 to the DNA double-strand break requires the formation of the BRCA1-PALB2-BRCA2 complex.

Answer 84

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

Answer 85

Loss of inhibitory dephosphorylation activity of PTEN (for PIP3) can result in a consituitively active PI3K pathway.

Answer 86

Cowden disease aka hamartoma syndrome. Autosomal dominant. BET – breast, endometrial and thyroid cancers.

Answer 87

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

Answer 88

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.

Answer 89

DNA damage Aberrant growth signals Cell stress- radiation, drug, hypoxia, nucleotide depletion

Answer 90

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)

Answer 91

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

Answer 92

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)

Answer 93

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

Answer 94

p53 stimulates production of MDM2 | Low amounts of p53 will reduce transcription of MDM2

Answer 95

DSB -> stimulates ATM -> phosphorylates and activates Chk2 -> ATM + Chk2 phophorylate amino-terminal sites of p53 -> interferes with binding of MDM2

Answer 96

Cell stress -> activates ATR -> casein kinase II -> phosphorylates p53

Answer 97

Activated oncogenes eg Ras -> activity of protein p14arf -> sequesters MDM2 to nucleolus of nucleus

Answer 98

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

Answer 99

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

Answer 100

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

Answer 101

>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.

Answer 102

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.

Answer 103

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

Answer 104

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

Answer 105

Adenovirus E1A, papillomavirus E6 & E7 etc inactivate Rb and p53. Some do this using uquitin-proteosome system to degrade it.

Answer 106

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

Answer 107

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 >> cascade of amplification >> 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

Answer 108

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

Answer 109

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.

Answer 110

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 >> Caspase 8 >> cascade of Caspase activation >> activation of executioner Caspases 3/6/7 - Inhibited by c-FLIP which blocks Procaspase 8 recruitment and activation

Answer 111

c-Flip | Binds to FADD or caspase 8 and inhibits caspase 8 recruitment and activation

Answer 112

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 >> BIM + BID bind to and activate BAX >> conformal change in BAX >> BAX monomers insert into Mitochondrial membrane >> form oligomers >> 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 >> activation to (initiator) Caspase 9 >> begins caspase cascade >> 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 >> generates ceramide >> second messenger to activate apoptosis via mitochondria

Answer 113

Cause dissociation of Bax oligomers from the mitochondrial membrane

Answer 114

BH3- only, pro apoptotic - Bad - Bik - Bid - Bim - Noxa - Puma Pro apoptotic other members - Bax - Bok - Bak

Answer 115

``` Bcl-2 Bcl-w A1 Boo Mcl-1 Bind to and inhibit pro-apoptotic proteins ```

Answer 116

MOMP | Mitochondrial Outer Membrane Permeabilisation

Answer 117

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

Answer 118

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

Answer 119

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.

Answer 120

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

Answer 121

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 >> activates AKT, AKT blocks pro-apoptotic BIM + BAD - Therefore PI3K activation inhibits apoptosis >> increased risk of proliferation and cancer

Answer 122

Use alternative proteases eg calpains, cathepsins, serine proteases eg autophagy, mitotic catastrophe

Answer 123

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

Answer 124

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

Answer 125

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.

Answer 126

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

Answer 127

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)

Answer 128

Wnt1 is a proto-oncogene | Mutations constiutively activated Wnt pathway

Answer 129

Colorectal , >90% | Most mutations inactivate APC or activate β-catenin (rarely affect Wnt)

Answer 130

Genetic Abnormality: autosomal dominant – mutation of APC tumour suppressor gene (usually truncating mutation) chromosome 5q21. Activates Wnt pathway Mechanism of Action: APC binds and regulates B-catenin activity, unregulated B catenin >> constitutive activation of Tcf = uncontrolled growth and proliferation Associated cancers: Multiple adenomatous polyps from adolescence – 100% risk of colorectal cancer. Have prophylactic colectomy before age 30. Loss of APC (hyperplastic) >> DNA hypomethlylation (early adenoma) >> K-Ras activation (intermediate adenoma) >> LOH 18q ?TSG (late) >> loss of p53 (carcinoma) polyps + medulloblastoma = turcot syndrome; polyps + benigns omas e.g. osteomas = gardner syndrome.

Answer 131

Role in embryonic development, tissue self renewal and carcinogenesis Cilia

Answer 132

3 members Hh family: Sonic, Desert and Indian = INTERcellular signalling molecules 2 transmembrane receptors: patched and smoothened (related to Frizzled)- responsible for signal transduction by Hh- interaction regulated within cilia.

Answer 133

Patched is localised in the cilia (at top) and inhibits smoothened (from coming to top of cilia) Gli (zinc fingered transcription factor) - sequestered by a protein complex in cytoplasm which induces cleavage of Gli by proteosomes -> results in a repressor -> nucleus and inhibits transcription of Hh target genes

Answer 134

Sonic, desert or Indian bind to patched Patched is translocated from cilia allowing smoothened to relocate there. Smoothened tranduces a signal into cell - causes large protein complex to dissociate and release Gli Gli moves to nucleus and regulates expression of Hh genes (VEGF, cyclin D, Bcl2, Snail)

Answer 135

Patched is a tumour suppressor gene

Answer 136

Gorlin syndrome- germline mutation in one copy of patched -> BCC, medulloblastoma, rhabdomyosarcomas All sporadic BCCs 30% sporadic medulloblastomas

Answer 137

Specific cancers metastasise to particular sites Many can be explained by direction of blood flow 1/3 cannot > seed and soil Seed & Soil Analogy: Seeds are carried in all directions but can only live and grow if they fall on congenial soil: - E.g. Spleen and Kidneys – rich blood flow but rare sites of metastases - E.g. Breast and prostate metastasise to bone more than expected - E.g. Unusual sites for metastaes such as Ocular Melanoma to Liver

Answer 138

Can be monoclonal (seeded by one cell) Or polyclonal (seeded by two or more cells) Can also be seeded from other metastases 1. Classical theory: clonal selection during cancer progression * Primary tumour heterogenous, cells seeded continuosly, cells may not initially have full metastatic potential but can remain dormant and evolve with additional genetic/epigenetic changes independent of but parallel to primary tumour progression * Primary tumour can seed >2 mets at once and mets can further seed mets 2. Predetermined genetic predisposition theory * Metastatic potential is an intrinsic property of each tumour from the start – therefore should be predictable from the primary tumour and detectable at first diagnosis – thus aiding management decisions 3. Host predisposition theory * Host genotype (and microenvironment) is predisposed to be metastases permissive

Answer 139

EMT- epithelial-mesenchymal transition. Transient loss of closely connected epithelial characteristics and acqusition of a more motile (fibroblast-like), mesenchymal phenotype. An important process in embryonic development As a result, cancer cells are more easily able to cross the extra-cellular matrix and basement membrane borders between organs

Answer 140

Loss of polarity and cytoskeleton rearrangements Deconstruction of epithelial cell-cell junctions Changes in cell shape Downregulation of epithelial markers eg e-cadherin - the predominant cell-cell adhesion molecule which acts as a TSG by securing cell-cell adhesion and suppressing metastases. EGFR binds SRC via its SH2 domain; SRC promotes motility; FAK activates SRC; The (FAK-SRC)- complex breaks E-Cadherin-Catenin bonds Upregulation of mesenchymal proteins eg n-cadherin and vimentin Secretion of specific proteases Increased motility and invasive potential, increased matrix deposition and resistance to apoptosis

Answer 141

Can be induced via a variety of transcription factors which can be activated via growth factors (eg HGF, EGF, PDGF, TGF) via MAPK, PI3K pathway Ultimately specific transcription factors eg Twist, snail, slug (Twist is a key regulator of metastasis) Reversed once tumour cell establishes secondary sites Key regulators: o Envirnomental factors e.g. hypoxia and inflammation o Wnt, Notch and Hedgehog pathways

Answer 142

CAMS and cadherins Cadherins are calclium dependent transmembrane glycoproteins that have an extracellular hook and bind to other caherins on cell next to them. They interact via their intracellular catenins. Catenins can also bind transcription factors and induce gene expression in nucleus

Answer 143

E-cadherin - secure cell-cell adhesion and suppresses metastasis. Mutations of extracellular domain and methyltion in promoter region of E-cadherin gene have been identified in gastric/prostate cancer

Answer 144

Integrins – cell interactions with the extracellular matrix e.g. collagen/laminins/fibronectins. Inhibit anoikis (apoptosis) when bound to ECM. As well as breaking free from caherins. Cells must break free from normal molecular constraints by ECM and avoid anoikis. Integrin receptors are a family of >24 heterodimers made up of a range of alpha and beta subunits that mediate cell-ECM interactions. They also help with some inside -> outside and outside -> inside signalling. Induce conformational change in the extracellular domain changing the affinity of integrins for their ECM ligands

Answer 145

ECM components eg collagen, fibronectin, laminin Recognised by integrin receptors Upon ligand binding the integrins clusster in the membrane and affect the cytoskeleton through interaction with actin-binding proteins and kinases like focal adhesion kiase(FAK)

Answer 146

Altered expression of receptors observed in tumour cells during EMT. Have to avoid integrin-induced anoikis in response to loss of ECM contact.

Answer 147

Invasion of tumour cells into surrounding tissue requires the action of specific proteases that degrade a path through the ECM and stroma MMPs can cleave the extracellular domain of E-cadherin and so contribute to loss of epithelial cell-cell junctions. Tumour cells produce proteases and induce surrounding stromal cells to do so also Plasminogen and Collagenase also involved in ECM degredation

Answer 148

Carefully regulated as they are synthesised as latent enzymes requiring cleavage. TIMPs regulate their function. Tissue inhibitors of metalloproteinases (TIMPs) Extracellular matrix metalloproteinase inducer (EMMPRIN) is upregulated on membrane of tumour cells and induces production of MMPs in adjacent stromal cells.

Answer 149

1. Cell must attach to the stromal face of vessel 2. Degrade basement membrane (using MMPs/serine proteases) 3. Pass between endothelial cells (transendothelial migration) into the blood stream. (tumour associated neovascularisation results in leaky tortuous blood vessels allowing easy access) tumour microenvironment of metastasis doorway (perivascular macrophage activated by tumour cell via colony stimulating factor-1) which activates tumour cell via EGF secretion – migrates along collagen fibres towards blood vessel following the HGF gradient from endothelial cells (chemotaxis) – once gets to the endothelial cell VEGF released by macrophages induces vascular permeabilization and tumour cell gets through

Answer 150

Tumour associated macrophages Involves colony-stimulating factor 1 (CSF 1) receptor on macrophages and EGFR on tumour cells. Macrophage associate with blood vessels and produce EGF -> binds to EGFR on tumour cells -> tumour cells produce CSF 1 which interacts with macrophages and leads to chemotaxis-mediated co-migration along HGF gradient.

Answer 151

Tumour cells within the bloodstream | Can travel singly or in clumps together with platelets as emboli

Answer 152

1. Tumour cell must attach to the endothelial side of the blood vessel. tumour cells bind to endothelial cells based on their selectins (Different endothelial cells have different selectins – seed and soil) and do transendothelial migration). 2. Pass through the endothelial cells and basement membrane (transendothelial migration). Proteases break BM. 3. Migrate into the surrounding stroma 4. Either grow or remain dormant depending on metastatic niche

Answer 153

E- selectin is important Binding to endothelium via e-selectin receptors on endothelium mediates adhesion and also triggers a signal cascade by activating stress-activated protein kinase 2 (SAPK2) - necessary for transendothelial migration.

Answer 154

Disseminated tumour cells | cells that have spread but have not yet colonised.

Answer 155

A site of future mets that is alerted as a result of factors released by primary tumour in preparation for the arrival of tumour cells. Supports the seed and soil theory Pathfinder cells (systemic mediators) e.g VEGF, TGF-B, precondition the distant soil ready for arrival of metastatic cells

Answer 156

Small vesicles that carry protein and nucleic acids which can travel in the circulation. They can carry DNA, RNA and protein to cells to which they can fuse- called horizontal transfer -> role in promoting mets. Can provide "education" to and change behaviour of receiving cells.

Answer 157

Definited by ability to inhibit overt metastases without affecting growth of primary tumour 23 identified - NM23 -MKK4 Both promote dormancy/apoptosis of micromets

Answer 158

Non specific growth factors - EGF, HGF, FGF, PDGF Vascular endothelium specific growth factors - VEGF & VEGF-R - MAIN PLAYER - angiopoeitins - Tie receptors

Answer 159

5 members VEGF-A-D and placental growth factor (PIGF) 3 VEGF tyrosine kinase receptors - VEGFR -1, -2, -3 (VEFG-A/VEGFR-2) complex key to angiogenesis

Answer 160

VEGFR-1 acts as a decoy by regulating the amount of VEGF-A available to VEGFR-2 as the binding affinity for VEGFR-1 is higher but its kinase activity lower so it restricts angiogenic response. Competitive inhibition.

Answer 161

Binds to VEGFR-3 and plays a role in lymphangiogenesis. VEGFC produced by tumours so promotes expansion of lymphatic vessels in the tumour periphery, increasing risk of lymphatic spread

Answer 162

VEGF-A produced by tumour cells and can be induced in surrounding normal cells. - One VEGF-A molecule Binds to two VEGFR-2 receptors -> dimerisation and phosphorylation. - intracellular proteins containing SH2 domains bind to phosphorylated receptor -> trigger - Ras-Raf-MAPK pathway - stimulated VEGF genes - PI3K pathway - AKT leads to inhibition of apoptosis (by inhibition of BAD)and production of endothelial nitric oxide synthase (eNOS) that stimulates endothelial permeability VEGF responsive genes include EGFR ligand, epiregulin, COX2, MMP1, MMP2

Answer 163

Angiostatin (binds annexin II) Endostatin (blocks MAPK and MMPs) Prolactin P53 Thrombospondin-1, -2 Notch Top1

Answer 164

Plasminogen can be cleaved by proteinases including several MMPs to release angiostatin. Angiostatin binds to endothelial cell surface receptor, annexin II to exert its inhibitory effects on angiogenesis

Answer 165

Fragment of collagen XVIII Can be proteolytically released by elastase and cathepsin Blocks MAPK activation in endothelial cells and also MMPs

Answer 166

? production of anti-angiogenic factors by certain tumours inhibits growth of micro-mets Surgery induces angiogenic growth factors

Answer 167

HIF is a heterodimic transcription factor comprising of one HIF-1α and HIF-1β Activity of HIF is regulated by oxygen concentration, not mRNA expression via HIF-1α HIF is a driver of tumour progression – transcriptionally activates genes linked to invasion, metastases and resistance. HIF regulates glycolysis (e.g GLUT-1), angiogenesis (e.g. VEGF), erythropoiesis (e.g. EPO), and pH (e.g. CA9).

Answer 168

HIF-1α hydroxylated by prolyl-4 hydroxylase - acts as a direct oxygen sensor by linking molecular oxygen to specific proline residues VHL binds to hydroxylated HIF-1α and activates proteins which ubuitinate it -> degraded -> target genes are not activated

Answer 169

Enzymes prolyl-4 hydroxylase is inactivated. HIF-1α is rapidly stabilised and transported to nucleus Heterodimeric HIF transcription factor can activate its target genes via hypoxia response element (HRE) Most notable target is VEGF gene which has a HRE in its promoter region

Answer 170

HIghly vascularised tumour caused by herpes virus three protein products of viral genome increase HIF-1α half life, nuclear localisation and transactivation under normoxic conditions mimicking hypoxia.

Answer 171

EGFR - receptor tyrosine kinase Src - intracellular tyrosine kinase Ras - intracellular transducer Fos and Jun - transcription factors p53- normally binds to and activates the promoter of thrombospondin- 1 gene (anti-angiogenic), when p53 is mutated it does not activate it

Answer 172

In response to angiogenic signals, endothelial cells extend filopodia and migrate towards signal At location of highest concentraion of VEGFA, VEGFR-2 is activated. Signal enhanced by co-receptor neuropilin-1 (Nrp-1) and transduced via MAPK cascade Stimulates formation of tip cell at forefront of a sprout Behind tip cell are the proliferating stalk cells that extent the sprouting vessel Upon VEGFR2 activation tip cells produce and release Notch ligand called Delta-like 4 (DLL4) DLL4 binds to notch receptor on neighbouring cells -> NICD (notch intracellular domain) is released intracellularly -> travels to nucleus and represses VEGFR2 expression and increases VEGFR1 expression. VEGFR-1 reduces concentration of VEGF available for VEGFR-2 therefore the growing sprout moves along a VEGF gradient

Answer 173

Farmed salmon - contains polychlorinated biphenols and other pesticides, eating >once a month increases cancer risk Red meat - cooking at high temps produces heterocyclic aromatic amines -> metabolised by p450 N-acetyltransferase which turns to carcinogen ->DNA adducts -> base substitutions and mutations. N-acetyltransferase polymorphisms affect colon cancer risk. Peanuts-> contaminated with aflatoxin B, a fungal product of aspergillus flavus -> cause GC->TA transversions -> HCC Nitrosamines in food preservatives such as sodium nitrite -> O6 guanine alkylation.

Answer 174

Vitamin B9 CAn accept or donate one carbon units in metabolic reactions Critical co-enzyme for nucleotide synthesis and DNA methylation (methionine ->SAM)- depletion can interfere with this and cause cancer (associated with colorectal cancer).

Answer 175

dTMP synthesis is inhibited in a low folate state and imbalance of nucelotides results in incorporation of uracil into DNA -> DNA strand breaks trying to repair this Also get disruption in DNA methylation may cause genomic hypomethylation - methyl groups used for methylation are supplied by folate -> decrease in synthesis of methionine and so genomic hypomethylation MUTATION AND HYPOMETHYLATION

Answer 176

Adipose tissue is an endocrine tissue that can release free fatty acids, peptide and steroid hormones. Obesity is associated with breast, endometrial, colon, kidney, liver, pancreas and oesophageal cancer. ALTERED SEX HORMONE METABOLISM - synthesis of oestrogen from androgens using aromatase - elevated cholesterol - 27-hydroxycholesterol is a ligand for oestrogen receptor INCREASED PRODUCTION OF FAT CELL HORMONES DUE TO ADIPOCYTE HYPERTROPHY - ADIPOKINES - causes chronic inflammatory response -> IL6, TNF INCREASED INSULIN SIGNALLING PATHWAYS - insulin resistance increases insulin levels and IGFs - promotion of proliferation and inhibition of apoptosis DIETARY ALTERATIONS OF GUT MICROBIOTA (dysbiosis) - promotes liver cancer -> more gram +ve => causes increase in bacterial metabolite deoxycholic acid made from cholesterol-derived bile acids -> chronic DNA damage in liver as human liver cannot metabolise it ASSOCIATED WITH NAFLD -> HCC probably due to cirrhosis/inflammation (Dietary effects on cancer: caloric restriction reduces cancer. High fat high carb diets = cancer. High fibre = reduced colorectal cancer) (Physical activity also associated with reduced cancer - perhaps due to improved insulin resistance.)

Answer 177

Metabolised by enzyme alcohol dehydrogenase to form acetaldehyde -> binds to DNA -> forms adducts Get more acetaldehyde in saliva (10-100x higher) as bacteria in saliva convert it (hence higher risk of mouth cancer). Acetaldehyde is oxidised by enzymes acetaldehyde dehydrogenase (single nucleotide polymorphism of gene causes intolerance and increased risk of oesophageal cancer) Alcohol dehydrogenase can metabolise oestrogen - excess alcohol competes with oestrogen for metabolism so more metabolism levels -> breast cancer. Liver cirrhosis (inflammation) -> HCC Causes oropharynx, larynx, oesophagus, liver. Increased risk of breast/colorectal cancer

Answer 178

Inverse assoc between total fruit and veg intake and risk of cancer Microconstituents of food play a role in cancer prevention directly via free radical scavenging or indirectly regulating expression of genes that code for phase I and II reactions Phase I P450 and II conjugation reactions are major defence mechanism against xenobiotics (foreign substances) - often phase I converts to ultimate carcinogen and phase II allows removal.

Answer 179

Water soluble vit C can donate electrons to a free radical (e.g.superoxide, hydrogen peroxide - note hydroxyl radicals are too reactive to be affected by antioxidants) Oxidised vit c forms ascorbyl radical that is fairly stable and unreactive Vit C reductase can regenerate vit C from ascorbyl radical for reuse or ascorbyl radical may lose another electron and become degraded Vit C needs to be replenished daily Lipid soluble vit E acts as a free radical scavenger in a similar way Note supplemental vitamin C has no effect on cancer. Vitamin D also supposed by good for cancer but supplemental vitamin D has no effect. Steroid hormone receptor family.

Answer 180

Antioxidant response element (ARE) in promoter region of several genes encoding detoxification and antioxidant enzymes (eg glutathione s-transferase, NADPH:quinone oxidoreductase, phase II reaction enzymes) KEAP1 inhibits NRF2 by forming complex with ubiquitin ligase. ROS interact with cysteines of KEAP1, stopping it interacting with NRF2. Therefore NRF2 drive transcription of ARE genes. Antioxidants also inhibit KEAP1. Nrf2 binds to Maf (member of basic leucine zipper family) and activates transcription of detoxification enzymes via ARE Antioxidants: - Isopthiocyanates in broccoli - EGCG in green tea Nrf2 antioxidant pathway often upregulated in cancer - perhaps protects cells/DNA from chemotherapy/ROS induced damage. Therefore Nrf2 pathway is to inhibit carcinogenesis but gets hijacked by cancer cells to protect themselves and promote tumour survival.

Answer 181

Observation that cancer cells carry out aerobic glycolysis, converting glucose to lactate in presence of oxygen.

Answer 182

1. Glucose undergoes glycolysis in cytoplasm to pyruvate x2 2. Pyruvate converted to acetyl coA by pyruvate dehydrogenase - oxidative process, NADH and CO2 formed. 3. Citric acid/Krebs cycle - series of reactions breaks down acetyl coA - > forms ATP, NADPH, FADH2 4. Electron transport chain - series of mitochondrial membrane proteins that transfer electron donors to electron receptors

Answer 183

1st line = skin and mucous membranes 2nd line = no memory, no prior exposure required, quick, non-specific - innate immune system - macrophages - phagocytose foreign substances + dead host tissue - constantly processing their microenvironment - present antigens via MHC class II - NK cells - targets abnormal host cells e.g. tumour, virus-infected - cytotoxicity through granzymes and inflammation through interferon gamma - neutrophils (granulocyte and phagocyte - target bacteria) - eosinophils (granulocyte - parasite) - basophils (granulocyte - allergy e.g. hayfever/anaphylaxis) - dendritic cells (APCs to activate the adaptive immune system) - links the innate and adaptive immune system - inflammation - complement - cytokines 3rd line = specific and adaptive; recombination required; has memory - b cells - antibodies (humoral immunity) - t lymphocytes - CD4 T helper (activates B cells and CD8 cells) and CD8 T killer cells (cell mediated immunity - infected/abnormal host cells through granzymes/perforin/granulysin) and CD4 T reg cells (immunosuppressive) - CD4 cells activated by antigen presenting cells - dendritic cells and macrophages

Answer 184

Pattern recognition e.g. toll like receptors Mainly found on antigen presenting cells (APCs) like dendritic cells, monocytes, NK cells, also found on other non-immune cells. Recognise PAMPs (pathogen assoc molecular patterns) and TFs INNATE IMMUNITY When PAMP binds to PRR -> recruitment of APCs, upregulation of MHC and secretion of cyto/chemokines

Answer 185

Innate immunity Classical pathway -> activated by antigen/antibody complexes -> starts at C1 Alternative pathway -> activated by cell membranes -> starts at C3b Lectin pathway -> activated by carbohydrate structures -> starts at MBL OUTCOMES: C3b -> causes OPSONISATION (phagocytosis) C3a & C5a -> inflammation, mast cell activation/chemotaxin MAC -> LYSIS

Answer 186

Activation of complement cascade. Opsonisation = tagging a target with an opsonin allowing it to be phagocytosed by phagocytes. | Specifically C3b

Answer 187

IL6, IL1, TNF-α -> cause acute phase proteins to be made -> CRP, mannan-binding lectin, serum amyloid protein A Maximise activation of complement system CRP and serum amyloid protein A bind to DNA and other nuclear material from cells helping in their clearance

Answer 188

Produced in reponse to viral infection. Inhibit protein synthesis Interferon type I (alpha and beta): produced in response to viral molecular patterns e.g. by dendritic cells and macrophages. Antagonise viral replication. Recruit NK cells. Interferon type 2 (gamma): produced by NK cells.

Answer 189

GF for lymphocytes eg IL2, IL4, influence nature of immune reponse IL2 and IL9 are produced by T cells to stimulate T cell growth. IL4 and IL5 are produced by T cells to stimulate B cell growth.

Answer 190

Critical to immune defence and inflammation eg IL1, TNF, IL6- activate lymphocytes, increase body temp, activate mobilise phagocytes, activate vascular endothelium. IL1 = fever. IL6 = acute phase proteins. TNFalpha = both, general inflammation.

Answer 191

Activate and direct effector cells expressing appropriate chemokine receptors to sites of tissue damage and regulate leukocyte migration to tissues. eg CXCL-8, CCL2

Answer 192

Most nucleated cells. Present intracellular antigen (peptide fragments bound to MHC class I in the rough endoplasmic reticulum) to cytotoxic CD8 T cells (via MHC receptor). MHC I – 3 alpha units and a beta-2 microglobulin; MHC II – 2 alpha and 2 beta chains

Answer 193

B cells, macrophages and dendritic cells. Antigen presenting cells. Present antigen to helper CD4 T cells. Extracellular antigens are endocytosed/phagocytosed and degraded by lysosomal enzymes. Bound to MHC class Ii in the lysosome and transferred to cell membrane. MHC I – 3 alpha units and a beta-2 microglobulin; MHC II – 2 alpha and 2 beta chains

Answer 194

CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor. SPECIFIC FOR MHC CLASS I CD8+ cells become cytotoxic T cells on activation. They turn to memory T cells after activation and clearance of infection. Overtime, if they are not reacted, they lose their function = T cell exhaustion.

Answer 195

CD4 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor. SPECIFIC FOR MHC CLASS II CD4 cells can be T helper cells, monocytes, macrophages, dendritic cells.

Answer 196

HLA genes on chromosome 6

Answer 197

2 different protein chains - heterodimer - 95% are α/β chains - 5% are γ/δ chains Transmembrane region Constant region Variable region at top with antigen binding site

Answer 198

1. Antigen presented by APC via MHC recognised by TCR (e.g. MHC class II and CD4) 2. Co-stimulatory activation by checkpoint molecules eg CD28 (t cell) and B7 (APC) - this is inhibited by CTLA4 on T cells competing to bind B7 instead CD28 3. Cytokines

Answer 199

CTLA4- co-inhibitory molecule is rapidly mobilised from intracellular vesicles within T cell membrane It competes for B7 (CD80/CD86) ligand (on APC) with CD28, it has a higher affinity for it so inhibits it - ipilimumab PD1 (pembrolizumab/nivolumab) and its ligands PDL1 (atezolizumab/durvalumab) and PDL2 are also co-inhibitory. Interaction of PDL1 on tumour cells and its receptor on activated effector T cells causes inhibition. Inhibition is due to recruitment of SHP-2 phosphatases inactivating PI3K and protein kinase C activation. Blocks production and secretion of molecules required for cytotoxic response. . Naive and memory T cells express high levels of cell surface CD28 but do not express CTLA4 on their surface. Instead, CTLA4 is sequestered in intracellular vesicles. After the TCR is triggered by antigen encounter, CTLA4 is transported to the cell surface. The stronger the stimulation through the TCR (and CD28), the greater the amount of CTLA4 that is deposited on the T cell surface. Therefore, CTLA4 functions as a signal dampener to maintain a consistent level of T cell activation in the face of widely varying concentrations and affinities of ligand for the TCR. By contrast, the major role of the programmed cell death protein 1 (PD-1) pathway is not at the initial T cell activation stage but rather to regulate inflammatory responses in tissues by effector T cells recognizing antigen in peripheral tissues. Activated T cells upregulate PD-1 and continue to express it in tissues. Inflammatory signals in the tissues induce the expression of PD-1 ligands, which downregulate the activity of T cells and thus limit collateral tissue damage in response to a microorganism infection in that tissue. The best characterized signal for PD-1 ligand 1 (PD-L1; also known as B7 H1) induction is IFNg, which is predominantly produced by T helper 1 (TH1) cells, although many of the signals have not yet been defined completely. Excessive induction of PD-1 on T cells in the setting of chronic antigen exposure can induce an exhaustive or anergic state.

Answer 200

PDL1 - tumour cells and many other types of cells after exposure to interferon gamma. PDL2 - APCs PD1 - T cells

Answer 201

Antigen processed - peptides. Taken into the endoplasmic reticulum. Bind to CLASS I MHC -> golgi -> vesicle and transported to cell surface

Answer 202

Antigen taken up into cell via endocytosis Degraded by phagosomes in vesicle MHC class II released from ER Travels to vesicle and combine with antigen Travels to cells surface and presents it

Answer 203

Bind soluble antigens or can bind antigen on cell surface. Neutralisation e.g. stopping PD1 binding to PDL1 Opsonisation (Opsonization is an immune process which uses opsonins to tag foreign pathogens for elimination by phagocytes. Without an opsonin, such as an antibody, the negatively-charged cell walls of the pathogen and phagocyte repel each other.) Complement activation Antibody-dependent cellular cytotoxicity (granzymes/perforin - NK cells/CD8 T cells)

Answer 204

Constant region (most of the antibody): 2 identical heavy chains linked by a disulphide bond, 2 identical light chains on outer side bound by disulphide bond to heavy chain = these are the same in all types of immunoglobulins. Variable region is the tip = antigen binding site. Produced by genetic recombination (NHEJ) Fc = fragment crystallisable = the stick of the Y. Fab = fragment antigen binding = the arms of the Y = includes the light chains and the antigen binding site.

Answer 205

IgM (6%) - pentamer- activates complement cascade = initial response IgG (80%) - monomer - neutralises toxins, opsonisation, complement, binds to phagocytes = memory response IgA (13%)- dimer- secreted in tears, mucus, saliva IgE (0.002%)- monomer- allergy, binds to mast cells/basophils IgD (1%) - monomer- B cell receptor

Answer 206

Antigen presented via MHC class II on APC + CD4 T cell cells = activation to plasma cells that produce antibodies. B cells/T cells interaction occurs in lymph nodes. Turns to memory B cell. Produces one type of antibody (multiple B cells target different part of the foreign protein = polyclonal).

Answer 207

Central - thymus - killed if they respond to host tissue too strongly - negative selection - receptor editing - generation of regulatory t cells Peripheral - suppress autoreactive T cells - clonal anergy (unresponsive, prevent overactivation) - clonal deletion - regulatory t cells - T-T cell interactions Maturation happens in response to antigen

Answer 208

Type of differentiated T helper cell -> T regulatory cell -> influence tolerance and immune suppression Secrete immunosuppresive cytokines (IL10, TGFb) Block T cell proliferation, inhibit NK function High levels of Tregs = poor prognosis in cancer

Answer 209

Elimination -> equilibrium -> escape Elimination of cancer; equilibrium where less immunogenic cancer cells are selected for; escape where tumour evades immune system. Evidence to support this is that immunodeficient hosts produce more immunogenic tumours than immunosufficient hosts; the number and type of tumour infiltrating lymphocytes gives an immunoscore which correlates with cell survival General cancer immune avoidance facts: LET survival curve is exponential. The process by which tumor cells change phenotype in response to an immune response (cytotoxicity or inflammation) in an attempt to avoid recognition (i.e. the induction of PD-1, PD-L1, and IDO following antigen recognition and the production of IFNγ) is characteristic of an adaptive immune response. Epitope spreading (or antigen cascade, antigen spread, determinant spread) describes a phenomenon where the immune response evolves and expands from focusing on a single antigenic epitope, into a multi-epitopic response be it naturally or following therapeutic intervention e.g. vaccination or radiotherapy. This process is dynamic and may continue to expand over time. Antigen spreading of the anti-tumor immune response from one antigen to another antigen has been linked to superior clinical outcome with the assumption that it counteracts tumor immune evasion. The somatic mutation load of cancers is thought to be a major determinant of response to immune checkpoint blockade through the generation of neoantigens targetable by the immune system.

Answer 210

T infiltrating lymphocytes (TILs) - can predict favourable outcome in some tumour types - infiltrating NK and CD8 T cells assoc with favourable prognosis o Immune desert (no T cells in tumour); immune excluded (T cells surround tumour but cannot infiltrate for unknown reason); immune inflamed (T cells infiltrate tumour). You want tumour to be immune inflamed to induce tumour death.

Answer 211

Peripheral tolerance is required to regulate self reactive T cells and NK cells- these regulatory mechanisms are often exploited by tumours to evade immune recognition TUMOUR IMMUNE INVASION VIA: - MODIFYING MICROENVIRONMENT- secrete immunosuppressive agents eg TGF-B, IL-10, VEGF - EVADES T CELL RECOGNITION- downregulates class I MHC (NK cells should still recognise cells that have lost MHC) - DYSREGULATION OF ANTIGEN PROCESSING/PRESENTATION PATHWAYS

Answer 212

CD8 T cells, NK cells, CD4 (Th1), M1 polarised macrophages (encourage inflammation)

Answer 213

CD4 Tregs, Myeloid-derived suppressor cells (MDSCs), tumour-associated macrophages (TAM), M2 macrophages (decrease inflammation and encourage tissue repair)

Answer 214

Can be unique to cancer- neoantigens e.g. mutated p53 peptides. Can be overexpressed normal antigens eg HER2 Differentially expressed antigens eg cancer testis antigens which are normally restricted to testis germ cells Presented to T cells via APCs. Neoantigens will be particularly immunogenic = more T infiltrating lymphocytes.

Answer 215

Type of APC - constantly sampling environment for foreign substances and activate both innate and adaptive immune system Can be activated by toll like receptors. Cause inflammation by releasing cytokines. Recruit NK cells to do cell mediated cytoxicity via interferons. Perform phagocytosis to present antigens to T cells. Have class I and II MHC Immature dendritic cells - specialised for antigen acquisition, highly phagocytic, low in class II, co-stimulatory, no NK activation Mature DC: antigen and danger signal specialised for antigen presentation of T cells, less phagocytic, high class II, co-stimulatory molecules, activate NK cells

Answer 216

Tumour necrotis cores Hypoxia Cell derived danger signals - heat shock proteins, ATP, RNA, DNA, uric acid, hyaluronic acid, NK ligands, IFN-a

Answer 217

senses presence of cytosolic DNA -> activates CGAS -> STING (stimulator of interferon genes) Inhibited by TREX1

Answer 218

Poor prognosis Secrete IL10, TGFb Enhance tumour proliferation through NFKB/STAT signalling

Answer 219

Based on idea that the immune system does not differentiate from self/non-self but by things that might cause danger via danger signals. Explains why immunosuppressed patients get more cancers. Immunosurveillance is a term used to describe the processes by which cells of the immune system look for and recognise foreign pathogens, such as bacteria and viruses, or pre-cancerous and cancerous cells in the body.

Answer 220

That APCs are activated via pattern recognition receptors (PRRs) which recognize evolutionary distant conserved patterns. These pathogen-associated molecular patterns (PAMPs) on such organisms as bacteria are recognized as infectious non-self, whereas PRRs are not activated by non-infectious self. DOES NOT EXPLAIN ANTI-TUMOUR IMMUNITY

Answer 221

Necrosis releases endogenous DAMPs (damage-associated molecular patterns), starts the cycle of chronic inflammation and recruits MDSC Produce NO and ROS may cause DNA damage and mutations Support tumour progression by increasing angiogenesis, tumour cell stemness, metastasis Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid progenitor cells that fail to differentiate into granulocytes, macrophages, or dendritic cells. These immature cells have the capacity to suppress immune responses mediated by natural killer (NK) cells and NKT cells, as well as CD8+ and CD4+ T cells. MDSCs have been suggested to have a causative role in promoting tumor-associated immunosuppression. They accumulate in the blood, bone marrow, and secondary lymphoid organs of tumor-bearing mice and cancer patients, where circulating levels of these cells have been shown to correlate with clinical stage, metastatic burden, and chemoresistance.

Answer 222

DNA virus (herpes virus 4) Causes lymphoma eg Burkitts, nasopharyngeal cancer EBV encodes several viral proteins that affect host gene expression- oncoprotein LMP1 able to transform cells in culture Activates inflammatory transcription factors NFKB and Stat and inhibits apoptosis via TRADD

Answer 223

DNA virus (papovavirus). HPV 16 & 18 account for 70% HPV gene products E6 and E7 are major players that target tumour suppressors - both are required to immortalise cells in culture. - E7 binds to Rb at HDAC binding site and triggers ubiquitin-mediated degradation of Rb preventing sequestering of E2F -> constitutive action of cyclin A/E -E6 forms a complex with ubiquitin ligase binds to p53 and targets it for degradation. Also inhibits transactivation activity of p53. Polymorphisms in p53 at amino acid 72 lead to differences in risk of cervical cancer - if both alleles code of Arg they have 7 x risk cervical cancer than those with Pro at site - Arg at 72 is more susceptible to E6 degradation. Sexually transmitted: Can cause cervical (100% caused by HPV, lag time 10 years), oral (50% caused by HPV), penile, vulval, anal cancer HPV vaccine gardasil

Answer 224

RETROVIRUS (RNA virus) adult T cell leukaemia (2-5% affected individuals get leukaemia) - 100% cases of T cell leukaemia have the virus - prevalent in Japan. Transmission via breast milk, semen, blood Genomic RNA copied into DNA by reverse transcriptase before viral proteins are synthesised by the host cells machinery -TAX protein and HBZ are required to transform T cells in culture - work by protein-protein interactions. TAX activates NFKB pathway. HBZ activates Jun. To prevent they limit breastfeeding to 6 months in Japan

Answer 225

DNA virus. Integrates into host genome. Chronic infection causes HCC - 10-25x risk Causes cancer via: - Indirect mechanism: evoking immune response, chronic inflammation - liver necrosis and regeneration - Direct mechanism: insertional mutagenesis at specific sites-> integration into human telomerase reverse transcriptase (hTERT) gene occurs frequently, results in upregulation due to close proximity with viral enhancer - Direct mechanism: HBV X protein activates proto-oncogenes and inhibits tumour suppressor genes (main effector) - induces liver cancer in mice. (Hepatitis C also causes HCC - RNA virus) hep B vaccine to prevent, check blood products

Answer 226

Herpes virus 8 or kaposi's sarcoma assoc herpes virus DNA virus Thought to infect circulating endothelial cells. Often assoc with AIDs, immunosuppression. KSHV produces viral cyclin, viral anti-apoptosis proteins eg vBcl2, vFLIP. Also viral encoded mRNAs and viral protein LANA (main effector) which interferes with p53 and Rb. LANA has same mechanism as HPV. They require autocrine and paracrine factors for cell transformation - induce neighbouring cells to produce growth factors and cytokines (by releasing paracrine factor vGCPR) which stimulate NFKB and MEK/ERK pathways in the HHV8 infected cells - tumourigenesis by "remote control".

Answer 227

75% gastric cancers caused by H pylori Chronic superficial gastritis -> atrophic gastritis -> intestinal dysplasia -> gastric carcinoma. Gastric atrophy characterised by loss of normal glandular tissue and results in reduced acid production -> other bacteria colonise -> inflammatory response H pylori eradication - PPI/2 antibiotics

Answer 228

Code for protein called cytotoxin-associated antigen (Cag A)- effector protein injected into cells via integrin receptor -> phosphorylated by tyrosine kinases e.g. Src and ABL -> activates SH2 containing proteins e.g. SHP2 -> MAPK pathway Unphosphorylated Cag A interacts with e-cadherin causing it to release b-catenin -> nucleus and is transcriptional regulator of genes required for metaplasia Also H-pylori induced inflammation results in hypermethylation of promoter of e-cadherin, reducing its expression. Also general inflammation will upregulate NFKB etc. Cag-A expression induced in high salt conditions - high salt diet associated with gastric diet.

Answer 229

JAK/STAT pathway -> induced by IL6 - induces proliferation (cyclin D), survival (Bcl-xl), inflammation (IL6), angiogenesis (VEGF) NF-κB -> regulate pro-inflammatory factors Tumour associated macrophages: Macrophages produce ROS and RNS (Reactive nitrogen species) to help fight infection but can also cause DNA damage TNF-a can affect cell motility and tumour metastasis -> nitric oxide synthase is stimulated by TNF-a. IL-6 important in hepatocarcinogenesis - downregulation of IL6 by macrophages in response to oestrogen makes women less susceptible Inflammation can also attract stem cells e.g. chronic inflammation in prostate cayses recruitment of mesenchymal stem cells. Induce DNA damage/genomic instability; stimulate cell growth/angiogenesis/EMT/motility/breakdown of ECM. Effect is on tumour cell themselves and stromal cells.

Answer 230

Transcription factor. Produced by macrophages and tumour cells. Activated by specific inflammatory agents eg bacteria, viruses, cytokines eg TNF-a, stress, hypoxia, smoke, carcinogens, radiation Causes inflammation (Cox2, IL6, TNFa), promotes metastasis (MMP9, TWIST), angiogenesis (VEGF), promotes proliferation (cyclin D, MDM2) and **inhibits apoptosis** (bcl-xl, cIAP, c-FLIP) Target genes : c-Flip, Cox-2, MMP9, VEGF, IL6, TNF-a, cyclin D, MDM2 Normally bound to IκB proteins which inhibits NF-κB, if these are degraded by IKK phosphorylation then NF-κB released and travels to nucleus as a hetero or homodimer. Can get activating mutations of the NFKB gene and also deletions of IκB gene

Answer 231

1. Sustained proliferative signalling: Acquired mutations short-circuit growth factor pathways leading to unregulated growth (oncogenes). EGFR inhibitors target this. 2. Insensitivity to anti-growth signals: Acquired mutations or gene silencing interfere with growth inhibition pathways (tumour suppressor genes). CDK inhibitors. 3. Evasion of programmed cell death. Cancer cells evade apoptotic signals. Pro-apoptotic BH3 mimetics. 4. Replicative immortality: Replication of normal cells limited by telomere shortening. Altered regulation of telomere maintenance results in unlimited replicative potential. Telomerase inhibitors 5. Induced angiogenesis: Cancer cells alter the balance between angiogenic inducers and inhibitors to activate the angiogenic switch. VEGF inhibitors. 6. Activating invasion + metastasis: Genomic alteration affects enzymes involved in invasion or molecules involved in cell-cell/cellular-extracellular adhesion. Inhibitors of HGF/c-Met + emerging 7. Avoid immune destruction. Immunotherapy agents 8. Disregulating cellular energetics: Cancer cells carry out glycolysis even in the presence of oxygen – glycolysis intermediates are used in biosynthetic pathways = Warburg effect. Aerobic glycolysis inhibitors + enabling 9. Genome instability and mutations: Faulty DNA repair leads to genomic instability. PARP inhibitors. 10. Tumour promoting inflammation: Inflammation facilitates the ability to acquire core hallmarks of cancer and release of mutagenic oxygen species. Aspirin.

Answer 232

Change in a single base eg A-> G Most either synonymous or benign Specific SNPs in particular genes may be assoc with increased or decreased risk of cancer. Many don’t cause alteration in protein, may not even be in a disease associated gene. THey are useful as if they are associated with a disease (GWAS study) that usually means they are near a gene. Most SNPs are bi-allelic. Very useful as abundant and can by typed using microarray (hybridisation)

Answer 233

5 point classification - whether a change in amino acid changes function of protein 1. clearly not pathogenic 2. Likely non-pathogenic 3. Uncertain 4. Likely pathogenic- exchange hydrophobic for hydrophilic which changes structure of protein 5, clearly pathogenic

Answer 234

Genetic Abnormality: Autosomal Dominant – defects in MSH2 and MLH 1 account for 90% of cases, also PMS1, PMS2, MSH6 Mechanism of Action: Loss of protein products coded by these genes >> loss of MMR >> reduced fidelity of DNA replication by orders of magnitude = microsatellite instability. Associated cancers: Colorectal Cancer Amsterdam II criteria: >3 family members (one a first degree relative of other two), two successive generations, >1 diagnosed < 50 years, FAP excluded. Also endometrial, small bowel, ovarian serous cystadenoma. Don't tend to get polyps unlike FAP.

Answer 235

11 CPGs activated by mutations - all autosomal dominant ALK- generally only somatic EGFR- generally on somatic RET- common somatic, can be germline in MEN2

Answer 236

103 CPGs contain loss of function mutations TP53 BRCA1 MLH1 Herogenous - retinoblastoma (single gene)

Answer 237

single nucleotide polymorphism Contribute to increased risk but not high risk like CPGs Some protective, some increase risk Multiplicative effect as each one may only increase risk a small amount but together risk might be much higher

Answer 238

Breast cancer <30yrs esp if HER2 +ve FH sarcoma Choroid plexus tumours Very high rates of early onset solid tumours over multiple generations Annual MRI breast from age 20yrs ? annual whole body MRI

Answer 239

Mutation of MEN1 gene (tumour suppressor menin) AD PPP - Parathyroid -90% onset 20s - Pancreatic - 70% - gastrinoma>non-functioning>insulinoma - Pituitary - 40%, (non-functioning>prolactinoma)

Answer 240

Parathyroid- annual PTH, calcium, prophylactic parathyroidectomy (removal of 3.5/4 parathyroid glands) Pancreatic- annual gastrin, annual imaging e.g. CT/endoscopic USS Gastrinomas- PPI/resection Pituitary - annual prolactin and IGFR

Answer 241

RET gene - mutations in extracellular cysteines cause intermolecular disulfide bonds -> constitutive RET dimerisation and aberrant activation. ONCOGENE Autosomal dominant TAP (thyroid/adrenal/parathyroid) Medullary thyroid cancer- 90% Phaeochromocytoma 50% Parathyroid adenoma 20-30% Familial medullary thyroid cancer- 100% risk but don't get other cancers, 10 people in family with medullary thyroid cancer, same mutation.

Answer 242

Annual calcitonin Prophylactic Thyroidectomy (timing depends on severity of mutation) From age of 8 or 20 - annual calcium PTH, urinary metanephrines and MRI abdo if urinary metanephrine abnormal

Answer 243

RET mutation - alters substrate binding pocket of tyrosine kinase domain via substitution -> increase kinase activity. Autosomal dominant. usually denovo RET mutation M918T variant in 95% cases. More severe/earlier onset than MEN2a TAM (thyroid, adrenal, all the Ms) Medullary thyroid cancer >90% Phaeochromocytoma 50% Assoc abnormalities 50% - marfanoid - mucosal neuromas - megacolon

Answer 244

VHL gene mutation (tumour suppressor gene): ubiquitin ligase that marks HIF for degradation in presence of oxygen so no angiogenesis Autosomal dominant Clear cell renal cancer (bilateral, young, familial history) Cysts in kidneys, pancreas, genital tract Retinal angiomas - Haemangioblastomas Cerebellar or spinal haemangiomas Retinal haemangioblastomas (blindness) Phaeochromocytomas Endocrine pancreatic tumours Cystadenomas Endolymphatic sac tumours of inner ear Belzutifan for RCC in VHL patients – HIF2a inhibitor

Answer 245

Yes if any other features of VHL /other genetic syndrome e.g. cerebellar haemangioblastoma Or if : <40 Bilateral Multifocal First/second degree relative with renal cancer

Answer 246

CDKN2A mutations - gene contains instructions for making p16 and p14 CDK4 mutations Multiple primaries at young age of onset - at least 3 melanomas in family - risk pancreatic cancer

Answer 247

Major criteria (3 or more, must be macrocephaly) - Macrocephaly (>97th centile) ALWAYs - do they have trouble buying hats - Breast cancer - Non-medullary thyroid cancer - Endometrial cancer Minor criteria (2 major + 3 minor) - other thyroid lesions - IQ <75 - Hamartomatous intestinal polyps - fibrocystic disease of breast - GU tumours or malformations - uterine fibroids

Answer 248

PTEN. Also called hamartoma syndrome. Autosomal dominant. Activation of PI3K pathway constitutively.

Answer 249

Mutations in STK11/LKB1 gene- tumour suppressor gene AD (although usually spontaneous mutation) Increase in hamartomatous polyps throughout GI tract Dark brown macules around mouth and oral mucosa Cutaneous and GI symptoms 50% have cancer by age 60yrs- breast, GI, pancreas, ovary, uterus, testicle, oesophagus, lung, 15 x risk of colorectal cancer Surveillance

Answer 250

Mutated in peutz-jegher syndrome. Serine/threonine kinase 11 - a tumour suppressor that helps to polarise cells and promotes apoptosis

Answer 251

Subtype of FAP characterised by osteomas, dental anomalies, epidermal cysts and soft tissue tumours

Answer 252

Mutation of NF1 gene -> makes protein neurofibromin- acts as a tumour suppressor in oligodendrocytes, schwann cells. Neurofibromin is a negative regulator of the Ras pathway by stimulating the GTPase activity of Ras so turning it off. Non functioning neurofibromin cannot regulate cell growth and division -> tumours along nerves. Autosomal dominant. - neurofibromas - Lisch nodules (eyes) - cafe au lait and axillary/groin freckles - malignant peripheral nerve sheath tumours - optic glioma - learning difficulties - increased risk of brain tumours and leukaemias - pectus excavatum, scoliosis, short stature - sarcomas

Answer 253

Phenotype severity varies according to genetic defect. Extreme sensitivity to UV sunlight - x 1000risk skin cancer. Burn at young age, by age of 2 freckling in sun exposed areas 1/2 develop skin cancer by age 10 without sun protection (SCC, BCC or melanoma) Blindness/cataracts/corneal ulcerations/ketatitis and 30% get progressive neurological deficits involving eyes, ears, balance 20 x risk other cancers. Mental retardation, premature dementia Life expectancy 20-30s. Note – these patients do not show more radiosensitivity – therefore NER is not important in DNA repair post radiotherapy.

Answer 254

7 types of mutation. Mainly in NER (nucleotide exicision repair) - XPC, ERCC2, POLH account for most. XPA-G

Answer 255

Rare form of dwarfism + deafness and retinal atrophy, microcephaly UV sensitivity Age quickly, 3 types starting at birth or childhood AR mutation of ERCC6/8 gene -> involved in NER

Answer 256

Li-fraumeni - sarcomas Ataxia telangiectasia - most radiosensitive Bloom syndrome Nijemegen breakage syndrome Gorlin syndrome Fanconi syndrome

Answer 257

ATM mutation (Ataxia Telangiectasia Mutated kinase (ATM) mutation). Chromosome 11q22 ATM is an inactive homodimer >> activated by DSBs (ionising radiation) >> phosphorylated to active monomers >> phosphorylate P53 & Chk1 (G1/S checkpoint), Chk2 (G2/M checkpoint) causing either cell cycle arrest and DNA repair or if damage too severe, apoptosis. ATM deficient cells are therefore very radiosensitive and unable to repair DSBs. Severity of phenotypes varies depending on mutation - progressive cerebellar ataxia from childhood (mask-like face, slow eye movements, ataxia) - telangiectasia (skin, conjunctiva) - radiosensitive - cancer risk x 100 of lymphoma + leukaemia - immune defects - radiosensitivity - sterility - normal IQ 1/100-200 people are carriers- higher in askenazi jews - carriers have increased risk of breast cancer – it is included in the breast cancer genetic panel - 1/40000 have disease - life expectancy 25 years – death due to cancer (usually lymphoma/leukaemia)p

Answer 258

``` Mutation of NBN gene - part of MRN complex (MRE11, RAD50, NBS) used in HR and activated by ATM AR Short stature Mental retardation Microcephaly Facial dysmorphism Immunodeficiency Increased risk of breast cancer ``` RADIOSENSITIVITY

Answer 259

Mutation in BLM gene - codes for RecQ helicase - responsbile for maintaining DNA structure and unwinding it -> increased sister chromatid exchanges ``` Short stature Sun sensitive rash Bird like features high pitched voice COPD DM Immuno def Increased cancer risk - H+N, SCC Radiosensitivity ```

Answer 260

Upregulate glutamine -> enter TCA cycle Glutamine can do reverse TCA cycle and once converted to a-ketoglutarate this can undergo reductive carboxylation and form citrate -> major substrate for lipid synthesis. Glutamine important in producing carbon intermediates and reduced nitrogen for nucleotide synthesis.

Answer 261

PI3K/AKT pathway may regulate glucose metabolism by: 1. Regulating glucose transporter expression through AKT -> stimulates transcription of GLUT1 2. Enhancing glucose capture by HK2 and inducing aerobic glycolysis by promoting HK2 bidning to voltage dependent anion channels 3. Stimulates PFK1 activity

Answer 262

Following ionizing radiation exposure, virtually all proliferating cells (independent of p53 (TP53) status) will show a radiation dose-dependent arrest in the G2 phase. Typically, cells with functional wild-type p53 will also arrest in the G1 phase of the cell cycle, and some cells may also arrest in S phase after radiation.

Answer 263

Nucleotides are composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose or deoxyribose), and a phosphate group consisting of one to three phosphates. DNA (deoxyribonucleic acid) is an acid (from the phosphate group) - the base forms bonds with the complementary base so doesn't have basic activity.

Answer 264

IL10 (inhibits T cells/macrophages/dendritic cells) - via STAT3 pathways, TGF beta (Tumour cells also produce IDO (indoleamine-2,3-dioxygenase) - role in maternal tolerance to foetal tissue - causes reduction in tryptophan for T cells to use - potential target to restore immunity)

Answer 265

Part of innate immune system. Perform cell mediated immunity by release cytotoxins e.g. granzymes and perforin. 1) Recognise stress ligands on tumour cells surface e.g. MIC through their stress ligand receptor (e.g. NKG2D) 2) Abundant cytosolic nucleic acids in tumour cells (due to genomic instability) activates a sensor called STING (stimulator of interferon gene). This induces interferon production which recruits NK cells. 3) Can perform ADCC (antibody dependent cellular toxicity)

Answer 266

Genetic Abnormality: Mutation of Wt1 or Wt2 tumour suppressor gene (transcriptional regulator) - seen in some genetic syndromes such as Beckwith-Wiedemann syndrome, Frasier syndrom, WAGR syndrome Associated cancers: Nephroblastoma (kidney cancer in children aged 4-5 years - can metastasise to the lung - form from nephrogenic rests)

Answer 267

Genetic Abnormality: BRCA 1 (17q) and 2 (13q) Mechanism of Action: autosomal dominant – involved In HR pathway of DNA repair. In patients with mutated BRCA, normal tissues are heterozygous – no increased sensitivity. A spontaneous somatic mutation in other BRCA allele in later life causes cells to be deficient in HR repair >> genomic instability >> sensitivity to DNA crosslinking agents (e.g. Cisplatin) Synthetic lethality: - PARP used in BER to repair SSBs. If left unrepaired (e.g. in context of PARPis) – replication converts these to DSBs. BRCA used in HR to repair DSBs - Normal cells treated with PARPis – functional HR – viable - BRCA mutant tumour cells treated with PARPis - loss of BER and HR – synthetic lethality - death Associated cancers: breast, ovarian cancer, prostate, fallopian tube, leukaemia/lymphoma. BRCA2 also in Fanconi anaemia (recessive) In the NHS currently the genetic breast cancer screening panel includes - BRCA1 (HR), BRCA2 (HR), PALB2 (HR – aids BRCA2), ATM (DSB) and CHEK2 (DSB) PALB2: Tumour suppressor gene that is a partner of both BRCA1 and BRCA2 so part of HR pathway: Breast cancer (lesser extent ovarian and pancreatic cancer Chk2: familial breast cancer due to mutation 1100delC in Chk2, 37% risk of breast cancer at age 70 in women The prevalence of BRCA1 mutation is slightly lower than that of BRCA2 mutations in the general US population (1:500 for BRCA1 vs 1:222 for BRCA2). The breast cancer risks for carriers of BRCA1 and BRCA2 mutations are similar at about 70% by the age of 80, but with earlier age of disease onset for the BRCA1 mutation (peaks in 4th decade) compared to BRCA2 carriers (peaks in 5th decade).

Answer 268

Cancer is also known as the "wound that never heals" - chronic inflammation. Inflammation and infection cause 20% cancers. Potential treatment/prevention e.g. aspirin, celecoxib (used in FAP) - inhibits prostaglandin production from arachidonic acid by COX2. 1) Infectious agents e.g. hepatitis B 2) Tobacco smoke (also has 82 carcinogens) 3) Asbestos 4) Obesity - enlarged adipocytes (hypertrophy) are source of pro-inflammatory molecules 5) Inflammatory bowel disease = increased risk of colorectal cancer 6) Cirrhosis and HCC e.g. obesity/alcohol/infectious driven 7) Dysbiosis (imbalance in gut microbiota) may contribute to cancer in obesity 8) E coli, Strep bovis, H pulori = colorectal cancer

Answer 269

Schistosoma haematobium = bladder (liver fluke) Opisthorchi viverrini = liver cancer (liver fluke) Clonorchi sinensis = liver cancer (liver fluke)

Answer 270

HIV – Kaposi’s, non-hodgkins lymphoma, hodgkins Retrovirus.

Answer 271

Causes lung, oropharynx, larynx, oesophagus, pancreas, bladder cancer. 81 carcinogens: benzo [a] pyrene (polycyclic aromatic hydrocarbon) which causes G to T transversions and activated by CYP1A1; nitrosamines cause O6 guanine alkyation. Polymorphisms in CYP1A1 can vary how much carcinogen is activated so affects cancer-rates in smokers. Inflammation

Answer 272

* Occupational e.g scrotal and nasal cancer in chimney sweeps from soot, bladder TCC from aromatic amines and azo-dyes (textile industry), mesothelioma from asbestos * Toxic agents in urban air, organic compounds from agriculture, chlorination/contaminants of drinking water, UV light * Diet – stomach cancer from high-nitrate foods; liver cancer from mouldy grain (aflatoxins)

Answer 273

1) Smoking (note there is synergy between smoking and alcohol in carcinogenesis) 2) Alcohol 3) Obesity 4) Physical activity 5) Reproductive choices: having children, late menarche, early menopause, breastfeeding, not using oestrogen-based contraceptives = less breast cancer. (Oestrogen is a mitogen for breast cancer and also thought the oestrogen metabolites are genotoxic) 6) Sexual activity e.g. HPV 7) IV drug use e.g. Hep B/C = HCC, HIV = kaposis/lymphoma. 8) Age is the most important risk factor

Answer 274

Ionising radiation e.g. alpha/beta particles and gamma rays. Ionises by mobilising an electron. Can directly ionise DNA or with water (radiolysis) to form hydroxyl radical (OH) then hydrogen peroxide (H2O2) then superoxide radical (O2-). Causes dsDNA breaks = mitotic catastrophe. Leukaemia is most frequent ionising-radiation induced cancer. Solid tumours increase with dose in linear fasion. Thymol glycol [5,6-dihydroxy-5,6-dihydrothymine] and oxidized guanine [8-oxo-7,8-dihydroguanine (8-oxoG)] are DNA base lesions present in clustered DNA damage induced in cells by ionizing radiation. These other forms of DNA damage, however, are more readily repaired and are less likely to result in cell death. Ultraviolet radiation - UVB - cyclobutane pyrimidine dimers and 6,4 photoproducts. BCC/SCC/MM. Also DNA/protein crosslinks - mostly occur in euchromatin Tumour cells die by mitotic catastrophe in radiation. Lymphoid and some epithelial normal tissues die by apoptosis in ionising radiation - this occurs in interphase.

Answer 275

Paracrine signaling acts on nearby cells, endocrine signaling uses the circulatory system to transport ligands, and autocrine signaling acts on the signaling cell.

Answer 276

Small areas of hypermutation in cancer genomes. "Thunderstorm"

Answer 277

Tumour cells contain large circular extrachromosomal DNAs - rarely found in normal cells. They can replicate but do not have centromeres so are not equally distributed into daughter cells. Causes tumour cell heterogeneity. Is a source of amplified genes, chimeric rearrangements and reintegration into the linear genome of chromosomes. They can also be excluded from cells temporarily e.g. to cause drug resistance in presence of chemotherapy then reabsorbed once chemotherapy gone - allows flexibility to tumour. Non-mendelian inheritance. Chromatin in ecDNA is more open than conventional linear DNA

Answer 278

Regulatory DNA sequences that are position and orientation independent relative to the promoter. Important for tissue and stage specific expression. Clusters of enhancers = super-enhancers.

Answer 279

Driver mutations = confer a growth advantage. Passenger mutations = do not confer a growth advantage. Mutations in DNA repair = mutator phenotype.

Answer 280

Heterochromatin - highly compact chromatin, no transcription - "silenced". Euchromatin - open accessible chromatin, transcription occurs.

Answer 281

- DNA Helicase unwinds the helix, DNA Polymerases synthesise DNA, always in the 5’ >> 3’ direction - Copied in a semi-conservative manner, each strand encodes its own complimentary strand, therefore each DNA molecule contains one parental strand and one de novo synthesised strand

Answer 282

The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protein-coding genes and non-coding genes.

Answer 283

- Tandemly repeated DNA (also found as centromeric DNA) - Mini – up to 20kb length and repeat units up to 25bp, telomeric DNA (TTAGGG repeat) is an example - Micro – up to 150bp and repeat units of 1-4bp (function unknown)

Answer 284

Polymorphism, as related to genomics, refers to the presence of two or more variant forms of a specific DNA sequence that can occur among different individuals or populations. Used as DNA markers SNPs is common type. RFLP – restriction fragment length polymorphisms: Allelic variation in site for restriction enzyme cutting, Bi-allelic, so not that useful SSLP – simple sequence length polymorphisms: Array of repeat sequences that have variable lengths, Multi-allelic, Minisatellites – variable number of tandem repeats (VNTRs), less useful as tend to be found in telomeric regions and less good for PCR due to length, Microsatellites – simple tandem repeats (STRs), preferred

Answer 285

- Halting of DNA replication and cell cycle proliferation, via activation of pRB and p53 - Cells can only replicate a limited no. of times (Hayflick Limit) as they’re unable to copy chromosome caps (Telomeres) - loss of senescence (limitless replicative potential) is a cancer hallmark - p21 is temporary senescence, p16 is permanent

Answer 286

Family of transcription factors: MYC, MAX, MAD, MXI. Dimerise in different ways and lead to distinct biological effects of growth, differentiation and death. MYC promotes proliferation including E2F, hTERT, p21 and CDK4. Requires heterodimerisation with MAX to function via basic helix-loop-helix leucin zipper domains - bind to E-box in target genes. MAX and MAD/MXI repress transcription at E-box target genes so inhibit proliferation. - t(8:14) translocation places c-MYC under control of immunoglobulin heavy or light chain gene transcriptional promoter >> increased cell proliferation

Answer 287

Neoplasia: abnormal proliferation of cells. * Benign neoplasm: -oma. * Malignant neoplasm: cancer, -carcinoma, -sarcoma etc. –carcinoma = epithelial cell cancer – accounts for 85% of all cancers –sarcoma = mesodermal (muscle/bone) cancer –adenocarcinoma = secretary/glandular tissue e.g. breast

Answer 288

Stages of metastasis: 1. Primary tumour formation 2. Local tissue invasion 3. Intravasation 4. Transport in circulation 5. Lodgement in distant tissues 6. Extravasation 7. Micrometastasis 8. Metastasis – dormancy or outgrowth

Answer 289

- Pain (somatic, neuropathic) - Pressure and Obstruction - Ulceration and bleeding - Invasion – organ dysfunction e.g. seizures

Answer 290

Cancer cachexia (due to cytokines - NOT nutritional demands of cancer) Paraneoplastic syndromes: o Cushing’s syndrome (ACTH), SIADH (hyponatraemia) – SCLC o Hypercalcaemia – NSCLC, Breast, Prostate – PTHrp o Hypoglycaemia – HCC, sarcomas – Insulin/Insulin like substances o Myaesthenia, peripheral neuropathy – bronchogenic carcinoma o Acanthosis nigricans (gastric), Dermatomyositis photosensitive rash (bronchogenic) o Hypertrophic osteoarthropathy and clubbing o Venous thrombosis o Nephrotic syndrome o polycythaemia = EPO from RCC

Answer 291

Angiogenic Switch – tumour creates conditions of hypoxia leading to: - over-expression of angiogenic-inducers (e.g. VEGF, PDGF, Hepatocyte-derived Growth Factor, Fibroblast growth factor, Epidermal Growth Factor) - down-regulation of angiogenic-inhibitors (e.g. Angiostatin, Endostatin, p53, Thrombospondin 1/2)

Answer 292

- chaotic and inefficient resulting in poor-perfusion, limited access by cytotoxic agents, hypoxia and subsequent resistance to radiotherapy and promotion of further angiogenesis - immature vessels lack pericytes, therefore haemorrhage, tumour cell escape and ascites

Answer 293

In the absence of angiogenesis, tumors would only be expected to reach a diameter of about 2 mm 1. Activation of nearby vessels and induction of sprouting (angiogenic switch) 2. Co-option of existing vessels in the peri-tumoral area – tumour grows along vessel walls 3. vasculogenesis is the formation of blood vessels from circulating bone-marrow endothelial progenitor cells. 4. Vascular mimicry: channels lined by tumour cells – common in melanoma

Answer 294

Internal: - error-prone synthesis or repair, cellular metabolism, reactive oxygen species External: - Ionising radiation, UV light, chemicals, toxins, virus, chemotherapy

Answer 295

- Mechanism of survival under stress - Cell breaks down own organelles using lysosomes to provide for more essential processes - can be selective to the organelle (mitophagy, ribophagy etc) or non-selective to the cytoplasma (macroautophagy) - Lack of chromatin condensation - Accumulation of (double-membraned) autophagic vacuoles (autophagosomes) which fuse with lysosomes (autolysosomes) - Little or no uptake by phagocytic cells - whole process is tightly regulated through at least 30 Atg-autophagy genes Not taken up by phagocytic cells, no chromatin condensation. Beclin is a protein important in the development of the autophagosome for autophagy. Autophagy is a process in which cells generate energy and metabolites by digesting their own organelles and macromolecules and as such it is a survival mechanism. Autophagy allows a starving cell, or a cell that is deprived of growth factors to survive up to a point. Cells that do not receive nutrients for extended periods ultimately digest all available substrates and die through autophagic death. Supplying nutrients before this critical point would restore the cell’s health

Answer 296

1. Annexin V – most commonly used method via flow cytometry 2. Tunel assay – detects DNA fragments 3. DNA ladder – via gel electrophoresis of pooled DNA During the execution phase of apoptosis, nucleases are activated which cleave DNA into 180-200 base pair increments. Several assays are available to measure this phenotype. The TUNEL method identifies apoptotic cells by using terminal deoxynucleotidyl transferase (TdT) to transfer biotin-dUTP to strand breaks of cleaved DNA. The Annexin V Assay, a classical technique for detecting apoptosis, is the most commonly used method for detecting apoptosis by flow cytometry. Annexin V is a calcium-dependent phospholipid binding protein that has a high affinity for the phophatidylserine (PS), a plasma membrane phospholipid. One of the earliest features of apoptosis is the translocation of PS from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external environment. Annexin V binds to PS exposed on the cell surface and identifies cells at an earlier stage of apoptosis than assays based on DNA fragmentation. DNA ladder formation is detected by gel electrophoresis of pooled DNA. Diamidino-2-phenylindole (DAPI) is DNA-specific dye that displays a blue fluorescence. This dye could be used to assess the nuclear morphology of normal versus apoptotic cells by fluorescence microscopy.

Answer 297

Pauses cell cycle via p53 (and less extent CDC25) - p53 triggers apoptosis if needed. Activates DNA repair via H2AX, Nbs1, DNAPKc, BRCA2, BLM1 = HR ATM is activated following irradiation by auto-phosphorylation and conversion from an inactive homodimer to an active monomer

Answer 298

G2/M>G1>early S>late S HR needs second chromatid so only active in late S - fixes radiation induced dsDNA breaks. G2/M is beyond the DNA repair checkpoint so too late to fix radiation induced dsDNA breaks so mitotic catastrophe occurs

Answer 299

<1.8Gy/fraction Tumour control (Assuming high alpha/beta ratio) = no effect Early toxicity (high alpha/beta ratio) = no effect Late toxicity (low alpha/beta ratio) = reduced toxicity As alpha/beta ratio is inversely proportional to sensitivity to fractionation

Answer 300

>2Gy/fraction Tumour control = no effect Early toxicity = no effect Late toxicity = increased toxicity

Answer 301

Acceleration = >10Gy/week Tumour control = increased control (Assuming you went beyond T delay initially) as less repopulation Early toxicity = increased as less repopulation Late toxicity = no effect as no repopulation

Answer 302

Tumour control: no effect due to rapid DNA repair Early toxicities: no effect due to rapid DNA repair Late toxicities: increased as slow DNA repair

Answer 303

Tumour control = increased Early toxicity = increased Late toxicity = increased

Answer 304

NHEJ > HR for causing radiosensitivity if mutated. BER does not usually cause radiosensitivity if mutated (Apart from XRCC1 gene) ssDNA breaks (BER/PARP) - most common as ssDNA breaks more common than dsDNA breaks dsDNA break - NHEJ (throughout cell cycle, fast, inaccurate/mutagenic, most used by cells. most important in radiation repair) and HR (S/G2 phase, slow, accurate) Loss of NHEJ = increase in radiosensitivity and lose the alpha part of the curve. Loss of HR = sensitive to PARP inhibitors = synthetic lethality

Answer 305

Alpha = sensitivity to low doses of radiation, linear bit of the curve. Represents dsDNA breaks and/or lethal damage and/or DNA repair pathways not saturated and/or direct damage. Gray-1 Beta = sensitivity to high doses of radiation, exponential bit of curve. Represents multiple ssDNA breaks in one cells and/or multiple sublethal damage hits and/or DNA repair pathways saturated and/or indirect damage. Gray-2 Alpha/beta ratio = inversely proportional to sensitivity to changes in fractionation. High = straight, low = curved. Gray. Is where AD=BD2. Linear component of cell kill = quadratic component. Determines the bendiness of the survival curve. The A/B ratio is the dose at which the linear and quadratic contributions to cell killing are equal. High alpha/beta ratio in tumour/acute toxicities: usually highly proliferating tissues, tissues with more lethal damage and less sublethal damage Low alpha/beta ratio: late toxicities, lower proliferating tissues, tissues with more sublethal damage repair

Answer 306

Late toxicity: 3 (Spinal cord: 2) Prostate cancer: 1.5 (hypofractionate) Breast: 4-4.5 (hypofractionate) Early toxicity: 10 H+N: 10 (hyperfractionate) Skin: fibrosis = 3, erythema = 10

Answer 307

Surviving fraction at 2Gy A measure of radiosensitivity

Answer 308

Semi-logarithmic (dose on x axis, logarithm of survival fraction on y axis) = allows visualisation of low doses as you want to get to 0 or as low as possible Dose response curves = total dose on x axis, response on y axis = sigmoid shape (low dose doesn't cure much, then get a lot of cure, then not much cure). Radiosensitiser shifts the tumour control curve to the left. Hypoxia shifts to the right. Complication rate must be to the right otherwise you cannot give treatment. Is parallel to the tumour control. Radioprotection moves complication rate to the right (but also protects the tumour hence none are used in practice)

Answer 309

* Repair: DNA repair of sublethal damage - SLD mostly in low alpha beta tissues I e. late toxicities. Fractionation = time for late toxicity repair = better tolerability, no effect on tumour control. * Radiosensitivity: depends on tumour. Can increase by radiosensitisers e.g. chemotherapy, PARP inhibitors, treating anaemia. The law of Bergonie and Tribondeau states: The radiosensitivity of a cell is directly proportional to reproductive rate and is inversely proportional to its degree of differentiation. * Repopulation: early toxicities and tumour repopulate. Fractionation = more repopulation of tumour = worse control. * Redistribution: G2/M>G1>early S>late S. Catch cells at different phases by fractionation = better tumour control. * Reoxygenation: oxygen = ROS = more radiosensitive. normal tissues are not hypoxic so only applies to tumour Fractionation = opens temporarily closed vessels + changes architecture so cells get oxygenated + oxygen can get to inside cells = better tumour control.

Answer 310

Biologically effective dose on a specific effect e.g. tumour control or late toxicity BED = nd (1+d/alphabeta ratio)-K x (T-T delay) n = number of fractions d = dose/fraction (nd = total dose) K = constant for each effect = Gy/day - dose lost per day of repopulation Tdelay = days until repopulation occurs (Tdelay usually 28 days, K = 0.9 for H+N)

Answer 311

EQD2 = nd (d1+alpha/beta) / (d2+alpha/beta) 74 = nd (3Gy + 1.5)/(2Gy + 1.5) 74 (2+1.5)/(3+1.5) = nd Used to compare different regimens with different dose per fraction. EQD2 gives you a practical dose in 2Gy factions whereas BED gives you a theoretical biological effect dose.

Answer 312

o Early:keratoconjunctivitis o Late: cataracts – occurs at low doses but is easily treatable with surgery. Occur at 2Gy. TD5/5 10Gy. o Late: “dry eye” due to damage to lacrimal glands – this is dose limiting effect. Causes chronic corneal ulceration and loss of eye. TD5/5 is 30Gy of dry eye. A radiation-induced cataract is one of the few examples of a radiation injury which does have distinct pathognomonic characteristics that identify it as having been induced by ionizing radiation; radiation-induced cataracts typically begin in the posterior portion of the lens, unlike age-related cataracts.

Answer 313

o Early: otitis media o Late: permanent hearing loss if >30 Gy

Answer 314

o Get loss of taste due to direct damage and due to xerostomia (dry mouth) from salivary glands after 30Gy – usually resolves but may not.

Answer 315

o Epiphysial plate: very radiosensitive (4Gy) get loss of growth. o Adult cartilage: radioresistant but can get osteoradionecrosis as a late effect. (Femoral head TD5/5 is 52Gy) Bone marrow: aplasia, pancytopenia. TD5/5 2.5Gy

Answer 316

o Late: reversible changes in ECG that are not predictive; reversible pericarditis with pericardial effusion – usually asymptomatic and occurs within 2 years; radiation-induced cardiomyopathy with reduced EF or conduction blocks – takes 10-20 years to develop; ischaemic heart disease. Occurs at 50Gy in 2Gy fractions. o Exact damage thought to be due to volume effects and also sensitive substructures – in future may volume the heart in sections e.g. coronary arteries, valves. A number of large clinical trials, particularly those performed in Hodgkin’s disease patients, have indicated that the populations most at risk for RIHD are young females and the elderly, and that the important factors governing tissue tolerance are total dose, fraction size, and volume irradiated. (V40 = 50%) TD5/5 40Gy.

Answer 317

o Late effects: sensory and motor defects

Answer 318

o Earliest late effect: Lhermitte sign (reversible demyelinating reaction) – not predictive for other effects. o Late effect: permanent myelopathy (paralysis) – can be due to demyelination and necrosis of white matter or due to vasculopathy. o Tolerance of spinal cord dependent on size of dose per fraction so giving repair time doesn’t have much effect. o Generally radioresistant compared to other organs but very important so use a TD5 (5% complication level) TD5/5 = 45Gy

Answer 319

o Transient demyelination = somnolence syndrome. o Leukoencephalopathy. o Radionecrosis - Initially can white matter necrosis and demyelination then get vascular changes with telangiectasis and focal haemorrhages. o Children’s brains more sensitive than adults. o Generally radioresistant compared to other organs but very important so use a TD5 (5% complication level) The effects of radiation on the nervous system arise primarily as a consequence of damage to oligodendrocytes and glial cells TD5/5 = 45Gy

Answer 320

o Get reduced storage so increased urinary frequency both early and late but due to different pathologies o Early: hyperaemia and mucosal oedema. Worsened by UTI which can cause desquamation and ulceration. These early changes contribute to the severity of the late changes. o Late: progressive mucosal breakdown resulting in ulceration and fistulae. Can get fibrosis of bladder wall. Can get bleeding episodes due to telangiectasia. Can take up to 10 years – the higher the radiation dose, the quicker the formation of complications. Urethra TD5/5: 70Gy Bladder TD5/5: 75Gy

Answer 321

second most sensitive volume effect o Late: radiation nephropathy – proteinuria, hypertension, anaemia and impairment in urine concentration. Glomerular injury then glomerular sclerosis then tubule-interstitial fibrosis. RAAS system plays a role - may be mitigated/prophylaxis with ACEi. o Low alpha/beta ratio. o Dose tolerated by kidney reduces with time as you develop the radiation nephropathy – therefore retreatment is best avoided. o Dependent on volume effect – if only partially irradiated, the remaining kidney can hypertrophy to compensate for the damaged area. The kidney has a relatively low tolerance dose because of the limited number of clonogens within each nephron, although the cells comprising the functional subunits of the kidney are not particularly radiosensitive. The kidney exhibits substantial sparing with fractionation and displays little or no tolerance to re-irradiation. (V20 = 20%) TD5/5 23Gy.

Answer 322

most sensitive volume effect o Early: pneumonitis. SOB, dry cough, reduced gas exchange, reduced pulmonary compliance. o Late: lung fibrosis (chronic radiation pneumopathy). Very radiosensitive, significant volume effect (lung is the dose-limiting organ in total body irradiation). Increasde age and tamoxifen treatment increase risk of fibrosis. The best predictor for late effects has been found to be the V20/V30, that is, the percentage of normal lung volume that receives should not receive more than 20 Gy or 30 Gy, respectively. (V20 = 30%). T5/5 17.5Gy.

Answer 323

third most sensitive volume effect o Radiosensitive, with tolerance dose of 30Gy in 2Gy fractions, volume dependent e.g. in total body irradiation for bone marrow transplantation (liver acini are the FSUs). o Early: acute radiation hepatitis with venoocclusive disease (central vein thrombosis) causing liver enlargement and ascites o Late: chronic hepatopathy – fibrosis of liver. (V30 = 40%)* TD5/5 30Gy.

Answer 324

o Early: get initial increase in motility then atonic phase followed by loss of proliferating epithelium (atrophic villi) which causes diarrhoea (Water and electrolyte loss) and resulted absorption/resorption (affects oral drug absorption). This can cause sepsis. “Fixed” intestinal loops e.g. due to adhesions and the rectum are more at risk as they will be exposed to radiation on each dose as they cannot move. o Late: chronic ulcers; stenosis; ileus; telangiectasis causing bleeding. These changes are induced by the early damage. in the rectum, it is the percentage of the wall that has received 40-50 Gy that determines the likelihood of rectal bleeding, although the extent of reserve, unirradiated tissue is also a factor. The small intestine is the most sensitive component of the GI tract. TD5/5S: Small intestine: 40Gy Colon: 45Gy Rectum: 60Gy

Answer 325

o Early: gastritis o Late: ulceration due to vascular damage TD5/5 50 Gy

Answer 326

o Early: transient hypersalivation then stop salivation after 40Gy. Volume effect very pronounced so need to irradiate entire salivary gland to get these complications. o Late: chronic xerostomia if doses >60Gy. Parallel organ The best way to spare the parotid gland is to decrease the volume of the gland irradiated. The parotid exhibits relatively little sparing with fractionation so use of either a hyperfractionated or hypofractionated protocol would have only a modest impact. Prolongation or acceleration of treatment would have little effect on the parotid. The serous acinar cells of the parotid and submaxillary glands are Iconsidered to be the targets for radiation-induced salivary gland damage. Quantec 1 gland dose <20Gy

Answer 327

o Early: radiation caries due to direct damage at dentine-enamel border and due to xerostomia and changes in the microbiome of the mouth. o Patients should have dental treatment prior to radiation as if they have it after they can get osteoradionecrosis. Radiation tolerance of the mandible is also affected by pre-irradiation dental disease, fraction size and gender (males more susceptible).

Answer 328

o Early: oral mucositis and ulceration. Dose limiting in radical head and neck radiation. Oesophagitis causing dysphagia. o Late: mucosal atrophy and ulceration; telangiectasia (bleeding); strictures in the oesophagus causing chronic dysphagia. TD5/5 is 55Gy.

Answer 329

o Early: temporary erythema (1 day) dry desquamation (Radiodermatitis sicca) then moist desquamation (4 weeks) o Late: subcutaneous fibrosis (induration); telangiectasia. Can occur without any early changes as it occurs in different skin layer. o Hair loss: transient or permanent depending on dose – can grow back with discolouration. o Loss of sebaceous and perspiratory glands resulting in dry skin. Following irradiation of the skin, the dose and time course for epilation and loss of sebaceous gland secretion are similar. Following skin irradiation, the first visible evidence of damage is a transient erythema that is observed within 24 hours following irradiation, whereas moist desquamation would only be observed after a few weeks. Epilation is observed at doses similar to those that cause the main wave of erythema that is typically manifested about one week following irradiation. Pigment changes typically appear long after irradiation due to the low proliferation rate of melanoblasts. It is usually not possible to predict the extent of late reactions based upon the severity of early reactions because early reactions result from killing of epidermal stem cells, whereas late reactions likely occur due to vascular damage in the dermis.

Answer 330

The ideal procedure is to transfer all patients to a matched linear accelerator on the day of interruption. Where this is not possible the following approaches are recommended. – Where possible, there should be the facility that allows patients who have missed scheduled weekday treatments to be treated at the weekend. Departmental protocols must ensure that complex treatments can be safely delivered out of normal hours. – Patients can be treated twice daily, with a minimum of six hours between therapies. – Use of biologically equivalent dose (BED) calculations to derive an alternative schedule involving a modified number of treatment fractions with which to complete the radiotherapy course in the planned overall time, but perhaps accepting a higher BED in normal tissues. – The addition of extra treatment fractions where compensation cannot be achieved within the original overall planned time

Answer 331

Treatment duration MUST not be prolonged by more than two days over the original prescription. Radical therapies for: § Squamous cell carcinoma of the head and neck region § Non-small cell and small cell lung carcinoma § Squamous cell carcinoma of the cervix § Squamous cell and adenocarcinoma of oesophagus § Squamous cell carcinoma of skin, vagina or vulva § Squamous cell carcinoma of the anus § Medulloblastoma and primitive neuroectodermal tumours (PNET) § Patients with tumours with a short mass-doubling time

Answer 332

It is ADVISED that their treatment should not be prolonged by more than two days over the original prescription. Must not go above 5 days. Generally adenocarcinoma § adenocarcinoma of the breast § transitional cell carcinoma of the bladder § carcinoma of the prostate

Answer 333

These are patients being treated with palliative intent. Overall time is less critical in achieving the desired palliative outcomes. Prolonged interruptions, which may occur because of intercurrent illness, may require compensation, particularly if longer than seven days.

Answer 334

Radiobiologically speaking, a cell is ‘killed’ if it is rendered unable to divide and cause further growth.

Answer 335

Non-targeted, radiation-induced bystander effects are effects that appear in unirradiated cells in the presence of irradiated cells. Cells growing in the growth media of cells that have been irradiated demonstrate effects of radiation Ability of transfected cells to transfer death signals to neighboring tumor cells through GAP junctions e.g. reactive oxygen/nitrogen species or extracellular signalling via TGFbeta or cytokines.

Answer 336

1000ssDNA breaks and 40 dsDNA breaks

Answer 337

Neutral comic assay can only detect Double strand DNA, thus reflect the only double strand DNA breaks. Alkaline comic assay can detect both double and single stranded DNA, thus theoretically can be used to measure single strand breaks as well as double strand break. Pulse Filed Gel Electrophoresis (PFGE) can detect fragmented DNA to assess level of DNA double strand breaks. Neutral comet assay can measure DNA double strand breaks. Specific antibodies can be used to detect oxidated bases, such as 8-Oxoguanine (8-OxoG). Phosphorylated H2AX (gH2AX) can be used as a surrogate marker for DNA double strand breaks -, most accurate marker.

Answer 338

The abscopal effect is a phenomenon in the treatment of metastatic cancer where localized irradiation of a tumor causes, not only a shrinking of the irradiated tumor, but also a shrinking of tumors far from the irradiated area. Thought to be due to immune activation.

Answer 339

The lower the dose rate, the lower the cell kill even for the same total dose. This is due to increased DNA repair of sublethal DNA damage. Therefore if you inhibit DNA repair = no dose rate effect. The traditional dose-rate effect is most pronounced between dose rates of 1Gy/min and 0.3Gy/hour. Within this range, as dose rate decreases, cell killing decreases and the slope of the survival cure becomes more shallow, not steeper. The magnitude of dose-rate effect varies significantly among different cell types due to inherent differences in capacity for sublethal damage repair. High dose rate = less sublethal damage repair = the beta component of cell killing will increase. High alpha/beta ratio = beta component not very important = not much sublethal damage = no dose rate effect High LET = less sublethal DNA damage = less dose rate effect. If you go below 1cGy/min then death increases due to asynchronous cell population becomes synchronized as sensitive G2 cells: inverse dose rate effect.

Answer 340

PLDR is best demonstrated with a “delayed plating” experiment, and is operationally defined as an increase in the surviving fraction resulting from prolonged incubation of cells under non-growth conditions following irradiation. If non-cycling cells are forced to re-enter the cell cycle immediately after irradiation, rather than remaining quiescent, potentially lethal damage will be “expressed” and therefore the surviving fraction will be lower. PLD is believed to be complex double strand breaks (DSBs) that are repaired slowly as compared to simple DSBs. Therefore, cells that are left in stationary phase after irradiation display enhanced survival as they have time to repair complex DSBs before resuming progression through the cell cycle. Sublethal Damage Repair (SLDR) denotes the phenomenon that cell survival increases when irradiated with split doses compared to a single dose of irradiation. Demonstrated by a split dose experiment. Repair occurs mostly within the first hour, starting to plateau after 2h for most proliferating mammalian cells. Repair of DNA damage and rejoining of chromosome breaks presumably underlie both the sublethal and potentially lethal damage recovery

Answer 341

High LET = high RBE (until 100kev/um) = low OER = low energy = densely ionising = less sublethal damage repair = less dose rate effect = less fractionation sparing High LET particles have a higher RBE in hypoxic cells than oxygenated cells (as they are equally as effective in both cells unlike the reference radiation) High LET particles have a higher RBE in fractionated regimens than acute regimens as they don't show sparing caused by fractionation.

Answer 342

Tumor response following radiation into has been described to occur in 4 discrete stages where stage I involves massive endothelial apoptosis or at least stopping of endothelial proliferation, stage II involves tumor regression via tumor cell death, stage III involves early regrowth of vessels from remnant endothelial and tumor cells with some growth factor support from myeloid bone marrow-derived cells, and stage IV involves late recurrence due to the tumor bed effect mediated by defective neovascularization.

Answer 343

Dose fractionation increases the risk for sterility in the male; the TD5 and TD50 for sterility are 2 Gy and 8 Gy, respectively, for a single dose of X-rays, whereas these values decrease to 1 Gy and 2 Gy for fractionated irradiation. This effect results from spreading the dose over time permitting reassortment sensitization to occur for spermatogonia, which have a large variation in radiation sensitivity through the course of their cell cycle, and more than compensating for any repair that might occur between fractions. Thus, spermatogonia located in a relatively radioresistant portion of the cell cycle may progress into a more radiosensitive part of the cell cycle at the time of the second and subsequent irradiations. Spermatids and spermatozoa are relatively radioresistant, whereas spermatogonia are radiosensitive. A drop in testosterone levels would not be detectable following a scattered dose of 0.1 Gy to the testes, particularly to an adult. Following a moderate dose of radiation, which kills a large number of spermatogonia, there may be relatively little effect on the levels of spermatocytes, spermatids and spermatozoa initially, since a period of 67 days is required for maturation of a spermatogonial stem cell to a mature spermatozoan. Hence, there may be very little drop in sperm count for the first month following irradiation, although the sperm count will decrease at a later time. Full recovery of a normal sperm count following radiation-induced azoospermia caused by exposure of the testes to a dose of 6 Gy, would require a period of at least 2 years. Based on animal data, a minimum waiting period of 3-6 months is recommended for both men and women before attempting procreation following radiotherapy in order to reduce the risk of radiation-induced genetic effects. Hormone failure TD/5 is 30Gy as Leydig cells can tolerate much higher doses - these secrete testosterone. We therefore see sterility with minimal effect on libido.

Answer 344

When plotted as the log of the total dose to produce a given isoeffect as a function of the log of the dose per fraction (plotted on a reverse scale), most late responding normal tissues are characterized by steep isoeffect curves, whereas those for early responding normal tissues and most tumors tend to be shallow. They allow identification of the optimal range of fraction sizes to use for treatment based on isoeffect on tumour control probability and late normal tissue complication probability. Isoeffect curves are often plotted with the log of the total dose on the y-axis and the log of the fraction size (from high to low) on the x-axis. Tissues with a greater repair capacity will show greater sparing with increasing Fractionation (smaller fraction sizes) and therefore will have steeper isoeffect curves.

Answer 345

An exonuclease cleaves one nucleotide at a time beginning at the end of a DNA strand. Endonucleases produce nicks within intact DNA strands

Answer 346

Sterilization (different to testis – fixed number from birth - EXTREMELY RADIOSENSITIVE & IMMEDIATE) - TD5/5 2Gy Menopause (Hormonal secretion is associated with follicular maturation, sterilisation by radiation leads to a loss of libido and menopause) - TD5/5 20Gy

Answer 347

LD50/60 - radiation dose that leads to death within 60 days of 50% population. 3.5Gy without medical treatment (die of haematopoietic syndrome) 7Gy with medical care (die of GI syndrome)

Answer 348

Time course and severity of clinical features are a function of overall body volume irradiated, inhomogeneity of dose exposure, absorbed dose, dose rate and particle type. >=1Gy = prodromal reaction. 1Gy (mild)-2.5Gy (significant) = haematopoietic syndrome - time to death is 1-2 months. 5Gy (mild)-8Gy (significant) = GI syndrome = death in 3-10 days. >20Gy = cerebrovascular syndrome = death within 24-48hrs Treatment: Accidental 2Gy exposure: monitor at home, prophylactic antibiotics and monitor blood counts Accidental 3Gy exposure: haematopoietic syndrome - hospitalised in reverse air flow isolation with supportive care and observation for 2-4 weeks, prophylactic antibiotics, transfusion of blood products. Accidental 8Gy exposure: likely death due to GI syndrome. Supportive care includes symptomatic management with antiemetics, IV fluid and electrolyte replacement. Can consider bone marrow transplant if survive the GI syndrome Don't give steroids. Accidental 10Gy exposure = death.

Answer 349

Occurs mildly at 1Gy, significantly at 2.5Gy. The chronological order for decline of the components of peripheral blood after irradiation is lymphocytes, granulocytes (7-10days), platelets (20 days) and lastly erythrocytes (based on transit time so time to drop - as the cell that actually dies is the bone marrow stem cells. Death in 1-2 months with no treatment. Death due to infection and haemorrhage. Treat with antibiotics and blood product transfusions and reverse air room and supportive care.

Answer 350

Mild symptoms ats at 5Gy, significant symptoms at 8Gy, definite death at 10Gy. Nausea, vomiting, blood diarrhoea (due to depopulation of epithelial lining due to sterilisation of crypt cells). Death in 3-7 days. Small bowel paneth cells are most sensitive. Treatment is supportive e.g. antiemetics, IV fluids, IV electrolytes, psychological support. No steroids. (Note if they survive the GI syndrome then you have to manage the haematopoeitic syndrome e.g. with bone marrow transplant)

Answer 351

Occurs at 20Gy. Death within 24-48hrs - no treatment. Cerebral oedema, microvasculitis, brain necrosis.

Answer 352

Nausea, vomiting, anorexia, fatigue = sublethal dose. Fever, diarrhoea, headache, hypotension, apathy, skin erythema = supralethal dose Occurs quicker and more severe with higher doses - can get vomiting within a few minutes with high exposure - usually starts within 2-6hours. Duration of phase is inversely proportional to dose - at high doses go straight to the manifest symptoms e.g. cerebrovascular syndrome. Usually 1-3 days. Can get a latent phase with "apparent cure" after prodromal phase before the patient gets the manifest illness phase - this latent phase can last hours-weeks.

Answer 353

the minimum whole body dose that can be detected through measurement of dicentric chromosomes in peripheral blood lymphocytes is approximately 0.25 Gy. The most reliable approach to estimate dose one month following a radiation exposure is to karyotype peripheral blood lymphocytes to detect chromosomal aberrations, particularly dicentric chromosomes, which are normally not found in unirradiated people.

Answer 354

1. If the tolerance within a given tissue volume has already been exceeded during the first treatment, and loss of function is present or expected soon, then re-irradiation is not possible without loss of function. 2. For early effects, restitution of the original tolerance may be complete after low to moderate doses, and after tissue-specific and dose-dependent time intervals. At high doses, residual damage may remain for longer intervals, particularly at the stem cell level, which is not necessarily reflected by the number of differentiated cells in the functional tissue compartments (e.g. blood cell counts for bone marrow). 3. For some late-responding tissues, partial (central nervous system, lung) or complete (skin) restoration of tolerance is observed after low and moderate initial doses (60 per cent of the initial tolerance). For example, spinal cord can safely be re-irradiated to a total of 140 per cent of the initial EQD2tol. 4. In some late responding tissues (kidney, urinary bladder), progression of damage at a subclinical level must be expected, thus precluding re-irradiation without exceeding tolerance. 5. Alternative treatment options must be considered before re-irradiation. 6. If (curative) re-irradiation is to be administered, optimum treatment planning (dose conformation) and a proper choice of fractionation protocol (hyperfractionation) are required