Cancer Biology Flashcards
(182 cards)
1
Q
What is the difference between a tumor suppressor gene and an oncogene?
A
Oncogenes refer to those genes whose alterations cause gain-of-function effects, while tumour suppressor genes cause loss-of-function effects that contribute to the malignant phenotype.
2
Q
How do tumors suppressor genes drive tumours?
A
TSGs encode proteins that prevent inappropriate cell growth and division, and stimulate cell death
Both TSG alleles must be inactivated to promote tumour development (loss of function). Inactivation of 2nd copy can be mutation, silencing by methylation or loss of heterozygosity
Usually recessive at cell level but show dominant inheritance in familial cancer syndromes (2nd “hit” is somatic)
3
Q
What are the main categories of tumour suppressor genes?
A
- Gatekeeper Genes: Encode proteins that control the cell cycle (inhibit S phase and action of mitogen) and regulate cell proliferation e.g. p53, Rb, and APC
- Caretaker Genes: Maintain and protect the integrity of the genome. Involved in DNA repair and help to prevent the accumulation of mutations e.g. MLH1, MSH2 and RECQ helicases.
- Landscaper genes: create environments that control cell growth e.g. PTEN
4
Q
What mechanisms do tumour suppressor genes use to control cell growth and division?
A
Control progression within cell cycle
- includes cell cycle arrest and regulator proteins.
- RB1, TP53 and CDKN2A
Inhibit proliferation
- receptors for secreted hormones, e.g. TGF-β, suppresses c-myc expression.
Maintain the integrity of genome
- DNA repair proteins e.g. MMR
Apoptosis
- Stimulate cell death in cells deviating from normal growth, e.g. p53.
5
Q
What was the first tumour suppressor gene identified?
A
RB1
Through investigating childhood retinoblastoma
6
Q
What is retinoblastoma
A
An aggressive childhood cancer of the eye that is usually diagnosed <5yrs.
Can be unilateral (usually sporadic) or bilateral (these are always heritable).
Incidence ~1 in 15,000 to 1 in 20,000 live births. ~15% cases familial (transmitted from an affected parent)
15% of sporadic cases carry germline mutations
7
Q
What is the role of RB1?
A
RB has important roles in cell cycle progression, chromosome stability and regulation of apoptosis.
RB is hypophosphorylated in G1, binds activator E2F TFs, inhibits cell cycle progression.
pRB is phosphorylated by CDKs which inactivates pRB. E2Fs are released and activate transcription, this leads to cyclin E production and eventual entry into S phase.
8
Q
What is Knudson’s two hit hypothesis?
A
Derived investigating RB
Both alleles need to be lost requiring two hits (one to each RB gene)
Hereditary retinoblastoma:
- a mutant, loss of function germline allele is inherited (most are de novo). Later, somatic mutation inactivates 2nd allele
- This leads to variable penetrance and an apparently dominant mode of inheritance (called one-hit kinetics).
Sporadic retinoblastoma - 2 somatic mutation events must occur (two-hit kinetics).
- Second mutation may be LOH, gene silencing by promoter methylation, mutation or deletion.
9
Q
What is TP53?
A
Guardian of the genome
p53 is in the centre of a network of signalling pathways that are essential for cell growth, regulation and apoptosis - induced by genotoxic and non-genotoxic stresses e.g. oxidative stress, ribonucleotide depletion, double strand breaks, hypoxia
10
Q
What is the function of TP53?
A
Normal cells:
- p53 levels downregulated via binding of proteins such as the E3 ubiquitin ligase, MDM2.
- MDM2 binding causes p53 migration to cytoplasm and degradation via the ubiquitin/proteasome pathway.
Stressed cells:
- p53 becomes phosphorylated and acetylated.
- No longer acts with MDM2, p53 levels rise to act as a transcription factor.
- Causes increased transcription of genes such as p21 (inhibits CDKs=cell cycle arrest; allows DNA repair before mitosis) and PUMA (controls apoptosis).
11
Q
What do mutations in tp53 lead to?
A
Somatic mutations in TP53 in more tumour types than any other gene mutation (~50% of tumours); found in 23% of breast, 50% of cases of ovarian cancers, for example.
Li-Fraumeni syndrome, caused by p53 germline mutation. Results in a high incidence of cancer, particularly tumours of the adrenal cortex, breast, brain and osteosarcomas.
12
Q
What do mutations in PTEN lead to?
A
PTEN is a TP53 regulated gene
Even a subtle difference in PTEN levels and activity results in cancer susceptibility and favours tumour progression.
PTEN germ-line mutations cause PTEN hamartoma tumour syndrome and Cowden syndrome.
13
Q
What is the role of CDKN2A?
A
Encodes 2 gene products
p16INK4A : Inhibits CDK4/6, thus keeps RB dephosphorylated and bound to E2F = cell cycle arrest.
p14ARF : Destabilises MDM2 resulting in active p53 and cell cycle arrest at G1.
Inherited CDKN2A mutations mainly affect p16INK4A only e.g. familial melanoma, pancreatic cancer.
14
Q
Give some miRNAs which act as tumour suppressors
A
let-7
- Normally expressed in differentiated tissues, but frequently lost in non-small lung cancers
- let-7 negatively regulates multiple cell cycle oncogenes, such as RAS, MYC, and HMGA26–8
- Exogenous application of let-7 to human lung cancer cells reduces proliferation
miR-34 family
- Also lost in lung cancer
- Transcription is activated by p53, expression of miR-34a promotes p53 mediated apoptosis
- Acts as a TSG, represses multiple cell cycle and cell survival genes e.g. CDK4 and BCL2
- Required for a radiation response in vitro and in vivo.
15
Q
What is an oncogene?
A
A gene that is normally is involved with controlling cellular proliferation.
When altered/over-activated, oncogenes can help transform normal cells into tumour cells by promoting uncontrolled cell growth and/or inhibiting apoptosis.
16
Q
What are the three main methods of oncogene activation?
A
Point mutation
Translocation
Gene amplification
17
Q
How do point mutations drive oncogene activation?
A
Activating point mutations lead to hyper-activated protein usually produced in normal amounts
18
Q
How do RAS point mutations lead to oncogene activation?
A
HRAS, KRAS and NRAS mediate signaling by G-protein coupled receptors. Point mutations in these genes can lead to constitutive activation of downstream GTP-signaling by blocking integral GTPase activity (which acts as a molecular switch). These mutations are often identified in metastatic colorectal cancer, as well as cancers of the lung, breast and bladder.
19
Q
How does amplification lead to oncogene activation?
A
Multiple copies of growth factor or transcription factor receptors on structural wild type cell surface, which leads to over-production coding protein
e.g. HER2 in breast cancer and MYC in rhadomyosarcoma
20
Q
How do translocations lead to oncogene activation?
A
Translocation to create a novel chimeric gene
- the product of an acquired balanced translocation where a fusion gene with an oncogene is create
- e.g. BCR-ABL1in CML
Translocation into transcriptionally active region
- acquired balanced translocations between oncogene and region with high transcription regulatory elements, leading to the oncogene being upregulated in expression
- e.g. IGH-MYC in burkitt lymphoma where MYC is brought under the control of IGH promoter
21
Q
What are 5 classes of oncogenes?
A
Secreted Growth Factors
Growth Factor Receptors
Signal Transducers
Inhibitors of Apoptosis
Transcription Factors
22
Q
Give an example of a secreted growth factor oncogene
A
Constitutive activation of a growth factor gene can contribute to malignant transformation by inducing cell proliferation.
Platelet-derived growth factor (PDGF) is released from platelets during coagulation and wound healing and can induce proliferation of various adjacent cell types.
Over-expression of PDGF-b in tumour cells triggers unregulated cell growth via the RAS/PIK3/AKT/IKK/NFKB1 pathway and is involved in angiogenesis (formation of new blood vessels from existing ones)
23
Q
Give an example of a growth factor receptor oncogene
A
Epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC), activating mutations in exons 18, 19 and 21 of the EGFR gene, which code for part of the protein’s tyrosine kinase (TK) domain, cause increased kinase activity of this receptor.
Such activating mutations confer a dependence on the mutated kinase for survival of the tumour cells.
Mutations occur around the ATP-binding pocket of the receptor protein. Mutated kinases have a lower affinity for ATP that increases their sensitivity to selective EGFR-TKIs, which compete with ATP for binding to the catalytic site.
24
Q
Give an example of a signal transducer oncogene
A
PIK3CA acts as an oncogene by encoding a mutated, constitutively active form of the PI3K enzyme, leading to continuous activation of the PI3K/AKT/mTOR pathway, which promotes uncontrolled cell proliferation and survival. This persistent signaling supports cancer development by enhancing cell division, inhibiting apoptosis, and reprogramming cellular metabolism.
25
Give an example of an inhibitor of apoptosis oncogene
The BCL2 gene encodes a cytoplasmic protein that localises to the mitochondria and inhibits apoptosis.
Over-expression of BCL2 protein is involved in the initiation of almost all follicular lymphomas and some diffuse large B cell lymphomas.
In follicular lymphoma a t(14;18) translocation commonly occurs which places the BCL2 gene next to the immunoglobulin (Ig) heavy chain locus. This leads to the transcription of excessively high levels of BCL2, decreasing the propensity of these cells to undergo apoptosis.
26
How does is apoptosis triggered?
Two main pathways (stress response and death) lead to apoptosis:
The stress pathway (or intrinsic cell death pathway) is triggered by proteins containing the BCL2 homology 3 domain which inactivate BCL-XL and BCL2 (these normally inhibit apoptosis). This removal of suppression activates the caspases that induce apoptosis.
The death receptor pathway (or extrinsic pathway) is activated by binding of Fas ligand, TRAIL and Tumour Necrosis Factor a to receptors on the cell surface. This leads to activation of caspases and cell death.
27
Give an example of a transcription factors oncogene
EWSR1
Ewings sarcoma (tumours in the bones and soft tissues) occurs as a result of the translocation t(11;22)(q24;q12) which results in the translocation of the 3’ end of the Friend leukaemia integration 1 TF (Fli1) on chromosome 11 to the 5’ end of the EWS gene on chromosome 22 (80% cases).
The resulting novel chimeric oncoprotein produced, EWS/Fli1, acts as an aberrant transcription factor with strong transforming capabilities.
28
What are the stages of the cell cycle?
G0
Interphase
- G1
- S
- G2
Cell division
- Mitosis
29
What is G0 phase?
A resting phase where the cell has left the cycle and has stopped dividing. These cells are not dormant and often actively secrete proteins, may be highly mobile and can continue growing. In response to certain stimuli, these cells can re-join the cell cycle.
30
What is G1 phase?
Growth phase during which proteins and RNA are synthesised. Each chromosome exists as a single double stranded helix - at no point is DNA synthesised in this phase.
At the G1 checkpoint - the restriction point - the cell is committed to division and moves into the S phase
31
What is S phase?
DNA synthesis replicates the genetic material. Each chromosome now consists of two sister chromatids.
32
What is G2 phase?
Cell continues to grow. The G2 checkpoint ensures enough cytoplasmic materials necessary for mitosis and cytokinesis.
33
What is M phase?
The cell stops growing. Nuclear division (mitosis) followed by a cell division (cytokinesis). The Metaphase checkpoint in the middle of mitosis ensures that the cell is ready to complete cell division.
34
What are cell cycle checkpoints?
Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions ensuring critical evens are completed with high fidelity
35
What proteins regulate the cell cycle?
The cell cycle is regulated by heterodimeric protein kinases composed of:
Cyclins - these form the regulatory subunit and have no catalytic activity.
Cyclin-dependent kinases (CDKs) - proteins which are inactive in the absence of a partner cyclin. They are the catalytic subunit of an activated heterodimer which phosphorylates target proteins to coordinate entry into the next phase of the cell cycle.
CDKs are constitutively expressed whereas Cyclins are synthesised at specific stages in response to various external stimuli / molecular signals.
36
What are the cell cycle checkpoints and why are they needed?
Cell examines queues and decides whether to move forward. 3 main ones:
G1 checkpoint, at the G1/S transition
2. G2 checkpoint, at the G2/M transition
M checkpoint, also called Spindle checkpoint, at the Metaphase/Anaphase transition
Failure to activate checkpoints allows cells to divide when there is DNA damage or when chromosomes are incorrectly placed, thus causing genome instability
37
What is the G1 checkpoint?
G1 checkpoint
- Main decision point; once G1 checkpoint is passed and S phase entered, the cell becomes irreversibly committed to division
- Factors: DNA integrity, molecular signals, nutrients, cell size
- Cell growth enables CDK-cyclin D formation
- Phosphorylates retinoblastoma protein
- Relieves inhibition of E2F transcription factor
- Cyclin E now expressed, binds to CDK2
- Allows G1-S phase transition
38
What is the G2 checkpoint?
CDK1 is activated by phosphorylation and de-phosphorylation of specific amino acid residues by Cyclin-Activating Kinase (CAK) and the wee1 protein
Enables CDK1-cyclin B formation (aka MPF)
Allows G2-M phase transition
39
What is the M checkpoint?
Chromosomes assemble on metaphase plate
Cell examines whether sister chromatids correctly attached to spindle microtubules
Anaphase-promoting complex (APC) activated
Degrades cyclin B = MPF disassembly
Relieves inhibition of ‘separase’ (a cysteine protease) = spindle cut
Sister chromatid separation = anaphase entry
40
How can the cell cycle lead to tumourigenesis?
A negative control on cell cycle progression plays an important role in preventing tumorigenesis.
The arrest of cell proliferation in many cases takes place when integrity of the genome has been compromised. Failure to arrest results in the release of cells with highly unstable genomes, which could evolve into cancer cells.
41
What is the function of cyclin D1?
Cyclin D1 is a growth factor-responsive cyclin that plays an important role in regulating entry into and progression through G1. Deregulated expression of cyclin D1 could increase cyclin D1/CDK4 activity and drive transit of the checkpoint even in the absence of appropriate growth factors. Cyclin D1 expression can be induced by the Ras and PI3 kinase signalling pathways.
42
What is the role of p53 in the cell cycle?
it stops the cell cycle at the G1 checkpoint by triggering production of CDK inhibitor (CKI) proteins. The CKI proteins bind to CDK-cyclin complexes and block their activity, buying time for DNA repair. p53’s second job is to activate DNA repair enzymes. If DNA is not fixable, p53 will play its third role: triggering programmed cell death.
In normal cells, p53 is kept at low levels by the protein murine double minute 2 (MDM2), an ubiquitin ligase. MDM2 and p53 form a negative feedback loop, in which p53 induces the expression of MDM2, which in turn promotes the degradation of p53 and quenches cellular p53 activity.
43
What is the role of RB1 in cell cycle?
Active, dephosphorylated pRb binds and inactivates the cellular transcription factor E2F1, function of which is required for cell cycle progression. The G1/S checkpoint seems to be the most crucial for the cell cycle; 2-4 hours before the cell enters S-phase, pRB is phosphorylated. This releases the inhibition of E2F1 and allows the cells to proceed to S phase
44
What is the role of CDKN2A in the cell cycle?
Exons 1α, 2 and 3 encode the CDKN2A (p16INK4A) protein.
- CDKs inactivate pRB by phosphorylation, but CDKN2A inhibits the kinases. Thus, the loss of CDKN2A function leads to the loss of RB1 function and inappropriate cell cycling.
Exon 1β is spliced on to exons 2 and 3, but the reading frame is shifted, protein ARF (p19ARF) is encoded.
- ARF mediates G1 arrest by destabilising MDM2 and therefore ARF acts to maintain the level of P53. Loss of ARF function leads to excessive levels of MDM2, excessive destruction of P53, and loss of cell cycle control.
45
How does inactivation of CDKN2A drive tumourigensis?
Homozygous deletion of the CDKN2A gene inactivates both the RB1 and the p53 arms of the cell cycle control, and is a very common event in the development of many tumours (e.g. mesothelioma).
46
How can MDM2 be targeted therapeutically?
Blocking MDM2 expression – limits its interaction with p53, therefore preventing p53 degradation and resulting in higher levels of p53 in cells.
Inhibiting MDM2-p53 binding
- p53-MDM2 interaction has been demonstrated to involve only three amino acid residues which are inserted into deep hydrophobic pockets on the surface of the MDM2 protein- small molecules being investigated
47
When in the cell cycle can structural abnormalities be assessed by karyotyping?
Metaphase
48
Give an example of a structural rearrangement of cyclin D1 (CCND1) (genes and chromosomal abnormality) and the associated disease.
IGH::CCND1 t(11;14) – Myeloma or Mantle cell lymphoma or other
49
What are the three main growth factor signaling pathways?
RAS/MAPK, PI3K/AKT or JAK/STAT
50
What are receptor tyrosine kinases?
Cell surface receptors activated by ligand binding (generally GF)
Ligand binding triggers dimerization of receptors, which activates intracellular tyrosine kinase domains.
Intracellular TK domains phosphorylate their own tyrosine residues = activation of downstream signalling.
Examples include ALK, ROS, EGFR, HER2, KIT, PDGFRA, FLT3 and TRK
51
What is the structure and function of EGFR?
Structure: extracellular ligand binding domain, short hydrophobic transmembrane region and intracytoplasmic TK domain
Important role in regulating various cellular functions such as proliferation, motility and differentiation
Ligand binding causes dimerization, followed by autophosphorylation and activation of downstream signalling pathways
52
What is the role of RAS/MAPK signaling?
Key signalling pathway that regulates a wide variety of cellular processes, including proliferation, differentiation, apoptosis and stress responses
RAS mutations are detected in approximately 30% of all tumours
Mutations in RAS or RAF result in constitutively activated MAPK pathway, leading to uncontrolled cell proliferation and resistance to apoptosis-inducing drugs
53
Outline the RAS/MAPK pathway
Activated via two pathways:
- a ligand-dependent pathway e.g. EGFR
- a ligand-independent pathway, physical stressor, such as radiation, injury,
Activation of a receptor tyrosine kinase triggers phosphorylation of RAS (KRAS, NRAS or HRAS) which is a GTPase. Phosphorylation with adaptor proteins GRB2 result in inactivate RAS-GDP turning to active RAS-GTP.
This activates RAF (BRAF, ARAF or CRAF) which phosphorylates MEK and then ERK which are imported into the nucleus and activates transcription factors for proliferation, survival, apoptosis
54
Why are RAS proteins difficult to target therapuetically?
Structure or RAS- lack of well-defined druggable nooks and cavities on the RAS surface
Sortorasib targeting KRAS G12C is the first approved drug- NSCLC
55
What is the PIK3/AKT pathway?
Highly conserved pathway involved in cell survival, growth and proliferation.
Two major functional proteins: PI3K (plasma membrane-associated lipid kinases) and AKT
Pathway alterations occur in 50% of tumors and is the primary mechanism causing cancer cells to develop radiation resistance
56
Outline the PIK3/AKT pathway
PI3K-alpha (PI3Kα) is a heterodimeric protein complex
Activation of a receptor tyrosine kinase (RTK) triggers PI3K-mediated phosphorylation of PIP2 to PIP3.
PIP3 activates PDK1 which in turn activates AKT
Activated AKT phosphorylates TSC2, which is a negative regulator of mTOR and leads to downstream mitogenic signalling, MYC being one of these targets
PI3K requires multiple inputs for full activation, including binding by membrane-bound RTKs and Ras
PTEN negatively regulates this process
57
What is JAK/STAT pathway?
JAK-STAT pathway is essential for a wide of critical cellular events such as haematopoiesis, lactation, development of immune systems and mammary glands
Major role in transferring signals from cell-membrane receptors to nucleus
50-95% patients with myeloproliferative neoplasms (MPNs) have an activating mutation in JAK2 in the malignant clone
58
Outline the JAK/STAT pathway
Ligand binding triggers receptor dimerization- phosphorylation of specific tyrosine residues that activates JAK tyrosine kinase domain.
Active JAKs recruit signal transducers and activators of transcriptions (STATs)
The STATs form dimers that translocate to the nucleus. The STAT dimers bind specific promoter sequences and modulate transcription of genes controlling cellular processes including proliferation, differentiation and apoptosis
59
What is NOTCH signalling?
Notch signalling cascade is critical for development (eg. neurogenesis, development of haemotopoietic stem cells, B-cells an T-cells), differentiation, proliferatiom and homeostasis.
4 receptors: NOTCH 1-4
Mutations in Notch1 were detected in more than 50% of T-cell acute lymphoblastic leukaemias (T-ALL).
activating NOTCH1 mutations in 8-12% of cases - primarily found in patients with the more clinically aggressive nonmutated IGV(H) subtype
60
Outline the NOTCH pathway
Ligand binding promotes two proteolytic cleavage events in the Notch receptor.
The first cleavage is catalysed by ADAM-family metalloproteases, whereas the second is mediated by γ-secretase, an enzyme, PEN2 and APH1.
The second cleavage releases the Notch intracellular domain (Nicd), which then translocates to the nucleus and cooperates with CSL and its coactivator Mam to promote transcription.
61
What is the Wnt/B-catenin?
Highly conserved pathway essential for development and one of the most commonly dysregulated associated with tumorigenesis.
The Wnt pathway is commonly divided into β-catenin dependent (canonical) and independent (non-canonical) signaling.
Loss of APC is the main driver of Wnt signaling in colorectal cancer
62
What is the canonical WNT signalling pathway?
Upon binding of secreted Wnt ligands to Fzd receptors and LRP co-receptors, the destruction complex is inactivated and accumulation of β-catenin which then translocates into the nucleus.
There, β-catenin forms an active complex with LEF and TCF proteins by displacing TLE/Groucho complexes and recruitment of histone modifying co-activators
In the absence of Wnt ligands, β-catenin is phosphorylated by the destruction complex which contains the scaffold protein Axin, APC and the kinases GSK3β and CK1α. β-catenin is then ubiquitinated by β-TrC and targeted for proteasomal degradation, leading to repression of target genes.
63
What is HH signalling?
Plays an important role in the embryonic development - mostly inactive or poorly active in the adult organism
involved in the maintenance of somatic stem cells and pluripotent cells
Deregulation of the Hh signaling pathway is associated with developmental anomalies and cancer e.g. PTCH1 and gorlin syndrome, and sporadic cancers, BCC, medulloblastomas
64
Outline the HH pathway
Hh signalling is activated by binding of Hedgehog ligand to Ptch1. HH-Ptch1 complex is internalised and degraded by lysosome. This relieves Smo inhibition and Smo becomes phosphorylated by PKA and CK1.
The inhibitory effect of Sufu is removed and Gli activator is formed. The activator form of Gli travels to the nucleus and stimulates the transcription of the target genes by binding to their promoters.
65
What is TGF-B/SMAD pathway?
TGF-β/SMAD4 signaling pathway controls the signal transduction from cell membrane to nucleus, and is responsible for a wide range of cellular processes, including proliferation, differentiation, apoptosis, migration, as well as cancer initiation and progression
Dual role in cancer: TGF-β inhibits cell proliferation and stimulates differentiation in normal cells, thus acting as a tumor-suppressor factor. However,iIn advanced cancer TGF-β acts as an oncogene by inducing tumour progression and metastasis
66
Outline the TGF-B/SMAD pathway?
TGF-β binds to receptors at the cell surface, forming a bi-dimeric receptor complex and causes activation of TGF-β receptor transmembrane dual specificity kinase.
Upon ligand binding the type II receptor phosphorylates serine and threonine residues in the type I receptor, which subsequently propagates the signal through activation of Smad family of TFs.
67
What is NF-kB?
comprises a family of five transcription factors that functions to regulate expression of genes involved in proliferation, apoptosis, inflammation and immune response. Required at low level for normal haematopoiesis.
Constitutive NF-κB activity is found in many cancers due to inflammatory microenvironment and oncogenic variants – promotes tumour cell proliferation, suppresses apoptosis, attracts angiogenesis, induces EMT and facilitates distant metastasis.
Common in NHL
68
Outline the NF-kB pathwat
NF-κB can be activated by various stimuli, such as cytokines, growth factors UV and ionizing radiation, reactive oxygen species (ROS), and DNA damage and oncogenic stress from inside the cells.
Lead to the activation of a large cytoplasmic protein complex.
The activated IKK complex is responsible for the phosphorylation of IκB, marking it for degradation by the E3 ubiquitin ligase.
Following proteosomal degradation, the free NF-κB dimers can translocate from the cytoplasm to the nucleus, bind to DNA and regulate gene transcription.
69
How does DNA damage relate to cancer development?
DNA damage has been long recognized as a causal factor for cancer development.
Every cell experiences up to 10^5 spontaneous or induced DNA lesions per day.
When erroneous DNA repair leads to mutations or chromosomal aberrations affecting oncogenes and tumour suppressor genes, cells undergo malignant transformation resulting in cancerous growth
70
What are the causes of DNA damage?
Chemical carcinogens
Radiation
Oxidative stress
Replication errors
Viruses and oncogenic pathways
71
How do chemical carcinogens lead to DNA damage?
- They can be divided into two main categories: genotoxic and non-genotoxic.
- Genotoxic carcinogens directly interact with DNA, leading to alterations such as base modifications, cross-linking, and strand breaks.
- Non-genotoxic carcinogens induce cancer through mechanisms not directly related to DNA damage but may still lead to genomic instability and tumorigenesis.
72
How does radiation lead to DNA damage?
Ionizing radiation, such as X-rays, gamma rays, and certain types of ultraviolet (UV) radiation, can directly damage DNA by generating reactive oxygen species (ROS) and causing breaks in the DNA backbone.
- UV radiation primarily induces formation of cyclobutane pyrimidine dimers (CPDs), leading to mutations if not repaired properly.
73
How does oxidative stress lead to DNA damage?
Oxidative stress results from an imbalance between the production of reactive oxygen species (ROS) and the body's antioxidant defences. causing base modifications, single-strand and double strand breaks
- Mitochondria, the primary site of ROS production within cells
- Chronic oxidative stress, often associated with inflammation and metabolic disorders, can lead to DNA damage accumulation and increase the risk of cancer development.
74
How do replication errors led to DNA damage?
- Replication errors can result in base substitutions, insertions, deletions, and replication slippage, leading to mutations and genomic instability.
- Mismatch repair (MMR) and proofreading by DNA polymerases help correct replication errors, but deficiencies in these mechanisms can increase the risk of cancer by allowing the accumulation of mutations over time.
75
How can viruses lead to DNA damage?
- Certain viruses, such as HPV, EBV, and hepatitis B and C viruses HBV, HCV, can integrate their DNA into the host genome and disrupt cellular pathways involved in DNA damage repair and cell cycle regulation.
- Viral oncoproteins produced during infection can interfere with tumor suppressor proteins (e.g., p53 and RB), promote cell proliferation, and inhibit apoptosis, contributing to malignant transformation.
76
What are the main DNA repair mechanisms?
Direct Reversal Repair
Nucleotide excision repair (NER)
Mismatch repair (MMR)
Non-Homologous End Joining (NHEJ)
Homologous recombination (HR)
Translesion Synthesis (TLS)
77
What is direct reversal repair?
Correct DNA lesions by reverting the damage to its original form, without the need for nucleotide excision or synthesis.
Examples include photoreactivation, which employs photolyase enzymes to reverse UV-induced thymine dimers, and MGMT, which removes alkyl groups from guanine residues.
78
How does MGMT relate to cancers?
Silencing or inactivation of MGMT due to epigenetic modifications or promoter methylation is frequently observed in various cancers, including glioblastoma multiforme (GBM) and colorectal cancer.
Loss of MGMT function confers resistance to alkylating chemotherapeutic agents, such as temozolomide, thereby compromising treatment efficacy and contributing to tumor progression and therapeutic resistance.
79
What is base excision repair?
Single-strand breaks and base damage by excising and replacing damaged bases.
Subsequent steps involve AP endonuclease activity, DNA polymerase β-mediated gap filling, and ligation by DNA ligase.
80
What is nucleotide excision repair?
Repair of bulky DNA lesions caused by factors like UV radiation or chemical crosslinks by removing and replacing stretches of damaged DNA
Two distinct sub-pathways:
global genomic NER (GG-NER) - initiates with lesion recognition by a multiprotein complex comprising XPC and RAD23B
transcription-coupled NER (TC-NER) - initiated when RNA polymerase encounters a lesion during transcription.
81
What are mutations in the NER pathway associated?
Implicated in various hereditary cancer predisposition syndromes, such as xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD).
82
What is the MMR pathway?
The DNA MMR apparatus recognizes errors that elude the proofreading function of DNA polymerase.
The MSH2-MSH6 (MutSα) complex preferentially repairs single base mismatch or mononucleotide repeats.
The MSH-2-MSH3 complex (MutSβ) preferentially recognises larger loop out errors such as dinucleotide repeats.
MutS heterodimers signal the site for mispairing to MLH1-PMS2 (MutL). MutL has endonucelease activity which targted DNA between the mismatch and an adjacent nick to be excised by exonuclease 1 (EXO1) and the excised strand is re-synthesised and repaired by DNA polymerase β
83
What are dysfunctions in MMR assocaited with?
Lynch syndrome
- Mutations in key MMR genes, such as MLH1, MSH2, MSH6, and PMS2, predispose individuals to a heightened risk of developing colorectal, endometrial, ovarian, and other cancers.
84
What is homologous recombination?
Homologous recombination repairs double stranded breaks and is utilized during the S and G2 phases of the cell cycle, when a sister chromatid or homologous chromosome is available as a template for repair.
BRCA1 mediates resection of the DSB ends, generating 3'-overhangs
This recruits BRCA2, PALB2 and RAD51 to the DSB. BRCA2 and PABL2 stabilise RAD51 which mediates the 3’ overhand invading the homologous chromosome
DNA synthesis then occurs, using the intact homologous DNA strand as a template,
Following DNA synthesis, the newly synthesized strand undergoes branch migration and Holliday junction resolution, resulting in the formation of a repaired DNA duplex identical to the intact template.
85
What is Non-Homologous End Joining (NHEJ)?
Non-homologous end joining represents a rapid but error-prone DSB repair mechanism employed throughout the cell cycle, particularly in the G1 phase when sister chromatids are unavailable.
NHEJ involves the direct ligation of broken DNA ends, often resulting in the loss or addition of nucleotides at the repair junction.
86
What is Translesion Synthesis (TLS) ?
TLS serves as a last resort mechanism to bypass DNA lesions that cannot be repaired by high-fidelity pathways
Specialized DNA polymerases, such as Pol η, Pol ι, and Pol κ, can replicate across damaged bases, albeit with reduced fidelity, allowing DNA synthesis to proceed at the expense of introducing mutations.
87
What is Personalised Medicine?
a targeted approach to the prevention, diagnosis and treatment of disease based on an individual’s specific profile’. The main aim of PM is to implement/use measures/technologies to treat the right patient (meaning correctly diagnosed) with the right medicine at the right time.
88
What is Stratified Medicine?
defines sub-populations (a group or proportion of patients) according to which disease sub-type an individual has been diagnosed with.
Stratification identifies patients with distinct mechanisms of disease, or the likelihood of particular responses to treatment, allowing the identification and development of treatments that are effective for specific groups of patients.
89
What are the two main types of targeted therapies?
monoclonal antibodies (mAbs) and small molecule kinase inhibitors (SMKIs).
90
What are small molecule inhibitors?
Small-molecule inhibitors which end with the stem “-ib”, are usually ≤500 Da in size, allowing translocation through the plasma membrane to interact with the cytoplasmic domain of cell-surface receptors or intracellular signalling molecules.
Most small-molecule inhibitors have been designed to interfere with enzymes, most notably the receptor tyrosine kinases (RTKs).
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How do small molecule kinase inhibitors block enzymatic activity?
Type I: bind the ATP pocket of the acitve confirmation
Type II: bind the ATP pocket of the inacitve confirmation
Type III: Bind to allosteric site
Type V: bivalent binding 2 positions
Type VI: covalent inhibtors
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What are monoclonal antibodies?
Antibodies that are specific to a single antigen.
Used in the treatment of many diseases, including cancer.
These can be recognised by the stem “-mab”, with a further sub-stem designating the source of the compound, e.g., “-mumab” for fully human antibodies.
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What is the structure of an antibody?
Antibodies all have the same basic structure consisting of two heavy and two light chains forming two Fab arms containing identical domains at either end attached by a flexible hinge region to the stem of the antibody, the Fc domain, giving the classical ‘Y’ shape.
The Fab domains consist of two variable and two constant domains, with the two variable domains making up the variable fragment (Fv), which provides the antigen specificity of the antibody, with the constant domains acting as a structural framework.
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How to monoclonal antibodies work?
While one part of the antibody, the antigen binding fragment (Fab), recognises the antigen, the other part of the antibody, known as the crystallizable fragment (Fc), interacts with other elements of the immune system, such as phagocytes or components of the complement pathway, to promote removal of the antigen.
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What are the types of mABs?
There are several types of mAbs, including naked, conjugated, and biphasic. The most common of these are the naked-mAbs, which do not have an attached drug or radioactive agent
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What is an advantage of mABs?
bind with high specificity, interacting with one small region on the target
Small molecules have more promiscuous binding as enzymatic active sites are frequently similar
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What is an example of a mAB?
Cetuximab
use for the treatment of metastatic CRC, metastatic NSCLC, and head and neck cancer.
binds the EGF ligand-binding site, thereby preventing dimerization; inducing receptor internalization, downregulation and degradation; inhibiting cell cycle progression through the G0/G1 phase; and increasing expression of pro-apoptotic proteins.
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What is the Cancer Research UK (CRUK) Stratified Medicine Programme?
An £18 million partnership – between the government, the NHS, pharma companies and CRUK – to streamline and standardise genetic testing of tumour samples across the UK’s health service.
Phase 2 (SMP2) between 2015 and 202- creating a national genetic pre-screening programme and advancing treatment for people with late stage non-small cell lung cancer (NSCLC). It was a collaboration between Cancer Research UK, the NHS and two drug companies—AstraZeneca and Pfizer.
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What are the benefits of stratified medicine?
Safer more effective medicines
Reduced chance of adverse events
More cost effective due to less sider effects and improved response
More accurate prognoses
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What is pharmacogenetics?
Pharmacogenetics represents a study of individual gene-drug interactions, usually one or two genes that have dominant effect on a drug response (SIMPLE relationship).
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What is pharmacogenomics?
Pharmacogenomics is genome wide analysis of genetic determinants of drug efficacy and toxicity. Pharmacogenomics represents a study of genomic influence on drug response, often using high-throughput data - sequencing, SNP array, expression, proteomics (COMPLEX interactions).
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Why do drugs work differently in different people due to genetics?
Drug activation
- Many drugs that treat cancer need to be activated by enzymes in order to work. Each person carries different variants in the genes that affect how fast a drug changes into its active form. This may mean standard dose of treatment may not work as well.
Drug deactivation
- Drugs also need to be deactivated by enzymes to limit the drug’s exposure to healthy tissues. Some variants cause enzymes to work more slowly and as a result, high levels of the drug may remain in their bodies for a long time. These individuals may therefore experience more side effects from the drug.
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What is pharmacokinetics and what impacts it?
How much drug is needed
Encompasses:
Absorption into the blood stream after administration (unless IV)
Distribution of the drug and its availability at the target site
Metabolism of the drug, potentially to the/another pharmacologically active form
Excretion/elimination of the drug from the body
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What is pharmacodynamics and what impacts it?
Considers the effect of a drug on its target
SNPs in alleles in affecting metabolic enzymes, drug transport genes, tumour hallmark genes, drug toxic genes
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What is immunopharmacogenomics?
uses NGS-based analysis of T- and B-cell receptors, and HLA analysis, to determine not only the molecular mechanisms of disease and immune response but also the associated drug responses.
Specific HLA alleles may increase susceptibility to immune-mediated adverse responses
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What is the clinical utility of pharmacogenomics?
greater efficacy of the drug, fewer side effects/adverse drug reactions (ADRs), greater tolerance of therapeutic regimens, reduced dependence, better compliance and overall improved patient quality of life
fewer and shorter hospital admissions (and reduction in associated nosocomial infections), fewer mortalities, less demand for cross-speciality care, improved disease management and monitoring, reduced cost
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What is DPYD?
DPYD is essential for metabolism of fluoropyrimidines, commonly used in treatment of colorectal cancer, such as 5-fluorouracil, capecitabine and tegafur.
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What is the significance of DPYD variants?
DPYD variants may confer poor or intermediate metabolism of the drug and, as such, dose-management is critical to preventing toxicity that can be severe and life-threatening.
Heterozygotes are often asymptomatic until challenged (treated), however homozygotes may present a host of symptoms including epilepsy, motor retardation and increased urinary levels of uracil and thymine.
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How do fluorouracil (5-FU) chemotherapies work?
Fluorouracils work as antimetabolites, which prevent cell proliferation. They primarily inhibit the enzyme thymidylate synthase blocking the thymidine formation required for DNA synthesis. DNA therefore cannot be repaired and replicated and so cells can no longer proliferate
After administration, a small proportion (around 5%) of the 5-FU is converted intracellularly to the active, cytotoxic enzymes. But the majority of the administered dose is converted into an inactive metabolite by the DPD enzyme
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What are the affects of DPD efficiency on 5-FU?
12 to 30% of patients who are given this treatment suffer from severe toxicity. This can cause diarrhoea, vomiting, cardiac problems, neurological problems and in some cases fatality.
One of the main reasons as to why many patients were suffering with toxicity was due to DPD deficiency.
Patients with DPD deficiency have reduced inactivation of 5-FU, thereby increasing their exposure to active metabolites and these individuals therefore have an increased risk of severe or even fatal toxicity.
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What variants are seen in DPYD?
The majority of variants within this gene are not known to affect protein function. However, four specific variants have been identified that are known to affect protein function and lead to an increased risk of toxicity to 5-FU
One of the variants is a haplotype ( 3 variants seen together in cis)
HapB3
1905+1G>A
I560S
D949V
These give a DPD activity level of 0-0.5 (when normal is 1)
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What is Clinical Pharmacogenetics Implementation Consortium?
CPIC is an international consortium of professionals addressing the barriers to implementing routine pharmacogenetic testing and its translation into drug delivery.
Provide guidelines for pharmogenomics
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What do CPIC guidelines say about the dosage of 5-FU when DPYD variants are present?
The total DPD activity value is then categorised into a normal, intermediate or poor metaboliser.
Current guidelines for poor metabasolisers is an alternatice treated.
Intermediate metabolisers should have dosage reduced to 50% of the standard dose. Dose is then titrated based on toxicity.
If there is no toxicity in the first two cycles the dose should be increased to maintain efficacy. If the starting dose is not tolerated the dose should be reduced in order to minimise toxicity.
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What testing is done for DPYD?
All patients who will be treated with 5-FU are eligible for germline screening of the 4 variants prior to treatment
TaT of 5 days
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What is Irinotecan?
A topoisomerase I inhibitor used in the treatment for colorectal and small cell lung cancer, severe side effects such as neutropenia are observed in 30-45% of patients and strongly associated with UGT1A1 genotype.
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What is the role of UG1TA1?
UGT1A1 present in the liver metabolizes SN-38 by glucuronidation to produce SN-38 glucuronide, which is removed in the bile and urine.
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How does UG1TA1 impact treatment with irinotecan?
UGT1A1 inactivates SN-38, the active metabolite of irinotecan and certain genotypes (*28/*28, UGT1A1*93) are predictive of toxicity- abnormal dinucleotide repeat sequences within the TATA box of the UGT1A1 gene promoter.
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How to UGT1A1 variants vary between populations?
TA)n TATA promoter polymorphisms are more frequent in Caucasians than in Asians which have more missense polymorphisms in the exons.
Inter-ethnic variation may limit predictive efficacy with targeted testing
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What is tamoxifen?
Routinely used in ER+ breast cancer
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What is CYP2D6?
One of the cytochrome P450 (CYP) drug-metabolising enzymes found in liver
Metabolizes multiple drugs including Tamoxifen
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How do CYP2D6 impact tamoxifen?
CYP2D6*4, *5, *10 and *41 as associated with impaired CYP2D6 activity and increased likelihood of adverse outcomes.
Poor metabolizer genotypes can alter treatment actions but the UK MHRA does not recommend routine testing
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What are thipurines (6-MP) and how do they work?
Use in organ transplants, autoimmune disease (IBD, rheumatoid arthritis) and ALL
6-MP is an oral prodrug that is metabolically converted to active cytotoxic thioguanine nucleotides (TGN). TGNs are incorporated into DNA instead of guanine nucleotides, leading to random methylation and base pair mismatching, resulting in double strand breaks and instability that triggers cell cycle arrest and apoptosis.
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What is TMPT?
Inactive thiopurines (6-MP)
TPMT catalyses S-methylation of 6-MP, so generating inactive methyl-mercaptopurine and resulting in less parent drug availability for anabolism to active TGNs.
myelotoxicity and leucopenia has been observed in TPMT-deficient patients
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How do TPMT variants vary in differnt populations
TPMT*3C common in Asian populations and TPMT*3A more common in Caucasians,
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Is TPMT tested routinely?
While genotyping has valid utility, phenotypic analysis of enzyme activity has also shown benefit in identifying potential for rare variants.
The UK widely adopts phenotypic assessment and it is this that is the determination of drug response. CPIC guidelines suggest that genotyping results should be used in accordance with phenotype guidelines
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How common is TPMT deficiency?
1 in 300 people are TPMT deficient; 11% have intermediate and 89% high enzyme activity. Patients with lower activity are far more likely to get an Adverse Drug Response at standard dose levels.
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What is NUDT15?
NUDT15 catalyses the conversion of cytotoxic and active metabolite T(d)GTP to less toxic T(d)GMP.
Variants result in reduced NUDT15-mediated degradation of TMTP and result in more available for incorporation into DNA, leading to DNA damage and apoptosis.
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What NUDT15 variants are common?
NUDT15 poor metaboliser variant alleles are less common than TPMT in certain populations, with <1% in people of African or European ethnicity.
However, this is increased to 2% in Asian and Hispanic populations. T
he most common poor metaboliser variant is NUDT15*3, c.415C>T, p.(Arg139Cys) and it is recommended that this variant is tested for before commencement of 6-MP therapy.
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What are some of the challenges of pharmacogenomic testing?
Non-genetic factors also affect drug response, such as age, height, body mass, indication for therapy and disease state
Limit the efficacy of single-gene or -target analysis.
Lack ethnic diversity in databases
Genotype-phenotype association data on clinical outcomes is often inconsistent
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What have the challenges of the implementation of pharmacogenomics been?
Many clinicians do not believe that pharmacogenetic tests have benefited their patients.
Lack of consistent clinical guidelines and limited awareness have led to reduced confidence
Commercial enterprises such as 23andme have developed pharmacogenetics assays that cover common alleles in a number of implicated genes- no clinical direction, no genetic support and may provide a false confidence in results i
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What is the major histocompatibility complex (MHC)?
Term used to describe a group of genes (including the human leukocyte antigen (HLA) genes) that encode cell surface proteins essential for the immune system.
Their primary function is to provide protection against pathogens. This is achieved through sophisticated pathways in which MHC class I molecules present endogenous antigens to CD8+ T cells and class II molecules present exogenous antigens to CD4+ T cells.
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What are HLA Class I?
HLA class I molecules are heterodimers composed of a polymorphic α chain of 45 kDa and a monomorphic β2 microglobulin of 12 kDa.
The α chain has 3 hypervariable Ig-like domains: two of these domains form the antigen-binding groove, and the third contacts the CD8 co-receptor on T cells and decides what peptides it binds to.
They are are expressed on all healthy nucleated cells and they present endogenous antigens to CD8+ T cells.
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What are HLA class II?
HLA class II molecules areexpressed on professional antigen-presenting cells like B cells, macrophages, and dendritic cells. They present extracellular antigens to CD4+ T cells.
Formed of α and β chains, each with two domains, forming a heterodimer.
Their expression can be upregulated by proinflammatory cytokines such as IFNγ and TNFα.
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Where is the human MHC located?
Contains hundred of genes including HLA gene
The human MHC is located on the short arm of chromosome 6 (6p21.3).
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What are HLA antigens?
HLA antigens are expressed on the surface of many cells and play a major role in self-recognition, evoking the immune response to an antigenic stimulus, and to the orchestration of cellular and humoral immunity.
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How are HLA genes inherited?
The HLA genes follow the principles of Mendelian genetics and the encoded antigens are co-dominantly expressed on the cell surface. In the absence of a recombination event, HLA genes are normally inherited en bloc from each parent due to their close proximity resulting in their close physical linkage. This results in 25% probability for two siblings to be genotypically HLA identical,
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What does the term HLA haplotype refer to?
An HLA haplotype is a combination of linked HLA genes transmitted on a single parental chromosome. (HLA-A, -B,-C,-DR, -DQ,-DP)
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What genes encode HLA class I?
The class I region contains the highly polymorphic ‘classical’ class I genes HLA-A, HLA-B, and HLA-C and two clusters of non-classical class I genes including HLA-E, -F, -G, -J, -X, and the MHC class I polypeptide-related (MIC) genes MICA, MICB, MICC, MICD, and MICE.
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What genes encode HLA class II?
The class II region contains genes encoding the HLA class II molecules: HLA-DP, DQ, and DR.
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What part of the HLA molecule determines peptide binding?
Based on the interaction that occurs between the amino acid residues of both the MHC groove and of the peptides. The groove of the HLA molecules contains different pockets, determining affinity for a specific peptide side chain.
Therefore, the HLA alleles differ from each other by polymorphisms resulting in substitutions in amino acid residues, contributing to the specific structure of the peptide-binding pockets and results in a different immune responses among individuals.
This variation generates distinct HLA types and also causes allograft rejection when tissues are transplanted.
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What happens to MHC transcription when interferons are present?
Interferons are produced early in viral infections as part of the innate immune response and so this effect increases the ability of cells to process viral proteins and present the resulting peptides at the cell surface.
There is a marked increase in transcription of MHC class I α-chain and β2-microglobulin genes, and of the proteasome, tapasin, and TAP genes. This helps to activate the appropriate T cells and initiate the adaptive immune response in response to the virus.
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What is the MHC Class III region?
The class III region is the most gene-dense region in the human genome. Although this region does not contain any of the HLA genes, it contains many genes of importance in the immune response, including the tumor necrosis factor (TNF) and complement gene loci. TNF has been shown to play an important role in regulating the inflammation of rheumatoid arthritis.
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How does HLA affect stem cell transplants?
Polymorphism of the classical HLA genes represents is a problem, leading physicians to select a HLA potential compatible donor, in order to reduce the risk of graft failure, graft-versus-host disease (GvHD)
Unfortunately, due to the great diversity of HLA alleles and haplotypes, the possibility of identifying an unrelated donor matched at allelic resolution remains a difficult challenge for most patients. A HLA genotypically identical sibling donor is, therefore, the best choice, although only 25–30% of the patients have this option available.
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How can NGS be used to improve HLA matched donors?
HLA typing by next-generation sequencing (NGS) methods is likely to improve donor–recipient matching by providing full sequence information on all HLA loci in a shorter period of time compared to other methods.
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What HLA molecules are most associated with poor transplant outcomes when mismatched?
HLA-C
- residue 116 in the F pocket of the peptide binding groove has been identified as having a high frequency of mismatches that are responsible for adverse clinical transplant outcome,
HLA-B
HLA-DRB1
- can be permissive or not-permissive
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What is the function of the TAP genes in antigen processing?
TAP genes encode proteins that transport peptides into the endoplasmic reticulum for loading onto MHC class I molecules.
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What is the nomenclature system for HLA alleles?
WHO uses: four digit typing
The system includes the gene name, an asterisk, and sets of digits separated by colons (e.g., HLA-A*01:01).
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What is V(D)J recombination?
V(D)J recombination is the mechanism of somatic recombination that generates the diverse repertoire of immunoglobulins and T cell receptors in B and T cells.
Involves rearrangements of the V (variable), D (diversity), and J (joining) gene segments.
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What encodes immunoglobulins?
Immunoglobulins are encoded from three loci: the immunoglobulin heavy (H) chain locus, the Kappa light chain locus and the lambda light chain locus.
Each region contains multiple copies of three different types of gene segments (VDJ) for the variable regions of the antibody proteins.
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How does heavy chain rearrangment occur?
Heavy chain rearrangement is a stepwise process. Specific D and JH segments are joined and the bridging DNA strand is excised (D-JH rearrangement). A second rearrangement then occurs between the VH and D-JH segments (V-DJH rearrangement), and the rearranged DNA strand is transcribed into mRNA after splicing out of additional redundant V and J regions.
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How does light chain rearrangement occur?
A: Light chains do not involve D segments and undergo only one rearrangement event (VL to JL).
An immunoglobulin light (L) chain gene is assembled from three types of gene segments: VL, JL and CL, but do not have D gene segments. Light chains undergo only one rearrangement event, VL to JL.
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How do N region insertions contribute to immunoglobulin diversity?
Nucleotides that are not encoded by genomic DNA may also be inserted at the gene segment junctions, and are designated as the N region. This process contributes to diversity by changing the length of the resultant protein; the reading frame can also shift depending on the length of insertion (if a non-multiple of three nucleotides).
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What is somatic hypermutation (SHM)?
SHM is a process where point mutations accumulate in the V regions of immunoglobulin genes after rearrangement, increasing diversity.
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What are the components of most T cell receptors?
An alpha (α) chain
- The alpha locus contains only V, J, and C gene segments
a beta (β) chain
- the beta locus includes V, J, C, and D segments.
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How does junctional diversity play a role in TCR diversity?
The addition of N regions at gene segment junctions and utilization of D genes in multiple reading frames enhance TCR diversity.
TCR genes do not undergo somatic hypermutation.
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What are immunoglobulins?
Antibodies
Composed of two identical heavy chains and two identical light chains, connected by disulfide bonds, with variable (V) and constant (C) regions.
Isotypes: IgG, IgA, IgM, IgE, and IgD.
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What is the role of the hinge region in Immunoglobulins?
A flexible hinge region is also found in IgG, IgA, and IgD (not IgM or IgE), located between CH1 and CH2. This is a proline-rich region, that allows the distance between the two antigen-binding sites to vary to allow better antigen accommodation. Proteases (such as papain or pepsin) cleave immunoglobulin molecules at this region, resulting in the Fab and Fc fragments.
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What is the role of the joining chain in Ig?
The joining (J) chain is a small polypeptide with cysteine residues that link the heavy chains of IgM to form a pentamer or hexamer, or the heavy chains of IgA to form a dimer by disulfide bonds
159
What is IgG?
75% of Ig
only isotype that crosses the placenta, binding to the neonatal Fc receptor, and providing immune protection to the developing fetus
The biological functions of the IgG subclasses include neutralization (blocking the binding of viruses to host cells), opsonization (binding of antibodies to the surfaces of a microbe or foreign particle to facilitate phagocytosis by neutrophils or macrophages via Fc receptors), complement activation, and antibody-dependent cellular cytotoxicity.
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What is IgA?
15% of Ig
It is predominantly found in the various mucous secretions of the respiratory, intestinal, and genitourinary systems
Helps protect mucosal surfaces from microbial invasion by coating microbes to prevent adherence to epithelial cells
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What is IgM?
10% of Ig
It is mostly intravascular, and is predominantly found in lymph fluid and blood
The first immunoglobulin class to be synthesized by the neonate and plays a role in the pathogenesis of some auto-immune diseases.
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What is IgE?
1% of Ig
IgE primarily defends against parasitic invasion and is responsible for allergic reactions.
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What is IgD?
<1%
IgD is a monomer expressed on the surface of mature, naïve B cells
Little is known about its function, but elevated levels are associated with an autoinflammatory disease.
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What does the T-cell receptor (TCR) complex recognize?
Antigens in the form of peptides bound to the major histocompatibility complex (MHC) on target cells.
165
What are the two main types of TCR heterodimers?
TCR alpha/beta and TCR gamma/delta
166
What TCR genes are there?
The human genome expresses four TCR genes known as TCRα, TCRβ, TCRγ, and TCRδ, which form two distinct heterodimers: TCR alpha/TCR beta or TCR gamma/TCR delta.
95% of circulating T cells express the T cell receptor containing the alpha (α) chain and a beta (β) chains (αβ T cells). A small portion of cells (0.5–5%) express receptors with the gamma (γ) and delta (δ) chains (γδ T cells).
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What is the structure of TCR?
TCR chains consist of an extracellular region, transmembrane region, and a short cytoplasmic tail.
The extracellular region contains two domains: a variable immunoglobulin-like (V) domain and a constant immunoglobulin-like (C) domain, and connecting peptide. The C domain is proximal to the cell membrane and used for the interactions with CD3 chains, while the V domain binds to the peptide/MHC complex.
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What co-receptors are associated with TCR?
CD4+ and CD8+
- mutually exclusive
- CD4+ cells recognize antigen presented by an MHC class II, whilst CD8+ cells recognize antigen presented by an MHC class I.
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What is CD3?
CD3 is the marker most widely used to identify total T cells on routine flow cytometry
They are required for TCR localization on the cell surface and signal transduction
169
What is the function of TCR?
The essential function of the TCR complex is to identify specific bound antigen derived from a potentially harmful pathogen and elicit an immune response.
Antigen is digested into peptides and bound to MHC class I or class II molecules on the surface of antigen presenting cells (APCs) for presentation to T cells.
When a TCR engages a relevant peptide major histocompatibility complex (pMHC), Lck is recruited to the TCR complex by CD4 or CD8 co-receptors and TCR signalling is initiated culminating in the generation of a T cell response.
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What causes rejection of a stem cell transplant?
T cell recognition of alloantigen is the primary event that will eventually result in rejection of a transplanted organ. There are three distinct pathways of allorecognition: direct, indirect, and semi-direct.
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What happens in direct allorecognition?
Recipient T-cells recognize intact allo-MHC molecules on the surface of donor cells.
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What occurs in indirect allorecognition?
Recipient T-cells recognize self-MHC molecules bound to peptides derived from foreign MHC molecules.
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What is semi-direct allorecognition?
It involves recipient APCs presenting both intact foreign MHC+peptide complexes and processed peptides, allowing both direct and indirect recognition.
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What is linkage disequilibrium?
When two loci are in close proximity to each other on the same chromosome, a set of alleles are often inherited together. These loci are said to be in ‘linkage disequilibrium’, with the frequency of the observed haplotype being significantly different than expected.
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What is the 8.1 ancestral haplotype (AH)?
The 8.1 ancestral haplotype (AH) is a long and conserved haplotype spanning the MHC region
This 8.1 AH is carried by approximately 10% of Northern Europeans and has been associated with both beneficial and detrimental effects. The 8.1 AH is associated with a number of autoimmune diseases, such as toxic diffuse goiter (Grave’s disease) and systemic lupus.
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What role does NGS play in immunogenetics?
It provides comprehensive genomic information, identifies immunogenic proteins, and improves HLA typing, providing high throughput, allele-level resolution critical for compatibility assessments.
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What is the role of NK cells?
Naural killer (NK) cells are immune cells that play a crucial role against viral infections and in the control of tumour growth and metastasis
Normal healthy cells express HLA class I molecules, inducing the activity of inhibiting receptors, which keep NK cells silenced. Transformed or infected cells have decreased expression of the HLA molecules, which allows NK cell activation through activating receptors and the potential to kill the target cells.
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What are Killer cell immunoglobulin-like receptors (KIRs)?
A group of polymorphic receptors on NK cells that recognize specific HLA allotypes.
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What are Immunoglobulin-like transcripts (ILTs)?
Constitute a group of NK cell receptors, including the inhibitory receptors ILT2, ILT3, and ILT4. As they can potentially suppress T cell responses and proliferation, they are thought to have a role in allograft tolerance during transplantation and cancer
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What are NKG2D receptor?
The NKG2D receptor is a natural killer group 2 member D protein that controls cells’ activity in both adaptive and innate immunities
Recognizing tumor cells and inducing cytotoxic effects.
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How do tumors escape T cell-mediated immune responses?
The downregulation or loss of HLA class I molecules can prevent tumour cells from being recognized by CD8+ cytotoxic T lymphocytes (CTLs).
Loss can be total or partial and occurs in 60-90% of cases
