Cancer Bio Flashcards
Inhibitors of Angiogenesis
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Prolactin, P53, Thrombospondin 1/2, Endostatin (blocks MAPK & MMPS), Angiostatin (binds anexin II), Top1, Notch
Angiogenic Inducers
GROWTH FACTORS
VEGF, PDGF (plt derived growth factor) hepatocyte derived growth factor, fibroblast growth factor, epidermal growth factor
VEGF Signalling Pathway (diagram)
VEGF A Signalling pathway (words)
1) 1 VEGFA binds to 2 VEGFR - dimerisation and autophosphorylation.
2) Activates Ras-raf-mek-mapk cascade - transcription factor activation & gene expression
3) also activates PI3K - AKT - inhibition of apoptosis (blocks BAD which is pro apoptotic) & stimulation of endothelial NO synthease + vascular permeability via NO production.
VEGF & VEGFR
5 ligands VEGF-A - D.
3 receptors VEGFR 1 -3
VEGF-A & VEGFR key to angiogenesis
VEGF1 regulates amount of VEGFA able to bind to VEGFR by competitively binding and so is inhibitory.
VEFGR3 - involved in lymph system - promotes expansion of lympahtic vessels and increases lymph spread risk.
Angiogenic sprouting
- Tip formation
- Stalk elongation
- Anastomosis
- Canalisation
- highest signal - vegfr2 & Vegfa stimulate formation of tip cell
- once vegf2 activated - tip cell induce expression and release of NOTCH ligand, delta like 4 ligand (DLL4) which bind to Notch R on neighbouring cell.
Notch intracellular domain released and transported to nucleus - acts as transcription factor to repress VEGFr2 and induce VEGFR1
VEGFR1 decreases conc of VEGF available for VEGFR2 therefore growing sprout moves along VEGF gradient.
Hypoxia & Hypoxxia inducible factor
Single strand Breaks
- Direct Repair
- Base excision repair
- Nucleotide excision repair
- Mismatch Repair
Detected by alkaline solution assay
Direct Repair
damage recognised by 06 Alkyl guanine DNA alkyl transferase (AGT) & directly chemically reversed by removing an alkyl group from 06 atom of guanine.
Base Excision Repair
Removal of single damaged base - can repair damage by deaminations, alkylations or oxidations of DNA Base
- DNA glycosydases recognise and remove damaged base
- APE2 cuts DNA back bone leaving SS break
- PARP1 detects SSB + promotes recruitment of SSB proteins to DNA damage site
- DNA polyerase B syntheseses new DNA
- DNA ligase II joins end of the newly synthesised DNA to fill the gap
- XRCC1 acts as a scaffold
Nucleotide excision repair
specific for helix distorting lesions (eg. pyrimidine dimers, bulky DNA adducts)
1. XPC recognition of helix distortion
2. recruitment of a number of proteins
3. Endonucleases remove a sequence of oligonucleotides
4. DNA polymerase fiills the gap
5. DNA ligase seals the nick.
Mismatch Repair
corrects replication errors that have resulted in mismatching bases that have escaped editing by polymerases (esp in microsatelite areas).
1. Recognition - MSH2 & MSH3/6 binding to affected DNA sites & recruiting MLH1 & PMS2
2. Excision - recruitment of endonucleases which excise the damaged strand
3. Substitution by DNA polymerase
4. Religation by DNA ligase
Double Strand Breaks
NHEJ
Homologous recombination repair
Detected by alkaline comet assay
Non Homologous End Joining
predominant DNA DSB repair pathway, DNA directly repaired w/o need for homologous DNA molecule
Error prone, can lose DNA
- DNA ends recognised by KU70/80 proteins (aka XRCC5/6) that recruit DNAPK which activates artemis & holds end together and creates stable scaffold.
- ARTEMIS (endonuclease) processing enzymes that tidies the ends to prepare for ligation
- If DNA ends are compatible they are directly ligated otherwise ends are resected until compatible.
- DNA ligase IV and XRCC4 join the DNA together
Homologous recombination Repair
requires sister chromatide, slower, generally S/G2 phase, 2 copies of intact DNA molecules produced with rarely any errors
- Detection of DSB break by aTM protein & MRN complex - excises DNA to create single strand 3 ends
- RPA protein binds to and stabilises the SSDNA
- BRCA1 & 2 aids nuclear transport of RAD51 onto ss DNA
- RAD52 facilitates RAD51 binding to exposed ends - once bond formed nucleoprotein filament that invades SSDNA to look for a homologous sequence
- Once found this is copied & followed either by DSB repair or synthesis dependent strand annealing
- RAD52 anneals the stands, BCM proteins help migrate the junctions toward each other.
Extrinsic pathway of apoptosis
- Death signals, TNF & Fas activate their death receptors TNF receptor & fas receptor respectively
- Binding causes change in shape & trimerisation of receptors
- Leads to aggregation of procaspase 8 (by adaptor proteins)
- leads to CASPASE 8 ACTIVATION
- initiates caspase cascade, proteolysis & apoptosis.
(protein c-flip can inhibit interation of procaspase 8 with the adaptor proteins)
Intrinsic pathway of apoptosis
- Cell stress triggers the BH3 only protein Bid to transiently bind to and activate Bax
- BAx undergoes conformational change - inserts into the outer mitochondrial membrane & oligomerises (6-8 molecules)
- important regulators are released from the intermembrane space
- cytochrome c joins the adaptor protein, apoptotic protease activating factor (APAF-1) & recruits procaspase 9 to form the APOPTOSOME
- caspase aggregation leads to activation of procaspase 9 & caspase cascade
(smac & diablo released from mitochondria inhibit IAPs that normally act to block caspases).
oncogenes
mutated or damaged proto-oncogenes, act in dominant manner
EG. RAS, Her2, BCR-ABL, C-myc
RAS oncogene
RAS-GTP (active
RAS-GDP (inactive)
Gaps inactivate
SOS activates
if mutation in RAS can’t use GAP to inactivate ras so permanently ON - increased signalling pathway through MAPK pathway downstream
- most commonly mutated oncogene
HER2 Oncogene
elevated expression - induces her2 family dimerisation - pro-prolfieration/anti-apoptotic signalling cascade
BCR-ABL oncogene
translocation
causing constitutively active tyrosine kinase - increased cell proliferation & dimerisation.
C-myc oncogene
nuclear protein, transcription factor.
Increased E2F, cyclin D & SCF transcription and therefore passage into cell cycle
CHr 8/14 translocation - incr. expression of c-myc - inc. transcription of c-myc related genes - seen in burkitts lymphoma
TSG
gene whose protein product directly/indirectly prevent cell division or induce cell death
recessive
mutation causes loss of function
Eg. P53,, RB, WT1
RB TSG
regulates activity of E2f which is crucial for expression of genes needed for S phase
- in hypophosphorylated state RB binds to E2F preventing it from interacting with transcription factors.
- hyperphosphorylation of RB - release of E2F - gene transcription - s phase
P53 tsg
continually monitors for metabolic disorder/genetic damage - may arrest cell cycle & initiate damage repair or induce apoptosis - mutated in 50% of cancers
- in norm conditions, quickly turned over but levles rise rapidly in response to stress (regulated by protein degradation)
- in response to DNA damage p53 is stabilised by ATM (acetylation)
- MDM2 attaches ubiquitin to P53 flagging it for proteolysis & binds to and inhibits the P53 transactivation domain - transfers p53 to cytoplasm and out of nucleus. is a negative regulator of p53.
- low amounts of P53 - lower mdm2 gene expression so reduction in negative regulation of p533 hence inc. p53.
-P14 inhibits MDM2 mediated degradation of P53 - hence higher levels of P53.
- in non stressed cells p53 associates with partner mdm2 leading to degradation.
- in DNA damage - ATM phosphorylates MDM2 & P53 which prevents their interaction - p53 is stabilised and acts as transcription factor.
EGFR pathway
innate system
no memory, can differentiate self from non self, co-ordinates the adaptive system
1st line defence - response is the same each and every time
NON specific - can’t differentiate between specific viruses and rely on recognition of danger cells
Danger signals are PAMP and DAMP (pathogen associated molecular patterns and damage associated molecular patterns) via pattern recognition receptors.
Pattern recognition - TOLL like receptors - recongnise PAMPs and some DAMPS
basophil, eosinophil, mast cell
role in both parasitic & allergic responses
cells degranulate on activation to release heparin and histamine
neutrophil
most abundant of the granulocytes (70% of all white blood cells)
phagocytic
Monocyte, macrophage, dendritic cell
phagocytes that engulf infected/dying cells. Critical role in antigen presentation.
Potent APCs
recongise pathogens through TLRs
has complement and Fc receptors
M1 - inflammatory, drives costimulation & T cell activation
M2 - associated with wound healing response - angiogensis, vegfr etc. Immunosuppressive, dampen inflammation and encourage healing.
Natural killer cell
major role in viral immune response
activation of Nk cell depends on the balance of stimulatory & inhibitory signals
generally require signals from accessory cells for activation
Adaptive immune system
T & B cells
Cells don’t rely on recognition of PAMPs like innate system
express receptors that can differentiate between self and non self by sequence specific recognition of antigen
compromised of B&T lymphocytes - both derived from the bone marrow.
adaptive - ability to learn and respond more efficiently during subsequent infection
B Cells
has antigen binding site
FAB region on both arms
FC region on tail
Cytotoxic T Cell CD8
effector immune cells that can identify and kill infected/neoplastic cells
Helper t Cell CD4
produce cytokines / signals that can promote CD8 (eg. IL2) or B cell activation + function (e.g IL4)
Regulatory T cell CD4, CD25, FoxP3
mediate peripheral tolerance of CD8 T cells through production of both solbule factors eg. tgf-b or cell cell contact (ctla4)
immunological memory
following activation both B & T lymphocytes produce relatively short lived effector & long lived memory cells
what are T cell antigens?
T cell antigens are peptides - typically 9-12 AA in length. Recognition is sequence specific. Bound in the groove of an antigen-presenting Major Histocompatibility antigen.
Antigens are presented by specialised antigen presenting cells (APC)
T cells synapse with antigen presenting cells
T cell receptor & MHC interaction
T cell doesn’t recognise MHC antigens alone
TCR sees both MHC & the peptide complexed with it
The whole complex defines a TCR specifically
MHC
region on short arm of chromosome 6, contains the genes for many proteins of importance for the immune response
of primary relevance for T cell immunity are HLA antigens (MHC antigens)
Class 1 - HLA-A, HLA-B, HLA-C
Class II - HLA-Dr, HLA-DP, HLA-DQ
Each person expresses 2 variants of each antigen HLA type
Class 1 - present on virtually every nucleated cell. peptide 8-12 aa.
Class II - peptide 12-24 Aa. more open structure - longer peptide can fit. present on all antigen presenting cells eg. B cells.
MHC Class 1
binds intracellular peptide antigens
mhc1 presents viral/mycobacterial/self & mutated peptides
MHc1 expressed by virtually all nucleated cells
antigens are processed into peptides in the immunoproteosome and shuttled into the ER via the transported associated with Ag processing.
PEPTIDES ARE LOADED ONTO THE MHC 1 AND CAN PRESENT ANTIGEN ONLY TO CD8 T CELL
MHC Class II
binds extracellular peptide antigens
MHC-II presents exogenous antigens following phagocytosis
expression of MHC II is restricted to professional APCs
antigens are processed through the endolysosomal pathway and loaded onto MHC II
peptides bound to MHC II can PRESENT ANTIGEN TO ONLY CD 4 T CELL
professional APCs can load exogenous antigen onto MHC-1 for presentation to CD8 T cell (cross presentation - only APCs can do)
T cell activation
Requires 2 signals
1. peptide MHC complexes
2. Co stimulation CD 28
(requires both membrane recognisation & intracellular signalling through TCR + co stimulatory molecule)
