Hard stuff for exam Flashcards
Proteins at G1 (restriction point)
Key proteins at this checkpoint are cyclin D4 and pRB
Retinoblastoma syndrome and Li Fraumeni proteins
- RB1, Retinoblastoma syndrome -> Ocular, melanoma, sarcoma
- TP53, Li Fraumeni syndrome -> lymphoma, sarcoma, glioma, breast
Antigen Receptor Gene (IGH, TCR) Recombination
During development, in B and T lymphocytes, antigen receptor genes assemble through random splicing of VDJ elements. There many combinations possible.
If you analyse a blood sample (with mixed lymphocytes in it), these will have a polyclonal population - containing with different versions of VDJ gene recombination
BUT
In lymphoma, the VDJ gene is monoclonal - indicating that all of the cancer cells arose from a single cell progenitor
Partial Gene Deletions e.g EGFR
e.g. Oncogene - Epidermal Growth Factor Receptor (EGFR)
* With no ligand, EGFR inhibits itself
* Dimer binds ligands (EGF and TGFa)
* Activates downstream signaling through
phosphorylation via the intracellular tyrosine kinase domain -> cell proliferation
In cancer, EGFR signalling is activated
- gene amplification -> more protein
- deletion of exons 2-7 -> removes inhibitory ligand binding domain -> ON tyrosine kinase activity without ligand binding (activating mutation)
Translocations (CCND1)
Rearrangements of genetic material to juxtapose gene promotors/regulatory elements to drive inappropriate gene expression (activating mutations).
Example:
* Translocation of CCND1 gene downstream of an active IGH enhancer element
* Switches ON CCND1 gene -> excess cyclin D1 protein, deregulation of cell cycle at R point
* Occurs in Mantle Cell Lymphoma
Oncogenes
Overactive proteins
EGFR, RAS, BRAF, CCND1
Tumour Supressor Genes
Inactivated proteins
RB1, TP53, BRCA1/2, APC
Growth factor cell signalling proteins
EGFR, RAS, BRAF
Cell cycle proteins
CCND1, RB1
Cell adhesion molecule
APC
Cyclins and CDKs
-Cyclins and Cyclin Dependent Kinases (CDK) are regulated by phosphorylation
-Cyclins bind to and increase the kinase activity of CDK allowing progression of the cell cycle
Cyclin-CDKs regulate
- the R restriction point
- the G1/S transition
- induction of DNA synthesis in S phase
- the G2/M transition
The cell cycle is paused by the activity of inhibitors of the CDKs (e.g. p21cip) and checkpoint proteins including pRb and p53
Ras Pathway
Ras is an intracellular protein that is central to integrating GFR signals
GFR activation -> Ras binds GTP and recruits and activates signalling proteins (e.g. Raf, PI3K)
Signalling stops when Ras converts GTP to GDP+phosphate (via its GTPase activity)
Cancers with mutant Ras have reduced GTPase activity -> remains active.
Ras activates transcription of CCND1, cyclin D which activates CDK4 to stimulate cell cycle progression.
Excess oncogenic cyclin D or CDK4 (by activating mutations/amplification) occur in cancers.
Retinoblastoma Protein (pRB) phosphorylation
The RB protein exists in two forms during the cell cycle
* Weakly phosphorylated - at G0 and G1 - active - halts cell cycle at the Restriction point by suppressing transcription factor signalling
Strongly phosphorylated (ppRb) - during rest of cell cycle - inactivates the protein so it does not signal and therefore cell cycle progresses
Inactivating phosphorylation of pRB is by CyclinD-CDK4
Loss of RB protein (e.g. by gene deletion/inactivating mutation/methylation) leads to uncontrolled cell cycle progression (skipping the G0/G1 R checkpoint)
Apoptosis drives wound healing by
Effector caspase-3 cleaves phospholipase A2 (iPLA2) from cell membrane lipids.
Produces arachidonic acid which is metabolised by COX2 into prostaglandin (PGE2) -> ++ cancer cell proliferation
Exposed phosphatidylserine (PS) on apoptotic bodies (‘eat me’), ingested by macrophages which then release
* VEGF -> angiogenesis
* MMPs, PDGF, TGFß -> extracellular matrix remodelling/fibrosis
Death Receptors
Extrinsic apoptosis is driven by external signals detected
by cell surface receptors e.g. Fas Receptor
Fas receptor binds to extracellular FasL (ligand)
Fas receptor binds an intracellular adaptor molecule FADD via a ‘death domain’ then activates an initiator caspase
The initiator caspase activates an effector caspase which leaves target proteins triggering the cell apoptosis process.
Switching off Fas and FADD
Fas receptor and FADD can be switched off by mutations in the death domain (inactivated protein) or through promoter methylation (less transcription)
Initiator caspase expression can also be
switched off in cancers by methylation
Leads to:
* resistance to pro-apoptotic signals
* death of tumour infiltrating
lymphocytes cells by secretion of FasL from resistant cancer cells (counter- attack).
BCL-2
Key sensor of cell stress.
It is an ANTI-apoptotic protein that is overexpressed by chromosome translocation to the IGH locus in B cell follicular lymphoma. This oncogene extends the life of B cells but does not affect the cell cycle (proliferation rate).
BCL-2 and Bax
Regulate the movement of pro-apoptotic proteins like Cytochrome C from mitochondria so they do not interact with and activate the APAF-1 apoptosome.
- BCL-2 closes membrane pores (no release of CytC, anti-apoptotic),
- Bax opens membrane pores (release of CytC, pro apoptotic)
Oncogenic, Anti-apoptotic BCL-2 is overexpressed in 50% of all cancers
Activating mutant EGFR protein (delEx2-7) can also induce Anti-apoptotic BCL-XL expression (as well as activating the cell cycle) leading to treatment resistance
PRO-apoptotic Bax, a tumour suppressor gene, is inactivated in colon and stomach cancers
LOTS of ways a cancer cell can avoid apoptosis!
Activation of p53
p53 is activated by DNA damage (by carcinogens
or chemotherapy) or abnormal cell cycle (Ras
mutation, pRB inactivation)
Activation of p53 leads to regulation of the cell cycle and/or activation of apoptosis via transcriptional programmes
p53 will pause the cell cycle to allow DNA repair,
if too damaged it will initiate apoptosis.
