Cell Cycle and Cell Death Flashcards
how many cells are replaced in humans every day?
50 billion
what are the cells in the G0 phase typically like?
smaller with reduced metabolic activity
why will cells exit the cell cycle to G0?
if no mitogens are present
what causes cells to go from G0 to G1?
mitogens providing the stimulatory signal
what is the point of no return in G1?
START/the Restriction Point/Commitment Point- after which cell is committed to progression through cell cycle
what is important for cells to pass through START in G1?
hyperphosphorylation of RB family proteins by CDK4/cyclin D
what happens in S phase?
DNA replication
what happens in G2?
replicated DNA monitored, organelles replicated
what happens in the M phase?
cell undergoes mitosis and divides
what are the 3 checkpoints in the cell cycle?
G1/S, G2/M and spindle assembly checkpoint
what are the key molecular regulators of the eukaryotic cell cycle?
the cyclin-dependent kinases (CDKs)
what are the CDKs involved in G1?
CDK4 and CCDK6
what are the CDKs involved in G1/S and S?
CDK2
what are the CDKs involved in mitosis?
CDK1
what are the cyclins involved in G1?
cyclin D1. D2, D3
what are the cyclins involved in G1/S and S?
cyclin E
what are the cyclins involved in S phase?
cyclin A
what are the cyclins involved in M phase?
cyclin B
what is cyclin/CDK activity regulated by?
transcriptional control, ubiquitin mediated proteolysis, inhibitory phosphorylation, small CDK inhibitor proteins
which E2F transcription factors activate cyclin transcription?
E2F1, E2F2, E2F3
which E2F transcription factors repress cyclin transcription?
E2F4 and E2F5
what family of repressors regulates activity of E2F transcription factors?
the RB family
what does the RB protein do?
unphosphorylated RB binds to and inhibits E2F-dependent transcription, e.g. of cyclin E
what do p107 and p130 repress?
E2F4 and E2F5
what does Cyclin D-CDK4 phosphorylate and inhibit?
RB family proteins
what is addition of a covalently linked chain of Ub molecules to a protein a signal for?
for the protein to be degraded
what attaches ubiquitin to proteins?
the ubiquitine ligase complex APC/C-Cdh1
what is the APC/C complex?
anaphase-promoting complex/cyclosome
what does the ubiquitin ligase complex APC/C-Cdh1 do?
sticks ubiquitin onto proteins, degrades cyclin A and geminin in G1 phase (prevents premature entry into S phase)
what does the ubiquitin ligase complex APC/C-Cdc20 do?
degrades cyclin B in mitosis to allow chromosomes to segregate
which is a faster method of gene expression control, protein degradation or transcription regulation?
protein degradation
what protein is responsible for inhibitory phosphorylation of CDKs?
Wee1 kinase
when is inhibitory phosphorylation of CDKs particularly important?
the G2/M checkpoint
what is inhibitory phosphorylation of CDKs counteracted by?
Cdc25 phosphatase
which small CDK inhibitor proteins are particularly important for keeping cells in the G0/G1 phase?
p21, p27, p15/16
what does p21 inhibit?
Cdk2
what does p27 inhibit?
Cdk2
what does p15-INK4b inhibit?
CDK4/6
what does p16-INK4a inhibit?
CDK4/6
what are mitogens?
the cue that drives cell cycle progression
in what proportion of non-small cell lung carcinomas is EGFR mutated to be hyper-activated?
20%
in what proportion of human tumours are Ras mutations found?
20-25%
in what proportion of cancers is Myc overexpressed?
4%
how do cancer viruses override the G1 checkpoint?
express viral proteins that directly inhibit Rb
what is the function of myostatin as an anti-mitogen?
activates co-repressors of the SMAD family
what is the function of SMADs?
inhibit E2F target genes and activate CDK inhibitors like p15
what mutation do Belgian blue cows have?
mutations in the anti-mitogen myostatin, which functions to stop production of too much muscle, so Belgian blues have hyper-proliferation of muscle
what is Cyclin A/Cdk2 essential for in the cell cycle?
activating the DNA replication helicase and recruiting the polymerase to replicate the genome
what are Cyclin A/Cdk2 and geminin inhibitors of?
DNA replication helicase loading
what ensures the genome is only replicated once?
once initiation has begun APC/C is absent and cyclin A-Cdk2 is present, so no further helicase loading can take place
what does chromosome segregation require?
linkage between newly replicated sister chromosomes, alignment of linked sisters in the middle of the cell, chromosomes under tension
what are the ways newly replicated sister chromosomes are linked?
topologically intertwined, chromosome cohesion
how does topological linkage of sister chromosomes work?
DNA supercoiling during replication is resolved by the action of topoisomerases and rotation of the chromosome, leads to sisters intertwining
how does chromosome cohesion work?
caused by the cohesin complex- cohesin is a ring with a gap closed by Scc1
how are linked sister chromosomes aligned in the middle of the cell?
mitotic spindle machinery- microtubules from spindle to cell cortex, position spindle in middle of cell, kinetochore is where microtubules attach to chromosome
what region of the chromosome assembles the kinetochore?
the centromere
what is the MT organising centre?
centrosome
what mediates cleavage of cohesion once chromosomes aligned in middle of the cell?
separase- a protease
what causes separase to be inactive?
CDK phosphorylation and binding to inhibitor called securin
how does APC-Cdc20 activate separase?
degrades cyclin B and securin
when does separase cleave cohesin?
at the metaphase to anaphase transition
what are the phases of mitosis?
prophase, prometaphase, metaphase, anaphase, telophase
what happens in prophase?
chromosomes duplicated, cohesed and condensed. centrosomes move toward opposite poles, microtubules gradually assemble
what happens in prometaphase?
nuclear membrane breakdown allows spindle microtubules (MTs) to access the chromosomes. MTs attach to chromosomes at their centromeres via a protein complex called the kinetochore
what happens in metaphase?
chromosomes align along the cell equator, every chromosome has at least 1 microtubule connected to each centrosome
what happens in anaphase?
sister chromatids separate after breakdown of cohesin
what happens in telophase?
the chromosomes arrive at the cell poles, new nuclear membrane forms around each group of chromosomes
what happens in cytokinesis?
the physical process the splits the parent cell into 2 identical daughter cells
what activates the G2/M checkpoint?
incomplete replication
what do the G2/M checkpoint kinases do?
stabilise activate Wee1 kinase, repress Cdc25 phosphatase- leading to inhibition of Cyclin B/Cdk1
what are the G2/M checkpoint kinases?
ATM/ATR and Chk1/Chk2
what does the spindle assembly checkpoint do?
prevents APC/C-Cdc20 activation until all chromosomes correctly attached to the mitotic spindle
what activates the spindle assembly checkpoint?
if a kinetochore isn’t attached to a MT, if tension isn’t correct
what does the mitotic checkpoint complex do?
binds and inhibits Cdc20 when kinetochores are not attached to MTs, and chromosome isn’t under tension
what % of cancers are aneuploid?
90%
what % of cancer mutations are caused by random mutations during replication?
66%
what can high levels of cyclin E cause?
centrosome over-duplication
what are reasons for increased segregation problems in women?
sister chromatids held together by cohesin, oocytes arrest in prophase I and may not be used for 20-25 years, older oocytes have less cohesin so less cohesion
what does the drug ribociclib do?
inhibits CDK4 and CDK6
what do DNA damage agents do? (type of chemotherapy)
inhibit DNA replication
what do anti-metabolites do? (type of chemotherapy)
reduce metabolites- deplete dNTPs
what do vinca alkaloids do? (type of chemotherapy)
inhibit microtubules so no chromosome segregation
what do intercalating agents do? (type of chemotherapy)
inhibit replication by distorting DNA
what do topoisomerase inhibitors do? (type of chemotherapy)
inhibit replication/segregation
what do taxanes do? (type of chemotherapy)
inhibit MTs so prevent segregation
what is the target of erlotnib?
EGFR, to block activation of Ras signalling, prevent G1-S transition
what is the main form of programmed cell death?
apoptosis
how is development of fallopian tubes, uterus and upper vagina arrested in male foetuses?
anti-Mullerian hormone expressed to inhibited Mullerian duct development, Mullerian ducts then destroyed by apoptosis
what is the difference between programmed cell death and necrosis?
necrosis is tissue wide not cell-specific, and is due to trauma not programmed
how is the body plan sculpted by apoptosis?
in hand interdigital cells die by apoptosis, in tadpoles apoptosis in tail before they become frogs
what does failure to induce apoptosis in the Mullerian duct in the male result in?
Persistent Mullerian duct syndrome causing hermaphroditism
how does apoptosis resolve effects of drug treatments/heavy drinking on the liver?
these cause hyperplasia in liver which apoptosis resolves after drug clearance
how is apoptosis involved in neural development?
neurons which don’t form connections with other neurons are eliminated by apoptosis
how is apoptosis involved in immune cell development?
eliminates B and T cells which don’t recognise any foreign bodies or recognise self cells
how is apoptosis involved in the immune response after an infection is destroyed?
apoptosis needed to reduced lymphocyte populations so only a few memory cells survive
what does apoptosis result in?
phagocytosis of the dead cell by specialised phagocytes including macrophages and microglia, and non-professional phagocytes such as hepatocytes in the liver
what is apoptosis mediated by?
proteases called caspases
why are caspases called that?
cysteine aspartic acid proteases
what are the 2 types of caspase?
initiator caspases and executor caspases
what are the initiator caspases?
Caspase8 and Caspase9
what are the executor caspases?
Caspase3 and Caspase7
what happens in apoptosis? (overview)
cell shrinks, chromatin condenses, membrane starts blebbing, organelles disintegrate, loss of cell-cell contact, nucleus and organelles collapse, membrane blebbing continues, DNA digested, apoptotic bodies form, cell fragmentation, macrophages phagocytose apoptotic cell
what is the critical amino acid in the active site of caspases?
Cys residue
where do caspases cleave peptide sequences?
immediately after an aspartic acid
how are inhibitor caspases activated?
by dimerisation and self-cleavage
what is initiator caspase activation induced by?
the intrinsic or extrinsic pathway
how are executioner caspases activated?
proteolytic cleavage of inhibitory domain by initiator caspases
what ensures apoptosis induction is self-amplifying and irreversible?
1 initiator caspase initiates many executor caspases
what 4 key features of apoptosis does caspase activation define?
normally inactivated, destructive, self-amplifying, irreversible
how many cellular proteins do caspases cleave to ensure cell death?
over 2000
how do caspases degrade DNA?
by destroying iCAD (inhibitors of Caspase-inactivated DNase), which activates CAD (cuts DNA)
what does CAD do?
cuts DNA
what outer membrane protein do caspases destroy?
Flippase
what does Flippase do?
keeps phosphatidylserine (PS) on the inner membrane
what does loss of Flippase due to destruction by caspase cause?
phosphatidylserine to move to the outer membrane- the ‘eat me signal’ for phagocytes
what is the ‘eat me signal’ for phagocytes?
phosphatidylserine on the outer membrane
what does destruction of cadherin by caspase lead to?
loss of cell adhesion
what does destruction of actin by caspase lead to?
loss of cell shape
what does destruction of lamin A by caspase lead to?
loss of nuclear membrane
what does destruction of iCAD by caspase lead to?
digestion of DNA
what does destruction of DNA repair enzymes (PARP) by caspase lead to?
no DNA repair
what does destruction of MDM2 by caspase lead to?
activation of p53
what does destruction of Flippase by caspase lead to?
phagocytosis due to PS exposure on outer membrane
how do checkpoint kinases activate p53?
by inhibiting the interaction between p53 and an E3 ubiquitin ligase (Mdm2)
what does Mdm2 do?
binds to and degrades p53
what is PUMA?
p53 unregulated modulator of apoptosis
why is hyperactive p53 lethal?
healthy cells are induced to arrest the cell cycle and commit apoptosis
what does PUMA inhibit?
B-cell lymphoma 2
what is BCL-2?
an oncogene that is overexpressed in approx. 45% of all cancers
what does BCL-2 do?
binds to the outer mitochondrial membrane where it inhibits a pore-forming protein called BAX
what is BAX?
BCL-2 associated protein X
what does PUMA binding to Bcl-2 allow BAX to form?
a pore in the outer mitochondrial membrane with the initiator Caspase9
what do BAX and Caspase9 form?
complex called the apoptosome
what does the apoptosome do?
dimerises and activates Caspase9 triggering apoptosis
what is Li-Fraumeni Syndrome?
patients inherit 1 mutant p53 gene and have a near 100% risk of developing cancer- as p53 blocks cell division and promotes apoptosis
what is the action of Venetoclax?
inhibits BCL-2 resulting in activation of BAX and restoring the intrinsic apoptosis pathway to p53 mutant cancers
how can apoptosis be induced extrinsically?
cell-to-cell contact between death receptors, and by cytokines
what are cytokines?
small proteins released/expressed mostly by immune cells
what is the TNF family?
tumour necrosis factor family- cytokines that mediate apoptosis through the extrinsic pathway
what does TNF bind to?
the TNF receptor (TNFR) which is expressed on many cell types
what is FAS ligand (FASL) a member of?
the TNF cytokine family
what is the receptor for FASL?
FAS
what is FAS expressed on?
immune cells such as macrophages
what is the other name for the TNF family?
death receptors
what are TNFR family proteins?
transmembrane domain proteins with a death domain on their cytoplasmic side
what does binding of TNF to TNFR/FASL to FAS cause?
structural changes in the death domain which trigger the recruitment of multiple proteins including the inactive form of the initiator Caspase8 to form the DISC complex
what is the DISC complex?
death induced signaling complex
what does the DISC result in?
dimerisation and activation of Caspase8 which triggers activation of executor caspases and cell death
what protein counteracts the effect of DISC activation?
FLIP protein (Flice inhibitory protein)
how does FLIP work?
FLIP is very similar to the Caspase8 protein except it lacks any protease activity so it acts as a Caspase8 dummy in the DISC complex preventing Caspase8 from being activated
when is FLIP important?
in protecting expanding mature lymphocyte populations from apoptosis
what is excessive activation of TNF signaling associated with?
chronic inflammation, can lead to development of autoimmune and immune-mediated disorders including rheumatoid arthritis. ankylosing spondylitis, inflammatory bowel disease, refractory asthma
how does constant TNF signaling induce chronic inflammation?
cell death in epithelia increasing penetration of pathogenic molecules and microbes, Caspase8 can induce expression of proinflammatory genes, TNFR signaling can invoke other modes of cell death such as necroptosis- these result in release of cellular contents- induces inflammation
what % in people in the US suffer from arthritis and joint disease?
20%
what is a TNF inhibitor drug that is a monoclonal antibody against TNF?
infliximab
what is etanercept?
a TNFR-antibody fusion, drug that inhibits TNF
what causes autoimmune lymphoproliferative syndrome (ALPS)?
mutations in the FAS gene
what are the symptoms of ALPS?
chronic non-malignant lymphoproliferation, autoimmunity, cancers, lymphadenopathy, splenomegaly
why do mutations in the FAS gene lead to ALPS?
due to failure to cause apoptosis in B and T cells
what does adenovirus release a protein to inhibit?
p53
what does HPV release a protein to promote?
p53 degradation
how can enveloped viruses infect phagocytes?
coat themselves in phosphatidylserine to get phagocytes to envelop them
how does HIV avoid immune detection?
upregulates FAS and TNF in T-cells resulting in T-cell death
how does Trichomonas vaginalis utilise apoptosis?
induces apoptosis to allow it to invade the vaginal/urethral mucosa, induces apoptosis in immune cells such as macrophages to promote survival
how do T cells utilise FASL to kill foreign/diseased cells?
FASL mediated interaction with the FAS death receptor on the target cell to induce apoptosis
what are key immune checkpoint proteins expressed on T cells?
CTLA-4 and PD-1
what is CTLA-4?
cytotoxic T-lymphocyte associated protein 4
what does CTLA-4 bind to?
the B7 receptor
what is PD-1?
programmed death protein 1
what does PD-1 bind to?
PD-L1 (programmed death ligand 1)
what is the function of CTLA-4?
regulates T-cell proliferation early in an immune response, primarily in lymph nodes
what is the function of PD-1?
suppresses T cells later in an immune response, primarily in peripheral tissues
what do CTL-4 mutant mice display?
aggressive early onset autoimmune disease
what do PD-1 mutant mice suffer from?
late-onset autoimmune diseases like arthritis
how do tumours avoid destruction by T cells?
upregulating immune checkpoint proteins (PD-L1 particularly)- by amplifying the locus of the PD-l1 gene, upregulation of PD-l1 expression due to loss of Rb/p53, oncogenic transcription of PD-L1 by Myc
