Cancer & Cell Cycle Flashcards
basis of signalling
first messengers (cytokines, growth factors, hormones, neurotransmitters, NO, histamines, eicosanoids, nucleotides) act on receiver cells, which synthesise secondary messengers (cyclic nucleotides & lipids)
cytokines
peptides/proteins derived from leukocytes that act to cause movement, growth or proliferation in cells by binding to cell surface receptos
dissociation constant
concentration at which 50% of receptors are occupied at equilibrium
Kd = koff/kon
ratio of dissociation rate and binding constant (specific to molecule and weight)
Ga size
39-46kDa; has major structural variation due to coding by ~20 genes
GTPase activity
a molecular clock, as conversion rate is known (kcat = 0.05sec^-1)
- can be increased by GAPs (GTPase activating proteins)
Gb size
37kDa
Gy size
8kDa
Receptor tyrosine kinase (RTK) mechanism
binding of ligand causes conformation change resulting in self-phosphorylation to create a binding site for SHC/Grb2/SOS. These activate Raf which activates MAPK phosphorylation cascade, resulting in phosphorylation of transcription factors to change gene expression
Ras GTPase superfamily
Can be activated by RTK, cytokine receptor, or the beta-gamma subunit of G proteins.
Causes MAPK phosphorylation cascade, which is facilitated by scaffold proteins.
MAPK pathway functions
mitosis, inflammatory response, differentiation, apoptosis
MAPK common activation loop
T-x-Y (Threonine - x - Tyrosine)
ERKs (TEY)
SAPKs (TPY)
p38 homologs (TGY)
Extracellular MAPK initiation
Ligand binding to RTK -> SHC/GRB2/SOS -> Ras GTP activation
-> Raf/MAPKKK -> MEK/MAPKK -> ERK -> MAPK ->
differentiation & cell division related genes
Stress MAPK initiation
Stress (UV/cytokine/heat) activate RasGTP -> MEK4 -> JNK/SAPs
= cell division stops, stress response
PAK
p21 activated kinase
SH2
domain that binds phospho-tyrosine
- present in STAT and also in Shc/Grb2/SOS complex
SH3
pro-line rich domain commonly found in cytoskeleton, allows localisation of proteins to membrane
paracrine
sender and receiver are in close proximity (tissue transmission)
juxtacrine
sender and receiver are next to each other (contact transmission)
autocrine
The same cell both secretes and receives the messenger (self transmission)
SHC
adaptor protein containing Sh2 sequence; binds to phospho-Tyr
Number of Tyrosine phosphorylated in RTKs
Tyr68
Grb2
growth factor receptor binding protein 2; an adaptor protein that binds to phosphorylated SHC protein via SH2 domain
p21Ras
subtypes of small monomeric GTP binding proteins.
Activated through guanine nucleotide exchange factor Sos and inactivated through GAP (GTPase activating protein).
Sos
son of sevenless; recruited to membrane by binding to SH3 domain of Grb2 to cause cause p21 Ras GTP exchange
Raf/MEKK/MAPKKK
ser/thr protein kinase activated by Ras-GTP and translocated to membrane. Alsoknown as MEKK or MAP kinase kinase kinase or MAP 3kinase (3 being number of downstream kinases)
MEK/MAPKK
dual specificity kinase activated by phosphorylation on 2 serine residues by Raf/MAPKKK/MEKK
ERK/MAPK/SAP
ser/thr kinase activated by phosphorylation on threonine and tyrosine residues by MEK and interacts with transcription factors to control gene expression
the cell cycle
controls growth, development, repair
heavily regulated by kinase activity and growth factors
cdks
cyclin-dependent kinases; activated by cyclins
g0
gap phase: cells are metabolically active but no growth occurs
G1
Growth of proteins and organelles necessary for growth processes and DNA replication processes.
Only phase that integrates external signals such as nutrients, growth factors, and suppressive factors with internal signalling.
G1 checkpoint
The restriction point assess whether there is sufficient nutrients and growth factors, it also checks DNA for any damage.
G1 regulation
Cyclin D
1. Links external and internal signals (Cyclin D generated by growth factor)
2. forms the cyclinD-cdk4/6 complex, allows cyclin E-cdk2 complex to form to drive through G1
3. Prepares for DNA replication through pocket proteins #RB protein activation
pocket proteins (RB)
RB, or retinoblastoma protein, acts to regulate whether gene transcription can occur.
- Couples cell cycle proteins #cyclin to the expression of genes required for cell cycle progression and DNA synthesis
- Also couples growth factors to gene expression
- mutations in RB account for 90% of Cancer
Pocket protein mechanism
Normally bind E2F, which prevents transcription. Phosphorylation by cdk4/6-cyclinD then cdk2-cyclin E allows E2F release, and gene expression of
- cyclin A & E
- DNA polymerase & helicase
- dihydrofolate reductases (purine synthesis)
- thyidilate synthase (pyrimidase)
Oncolytic adenovirus
Hijacking of adenovirus action: E1A frees Rb and E2F, driving into S phase, transcription of viral DNA = cell death and infection of nearby cells
removal of E1A gene means the virus can only act on cells with free E2F ie: will mainly target cancer cells with uncontrolled replication
S
DNA replication phase
G2
Growth of structures necessary for mitosis
G2 checkpoint
Checks that ‘S’ phase completed properly by sensing DNA damage and DNA replication integrity
G2 regulation
Nuclear envelope disintegration causes cyclin A degradation. Cyclin B synthesis rises until a threshold is reached.
Once sufficient cyclinB-cdk1 is reached, mitosis occurs
- Acts to condense DNA, disassemble nuclear envelope, rearrange cytoskeleton into spindles
- through phosphorylation of:
- Histones H1/H3 (to unwind DNA)
- Lamin (cytoskeleton)
- nucleolin (nucleus protein)
M
mitosis
M checkpoint
Senses spindle assembly, and that the chromosomes have separated correctly
Once chromosomes confirmed to be alg, cyclin B is degraded
M regulation
Controlled by cdk1 phosphorylation:
single phosphorylation by CAK/cdc25 = active, cells cannot exit from mitosis
triple phosphorylation (via Myt1/Wee1) = inactive = can exit from mitosis
regulation of Cdk
Temporally regulated by cyclin availability. Physically regulated by CKIs (cyclin-specific kinase inhibitors) and phosphorylation.
KIP/CIP
form complexes with cycling and cdks to prevent activation
= p21, p27, p57
INK4
binds cdk4/6
= p15, p16, p18, p19
cdk phosphorylation sites
threonine 14 & tyrosine 15 = inhibitory phosphorylation
threonine 160 = activating phosphorylation
p53
A transcription factor that detects damage in DNA. If damage is noticed, concentration of p53 increases and p21 is induced.
Expression of cdk is constitutive (always present), and increases in p53 are equally rapidly terminated
characteristics of cancer
- sustained proliferative signalling
- evading growth suppressors
- tissue invasion and metastasis
- replicative immortality
- inducing angiogenesis
- evading apoptosis
growth factors
act on RTKs to change gene transcription, typically result in cell cycle initiation
common mutations in cancer
kinases (autophosphorylation)
transcription factors
cyclin
p53
= continual cell cycle
multi-step development
The idea that accumulation of mutations- caused by either toxins from environment, or by general errors in DNA replication- leads to increased liklihood of cancer development
Key event is a series of mutations that alter the cells ability to detect and repair damaged DNA.
= This allows the cells to survive and divide, causing a cascade of mutations that accumulate to become a malignant phenotype
chemotherapy
often used as an adjunct therapy to surgery (tumour removal) and radiotherapy in order to reduce the chances of metastasis
often the only remaining treatment option once metastasis has occurred
used on a long term basis to reduce the chances of remission (re-occurrence of the cancer after a long period of time)
log-kill hypothesis
based on the assumption that tumour size doubles every three months
- 2.5 years for a tumour to develop from 1g to 1kg resulting in death
cytotoxic drugs kill a certain percentage of cells (proportional to dose)
- tumour cells don’t rapidly recover, however healthy tissue does
issues with chemotherapy treatment
even an extremely effective drug that kills 50% of a tumour will take two years to eradicate the tumour
- side effects on healthy cells can lead to death
- tumours can acquire drug resistance
pulse therapy
administration of anti-cancer drugs with a rest phase to allow recover of healthy tissue
- minimise organ/immune damage and maintain tumour depletion
combination therapy
administration of a drug ‘cocktail’ with different mechanisms of action and toxicities
- dispersal of side effects allows greater dose
- decreased chance of resistance
- synergistic effect
cisplatin toxicity
renal
bleomycin toxicity
pulmonary
doxorubicin toxicity
cardiac
vincristine/paciltaxel toxicity
neurological effects (impact microtubules)
MTX/cyclophosphamide toxicity
immunosuppression
drugs used to minimise side effects of chemo
anti-nausea, anti-emetics (anti-vom/nausea), analgesics, anti-wasting drugs
alkylating agents
series of compounds based on sulphur gases that act to alkylate nitrogen atoms of guanine/nucleophilic centres in DNA to prevent or disrupt separation during replication
- single lesions (less effective, promote mutations)
- double lesions (link between/within strands to permanently distort DNA structure
abnormality (alkyl group) detected = apoptosis
cyclophosphamide
an alkylating agent (targets DNA replication) that has good solubility, thus good action as a drug
chlorambucil
an alkylating agent (targets DNA replication) that has a greater ability to pass through metabolism, that can be further distributed throughout the body
nitrogen mustard
an alkylating agent (targets DNA replication) that causes inter-strand joining of DNA to cause apoptosis
mitomycin C
an alkylating agent (targets DNA replication) that causes inter-strand joining of DNA to cause apoptosis
cisplatin
an alkylating-like agent that distorts DNA causes cell death when DNA is separated for replication
context of anti-folates
In order to form DNA, there must be sufficient nucleotides. Purines undergo synthesis through carbon addition via tetrahydrofolic acid (THFA)
- THFA is formed by reduction of folic acid, which is catalysed by dihydrofolate reductase
- Anti-folate drugs bind to inhibit dihydrofolate reductase
MTX
an anti-folate (target purine synthesis);
a synthetic analogue of Folic acid that binds to dihydrofolate reductase with greater affinity than folic acid to inhibit it.
This limits formation of THFA, thus limiting nucleotide formation, thus DNA replication is arrested
+ Can also act as an immunosuppressant, as it acts on rapidly dividing cells such as white blood cells.
This decreases the speed of immune response, which is effective in conditions such as autoimmune disease
anti-metabolites
- Interfere with base precursors by acting as analogues of bases or base precursors
- Act as pseudo feedback inhibitors
1. In order to conserve energy, cells have feedback mechanisms to determine how many bases are present; and how many need to be synthesised. The analogues trick the cell into believing there are sufficient bases, and thus base synthesis is closed.
2. Enzymes are unable to add natural bases to the synthetic ones, thus biosynthesis is blocked by the analogue
6-fluorouracil
an anti-metabolite that acts to misleadingly convey negative feedback that sufficient endogenous bases are present, and blocks biosynthesis as cannot be incorporated into DNA
anti-mitotic drugs
Interfere with microtubules requires for mitosis.
Can be used to synchronise cell replication by locking cells in mitotic phase, creating increase susceptibility of cells.
Cells populations are normally heterogenous; all cells are at different phases of cell cycle. This decreases the maximal of drugs that act in specific phases of the cell cycle. Anti-mitotic drugs drive cell populations to become homogenous.
(vinca alkaloids, taxanes)
vinca alkaloids
anti-mitotic drugs that block microtubule assembly by inhibiting tubulin polymerisation, thus arrest mitosis at metaphase
Eg: vinblastine + vincristine
vinblastine and vincristine
vinca alkaloids that block microtubule assembly by inhibiting tubulin polymerisation, thus arrest mitosis at metaphase
taxanes
anti-mitotic drugs that block microtubules disassembly by binding preferentially to assembled tubulin to enhance the assembly of microtubules and stabilise them against depolymerisation
Mitosis is stopped when the stable, nonfunctional microtubules fail to form a normal mitotic apparatus
(eg: paciltaxel, docetaxel)
actinomycin D
an antibiotic drug that inhibits the process of transcription in protein synthesis by binding to DNA (via three conjugated aromatic rings), preventing the movement of RNA polymerase (via chunky side chains)
topoisomerase inhibitors
Bind to dsDNA and inhibit topoisomerase by stabilising the DNA complex, causing disrupting of replication
topoisomerase
Topoisomerase is an enzyme that cuts (ssDNA or dsDNA) at ‘knots’ to relieve tension. Once ‘knot’ has been ligated must unravel.
Only ‘enzyme’ that does not chemically modify
Type I: Cuts one strand of DNA, increasing flexibility
Type II: cuts DNA through both strands
- disrupts ligation of dsDNA
common side effects of chemotherapy
- Alopecia (hair loss)
- Gastrointestinal disorders (nausea, vomiting, anorexia, diarrhoea), and mucositis (inflammation of the mucous membranes of the GI tract and mouth) can be very severe and cause non-compliance
- Fever, extravasation (dispersal of IV solution out of vessels and into tissues, causing necrosis), impaired immune function and wound healing
- Long-term toxicity can include infertility and carcinogenic and teratogenic effects
reasons why cell death is initiated
- exposure to toxins, infection, injury, ischaemia, inflammation
- maintain cell homeostasis
extrinsic/death receptor apoptosis pathway
- ligand (FasL/ TNF-a, TRAIL) binds to death receptor (Fas/TNFR1/DR5)
- recruitment of death domains, which recruit adaptor proteins (DEDs)
- DED binds pro-caspase 8
- cleavage of pro-caspase 8 and dimerisation to form caspase 8
- activation of caspase 3, 6, & 7 cause apoptosis by cleaving DNA and nuclear enzymes
Death receptor ligands
FasL, TNF-a, TRAIL
death receptors
Fas, TNFR1, DR5
Pro-caspase 8
constitutively present in cytoplasm, implicated in extrinsic death pathway, requires cleavage and dimerisation to be activated
action of caspase 8
activates caspase 3, 6 & 7 = cause cleavage of DNA and nuclear enzymes to cause apoptosis
+ truncates Bid -> tBid
intrinsic/mitochondrial apoptotic pathway
pro-apoptotic Bcl proteins form transition pores in the mitochondria membrane
- Release of cytochrome C from inner membrane of mitochondria to cytoplasm
- formation of apoptosome by binding of Cyt. C and Apaf-1 x 7
- apoptosome binds caspase 9
- dimerisation of caspsae 9
- activation of other caspases, including caspase 3
- apoptosis
apoptosome
formed from 7 units made up of cytochrome C and apaf-1 ATP-coupled binding
requirement for 7 subunits acts a threshold requiring sufficient damage to occur before apoptosis
differentiating apoptosis vs necrosis
apoptosis requires ATP, necrosis does not.
Double staining with fluorescent Annexin-V and propidium iodide
= vertical; necrosis
= horizontal; apoptosis
(secondary necrosis always occurs in cell cultures due to limited ATP)
apoptosis plasma membrane remains intact, in necrosis it breaks apart
Bcl-2 family
family of pro/anti-apoptotic molecules
- all contain a BH3 domain for ligand binding
pro-apoptotic Bcl proteins
Bim, Puma, tBid, Bax, Bak, Noxa, Bad
anti-apoptotic Bcl proteins
Bcl-2, Bcl-xL, Bcl-W, Mcl-1, A1
shared features of extrinsic/intrinsic apoptosis
- caspase 8 (from extrinsic) converts Bid to tBid (a pro-apoptotic Bcl) which stimulates Bax/Bak to form pores in mitochondria
- both activate caspase 3
apoptotic changes
DNA fragmentation, phosphatidylserine exposure, changes in cell shape, cell breaks apart & undergoes phagocytosis
DIABLO-Smac
acts as a handbrake in intrinsic pathway: inhibits IAP, which inhibits caspase 3/9 (disinhibition)
phosphatidylserine exposure
inversion of phospholipid of cell membrane causing membrane disintegration
caspase enzymes
cysteine aspartate protease family that cleaves proteins after an aspartate residue
- secreted as zymogens/pro-enzymes thus are constitutively expressed and inactive
- cysteine site must be reduced for activation (caspases are inactive in highly oxidative environments)
morphological changes in apoptosis
- cell shrinkage
- chromatin condensing
- intact plasma membrane
- formation of ‘blebs’ which package cellular contents into apoptotic bodies for phagocytosis
- DNA fragmentation
- nucleus forms pyknosis shape
measurement of caspase activation
western blot
measurement of mitochondrial changes
- loss of outer membrane permeability = flow cytometry/TMRE (red mitochondrial stain)
- release of proteins (cyt. C) = western blot comparing mitochondrial levels to cytosol
- Bcl levels (western blot)
measurement of DNA fragmentation
- propidium iodide dye on fixed cells
- flow cytometry
- if peak below G2 (less than two chromosomes) indicates DNA fragmentation
measurement of phosphatidylserine exposure
- double staining with annexin-C and propidium iodide
- flow cytometry
= upwards movement = high PI, high A = necrosis
= horizontal movement = low PI, high A = apoptosis
non-classical apoptosis
Mechanisms of cell death that exist on the spectrum between classical apoptosis and necrosis.
Have similar processes, but with different involvement of certain enzymes, organelles, etc.. Eg: caspase-independent, lysosomal, autophagic
necrosis
regulated by PARP & RIP1 kinase
- cell swells and bursts releasing intracellular contents including proteases and other damaging enzymes
- causes damage to surrounding tissue, thus provokes immune response
- cell debris often poorly cleared due to lack of scavenger receptors
morphology of necrosis
(measure nucleus via Hoescht/DAPI or whole cell via electron microscopy)
- release of cellular contents
- DNA becomes diffuse
- cell swelling
- plasma membrane lysis
biochemical detection of necrosis
- low ATP
- RIP1 kinase activation (western blot)
- Trypan blue/techniques that show compromised membrane
- annexin V + propidium iodide = high for both means necrosis
NO synthesis
Arginine converted to hydroxy-arginine, converted to citrulline with NO as a biproduct
- mediated by H4B and CaM
H4B
tetrahydrobiopterin
Calmodulin
Binding of Ca2+ to calmodulin allows reductase and oxygenase domains of NOS to be in close proximity.
Allows electron flow (from NADPH) through flavin centres to Fe/H4B, which facilitates binding of oxygen to arginine
NO diffusion time
0.001 second to cross cell membrane
NOS-3
eNOS (133kDa)
constitutively active, calcium dependent, angiogenesis & smooth muscle relaxation
NOS-2
iNOS (131kDa)
inducible, calcium independent, associated with immune defence via ONOO-
NOS-1
nNOS (160kDa), constitutively active, calcium dependent, retrograde messenger
NO downstream effects
binds to soluble guanylyl cyclase to increase cGMP, which phosphorylates PKG to promote the open state of cation channels
eNOS mechanism
smooth muscle relaxation
- calmodulin (bound to Ca2+) activates myosin kinase, causing contraction
- NO binds to guanylyl cyclase, increasing cGMP
- cGMP activates PKG
- PKG pumps Ca2+ into intracellular domains, decreasing cGMP
= less Ca2+ = less calmodulin activation on myosin kinase + more phosphodiesterase = relaxation
phosphodiesterase
dephosphorylates
peroxynitrite (ONOO-)
synthesised when levels of NO are high
- DNA damage = p53 activation (apoptosis) & PARP activation
- mitochondrial damage = ATP depletion (due to damage and PARP activation) + calcium dyshomeostasis = necrosis
NO as a retrograde messenger
NO (from nNOS) can diffuse to act on pre-synaptic terminal, enhancing glutamate release
and promoting dendritic growth by actions on guanylyl cyclase
NO donors
used clinically for cardiovascular disease, pulmonary hypertension, topical wound healing
used experimentally for anti-inflammation, anti-cancer, anti-viral, and anti-bacterial action
organonitrites
composed of ONO2 groups held together by alkyl group
used during cardiac surgery and in angina treatment
GTN
nitroglycerin/glyceryl trinitrate
an organonitrite
side effects of organonitrites
decreased blood pressure = headache
NO as a cytotoxic drug
Nanoparticles are used to encapsulate NO donors.
includes polymeric carrier POEGMA-b-PPA to form protected NO donor (P-NO)
delivery micelle = P-NO-PMs
mechanism of P-NO-PMs
nanoparticle encapsulate protected NO donors injected into blood, which travel to tumour via leaky blood vessels
- endocytosis into tumour cells
- nanoparticle degraded by acidic endosomes
- release of NO in presence of glutathione
IAPs
inhibitor of apoptotic proteins
