Control of Cell numbers Flashcards
four main phases of the eukaryotic cell cycle
- M phase (mitosis and cytokinesis)
- G1 (cell growth and partial doubling of proteins and organelles)
- S (DNA replication)
- G2 (cell growth and remaining doubling of proteins and organelles)
three major checkpoints
- G2/M –> enter mitosis
- metaphase-anaphase –> trigger anaphase and proceed to cytokinesis
- start checkpoint –> enter cell cycle and proceed to s phase
Cdks
Cyclin dependent kinases
- protein kinases with targets that control the cell ccle
- activity allows passage through a checkpint
- dependent on cyclin binding and other modifications
Cdk checkpoints
M phase
- binds to M-cyclin to create M-cdk
- triggers mitosis machinery
- degraded to stop mitosis
S phase
- binds to S-cyclin to create S-cdk
- triggers DNA replication machinery
- degraded to stop replication
cyclins in yeast and vertebrates
VERTEBRATES
- different cyclin
- sometimes the same Cdk
YEAST
- different cyclin
- always the same Cdk (Cdk-1)
how is cdk activated
inactive –> t-loop blocks the active site
low/ no activity –> t-loop a part of the cyclin
full activity –> CAK comes in and phosphorylates
S-cdk and M-cdk
S-Cdk promotes DNA replication
M-Cdk phosphorylates multiple targets required to start mitosis
what turns cdk off?
targeted degradation of cclins
- e.g. anaphase promoting complex (APC) targets M-cyclin to the proteasome allowing the completion of mitosis
- E3 adds a polyubiquitin tail causing degradation
CKI
Cdk inhibitor proteins
- regulate Cdk activity by clamping on the protein and inactivating it
- the active site is distorted and the ATP binding site is blocked
Cdk regulation by phosphorylation
Wee1 kinase phosphorylates cdk on a different spot, causing inhibitory phosphorylation
Cdc25 phosphatase removes the inhibitory phosphate
- see lecture 10 for summary slide
Rb
Retinoblastoma protein
- tumour suppressor
- inhibits cyclin synthesis and blocks G1 progression and S phase
- without it, E2F is always active and DNA proliferation goes on uncontrolled
mitogen activation of the cell
mitogen signaling induces Myc transcription via a Ras-MAP kinase signaling cascade
- increases cyclin synthesis and CKI degradation by regulating transcription
Myc
an oncogene
- overactivity leads to cancer due to excess cell proliferation
temporal feedback promoting M phase
M-cyclin + Cdk-1 –> inactive M-Cdk –> CAK + Wee1 –> M-Cdk has both inhibitory and activating phosphates –> Cdc25 is activated by polo kinase –> active M-Cdk
positive feedback for Wee1 and Cdc25 phsophorylation
p-53
tumour suppressor
- loss of p53 leads to cancer
- stops cell cycle in response to DNA damage
DNA damage –> phosphorylation of p53 –> Mdm2 can’t bind –> active p53 binds to regulatory region of p21 gene –> transcription of clamp protein
apoptosis
programmed cell death
- sculpts fingers and toes
- kills dangerous cells
necrosis
accidental cell death
- can lead to a damaging inflammatory reaction
how does apoptosis avoid inflammatory reactions?
- shrinking the cell
- collapsing the cytoskeleton
- fragmenting the NA
- signaling to macrophages for cell removal by engulfment
how is apoptosis triggered?
- cysteine proteases cleave target proteins at SPECIFIC aspartic acid residues
- synthesized as procaspase precursor molecules (ready to be activated)
- procaspases are cleaved and activated by other caspases
- leads to an amplified cascade of caspase activity
individual caspase activation
procaspases activated by cleavage
- two cleavage sites
- one multiprotein complex made of small and large subunits
- caspase-8, -9, -10
- results in cleavage of cytosolic protein and nuclear lamin
detecting fragmented DNA in a gel after induction of apoptosis
- Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL)
- cells have phospatidylserine in the outer leaflet instead of the inner
- time is dependent on stimulus
what activates caspase cascades
extrinsic signals (e.g. via death cell receptors)
- Fas (death) ligand binds to Fas receptor
- assembly of DISC (death inducing signaling complex)
- activation and cleavage of procaspase-8, -10, or both
- activation of executioner caspases
intrinsic (e.g. triggered from within)
- apoptotic stimulus causes release of cytochrome c
- activation of Apaf1, dATP –> dADP
- assembly of apoptosome triggered by release of dADP in exchange for dATP
- recruitment and activation of procaspase-9 which cleaves and activates executioner procaspases
extracellular inhibitors for apoptosis
decoy receptors act by competitive inhibition
- have a ligand-binding domain but not a death domain
- out-compete functional Fas death receptors
intracellular inhibitors for apoptosis
examples of competitive inhibition
- e.g. mimic of an initiator caspase that lacks a proteolytic domain
Bcl2
inhibits channel formation in the outer mitochondrial membrane
- stops BH123 proteins from being released by apoptotic stimulus
IAPs
inhibitors of apoptosis
- block caspase activity in the cytoplasm
- bind to caspases
- stopped by anti-IAPs to activate apoptosis
cells can choose to induce or prevent apoptosis
- default state kills off cells if they leave their protective environment
- survival factors are used to control proper cell numbers in body tissues
molecular machinery and pathways linked to cancer
oncogenes and tumour suppressors often function in the same pathway
basic bio of cancer
cancer develops from a cell gaining a mutation allowing it to survive and divide forming a tumour
- clonal origin of cancer
carcinoma
epithelial cell cancers
sarcoma
connective tissue or muscle cell cancers
leukemia
blood cell cancers
tumour (neoplasm)
uncontrolled growing mass of abnormal cells
- not all tumours are cancers and not all cancers have tumours
benign vs malignant tumours
benign
- growing mass is self-contained
- still has cell-cell adhesion and basal lamina
malignant
- aggressive tumor that has broken free and invaded surrounding tissue
- destroys surrounding area
how are tumours categorized?
by stage
- metastatic tumour: cancer invading other tissues
cancerous cell DNA
DNA is disrupted
- large scale chromosomal abnormalities
- point mutation (can’t be detected by karyotypes)
what causes mutations leading to cancer?
chemical mutagens
- can be either natural or man-made
the Ames test
- mix test compound, histidine-dependent salmonella, and homogenized liver extract
- plate out on agar medium lacking histidine
- nothing should grow if there’s no mutagen
experiment must be done with and without the liver extract
oncogenes
cancer arises from a gain of function mutation
- normal form of the gene is a proto-oncogene (e.g. Ras, Myc)
- mutations are dominant
tumour suppressor genes
cancer arises from a loss of function mutation (p53, Rb)
- mutations are recessive
Philadelphia chromosome
- example of producing an oncogene
- Bcr gene on chromosome 22 (ser-thr kinase) + Abl gene on chromosome 9 (tyrosine kinase)
- translocation fuses the two
- transcription and translation creates an oncogene causing chronic myelogenous leukemia
producing tumour suppressor genes
inactivating mutation event + inheritance/environment
how do mutations cause cancer
abnormal cell cycle
- leads to more cells in the tumour
- missed checkpoints permit further chromosomal aberrations
abnormal cell death
- loss of control of cell number
abnormal cell differentiation
- cells may live longer and acquire additional mutations
abnormal cell-cell interactions
- low cell adhesion contributes to metastasis
gleevec
drug treatment for chronic myelogenous leukemia targeting Bcr-Abl
- binds to Bcr-Abl
- cannot bind to substrate protein
- no signal
- no lelukemia
multi drug treatments vs sequential treatments
multi-drug treatments more effective
- sequential ones may allow for uncontrollable cancer resistant to both drugs
- simultaneous creates a cell resistant to only one drug
- side effects may aggregate
