Control of Cell numbers

Question 1

four main phases of the eukaryotic cell cycle

Answer 1

• 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)

Question 2

three major checkpoints

Answer 2

1. G2/M –> enter mitosis
2. metaphase-anaphase –> trigger anaphase and proceed to cytokinesis
3. start checkpoint –> enter cell cycle and proceed to s phase

Question 3

Cdks

Answer 3

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

Question 4

Cdk checkpoints

Answer 4

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

Question 5

cyclins in yeast and vertebrates

Answer 5

VERTEBRATES

• different cyclin
• sometimes the same Cdk

YEAST

• different cyclin
• always the same Cdk (Cdk-1)

Question 6

how is cdk activated

Answer 6

inactive –> t-loop blocks the active site
low/ no activity –> t-loop a part of the cyclin
full activity –> CAK comes in and phosphorylates

Question 7

S-cdk and M-cdk

Answer 7

S-Cdk promotes DNA replication

M-Cdk phosphorylates multiple targets required to start mitosis

Question 8

what turns cdk off?

Answer 8

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

Question 9

CKI

Answer 9

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

Question 10

Cdk regulation by phosphorylation

Answer 10

Wee1 kinase phosphorylates cdk on a different spot, causing inhibitory phosphorylation

Cdc25 phosphatase removes the inhibitory phosphate

• see lecture 10 for summary slide

Question 11

Rb

Answer 11

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

Question 12

mitogen activation of the cell

Answer 12

mitogen signaling induces Myc transcription via a Ras-MAP kinase signaling cascade
- increases cyclin synthesis and CKI degradation by regulating transcription

Question 13

Myc

Answer 13

an oncogene

- overactivity leads to cancer due to excess cell proliferation

Question 14

temporal feedback promoting M phase

Answer 14

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

Question 15

p-53

Answer 15

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

Question 16

apoptosis

Answer 16

programmed cell death

• sculpts fingers and toes
• kills dangerous cells

Question 17

necrosis

Answer 17

accidental cell death

- can lead to a damaging inflammatory reaction

Question 18

how does apoptosis avoid inflammatory reactions?

Answer 18

• shrinking the cell
• collapsing the cytoskeleton
• fragmenting the NA
• signaling to macrophages for cell removal by engulfment

Question 19

how is apoptosis triggered?

Answer 19

• 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

Question 20

individual caspase activation

Answer 20

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

Question 21

detecting fragmented DNA in a gel after induction of apoptosis

Answer 21

• 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

Question 22

what activates caspase cascades

Answer 22

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

Question 23

extracellular inhibitors for apoptosis

Answer 23

decoy receptors act by competitive inhibition

• have a ligand-binding domain but not a death domain
• out-compete functional Fas death receptors

Question 24

intracellular inhibitors for apoptosis

Answer 24

examples of competitive inhibition

- e.g. mimic of an initiator caspase that lacks a proteolytic domain

Question 25

Bcl2

Answer 25

inhibits channel formation in the outer mitochondrial membrane - stops BH123 proteins from being released by apoptotic stimulus

Question 26

IAPs

Answer 26

inhibitors of apoptosis - block caspase activity in the cytoplasm - bind to caspases - stopped by anti-IAPs to activate apoptosis

Question 27

cells can choose to induce or prevent apoptosis

Answer 27

- default state kills off cells if they leave their protective environment - survival factors are used to control proper cell numbers in body tissues

Question 28

molecular machinery and pathways linked to cancer

Answer 28

oncogenes and tumour suppressors often function in the same pathway

Question 29

basic bio of cancer

Answer 29

cancer develops from a cell gaining a mutation allowing it to survive and divide forming a tumour - clonal origin of cancer

Question 30

carcinoma

Answer 30

epithelial cell cancers

Question 31

sarcoma

Answer 31

connective tissue or muscle cell cancers

Question 32

leukemia

Answer 32

blood cell cancers

Question 33

tumour (neoplasm)

Answer 33

uncontrolled growing mass of abnormal cells | - not all tumours are cancers and not all cancers have tumours

Question 34

benign vs malignant tumours

Answer 34

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

Question 35

how are tumours categorized?

Answer 35

by stage | - metastatic tumour: cancer invading other tissues

Question 36

cancerous cell DNA

Answer 36

DNA is disrupted - large scale chromosomal abnormalities - point mutation (can't be detected by karyotypes)

Question 37

what causes mutations leading to cancer?

Answer 37

chemical mutagens | - can be either natural or man-made

Question 38

the Ames test

Answer 38

- 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

Question 39

oncogenes

Answer 39

cancer arises from a gain of function mutation - normal form of the gene is a proto-oncogene (e.g. Ras, Myc) - mutations are dominant

Question 40

tumour suppressor genes

Answer 40

cancer arises from a loss of function mutation (p53, Rb) | - mutations are recessive

Question 41

Philadelphia chromosome

Answer 41

- 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

Question 42

producing tumour suppressor genes

Answer 42

inactivating mutation event + inheritance/environment

Question 43

how do mutations cause cancer

Answer 43

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

Question 44

gleevec

Answer 44

drug treatment for chronic myelogenous leukemia targeting Bcr-Abl - binds to Bcr-Abl - cannot bind to substrate protein - no signal - no lelukemia

Question 45

multi drug treatments vs sequential treatments

Answer 45

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