BIO230 Lecture 10-12 (How are cell numbers controlled)

lecture 10,

what are the 3 checkpoints of the cell cycle

1. start transition

2. G2/M transition

3. metaphase to anaphase transition

what do cyclin dependent kinases required/ depended on to be active

cyclins

what do cyclin dependent kinases do

phosphorylate target protiens for the cell cycle process - they mediated checkpoints

what do cyclin-Cdk combinations control

which target proteins are phosphorylated , they are made and broken down at different cell cycle stages 

changing cyclins changes what gets phosphorylated

one cdk may work with multiple cyclins but cyclins only work at one stage (just remember)

one cdk may work with multiple cyclins but cyclins only work at one stage

how is cyclin-cdk regulated

1. activating phosphorylation, cyclins = allow passing of checkpoint

2. inhibitory phosphorylation, inhibitory proteins = do not allow passing of checkpoint

also regulated by synthesis and degradation of cyclins

• polyubiquitination marks for degradation

  • if broken down cdks no longer active

what do APC/C and Cdc20 do

promote M-cyclin degradation- they act as a E3 ubiquitin ligase

how are Cdks activated- 2 ways and how

• cyclins - exposes the T loop

• Cdk-activating kinase (CAK) - phosphorylates cdk t loop to further activate

what do protein phosphatases do (PP2As)

they reverse effects of cyclin- cdks by removing phosphorylation from proteins

how can cyclin-cdks be inactivated

Binding of Cdk inhibitor proteins (CKIs): CKIs such as p27 and p21 bind to Cyclin-Cdk complexes, blocking the ATP binding site and distorting the active site. This prevents the Cdk from phosphorylating its substrates, effectively halting cell cycle progression.

Inhibitory phosphorylation: The kinase Wee1 adds an inhibitory phosphate group to a specific site on the Cdk, which inactivates the Cyclin-Cdk complex. This acts as a "red light" for cell cycle progression.

Protein phosphatases: Enzymes like protein phosphatase 2A (PP2A) remove phosphate groups from proteins that were previously phosphorylated by cyclin-CDKs. By dephosphorylating these targets, PP2A reverses cyclin-CDK-driven events and applies a brake on cell cycle transitions.

what does phosphatase Cdc25 do

removes inhibitory phosphorylation on M-cdk and reactivates it (if it has a phosphorylation also in active site) 

how are cells retained in G1 by Rb inhibition of E2F

• E2F is a transcription factor required for S-phase gene expression

• RB binds and inhibits E2F

• RB can be inhibited via phosphorylation 

so if RB is bound to E2F cannot express genes

if RB is not bound to E2F (ie is phosphorylated) then E2F can express genese

what are mitogens

extracellular signaling molecules

how can mitogens trigger production of Myc

they can activate signaling pathways including RTKs, Ras and MAP kinase cascade or upregulate Myc expression

what is Myc

the key transcription factor involved in cell cycle control - inhibits Rb

how does Myc inhbit Rb

1. it activates G1-cyclin gene expression which phosphorylates Rb which then releases E2F

2. E2F activates expression of S-phase genes

apoptosis

programmed cell death - highly regulated, reproducable - careful removal of cell signals by engulfment

necrosis

accidental, uncontrolled cell death and can cause inflammation

what are caspase proteasses, how are they originally made

proteases cleave specific target proteins to trigger apoptosis. synthesized as inactive procaspases and signals initiate caspase cleavage to form active caspase dimers 

how are caspase cascades made

some caspases can cleave and activate other caspases to create and amplified caspase cascade

initiator caspases- give two examples

cleaved and activated in response to apoptotic signals

caspase-8, 9

executioner caspases

cause a caspase cascade when cleaved/activated by initator caspases

caspase-3, 6, 7

• they indirectly cause DNA breakdown 

• alter cell surface lipid composition

how do executioner caspases alter cell surface lipid composition

apoptotic cells fail to maintain healthy lipid bilayer balance so caspase cleaves flippase to inactivate it and it activates scarmblase to move more lipids to the outer layer 

what does apoptosis depend on when triggered intrinsically? extrinsically?

intrinsically: depends on intracellular receptors (cytochrome C)

extrinsically: depends on cell surface receptors binding to an extracellular signal molecule to activate executioner caspases

how does the extrinsic apoptosis pathway work

The extrinsic apoptosis pathway is triggered by extracellular death signals binding to cell surface death receptors.

• Example: Killer lymphocyte expresses Fas ligand (FasL), which binds to the Fas receptor on the target cell.

• Binding exposes the intracellular death domain of the Fas receptor.

• The exposed death domain recruits adaptor protein FADD, which has both a death domain and a death effector domain.

• FADD binds its own death domain to the Fas receptor and uses its death effector domain to recruit procaspase-8 molecules.

• Two procaspase-8 molecules undergo cross-cleavage and activation, forming active caspase-8 in a signaling cluster called Death-Inducing Signaling Complex (DISC).

• Activated caspase-8 initiates a cascade by cleaving and activating executioner caspases like caspase-3.

• Executioner caspases systematically dismantle cellular components leading to programmed cell death.

how do healthy cells avoid apoptosis

they express decoy receptors to evade extrinsic apoptosis pathway signals- decoy receptors lack the intracellular death domain

no DISC assembles when Fas ligand binds a decoy receptor therefore apoptosis is not triggered

how does the intrinsic apoptosis pathway work

The intrinsic apoptosis pathway is a form of programmed cell death relying on intracellular signals, particularly from mitochondria.

• Cytochrome c binds to Apaf1 along with dATP in the cytosol, exposing Apaf1's CARD domain.

• This binding enables Apaf1 to oligomerize into a wheel-like apoptosome structure.

• The CARD domains of Apaf1 align with the CARD domain of caspase-9 within the apoptosome, recruiting and activating caspase-9.

• Activated caspase-9 triggers a cascade by cleaving executioner caspases such as caspase-3.

• Executioner caspases systematically dismantle cellular components by cleaving specific substrates:

• Inactivate ICAD to allow DNA fragmentation by CAD

• Alter flippases/scramblases to disrupt membrane lipid asymmetry

• This disruption acts as an "eat me" signal for phagocytes to engulf dying cells

where is cytochrome c usually found, how does it enter the mitochondiral membrane

usually in the intermembrane space of the mitochondira

apoptotic stimuli trigger Bak and Bax proteins to form a channel to promote mitochondiral outer membrane permeabilization (MOMP)

what do Bcl2 and BclxL do

they can bind to Bak/Bax and prevent them from forming channels

what are the anti-apoptotic proteins 

Bcl2, BclxL → because they block MOMP

XIAP → can directly block both initiator and executioner caspases 

inhibitors of apoptosis (IAPs) → block apoptosis form accidentally occuring 

what are the pro-apoptotic proteins

Bad → binds to Bcl2 and BclxL to promote MOMP

Anti-IAP → inhbiit XIAP allowing the caspase cascade to trigger apoptosis; they are relased with cyt c when MOMP is triggered 

what are cell survival factors for

• they are required in order for a cell to continue living, a cell will undergo apoptosis if not continually supplied with survival factors

• cells that leave their correct environment will not longer be protected by survival factors 

carcinoma cancer

cancers from epithelial cells

sarcoma cancer

cancers from connective tissue and muscle

leukemia cancer

cancer from blood cells

tumor vs cancer

tumor → cells that survive, grow and dirivde inappropriately

cancer → malignant tumor with cells that have invaded surrounding tissue 

what does metastasis allow for

tumor cells to colonize additional tissues; benign tumor forms then becomes cancerous and invades and circulates the body and forms a metastatic (secondary) tumor

NOTE: only one cell needs to survive for this to occur

what causes tumor growth

decreased apoptosis and increased cell division

(can be one or other or both)

also changes in cell adhesion 

what are two types of genetic instability that allows cancers to gain additional mutations quickly

• large scale rearrangements

• point mutations 

why does mutagens increase probability of mutations

because they introduce additional sources of DNA damage beyond the natural errors that occur during DNA replication and repair.

e.g. UV, x rays 

what is the ames test

test to detect chemical mutagens

• need test compound and a test with and without homogenized liver extract

  • bc want to know if liver detoxifies it or makes it worse

what are oncogenes vs tumor-suppressor genes

oncogenes→ continue to divide (gain of function, dominant)

tumor-suppressor → prevent cell proliferation excessively  (loss of function, recessive)

what does myc oncogene transcription factor do

promotes cell cycle progression → overexpression can be bad and cause cells to move through their cycle too quickly 

Abl

a tyrosine kinase involved in cell signaling

philadelphia chromosome

chromosomal rearrangement that causes fusion between Bcr-Abl which removes the regulatory component of Abl and makes it hyperactive to keep on surviving 

tumor suppressor gene are _______ in tumor cells. Oncognes are _________ in tumor cells. (inactive/active)

inactive, active 

what does gleevec do

targets the Bcl-Abl kinase

what factors contribute to cancer

1. genetics

2. environment

3. probability 

what is p53

a tumor supressor that is activated when the cell might be in danger- it leads to cell-cycle arrest, senescence or apoptosis

loss of p53 allows cells to survive when they shouldn’t