Cell Birth And Death

Front 1

What is the purpose of flow cytometry?

Back 1

To determine the number of cells in G1, S, G2-M.

Add propidium iodide to cells. Cells take up the dye and intercalate with DNA. The amount of stain is proportional to the amount of DNA

Front 2

What are cell cycle regulators?

Back 2

cyclins, cdks, cyclin-dependent kinase inhibitors (cki's)

Front 3

What regulates cyclins?

Back 3

transcription, translation, proteosomal degradation

Front 4

What regulates cdk's?

Back 4

phosphorylation, dephosphorylation

Front 5

What do cki's do?

Back 5

Binds cyclin or cyclin/cdk and causes inactivation of cdk activity

Front 6

what are the two families of Cki proteins?

Back 6

CIP/KIP - bind to cyclin/cdk complex (ternary - binds to something that binds to the cdk)

INK4 - bind cdk - binary (binds directly to the cdk)

Front 7

what is the purpose of cell cycle check points?

Back 7

to make sure that damaged chromosomes dont continue on to get replicated and passed onto daughter cells. Also, to make sure that genome is only replicated once per cell cycle.

Front 8

What happens if you lose both copies of the Rb tumor suppressor gene?

Back 8

uncontrolled cell growth, tumor

Front 9

What are the two forms of control at the G1 checkpoint? (to block G1 to S)

Back 9

1. Low level of DNA damage: induces low-level p53 expression --> growth arrest --> upregulation of p21 expression --> p21 binds to CDK, blocks G1/S progression
= cytostatic function


2. High level of DNA damage: induces high level p53 expression --> induction of Bax protein --> cell death
= intrinsic pathway of apoptosis

Front 10

What is Bax?

Back 10

Member of Bcl2 family of proteins
acts at mitochondria to induce cell death by blocking the action of Bcl2, so that the membrane is permeable to cytochrome c (a potent death effector)

Front 11

How is apoptosis triggered?

Back 11

Bax or Bak induces the release of death-promoting factors (ie. cytochrome c) . death inducers (like cytochrome c) are stored in intermembrane space of the mitochondria. cytochrome c activates initiator caspase 9.

cytochrome c activates adaptor protein --> assembly of adaptor proteins --> recruitment of procaspase 9 molecules --> apoptosome

Front 12

How is procaspase activated?

Back 12

Cleaved (into caspase molecules)

Procaspase = inactive enzyme (proenzyme)

Front 13

What is caspase?

Back 13

cystine-aspartase protease

Cleaves other proteins - required for apoptosis.
Can cleave and activate other caspases. (so initiator caspases cleave and activate executionor caspases)

Caspases also activate the endonucleases that cleave DNA.

Front 14

What is caspase 9?

Back 14

Initiator caspase, activated in response to cytochrome c release

Front 15

What are the anti and pro-apoptotic members of the Bcl2 family?

Back 15

anti: Bcl2, BclXL
pro: Bax, Bak, Bid

exist as dimers, which affects function. Ie. Bcl2 + Bax = not pro-apoptotic

Front 16

How do pro-apoptotic proteins work?

Back 16

Form channels or pores in the mitochondria that allow cytochrome c release

Front 17

What happens in B cell lymphoma?

Back 17

Bcl2 driven by a very active promoter (due to chromosomal translocation), which blocks cell death after massive clonal expansion.
Bcl2 = first apoptotic gene implicated in cancer

Front 18

What morphological changes occur in an apoptotic cell?

Back 18

- At the very end, you see TB (trypan blue), which can only pass through a compromised cell membrane (so no longer in tact)

cells shrinkage, blebbing, chromatin condensation, DNA degradation, fragmentation of nucleus (then fragmentation of cell..), apoptotic bodies

Front 19

What are apoptotic cells? What happens to them?

Back 19

organelles, degraded DNA, other cellular material wrapped in a cellular membrane.

Engulfed by neighboring cells/macrophages

Front 20

What is the signal for macrophages to engulf dying cells?

Back 20

Phosphitadylserine (phospholipid) flipped to outer leaflet

(PS can be tagged with annexin V, a Ca-dependent protein which has a high affinity for PS)

Front 21

How can you tell if a cell is dying of apoptosis or necrosis?

Back 21

Cell morphology
DNA fragmentation patterns
Phosphatidylserine localization to outer leaflet

Front 22

How is chromosomal DNA fragmented/degraded? When does this occur in apoptosis (beginning or end)?

Back 22

In a DNA ladder pattern
- this is the final stage of apoptosis

Front 23

Name 3 chemotherapeutic agents (to treat cancer). How do they act?

Back 23

etoposide (topoisomerase II) -
camptothecin (topoisomerase I) - natural product drug
taxol
- induce endonucleolytic DNA cleavage

Front 24

what is cytostatic? cytotoxic?

Back 24

Cytostatic - induces growth arrest
Cytotoxic - induces cell death (apoptosis)

Front 25

How can you measure the efficacy of camptothesin, other chemotherepeutic agents, to kill cancer cells?

Can you always use this method to measure apoptotic cell death?

Back 25

Flow cytometry shows new population of cells in Sub-G1 - b/c they have less than 2n content of DNA during apoptosis

No - only can be used for intrinsic apoptotic pathway.

Front 26

What is the extrinsic pathway of apoptosis?

Back 26

Receptor/ligand interaction can activate caspace 8,10 without cytochrome c release from mitochondria.

caspase 8 activates downstream executioner caspaces (3,6,7), endonucleases, etc.

Front 27

How does the TRAIL induction of death receptors occur?

Back 27

- ligand docks on trimerized receptor
- Formation of DISC (Death-inducing signaling complex c)
- Induction of FADD (Fas associated death domain)
- Release of active caspase 8,10
- Activation of effector caspases 3,6,7

Front 28

How is Rb regulated?

Back 28

Central Control system communicates with the environment via growth factor signaling to regulate Rb

Front 29

Where is the Rb restriction point?

Back 29

Before S (DNA replication)

*between G1 and S

Front 30

How is Rb inactivated?

Back 30

phosphorylated by Cdk

Front 31

What happens if a cell does not express Rb?

Back 31

uncontrolled cell growth regardless of the nature of the environment (--> mutations)

Front 32

What does Cyclin D do?

Back 32

Major cyclin controlling progression from G1 to S

Front 33

How many D cyclins are in mammals?

Back 33

3 (Cyclin D1, D2, D3)

Front 34

What does p21 CKI binding result in?

Back 34

Blocks CDK activity
Rb remains active

Front 35

What percent of cancers have lost Rb function?

Back 35

50%

Front 36

What is the initiator caspase?
executioner caspases?

Back 36

initiator: Caspase 9, 8, 10
executioner: Caspase 3, 6, 7

Front 37

What can caspases cleave?

Back 37

- endonucleases to activate them
- other caspases
- important structural proteins, like lamins

Front 38

How do anti-apoptotic members of the Bcl2 family work?

Back 38

bind to pro-apoptotic proteins and prevent them from working

Front 39

What does the initiator caspase do?

Back 39

Cleave other caspases, which then become executioners which kill the cell

Front 40

What happens in necrosis?

Back 40

Cell membrane compromised entirely.
Genomic DNA spills out into external millieu (tissue, blood...)
Sets up inflammatory response in body

Front 41

What does FLIP do?

Back 41

In extrinsic apoptotic pathway...FLIP blocks caspase 8,10 activation (so prevents apoptosis)

Front 42

Both the intrinsic and extrinsic apoptotyic pathways have crosstalk through what protein?

Back 42

Bid

(can cause release of cytochrome c)