Mechanism of Cell Death: Apoptosis, Autophagy, and Necrosis Flashcards Preview

BMS 2 Week 3 > Mechanism of Cell Death: Apoptosis, Autophagy, and Necrosis > Flashcards

Flashcards in Mechanism of Cell Death: Apoptosis, Autophagy, and Necrosis Deck (26):
1

Apoptosis

The cell itself senses that there is something wrong with it and it decides to kill itself

2

Autophagy

Catabolic process where cell physically eats itself.

3

Necrosis

Premature death of cell by external factors accident or murder

4

Normal Functions of Apoptosis

1. Normal development: cell death needed for tissue sculpting

2. Immune system- removal of B and T cells after infection

3. Maintain homeostasis: balance between proliferation and cell death

4. Eliminating cells damaged by UV, chemical toxins, or viral infections

5

Inhibiting apoptosis results in:

1. Syndactily: finger webbing

2. Abnormal brain development

3. Tail resorption

6

Morphological markers of Apoptosis

1. Electron dense nuclei

2. Nuclear fragmentation

3. Intact cell membrane but there is blebbing due to actin cytoskeleton degradation

4. Disorganized cytoplasmic organelles.

5. Large clear vacuoles

6. Loss of cell-cell adhesion

7. Apoptotic bodies (contain DNA)

8. Chromatin condensation (DAPI)

9. Cell shrinkage

7

Phosphotidyl Serine

1. In early stages of apoptosis , phosphotidyl serine which is normally on cytoplasmic side flips to the outside of the cell.

  1. Can be stained with Anexin V antibody coupled with fluorophor
  2. Can use flow cytometry to see number of cells undergoing apoptosis by using column that contains Anexin 5 antibody to phosphotidyl serine

8

TUNEL assay

-Normal DNA is not nicked

-DNA that is in process of getting degraded has nicks

    *Terminal transferase is used to add labeled dUTP to

      the 3’end of the DNA fragments.

9

TUNEL assay Advantages and Disadvatages

Advantages

*High sensitivity so can detect fewer than 100 cells that are undergoing cell death

*Fast: can be completed in 3 hours

*High reproducibility with good precision

 

Disadvatages

*We don’t know minimum number of strand breaks necessary for detection so you may miss early stages of apoptosis

*Necrotic cells also have nicks so can’t really tell the difference

*Detergent used to permeabilize cells can make cell fragile

10

DNA laddering

If you take DNA from apoptosis and smear it, you get laddering with fragments of 180-185 bp due to DNA being cleaved by caspaces at the internucleosomal linker region

11

Initiator Caspaces

-2,8,9,10

-Give order to have the effector capsules kill the cell

12

Effector Caspaces

-3,6,7

-Caspaces kill the cell

13

Caspace activation

-Initiator caspaces are cleaved in two spots and become activated.

-Initiator caspaces cleave effector caspaces in 2 spots which activate them.

-Effector caspaces cleave:

   *nuclear lamins

   *inhibitors of DNAses

   *actin

*Inactive caspace= 33 kd *Active caspace= 17 kd

14

How does caspace activation result in DNA fragmentation?

-Executioner caspaces 3 and 7 cleave DFF45 and DFF40 dimer (DNA fragmentation factor)

     *In dimer form, they are inactive.

-DFF45 is released from DFF40 when executioner caspaces 3 and 7 cleave

-DFF40 oligomerizes and in that form, it is an active DNAse

-Oligomeric DFF40 cleaves DNA at the intranucleosomal area

-DNA fragments of 180-185 bp typically resulting in a DNA ladder on agarose gel

15

Intrinsic Apoptotic pathway

-Some signal tells cell to kill itself

-DNA damage is sensed by p53 -P53 up-regulates PUMA/NOXA

-PUMA/NOXA activate BAX and BAK (pro-apoptotic)

-BAK and BAX antagonize BLC-2 and bind to mitochondria making them leak and causing release of cytochrome C

-Cytochrome C activates APAF1 -APAF1 (apoptosis peptidase activating factor) form apoptosome

-Apoptosome activates initiator caspase 9

-Initiator capsace activates effector capsaces 3,6,7

-Caspaces 3,6,7 result in apoptosis

16

In Normal Cell

1. BCL-2 plugs up mitochondria preventing leakage of cytochrome C.

17

P53 has many mechanisms of anticancer function

-It can activate DNA repair proteins when DNA has sustained damage

-It can arrest growth by holding cell at the restriction point after G1

-It can initiate apoptosis if DNA damage proves irreparable

18

p53 activity is in unstressed cells

-P53 activity is low

-It associates with MDM2

-MDM2 acts as Ub-ligase and covalently attaches Ub to p53

-p53 is degraded by proteosome

-MDM2 also transport p53 from nucleus to cytosol

19

p53 activation by stress

-P53 N-terminal domain is activated by

    *MAPK

    *Checkpoint kinases

    *Oncogenes ( p14ARF) → p53

-P53’s association with MDM2 is inhibited

-P53 can translocate to nucleus and upregulate genes including itself that are involved in:

      *DNA repair

      *halting cell cycle

      *apoptosis

20

Antagonism between BH3 proteins

1. Bax and Bak are pro-apoptotic

    -They promote pore formation

    -Release cytochrome C

2. BCL-2 is antiapoptotic

     -BLC-2 binds Bax and Bak preventing them from

       translocating and opening pores

3. Bid and Bad

      -Normally found in cytosol

       -Can translocate to mictohondria membrane and

       stimulate apoptosis by tieing up BCL-2 thereby    

       allowing Bax and Bak to open mitochondrial

       membrane to release cytochrome C, assembly of

       APAF1 and apotosome, activating initiator Caspase

       9, which then activates effector caspaces 3,6,7 that

       lead to apoptosis

21

Bcl-2 and cancers

-chromosomal translocation commonly occurs between the 14th and 18th chromosome which places the Bcl-2 gene next to the IgG heavy chain locus

-high transcription of Bcl-2 thereby preventing cells from undergoing apoptosis even when they have mutations

-Cancer results

22

Extrinsic Pathway

-Activating ligand is Apo2L/Trail

-Binds to death receptors (DR4 and DR5) which are transmembrane proteins

-When DR4 and DR5 bind to Apo2L/Trail, you get an activation of the receptor in the cytoplasmic domain

-The death receptor fast associated death domain or (FADD) forms

-When FADD is activated, you get an assembly of DISCs (death-induced signaling complex)

-FADD recruits pro-caspases 8 and 10 to DISC

-DISC undergoes autocatalysis so that capsaces 8 and 10 become activated caspaces

-Activated intiator capscases activate effector caspases 3.6.7

-Effectors caspaces leads to apoptosis of the cell

23

Convergence of Extrinsic and Intrinsic apoptosis pathway

1. Converge at effector caspases 3,6,7

2. BID

    -Caspace 8 and 10 activate BID that ties up BCL-2

24

Cellular function of Autophagy

1. Response to cellular starvation

2. Housekeeping process

3. Role in normal cell processes

25

Autophagosome formation

1. Induction by starvation conditions

    -mTOR is inactivated

    -mTOR inactivation leads to downstream

      dephosphorylation events

     -Transcriptional activation of autophagy related genes

       occurs which are involved in autophagosome

      formation and fusion

2. Formation

      -Many genes are upregulated in response to mTOR

      deactivation which participate in autophagosome

      formation

      -Step involves the formation of a membrane around

       a targeted portion of the cell

3. Lysosome fusion

4. Autophagosome breakdown

26

Morphological features of necrosis

Early

-Cell swells

-Chromatin digestion

-Plasma membrane and organelle are disrupted

Late

-Extensive DNA hydrolysis

-Vacuolation of ER

-Organelle breakdown

-Cell lysis

-Release of intracellular contents causes inflammatory response in necrosis