Cell cycle and programmed cell death

1. Cell Cycle and Apoptosis
R. C. Gupta
M.D. (Biochemistry)
Jaipur, India

2. Cell cycle
Eukaryotes have different types of cells
The somatic cells divide by mitosis
All the cells do not divide at the same
rate and at the same time

3. The time period between one mitotic
event and the next constitutes the
‘cell cycle’

4. The cell cycle can be divided
into four phases:
Mitotic or M phase
Gap1 or G1 phase
Synthetic or S phase
Gap2 or G2 phase

5. G1
SG2
M

6. DNA is synthesized in the S phase
The cell divides in the M phase
Progression of cell cycle from one phase
to the next is precisely regulated

7. Hartwell, Hunt and Nurse showed how
the cell cycle progresses
They discovered the key regulators of
cell cycle
Hartwell Hunt Nurse

8. The regulators of cell cycle are some
proteins and some enzymes
Cyclin levels keep on changing during
the cell cycle
The cyclins act through some enzymes,
called cyclin-dependent kinases (CDKs)
The regulatory proteins are cyclins

10. At a certain time in the cell cycle, level of a
particular cyclin increases
The cyclin activates a particular CDK
Active CDK phosphorylates some proteins
These proteins push the cell cycle from
one phase to the next

11. G2
M
Cyclin A CDK 1 (inactive)
Cyclin A CDK 1 (inactive)
Active CDK 1 phosphorylates
some proteins that results in:
• Breaking of nuclear membrane
• Reorganization of cytoskeleton
• Chromosome condensation

12. G1 S G2
M
Cyclin D-
CDK 4 & 6
Cyclin E-
CDK2
Cyclin A-
CDK 1 & 2
Cyclin B-
CDK1
Cyclin-cdk complexes regulate the cell cycle

13. Some ccheckpoints are designed to
monitor progression of the cell cycle
Certain events are essential before the cell
cycle proceeds from one phase to the next
Checkpoints

14. The cell cycle cannot go to the next phase
until checkpoint requirements have been
met
The checkpoints verify that all the events
required for progression to the next phase
have occurred

15. Two major
checkpoints are:
G1/S checkpoint
G2/M checkpoint

16. Check points
in cell cycle
Entry into M
phase is blocked
if replication is
not complete
Entry into S phase
is blocked if
genome is
damaged
G2/M checkpoint
G1/S checkpoint

17. G1/S checkpoint monitors whether all the
requirements for DNA synthesis have been
met
G1/S transition is the rate-limiting step in
cell cycle
This is also known as the restriction point
G1/S checkpoint

18. p53 gene plays a key role at G1/S
restriction point
p53 protein halts the cell cycle in G1
phase if it detects any damage to DNA
This is known as G1 arrest
The product of p53 gene is p53 protein

19. Normally, cyclin D level rises in G1 phase
which activates CDK4 and CDK6
This pushes the cell cycle into S phase
In G1 arrest, CDK4 and CDK6 remain
inactive

20. G1 arrest gives more time to DNA repair
systems to repair the damage
If the damage is repaired, the cell cycle
is allowed to proceed to the S phase
If the damage cannot be repaired, the
cell is pushed into apoptosis

21. G2/M checkpoint monitors whether DNA
has been replicated completely and
accurately
If the replication is found to be defective,
the cell is pushed into apoptosis
G2/M checkpoint

22. Apoptosis

23. Apoptosis is a Greek word that means
“dropping off”
It is dropping off of leaves from a tree or
petals from a flower
In biology, apoptosis is a form of cell death
The other form of cell death is necrosis

24. Necrosis Apoptosis
Results in disease,
if excessive
Generally confers
some advantage
Unnatural cell
death
Natural cell death
Cell is programmed
to die
Acute injury results
in cell death

25. Unnecessary cells are removed during
embryonic and foetal life by apoptosis
Their removal is essential for normal
development

26. Another example of apoptosis is seen in
infections
After an infection is over, the activated B
cells and T cells are no longer required
They are removed by apoptosis

27. Apoptosis continues throughout life
Nearly 60 billion cells undergo apoptosis
every day in an adult human being
These are replaced by new cells

28. The apoptotic reactions are catalysed by
enzymes known as caspases
Caspases are cysteine proteases
Their name is derived from cysteine-
dependent aspartate-specific protease

29. A cysteine residue of caspase is involved
in catalysis
The enzyme hydrolyses peptide bonds
next to aspartate residues in the protein
Glu‒Ala‒Val‒Asp‒Leu‒Arg‒Phe‒Ser
Caspase

30. Caspases are initially synthesised as
inactive pro-enzymes (pro-caspases)
These are converted into active enzymes
upon receiving appropriate signals

31. Caspases play a crucial role in:
Apoptosis Inflammation
Accordingly, the caspases can be:
Apoptotic Pro-inflammatory

32. Caspases 2, 3, 6, 7, 8, 9, 10 and 14 are
apoptotic caspases
Pro-inflammatory caspases are caspases
1, 4, 5 and 13

33. The apoptotic caspases can be divided
into:
Initiator caspases Effector caspases
Initiator caspases start the apoptotic
cascade
Effector caspases execute actual cell
death

34. Caspases 2, 8, 9 and 10 are initiator
caspases
Caspases 3, 6, 7 and 14 are effector
caspases

35. The initiator caspases are activated by
dimerization
In the inactive state, initiator caspases are
monomers
After receiving an apoptotic signal, some
adaptor molecules bind the monomers
This binding results in proximity-induced
dimerization

36. Caspase
Procaspase
Adaptor protein
Adaptor protein induces dimerization
of procaspases

37. The active initiator caspases activate the
effector caspases
Activation occurs by proteolytic cleavage
of effector procaspases

38. Effector
procaspase
Effector
caspase
Initiator
caspase
Activation of an effector procaspase

39. The activated effector caspase activates
some downstream effector caspases
The active effector caspases degrade a
host of intracellular proteins
This results in cell death

40. Cell death by apoptosis

41. Activation of initiator caspases
requires some signals
The signals may be:
Extrinsic
(extracellular)
Intrinsic
(intracellular)

42. The extracellular signals include toxins,
growth factors, hormones, cytokines etc
Their effect on apoptosis may be positive
or negative
Positive signals trigger apoptosis while
negative signals inhibit apoptosis

43. The intracellular signals include different
types of stress
Examples are hypoxia, heat, radiation,
nutrient deprivation etc

44. This pathway begins with the binding of a
ligand to its receptor
The ligand is extracellular and the receptor
is a cellular trans-membrane protein
Extrinsic (extracellular) pathway

45. One trans-membrane receptor is Fas, and
its ligand is Fas ligand (FasL)
FasL is a member of the tumour necrosis
factor (TNF) family of cytokines
Fas is a member of TNF receptor family

46. Another receptor is TNFR-1, and its ligand
is TNF
Fas-FasL binding is the more significant
pathway
The ligands for these receptors exist in the
form of trimers
Binding of ligands to receptors induces
trimerization of the receptors

47. Cytoplasmic portions of these receptors
possess a common motif
This motif is known as ‘death domain’
This domain is the site for binding of an
adaptor protein

48. The adaptor protein for Fas is known as
Fas-associated death domain (FADD)
protein
The adaptor protein for TNFR-1 is TNF
receptor-associated death domain
(TRADD) protein

49. The receptor-adaptor complex attracts
pro-caspase 8
Pro-caspase 8 is converted into active
caspase 8 by dimerization
Active caspase 8 converts pro-caspase 3
into caspase 3 by proteolysis

50. Activation of caspase 3 starts a protease
activation cascade
Activated caspases cleave and activate a
succession of downstream caspases
The activated caspases hydrolyse their
substrate proteins in the cell

51. Apoptosis
Procaspase 3 Caspase 3
Caspase 9
Procaspase 9
FADD protein
Fas
FasL
Extrinsic pathway of apoptosis
Caspase cascade

52. At the final stage of the pathway, a
caspase activates a deoxyribonuclease
The deoxyribonuclease enters the nucleus
and cleaves and fragments DNA

53. Mitochondria are essential for multicellular
organisms
A cell will cease to respire aerobically in
the absence of mitochondria
Apoptotic signals acting through mito-
chondria either increase or decrease the
permeability of mitochondrial membrane
Intrinsic (mitochondrial) pathway

54. Membrane permeability is influenced by
proteins encoded by Bcl-2 family of genes
Some proteins of this family, e.g. Bax and
Bak, increase permeability
Some other proteins, e.g. Bcl-2 and Bcl-
xL, decrease permeability

55. When membrane permeability increases,
some mitochondrial proteins leak into
cytosol
These include Small Mitochondria-derived
Activators of Caspases (SMACs) and
cytochrome c

56. SMAC
Cytochrome c

57. The SMACs bind to a protein known as
Inhibitor of Apoptosis Protein (IAP)
Normal function of IAP is to arrest the
apoptotic process by inhibiting caspases
Inhibition of IAP by SMACs allows
activation of apoptotic procaspases

58. SMACs
IAP
Θ
Θ
Procaspase
Caspase
Release of SMACs inhibits IAP and allows
activation of procaspases

59. Cytochrome c released from mitochondria
starts an activation cascade
Cytochrome c binds to Apoptotic protease
activating factor-1 (Apaf-1) and ATP/dATP
This complex binds pro-caspase 9 and
converts it into active caspase 9

60. ATP/dATP
Oligomerization
Caspase 9 (dimer)
Procaspase 9 (monomer)
Apaf-1 (rigid conformation)Apaf-1 (open)
Apaf-1
(closed)
Cytochrome c
Release of cytochrome c leads to
activation of procaspase 9

61. Active caspase 9 converts pro-caspase 3
into active caspase 3
Caspase-3 activates some downstream
caspases
The activated caspases hydrolyse cellular
proteins resulting in apoptosis

62. A cell undergoing apoptosis shows some
characteristic morphological features
The cell shrinks because of breakdown
of its cytoskeletal proteins by caspases
The cytoplasm appears dense, and the
organelles appear tightly packed
Morphology of apoptotic cells

63. The cell membrane shows irregular buds
known as blebs
Chromatin is condensed
Nuclear envelope becomes discontinuous
The nuclear DNA is fragmented

64. The cell breaks up into vesicles called
apoptotic bodies
Phosphatidylserine is normally present on
cytosolic surface of cell membrane
It reaches extracellular surface during
apoptosis and attracts macrophages
Macrophages engulf and destroy the
remains of the dying cell

65. Cell undergoing apoptosis Blebs formed
Apoptotic bodies formedPhagocytosis of remains