The document discusses the processes of cell cycle and apoptosis, detailing how cells transition through division, quiescence, and programmed death. It emphasizes the roles of cyclins and cyclin-dependent kinases (cdks) in regulating the cell cycle, checkpoints, and mechanisms of apoptosis, distinguishing it from necrosis. Key players in these processes include p53, which regulates DNA damage responses and can induce apoptosis, along with various signaling pathways that lead to cell death.

1. CELL CYCLE AND
REGULATION
17BCB0087 A.G.Harith Laxman

2. CELL CYCLE AND APOPTOSIS
 Cells undergo 3 controlled processes
 The first two are part of the cell cycle, the last an exit from the cell cycle
 Division (the cell cycle)
 Quiescence
 This is where most of the work of being a cell lies
 During division the energy of the cell is devoted to making a new cell
 Death
 This can be a normal process creating a final functional form or an induced suicide
 Epithelium and reticuloendothelial cells undergo active transitions towards terminally
differentiated states in which the final forms are unable to divide
 The stratum corneum consists of cells that have become bags of crosslinked keratin protein with
no internal metabolism
 Suicide can be induced because the organism senses a threat to the entire organism
 Infection, cancer, avoidance of autoimmunity
17BCB0087 A.G.Harith Laxman

3. CONTROL OF ENTRY INTO CELL CYCLE
AND APOPTOSIS
 Cell cycle is initiated by
phosphorylation of transcription
factors
 These activate transcription of a set of
proteins known as cyclins
 The appearance of cyclins is
progressive and determines the types
of proteins that will be phosphorylated
at a particular point during the cell
cycle
17BCB0087 A.G.Harith Laxman

4. CYCLINS AND CDK’S  CDK levels don’t change while
cyclins are destroyed at the end
of each phase
 There are 3 general groups of
each
 G1 cyclins
 Cyclin D
 S-phase cyclins
 Cyclin A
 G2 cyclins
 Cyclin B (maturation promoting
factor MPF)
 Cyclin E is shared between G1
and M phase
 Cyclin A is shared between M
phase and G2
17BCB0087 A.G.Harith Laxman

5. CYCLINS BIND
CDK’S
 CDK’s are Cyclin Dependent Kinases
 Association with cyclins activates
their kinase function
 A cyclin tethers a target protein to the
CDK
 The targets of CDK’s are
transcription factors among other
proteins
 CDK’s are serine/threonine kinases
17BCB0087 A.G.Harith Laxman

6. THE EXIT FROM G0
 G0 is a quiescent state
 Activation of G1 CDK occurs due to a
rising level of G1 cyclins
 G1 cyclins are transcriptionally activated
by growth factors
17BCB0087 A.G.Harith Laxman

7. EVENTS DURING G1
 A rising level of G1 cyclins increases
the activity of G1 CDK’s
 CDK’s in turn activate proteins and in
turn genes that prepare the cell to
begin DNA replication
 At the G1 S boundary, the cell
encounters a checkpoint
17BCB0087 A.G.Harith Laxman

8. G1/S
CHECKPOINT
 This is controlled by the activity of the
transcription factor E2F
 E2F is a family of related proteins (E2F 1 to E2F5)
 E2F is found complexed throughout the cell cycle
to another family of proteins: Rb
 At the G1/S checkpoint, Rb is phosphorylated by
CDK2/cyclinA
 E2F is freed from sequestration and activates
transcription at genes containing an E2F consensus
sequence
17BCB0087 A.G.Harith Laxman

9. AND THOSE GENES ARE
 Three groups
 Cell cycle regulators
 Cyclin A
 E2F, Rb, myc, myb
 Note that these are not all positive
regulators of cell cycle
 Enzymatic machinery for DNA
synthesis
 DNA polymerase
 PCNA
 Enzymes involved in nucleotide
metabolism
 DNA synthesis regulators
 Enzymes that recognize the origins
of replication for example17BCB0087 A.G.Harith Laxman

10. OTHER CHECKPOINTS
 These monitor the completion of
DNA synthesis
 The presence of Okazaki fragments
prevents entry into G2
 DNA damage
 This occurs before, during and after
completion of S phase
 Spindle attachment
 Failure to attach spindle to
centromere results in blockage of
mitosis at metaphase
 Failure to align the spindle during
cytokinesis results in blockage at
anaphase17BCB0087 A.G.Harith Laxman

11. DOWNREGULATION OF CYCLIN INFLUENCED CDK ACTIVITY
cyclin B
cyclin A
ribonucleotide
reductase
Mitosis MitosisInterphase Interphase
Time
Newly synthesized proteins labeled with 35S-methionine:
 This is accomplished through proteolysis
of the cyclins
 G1 phase cyclins disappear during S and G2
phase
 M-phase promoting factor (CDK2 + cyclin
B) concentrations rise just prior to onset of
mitosis
 Cyclins associated with MPF are degraded by
anaphase promoting complex
 Cyclin B levels peak at G1/M
 Degradation during anaphase
 APC promotes polyubiquitination of cyclin B
 Ubiquitinated cyclin B is degraded by a
proteosome
 Cyclin transcription is also turned off and
the mRNA is unstable
 So no new cyclin is made until transcription
is restored
17BCB0087 A.G.Harith Laxman

12. 17BCB0087 A.G.Harith Laxman

13. IN THE OVERALL
 Stimulated entry into G1 results in
appearance of an initial level of
cyclins that promote the progressive
activation of genes enabling the cell
to synthesize DNA
 A series of progressive steps result in
 Activation of genes further into the
cycle
 Degradation of cyclins that promoted
earlier steps
 Passage through checkpoints that
insure mechanistic fidelity of each step
17BCB0087 A.G.Harith Laxman

14. APOPTOSIS
 This is programmed cell death
 Distinguish it from necrosis
 Necrosis results from traumatic forces
outside the cell
 Necrotic tissue provokes
inflammation as the immune system
moves in to clear out damaged and
dead cells
 Apoptosis is an ordered stepwise
self-destruction that permits
surrounding cells to utilize the
breakdown products of the dead
cell
 There is no inflammation involved17BCB0087 A.G.Harith Laxman

15. THE APOPTOTIC CELL
 Mitochondria break open
 DNA fragments in a regular way
 The cell loses a regular shape
 Undergoes blebbing
 This is an irregular bubbling appearance of
the plasma membrane
17BCB0087 A.G.Harith Laxman

16. THE MECHANISMS OF APOTOSIS
 Can be classified as externally or
internally signaled
 One internal route involves p53
 p53 is a transcription factor that is
involved in cell cycle control and
sensing the presence of DNA damage
 The central role p53 plays is at the
G1/S checkpoint
17BCB0087 A.G.Harith Laxman

17. P53 CONTROLS ENTRY INTO S-
PHASE
 P53 can sense DNA damage by binding
mismatches
 In the presence of damage, p53 activates
transcription of p21
 P21 binds and inactivates CDK2-cyclin E complexes
 The complex is unable to phosphorylate Rb and free E2F
 Thus entry into S phase is inhibited
 If the damage is repaired, p53 levels and p21 levels drop
and S phase ensues
17BCB0087 A.G.Harith Laxman

18. BUT IF THE DNA DAMAGE IS
EXTENSIVE
 P53 induces apotosis by activating transcription of Bax
 BAX protein competes with BCL-2 to form pores in
mitochondrial membranes
 BCL-2 prevents the release of cytochrome c from mitochondria into the
cytoplasm
 BAX permits release of cytochrome c
 When released, cytochrome c stimulates caspase activation
17BCB0087 A.G.Harith Laxman

19. THE CASPASES
 These are proteolytic enzymes that
are held in check by external or
internal inhibitors
 Activation results in an explosive
proteolytic cascade
 Caspase 9 cleaves and activates
other caspases
 The caspases also activate
quiescent nucleases
17BCB0087 A.G.Harith Laxman

20. EXTERNAL APOPTOTIC MECHANISMS
 Involve external “death signals”
 Cells may be recognized as a threat to
the whole organism
 The immune system moves in to kill them
 One mechanism of killing involves a
command to the cell to initiate apoptosis
17BCB0087 A.G.Harith Laxman

21. FAS/FAS LIGAND SIGNALING
 Fas ligand (FasL) is a membrane
bound cell surface protein
 It binds to Fas receptor
 Binding results in trimerization
and activation of APAF
 APAF in turn activates caspase 8
by proteolysis of a caspase 8
zymogen
 Caspase 8 cleaves a BCL-2 family
member BID
 BID translocates to the mitochondria
and binds BAX
 Bax permits leakage of cytochrome
c and activation of the caspase 9
cascade via APAF-1 again
17BCB0087 A.G.Harith Laxman