1. Human Physiology: Unit-1
Regulation of Cell Division/Proliferation BY
DR BOOMINATHAN Ph.D.
M.Sc.,(Med. Bio, JIPMER), M.Sc.,(FGSWI, Israel), Ph.D (NUS, SINGAPORE), PDF (USA)
PONDICHERRY UNIVERSITY
Sixth Lecture
21/August/2012
Source: Collected from different sources on the internet and modified by Dr Boominathan Ph.D.

2. Regulation of Cell Division/Proliferation

3. Learning objectives Cell Division Cell cycle
Cyclin-CDKs that control cell cycle progression
Checkpoints
p53/RB
Control of cell cycle/division/proliferation
Cancer

4. THINK: Of how many cells are you composed?
When an organism grows bigger do you get more cells or just bigger cells or both?
When do your cells divide the fastest? Slowest?
Do cells ever stop dividing?
Are all cells capable of division and replacement?

5. Cell division

6. How the cell cycle works
works.html
Mitosis and Cytokinesis:
chapter2/animation__mitosis_and_cytokinesis.html

7. Comparision of mitosis and meiosis
Control of cell cycle

8. Coordination of cell division
A multicellular organism needs to coordinate cell division across different tissues & organs
critical for normal growth, development & maintenance
coordinate timing of cell division
coordinate rates of cell division
not all cells can have the same cell cycle

9. Frequency of cell division
Frequency of cell division varies by cell type
embryo
cell cycle < 20 minute
skin cells
divide frequently throughout life
12-24 hours cycle
liver cells
retain ability to divide, but keep it in reserve
divide once every year or two
mature nerve cells & muscle cells
do not divide at all after maturity
permanently in G0 G2 S G1 M
metaphase
prophase
anaphase
telophase
interphase (G1, S, G2 phases)
mitosis (M)
cytokinesis (C) C

10. Control of the Cell Cycle /Cell proliferation

11. Interphase Interphase ~ 90% of the time.
Ø   G1: Little new cell absorbs nutrients and grows larger. Does protein synthesis, its job.
Ø   S phase: Synthesis of new DNA (DNA replication) for daughter cells in preparation for mitosis.
Ø   G2: Cell continues to grow, do protein synthesis, do its job. Gets too large, needs to divide.

12. Cyclin & Cyclin-dependent kinases
CDKs & cyclin drive cell from one phase to next in cell cycle
proper regulation of cell cycle is so key to life that the genes for these regulatory proteins have been highly conserved through evolution
the genes are basically the same in yeast, insects, plants & animals (including humans)
CDK and cyclin together form an enzyme that activates other proteins by chemical modification (phosphorylation). The amount of CDK molecules is constant during the cell cycle, but their activities vary because of the regulatory function of the cyclins. CDK can be compared with an engine and cyclin with a gear box controlling whether the engine will run in the idling state or drive the cell forward in the cell cycle.

13. The cell cycle engines Cyclin Dependent Kinases (CDKs) CDK cyclin
substrate
ATP P
product
+ ADP

14. Cyclin D-CDK4
Cyclin E-CDK2
Cyclin A-CDK2
Cyclin B-CDC2

15. CDK activity

16. Mitogens stimulate the onset of the cell cycle

17. Mitogens control cyclin D expression

18. Mitogens control cyclin D expression
- Mitogens act by activating the D-Cdk4/6 complexes
- Mitogens act by inhibiting CKIs
- Mitogen signaling is correlated with growth, answering the question: “have I grown enough?”

19. We actually have: 3 D-type cyclins 2 E-type cyclins 2 A-type cyclins
3 B-type cyclins

20. cyclin D and growth
w/o growth signals, sub-threshold levels of enzymes will lead to quiescence (G0)
START/Restriction point

21. - Activated D-Cdk4/6 initiates transcription of cyclin E and activation of E-Cdk2
- Activated E-Cdk2 allows progression through START
- From here on, it’s a cell cycle clock game

23. APC = anaphase-promoting complex
The different cyclins are degraded by two different E3 ligases
e.g. cyclin B, the G2/M cyclin is degraded by
APC = anaphase-promoting complex

24. cyclins must be removed for mitosis to be completed
Remember?
cyclins must be removed for mitosis to be completed
Protein
Level
Time
cyclin A
cyclin B M

25. CDKs are positively regulated by cyclins
For example, Cyclin-X promotes synthesis of the next cyclin that in turn, promotes destruction of the Cyclin-X
These regulatory activities are indirect

26. Mechanisms of CDKs regulation
1. Abundance of cyclins
2. CDK phosphorylation
3. Binding to CKIs (inhibitory proteins)
CDK
Cyclin
active +
inactive
CDKI
CDKI

27. Activating phosphorylation is catalyzed by Cdk-Activating Kinases (CAKs). However, they are abundant and not regulated 1 3
Cdk
Phosporylation of Thr by CAK 2 4
Cyclin
Substrate binding to the kinase

28. Mechanisms of CDKs regulation
1. Abundance of cyclins
2. CDK phosphorylation
3. Binding to CKIs (inhibitory proteins)
CDK
Cyclin
active
CDKI +
inactive
CDKI

29. Cdk inhibitor proteins (CKIs)
- Discovered by asking : “what binds to CDKs”?
- The INK4 family proteins bind to CDK4/6, blocking cyclin D binding
- The Cip/Kip family proteins bind to blocking active site of multiple CDKs
- CKIs normally regulate entry into S phase

30. CKIs Regulate the G1-S Transition

31. Overview of Cell Cycle Control
There’s no turning back,
now!
Overview of Cell Cycle Control
Two irreversible points in cell cycle
replication of genetic material
separation of sister chromatids
Checkpoints
process is assessed & possibly halted
centromere
sister chromatids
single-stranded
chromosomes
double-stranded  

32. Checkpoint control system
Checkpoints
cell cycle controlled by STOP & GO chemical signals at critical points
signals indicate if key cellular processes have been completed correctly   

33. Checkpoint control system
3 major checkpoints:
G1/S
can DNA synthesis begin?
G2/M
has DNA synthesis been completed correctly?
commitment to mitosis
spindle checkpoint
are all chromosomes attached to spindle?
can sister chromatids separate correctly?

34. G1/S checkpoint G1/S checkpoint is most critical
primary decision point
“restriction point”
if cell receives “GO” signal, it divides
internal signals: cell growth (size), cell nutrition
external signals: “growth factors”
if cell does not receive signal, it exits cycle & switches to G0 phase
non-dividing, working state

35. Progression of the cell cycle is regulated by feedback from intracellular events

36. G0 phase G0 phase non-dividing, differentiated state
most human cells in G0 phase
liver cells
in G0, but can be “called back” to cell cycle by external cues
nerve & muscle cells
highly specialized
arrested in G0 & can never divide

37. Activation of cell division
How do cells know when to divide?
cell communication signals
chemical signals in cytoplasm give cue
signals usually mean proteins
activators
inhibitors
experimental evidence: Can you explain this?

38. “Go-ahead” signals Protein signals that promote cell growth & division
internal signals
“promoting factors”
external signals
“growth factors”
Primary mechanism of control
phosphorylation
kinase enzymes
either activates or inactivates cell signals
We still don’t fully understanding the regulation of the cell cycle. We only have “snapshots” of what happens in specific cases.

39. Cell cycle signals Cell cycle controls cyclins Cdk’s
inactivated Cdk
Cell cycle controls
cyclins
regulatory proteins
levels cycle in the cell
Cdk’s
cyclin-dependent kinases
phosphorylates cellular proteins
activates or inactivates proteins
Cdk-cyclin complex
triggers passage through different stages of cell cycle
activated Cdk

40. Cyclins & Cdks
Interaction of Cdk’s & different cyclins triggers the stages of the cell cycle
There are multiple cyclins, each with a specific role. Cyclins are unstable. Some are triggered for destruction by phosphorylation. Others are inherently unstable and are synthesized discontinuously during the cell cycle.
Oscillations in the activities of cyclin-dependent kinases (CDKs) dictate orderly progression through the cell division cycle. In the simplest case of yeast, a progressive rise in the activity of a single cyclin-CDK complex can initiate DNA synthesis and then mitosis, and the subsequent fall in CDK activity resets the system for the next cell cycle.
In most organisms, however, the cell cycle machinery relies on multiple cyclin- CDKs, whose individual but coordinated activities are each thought to be responsible for just a subset of cell cycle events.
Leland H. Hartwell
Checkpoints
Tim Hunt
Cdks
P. Nurse
Cyclins

41. Nobel Prize
Leland H. Hartwell
Checkpoints
Tim Hunt
Cdks
P Nurse
Cyclins
They won the Nobel Prize in Physiology or Medicine for their discoveries of protein molecules that control the division (duplication) of cells

42. Spindle checkpoint G2 / M checkpoint M cytokinesis C G2 mitosis G1 S
Chromosomes attached at metaphase plate
Replication completed
DNA integrity
Inactive
Active
Active
Inactive
Cdk / G2 cyclin (MPF) M
APC
cytokinesis C G2
mitosis G1 S
Cdk / G1 cyclin
Inactive
MPF = Mitosis Promoting Factor
APC = Anaphase Promoting Complex
Active
G1 / S checkpoint
Growth factors
Nutritional state of cell
Size of cell

43. Summary
- The cell cycle is controlled by Cdks, activated by cyclins and CDK activating Kinases, and inhibited by CKIs
- Cyclins are positively and negatively regulated by cyclin-Cdks complexes
- Any process in the cell cycle is dependent on the previous one
- The cell cycle progresses in the right order

44. 70kg human ~ 1013 cells

45. Growth factor signals growth factor cell division cell surface
nuclear pore
nuclear membrane P P
cell division
cell surface
receptor
Cdk
protein kinase
cascade P
E2F P
chromosome Rb P
E2F
cytoplasm Rb
nucleus

46. Example of a Growth Factor
Platelet Derived Growth Factor (PDGF)
made by platelets in blood clots
binding of PDGF to cell receptors stimulates cell division in fibroblast (connective tissue)
heal wounds
Erythropoietin (EPO): A hormone produced by the kidney that promotes the formation of red blood cells in the bone marrow. EPO is a glycoprotein (a protein with a sugar attached to it).
The kidney cells that make EPO are specialized and are sensitive to low oxygen levels in the blood. These cells release EPO when the oxygen level is low in the kidney. EPO then stimulates the bone marrow to produce more red cells and thereby increase the oxygen-carrying capacity of the blood.
EPO is the prime regulator of red blood cell production. Its major functions are to promote the differentiation and development of red blood cells and to initiate the production of hemoglobin, the molecule within red cells that transports oxygen.
EPO has been much misused as a performance-enhancing drug (“blood doping”) in endurance athletes including some cyclists (in the Tour de France), long-distance runners, speed skaters, and Nordic (cross-country) skiers. When misused in such situations, EPO is thought to be especially dangerous (perhaps because dehydration can further increase the viscosity of the blood, increasing the risk for heart attacks and strokes. EPO has been banned by the Tour de France, the Olympics, and other sports organizations.

47. Growth Factors and Cancer
Growth factors can create cancers
proto-oncogenes (normal)
normal growth factor genes that become oncogenes (cancer-causing) when mutated
stimulates cell growth
if switched “ON” can cause cancer
Eg., Myc (activates cyclins)
tumor-suppressor genes
inhibits cell division
if switched “OFF” can cause cancer
Eg., : p53, RB

48. Cancer & Cell Growth
Cancer is essentially a failure of cell division control
unrestrained, uncontrolled cell growth
What control is lost?
lose checkpoint stops
gene p53 plays a key role in G1/S restriction point
p53 protein halts cell division if it detects damaged DNA
options:
stimulates repair enzymes to fix DNA
forces cell into G0 resting stage
keeps cell in G1 arrest
causes apoptosis of damaged cell
ALL cancers have to shut down p53 activity
p53 is the Cell Cycle Enforcer
p53 was discovered mainly by Drs. Arnold Levine & David Lane
Bert Vogelstein, Moshe Oren, Varda Rotter…

49. p53 — master regulator gene
NORMAL p53
p53 allows cells
with repaired
DNA to divide.
p53
protein
DNA repair enzyme
p53
protein
Step 1
Step 2
Step 3
DNA damage is caused
by heat, radiation, or
chemicals.
Cell division stops, and p53 triggers enzymes to repair damaged region.
p53 triggers the destruction of cells damaged beyond repair.
ABNORMAL p53
abnormal
p53 protein
cancer
cell
Step 1
Step 2
DNA damage is
caused by heat,
radiation, or
chemicals.
The p53 protein fails to stop
cell division and repair DNA.
Cell divides without repair to
damaged DNA.
Step 3
Damaged cells continue to divide.
If other damage accumulates, the
cell can turn cancerous.

50. Rb inhibits G1 cell cycle progression
Rb and G1 cell cycle progression. Rb exists in different states of phosphorylation: unphosphorylated, hypophosphorylated, and hyperphosphorylated. The hypophosphorylated state occurs after phosphorylation by cyclin D–CDK4/6, and this leaves Rb associated with E2F family transcription factors such that E2F is unable to activate transcription. Rb becomes hyperphosphorylated after the activation of cyclin E–CDK2. At this point, E2F dissociates from E2F and can initiate the transcription of genes required for progression into S phase. One of the genes activated by E2F is cyclin E generating a positive feedback loop to aid in the progression through G1-ps.
Foster D A et al. Genes & Cancer 2011;1:
Copyright © by SAGE Publications

51. Positive Feedback Loop

52. Restriction point control and the G1-S transition.
Restriction point control and the G1-S transition. As cells enter the division cycle from quiescence, the assembly of cyclin D-dependent kinases in response to mitogenic signals requires Cip/Kip proteins, which are incorporated into catalytically active holoenzyme complexes. The cyclin D-dependent kinases initiate Rb phosphorylation, releasing E2F from negative constraints and facilitating activation of a series of E2F-responsive genes, the products of which are necessary for S-phase entry. Activation of cyclin E by E2F enables formation of the cyclin E-cdk2 complex. This is accelerated by the continued sequestration of Cip/Kip proteins into complexes with assembling cyclin D-cdk complexes. Cyclin E-cdk2 completes the phosphorylation of Rb, further enabling activation of E2F-responsive genes, including cyclin A. Cyclin E-cdk2 also phosphorylates p27Kip1, targeting it for ubiquitination and proteasomal degradation. The initiation of the self-reinforcing E2F transcriptional program together with degradation of p27Kip1 alleviates mitogen dependency at the restriction point and correlates with the commitment of cells to enter S phase. In subsequent cycles, cyclin D-dependent kinases remain active as long as mitogens are present, and levels of p27Kip1 remain low. All p27Kip1 in cycling cells is complexed with cyclin D-cdk complexes. Mitogen withdrawal results in cyclin D degradation, liberating p27Kip1 from this latent pool. The resulting inhibition of cyclin D- and E-dependent kinases leads to cell cycle arrest, usually within a single cycle.
Sherr C J Cancer Res 2000;60:
©2000 by American Association for Cancer Research

53. How tumor suppressor genes block cell division

54. Development of Cancer
Cancer develops only after a cell experiences ~6 key mutations (“hits”)
unlimited growth
turn on growth promoter genes
ignore checkpoints
turn off tumor suppressor genes (p53)
escape apoptosis
turn off apoptotic genes
immortality = unlimited divisions
turn on chromosome maintenance genes
promotes blood vessel growth
turn on blood vessel growth genes
overcome anchor & density dependence
turn off touch-sensor gene

55. What causes these “hits”?
Mutations in cells can be triggered by
UV radiation
chemical exposure
radiation exposure
heat
cigarette smoke
pollution
age
genetics

56. Tumors Mass of abnormal cells Benign tumor Malignant tumors
abnormal cells remain at original site as a lump
p53 has halted cell divisions
most do not cause serious problems & can be removed by surgery
Malignant tumors
cells leave original site
lose attachment to nearby cells
carried by blood & lymph system to other tissues
start more tumors = metastasis
impair functions of organs throughout body

57. Traditional treatments for cancers
Treatments target rapidly dividing cells
high-energy radiation
Inhibits/apoptose rapidly dividing cells
chemotherapy
stop DNA replication
stop mitosis & cytokinesis
stop blood vessel growth

58. Resources Mitosis  CANCER
How Cancer grows
Mitosis:
Cell Cycle & Cancer Animations:
Cell Biology & Cancer Animations:
Mitosis & Meiosis Interactive Exercise:
Mitosis Animations:
Plant Cell Mitosis:

59. Questions??