HOW TUMOR SUPRESSOR GENES REGULATE CELL CYCLE. ON MUTATION OF THE GENES LEADS TO CANCER.

1. ROLE OF P53 AND Prb PROTEIN
IN CELL CYCLE REGULATION
Presented by:-
BAPI MAKAR- DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR
BIOLOGY- PONDICHERRY UNIVERSITY

4. R point
Cell cycle control involves several checkpoints and
checkpoint (molecular breaking) mechanisms

5. Cyclins
G0 G1 S G2 M
Cyclin D
Cyclin E Cyclin A Cyclin B
determined
by
mitogenic
growth
factors
• Cyclines and cyclin-dependent kinases (cdks)
– The cyclines have oscillating levels during cell cycle
– The cyclines are regulatory subunits of the CDK-kinases
• cyclines+ cdk  cell cycle-dependent variations in the activity
of the kinases

7. M
G1
G2
S
R
E2F released
S-phase genes expressed
Rb
Cdk4/6
Cyclin D
Gate keeper model

8. pRb: What does it do?
pRb is a nuclear protein that undergoes
phosphorylation and dephospharylation in concert
with the cell cycle

9. Hypo-phosphorylated
or un-phosphorylated
pRb inhibits the cell
from entering a new
cell cycle
The guardian of the cell at early-mid G1
Upon further phosphorylation at the R point,
hyper-phosphorylated pRb becomes inert and the
cell cycle can proceed

10. Hypo-phosphorylated Rb inhibits activity of
the E2F family of transcription factors
Hyper-phosphorylation
of Rb sequesters Rb, and
releases E2Fs
E2Fs are needed for
transcription of genes
that are essential for
the cell to enter the
cell cycle

11. Hypo-phosphorylated Rb
binds to E2Fs and:
- Inhibits their transcription
activation sites
- Recruits proteins that will
“close” the chromatin down

12. PLGA dissolved in acetone (for
ENP).PLGA+ DRUG(10:1)
dissolved in acetone for
CNP/BDMCANP)
Synthesis
GO FOR
CHARACTERISATION
PREPARATION
OF
NANO-
PARTICLE

13. Releasing Rb from the
E2Fs leads to:
- Release of their transcription
activation sites
- Recruitment of proteins that
will “open up” the chromatin

14. Rb, the retinoblastoma protein
regulates the cell cycle
Cell cycle = OFF
Rb binds to E2F: no
transcription, no entry
into S phase
Cell cycle = ON
Rb does not bind to
E2F: transcription and
entry into S phase

15. Rb activity is tightly regulated by the cell
cycle clock
Hypo-phosphorylation is
catalyzed by cycD-CDK4/6
Hyper-phosphorylation is
catalyzed by cycE-CDK2

16. pRb is hyper-phosphorylated and inhibited (and
released from its role as a guardian), only upon
cycE expression

17. Rb activity is tightly regulated by the cell
cycle clock
However, E-CDK2 can phosphorylate Rb, only
AFTER Rb is phosphorylated by cycD-CDK4/6

18. Only after we have enough mitogen signaling (and, as a
result, enough cycD-CDK4/6 activity), cycE can
phosphorylate Rb and allow entry to the cell cycle

19. E2Fs have more than 100 target
genes, mostly involved in the
first steps of DNA replication
One of the targets:
the cycE gene
Transcription of cycE
starts a positive
feedback loop

20. As E2Fs are necessary for expression of cycE,
think how critical negative regulation by Rb
is for cell cycle control
E2Fs

21. Uncontrolled crossing of the R site can be due to loss
of Rb function (e.g. mutation), loss of CKIs or
oncogenic activity of cyclins E and D

22. Rb gene alteration is involved many tumors
In the majority of tumors you will find mutation
involved in the R site

23. p53
The Master Guardian

24. Effect of mutant p53 alleles in the mouse germ
line

26. p53 is a transcription factor, active only as a
homotetramer

27. In normal cells we find only low
concentrations of the p53 protein
- p53 protein is actually
synthesized all the time,
but is degraded very fast
via the ubiquitine system

28. p53 protein is ubiquitinated by the E3 ligase
MDM2

29. p53
DNA
damage
Hyperproliferative
stress
Many agents induce p53 activity
Grouped into two classes

30. DNA damage is sensed by sensor proteins and
repaired by the DNA repair machinery
Extensive DNA damage recruits the
DNA damage response machinery
Two key players:
The protein kinases ATM and ATR
Ataxia Telangiectasia Mutated
Ataxia Telangiectasia and rad3
related

31. ATM and ATR are recruited to distinct sites and
phosphorylate downstream effectors

32. ATR is recruited to single - stranded DNA
- ATR-dependent phosphorylation of
the Rad9 adaptor protein is needed
for activating Chk2
- Activated Chk2 is released to phosphorylate its effectors
ATR
Rad9
Chk2
(inactive)
Chk2
(active)

33. Phosphorylation of p53 (by
ATM/ATR and/or Chk2)
makes it insusceptible to
MDM2 binding
Phosphorylated p53 acts
as a transcription factor

34. DNA damage response activates p53 by
stabilizing the protein via phosphorylation
(and additional mechanisms)
Additional inhibitory
phosphorylation of MDM2

35. p53
DNA
damage
Hyperproliferative
stress
E2Fs
e.g. high activity of E2Fs

36. Hyperproliferative stress response is mediated
through the ARF protein
- E2Fs induce transcription
of the ARF gene
- ARF binds to and sequesters MDM2
- p53 is stabilized

37. Over activity of oncogenes stimulates apoptosis
through ARF

38. Two reactions for the price of one

39. - p53 is a transcription factor, acting
as a homotetramer
Summary
- Transcribed constitutively, but
has a very short half life
- DNA damage and a stalled replication fork
induce p53 phosphorylation and activation
- Hyperproliferative stress (e.g. oncogenic
signaling, hypoxia) activates p53 via ARF
- Ubiquitinated by the E3 ligase MDM2

40. p53
DNA
damage
Cell cycle arrest Apoptosis
What about outputs?
Hyperproliferative
stress