Download Role of the p21 Protein in the Fanconi Anemia Pathway

Role of the p21 Protein in the
Fanconi Anemia (FA) Pathway
Mae Shen
Faculty Sponsor: Dr. Niall Howlett
Fanconi Anemia - Clinical Description
Congenital malformations
–
–
–
–
–
Short stature
Microcephaly (small head)
Micropthalmia (small eyes)
Hypo/hyperpigmentation
Abnormal thumbs
Hematological abnormalities
– Bone marrow failure
– Acute myeloid leukemia (x800)
Non-Hematological abnormalities
– Head and neck squamous cell carcinomas (x2000)
– Gynecologic squamous cell carcinomas (x4000)
– Benign and malignant liver, brain and renal tumors
Fanconi Anemia - Inheritance
• Recessive genetic disorder
– Biallelic mutations in any one of thirteen genes results in FA
– Autosomal and X-linked
• Incidence: 1 in 200,000 – 400,000 live births
– Higher incidence rate in certain populations such as Spanish
Gypsies and Ashkenazi Jews.
Why Study Fanconi Anemia?
• Link to Cancer
– FA is strongly linked to cancer in the general (non-FA)
population.
– Four FA genes are breast cancer susceptibility genes in the
heterozygous state, for example FANCD1/BRCA2.
• Target for Cancer Chemotherapy
– The FA pathway is a major cellular determinant of resistance to
DNA crosslinks.
– Oxaliplatin and cisplatin are DNA-crosslinking agents commonly
used in cancer chemotherapy.
– Drugs that inactivate the pathway could be used in adjuvant
cancer chemotherapy.
The Fanconi Anemia Pathway
G
F
C
A
E
24
M
L 100
Ub
UBE2T Ub
Ub
D2
ATM
B BLM
I
ATR
?
D2
Ub
USP1 Ub
UAF1
ATRIP
P
Ub
PCNA
P   Ub
I NBS1
BRCA1
J/BRIP1
D1/BRCA2
51
N/PALB2
51 51
p21 is Required for DNA damage-inducible
FANCD2 monoubiquitination
HCT116
p21+/+
Hours post UV (20 J/m2)
FANCD2-Ub
FANCD2
0 0.1 0.3 0.6 1
p21
p21 -/2
6
24
*
0 0.1 0.3 0.6 1
2
6 24
*
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
What is p21?
• Major regulator
of the cell cycle
• Arrests cell cycle
1) Inhibit CDKs
2) Interact with PCNA to halt DNA
replication
• Major target of the tumor
suppressor transcription
factor p53
Figure 8.4 The Biology of Cancer (© Garland Science 2007)
My Project
Goal:
Characterize the interaction between
FANCD2/FANCD2-Ub and p21
Specifically:
1) Are FANCD2 monoubiquitination and p21
coordinately regulated following DNA damage?
2) Is p21 required for resistance to DNA crosslinks?
3) Does inhibition of p21 degradation affect FANCD2
monoubiquitination?
4) Is the CDK-inhibiting function of p21 required for
FANCD2 monoubiquitination?
1) Are D2 mono-Ub and p21 Coordinately
Regulated following DNA Damage?
Rationale:
– If the kinetics of D2 mono-Ub and p21 expression are
similar, this would be suggestive that both proteins
are involved in the same cellular process.
Methods:
– Treat cells with DNA damaging agents.
• UV irradiation (TT dimer)
• Mitomycin C (DNA crosslink)
– Observe levels of monoubiquitinated D2 (D2-Ub) and
p21 over time.
1) Are D2 mono-Ub and p21 coordinately
regulated following DNA damage?
U2OS
Hours post-UV (20J/m2 )
0
0.3
1
2
6
24
1
2
3
4
5
6
0
0.3
1
2
6
24
1
2
3
4
5
6
FANCD2-Ub
FANCD2
*
p21
U2OS
Hours post-MMC (100nM)
FANCD2-Ub
FANCD2
p21
*
FANCD2 and p21 do not appear to be coordinated following DNA damage
2) Is p21 required for
resistance to DNA crosslinks?
Rationale:
• FA patient cells are hypersensitive to DNA-crosslinks.
• Since cells lacking p21 have impaired monoubiquitination of
D2, they should exhibit a similar sensitivity.
Methods:
• Treat HCT116 p21+/+ and HCT116 p21-/- cells with the DNAcrosslinking agent mitomycin C (MMC).
• Allow cells to grow for about 10 days.
• Stain with crystal violet to quantify cells that survived.
• Observe whether cells lacking p21 (HCT116 p21-/-) are more
sensitive to MMC than wild type cells (HCT116 p21+/+).
2) Is p21 required for
resistance to DNA crosslinks?
100
p21+/+
p21 -/-
% Viability
80
60
40
20
0
0
100
200
300
400
MMC (nM)
p21 does not appear to be required for resistance to MMC
3) Does inhibition of p21 degradation affect
FANCD2 monoubiquitination?
• Rationale:
– p21 is degraded by the proteasome following UV
irradiation.
– If p21 degradation is inhibited, how is D2 mono-Ub
affected?
• Methods:
– Treat cells with the proteasome inhibitor MG-132 prior to
UV irradiation to inhibit p21 degradation.
– Observe levels of FANCD2 monoubiquitination.
3) Does inhibition of p21 degradation affect
FANCD2 monoubiquitination?
Control
Hours post-UV
(20J/m2 )
No UV
2
MG-132 (10μM)
6
2
6
BJ-Tert
FANCD2-Ub
FANCD2
*
p21
1
2
3
4
5
Hela
FANCD2-Ub
FANCD2
p21
U2OS
FANCD2-Ub
FANCD2
*
p21
1
2
3
4
5
FANCD2 monoubiquitination decreases while p21 increases
consistently after treatment with MG-132.
4) Is the CDK-inhibiting function of p21
required for FANCD2 monoubiquitination?
Rationale:
– One of the functions of p21 is to inhibit CDK.
– Is this function required for FANCD2 mono-Ub?
Methods:
– Treat HCT116 p21-/- cells with roscovitine (a CDK
inhibitor) prior to UV irradiation.
– Observe whether roscovitine can substitute CDKinhibiting role of p21 and restore D2
monoubiquitination following DNA damage.
4) Is the CDK-inhibiting function of p21
required for FANCD2 monoubiquitination?
Roscovitine (5 μM)
Control
Hours post-UV (20J/m2)
0
2
6
24
0
2
6
24
FANCD2-Ub
FANCD2
HCT116 p21+/+
FANCD2-Ub
FANCD2
HCT116 p21- / 1
2
3
4
5
6
7
8
1.4
Ratio Ub/Non-Ub
1.2
1
p21+/+ NT
0.8
p21+/+ Rosc
0.6
p21-/- NT
0.4
p21-/- Rosc
0.2
0
0
2
6
Hours Post-UV
24
Roscovitine does not restore UV-inducible FANCD2 monoubiquitination
Conclusions
p21 is clearly important to the FA pathway.
However the interaction between FANCD2
and p21 is complex.
The mechanism by which p21 regulates the
monoubiquitination of FANCD2 remains to
be uncovered.
Thank You!
RI-INBRE
Leukemia Research Foundation
Fanconi Anemia Research Fund
Dr. Howlett
Julie
Jeanne
Meg
Fred
Bill
Sarah