expression levels of dna repair proteins modulate their response to radiotherapy, in glioblastoma...
TRANSCRIPT
Expression levels of DNA repair proteins modulate their response to radiotherapy, in
glioblastoma cell lines
Natalie Mayhead
Outline of talk
• Introduction to glioblastoma• Repair pathways of interest• Expression data• Survival data• Preliminary conclusions
What is Glioblastoma?Most invasive type of glial tumourCommon among men and women in their 50s-70sPrognosis is poor, with median survival at 14 months post diagnosis.Treatment includes; surgery, chemotherapy and radiotherapy.
Chemotherapy- Temozolomide
N7-G N3-A O6-G
70% of adducts 10% of adducts 6% of adducts
TMZ
ME-base
O6G
BERMGMT/MMR
5’drp
MGMT
Mismatch repair
In cells that do not express MGMT MMR deals with O6MG
MMR can cause a futile repair loop leading to cell death
Repaired DNA
ME-base
AAG
APEXRCC1 Pol β
Lig 3
PARP
Repaired DNA
Repair pathways of interest
What is the problem with MGMT?• The lethal lesion of TMZ is methylated guanine at the
O6 position, which leads to cell death. • MGMT is a single acting protein which removes this
lesion greatly reducing the efficacy of TMZ. • 50% of GBM patients express this protein leaving TMZ
ineffective. • However, the addition of TMZ has increased survival
statistics from 12 months after diagnosis to 14 months.
MGMT No MGMT
Gene expression levels
A172
LN18T98
GU25
1U87
0
5
10
15
20
Cell LinesR
elat
ive
Qu
anti
fica
tio
n
AAG levels in cell lines
A172
LN18T98
GU25
1U87
0
2
4
Cell Lines
Rel
ativ
e Q
uan
tifi
cati
on
PolB levels in cell lines
A172
LN18T98
GU25
1U87
0
5
10
Cell Lines
Rel
ativ
e Q
uan
tifi
cati
on
Lig 3 levels in cell lines
TMZ
ME-base
O6MG
XRCC1
ME-base
AAG
APE
5’drp
Pol β
Lig 3
PARP
MGMT
Expression A172 LN18 T98G U251 U87
MGMT - - -
AAG
APE
PolB
PARP
LIG3
XRCC1
Expression levels summarised
High No expression
Average
Symbol -
All Cell Survival after TMZ treatment
Survival for MGMT +ve with X-rays
Survival MGMT –VE with X-rays
Cell lineMGMT expression AAG expression IC50- TMZ IC50- X-rays Gy
IC50 combined Gy
A172 0 1.70 29 1.766 1.247
LN18 0.99 12.88 2.05 1.952
T98G 1 1 2.38 2.604
U251 0.03 0.74 48 2.42 1.841
U87 0 15.83 43 2.46 0.05947
Expression versus IC50
Methoxyamine
ME-base
AAG
APE
5’drp
Pol β
Lig 3
PARP
Addition of MX with/without X-rays
APE
Treatment with TMZ or X-rays
Survival after treatment with inhibitors
MX with higher TMZ concentrations MGMT +ve cell lines
MX with higher TMZ concentrations MGMT -ve cell lines
X-rays with MX and TMZ
~10% change
Preliminary conclusions
• AAG expression levels vary greatly but other BER protein expression levels are similar.
• AAG levels are high in U87 correlating with greater sensitivity to TMZ and X-rays combined.
• Cells treated with MX and TMZ at 25 µM show little difference in survival
Future/current work
• Create flourescently tagged repair proteins:-XRCC1-GFP (example a)-53BP1-GFP (example b)
Measure in live cells strand repair
A B
Overexpress AAG
• Measure strand breaks in live phase with/out OE AAG
• X-ray clonogenics, higher levels of AAG greater combination effect?
• Temozolomide and inhibitor clonogenics
Lamp promoter AAG gene Red flourescent
ProteinAntibiotic
resistance genes
Future work
• Live-time cell break• Cell cycle arrest measurements• Apoptosis induction
1 year = too much work to do!