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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!