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!