Development of a novel
assay to investigate changes
to global DNA methylation
levels in response to
radiation exposure in ...
Linear No-Threshold model for radiation risk
assessment predicts cancer risk due to radiation
exposure
Risk (excess cancer...
DNA damage increases mutations
and cancer risk
• Increase in cancer risk could be due to increased DNA damage 
and mutatio...
Inappropriate DNA methylation is a form
of DNA damage
Or:
Adapted from: http://www.nature.com/nature/journal/v441/n7090/im...
• Aberrant DNA methylation is linked with cancer
• Incorrect DNA methylation can be one of the initiating events 
in tumou...
• High doses of radiation have been reported to cause a loss in 
global DNA methylation
• Based on LNT, low dose radiation...
• Low doses of radiation have been shown to:
– reduce cancer risk and DNA mutations
– increase tumour latency
– also preve...
Novel application of an assay to detect
global DNA methylation changes
• Needed an assay that:
– is sensitive
– high throu...
ATCGTA
TAGCAT
CH3
=≠
Temperature →
Double Stranded
Single Stranded
CH3
ATUGTA
TAGUAT
ATCGTA
TAGCAT
Bisulphite Modification...
LINE1 methylation assay can detect
chemical demethylation
100% methylated
Vehicle
0.125 µM 5‐aza
0.5 µM 5‐aza
0 % methylat...
Preliminary data – the temporal effect of 1 Gy and
10 µGy on spleen tissue global DNA methylation levels
n=5
*p<0.05
*
*
• Is this a phenomenon that occurs only at LINE1 elements?
– other repeat elements (SINE, LTR)
• Investigate total methyla...
Future directions
If a single exposure (high or low) has long
term effects on DNA methylation
young mice old mice
if a low...
Summary and Significance
• This is the first example where temperature differences have 
been used to detect global DNA me...
• Highlights the importance of conducting longitudinal studies
– to assess the long term impact of a single exposure
• Und...
• DOE Group :
– Supervisors: Pam Sykes and Rebecca Ormsby
– Lab Members: Ben Blyth, Mark Lawrence, Alex Staudacher, Ami 
C...
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Development of a novel assay to investigate changes to global dna methylation levels in response to radiation exposure in vivo newman

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Development of a novel assay to investigate changes to global dna methylation levels in response to radiation exposure in vivo newman

  1. 1. Development of a novel assay to investigate changes to global DNA methylation levels in response to radiation exposure in vivo Michelle Newman
  2. 2. Linear No-Threshold model for radiation risk assessment predicts cancer risk due to radiation exposure Risk (excess cancers) Dose above background High dose epidemiological data Extrapolated low dose data 200 mGy
  3. 3. DNA damage increases mutations and cancer risk • Increase in cancer risk could be due to increased DNA damage  and mutations following radiation exposure strand breaks
  4. 4. Inappropriate DNA methylation is a form of DNA damage Or: Adapted from: http://www.nature.com/nature/journal/v441/n7090/images/441143a‐i2.0.jpg CH3 CH3 CH3 CH3 DNA methylation • Is a chemical modification to DNA  – occurs at cytosine residues • DNA methylation is involved in the     control of gene expression – affects the accessibility of DNA – changes structure of DNA • Can be altered by diet and exposure to   carcinogens
  5. 5. • Aberrant DNA methylation is linked with cancer • Incorrect DNA methylation can be one of the initiating events  in tumour progression Gene Gene X loss of methylation increased methylation expression of genes that  promote tumour growth suppression of genes that  prevent tumour growth DNA methylation is a chemical modification to DNA
  6. 6. • High doses of radiation have been reported to cause a loss in  global DNA methylation • Based on LNT, low dose radiation exposure should cause a  loss in DNA methylation – but on a lower scale than high dose radiation exposure • Lowest dose studied (500 mGy) has been shown to cause: – an increase or a decrease in DNA methylation (Kovulchuk et al., 2004; Raiche et al., 2004; Pogribny et al., 2005) – this was dependent on tissue investigated and sex of mice used in the  study – doses similar to medical exposures have not been studied DNA methylation can be altered by radiation exposure
  7. 7. • Low doses of radiation have been shown to: – reduce cancer risk and DNA mutations – increase tumour latency – also prevent the damage induced by high dose radiation • if given prior to high dose • Aim: – to investigate if DNA methylation is affected by low doses of radiation  in vivo Is DNA methylation affected by low dose radiation exposure?
  8. 8. Novel application of an assay to detect global DNA methylation changes • Needed an assay that: – is sensitive – high throughput – robust – reproducible • Most methods detect changes in methylation of single genes • We wanted to detect (global) methylation changes throughout the  genome resulting from radiation exposure • Other global assays are either not high throughput, reproducible or  sensitive • Utilising a chemical that allows the distinction between methylated and  unmethylated DNA and  – applying to sequences of DNA known as LINE1 elements, which are  found throughout the genome
  9. 9. ATCGTA TAGCAT CH3 =≠ Temperature → Double Stranded Single Stranded CH3 ATUGTA TAGUAT ATCGTA TAGCAT Bisulphite Modification Bisulphite Modification 0% Methylation 100% Methylation x % Methylation
  10. 10. LINE1 methylation assay can detect chemical demethylation 100% methylated Vehicle 0.125 µM 5‐aza 0.5 µM 5‐aza 0 % methylated ↑ methylated*p<0.05 n=5 ↓methylated Double stranded DNASingle stranded DNA
  11. 11. Preliminary data – the temporal effect of 1 Gy and 10 µGy on spleen tissue global DNA methylation levels n=5 *p<0.05 * *
  12. 12. • Is this a phenomenon that occurs only at LINE1 elements? – other repeat elements (SINE, LTR) • Investigate total methylated cytosine content using another  technique • Tissues, genetic background, sample size DNA methylation does not appear to be altered by low doses of radiation
  13. 13. Future directions If a single exposure (high or low) has long term effects on DNA methylation young mice old mice if a low dose can prevent the changes in methylation induced by high dose
  14. 14. Summary and Significance • This is the first example where temperature differences have  been used to detect global DNA methylation levels • Results indicate: – high dose exposure alters DNA methylation levels and this change is  dynamic over time – low doses may not alter methylation levels • effects may be subtle
  15. 15. • Highlights the importance of conducting longitudinal studies – to assess the long term impact of a single exposure • Understanding changes to DNA methylation in response to  relevant radiation exposures is important – given that aberrant DNA methylation changes is associated with  cancer Summary and Significance
  16. 16. • DOE Group : – Supervisors: Pam Sykes and Rebecca Ormsby – Lab Members: Ben Blyth, Mark Lawrence, Alex Staudacher, Ami  Cochrane, Katrina Bexis • RAH:  – Eva Bezak, Medical Physicist • Funding: – Low Dose Radiation Research Program, U.S. Department of Energy  – BHP Billiton/FMC Low Dose Radiation Research Scholarship Acknowledgements

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