The effects of Heat Shock Protein 90a and ATM on DNA repair

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The effects of Heat Shock Protein 90a and ATM on
DNA repair
Prolactin promotes the survival of breast cancer
cells
By Jennifer Procyshen CMMB 530 Honours Research
Project Undergraduate Student Research Program
USRP Supervisor Dr.C.Shemanko
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Outline

• Prolactin
• ATM and Heat Shock Protein 90a
• Histone 2AX
• Project Questions and Hypothesis
• Methods
• Results
• Conclusion and Future Directions

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Prolactin and Cancer

• PRL is produced in both the pituitary and mammary
  gland and regulates mammary development
• PRL overexpression in mice increased the
  frequency of both spontaneous and
  irradiation-induced mammary carcinomas
• PRL has been associated with an increased risk of
  breast cancer in post-menopausal women

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My Project

• Given prolactins importance in mammary
  development and its likely contribution to
  cancer, I am interested in studying the
  involvement of this hormone and its potential
  target genes in DNA repair

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Heat Shock Protein 90a and ATM

• Heat shock protein 90a is an important chaperone
  protein that is up-regulated in response to
  prolactin

• ATM Ataxiatelangiectasia mutated is a protein
  kinase activated in cells following
  irradiation-induced damage

Hsp90a
Hsp90a
Chaperone
ATM
DNA Repair
Clevenger et al., 2003
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Histone 2AX
Abraham R. Genes and Development. 2001.
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Histone 2AX
Abraham R. Genes and Development. 2001.
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Histone 2AX
Abraham R. Genes and Development. 2001.
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Geldanamycin (17-AAG)

• 17-AAG a less toxic derivative of geldanamycin
  and is currently in clinical trials for cancer
  therapies
• Binds Hsp90 inhibiting its chaperone ability,
  causing its substrates to be degraded
• Our lab has found that treatment with 17-AAG
  induces radiosensitivity

GA
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Project Questions!!

• Does expression of Hsp90a influence the
  effectiveness of ATM in DNA repair?
• Does prolactin affect DNA repair through the
  up-regulation of Hsp90a?

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Hypothesis

• Prolactin upregulates Hsp90 which then stabilizes
  ATM and allows for more efficient phosphorylation
  of repair proteins and hence increases the
  phosphorylation of H2AX.

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Methods

• Analysis
• 1) Immunofluorescence observe DNA foci and
  thus quantify the amount of DNA damage
• 2) Fluorescence Activated Cell Sorting (FACS)
  analysis quantify fluorescence in treated
  samples and compare to cell cycle position

pH2AX
DAPI
30 min Etoposide 4hr repair
30 min Etoposide 6hr repair
30 min DMSO- 0hr repair
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Cell cycling contributes to replicationinduced
DNA foci
pH2AX
30 min Etoposide 2hr repair
DAPI
30 min DMSO 2hr repair

• The replication induced DNA foci have made it
  impossible to detect damage-induced foci

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Hsp90a inhibition does not prevent H2AX
phosphorylation in actively cycling cells
pH2AX
DAPI
DMSO 17-AAG 1hr repair
ATM is not the only kinase responsible for
replication-induced H2AX phosphorylation or
possibly damage-induced foci
Etoposide 17-AAG 1hr repair
Etoposide 17-AAG 1hr repair
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Switching Directions.

• Replication-induced foci did not appear to be due
  to ATM activity and prevented detection of
  damage-induced foci
• Use of a different damaging agent and I switched
  to irradiation to examine ATMs role in
  damage-induced DNA foci

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ATM is essential for H2AX phosphorylation at 2Gy
irradiation
pH2AX
DAPI
2Gy irradiation - 17-AAG 1hr repair
2Gy irradiation 17-AAG 1hr repair
ATM is essential for DNA repair at 2 Gy
irradiation yet other kinases may be responsible
for phosphorylation at higher doses
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Summary of Findings.

• High levels of replication-induced DNA foci in
  un-treated samples likely due to active cell
  cycling
• Etoposide treatment initiated cell cycle arrest
  yet did not generate an increase in foci that
  could be detected by Immunofluorescence or FACS
• ATM is not the only kinase contributing to basal
  or possibly damage-induced DNA foci
• ATM contributes to H2AX phosphorylation at
  irradiation doses of 2 Gy yet other kinases
  appear to be involved in H2AX phosphorylation at
  higher doses

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Future Directions

• Replicate irradiation experiments with lower
  concentrations of 17-AAG and subsequently /- Prl
  to observe possible rescue of ATM activity
• Establish a consistent method of detecting
  damage-induced DNA foci and scoring based on foci
  intensity
• Specifically examine the role of Hsp90a using an
  additional cell line in combination with
  irradiation

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Thanks!!!
USRP in Health and Wellness

• Dr. Shemanko
• Dr. Christian Perotti
• Rachel Liu
• Tom Wiedl
• Caroline White
• Anna Urbanska
• Stephanie Moffat
• Dr. Lohka
• Laurie Robertson Flow Lab
• Dr.S.Lees Millers lab Irradiation source
• Dr.Hansen and the entire Hansen Lab!!!!!
• Undergraduate Student Research Program - USRP