Project 3 Mitochondrial DNA Mutations in Prostate Tumorigenesis and StromalEpithelial Interaction

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Project 3Mitochondrial DNA Mutations in Prostate
Tumorigenesis and Stromal-Epithelial Interaction

• PI John Petros

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Fluorescent Dyes MitoSOX Red superoxide
indicator for live cells. It is rapidly and
selectively targeted to mitochondria. APF
aminophenyl fluorescein is a new ROS indicator.
It becomes fluorescent in the presence of
hydroxyl radical (.OH), peroxyntrite anion
(ONOO-), and hypochlorite anion (-OCl). DAF-FM
4-amino-5-methylamino ,7-difluorofluorescein
becomes fluorescent when it reacts with
NO. Inhibitors FCCP (Carbonyl cyanide
4-(trifluoromethoxy)phenylhydrazone) FCCP is a
protonophore (H ionophore) and uncoupler of
oxidative phosphorylation in mitochondria. It is
capable of depolarizing plasma and mitochondrial
membranes. Stigmatillin - Inhibitor of electron
transport. Acts at the Qo center of the bc1
complex, binds to the heme b1 domain of
cytochrome b as well as to the iron-sulfur
protein1
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Paper 1 Increase ROS Levels Due to Mutations in
Mitochondrial DNA

• Show definitively that certain mutations in the
  mitochondrial DNA can affect levels of superoxide
  and hydrogen peroxide in the cell.
• Various techniques
• EPR (multiple probes)
• DCF

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Cytochrome C Assay of mitopreps from selected
lymphoblasts
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EPR Experiment to measure superoxide internally
and externally
Probe - CMH
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DAF-FM indicator of NO levels.
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APF indicator of the presence of hydroxyl
radical (.OH), peroxyntrite anion (ONOO-), and
hypochlorite anion (-OCl).
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MitoSOX Red superoxide indicator for live cells
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CM-DCFDA indicator of peroxide levels
Not shown stigmatellin had no detectable
effect on peroxide levels.
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Currently

• Looking at superoxide exclusively on the outside
  of the cell using a different spin probe (Cat-1)
  in an attempt to determine if changes in ROS
  levels is due to NADPH oxidase as well as
  Mitochondrial DNA mutations.

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Superoxide generated externally
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Where to go from here

• More detailed studies using CM-DCFDA to examine
  peroxide levels.
• Characterize additional lymphoblasts with other
  mitochondrial mutations.

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We all remember the famous Farach-Carson Film

• Watching the cells exchange material stimulated
  some ideas.
• Could we use mitochondrial mutations to track the
  exchange.
• Co-culture prostate epithelial and bone stromal
  cells and then separate and analyze mitochondrial
  DNA
• Does the exchange have an effect on cell behavior
• Changes in ROS levels
• Changes in message levels
• Both of these changes could occur in the presence
  of co-culture without exchange of material.
• Is this physiologically relevant? Relevant to
  metastasis?

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Co-cultured cells and measured peroxide levels
Use a cell tracker and separate C4-2B population
from HS5 population and measure individual DCF
levels. Need to consider cell number,
confluency, incubation time
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C4-2B and H5 Co-culture experiment
160
40
140
35
120
30
100
25
80
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Relative Red Fluorescence
Relative DCF Fluorescence
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60
10
40
5
20
0
0
HS5
C4-2B
HS5
C4-2B
HS5 no dye
HS5 no dye
C4-2B/HS5 combined
C4-2B/HS5 combined
C4-2B/HS5 combined
C4-2B/HS5 combined
C4-2B/HS5 co-cultured
C4-2B/HS5 co-cultured
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Directions we are headed

• Use cell tracker in both epithelial and stromal
  cells (like Cindy did) and co-culture followed by
  separation in epithelial from stromal cells.
• Analyze individual populations for mitochondrial
  DNA (help from Dave Martin)
• Look at message levels

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How are we doing with regards to our Specific
Aims?

• Specific Aim 1. We are continuing to pursue the
  development of fusion protocols to make
  additional cybrids.
• Specific Aim 2. We have begun studies with our
  existing cybrids to determine the effects of
  co-culture of these cells with bone stromal
  cells.
• Specific Aim 3. I will begin the analysis of the
  correlation between mitochondrial DNA mutations
  and apoptosis related proteins in prostate tissue
  within the next week or so.

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Coming up next time we present!

• Work by Carrie Sun
• Co-culture of cybrids with bone stromal
• Growth effects in culture
• Growth effects in mice
• ROS levels