By: Chris Carr

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• By Chris Carr

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Background Information

• Theileria parva is an intracellular protozoan
  parasite that is transmitted by ticks and causes
  East Coast fever, a disorder of cattle in East
  and Central Africa
• They invade and immortalize bovine lymphocytes

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The major players

• C-Myc a transcription factor that binds to
  specific sites in the promoters of at least 1382
  different genes
• JAK2/STAT3 a signalling pathway that
  contributes to c-Myc transcription
• Mcl-1 target gene of c-Myc
• CK2 a factor that increases the stability of
  c-Myc
• BW720c a drug that kills Theileria
• AG 490 a drug that inhibits JAK2 activity
• PARP poly(ADP-ribose) polymerase
• Z-VAD Caspase inhibitor

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What on earth is this about?

• Theileria initiates a strong and sustained
  induction of c-Myc
• This is caused by intervening with both c-Myc
  transcription and stability
• Infection then results in a c-Myc mediated
  antiapoptotic response

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How is this possible?

• Activation of CK2
• Activation lf NF-?B signalling pathway
• Induction of anti-apoptotic proteins
• Decrease in the number of pro-apoptotic proteins
• Infected lymphocytes use autocrine loops
• -TNF autocrine loop NF-?B activation
• -GM-CSF autocrine loop

P13K activation
AP-1 induction
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What are autocrine loops?

• A process of cell signaling whereby a cell both
  releases and responds to a soluble factor
• The loops refer to the signal both originating
  and ending in the same location

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Experiment 1 Theileria infection leads to
phosphorylation of STAT3, and induction of c-Myc
and Mcl-1 (part 1)

• Theileria pursuade lymphocytes to proliferate
  uncontrollably via GM-CSF autocrine loop.

Non-infected
Infected
Does this proliferation occur in tandem with a
signalling pathway? Is the end result of this
signalling pathway an increase in c-Myc levels?
YES
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Experiment 1 Part 2

• Are the phosphorylation of STAT3 and the
  induction of c-Myc due directly to infection of
  Theileria?

Note Stat3 is a protein that is present
consistently with or without infection. However,
the phosphorylation of this protein is
dramatically increased upon infection. BW 720c
was used to kill Theileria
Of Course
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Wait theres moreExperiment 1 Part 3

• Does STAT3 signalling to c-Myc involve the JAK2
  kinase?

AG490 was used to inhibit JAK2 Over the course
of the experiment, blocking JAK2 signalling
resulted in a greater decrease in STAT3
phosphorylation than eliminating Theileria
altogether
Indubitably
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Experiment 2 Theileria dependent
post-translational stabilization of c-Myc
involves CK2

• Does parasite infection affect the stability of
  the c-Myc protein?

BW 720c was used to kill Theileria Cyclohexamide
was used to inhibit the transcription of
c-Myc In B-cells containing Theileria, c-Myc
half-life was significantly prolonged
Yes
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Experiment 2 continued
Apigenin was used as a CK2 inhibitor

• The presence of live Theileria parasites leads to
  a CK2-mediated increase in the stability of c-Myc

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Experiment 3 Theileria-induced transcriptional
activation of c-Myc is mediated in part by GMC-SF
via activation of STAT3

• Does Theileria induce high c-Myc levels by
  transcriptional regulation?

Theileria induces a four-fold increase in c-Myc
driven luciferase activity
Yup
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Experiment 3 Continued
C-Myc transactivation is dependent on live
parasites
C-Myc binds to the luciferase gene which results
in luciferase activty
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Experiment 3 Yes, there is more
Addition of recombinant GM-CSF to infected cells
increased endogenous c-Myc transactivation
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Experiment 3 further continued
A promotor containing 4 E-box elements were
introduced upstream from the luciferae gene.
Luciferase activity is decreased because there
are an increased number of binding sites for
c-Myc to attach to.
Thus, target gene transcription is reduced.
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Experiment 3 Wait, there is more
control
Kinase dead JAK2
The co-transfection of different mutants
significantly lowered c-Myc driven luciferase
activity Therefor, transcriptional induction of
c-Myc clearly involves JAK2 and STAT3
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Experiment 4 Theileria-transformed B cell
survival is c-Myc dependent

• How significant is the contribution of c-Myc to
  the survival of Theileria-infected B cells?

Anti-sense
C-Myc transcription was interfered with by the
addition of antisense oligonucleotides
Non-sense
Very Significant
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Experiment 4 continued
Cell death correlated to a loss of c-Myc levels,
Mcl-1 expression, and PARP cleavage Thus, Mcl-1
can be directly attributed to c-Myc activation
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Experiment 5 Inhibition of JAK2 results in
caspase-dependent apoptosis of Thjeileria-
transformed B cells
AG 490 induced apaptosis is caspase dependent
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Experiment 5 continued
LETD-AFC is a caspase 8 substrate DEVD-AFC is a
general caspase substrate
There is no LETD activity which means caspase 8
is not a part of this mechanism of cell death
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Experiment 5 Can you handle it?
In correlation with caspase 9 activation, there
is PARP cleavage (fig 5c) There is also
Annexin-V positivity and nuclear fragmentation
(fig 5d)
Therefore, the inhibition of the JAK2 signalling
pathway leads to B-cell apoptosis
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Experiment 6 Ectopic expression of c-Myc
rescues Theileria infected B cells from AG 490
mediated apoptosis
Apoptosis results when AG 490 blocks JAK2
activity, however, the addition of ectopic c-Myc
reverts cell death.
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Experiment 6 continued
Ectopic expression of CMV-cMyc augments c-Myc
levels, but does not effect STAT3 or its
phosphorylation status
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Results Yes, the experiments are finally over
Theileria infection leads to increased levels of
c-Myc The life of the c-Myc transcription
factor is prolonged due to CK2-mediated
phosphorylation A JAK 2/STAT3 signalling pathway
also contributes to increased c-Myc
transcription This anti-apoptotic process can be
inhibited at several junctures (Apigenin, BW
720c, AG 490)
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Further studies

• Other cytokines may be secreted that activate
  JAK2 and c-Myc by yet-to-be described autocrine
  loops
• How do P13K activation and AP-1 induction lead to
  the phosphorylation of STAT3

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The End
Now you may start clapping