2. TEP1
• Yeast homolog of PTEN/MMAC1/TEP1
Mutations can lead to prostate, endometrial, melanoma
• Tensin-like phosphatase
• Dephosphorylates PIP3 inactivating Akt signaling
pathway
3.
4. Wortmannin
• Steroid metabolite of Penicillium funiculosum
• In vitro inhibitory concentration of around 5 nM
• Irreversible inhibitor
Lithium Chloride
• Exothermic
• Less potent than wortmannin
Inhibitory concentration around 1.4 uM
• Reversible
5. Enzymes responsible for cell growth and
proliferation
What happens to the growth rate of yeast
when PI3K inhibitors are introduced?
• Wild-type
• TEP1 knockout
6. Introduce various concentrations of each
inhibitor
Measure absorption every 4 hours for 32
hours
The higher the absorption, the more cells
• Inhibition lowers absorption
Determine growth curve and compare
strains
7. 0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0 4 8 12 16 20 24 28 32
Absorbance
Time (hrs)
Wild-type Mat Alpha
Control 6 uM 24 uM 60 uM
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0 4 8 12 16 20 24 28 32
Absorbance
Time (hrs)
Wild-type Mat A
Control 6 uM 24 uM 60 uM
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0 4 8 12 16 20 24 28 32
Absorbance
Time (hrs)
TEP1 K/O Mat A
Control 6 uM 24 uM 60 uM
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0 4 8 12 16 20 24 28 32
Absorbance
Time (hrs)
TEP1 K/O Homo Dip
Control 6 uM 24 uM 60 uM
9. Determined growth rate of yeast
beforehand
Different spectrophotometer
• Level of solution in cuvettes
Vortex cells before measuring absorption
10. 0
0.2
0.4
0.6
0.8
1
1.2
0 4 8 12 16 20 24 28 32
Absorption
Time (hrs)
Wild-type Mat Alpha
Control 3 mM 20 mM 100 mM
0
0.2
0.4
0.6
0.8
1
1.2
0 4 8 12 16 20 24 28 32
Absorption
Time (hrs)
Wild-type Mat A
Control 3 mM 20 mM 100 mM
0
0.2
0.4
0.6
0.8
1
1.2
0 4 8 12 16 20 24 28 32
Absorption
Time (hrs)
TEP1 K/O Mat A
Control 3 mM 20 mM 100 mM
0
0.2
0.4
0.6
0.8
1
1.2
0 4 8 12 16 20 24 28 32
Absorption
Time (hrs)
TEP1 K/O Homo Dip
Control 3 mM 20 mM 100 mM
11. Much better results
• Very few outliers
TEP1 homozygous
Diploid
• Not expected
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 4 8 12 16 20 24 28 32
Absorption
Time (hrs)
TEP1 K/O Mat Alpha
Control 3 mM 20 mM 100 mM
12. Wortmannin
• PI3K inhibitor or activator?
Lithium Chloride
• Better results with indication of inhibition of the Akt
signaling pathway
Replications are unfortunately missing
• Time
• Money
13. Heymont, Jennifer, Ludmilla Berenfeld, Jennifer Collins,
and Alexandra Kaganovich. "TEP1, the Yeast Homolog of
the Human Tumor Suppressor Gene
PTEN/MMCAC1/TEP, Is Linked to the
Phosphatidylinositol Pathway and Plays a Role in the
Developmental Process of Sporulation." PNAS 97.23
(2000): 12673-2677. Print
"PI3K / Akt Signaling Pathway." Welcome to CST. Ed.
Michael Scheid. N.p., Sept. 2007. Web. 3 Nov. 2014.
14. I would like to thank Dr. Coleman for being
my mentor and also Miguel Zambrano and
Andrea Caselles for helping me with this
research.
