Molecular Signaling
Network of
Cripto-1
Biplab Bose
Department of Biotechnology
Indian Institute of Technology Guwahati
Complexity of Signaling Networks
1. Large number: molecules and their
interactions
2. Non-linear architecture
3. Integration of multiple paths
4. Extensive variations: Cell specific,
time specific
5. Adaptive in the face of noise
Human Oncofetal Protein Cripto-1
During Development:
Intracellular Signaling
Pathways:
• Nodal/ALK4/Smad-2
• Glypican-1/c-Src/MAPK
• Glypican-1/c-Src/AKT
• Patterning of the Anterior/Posterior axis
• Specification of mesoderm and
endoderm during gastrulation
In Adult :
• Mammary gland development
In Oncogenesis :
• Triggers proliferation
• Induction of cellular migration and invasion
• promotion of angiogenesis
• Stimulates EMT
Mitogenic Pathways of CR-1
• Soluble CR-1 and membrane
bound CR-1, both are functional
• Can signal through ErbB4 too.
• Can block growth inhibitory
signal of TGF-b
Open Questions in CR-1 Signaling
% Proliferation
HUVEC cells*
CR-1 (ng/ml)
HC-11 cells**
• Origin of Cell-specific effect
• Control elements: feedback,
involvement of multiple receptors
*J Natl Cancer Inst. 2005;97:132– 41
** Cell Growth Differ. 1997, 8(12):1257-66.
The Strategy to Look For Contextual Effect
Cripto-1
Cells with high
Glypican-1
• Cell Proliferation
• Activation of mitogenic patwhays
Cells with lower
Glypican-1
Selecting Suitable Cellular System
• Cripto-1 + Glypican-1
ERK1/2
and Akt
+p
Proliferation
• Glypican-1 has very low expression in HeLa cells in comparison to U-87 MG cells
+p
• Real Time PCR was done using cyber green as the reporter dye
• GAPDH, β-actin and PPIA used as endogeneous control.
• U-87 MG: Human Glioblastoma; HeLa: Cervical Adenocarcenoma
Expression of recombinant human Cripto-1 in E. coli
C-terminal truncated CR-1 cloned in pGEX-4T2
Cloning of CR-1-ΔC
Protein expressed in E. coli is purified
SDS - PAGE
cDNA clone of CR-1
1
PCR
BamHI
169
97kDa
66kDa
XhoI
43kDa
CR-1-ΔC
29 kDa
GST
20 kDa
Ptac
M
pGEX-4T-2
Ampr
pBR322
ori
CR1-GST
12% SDS-PAGE and
stained by silver staining
CR-1 Induces Proliferation of U-87 MG
MTT assay
• Cell line: U-87 MG
• 48 hr treatment on 104 cells/ well.
• Biphasic: Mitogenic at low concentration; inhibits at higher
CR-1 Reduces Proliferation of HeLa Cell
MTT assay
• Cell line: HeLa
• 48 hr treatment on 104 cells/ well.
Serum free
With serum
CR-1 Reduces Proliferation of HeLa Cell
Trypan Blue Exclusion Test
• Cell line: HeLa
• 72 hr treatment on 103 cells/ well.
• CR1-GST and GST: 200 ng/ml
One way ANOVA with pairwise multiple comparison: *, significantly different from other treatment groups, p < 0.01 and #,
not significantly different from untreated, p > 0.05.
CR-1 Reduces Proliferation of HeLa Cell
BrdU Assay
• Cell line: HeLa, 104 cells/ well.
• BrdU pulse: 3 hr before assay
• CR1-GST : 200 ng/ml
CR-1 Activates Mitogenic Pathways in U-87 MG
• Cripto-1 + Glypican-1
ERK1/2
+p
and Akt
+p
Proliferation
U-87 MG
Densitometry of WB
• Treatment : 200 ng/ml of CR1-GST and GST in serum free condition
CR-1 Fails to Activate Mitogenic Pathways in HeLa
• Cripto-1 + Glypican-1
ERK1/2
+p
and Akt
HeLa
• Treatment : 200 ng/ml of CR1-GST and GST in serum free condition
+p
Proliferation
Anti-Proliferative Effect in Other Cell Lines
Cell lines: HT-29 and HEK 293
Expression of Glypican-1 is low
Real-Time PCR
CR1-GST inhibit proliferation
MTT assay
Understanding The Mechanism of Reduced Cell Viability
In contrast to conventional wisdom, treatment with CR-1 reduces
number of viable HeLa cells
Apoptosis
Necrosis
Cell cycle Arrest
Slow cell cycle
CR-1 Does Not Induce Apoptosis
Flowcytometry : AnnexinV vs PI
• Cell line: HeLa
• 72 hr treatment on 105 cells/ well.
• CR1-GST and GST: 200 ng/ml; Cisplatine (3 μg/ml) as (+)ve control
Untreated
CR1-GST treated
PI
Annexin V
GST treated
Cisplatin treated
CR-1 Does Not Induce Apoptosis
Flowcytometry : AnnexinV vs PI
• Cell line: HeLa
• 72 hr treatment on 105 cells/ well.
• CR1-GST and GST: 200ng/ml, Cisplatine : 3μg/ml
*
*
*
*
One-way ANOVA , no significant difference among these treatment groups, p > 0.05.
CR-1 Does Not Induce Necrosis
LDH Assay
• Cell line: HeLa, 104 cells/ well.
• CR1-GST and GST: 200 ng/ml
• Triton X-100 (0.1%) as a +Ve control
CR-1 Does Not Induce Cell Death in HeLa
• HeLa cell treated with CR1-GST for 72 hr
•No morphological change
CR-1 Dose Not Arrest Cell Cycle
• HeLa cells were treated with CR1-GST or GST 200ng/ml 72 hr treatment
• Stained with propidium iodide (PI)
• Stained cells were analyzed by flow cytometry
CR-1 merely changes the cell cycle phases
Each bar represent mean of four independent experiments. * and ** represent significant
differences with untreated and GST treated cells, One-way ANOVA, p < 0.05.
Measuring Cell Proliferation by Dye (CFSE) Dilution
•CFDA-SE : Membrane permeable dye
•CFSE: Not membrane permeable, it can retain
inside cell upto 10 successive generation
Day 2
Count
Day 3
CFSE (FL1-H)
• CFDA –SE: carboxyfluorescein diacetate succinimidyl ester
• CFSE: carboxyfluorescein diacetate succinimidyl
Day 1
CR-1 Increases Doubling Time of HeLa Cells
• After CFSE (5 μM ) staining HeLa cells were treated with CR1-GST (200ng/ml), GST (200ng/ml) for
72 hr
•Flow cytometry was done at respective time points
Calculation of doubling time :
Mean doubling time, <Td> =
T/log2(<F0>/<FT>); here, <F0> =
geometric mean fluorescence
intensity at zero hour and <FT>=
geometric mean fluorescence
intensity at T hour.
CR-1 Increases Doubling Time of HeLa Cells
HeLa Cell
27 hrs
Each bar represents mean of three independent experiments.
*, significantly different from other treatment groups, One-way
ANOVA, p < 0.05.
CR-1 Treated HeLa
Cell
37 hrs
Effect of CR-1 on Cyclins
* Statistically significant p<0.05
Cell cycle figure credit: Nature Reviews Neuroscience, 2007, 8, 438-450
Effect of CR-1 on Cdk Inhibitors
* Statistically significant p<0.05
Cell cycle figure credit: Nature Reviews Neuroscience, 2007, 8, 438-450
Anti –proliferative Pathway of CR-1
Anti –proliferative Pathway of CR-1
Status of PTEN Based Pathway in U-87 MG cells
• U-87 MG is a PTEN-null cell line
Anti-proliferative Pathway of CR-1 is Translation Dependent
MTT Assay
• Cell line: HeLa
• 48 hr treatment: CR1-GST ( 200ng/ml) + Cyclohexamide
Two Opposing Pathways Determine The Fate
Deconstructing CR-1 Pathway
≡
Incoherent Feed-Forward
Deconstructing CR-1 Pathway
Non-adaptive System
Adaptive System
Incoherent Feedforward: Adaptive, Pulse generating, Ultra-sensitive switch
Funded by: MHRD, DBT & DST, Govt. of India