Department of BiotechnologyIndian Institute of Technology Guwahati
Molecular Signaling
Network of
Cripto-1
Biplab Bose
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
Intracellular Signaling Pathways:
• Nodal/ALK4/Smad-2
• Glypican-1/c-Src/MAPK
• Glypican-1/c-Src/AKT
During Development:
• Patterning of the Anterior/Posterior axis
• Specification of mesoderm and endoderm during gastrulation
In Oncogenesis :
• Triggers proliferation
• Induction of cellular migration and invasion
• promotion of angiogenesis
• Stimulates EMT
In Adult : • Mammary gland development
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-
** Cell Growth Differ. 1997, 8(12):1257-66.
HC-11 cells**
HUVEC cells*
*J Natl Cancer Inst. 2005;97:132– 41
% P
rolif
erat
ion
CR-1 (ng/ml)
• Origin of Cell-specific effect
• Control elements: feedback,
involvement of multiple receptors
Open Questions in CR-1 Signaling
Cripto-1
Cells with high Glypican-1
Cells with lower Glypican-1
• Cell Proliferation• Activation of mitogenic patwhays
The Strategy to Look For Contextual Effect
Selecting Suitable Cellular System
• Cripto-1 + Glypican-1 ERK1/2 and Akt
• Glypican-1 has very low expression in HeLa cells in comparison to U-87 MG cells
• 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
+p +p Proliferation
Expression of recombinant human Cripto-1 in E. coli
C-terminal truncated CR-1 cloned in pGEX-4T2
GST
pGEX-4T-2
Ptac
AmprpBR322ori
1 169
BamHI XhoI
cDNA clone of CR-1
PCR
CR-1-ΔC
Cloning of CR-1-ΔC SDS - PAGE
Protein expressed in E. coli is purified
12% SDS-PAGE and stained by silver staining
M CR1-GST
97kDa
43kDa
29 kDa
66kDa
20 kDa
CR-1 Induces Proliferation of U-87 MG
• Cell line: U-87 MG
• 48 hr treatment on 104 cells/ well.
MTT assay
• Biphasic: Mitogenic at low concentration; inhibits at higher
• Cell line: HeLa
• 48 hr treatment on 104 cells/ well.
CR-1 Reduces Proliferation of HeLa Cell
MTT assay
Serum free With serum
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 Reduces Proliferation of HeLa Cell
CR-1 Activates Mitogenic Pathways in U-87 MG
U-87 MG
• Treatment : 200 ng/ml of CR1-GST and GST in serum free condition
Densitometry of WB
• Cripto-1 + Glypican-1 ERK1/2 and Akt +p +p Proliferation
CR-1 Fails to Activate Mitogenic Pathways in HeLa
• Treatment : 200 ng/ml of CR1-GST and GST in serum free condition
• Cripto-1 + Glypican-1 ERK1/2 and Akt +p +p Proliferation
HeLa
Anti-Proliferative Effect in Other Cell Lines
Cell lines: HT-29 and HEK 293
Expression of Glypican-1 is low CR1-GST inhibit proliferation
Real-Time PCR MTT assay
Understanding The Mechanism of Reduced Cell Viability
In contrast to conventional wisdom, treatment with CR-1 reduces number of viable HeLa cells
Cell cycle Arrest
Slow cell cycle
Apoptosis Necrosis
CR-1 Does Not Induce Apoptosis
• Cell line: HeLa
• 72 hr treatment on 105 cells/ well.
• CR1-GST and GST: 200 ng/ml; Cisplatine (3 μg/ml) as (+)ve control
Flowcytometry : AnnexinV vs PI
PI
Annexin V
Untreated
Cisplatin treatedCR1-GST treated
GST treated
• Cell line: HeLa • 72 hr treatment on 105 cells/ well.
• CR1-GST and GST: 200ng/ml, Cisplatine : 3μg/ml
Flowcytometry : AnnexinV vs PI
*
**
One-way ANOVA , no significant difference among these treatment groups, p > 0.05. *
CR-1 Does Not Induce Apoptosis
• 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 Necrosis
LDH Assay
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.
CFSE (FL1-H)
Co
un
t
Measuring Cell Proliferation by Dye (CFSE) Dilution
• CFDA –SE: carboxyfluorescein diacetate succinimidyl ester • CFSE: carboxyfluorescein diacetate succinimidyl
•CFDA-SE : Membrane permeable dye
•CFSE: Not membrane permeable, it can retain inside cell upto 10 successive generation
Day 1Day 2Day 3
• 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
CR-1 Increases Doubling Time of HeLa Cells
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.
Calculation of doubling time :
27 hrs 37 hrs
HeLa Cell CR-1 Treated HeLa Cell
Each bar represents mean of three independent experiments. *, significantly different from other treatment groups, One-way ANOVA, p < 0.05.
CR-1 Increases Doubling Time of HeLa Cells
Cell cycle figure credit: Nature Reviews Neuroscience, 2007, 8, 438-450
Effect of CR-1 on Cyclins
* Statistically significant p<0.05
Cell cycle figure credit: Nature Reviews Neuroscience, 2007, 8, 438-450
* Statistically significant p<0.05
Effect of CR-1 on Cdk Inhibitors
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
• Cell line: HeLa • 48 hr treatment: CR1-GST ( 200ng/ml) + Cyclohexamide
MTT Assay
Two Opposing Pathways Determine The Fate
≡
Incoherent Feed-Forward
Deconstructing CR-1 Pathway
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