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