Utility of Luminex technology to determine Utility of Luminex technology to determine growth factor activation and growth factor activation and

downstream signaling of factor (X) downstream signaling of factor (X)

Alvin AyalaAlvin AyalaAmgen IncAmgen IncApril 2006April 2006

Utility of Luminex technology to determine Utility of Luminex technology to determine growth factor activation and growth factor activation and

downstream signaling of factor (X) downstream signaling of factor (X)

Alvin AyalaAlvin AyalaAmgen IncAmgen IncApril 2006April 2006

Outline Outline

To confirm the activation status of a growth factor receptor stimulated by a known ligand (X) in various solid tumors

– Gene expression of the receptor in various tumor cell lines

– Perform In vitro proliferation assays– IP/Western blot to confirm activation of the

receptor– Luminex-downstream signaling

To confirm the activation status of a growth factor receptor stimulated by a known ligand (X) in various solid tumors

– Gene expression of the receptor in various tumor cell lines

– Perform In vitro proliferation assays– IP/Western blot to confirm activation of the

receptor– Luminex-downstream signaling

Experimental approachExperimental approach

Screen for human tumor lines that express the Screen for human tumor lines that express the receptor for factor Xreceptor for factor X

Receptor gene expression profilingReceptor gene expression profiling

Design primers and probesDesign primers and probes

For mRNA detectionFor mRNA detection

In vitro proliferation assays adding factor XIn vitro proliferation assays adding factor X

In vitro IP/Western blot of p-Y of the receptor after stimulationIn vitro IP/Western blot of p-Y of the receptor after stimulation

Confirm downstream signaling triggered by factor additionConfirm downstream signaling triggered by factor addition

• Expression via quantitative RT-PCR (TaqMan) (23 cell lines).

• Assessed as a ratio of expression of target gene over beta actin.

• Data later expressed as ‘fold change’ of gene expression relative to a single sample whose gene expression has been set to a value of 1.

• Error bars represent SEM using three independent replicates.

Tumor receptor expression screeningTumor receptor expression screening

Human tumor cell line Human tumor cell line gene expression of receptor Xgene expression of receptor X

Human tumor cell line Human tumor cell line gene expression of receptor Xgene expression of receptor X

Response to factor X addition In vitroResponse to factor X addition In vitro

• To confirm the activation status of the receptors expressed in these cell types

• Two medium expressing cell lines were chosen for further analysis

– HT-29 (Colorectal)

– FADU (Head and Neck)

• These cells lines were grown in vitro and stimulated with factor X in the presence or absence of serum

• To confirm the activation status of the receptors expressed in these cell types

• Two medium expressing cell lines were chosen for further analysis

– HT-29 (Colorectal)

– FADU (Head and Neck)

• These cells lines were grown in vitro and stimulated with factor X in the presence or absence of serum

HT-29 cells response to factor XHT-29 cells response to factor XHT-29 cells response to factor XHT-29 cells response to factor X

Proliferation of HT-29 in the presence of GF

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

No serum Fullserum

0 1 10 50 100 1K 10K

GF ng/ml

Fo

ld c

ha

ng

e

Starve

Full

FADU cells response to factor XFADU cells response to factor XFADU cells response to factor XFADU cells response to factor X

Proliferation of FADU in the presence of GF

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

No Serum Fullserum

0 1 10 50 100 1K 10K

GF ng/ml

Fo

ld c

ha

ng

e

Starve

Full

In vitro proliferation poor

Imuno-precipitation / Western blotImuno-precipitation / Western blot• Effort was made to detect phosphorylation of the receptor using specific

receptor antibodies coupled to generic anti phospho-tyrosine antibodies

• This approach failed to demonstrate a differential level of phosphorylation

between stimulated and un stimulated cells from either HT-29 or FADU cells

• Effort was made to detect phosphorylation of the receptor using specific

receptor antibodies coupled to generic anti phospho-tyrosine antibodies

• This approach failed to demonstrate a differential level of phosphorylation

between stimulated and un stimulated cells from either HT-29 or FADU cells

Factor X receptor

Pos

CH

O

Neg

Pos

Pos

CH

O

Neg

Pos

Pos = GF 100ng/ml 10’Neg = UnstimulatedPos CHO (stable CHO line)

Ig heavy and light chains

HT-29 cells FADU cells

IP: anti factor X receptorWB: anti-p-Y

Assessing downstream signaling P-Akt status after factor additionAssessing downstream signaling P-Akt status after factor addition

Experimental design

5 10 20 30 50 60

GF addition 100ng/ml

Cells plated 5 million/well

Serum starvation48 hours

AssayCell lysis

Time (mins)

Assessing downstream signaling p-Akt status after factor X additionAssessing downstream signaling p-Akt status after factor X addition

Ser 473 p-Akt after factor X stilmulation

0

5

10

15

20

25

30

35

40

45

50

No GF 5 min. 10 min. 20 min. 30 min. 50 min. 60 min.condition

Rat

io p

ho

sph

o/t

ota

l

FADU

HT-29

Imuno-precipitation / Western blotImuno-precipitation / Western blot• Effort was made to detect phosphorylation of the receptor using specific

receptor antibodies coupled to generic anti phospho-tyrosine antibodies

• This approach failed to demonstrate a differential level of phosphorylation

between stimulated and un stimulated cells from either HT-29 or FADU cells

• Effort was made to detect phosphorylation of the receptor using specific

receptor antibodies coupled to generic anti phospho-tyrosine antibodies

• This approach failed to demonstrate a differential level of phosphorylation

between stimulated and un stimulated cells from either HT-29 or FADU cells

Factor X receptor

Pos

CH

O

Neg

Pos

Pos

CH

O

Neg

Pos

Pos = GF 100ng/ml 10’Neg = UnstimulatedPos CHO (stable CHO line)

Ig heavy and light chains

HT-29 cells FADU cells

IP: anti factor X receptorWB: anti-p-Y

Data SummaryData SummaryData SummaryData Summary

• Gene expression of factor X receptor at the cDNA level may not be representative of function

• In vitro proliferation responses of HT-29 and FADU cells to factor X stimulation are a poor measure of activation

• Poor availability of tools to investigate biology can restrict the type of assays established to answer specific scientific questions

• Luminex technology provided a high throughput and sensitive measure of signal transduction triggered by factor X addition in HT-29 and FADU cells

• Gene expression of factor X receptor at the cDNA level may not be representative of function

• In vitro proliferation responses of HT-29 and FADU cells to factor X stimulation are a poor measure of activation

• Poor availability of tools to investigate biology can restrict the type of assays established to answer specific scientific questions

• Luminex technology provided a high throughput and sensitive measure of signal transduction triggered by factor X addition in HT-29 and FADU cells

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