Introduction genomic studies. This because a proteins function, structure

Introduction

Reverse phase protein array (RPPA) is a
proteomic array designed to measure the protein levels and protein
modifications (e.g. phosphorylation) of a number of samples, simultaneously. Proteomics
is the large scale study of a proteins’ structure, function and interactions.
The study of proteins has proven difficult compared to genomic studies. This
because a proteins function, structure and post translational state fluctuates
greatly from cell to cell and time to time. Proteomic studies can be extremely
useful in understanding diseases, their progression and in developing targeted
therapies. Thus, this review will examine how RPPA has been utilized to shed
light on cancer progression, a complicated multistep disease. Cancer can
develop due to genetic or epigenetic susceptibilities. Genomics has proven
useful in determining key oncogenes (e.g. BRCA1) which can lead to the
development of cancer. However, not all cancers are due to the presence of an
oncogene or the absence of a tumor suppressor (e.g PTEN). Deregulated signaling
pathways have now gained many cancer researchers attention. Hijacking signaling
pathways has proven a successful mode in attaining immortality. These cellular
pathways which can be activated by growth factors, generally proceed through a
signaling cascade, often causing successive phosphorylation of downstream
proteins. Unfortunately, transcriptional or genomic arrays can only detect a
cells protein level and do not inform on the proteins actual activity (or
phosphorylation state).  This is a
crucial piece of knowledge that could aid in drug targeted therapies. RPPA can
be and has been used for just this, detecting active, over-active, phosphorylated
proteins that may be aiding in cancer proliferation and migration. These
proteins can then be targeted by an anti-protein drug that can target the
pathway or protein itself.

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Background

The principle behind this robust and
sensitive approach is relatively simple compared to other immunoassays like
ELISA. ‘Reverse phase’ refers to the sample being immobilized to a solid phase.
This differs to other immunohistochemical assays whereby the antibody is
generally immobilized to the solid surface. A primary antibody is then applied
in solution phase and allowed incubate overnight. A secondary antibody is then
used for detection. This method is a cost effect approach that only requires
one primary antibody. Other assays normally require two highly specific primary
antibodies and thus a large sample is needed. Large samples is not viable for
clinical trials where the sample can only be got via a biopsy. It allows for a
large multitude of samples to be tested for specific protein/phosphoprotein under
the same experimental conditions, at the same. The general procedure can be
visualized in figure 1.1. The samples to be analyzed may be cellular lysates (Nishizuka et al., 2003), tissue samples  (Agarwal et al., 2009), or
even bodily fluids (CSF, serum, urine etc.). For quantification purposes cell
lysates are serially diluted. Lysate material, roughly 1nL of lysate is then
printed onto a nitrocellulose glass slide via an automatic pin based microarrayer.
This miniscule amount of lysate sample proves to be a great advanatage of RPPA
over other assays. Each slide is then probed with a primary antibody which can
be detected by fluorescent of chemiluminescent assays. Software then quantifies
signal intensities for each spot. The DI25 algorithim is used to eliminated
background effects.

 

 

Figure
1.1

(Spurrier, Ramalingam, & Nishizuka, 2008)

Central to RPPA preparation is antibody
screening. Due to the required sensitivity of antibodies, all must first
undergo a stringent validation. Western blots are performed using the same
material as used in RPPA. Importantly, antibodies must display a single band
for expected protein. Antibodies against phosphorylated proteins must show
specificity against stimulated (growth factors) or inhibited proteins
(inhibitors). In many cases antibody concentration, compared to that used in
WB, will be increased in RPPA. Some antibodies may also need a longer
incubation time.

 

Discussion

RPPA was first developed and utilized by (Paweletz et al., 2001). This
study showed the effective use of RPPA in determining activated proteins
(phosphorylated proteins) that may be involved with the development of prostate
cancer. The PI3k/Akt pathway, previously implicated in cancer proliferation (Fresno Vara et al., 2004),
involves the activation of downstream proteins by successive phosphorylation.
Akt is one such downstream protein in which RPPA was used to demonstrate the
increased phosphorylation states of Akt in prostate cancer cells. Protein
levels of Akt remained constant across samples, but phosphorylation increased.
Activated or phosphorylated Akt has a wide range of pro- survival effects that
inhibit apoptosis. 

Introduction

Reverse phase protein array (RPPA) is a
proteomic array designed to measure the protein levels and protein
modifications (e.g. phosphorylation) of a number of samples, simultaneously. Proteomics
is the large scale study of a proteins’ structure, function and interactions.
The study of proteins has proven difficult compared to genomic studies. This
because a proteins function, structure and post translational state fluctuates
greatly from cell to cell and time to time. Proteomic studies can be extremely
useful in understanding diseases, their progression and in developing targeted
therapies. Thus, this review will examine how RPPA has been utilized to shed
light on cancer progression, a complicated multistep disease. Cancer can
develop due to genetic or epigenetic susceptibilities. Genomics has proven
useful in determining key oncogenes (e.g. BRCA1) which can lead to the
development of cancer. However, not all cancers are due to the presence of an
oncogene or the absence of a tumor suppressor (e.g PTEN). Deregulated signaling
pathways have now gained many cancer researchers attention. Hijacking signaling
pathways has proven a successful mode in attaining immortality. These cellular
pathways which can be activated by growth factors, generally proceed through a
signaling cascade, often causing successive phosphorylation of downstream
proteins. Unfortunately, transcriptional or genomic arrays can only detect a
cells protein level and do not inform on the proteins actual activity (or
phosphorylation state).  This is a
crucial piece of knowledge that could aid in drug targeted therapies. RPPA can
be and has been used for just this, detecting active, over-active, phosphorylated
proteins that may be aiding in cancer proliferation and migration. These
proteins can then be targeted by an anti-protein drug that can target the
pathway or protein itself.

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For You For Only $13.90/page!


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Background

The principle behind this robust and
sensitive approach is relatively simple compared to other immunoassays like
ELISA. ‘Reverse phase’ refers to the sample being immobilized to a solid phase.
This differs to other immunohistochemical assays whereby the antibody is
generally immobilized to the solid surface. A primary antibody is then applied
in solution phase and allowed incubate overnight. A secondary antibody is then
used for detection. This method is a cost effect approach that only requires
one primary antibody. Other assays normally require two highly specific primary
antibodies and thus a large sample is needed. Large samples is not viable for
clinical trials where the sample can only be got via a biopsy. It allows for a
large multitude of samples to be tested for specific protein/phosphoprotein under
the same experimental conditions, at the same. The general procedure can be
visualized in figure 1.1. The samples to be analyzed may be cellular lysates (Nishizuka et al., 2003), tissue samples  (Agarwal et al., 2009), or
even bodily fluids (CSF, serum, urine etc.). For quantification purposes cell
lysates are serially diluted. Lysate material, roughly 1nL of lysate is then
printed onto a nitrocellulose glass slide via an automatic pin based microarrayer.
This miniscule amount of lysate sample proves to be a great advanatage of RPPA
over other assays. Each slide is then probed with a primary antibody which can
be detected by fluorescent of chemiluminescent assays. Software then quantifies
signal intensities for each spot. The DI25 algorithim is used to eliminated
background effects.

 

 

Figure
1.1

(Spurrier, Ramalingam, & Nishizuka, 2008)

Central to RPPA preparation is antibody
screening. Due to the required sensitivity of antibodies, all must first
undergo a stringent validation. Western blots are performed using the same
material as used in RPPA. Importantly, antibodies must display a single band
for expected protein. Antibodies against phosphorylated proteins must show
specificity against stimulated (growth factors) or inhibited proteins
(inhibitors). In many cases antibody concentration, compared to that used in
WB, will be increased in RPPA. Some antibodies may also need a longer
incubation time.

 

Discussion

RPPA was first developed and utilized by (Paweletz et al., 2001). This
study showed the effective use of RPPA in determining activated proteins
(phosphorylated proteins) that may be involved with the development of prostate
cancer. The PI3k/Akt pathway, previously implicated in cancer proliferation (Fresno Vara et al., 2004),
involves the activation of downstream proteins by successive phosphorylation.
Akt is one such downstream protein in which RPPA was used to demonstrate the
increased phosphorylation states of Akt in prostate cancer cells. Protein
levels of Akt remained constant across samples, but phosphorylation increased.
Activated or phosphorylated Akt has a wide range of pro- survival effects that
inhibit apoptosis. 

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