Impact of imatinib on

the expression of Oct-1

Sandhya Sreenivasan
M.Med.Sc

Department of Haematology and Cell Biology

Tel: 051 405 3198
E-mail: sreenivs@ufs.ac.za
Imatinib – the wonder drug
The first targeted therapy
for cancer
 Tyrosine kinase inhibitor
 80% respond favourably
 Often referred to as the
“gold standard” of
treatment
 Potently inhibits BCR-ABL
tyrosine kinase
Deregulated activity of Bcr-Abl

signal transduction
Bcr-Abl cascade
Substrate
Tyrosine
kinase cell
proliferation
ATP cell
P survival
Altered cell
adherence
Mode of action of Imatinib

Substrate

Bcr-Abl
protein
ATP

Imatinib
 Response to imatinib
All patients do not respond similarly
 80% respond favourably
 Approximately 20% of CML patients do not
respond to treatment with imatinib
 Primary resistance
 Acquired resistance
Predicting response to imatinib
Studies have shown that patients with low
intracellular uptake and retention (IUR) of
imatinib do not respond favourably to
imatinib
Whereas patients with higher intracellular
levels of imatinib respond favourably
Therefore, intracellular concentration of
imatinib is a prognostic marker to predicting
response
 Intracellular imatinib

X X

Low intracellular [imatinib] High intracellular [imatinib]

Non-responders to treatment Responders to treatment
Transport of imatinib into cells
The intracellular uptake of imatinib is
actively mediated by a membrane protein,
OCT-1
Since Oct-1 transports imatinib into cells,
patients with high Oct-1 activity may be able to
achieve adequate intracellular concentration of
imatinib and favourable response unlike
patients with low Oct-1 activity
 A measure of Oct-1 activity may give an idea of
clinical outcome with imatinib therapy
 Significance of OCT-1
Currently there are two hypotheses in
literature regarding what constitutes as a
measure of OCT-1 activity
 A measure of OCT-1 mRNA determines the
potential amount of OCT-1 protein present and
is a measure of OCT-1 activity
 The intracellular level of imatinib achieved is a
measure of OCT-1 activity
 Measuring Oct-1 activity
Measures the potential amount of OCT-1
protein present
Levels of Oct-1 mRNA is a prognostic
marker for response to imatinib therapy
(Wang et al. 2008)
Baseline expression of OCT-1 is lower
(1/8) in non responders compared to
responders (Crossman et al. 2006)
 Measuring Oct-1 activity
High OCT-1 mRNA
High OCT-1
protein;
High [im]

Responders to treatment

Low OCT-1 mRNA

Low OCT-1
protein;
Low [im]
Non-responders to treatment
 Measuring Oct-1 activity
Activity of Oct-1 measured as intracellular
accumulation of imatinib can predict response to
treatment (White et al. 2008)
 [IUR of imatinib without prazosin – IUR of
imatinib with prazosin]
 Greater the difference, greater is OCT-1 activity
IUR without prazosin IUR with prazosin

X
X
 Oct-1 mRNA vs. IUR [im]
Patients displaying increased intracellular
accumulation of imatinib do not necessarily
show increased OCT-1 mRNA levels
In addition, effect of exposure to imatinib on
OCT-1 mRNA is unclear (Crossman et al.
2006)
This led White et al. (2008) to question
whether measuring OCT-1 mRNA is indeed
a true reflection of the amount of OCT-1
protein expressed on cells
 Oct-1 mRNA vs. IUR [im]
It would be ideal to implement Oct-1 mRNA
method as a prognostic marker
 Real time q-PCR for determining Oct-1 mRNA
will be easier, quicker and cost-effective
 The IUR assay is technically complex and not
feasible as a routine diagnostic test
However, if levels of OCT-1 mRNA are
influenced by imatinib without subsequent
impact of the uptake of imatinib, this poses a
problem
It is important to determine whether OCT-1
mRNA corresponds to OCT-1 protein
 Measuring OCT-1 protein
 Already established a method to determine
OCT-1 mRNA but it is a challenge to
determine OCT-1 protein
 Traditional methods such as western
blotting, flow cytometry etc
 Semi-quantitative and tedious
 Requires the use of monoclonal antibodies
 Membrane protein challenges
 Will not enable simultaneous mRNA AND
protein determination from the same
sample set
 TaqMan protein assay
Shannon et al. (2009) described a real-time
Taqman protein assay
 Detection and quantification of cellular protein
using real-time quantitative PCR
 Use of polyclonal and/or monoclonal
antibodies
Overview of assay principle

 2 antibodies bind to different sites on target protein

 Each antibody has an oligo attached, which, when
the antibodies are bound in correct proximity, serve
as a template for real-time PCR
 Fluorescence measured is indicative of [protein]
TaqMan protein assay

Total Time
Hands on Time ~1.5 hrs
Time to Results ~3.5 hrs
 Experiment
K562 cells treated with different
concentrations of imatinib and incubated for
24 hours, 48 hours and 72 hours
Determine amount of OCT-1 mRNA and
protein present using real time quantitative
PCR and the TaqMan assay respectively
Investigate the impact of imatinib on OCT-1
 Genetics of Oct-1
Belongs to largest super family of
transporters
 SLC (solute carrier) super family
 Encoded by SLC22A1 gene
 Located on chromosome 6
 ~36.9 kb with 11 exons
 Alternative splicing of OCT-1 mRNA
Isoform a
1 2 3 4 5 6 7 8 9 10 11
554 amino acids
Functional transporter

1 2 3 5 6 7 8 10 Isoform b
4 11
Lacks exon 9
504 amino acids
Function unknown
 Methodology
 Real time quantitative PCR
 Primers that amplify only functional isoform of Oct-1
are incorporated (spanning exon 7 and 9)
1 2 3 4 5 6 7 8 9 10 11

 SYBR Green detection assay is performed

I II III IV
Step 1: Bind antibody to oligo

3’ oligo linked with 5’ oligo linked with

streptavadin streptavadin

Antibody A Antibody B
linked to 3’ linked to 5’
oligo oligo
 Step 2: Cell lysis

CELL LYSIS

OCT-1

 The assay is performed on crude cell

extracts and does not require any protein
extraction and purification
StepStep
3: Antibody tothe
4: Ligate target protein
oligos
Antibody A Antibody B

DNA Ligase

OCT-1
Step 5: Real-time PCR
Connector oligo

OCT-1
 Experimental outcomes
Oct-1 mRNA OCT-1 protein Comment
With increasing concentrations of
Unaffected Unaffected OCT-1 mRNA is valid
as a prognostic marker

Changes Changes OCT-1 is not the

limiting step in uptake
imatinib

of imatinib into cells

and other associated
protein interactions
may be at play to
determine uptake of
imatinib
Changes Does not Levels of OCT-1 mRNA
change are not prognostic
 Research Outcomes
 Knowledge of the impact of imatinib on Oct-1 mRNA
and protein expression will help clarify the role of
Oct-1 in CML treated with imatinib
 This in turn will help evaluate the use of Oct-1 mRNA
as a prognostic marker and diagnostic marker
depending on whether imatinib affects expression of
Oct-1 in any way
 M.Med.Sc degree
 Study will be published as an article in accredited
journal and presented at the faculty forum and
conferences
Change in SLC22A1 expression in K562 cells treated with 0.1 µM
to 10 µM imatinib compared to the untreated control. Statistically
significant differences (p < 0.05) between control and treated cells
determined using ANOVA and Student’s t-test are indicated with
asterisk (*).
THANK YOU !!!