Chronic myeloid leukaemia (CML) is a haematopoietic stem cell disorder
characterised by the BCR-ABL fusion gene. The BCR-ABL fusion gene encodes a
constitutively active BCR-ABL tyrosine kinase, which is the driving force of the
malignancy. Otherwise fatal, the use of imatinib mesylate has proved highly
effective in the treatment of this disease in up to 85% of CML patients. However,
approximately 25% of CML patients appear to respond suboptimally or experience
treatment failure with imatinib. Suboptimal response in CML patients has been
attributed to inadequate BCR-ABL kinase inhibition as a result of reduced
intracellular accumulation of imatinib in target leukemic cells. The cellular influx of
imatinib is mediated by the influx transport protein, SLC22A1. Therefore, its
activity is considered a clinical determinant of imatinib uptake, and hence patients
response to therapy.
A number of studies use levels of SLC22A1 mRNA as a measure of SLC22A1
activity. It has been reported that cells over expressing levels of SLC22A1 mRNA
showed significantly increased uptake of imatinib, thus, suggesting that levels of
SLC22A1 mRNA can be used as a measure of SLC22A1 activity. However, there
is a concern that imatinib may affect SLC22A1 expression. This consideration,
however, is based on two studies involving a limited patient cohort and although
widely accepted, has not been proven conclusively. Should it be proven that
imatinib does influence SLC22A1 expression, levels of SLC22A1 mRNA may not be a reliable indicator of SLC22A1 activity. It is therefore important to understand
the effect of treatment with imatinib on SLC22A1 gene expression.
The data from this study demonstrated that imatinib induces expression of
SLC22A1 mRNA in a non-linear dose dependent manner. It was also observed
that expression of SLC22A1 was not dependent on time of exposure to imatinib.
These results explain the differential expression of SLC22A1 mRNA reported in
CML patients on a standard dose of 400 mg/day of imatinib. The trough plasma
levels of imatinib achieved between patients after 24 hours of exposure to the
same dose of imatinib may vary owing to inter individual differences. Since
SLC22A1 expression is dependent on plasma levels of imatinib, therefore, patients
administered the same dose of imatinib may show differential expression of
SLC22A1. These findings suggest that imatinib does affect SLC22A1 mRNA
expression and that the change in SLC22A1 expression observed at any particular
time is dependent on the intracellular levels of imatinib achieved in CML patients
within 24 hours of exposure to the drug.
One of the challenges in this study was the availability of suitably qualified
SLC22A1 antibodies for use in the Taqman protein assay to quantify SLC22A1
protein. Antibodies used in the Taqman assay have to fulfil specific criteria and
out of 55 commercially available antibodies, only three SLC22A1 antibodies met
the minimum requirements for use in the assay. However, despite various efforts
focused at optimising the assay, the range of the assay was very limited and
hence it was not possible to quantify SLC22A1 protein. We hypothesize that one of the reasons for assay failure could be as a result of antibodies not binding to the
target protein at the required spatial distance to facilitate amplification by real-time
PCR. Since the antibodies used in the assay have not been epitope mapped, it is
uncertain whether they fulfil this requirement. Future research will be aimed at
antibody production for manufacturing SLC22A1 antibodies suitable for use in the
Taqman protein assay to enable successful quantification of SLC22A1 protein.
In conclusion, this is the first study which specifically aimed to investigate the
influence of imatinib on SLC22A1 expression. This is also the first study to
demonstrate that expression of SLC22A1 is not time dependent, but follows a nonlinear
correlation to imatinib concentration. Although it would have been useful to
investigate the effect of increasing levels of SLC22A1 mRNA on intracellular
uptake of imatinib in K562 cells, unfortunately, the latter technique requires the use
of radio-labelled imatinib and specialized equipment which made it a limiting factor
for use in this study. While this study does not invalidate the use of levels of
SLC22A1 mRNA as a prognostic marker for treatment outcome, these findings
suggest that levels of SLC22A1 mRNA as a measure of SLC22A1 activity is only
applicable to newly diagnosed imatinib naive or previously untreated CML patients.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-11212013-155220 |
| Date | 21 November 2013 |
| Creators | Sreenivasan, Sandhya |
| Contributors | Prof CD Viljoen |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-11212013-155220/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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