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dc.contributor.advisorViljoen, C. D.
dc.contributor.authorSreenivasan, Sandhya
dc.date.accessioned2015-11-25T08:28:26Z
dc.date.available2015-11-25T08:28:26Z
dc.date.issued2013
dc.identifier.urihttp://hdl.handle.net/11660/1907
dc.description.abstractEnglish: 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.en_ZA
dc.description.abstractAfrikaans: Chroniese meyloid leukemie (CML) is ʼn hematopoïetiese stamsel siekte wat gekenmerk word deur die BCR-ABL fusie onkogeen. Hierdie BCR-ABL geen kodeer vir ʼn aktiewe tiroksienkinase wat die maligniteit veroorsaak. Die gebruik van imatinib is doeltreffend in ongeveer 85% van CML pasiënte, waarsonder CML ‘n dodelike siekte is. Tog blyk dit dat 25% van CML pasiënte nie optimaal teenoor behandeling reageer nie. Sub-optimale response in CML pasiënte is toegeskryf aan ondoeltreffende inhibisie van BCR-ABL-kinase as gevolg van verlaagde intrasellulêre vlakke van imatinib in teiken leukemie selle. Die opname van imatinib binne die sel word bepaal deur die vervoer proteïen, SLC22A1. Die aktiwiteit van hierdie proteïen word dus gebruik as ʼn maatstaf van hoe pasiënte gaan reageer teenoor behandeling met imatinib. Verskeie studies beskryf die gebruik van die vlak van SLC22A1 boodskapper-ribonukleïensuur (bRNS) as ʼn aanduiding van SLC22A1 aktiwiteit. Daar is gevind dat selle wat SLC22A1 in oormaat uitdruk verhoogde opname van imatinib ondergaan wat die veronderstelling steun dat SLC22A1 boodskapper-ribo-nukleïensuur as ʼn aanduiding van SLC22A1 aktiwiteit beskou kan word. Tog is daar aanduidings dat imatinib die geenuitdrukking van SLC22A1 mag beïnvloed. Hierdie oorweging is net op twee studies met ‘n beperkte aantal pasiënte gebaseer, en alhoewel dit algemeen aanvaar word, is dit nie onweerlegbaar bewys nie. Sou dit wel bewys word dat imatinib die geenuitdrukking van SLC22A1 beïnvloed, mag dit ook beteken dat vlakke van SLC22A1 boodskapper-ribo-nukleïensuur nie as ‘n aanduiding van SLC22A1 aktiwiteit kan dien nie. As gevolg hiervan is dit dus belangrik om die invloed van imatinib op SLC22A1 geenuitdrukking te verstaan. Die resultate van hierdie studie het gewys dat SLC22A1 geenuitdrukking wel deur imatinib beïnvloed word deur ʼn ongelinieerde verwantskap. Dit is ook gevind dat die invloed van imatinib op SLC22A1 geenuitdrukking nie van die tyd van blootstelling afhanklik is nie. Hierdie resultate verduidelik die differensiële vlakke van SLC22A1 aangedui in pasiënte uit verskillende studies. Dus mag die plasmavlakke van imatinib van pasiënte op dieselfde dosering wissel as gevolg van verskille tussen individue. Gevolglik toon pasiënte op dieselfde dosering van imatinib verskillende vlakke van SLC22A1 geenuitdrukking . Dus word die voorstelling gemaak dat imatinib die wel die geenuitdrukking van SLC22A1 beïnvloed en dat dit na 24 uur nie afhanklik is van tydsduur van blootstelling nie. Een van die uitdagings in hierdie studie is die beskikbaarheid van bruikbare antiliggame vir gebruik in die “Taqman” proteïen-toets. Die antiliggame wat gebruik word moet voldoen aan spesifieke vereistes en uit die 55 wat beskikbaar is, het slegs drie aan die nodige vereistes voldoen. Alhoewel daar sorg geneem is om die toets te optimaliseer, was dit steeds nie moontlik om die hoeveelheid SLC22A1 proteïen te bepaal nie. Ons vermoed dat die rede hiervoor die onvermoë van die antiliggame is om binne die vereiste afstand op die teiken proteïen te bind. Omdat die antiliggame wat in hierdie toets gebruik is nog nie teenoor die epitoop gekarteer is nie, was dit onmoontlik om bindings inligting te bekom Toekomstige navorsing moet gemik word op die ontwikkeling van toepaslike antiliggame vir gebruik in die “Taqman”-proteïen-toets. Ten slote, hierdie is die eerste studie wat probeer het om die invloed van imatinib op SLC22A1 geenuitdrukking te bestudeer. Dit is ook die eerste studie wat bewys dat die invloed van imatinib op SLC22A1 geenuitdrukking nie verbonde is aan tyd van blootstelling deur ʼn ongelinieer verwantskap nie. Dit sou toepaslik gewees het om die invloed van SLC22A1 bRNS vlakke op die opname van imatinib te bestudeer, maar die metode daarvoor was egter nie beskikbaar vir hierdie studie nie. Alhoewel hierdie studie nie die gebruik van SLC22A1 bRNS as prognostiese merker in CML pasiënte uitsluit nie, is hierdie bevindings wel aanduidend dat dit toepaslik is as ‘n maatstaf van SLC22A1 aktiwiteit slegs op pasiënte wat nie voorheen met imatinib behandel is nie.af
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectSLC22A1en_ZA
dc.subjectGene expressionen_ZA
dc.subjectTaqman protein assayen_ZA
dc.subjectImatiniben_ZA
dc.subjectBCR-ABLen_ZA
dc.subjectChronic myeloid leukaemiaen_ZA
dc.subjectBlood -- Diseasesen_ZA
dc.subjectChronic myeloid leukemia -- Genetic aspectsen_ZA
dc.subjectChronic myeloid leukemia -- Treatmenten_ZA
dc.subjectGene expressionen_ZA
dc.subjectDissertation (M.Med.Sc. (Haematology and Cell Biology))--University of the Free State, 2013en_ZA
dc.titleImpact of imatinib mesylate on SLC22A1 gene expression in chronic myeloid leukaemia cell line, K562en_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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