Authors: Anna Fyrberg Kourosh Lotfi
Publish Date: 2010/10/19
Volume: 62, Issue: 6, Pages: 497-507
Abstract
In order to study nucleoside analog activation in the CEM cell line a transfection protocol had to be optimized in order to silence an enzyme involved in nucleoside analog activation Hematopoetic cell lines can be difficult to transfect with traditional lipidbased transfection so the electroporation technique was used Field strength pulse length temperature electroporation media siRNA concentration among other conditions were tested in order to obtain approximately 70–80 mRNA and enzyme activity downregulation of the cytosolic enzyme deoxycytidine kinase dCK necessary for nucleoside analog activation Downregulation was assessed at mRNA and enzyme activity levels After optimizing the protocol a microarray analysis was performed in order to investigate whether the downregulation was specific Additionally two genes were differentially expressed besides the downregulation of dCK These were however of unknown function The leakage of intracellular nucleotides was also addressed in the electroporated cells since it can affect the DNA repair mechansism and the efficiency of nucleoside analogs Three of these pools were increased compared to untreated unelectroporated cells The siRNA transfected cells with reduced dCK expression and activity showed reduced sensitivity to several nucleoside analogs as expected The multidrug resistance to other drugs as seen in nucleoside analoginduced resistant cells was not seen with this model
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