Overexpression Lab Report

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Overexpression of AC alters ceramide signaling. AC is mainly located in lysosomes and is considered a major enzyme used by a number of cancers to limit intracellular ceramide accumulation levels. Under normal physiological conditions, ceramide converts AC into sphingosine, which is then converted by sphingosine kinase to become sphingosine-1-phosphate, therefore inducing cell proliferation, as shown in figure 1. However, when cells are under stress, such as lack of oxygen, or following radiation or chemotherapy, ceramide is usually produced as a pro-apoptotic response (cell death). AC overexpression mediates ceramide hydrolysis to generate sphingosine and a fatty acid. Sphingosine can then be phosphorylated by sphingosine kinase forming …show more content…
To confirm PPC1-AC-V5 was overexpressed, Western-blotting and SDS PAGE measured AC protein levels. In figure 2 prostate cancer cell lines PPC1 – LacZ- V5 and PPC1 – AC- V5 illustrated an overexpression of AC in PPC1 – AC- V5, whereas the control PPC1 – LacZ- V5 does not. Also, the uniformed cellular levels of actin showed equal protein loading in both lanes.
AC over-expression increases resistance to ceramide. To assess the effect of AC expression on cell growth potential, PPC1 – AC- V5 and control PPC1 – LacZ- V5 were plated in 96-well plates and treated with ceramide. A significant difference existed between PPC1 – AC- V5 and PPC1 – LacZ- V5 cell lines, as shown in figure 3, PPC1 – AC - V5 had a lesser response to ceramide treatment. After 48 hours, AC overexpressing cells were less sensitive than PPC1 – LacZ- V5 especially at the 2.5 micromolar ceramide …show more content…
Protein expression was analyzed for ACsiRNA (transfected PPC1 – AC- V5), PPC1 – AC- V5, SCRsiRNA, and PPC1 – LacZ- V5 by Western-blotting and SDS PAGE. The SCRsiRNA served as a negative siRNA control for comparison in knockdown efficiency of ACsiRNA protein levels. Furthermore, the untreated prostate cancer cell blots PPC1 – LacZ- V5 and PPC1 – AC- V5 illustrated the previous results from figure 2 confirming reduced AC overexpression in AC-siRNA protein levels. Figure 4 showed AC-siRNA protein levels were reduced to the level of the control PPC1 – LacZ- V5 cell and the uniformed cellular levels of actin showed equal protein loading in both
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