The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells.
Bottom Line: A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored.Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein.APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment.
Affiliation: Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, UK. Electronic address: firstname.lastname@example.org.Show MeSH
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Mentions: Next we sought to determine which parts of the APP molecule were prerequisites for the mitigation of copper-induced growth inhibition. Initially we examined whether soluble forms of the protein could exert a similar effect to that of full-length APP using constructs analogous to sAPPα (truncated C-terminally to lysine612) and sAPPβ (truncated C-terminally to methionine 596) (Fig. 2A). Note that all molecular constructs were based on the 695 amino acid APP isoform. Unfortunately, when these soluble constructs were expressed in DU145 cells, the products were aberrantly processed intracellularly (data not shown). However, when expressed in alternative cell lines (HEK or SH-SY5Y) the two constructs were secreted as the predicted fragments of approximately 110 kDa (Fig. 2B and C). We, therefore, cultured mock-transfected DU145 cells in pre-conditioned medium from the HEK or SH-SY5Y stable transfectants (see Section 2) in the absence or presence of copper. The results (Fig. 2D) show that the growth of cells was unaffected by the presence of exogenous sAPPα or sAPPβ in the medium. Also of note is the fact that α- and β-secretase inhibitors had no effect on the growth of wt-APP-transfected DU145 cells in the presence of copper (data not shown). Collectively, these data indicate that the full-length form of APP is a prerequisite for the mitigation of copper-induced growth inhibition in DU145 cells.
Affiliation: Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, UK. Electronic address: email@example.com.