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: The fact that soluble forms of APP failed to mitigate copper-induced growth inhibition suggested that the intracellular domain (ICD) of the protein might be a prerequisite in this respect. Therefore, we generated a construct (APPΔICD) truncated C-terminally to residue 648 (Fig. 3A). In order to narrow down any potential involvement of the APP ICD even further, we also examined the role of two cytosolic tyrosine residues within this domain (tyrosines682 and 687) the phosphorylation of which might be linked to cell signalling. To this end, three further constructs were generated; APP Y682G, APP Y687G and APP Y682 + 687G (Fig. 3A). Following transfection of the constructs into DU145 cells, the holoprotein expression levels and generation of sAPPα were indistinguishable from those of wt-APP695 (Fig. 3B and C). However, no sAPPβ was generated from APPΔICD (Fig. 3B) which was probably indicative of the fact that the APP ICD is required for internalisation of the protein and subsequent β-secretase cleavage. Reduced levels of sAPPβ generation were also observed in the case of the tyrosine mutant constructs (Fig. 3C) indicating that these residues are specifically linked to the generation of this fragment. However, given our previous results showing that sAPPβ had no effect on DU145 cell growth in the presence of copper (Fig. 2) the reduced generation of this fragment from the cytosolic domain constructs is unlikely to have impacted on cell growth. The reduced growth of cells expressing these constructs in the presence of copper (relative to wt-APP-transfected cells) (Fig. 3D) was, therefore, most likely due to changes in the nature of the APP intracellular domain.
Affiliation: Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, UK. Electronic address: email@example.com.