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The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells.

Gough M, Blanthorn-Hazell S, Delury C, Parkin E - Biochem. Biophys. Res. Commun. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, UK. Electronic address: m.gough1@lancaster.ac.uk.

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The effect of the APP E1 copper binding domain on the growth of DU145 cells in the presence of copper. (A) The APPΔCuE1 construct possesses three histidine to alanine mutations at positions 147, 149 and 151 within the E1 copper binding domain (CuBD) of the protein. (B) Immunoblots of cell lysates and conditioned medium from Mock-, wt-APP695- and APPΔCuE1-transfected cells demonstrating APP holoprotein and actin expression in lysates and sAPPα (antibody 6E10) and sAPPβ (antibody 1A9) levels in conditioned medium. (C) DU145 cells stably transfected with either vector alone (Mock) or the indicated APP construct were cultured over a 7 day period in the presence of copper (150 μM). At 7 days cell viability was determined as described in the Section 2. Results are expressed relative to the viability of Mock-transfected cells cultured in the presence of copper at day 7 and are means ± S.D. (n = 3). ** Denotes significance at P = 0.01.
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f0020: The effect of the APP E1 copper binding domain on the growth of DU145 cells in the presence of copper. (A) The APPΔCuE1 construct possesses three histidine to alanine mutations at positions 147, 149 and 151 within the E1 copper binding domain (CuBD) of the protein. (B) Immunoblots of cell lysates and conditioned medium from Mock-, wt-APP695- and APPΔCuE1-transfected cells demonstrating APP holoprotein and actin expression in lysates and sAPPα (antibody 6E10) and sAPPβ (antibody 1A9) levels in conditioned medium. (C) DU145 cells stably transfected with either vector alone (Mock) or the indicated APP construct were cultured over a 7 day period in the presence of copper (150 μM). At 7 days cell viability was determined as described in the Section 2. Results are expressed relative to the viability of Mock-transfected cells cultured in the presence of copper at day 7 and are means ± S.D. (n = 3). ** Denotes significance at P = 0.01.

Mentions: Finally, we examined the role of the copper binding domain (CuBD) in the E1 extracellular domain of APP. We generated a construct (APPΔCuE1) in which three histidines were mutated to alanine in a key area of the protein associated with copper binding [18] (Fig. 4A). This construct was expressed and processed in DU145 cells in a manner identical to that of wild-type APP695 (Fig. 4B). However, APPΔCuE1 failed to mitigate copper-induced growth inhibition (Fig. 4C) indicating that the extracellular E1 CuBD, in addition to the cytosolic domain, of APP were prerequisites in this respect.


The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells.

Gough M, Blanthorn-Hazell S, Delury C, Parkin E - Biochem. Biophys. Res. Commun. (2014)

The effect of the APP E1 copper binding domain on the growth of DU145 cells in the presence of copper. (A) The APPΔCuE1 construct possesses three histidine to alanine mutations at positions 147, 149 and 151 within the E1 copper binding domain (CuBD) of the protein. (B) Immunoblots of cell lysates and conditioned medium from Mock-, wt-APP695- and APPΔCuE1-transfected cells demonstrating APP holoprotein and actin expression in lysates and sAPPα (antibody 6E10) and sAPPβ (antibody 1A9) levels in conditioned medium. (C) DU145 cells stably transfected with either vector alone (Mock) or the indicated APP construct were cultured over a 7 day period in the presence of copper (150 μM). At 7 days cell viability was determined as described in the Section 2. Results are expressed relative to the viability of Mock-transfected cells cultured in the presence of copper at day 7 and are means ± S.D. (n = 3). ** Denotes significance at P = 0.01.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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f0020: The effect of the APP E1 copper binding domain on the growth of DU145 cells in the presence of copper. (A) The APPΔCuE1 construct possesses three histidine to alanine mutations at positions 147, 149 and 151 within the E1 copper binding domain (CuBD) of the protein. (B) Immunoblots of cell lysates and conditioned medium from Mock-, wt-APP695- and APPΔCuE1-transfected cells demonstrating APP holoprotein and actin expression in lysates and sAPPα (antibody 6E10) and sAPPβ (antibody 1A9) levels in conditioned medium. (C) DU145 cells stably transfected with either vector alone (Mock) or the indicated APP construct were cultured over a 7 day period in the presence of copper (150 μM). At 7 days cell viability was determined as described in the Section 2. Results are expressed relative to the viability of Mock-transfected cells cultured in the presence of copper at day 7 and are means ± S.D. (n = 3). ** Denotes significance at P = 0.01.
Mentions: Finally, we examined the role of the copper binding domain (CuBD) in the E1 extracellular domain of APP. We generated a construct (APPΔCuE1) in which three histidines were mutated to alanine in a key area of the protein associated with copper binding [18] (Fig. 4A). This construct was expressed and processed in DU145 cells in a manner identical to that of wild-type APP695 (Fig. 4B). However, APPΔCuE1 failed to mitigate copper-induced growth inhibition (Fig. 4C) indicating that the extracellular E1 CuBD, in addition to the cytosolic domain, of APP were prerequisites in this respect.

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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, UK. Electronic address: m.gough1@lancaster.ac.uk.

Show MeSH
Related in: MedlinePlus