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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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Copper-induced growth inhibition and wild-type APP expression in DU145 cells. (A) Untransfected DU145 cells were cultured over a 7 day period in the absence/presence of the indicated copper concentrations. At various time points, cell viability was assayed as described in the Section 2. Results are expressed relative to viability at day 0. (B) Immunoblot of APP and actin expression in lysates prepared from untransfected DU145 cells cultured for 7 days in medium containing the indicated copper concentrations. (C) Quantification of endogenous APP expression in DU145 cells cultured for 7 days in the absence or presence of copper (50 μM). Results are expressed relative to APP expression levels in the untreated control (no metal). (D) DU145 cells stably transfected with either vector alone (Mock) or wt-APP695 (WT-APP) were cultured over a 7 day period in the absence or presence of copper (150 μM). At the various time points, cell viability was assayed as described in the Section 2. Results are expressed relative to viability at day 0. (E) DU145 cells stably transfected with either vector alone (Mock) or the indicated APP isoform were cultured over a 7 day period in the presence of copper (150 μM). At 7 days lysates were prepared and immunoblotted for APP and actin and 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. All results are means ± S.D. (n = 3). *, ** and *** Denote significance at P = 0.05, P = 0.01 and P = 0.005, respectively.
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f0005: Copper-induced growth inhibition and wild-type APP expression in DU145 cells. (A) Untransfected DU145 cells were cultured over a 7 day period in the absence/presence of the indicated copper concentrations. At various time points, cell viability was assayed as described in the Section 2. Results are expressed relative to viability at day 0. (B) Immunoblot of APP and actin expression in lysates prepared from untransfected DU145 cells cultured for 7 days in medium containing the indicated copper concentrations. (C) Quantification of endogenous APP expression in DU145 cells cultured for 7 days in the absence or presence of copper (50 μM). Results are expressed relative to APP expression levels in the untreated control (no metal). (D) DU145 cells stably transfected with either vector alone (Mock) or wt-APP695 (WT-APP) were cultured over a 7 day period in the absence or presence of copper (150 μM). At the various time points, cell viability was assayed as described in the Section 2. Results are expressed relative to viability at day 0. (E) DU145 cells stably transfected with either vector alone (Mock) or the indicated APP isoform were cultured over a 7 day period in the presence of copper (150 μM). At 7 days lysates were prepared and immunoblotted for APP and actin and 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. All results are means ± S.D. (n = 3). *, ** and *** Denote significance at P = 0.05, P = 0.01 and P = 0.005, respectively.

Mentions: Initially we examined the inhibitory effect of copper on the growth of untransfected DU145 cells by culturing them in complete growth medium containing various metal concentrations (results were identical whether metal was added to growth medium in the form of free CuCl2 or glycine-complexed copper). DU145 cells were fundamentally very resistant to copper with no significant inhibition of growth being apparent at metal concentrations of 100 μM and lower (Fig. 1A). At 150 μM copper, growth inhibition became apparent at 4 days and persisted for the entire 7 day growth period whilst very little growth occurred at all in the presence of 200 μM copper. The authors concede that levels of copper within the tumour microenvironment are unlikely to exceed 30 μM [1] at which concentration tumour cells are clearly able to maintain copper homeostasis through a range of possible mechanisms [16]. However, it is only when these mechanisms are overloaded at higher metal concentrations (as in the current study) that individual elements of these mechanisms can be tested.


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)

Copper-induced growth inhibition and wild-type APP expression in DU145 cells. (A) Untransfected DU145 cells were cultured over a 7 day period in the absence/presence of the indicated copper concentrations. At various time points, cell viability was assayed as described in the Section 2. Results are expressed relative to viability at day 0. (B) Immunoblot of APP and actin expression in lysates prepared from untransfected DU145 cells cultured for 7 days in medium containing the indicated copper concentrations. (C) Quantification of endogenous APP expression in DU145 cells cultured for 7 days in the absence or presence of copper (50 μM). Results are expressed relative to APP expression levels in the untreated control (no metal). (D) DU145 cells stably transfected with either vector alone (Mock) or wt-APP695 (WT-APP) were cultured over a 7 day period in the absence or presence of copper (150 μM). At the various time points, cell viability was assayed as described in the Section 2. Results are expressed relative to viability at day 0. (E) DU145 cells stably transfected with either vector alone (Mock) or the indicated APP isoform were cultured over a 7 day period in the presence of copper (150 μM). At 7 days lysates were prepared and immunoblotted for APP and actin and 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. All results are means ± S.D. (n = 3). *, ** and *** Denote significance at P = 0.05, P = 0.01 and P = 0.005, respectively.
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Related In: Results  -  Collection

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f0005: Copper-induced growth inhibition and wild-type APP expression in DU145 cells. (A) Untransfected DU145 cells were cultured over a 7 day period in the absence/presence of the indicated copper concentrations. At various time points, cell viability was assayed as described in the Section 2. Results are expressed relative to viability at day 0. (B) Immunoblot of APP and actin expression in lysates prepared from untransfected DU145 cells cultured for 7 days in medium containing the indicated copper concentrations. (C) Quantification of endogenous APP expression in DU145 cells cultured for 7 days in the absence or presence of copper (50 μM). Results are expressed relative to APP expression levels in the untreated control (no metal). (D) DU145 cells stably transfected with either vector alone (Mock) or wt-APP695 (WT-APP) were cultured over a 7 day period in the absence or presence of copper (150 μM). At the various time points, cell viability was assayed as described in the Section 2. Results are expressed relative to viability at day 0. (E) DU145 cells stably transfected with either vector alone (Mock) or the indicated APP isoform were cultured over a 7 day period in the presence of copper (150 μM). At 7 days lysates were prepared and immunoblotted for APP and actin and 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. All results are means ± S.D. (n = 3). *, ** and *** Denote significance at P = 0.05, P = 0.01 and P = 0.005, respectively.
Mentions: Initially we examined the inhibitory effect of copper on the growth of untransfected DU145 cells by culturing them in complete growth medium containing various metal concentrations (results were identical whether metal was added to growth medium in the form of free CuCl2 or glycine-complexed copper). DU145 cells were fundamentally very resistant to copper with no significant inhibition of growth being apparent at metal concentrations of 100 μM and lower (Fig. 1A). At 150 μM copper, growth inhibition became apparent at 4 days and persisted for the entire 7 day growth period whilst very little growth occurred at all in the presence of 200 μM copper. The authors concede that levels of copper within the tumour microenvironment are unlikely to exceed 30 μM [1] at which concentration tumour cells are clearly able to maintain copper homeostasis through a range of possible mechanisms [16]. However, it is only when these mechanisms are overloaded at higher metal concentrations (as in the current study) that individual elements of these mechanisms can be tested.

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