Table 1

Effect of M30 and HLA20 on APP 5'UTR- conferred translation of a luciferase reporter mRNA


Inhibition (% of control)

M30 (20 μM)

7.1 ± 1.07 *

M30 (100 μM)

33.7 ± 8.3

HLA20 (20 μM)

16.2 ± 2.4

HLA20 (100 μM)

27.3 ± 2.9 *

VK28 (20 μM)

26.6 ± 7.7

VK28 (100 μM)

40.2 ± 7.5

Since there is a functional IRE in the 5'UTR of APP mRNA, we further investigated the efficacy of the iron chelator drugs M30, HLA20 and VK28 to modulate the translation of a luciferase reporter gene driven by APP 5'UTR sequences. The assay was performed essentially as described in Reznichenko et al. [56]. The pGALA construct was generously provided by JT Rogers (Massachusetts General Hospital, Boston, MA, USA). Human U-87-MG glioma cells, selectively chosen for their high transfection efficiency, were grown in flasks (100 mm2) and transfected with 7 μg of DNA from the parental vector pGL-3 or pGALA constructs and co-transfected with 3 μg of DNA from a construct that expresses green fluorescent protein (GFP) to standardize for transfection efficiency. The pGALA consists of a pGL-3 backbone to which the APP 5'UTR sequences (containing the IRE) were inserted in front of the luciferase gene start codon and the complete APP 3'UTR sequences immediately downstream of the luciferase, to provide the natural arrangement of the APP gene 5' and 3'. After 12 h, the cells were split equally into 96-well plates and grown without (control) or with the iron chelator drugs M30, HLA20 or VK28 for 48 h. Cell viability was established by a fluorescent microscopic examination of each well, quantified for GFP activity at 480/509 nm wavelength (using an automatic Wallac-1420 multilabel counter) and then lysed to determine luciferase activity. For statistical analysis one-way ANOVA followed by Student's t-test was performed. Quantitative values are mean ± standard error of the mean (n = 3). P < 0.05, *P < 0.01 versus control was considered significant.

Avramovich-Tirosh et al. BMC Neuroscience 2008 9(Suppl 2):S2   doi:10.1186/1471-2202-9-S2-S2

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