On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y

Abstract

Background: The human cancer-related nucleoside triphosphatase (HCR-NTPase) is overexpressed in several tumour tissues including neuroblastoma. HCR-NTPase is an enzyme exhibiting a slow in vitro activity in hydrolysing nucleosidetriphosphates. However, its in vivo function is still unknown. To learn more about the physiological role of HCR-NTPase, we both overexpressed and silenced it in the neuroblastoma cell line SH-SY5Y.

Findings: No effect was observed when the expression of endogenously expressed HCR-NTPase in the cells was silenced by RNA interference. On the other hand, overexpression of HCR-NTPase led to cytotoxicity of the protein in SH-SY5Y cells. Even if the catalytic essential amino acid glutamate 114 was replaced by alanine (E114A-HCR-NTPase), the protein remained cytotoxic. The results could be confirmed by successfully rescuing the cells via RNA interference.

Conclusion: Although expressed in several tumours, at least in SH-SY5Y, HCR-NTPase is not essential for the cells to survive. Increased levels of the protein lead to cytotoxicity due to physical intracellular interactions rather than hydrolysis of nucleosidetriphosphates by its intrinsic residual enzymatic activity.

Figure 1

Positions of siRNAs used to silence HCR-NTPase in SH-SY5Y. ClustalW-alignment of the eight most diverse sequences out of a group of 29 species that have been found to contain Walker A and B motifs similar to those of HCR-NTPase [3]. The sequences were from Homo sapiens, Mus musculus, Drosophila melanogaster, Aquifex aeolicus (aaTHEP1), Thermophilum pendens, Methanopyrus kandleri, an uncultured archaeon, Sulfolobus tokodaii, and Aeropyrum pernix, respectively.

Figure 2

Silencing HCR-NTPase in SH-SY5Y has no effect. Normalised cell counts (in % of untransfected control) of SH-SY5Y transfected with plasmids containing GFP, RFP, siRNA1, siRNA2, siRNA3, and siRNA4, respectively. The data for the GFP- and RFP-controls are the same as those shown in table 1. The error bars indicate the standard deviations of at least 3 independently performed experiments.

Figure 3

Cytotoxicity of HCR-NTPase expressed in SH-SY5Y. Phase contrast and fluorescence images of SH-SY5Y cells are shown. The cells were either untreated (A) or transfected with Plasmids encoding HCR-NTPase (B), GFP (C), GFP + HCR-NTPase (D), RFP (E), and an HCR-NTPase-RFP fusion product (F).

Figure 4

Rescue of SH-SY5Y cells by RNA-interference. Normalised cell counts (in % of untransfected control) of SH-SY5Y transfected with plasmids encoding a fusion protein between HCR-NTPase and RFP alone (HCR-NTPase-RFP) or cotransfected with siRNA1, siRNA2, siRNA3, and siRNA4, respectively. Compared to the control, less than 1% of cells containing the HCR-NTPase-RFP fusion protein could be detected by fluorescence microscopy (data not shown). The data for the GFP-, RFP- and HCR-NTPase-RFP-controls are the same as those shown in table 1. The error bars indicate the standard deviations of at least 3 independently performed experiments.

Figure 5

Cytotoxicity of the E114A-HCR-NTPase mutant and rescue of SH-SY5Y cells by siRNA3. Normalised cell counts (in % of untransfected control) of SH-SY5Y transfected with plasmids encoding the E114A-HCR-NTPase mutant (E114A) alone or cotransfected with either GFP or siRNA3. Whereas fGFP + E114A were analysed by fluorescence microscopy, all other cells were counted via phase contrast microscopy. The data for the GFP-, RFP- and HCR-NTPase-controls are the same as those shown in table 1. The error bars indicate the standard deviations of at least 3 independently performed experiments.