On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line SH-SY5Y
© Kaufmann et al; licensee BioMed Central Ltd. 2009
Received: 20 August 2008
Accepted: 11 June 2009
Published: 11 June 2009
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.
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.
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.
A screen of the cancer genome anatomy project (CGAP) database [1, 2] for expressed sequence tags that compared to normal tissue are highly expressed in human tumours revealed the human cancer-related nucleoside triphosphatase (HCR-NTPase) . HCR-NTPase, the gene product of the mRNA NM_032324 (synonyms: MGC13186, LOC84284, C1orf57, GI:14150100) is described to exhibit an increased expression profile in liver cholangiocarcinoma when compared to normal tissue . In addition, as retrieved from SOURCE [4, 5] when this project was initiated, HCR-NTPase was stated to be expressed in many human tumours, several of them located in the brain such as medullablastoma, glioblastoma, and neuroblastoma.
A homology search using BLAST [6, 7] retrieved significant sequence homologies between HCR-NTPase and proteins assigned to COG1618 of the COG database . As a representative of COG1618 proteins, aaTHEP1 from the hyperthermophilic bacterium Aquifex aeolicus was biochemically characterised  and its crystal structure was resolved . Both HCR-NTPase and aaTHEP1 hydrolyse ATP and GTP in vitro with Km in the micromolar range and kcat in the range between 5 and 9 × 10-3 s-1 corresponding to approximately 20–30 molecules hydrolysed per hour and enzyme molecule. In addition, the proteins resemble each other in terms of structure. They belong to the class of P-loop NTPases, and their regular secondary structures can be superimposed with a backbone RMSD of 2.8 Å . Thus, aaTHEP1 and consequently COG1618 proteins may serve as model systems for HCR-NTPase.
Analysing the phylogenetic distribution of COG1618 proteins by phylogenetic COG ranking [11, 12] reveals that they are absent from almost all mesophiles and present in all thermophiles, most of them archeae. In addition, HCR-ATPase is present in many eucarya analysed thus far. Therefore, HCR-ATPase and COG1618 proteins are at least very similar to PACE proteins i. e. proteins from archaea without assigned function that are conserved in eukarya as described by Matte-Tailliez et al. . The authors argue that most of the PACE proteins are informational proteins and could be of strong biomedical interest.
The aim of this study was to obtain further insight into the role of HCR-NTPase in tumour cells. For that purpose we investigated the phenotype of the neuroblastoma cell line SH-SY5Y  as a model system for brain tumours under both conditions overexpressing and silencing HCR-NTPase.
Eucaryotic cell culture and transfections
SH-SY5Y neuroblastoma cells  were obtained from ATCC, Manassas, USA and grown at 37°C and 5% CO2 in Dulbecco's modified Eagle's Medium containing 1% penicillin/streptomycin and 15% fetal calf serum (growth medium; GIBCO, Eggenstein, Germany) in a humidified incubator. To keep the cells in logarithmic growth, confluent cells were washed with phosphate buffered saline (PBS), detached by 0.05% Trypsin and 0.5 mM EDTA in PBS, followed by washing and diluting them in fresh growth medium. 106 cells were transfected with 2 μg Plasmid by electroporation in 50 μl of Cell Line Nucleofector V solution (Amaxa, Cologne, Germany). After transfection, cells were washed in growth medium, transferred to 6-well plates and judged microscopically after 24 h of incubation at 37°C and 5% CO2 using an Axiovert100 microscope (Zeiss, Oberkochen, Germany). Red fluorescent cells were analysed at 584 nm and green fluorescent cells at 482 nm excitation. Three different fields of view were evaluated for each experiment and all experiments were performed independently in triplicate.
Silencing HCR-NTPase in SH-SY5Y by RNA interference
The expression of HCR-NTPase in SH-SY5Y cells was blocked via RNA interference  using pSilencer1.0-U6 (Ambion, Darmstadt, Germany). shRNA coding inserts flanked by ApaI and EcoRI restriction sites were constructed by phosphorylating 25 pmol of oligonucleotides (Operon, Cologne, Germany) using T4 poynucleotide kinase, followed by assembling complementary pairs by incubation for 5 min at 100°C and 60 min at 37°C. pSilencer1.0-U6 sequentially was cut by ApaI (Fermentas, St. Leon-Rot, Germany) and EcoRI (Fermentas, St. Leon-Rot, Germany), the inserts were inserted and the plasmids were amplified in E. coli SURE (Stratagene, San Diego, USA). Expression of HCR-NTPase was blocked by transfecting SH-SY5Y cells with purified plasmids. The following pairs of oligonucleotides were used:
5'-ATC CAT AAA GCC AGT GAT TCT CAA GAG AAA TCA CTG GCT TTA TGG ATC ATT TTT T-3' and
5'-AAT TAA AAA ATG ATC CAT AAA GCC AGT GAT TTC TCT TGA GAA TCA CTG GCT TTA TGG ATG GCC-3'
5'-AGA GCC TCC ACC TGG AAT TCT CAA GAG AAA TTC CAG GTG GAG GCT CTA ATT TTT T-3' and
5'-AAT TAA AAA ATT AGA GCC TCC ACC TGG AAT TTC TCT TGA GAA TTC CAG GTG GAG GCT CTG GCC-3'
5'-GAA TGC CGA CTG CAG CAT TCT CAA GAG AAA TGC TGC AGT CGG CAT TCC TTT TTT T-3' and
5'-AAT TAA AAA AAG GAA TGC CGA CTG CAG CAT TTC TCT TGA GAA TGC TGC AGT CGG CAT TCG GCC-3'
5'-TTC CTA AAG GAA AGC CAT TCT CAA GAG AAA TGG CTT TCC TTT AGG AAC TTT TTT T-3' and
5'-AAT TAA AAA AAG TTC CTA AAG GAA AGC CAT TTC TCT TGA GAA TGG CTT TCC TTT AGG AAG GCC-3'
Overexpressing HCR-NTPase in SH-SY5Y cells
HCR-NTPase cDNA was obtained from RZPD, Berlin, Germany. The gene was amplified and cloned into pET101/D-TOPO yielding pET101/D-TOPO/HCR-NTPase to express the protein in E. coli. This construct also served as the basis for all further cloning steps. HCR-NTPase was overexpressed using the plasmid pRc/CMV (Invitrogen, Karlsruhe, Germany). The gene was amplified from pET101/D-TOPO/HCR-NTPase using the primers
5'-AAA AGC TTA TGG CCC GGC ACG TGT TCC-3' and
5'-AAA ATC TAG ATC ACT TCC TGC TGC TCT G-3'.
The PCR-fragment was digested sequentially by XbaI (Fermentas, St. Leon-Rot, Germany) and HindIII (Fermentas, St. Leon-Rot, Germany), inserted into pRc/CMV and amplified in E. coli TOP10 (Invitrogen, Karlsruhe, Germany).
HCR-NTPase as a fusion with red fluorescent protein  (HCR-NTPase-RFP) was overexpressed using the plasmid pmaxFP-Red-C (Amaxa, Cologne, Germany). The gene was amplified from pET101/D-TOPO/HCR-NTPase using the primers
5'-AAA AGA TCT GGA GGA GGA GGA ATG GCC CGG CAC GTG TTC-3' and
5'-AAG GTA CCT CAC TTC CTG CTG CTC TG-3'.
Before digesting with BglII (Fermentas, St. Leon-Rot, Germany) and KpnI (Fermentas, St. Leon-Rot, Germany), the PCR-fragment was inserted into pCR2.1-TOPO (Invitrogen, Karlsruhe, Germany). The resulting fragment was inserted into pmaxFP-Red-C and amplified in E. coli TOP10. As a control for the efficiency of transfection, pmaxGFP (QIAGEN) was used. HCR-NTPase was overexpressed by transfecting SH-SY5Y cells with purified plasmids.
Construction of a E114A-HCR-NTPase mutant in pRc/CMV
An enzymatically inactive HCR-NTPase mutant was constructed by mutating a conserved catalytic glutamate . For that purpose, E114 of HCR-NTPase in pRc/CMV was replaced by an alanin using the Phusion™ Site-Directed Mutagenesis Kit (Finnzymes, Espoo, Finland) according to the instructions of the manufacturer. The utilised phosphorylated primers were
5'-P-CGT CAT CGA TGC GAT TGG GAA GA-3' and
5'-P-CAC ACT CTT TGC CCT GGG CCA-3'.
Inhibition of apoptosis
Apoptosis was inhibited by adding the caspase inhibitor Z-VAD-FMK (Promega, Mannheim) to the culture medium directly after transfection at a final concentration of 20 μM.
All investigations have been performed in accordance to German regulatory affairs.
Loss-of-function: silencing HCR-NTPase by RNA interference
Gain-of-function: overexpressing HCR-NTPase in SH-SY5Y
Quantitative analysis of the cytotoxicity of HCR-NTPase expressed in SH-SY5Y
GFP + HCR-NTPase6
GFP + HCR-NTPase-RFP3
Rescue of SH-SY5Y cells by RNA interference
The E114A-HCR-NTPase mutant
Effect of Z-VAD-FMK on the cytotoxicity induced by HCR-NTPase and E114A-HCR-NTPase in SH-SY5Y
GFP + Z-VAD-FMK
GFP + HCR-NTPase
GFP + HCR-NTPase + Z-VAD-FMK
GFP + E114A-HCR-NTPase
GFP + E114A-HCR-NTPase + Z-VAD-FMK
The authors would like to thank Daniela Kaufmann for her help in preparing the manuscript and Thomas Dittmar for affording the opportunity to use the AMAXA nucleofector device.
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