On the cytotoxicity of HCR-NTPase 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) [3]. 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 [3]. 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 [8]. As a representative of COG1618 proteins, aaTHEP1 from the hyperthermophilic bacterium Aquifex aeolicus was biochemically characterised [9] and its crystal structure was resolved [10]. 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 an