Development of an in vitro model for cysteine dioxygenase expression in the brain.
The development of an in vitro model for cysteine dioxygenase (CDO) expression in the brain would provide a useful model for determining the mechanisms for the regulation of CDO expression that does not involve the use of animals. Here we demonstrate the screening and characterization of a cell line that expresses CDO, the primary metabolizing enzyme of cysteine and the regulatory point of sulfate production. A panel of four commercially available tumor-derived human brain cell lines, each representing one major class of brain cell, were screened using western blotting and activity assay for cysteine dioxygenase expression. One cell line, TE 671 (human medulloblastoma) was found to express both a protein of approximately 70 kDa and CDO activity. Nuclease protection assay (NPA) of mRNA isolated from TE 671 showed the expression of a CDO mRNA. Reverse transcription-polymerase chain reaction of this mRNA and sequencing of the cDNA obtained showed that this was indeed CDO. Treatment of TE 671 cells with cysteine resulted in the upregulation of CDO mRNA, whereas treatment with tumor necrosis factor alpha resulted in the downregulation of CDO mRNA, as evidenced using NPA. The characterization of an in vitro model for CDO expression provides a useful tool for the investigation of this important enzyme, which may have an etiological role in the pathogenesis of Parkinson's disease.
Qusti
S
S
School of Biosciences, University of Birmingham, Edgbaston, UK.
Parsons
R B
RB
Abouglila
K D
KD
Waring
R H
RH
Williams
A C
AC
Ramsden
D B
DB
D000818:Animals; D001254:Astrocytoma; D001483:Base Sequence; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D001932:Brain Neoplasms / Q000201:enzymology; D002470:Cell Survival; D002528:Cerebellar Neoplasms; D050558:Cysteine Dioxygenase; D049308:Dioxygenases; D015971:Gene Expression Regulation, Enzymologic; D005909:Glioblastoma; D006801:Humans; D008297:Male; D008527:Medulloblastoma; D023421:Models, Animal; D008959:Models, Neurological; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D010105:Oxygenases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D010105:Oxygenases; D049308:Dioxygenases; D050558:Cysteine Dioxygenase