Tumour suppressor protein p53 regulates the stress activated bilirubin oxidase cytochrome P450 2A6.
Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5'-Luc constructs--down to -160bp from the TSS--showed p53 responsiveness in p53 overexpressed C3A cells. However, a further deletion from -160 to -74bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene--a well-known p53 activator--increased the expression of the p53 responsive positive control and the CYP2A6-5'-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5'-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6.
The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland, Australia. Electronic address: hao.hu1@uqconnect.edu.au.
Hao
H
Hu
The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland, Australia. Electronic address: t.yu2@uq.edu.au.
Ting
T
Yu
Institute of Biomedicine, Department of Pharmacology and Toxicology and Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland. Electronic address: Satu.Juhila@orion.fi.
Satu
S
Arpiainen
The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland, Australia. Electronic address: m.lang@uq.edu.au.
Matti A
MA
Lang
Institute of Biomedicine, Department of Pharmacology and Toxicology and Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland. Electronic address: Jukka.hakkola@oulu.fi.
Jukka
J
Hakkola
The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland, Australia. Electronic address: a.abubakar@uq.edu.au.
A'edah
A
Abu-Bakar
D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D065637:Cytochrome P-450 CYP2A6 / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D024202:Electrophoretic Mobility Shift Assay; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D012333:RNA, Messenger; C495901:TP53 protein, human; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase
Bilirubin; Bilirubin oxidase; CYP2A6; Oxidative stress; p53