Evidence for peroxynitrite-mediated modifications to p53 in human gliomas: possible functional consequences.
Based on previous findings of increased nitric oxide synthase (NOS) expression in human gliomas (4), we hypothesized that peroxynitrite, a highly reactive metabolite of nitric oxide (NO) and superoxide (O(*-)(2)), might be increased in these tumors in vivo. Here we demonstrate that nitrotyrosine (a footprint of peroxynitrite protein modification) is present in human malignant gliomas. Furthermore, we show that p53, a key tumor suppressor protein, has evidence of peroxynitrite-mediated modifications in gliomas in vivo. Experiments in vitro demonstrate that peroxynitrite treatment of recombinant wild-type p53 at physiological concentrations results in formation of higher molecular weight aggregates, tyrosine nitration, and loss of specific DNA binding. Peroxynitrite treatment of human glioma cell lysates similarly resulted in selective tyrosine nitration of p53 and was also associated with loss of p53 DNA binding ability. These data indicate that tyrosine nitration of proteins occurs in human gliomas in vivo, that p53 may be a target of peroxynitrite in these tumors, and that physiological concentrations of peroxynitrite can result in a loss of p53 DNA binding ability in vitro. These findings raise the possibility that peroxynitrite may contribute to loss of wild-type p53 functional activity in gliomas by posttranslational protein modifications.
Cobbs
C S
CS
Department of Surgery, Birmingham Veterans Affairs Hospital, Birmingham, Alabama 35294, USA.
Samanta
M
M
Harkins
L E
LE
Gillespie
G Y
GY
Merrick
B A
BA
MacMillan-Crow
L A
LA
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005909:Glioblastoma / Q000378:metabolism* / Q000473:pathology; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
D009841:Oligonucleotides; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine