Dominant-negative p53 mutations in rheumatoid arthritis.
OBJECTIVE
Studies were performed to determine if p53 mutations identified in rheumatoid arthritis (RA) synovial tissue are dominant negative.


METHODS
Site-directed mutagenesis was used to produce 2 RA-derived mutants: asparagine-->serine at codon 239 (N239S) and arginine-->stop at codon 213 R213*). HS68 dermal fibroblasts were transfected with either empty vector, wild-type p53 cDNA (wt), or the N239S or R213* mutant p53 cDNA clones. Interleukin-6 (IL-6) and bax gene expression were determined by Northern blot analysis. Bax transcription was determined using a bax promoter/reporter gene construct (bax-luc).


RESULTS
Transfection of HS68 cells with wt increased bax mRNA levels. This process was blocked by cotransfection with either mutant. The mutant p53 genes also increased IL-6 gene expression. Low levels of bax promoter activity were detected in HS68 cells co-transfected with bax-luc and empty vector, N239S, or R213*, indicating that the RA mutants lacked transcriptional activity. Transfection with wt and bax-luc led to a 10-fold increase in luciferase expression. When the wt gene was cotransfected with either of the mutants, there was a dose-dependent inhibition of bax promoter activity.


CONCLUSIONS
These data indicate that at least 2 of the p53 mutants identified in RA joint samples are dominant negative and suppress endogenous wild-type p53 function.
Han
Z
Z
University of California, San Diego, School of Medicine, La Jolla 92093-0656, USA.
Boyle
D L
DL
Shi
Y
Y
Green
D R
DR
Firestein
G S
GS
D001172:Arthritis, Rheumatoid / Q000235:genetics* / Q000473:pathology; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D000078404:Epidermal Cells; D004817:Epidermis / Q000201:enzymology; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D005799:Genes, Dominant / Q000235:genetics*; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000737:chemistry / Q000473:pathology; D014162:Transfection; D051028:bcl-2-Associated X Protein; D001616:beta-Galactosidase / Q000378:metabolism
C494826:BAX protein, human; D017931:DNA Primers; D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D008156:Luciferases; D001616:beta-Galactosidase