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, In terleukin-6 (IL-6) and bar gene expression were determined by Northern blot analysis. Bar transcription was determined using a bar promoter/reporter g ene construct (bax-luc), Results, Transfection of HS68 cells with wt increased bar mRNA levels. This process was blocked by cotransfection with either mutant. The mutant p53 g enes also increased IL-6 gene expression, Low levels of bar promoter activi ty were detected in HS68 cells cotransfected with bax-luc and empty vector, N239S, or R213*, indicating that the RA mutants lacked transcriptional act ivity. Transfection with wt and bax-luc led to a 10-fold increase in lucife rase expression. When the wt gene was cotransfected,vith either of the muta nts, there was a dose-dependent inhibition of bar promoter activity. Conclusion. These data indicate that at least 2 of the p53 mutants identifi ed in RA joint samples are dominant negative and suppress endogenous wild-t ype p53 function.