Regional analysis ofp53mutations in rheumatoid arthritis synovium

Abstract

The p53 tumor suppressor protein plays a central role in cell cycle regulation, DNA repair, and apoptosis. Recent studies indicate that DNA damage and somatic mutations in the p53 gene can occur because of genotoxic stress in many tissues, including the skin, colon, and synovium. Although somatic mutations in the p53 gene have been demonstrated in rheumatoid arthritis (RA) synovial tissue and synoviocytes, no information is available on the location or extent of p53 mutations. Using microdissected RA synovial tissue sections, we observed abundant p53 transition mutations, which are characteristic DNA damage caused by oxidative stress. p53 mutations, as well as p53 mRNA expression, were located mainly in the synovial intimal lining rather than the sublining (P < 0.01). Clusters of p53 mutant subclones were observed in some microdissected regions, suggesting oligoclonal expansion. Because IL-6 gene expression is regulated by wildtype p53, IL-6 mRNA expression in microdissected tissues was quantified by using real-time PCR. The regions with high rates of p53 mutations contained significantly greater amounts of IL-6 mRNA compared with the low mutation samples (P < 0.02). The microdissection findings suggest that p53 mutations are induced in RA synovial tissues by inflammatory oxidative stress. This process, as in sun-exposed skin and inflamed colonic epithelium, provides some of the mutant clones with a selective growth advantage. A relatively low percentage of cells containing p53 mutations can potentially affect neighboring cells and enhance inflammation through the elaboration of proinflammatory cytokines.

Serum macrophage inhibitory cytokine 1 in rheumatoid arthritis: A potential marker of erosive joint destruction

“…While not exclusively regulated by p53, MIC-1 transactivation and protein production are strongly up-regulated by activation of this pathway (47). Additionally, activation of the p53 pathway is associated with suppression of IL-6 production in rheumatoid synovium (48) and consequently could lead to decreased serum levels of CRP. The interrelationship between these 2 cytokines would seem to support the use of the compound variable MIC-1/CRP, which was associated with severe refractory RA and part of the RA severity score.…”

Suppression of thymic lymphomas and increased nonthymic lymphomagenesis in Trp53‐deficient mice lacking inducible nitric oxide synthase gene

“…Deregulation of cytokine production plays a central role in lymphomagenesis, 34 and p53 regulates anti-or proinflammatory cytokines in rheumatoid arthritis and cancer cell lines. 35,36 p53 functional loss can lead to autocrine IL-6 production. 35,36 Ohkusu-Tsukada et al 37 showed that accumulation of memory T cells (CD4 ϩ ) was spontaneously accelerated and Th2 cytokines such as IL-4, IL-6 and IL-10 were strongly induced by antigen stimulation in Trp53-deficient mice.…”

“…First-strand cDNA was synthesized with a commercial kit (Amersham) in a reaction volume of 15 l containing 5 g of total RNA and 0.2 g of random hexamer primers, according to the manufacturer's instructions. To detect cytokines, first-strand cDNA was amplified by PCR with the following sets of oligonucleotide primers: IL-2 forward, 5Ј-AACAGCGCAC-CCACTTCAA-3Ј; IL-2 reverse, 5Ј-TTGAGATGATGCTTT-GACA-3Ј; IL-4 forward, 5Ј-TAGTTGTCATCCTGCTCTT-3Ј; IL-4 reverse, 5Ј-CTACGAGTAATCCATTTGC-3Ј; IL-6 forward, 5Ј-GAACAACGATGATGCACTTGCAG-3Ј; IL-6 reverse, 5Ј-CCTTAGCCACTCCTTCTGTGAC-3Ј; IL-7 forward, 5Ј-GCCT-GTCACATCATCTGAGTGCC-3Ј; IL-7 reverse, 5Ј-CAGGAG-GCATCCAGGAACTTCTG-3Ј; IL-10 forward, 5Ј-TCAAACA-AAGGACCAGCTGGACAACATACTG-3Ј; IL-10 reverse, 5Ј-CTGTCTAGGTCCTGGAGTCCAGCAGACTCAA-3Ј; IL-12 (35) forward, 5Ј-ACCTGCTGAAGACCACAGAT-3Ј; IL-12(35) re-verse, 5Ј-GATTCTGAAGTGCTGCGTTG-3Ј; IL-12(40) forward, 5Ј-CTCACCTGTGACACGCCTGA-3Ј; IL-12(40) reverse, 5Ј-CAGGACACTGAATACTTCTC-3Ј; IL-15 forward, 5Ј-GTGAT-GTTCACCCCAGTTGC-3Ј; IL-15 reverse, 5Ј-CACATTCTTTG-CATCCAGA-3Ј; IL-18 forward, 5Ј-ACTGTACAACCG-GAGTAATACGG-3Ј; IL-18 reverse, 5Ј-TCCATCTTGTTGT-GTCCTGG-3Ј; IFN-␥ forward, 5Ј-TGAACGCTACACACTG-CATCTTGG-3Ј; IFN-␥ reverse, 5Ј-CGACTCCTTTTCCGCTTC-CTGAG-3Ј; TNF-␣ forward, 5Ј-AACTAGTGGTGCC-AGCCGAT-3Ј; TNF-␣ reverse, 5Ј-CTTCACAGAGCAAT-GACTCC-3Ј; GAPDH forward, 5Ј-TGAAGGTCGGTGT-GAACGGATTTGGC-3Ј; GAPDH reverse, 5Ј-CATGTAGGC-CATGAGGTCCACCAC-3Ј. Samples were amplified in 28 cycles for IL-18 and GAPDH and in 35 cycles for IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IFN-␥ and TNF-␣ (denaturation at 94°C for 30 sec, annealing at 57°C for 60 sec and extension at 72°C for 120 sec).…”

Regulation of p53 by macrophage migration inhibitory factor in inflammatory arthritis

“…Overexpression of p53 and functional p53 mutations have been detected in cells and tissue derived from RA patients (2). Difficulty clarifying the contribution of p53 mutations to synovial hyperplasia has resulted largely from discrepancies in the detection rate of p53 mutations within tissues and FLS derived from RA patients (9). Fluctuations in the levels or function of wild-type p53 protein unrelated to mutation could also have an impact on synovial proliferative capacity.…”