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A single-nucleotide polymorphism (SNP), identified at nucleotide position −844 in the 5′ promoter of the FasL gene, lies within a putative binding motif for CAAT/enhancer-binding protein β (C/EBPβ). Electrophoretic mobility shift and supershift assays confirmed that this element binds specifically to C/EBPβ and demonstrated that the two alleles of this element have different affinities for C/EBPβ. In luciferase reporter assays, the −844C genotype had twice the basal activity of the −844T construct, and basal expression of Fas ligand (FasL) on peripheral blood fibrocytes was also significantly higher in −844C than in −844T homozygous donors. FasL is located on human chromosome 1q23, a region that shows linkage to the systemic lupus autoimmune phenotype. Analysis of 211 African American systemic lupus erythematosus patients revealed enrichment of the −844C homozygous genotype in these systemic lupus erythematosus patients compared with 150 ethnically matched normal controls (p = 0.024). The −844C homozygous genotype may lead to the increased expression of FasL, to altered FasL-mediated signaling in lymphocytes, and to enhanced risk for autoimmunity. This functionally significant SNP demonstrates the potential importance of SNPs in regulatory regions and suggests that differences in the regulation of FasL expression may contribute to the development of the autoimmune phenotype.

Leflunomide, a novel immunosuppressive drug, is able to prevent and reverse allograft and xenograft rejection in rodents, dogs, and monkeys. It is also effective in the treatment of several rodent models of arthritis and autoimmune disease. In vitro studies indicate that leflunomide is capable of inhibiting anti-CD3- and interleukin-2 (IL-2)-stimulated T cell proliferation. However, the biochemical mechanism for the inhibitory activity of leflunomide has not been elucidated. In this study, we characterized the inhibitory effects of leflunomide on Src family (p56lck and p59fyn)-mediated protein tyrosine phosphorylation. Leflunomide was able to inhibit p59fyn and p56lck activity in in vitro tyrosine kinase assays. The IC50 values for p59fyn (immunoprecipitated from either Jurkat or CTLL-4 cell lysate) autophosphorylation and phosphorylation of the exogenous substrate, histone 2B, were 125-175 and 22-40 microM respectively, while the IC50 values for p56lck (immunoprecipitated from Jurkat cell lysates) autophosphorylation and phosphorylation of histone 2B were 160 and 65 microM respectively. We also demonstrated the ability of leflunomide to inhibit protein tyrosine phosphorylation induced by anti-CD3 monoclonal antibody in Jurkat cells. The IC50 values for total intracellular tyrosine phosphorylation ranged from 5 to 45 microM, with the IC50 values for the zeta chain and phospholipase C isoform gamma 1 being 35 and 44 microM respectively. Leflunomide also inhibited Ca2+ mobilization in Jurkat cells stimulated by anti-CD3 antibody but not in those stimulated by ionomycin. Distal events of anti-CD3 monoclonal antibody stimulation, namely, IL-2 production and IL-2 receptor expression on human T lymphocytes, were also inhibited by leflunomide. Finally, tyrosine phosphorylation in CTLL-4 cells stimulated by IL-2 was also inhibited by leflunomide. These data collectively demonstrate the ability of leflunomide to inhibit tyrosine kinase activity in vitro, and suggest that inhibition of tyrosine phosphorylation events may be the mechanism by which leflunomide functions as an immunosuppressive agent.

BACKGROUND Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid neoplasia and represents the end stage of thyroid tumor progression. In the current study, genetic alterations in a panel of ATC were profiled to determine the origins of ATC. METHODS Eight ATC were analyzed for BRAF mutation at codon 599 by using mutant‐allele‐specific polymerase chain reaction (PCR) and DNA sequencing of the PCR‐amplified exon 15. RAS mutation (HRAS, KRAS, and NRAS) at codons 12, 13, and 61 was analyzed by direct sequencing of PCR‐amplified exons 1 and 2 of the RAS gene. RET/PTC rearrangements and p53 mutation were monitored by immunohistochemical (IHC) staining by anti‐RET antibodies and an anti‐p53 mAb, respectively. RESULTS BRAF was mutated in 5 of the 8 ATCs tested. Histologic examination revealed that 4 of these 5 BRAF‐mutated ATCs contained a PTC component, suggesting that they may be derived from BRAF‐mutated PTC. Of the 3 ATCs with wild‐type BRAF, 2 had spindle cell features; one had follicular neoplastic characteristics mixed with papillary structures. Analysis of RAS mutation revealed only an HRAS mutation at codon 11, due to the transversion of GCC to TCC in one ATC with wild‐type BRAF. This leads to the substitution of valine to serine. IHC analysis of RET/PTC rearrangements revealed no positive staining of RET in any of 8 ATCs, suggesting that these ATCs are not derived from RET/PTC‐ rearranged PTC. In contrast, IHC analysis of p53 mutation revealed that p53 was detected in the nuclei of 5 of 5 BRAF‐mutated ATCs and 2 of 3 ATCs with wild‐type BRAF. p53 staining was present only in anaplastic thyroid tumor cells but not in neighboring papillary thyroid tumor cells. CONCLUSIONS These results suggest that many ATCs with papillary components are derived from BRAF‐mutated PTC, because of the addition of p53 mutation. Cancer 2005. © 2005 American Cancer Society.