Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


Functional Change of 5-Aminoimidazole-4-Carboxamide Ribonucleotide Formyltransferase in Dermal Fibroblasts from Methotrexate-Sensitive and Resistant Mice.

You2,  Xin, Cronstein2,  Bruce N., Williams2,  Adrienne, Dervieux1,  Thierry

Cypress Pharmaceuticals
NYU

Background:

Methotrexate (MTX) has been widely used in the treatment of RA but it is effective in only 60% of patients and its mechanism is complex. To date, the adenosine-mediated anti-inflammatory effect of MTX is best supported by the in vitro, in vivo, and clinical data. Pharmacogenetic studies indicate that genetic polymorphisms in adenosine pathway enzymes are associated with good response to MTX therapy in RA and animal experiments showed that MTX suppressed inflammation in air pouches of C57Bl/6 and BALBc, but not DBA/1J mice. To investigate the mechanism for genetically based resistance to the anti-inflammatory effects of MTX, functional change of a critical enzyme in MTX metabolism and the adenosine pathway, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICAR TF) was measured.

Methods:

Dermal Fibroblasts (DF) from BALBc and DBA/1J mice were cultured from skin. Following MTX incubation with DFs, AICAR TF activity before and after MTX treatment was analyzed after preparation of (6R,6S)-10-formyltetrahydrofolate from 5,10-Methenyltetrahydrofolate. Protein concentration was determined by the Bradford protein assay. AICARFT activity was monitored by the formation of FHat A for 4 min using a Perkin-Elmer Lambda2 spectrophotometer. In addition, MTX polyglumate (pg) concentration was measured and AICAR TF mRNA was quantited by real time (RT) PCR.

Results:

In silico analysis of the sequence of folyl polyglutamate synthase and AICAR TF indicate differing haplotypes of the enzymes in BALB/c and DBA1J mice. MTX treatment inhibited AICAR TF activity in DF from BALBc mouse in a dose-dependent manner (2.61 nmole/mg.min, 1.00 nmole/mg.min, and 0.9 nmole/mg.min at the dose of MTX 0, 10-7M, and 10-6M, respectively). Seventy percent decrease was observed at MTX 10-6M. AICAR TF activity was not reduced in DF from DBA1J mouse by MTX, and even increased after hydrogen peroxide stimulation with MTX (1.98 nmol/mg.min, 2.78 nmol/mg.min, and 3.83 nmol/mg.min at the dose of MTX 0, 10-7M, and 10-6M, respectively). The cellular concentration of MTXPG in BALBc fibroblasts is more than 2-fold greater than that observed in DBA1J fibroblasts at MTX 10-6M. There is no significant difference in AICAR TF mRNA in DFs between BALBc and DBA1J mice.

Conclusions:

Genetic polymorphisms of the critical enzymes involved in MTX metabolism and the adenosine pathway contribute to the different responses to MTX in the treatment of Rheumatoid Arthritis.

To cite this abstract, please use the following information:
You, Xin, Cronstein, Bruce N., Williams, Adrienne, Dervieux, Thierry; Functional Change of 5-Aminoimidazole-4-Carboxamide Ribonucleotide Formyltransferase in Dermal Fibroblasts from Methotrexate-Sensitive and Resistant Mice. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :827
DOI: 10.1002/art.28595

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