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Methotrexate increases expression of cell cycle checkpoint genes via JNK activation†
Article first published online: 25 MAY 2012
Copyright © 2012 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 64, Issue 6, pages 1780–1789, June 2012
How to Cite
Spurlock, C. F., Tossberg, J. T., Fuchs, H. A., Olsen, N. J. and Aune, T. M. (2012), Methotrexate increases expression of cell cycle checkpoint genes via JNK activation. Arthritis & Rheumatism, 64: 1780–1789. doi: 10.1002/art.34342
The Pennsylvania Department of Health Settlement Funds specifically disclaims responsibility for any analyses, interpretations, or conclusions herein.
- Issue published online: 25 MAY 2012
- Article first published online: 25 MAY 2012
- Accepted manuscript online: 19 DEC 2011 10:58AM EST
- Manuscript Accepted: 13 DEC 2011
- Manuscript Received: 11 AUG 2011
- NIH. Grant Numbers: R42-AI-53948, R01-AI-044924
- American College of Rheumatology Research and Education Foundation. Grant Number: Within Our Reach Program grant ACR124405
- Vanderbilt Ingram Cancer Center. Grant Number: P30-CA-68485
- Vanderbilt Digestive Research Center. Grant Number: DK-058404
- Core facility services and instruments used at Penn State University
- Pennsylvania Department of Health Settlement Funds
To assess defects in expression of critical cell cycle checkpoint genes and proteins in patients with rheumatoid arthritis (RA) relative to presence or absence of methotrexate (MTX) treatment, and to investigate the role of JNK in induction of these genes by MTX.
Flow cytometric analysis was used to quantify changes in levels of intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcription–polymerase chain reaction was used to identify changes in cell cycle checkpoint target genes.
RA patients expressed reduced baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and JNK total protein. The reduction in expression of mRNA for MAPK9, TP53, CDKN1A, and CDKN1B was greater in patients not receiving MTX than in those receiving low-dose MTX, with no difference in expression levels of CHEK2 and RANGAP1 mRNA between MTX-treated and non–MTX-treated patients. Further, JNK levels were inversely correlated with C-reactive protein levels in RA patients. In tissue culture, MTX induced expression of both p53 and p21 by JNK-2– and JNK-1–dependent mechanisms, respectively, while CHEK2 and RANGAP1 were not induced by MTX. MTX also induced ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocked these MTX-mediated effects.
Our findings support the notion that MTX restores some, but not all, of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells, via a JNK-dependent pathway.