title: Methotrexate increases expression of cell cycle checkpoint genes via JNK activation.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: Charles F
authors.initials: CF
authors.lastname: Spurlock
authors.forename: John T
authors.initials: JT
authors.lastname: Tossberg
authors.forename: Howard A
authors.initials: HA
authors.lastname: Fuchs
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Olsen
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Aune
mesh_terms: D000328:Adult; D000368:Aged; D018501:Antirheumatic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001172:Arthritis, Rheumatoid / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D059447:Cell Cycle Checkpoints / Q000187:drug effects* / Q000502:physiology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000235:genetics; D008297:Male; D008727:Methotrexate / Q000494:pharmacology* / Q000627:therapeutic use; D008875:Middle Aged; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D018501:Antirheumatic Agents; D017382:Reactive Oxygen Species; D008727:Methotrexate