2-Chloroadenosine but not adenosine induces apoptosis in rheumatoid fibroblasts independently of cell surface adenosine receptor signalling.
1. The apoptotic effect of adenosine and its analogues was studied in fibroblast-like synoviocytes derived from rheumatoid arthritis patients (RA-FLSs). Evoked cell death was quantitatively examined by assessing DNA fragmentation using an enzyme-liked immunosorbent assay and by measuring phosphatidylserine exposure through flow cytometric analysis of annexin V binding. 2. Exposing cells for 24 h to 2-chloroadenosine (2-CADO), a nonspecific, adenosine deaminase (ADA)-resistant, adenosine receptor (AdoR) agonist, induced DNA fragmentation, and thus apoptosis, in RA-FLSs at concentrations > or =50 microM. By contrast, incubation with adenosine for up to 72 h did not evoke DNA fragmentation, even in the presence of ADA inhibitor coformycin and nucleoside transporter inhibitor nitrobenzylmercaptopurin (NBMPR). Transcription of all four AdoR isoforms was detected in RA-FLSs; nevertheless selective AdoR agonists similarly failed to induce DNA fragmentation. 3. DNA fragmentation evoked by 2-CADO was inhibited by NBMPR and by 5'-iodotubercidin, an adenosine kinase inhibitor, but not by xanthine amine congener, an A(1) and A(2) receptor antagonist, or by selective AdoR antagonists. 4. The nonspecific caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone abolished the apoptotic effect of 2-CADO. 5. These results suggest that 2-CADO induces apoptosis in RA-FLSs independently of AdoR-mediated signalling. Instead, 2-CADO, but not adenosine, is taken up into RA-FLSs via human equilibrative nucleoside transporter-1, where it is phosphorylated by adenosine kinase. The resultant phospho-2-CADO induces DNA fragmentation by activating a caspase pathway.
Koshiba
Masahiro
M
Clinical Pathology and Immunology, Department of Biomedical Informatics, Kobe University Graduate School of Medicine, Kobe, Japan. mkoshiba@med.kobe-u.ac.jp
Kosaka
Hidekazu
H
Nakazawa
Takashi
T
Hayashi
Nobuhide
N
Saura
Ryuichi
R
Kitamura
Noriko
N
Kumagai
Shunichi
S
D015762:2-Chloroadenosine / Q000494:pharmacology* / Q000627:therapeutic use; D000241:Adenosine / Q000494:pharmacology*; D000700:Analgesics / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001172:Arthritis, Rheumatoid / Q000188:drug therapy / Q000473:pathology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000473:pathology*; D006367:HeLa Cells; D006801:Humans; D011956:Receptors, Cell Surface / Q000502:physiology*; D018047:Receptors, Purinergic P1 / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013583:Synovial Membrane / Q000166:cytology / Q000187:drug effects / Q000473:pathology; D014407:Tumor Cells, Cultured
D000700:Analgesics; D011956:Receptors, Cell Surface; D018047:Receptors, Purinergic P1; D015762:2-Chloroadenosine; D000241:Adenosine