DNA repair in mononuclear cells: role of serine/threonine phosphatases.
Treatment with cyclosporin A (CsA) in kidney-transplant recipients is associated with reduced DNA repair and enhanced cancer incidence. CsA is an inhibitor of the serine/threonine phosphatase calcineurin, also termed PP2B, which is a Ca(2+)/calmodulin-dependent phosphatase. In this study we sought to elucidate the role of calcineurin in DNA repair using CsA and tacrolimus; examine whether UV-induced DNA repair is associated with dephosphorylation; and investigate whether phosphatases other than calcineurin are active in DNA repair, in light of the fact that calcineurin inhibition only partially suppressed DNA repair. Peripheral blood mononuclear cells from healthy donors were used. In vitro, we assayed UV-induced DNA repair by measuring the incorporation of tritiated thymidine in UV-irradiated cells. We gauged phosphatase activity indirectly by measuring free inorganic phosphate (Pi) excreted into the medium. The phosphatase assay was performed under the same conditions and in parallel to the DNA-repair assay. Tacrolimus, like CsA, inhibited DNA repair in a dose-dependent fashion. DNA repair was associated with production of Pi, which correlated with the number of cells performing DNA repair. Phosphatase activity increased after UV irradiation. DNA repair correlated directly with phosphatase activity, whereas CsA reduced both DNA repair and Pi production. Inhibition of calmodulin by trifluoperazine and W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide] reduced DNA repair in part. We investigated the role of the Ca(2+)-independent phosphatases PP1 and PP2A using specific inhibitors. Calyculin A, which inhibits both phosphatases, reduced DNA repair. Endothall, a PP2A inhibitor, had no effect on DNA repair. Okadaic acid, which is mostly a PP2A inhibitor but also a weak inhibitor of PP1, reduced DNA repair only slightly. We suggest that DNA repair is mediated by way of Ca(2+)-dependent and Ca(2+)-independent pathways, with calcineurin and PP1 being the respective phosphatases involved in each pathway.
Herman
Michal
M
Institute of Nephrology and Hypertension, Rabin Medical Center, Golda-Hasharon Campus, 7 Keren Kayemet Street, Petah-Tikva 49372, Israel.
Ori
Yaacov
Y
Chagnac
Avry
A
Weinstein
Talia
T
Korzets
Asher
A
Zevin
Dina
D
Malachi
Tsipora
T
Gafter
Uzi
U
D019703:Calcineurin / Q000378:metabolism; D065095:Calcineurin Inhibitors; D016572:Cyclosporine / Q000009:adverse effects; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007166:Immunosuppressive Agents / Q000009:adverse effects; D066298:In Vitro Techniques; D016030:Kidney Transplantation / Q000009:adverse effects; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D008387:Marine Toxins; D009369:Neoplasms / Q000209:etiology; D010080:Oxazoles / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D016559:Tacrolimus / Q000009:adverse effects; D014466:Ultraviolet Rays
D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D008387:Marine Toxins; D010080:Oxazoles; D010710:Phosphates; C059041:calyculin A; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D016559:Tacrolimus