Cyclosporin A inhibits Ca²⁺‐mediated upregulation of the DNA repair enzyme DNA polymerase β in human peripheral blood mononuclear cells

Abstract

Alterations in gene expression may represent an underlying cause of undesired side-effects mediated by the immunosuppressant cyclosporin A (CsA). We employed the method of differential display PCR to identify new genes whose expression is modulated by CsA. Human peripheral blood mononuclear cells (PBMCs), or subpopulations thereof, were simultaneously stimulated with the phorbol ester 4b-phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. We identify the gene encoding the DNA repair enzyme DNA polymerase b (Pol b) as a novel CsA-sensitive transcription unit. Our data show that transcription of pol b mRNA is induced by Ca 2+ and that CsA significantly inhibits PMA/ionomycin-and ionomycin-mediated upregulation of both pol b mRNA and Pol b protein. The CsA-mediated inhibition of pol b upregulation is maintained for at least 21 h after gene activation and is exerted via the phosphatase calcineurin. FK506, another immunosuppressant that targets calcineurin, also inhibits pol b upregulation, while rapamycin competes with FK506 action. This work identifies Ca 2+ as an inducer of pol b gene activity in primary blood cells. The demonstrated CsA sensitivity of this process suggests a novel molecular mechanism that may contribute to the increased tumor incidence in patients receiving CsA treatment.Keywords: cyclosporin A; immunosuppression; DNA repair; Ca 2+ -mediated signal transduction.The immunosuppressant cyclosporin A (CsA) is a first choice drug for treating patients receiving allograft transplantation. The molecular mechanism by which CsA exerts its immunosuppressive function has been attributed to its inhibition of the phosphatase calcineurin [1,2]. CsA binds to a cyclophilin, forming a CsA±cyclophilin complex, which subsequently acts to inhibit the Ca 2+ -dependent protein phosphatase calcineurin. Active calcineurin dephosphorylates, for example, the cytosolic transcription factor nuclear factor of activated T-cells (NF-AT) [3], allowing its translocation into the nucleus and modulation of gene transcription. CsA affects the transcription of many genes, including interleukin-2, interleukin-3, granulocyte macropage colony stimulating factor and tumor necrosis factor-a (reviewed in [4]). We used the method of differential display [5] to find hitherto unidentified genes, whose expression in primary human cell populations (i.e. peripheral blood mononuclear cells; PBMCs) is affected by CsA.We demonstrate the ability of CsA to inhibit both the induced transcription and protein expression of the DNA polymerase b (pol b) gene. Pol b is the smallest (39 kDa, 339 amino acids) of the five known eukaryotic cellular DNA polymerases and is primarily involved in processive gap-filling syntheses associated with base excision repair of small (6 nucleotide) DNA gaps [6]. Pol b is the major enzyme in a base excision repair pathway responsible for correction of DNA damage induced by depurination [7]; formation of apurinic/apyrimidinic sites ranks ...

Ca2+‐induced p38/SAPK signalling inhibited by the immunosuppressant cyclosporin A in human peripheral blood mononuclear cells

“…7B). Of the transfectable established cell lines tested, none has confirmed the CsA sensitivity described in this paper [63]. This precluded the use of CsA in our transient transfection studies using pol β promoter constructs.…”

Topical Calcineurin Inhibitors Decrease the Production of UVB-Induced Thymine Dimers from Hairless Mouse Epidermis

“…On the other hand, it is conceivable that inhibitors of both Th1 and Th2 cytokines, such as pimecrolimus and tacrolimus, may have an impact on the repair of UV-mediated DNA damage, as the rate of DNA repair can be increased by cytokines such as interleukin 12 [19]. Prior studies with cyclosporine A suggest that calcineurin has a role in the DNA repair pathway [20, 21], and inhibition of the DNA repair activity by tacrolimus in mononuclear cells [22] has been linked to the inhibition of the DNA repair enzyme DNA polymerase β [23]. However, this enzyme is primarily implicated in base excision repair, while the predominant mechanism in UV-mediated DNA damage is nucleotide excision repair [10], the interactions of which with calcineurin inhibitors are still unclear.…”

CsA can induce DNA double-strand breaks: implications for BMT regimens particularly for individuals with defective DNA repair

“…[24][25][26][27] CsA has been proposed to inhibit DNA damage-induced overexpression of DNA polymerase b, which plays an important role in BER. 26,28 To examine whether CsA might enhance the persistence or formation of DSBs following replication, we examined the impact of CsA on DSBs arising following treatment with MMS, an agent that can induce SSBs in S-phase (Figure 3c). Following exposure to 1 mM MMS for 1 h and incubation for a further 24 h, a small increase in DSBs (5-8 53BP1 foci) was observed in WT, LIG4 syndrome and ART-SCID primary fibroblasts that had traversed S-phase (BrdU þ ive).…”

“…This is consistent with the notion that DSBs can arise following replication of SSBs but that under normal conditions they are rapidly repaired. Strikingly, when CsA (5 mM) was included with MMS, a significant increase in DSBs was observed, specifically in LIG4 syndrome fibroblasts (23)(24)(25)(26)(27)(28)(29)(30) foci) compared to WT or ART-SCID (5-10 53BP1 foci). This suggests that CsA-induced DSBs can form, likely indirectly, from SSBs during S-phase.…”