Repair of Cytokine-induced DNA damage in cultured rat islets of Langerhans

A.L. Rosales, James Cunningham, Adrian Bone, I.C. Green, M.H.L. Green

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1bgr (IL-1bgr), interferon ggr (IFN ggr) and tumour necrosis factor agr (TNF agr) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4 h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor NG-monomethyl-l-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during, the cytokine treatment and islets are incubated for 4 h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4 h in the presence of hydroxyurea (HU) and 1-bgr-d-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18 h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.
Original languageEnglish
Pages (from-to)665-674
Number of pages10
JournalFree Radical Research
Volume38
Issue number7
Publication statusPublished - Jul 2004

Fingerprint

Islets of Langerhans
DNA Damage
Cytokines
Arginine
Purines
Nitric Oxide
Hydroxyurea
Cytosine
Nitric Oxide Synthase
DNA Repair
Interleukins
Insulin-Secreting Cells
Interferons
Tumor Necrosis Factor-alpha

Keywords

  • Comet assay
  • DNA repair
  • Nitric oxide
  • Hydroxyurea
  • 1-beta-d-arabinosyl cytosine
  • NG-monomethyl-l-arginine
  • 8-OHG, 8-hydroxyguanine (7,8-dihydro-8-oxoguanine)
  • AraC, 1-beta-d-arabinofuranosyl cytosine
  • Fpg, formamidopyrimidine glycosylase
  • HU, hydroxyurea
  • IFNgamma, interferon gamma
  • IL-1beta, interleukin-1beta
  • MEM, minimum essential medium with Earle's salts
  • NMMA, NG-monomethyl-l-arginine monoacetate
  • TNFalpha, tumour necrosis factor alpha

Cite this

Rosales, A. L., Cunningham, J., Bone, A., Green, I. C., & Green, M. H. L. (2004). Repair of Cytokine-induced DNA damage in cultured rat islets of Langerhans. Free Radical Research, 38(7), 665-674.
Rosales, A.L. ; Cunningham, James ; Bone, Adrian ; Green, I.C. ; Green, M.H.L. / Repair of Cytokine-induced DNA damage in cultured rat islets of Langerhans. In: Free Radical Research. 2004 ; Vol. 38, No. 7. pp. 665-674.
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Rosales, AL, Cunningham, J, Bone, A, Green, IC & Green, MHL 2004, 'Repair of Cytokine-induced DNA damage in cultured rat islets of Langerhans' Free Radical Research, vol. 38, no. 7, pp. 665-674.

Repair of Cytokine-induced DNA damage in cultured rat islets of Langerhans. / Rosales, A.L.; Cunningham, James; Bone, Adrian; Green, I.C.; Green, M.H.L.

In: Free Radical Research, Vol. 38, No. 7, 07.2004, p. 665-674.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Repair of Cytokine-induced DNA damage in cultured rat islets of Langerhans

AU - Rosales, A.L.

AU - Cunningham, James

AU - Bone, Adrian

AU - Green, I.C.

AU - Green, M.H.L.

PY - 2004/7

Y1 - 2004/7

N2 - Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1bgr (IL-1bgr), interferon ggr (IFN ggr) and tumour necrosis factor agr (TNF agr) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4 h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor NG-monomethyl-l-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during, the cytokine treatment and islets are incubated for 4 h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4 h in the presence of hydroxyurea (HU) and 1-bgr-d-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18 h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.

AB - Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1bgr (IL-1bgr), interferon ggr (IFN ggr) and tumour necrosis factor agr (TNF agr) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4 h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor NG-monomethyl-l-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during, the cytokine treatment and islets are incubated for 4 h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4 h in the presence of hydroxyurea (HU) and 1-bgr-d-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18 h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.

KW - Comet assay

KW - DNA repair

KW - Nitric oxide

KW - Hydroxyurea

KW - 1-beta-d-arabinosyl cytosine

KW - NG-monomethyl-l-arginine

KW - 8-OHG, 8-hydroxyguanine (7,8-dihydro-8-oxoguanine)

KW - AraC, 1-beta-d-arabinofuranosyl cytosine

KW - Fpg, formamidopyrimidine glycosylase

KW - HU, hydroxyurea

KW - IFNgamma, interferon gamma

KW - IL-1beta, interleukin-1beta

KW - MEM, minimum essential medium with Earle's salts

KW - NMMA, NG-monomethyl-l-arginine monoacetate

KW - TNFalpha, tumour necrosis factor alpha

M3 - Article

VL - 38

SP - 665

EP - 674

JO - Free Radical Research

T2 - Free Radical Research

JF - Free Radical Research

SN - 1029-2470

IS - 7

ER -

Rosales AL, Cunningham J, Bone A, Green IC, Green MHL. Repair of Cytokine-induced DNA damage in cultured rat islets of Langerhans. Free Radical Research. 2004 Jul;38(7):665-674.