Ionizing radiation-induced NF-kappaB activation requires PARP-1 function to confer radioresistance

Abstract

Recent reports implicate poly(ADP-ribose) polymerase-1 (PARP-1) in the activation of nuclear factor kappaB (NF-kappaB). We investigated the role of PARP-1 in the NF-kappaB signalling cascade induced by ionizing radiation (IR). AG14361, a potent PARP-1 inhibitor, was used in two breast cancer cell lines expressing different levels of constitutively activated NF-kappaB, as well as mouse embryonic fibroblasts (MEFs) proficient or deficient for PARP-1 or NF-kappaB p65. In the breast cancer cell lines, AG14361 had no effect on IR-induced degradation of IkappaBalpha or nuclear translocation of p50 or p65. However, AG14361 inhibited IR-induced NF-kappaB-dependent transcription of a luciferase reporter gene. Similarly, in PARP-1(-/-) MEFs, IR-induced nuclear translocation of p50 and p65 was normal, but kappaB binding and transcriptional activation did not occur. AG14361 sensitized both breast cancer cell lines to IR-induced cell killing, inhibited IR-induced XIAP expression and increased caspase-3 activity. However, AG14361 failed to increase IR-induced caspase activity when p65 was knocked down by siRNA. Consistent with this, AG14361 sensitized p65(+/+) but not p65(-/-) MEFs to IR. We conclude that PARP-1 activity is essential in the upstream regulation of IR-induced NF-kappaB activation. These data indicate that potentiation of IR-induced cytotoxicity by AG14361 is mediated solely by inhibition of NF-kappaB activation.

FIGURE 1. Characterization of cell lines

(A) Western blots of whole cell extracts from untreated cells Blots were probed for PARP-1, p50, p65, IkBα, IkBβ and actin. (B)Western blots of nuclear extracts from untreated cells Blots were probed for p50, p65 and lamin. (C) Western blots of whole cell extracts of MDA-MB-231 and T47D at 48 h following transfection with vehicle alone, non specific siRNA or p65 siRNA. (D) PARP-1 activity. Open bars, basal activity in the absence of oligonucleotide; closed bars, oligonucleotide-stimulated activity. Results are the mean of three replicates from three independent experiments ± SE

FIGURE 2. Effect of IR ± AG14361 on NF-κB DNA binding

(A) P50 and p65 binding activity at 2 h following increasing doses of IR. A1, MDA-MB-231 cells; A2, T47D cells. (B) Kinetics of induction of p50 and p65 binding activity following 20 Gy IR. B1, MDA-MB-231 cells; B2, T47D cells. (C) Effect of AG14361 on IR-induced and constitutive p50 binding activity. C1, MDA-MB-231 cells 2 h following 20 Gy IR; C2 T47D cells 2 h following 20 Gy IR; C3, MDA-MB-231 cells in the absence of IR. (D) P50 binding activity 2 h following increasing doses of IR in PARP-1 ^+/+ and PARP-1 ^-/- cells

FIGURE 3. Effect of IR ± AG14361 on IkBα degradation and nuclear translocation

(A) Kinetics of degradation of IkBα following 20 Gy IR ± AG14361. A1, MDA-MB-231 cells; A2, T47D cells. Western blots of whole cell lysates. Blots were probed for IkBα. Bar charts:- relative density of a representative blot normalized to actin co-loading obtained by densitometry (B) Kinetics of nuclear translocation pf p50 and p65 following 20 Gy IR ± AG14361 in T47D cells. C1, T47D cells probed for p50; C2, T47D cells probed for p65. Western blots of nuclear extracts. Blots were probed for p50 or p65. Bar charts are normalized to lamin co-loading. (C) Kinetics of degradation of IkBα protein following 20 Gy IR in PARP-1^-/- cells. Western blots of whole cell lysates. Blots were probed for IkBα. Bar charts are normalized to actin co-loading. (D) Kinetics of nuclear translocation of p50 and p65 following IR in PARP-1^-/- cells. Blots were probed for p50 or p65. Bar charts are normalized to lamin co-loading.

FIGURE 4. Effect of IR ± AG14361 on NF-κB transcriptional activity in the cell lines

(A) Induction of luciferase activity 8 h following increasing doses of IR ± AG14361. A1, MDA-MB-231 cells; A2, T47D cells. (B) Induction of luciferase activity 8 h following increasing doses of IR in PARP-1 ^+/+ and PARP-1 ^-/- cells.

FIGURE 5. Effect of IR ± AG14361 on the levels of caspase-3 activity and XIAP protein

(A) Kinetics of induction of caspase -3 activity following 20 Gy IR A1, MDA-MB-231 cells; A2, T47D cells. (B) The effect of IR ± AG14361 ± p65 siRNA on the induction of caspase-3 activity at 24 h post-IR. B1, MDA-MB-231 cells; B2, T47D cells. (C).Induction of XIAP following 20 Gy IR ± AG14361. C1, MDA-MB-231 cells; C2, T47D cells; C3, p65^+/+ and p65^-/- cells. Blots were probed for XIAP. Bar charts:-relative density of a representative blot normalized to actin co-loading.

FIGURE 6. Effects of increasing doses of IR ± AG14361 on cell survival

A, MDA-MB-231 cells; B, T47D cells; C, p65 ^+/+ cells; D, p65 ^-/-. ■, IR alone ; □, IR + AG14361. Data are the mean of 3 independent experiments ± SE.