Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.566963
Title: The role of poly (ADP-ribose) polymerase-1 in the regulation of the stress inducible transcription factor, nuclear factor kappa-B
Author: Hunter, Jill Elizabeth
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2012
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Abstract:
The stress inducible transcription factor, NF-κB, induces genes involved in proliferation and apoptosis. Aberrant NF-κB activity is common in cancer and contributes to therapeutic-resistance. Moreover, constitutive NF-κB activation has been shown to contribute to malignant progression through the regulation of gene expression. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated during DNA strand break repair and is a known transcriptional co-regulator. The role of PARP-1 function during NF-κB activation using siRNA knockdown of either p65 or PARP-1, or the potent PARP-1 inhibitor, AG-014699, was investigated. Survival and apoptosis assays showed that NF- кB p65-/- mouse embryonic fibroblasts (MEFs) were more sensitive to ionising radiation (IR) than p65+/+ MEFs. Co-incubation with p65 siRNA, PARP siRNA or AG-014699 radio-sensitised p65+/+, but not p65-/- MEFs, demonstrating that PARP-1 mediates its effects on survival via NF-κB. Furthermore, a combination of p65 siRNA and AG- 014699 radio-sensitised p65+/+ MEFs to the same extent as either agent alone, strongly indicating that PARP-1 and NF-κB are mechanistically linked. PARP-1 is known to be vital for the repair of single strand breaks (SSBs). SSB repair kinetics, and the effect SSB repair inhibition by AG-014699 were similar in p65+/+ and p65-/- cells. Since inhibition of SSB repair did not radio-sensitise p65-/- cells, these data show that radiosensitisation by AG-014699 is due to downstream inhibition of NF-kB activation, and independent of SSB repair inhibition. PARP-1 catalytic activity was essential for IRinduced p65 DNA binding and NF-κB-dependent gene transcription, whereas for TNF- α treated cells, PARP-1 protein alone was sufficient. It can therefore be hypothesised that this stimulus-dependent differential is mediated via stimulation of the Poly(ADPribose) (PAR) polymer, which was induced following IR, not TNF-α. Poly(ADP-ribose) glycohydrolase (PARG) is the major enzyme responsible for the catabolism of the PAR polymer. In order to inhibit degradation of the PAR polymer a potent and specific PARG inhibitor was used. Increased polymer stability following IR, by virtue of inhibition of polymer degradation, led to the persistence of NF-κB DNA binding, an increase in anti-apoptotic gene expression and a protection against IR-induced cell death. These data confirm a role for the PAR polymer in the activation of NF-κB following DNA damage. Microarray analysis showed that the TNF-α driven transcription of NF-κBdependent inflammatory and immune response genes was unaffected by AG-014699, 4 suggesting that targeting DNA-damage activated NF-κB using AG-014699 may overcome toxicity observed with classical NF-κB inhibitors without compromising other vital inflammatory functions. An investigation into the role of PARP-1 in DNA damage activated NF-κB activation in glioblastoma cells was also undertaken to assess the potential utility of AG-014699 in tumour types known to express constitutively active NF-κB. PARP-1 activity was vital for both radio- and chemo-sensitisation of U251 glioblastoma cells, and in IR- or temozolomide- treated cells, PARP-1 mediated its effects on survival via NF-κB. Importantly, these data confirm the findings in the MEFs by demonstrating that radio- or chemo-sensitisation by AG-014699 is due to downstream inhibition of NF-kB activation, and independent of SSB repair inhibition. Hence, these data highlight the potential of PARP-1 inhibitors to overcome NF-κBmediated therapeutic resistance and widens the spectrum of cancers in which these agents may be utilised. The therapeutic potential of a potent inhibitor of NF-κB subunit DNA binding, PBS- 1086, was also assessed in the NF-κB p65+/+ and p65-/- MEFs and the MDA-MB-231, T47D and MCF7 breast cancer cell lines. DNA binding and luciferase reporter assays showed that PBS-1086 inhibited IR-induced p65 and p50 DNA binding and NF-κBdependent gene transcription in p65+/+ cells. Co-incubation with PBS-1086 or p65 siRNA radio-sensitised p65+/+, but not p65-/- cells, demonstrating that PBS-1086 mediates radio-sensitisation via the p65 NF-κB subunit. Gene expression analysis showed that PBS-1086 inhibited IR-induced transcription of known NF-κB-regulated anti-apoptotic genes. MDA-MB-231 cells were found to have the highest constitutive levels of DNA binding of all NF-κB subunits. PBS-1086 radio-sensitised all three breast cancer cell lines. In survival assays, all breast cancer cell lines tested were also sensitive to PBS-1086, however, MDA-MB-231 cells were the most sensitive to PBS-1086 alone. Thus, high NF-κB DNA binding activity appears to correlate with sensitivity to PBS- 1086. Collectively these data highlight the potential of modulating NF-κB activity, either by PARP-1, or directly via inhibition of subunit DNA binding, to restore radioand chemo-sensitivity in cancers with aberrantly active NF-κB, and to overcome NF-κB mediated therapeutic resistance.
Supervisor: Not available Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.566963  DOI: Not available
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