Role of PARP-1 in colitis-associated colorectal cancer induced by alkylating N-nitroso compounds
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Abstract
Poly(ADP-ribose) polymerase-1 (PARP-1), which catalyses the synthesis of poly(ADP-ribose), is implicated in a variety of cellular processes, such as chromatin remodelling and DNA repair. PARP-1 is well-known to promote base excision repair, a conserved pathway that removes DNA base modifications, including alkylated DNA bases. These lesions are induced by N-nitroso compounds (NOCs) that are tightly linked to the aetiology of colorectal cancer (CRC). It is further known that PARP-1-deficient mice display an enhanced sensitivity against alkylating agents in terms of toxicity and genomic instability.
The objective of this work was to assess the influence of PARP-1 on colitis-associated CRC induced by NOCs. PARP-1-proficient (WT) and PARP-1-deficient (PARP-1-/-) mice were treated with the colonotropic tumour initiator azoxymethane (AOM) followed by dextran sodium sulphate (DSS), which triggers colitis. Tumour formation was monitored by non-invasive mini endoscopy. WT mice displayed a significantly higher tumour number and tumour score compared to PARP-1-/- animals, which correlated with the AOM dose.
Analysis of initial DNA damage induction in both genotypes revealed an increased number of DNA strand breaks in PARP-1-/- animals, but comparable levels of O6-methylguanine (O6-MeG) DNA adducts in both genotypes. The latter is the critical lesion driving NOC-induced CRC. In line with this, the activity of O6-methylguanin-DNA methyltransferase (MGMT), which is responsible for the repair of O6-MeG DNA adducts, was depleted in both mouse strains in liver and colon. PARP-1-proficient and PARP-1-deficient mice did not display differences in basal cell proliferation or AOM-induced cell death in colorectal tissue.
As PARP-1 is a known coregulator of the pro-inflammatory transcription factor nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB), the acute DSS-induced inflammation was assessed in WT and PARP-1-/- mice by mini endoscopy. WT animals exhibited significantly stronger colitis in response to AOM/DSS, which is in line with a higher abundance of the NF-kB-inducible cyclooxygenase-2 (COX-2). IHC and confocal microscopy showed strongly decreased numbers of monocytes and macrophages in colon tissue of PARP-1-/- animals. Accordingly, expression of the cytokine high mobility group box 1 (HMBG1), which is tightly linked to inflammation, was reduced in PARP-1 k.o. mice. The number of cluster of differentiation 3 (CD3)-positive T-cells, associated with the adaptive immune response, was similar in both genotypes.
Our group recently demonstrated the high sensitivity of MGMT k.o. animals to NOC-induced CRC (Fahrer et al. 2015). Therefore, the role of inflammation driven tumour promotion was dissected in PARP-1 k.o. animals treated with the MGMT inhibitor O6-benzylguanine (O6-BG) prior to the AOM/DSS protocol. Inhibition of MGMT caused a clear increase in both tumour score and number, likely attributable to a higher induction of mutagenic O6-MeG DNA adducts in absence of MGMT. This was further studied with the help of a PARP-1-/-/MGMT-/- double knock out (DKO) strain. Interestingly, DKO mice displayed a higher tumour number but lower mean tumour size compared to MGMT-deficient animals.