Determination of an interaction between the DNA repair proteins MLH1 and sMBD4 and aspirin regulation of DNA repair gene and protein expression in colorectal cancer

Abstract

The base excision repair protein, MBD4 (also known as MED1) is known to be transcriptionally coupled to a mismatch repair protein MLH1. To date the significance of this coupling has not been elucidated and the significance of MBD4 within the mismatch repair system and apoptotic pathway is still being understood. Recently a novel alternatively spliced form of MBD4 has been identified and termed sMBD4. To date the significance of sMBD4 is unknown. MBD4 and sMBD4 share a common glycosylase domain and this is the domain through which MBD4 is reported to interact with MLH1. It was the aim of this study to determine if sMBD4 was also a binding partner of MLH1 to help elucidate a potential role of sMBD4 and to further characterise the binding domain between MLH1 and MBD4. Recombinant proteins were utilised in binding assays however, a specific protein – protein interaction could not be determined. Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of DNA mismatch repair (MMR) proteins in DNA MMR proficient cells. As MBD4 has previously been suggested to be coupled to MLH1 expression by a post‐translational mechanism the cytotoxicy of aspirin in relation to MBD4 expression was examined. This study reports that aspirin does not up‐regulate MBD4 gene transcription in vitro in the DNA mismatch repair proficient/p53 mutant colorectal cancer cell line SW480. However, MBD4 gene transcription was up‐regulated upon treatment with the aspirin precursor, salicylic acid. The suggested involvement of the DNA repair proteins in the mechanism of action of aspirin promoted the investigation into the expression of DNA damage signalling pathways genes upon aspirin exposure. This study utilised a commercially available PCR array to analyse the expression of 84 DNA damage signalling genes in the SW480 colorectal cancer cell line upon aspirin treatment. It is reported that treatment of the SW480 cell line with aspirin caused changes in mRNA expression of several key genes involved in DNA damage signalling including a significant down‐regulation in expression of the genes encoding ATR, BRCA1 and MAPK12 and increases in the expression of XRCC3 and GADD45α genes. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo‐protective effect of aspirin in vivo.Further to this, protein expression was analysed to determine if correlation could be established with the changes in mRNA expression observed. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45α, an increase in XRCC3 protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This study indicates that alterations in gene expression seen in microarray studies need to be verified at the protein level. Furthermore, this study reports the novel discovery of XRCC3 gene and protein expression being susceptible to exposure to the non‐steroidal anti‐inflammatory drug, aspirin.