The base excision repair (BER) that repairs oxidative damage is upregulated as an adaptive response in maintaining tumorigenesis of RAS-transformed cancer cells.

Despite having long telomeres, mouse embryo fibroblasts (MEFs) senesce more rapidly than human diploid fibroblasts because of the accumulation of oxidative DNA damage. The CUX1 homeodomain protein was recently found to prevent senescence in RAS-driven cancer cells that produce elevated levels of reactive-oxygen species. Here we show that Cux1−/− MEFs are unable to proliferate in atmospheric (20%) oxygen although they can proliferate normally in physiological (3%) oxygen levels. CUX1 contains three domains called Cut repeats. Structure/function analysis established that a single Cut repeat domain can stimulate the DNA binding, Schiff-base formation, glycosylase and AP-lyase activities of 8-oxoguanine DNA glycosylase 1, OGG1. Strikingly and in contrast to previous reports, OGG1 exhibits efficient AP-lyase activity in the presence of a Cut repeat. Repair of oxidative DNA damage and proliferation in 20% oxygen were both rescued in Cux1−/− MEFs by ectopic expression of CUX1 or of a recombinant Cut repeat protein that stimulates OGG1 but is devoid of transcription activation potential. These findings reinforce the causal link between oxidative DNA damage and cellular senescence and suggest that the role of CUX1 as an accessory factor in DNA repair will be critical in physiological situations that generate higher levels of reactive oxygen species.

IntroductionNew levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored.MethodsChanges in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2.ResultsIt was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2.Conclusionsmir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests that miR-24-2 is more effective in controlling H2AX gene expression, regardless of the change in gene copy number. Further, the study indicates that combination therapy with miR-24-2 along with an anticancer drug such as cisplatin could provide a new avenue in cancer therapy for patients with tumors otherwise resistant to drugs.

Background: CUX2 contains three Cut repeat domains and is expressed in postmitotic neurons. Results: Cut repeats stimulate the OGG1 DNA glycosylase, and lower or higher CUX2 expression, respectively, delays or accelerates repair of oxidative DNA damage. Conclusion: CUX2 functions as an accessory factor in base excision repair. Significance: CUX2 contributes to the maintenance of genome integrity in long lived neurons.