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The function of CUX1 in oxidative DNA damage repair is needed to prevent premature senescence of mouse embryo fibroblasts.

ABSTRACT: 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.

SUBMITTER: Ramdzan ZM

PROVIDER: S-EPMC4414141 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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The function of CUX1 in oxidative DNA damage repair is needed to prevent premature senescence of mouse embryo fibroblasts.

Ramdzan Zubaidah M ZM Pal Ranjana R Kaur Simran S Leduy Lam L Bérubé Ginette G Davoudi Sayeh S Vadnais Charles C Nepveu Alain A

Oncotarget 20150201 6

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 thr ...[more]

PMID: 25682875

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Project description:The Cut homeobox 1 (CUX1) gene is a target of loss-of-heterozygosity in many cancers, yet elevated CUX1 expression is frequently observed and is associated with shorter disease-free survival. The dual role of CUX1 in cancer is illustrated by the fact that most cell lines with CUX1 LOH display amplification of the remaining allele, suggesting that decreased CUX1 expression facilitates tumor development while increased CUX1 expression is needed in tumorigenic cells. Indeed, CUX1 was found in a genome-wide RNAi screen to identify synthetic lethal interactions with oncogenic RAS. Here we show that CUX1 functions in base excision repair as an ancillary factor for the 8-oxoG-DNA glycosylase, OGG1. Single cell gel electrophoresis (comet assay) reveals that Cux1?/? MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. In vitro base excision repair assays with purified components demonstrate that CUX1 directly stimulates OGG1's enzymatic activity. Elevated reactive oxygen species (ROS) levels in cells with sustained RAS pathway activation can cause cellular senescence. We show that elevated expression of either CUX1 or OGG1 prevents RAS-induced senescence in primary cells, and that CUX1 knockdown is synthetic lethal with oncogenic RAS in human cancer cells. Elevated CUX1 expression in a transgenic mouse model enables the emergence of mammary tumors with spontaneous activating Kras mutations. We confirmed cooperation between Kras(G12V) and CUX1 in a lung tumor model. Cancer cells can overcome the antiproliferative effects of excessive DNA damage by inactivating a DNA damage response pathway such as ATM or p53 signaling. Our findings reveal an alternate mechanism to allow sustained proliferation in RAS-transformed cells through increased DNA base excision repair capability. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway.

Project description:BackgroundDental pulp stem cells (DPSCs) are increasingly being advocated as viable cell sources for regenerative medicine-based therapies. However, significant heterogeneity in DPSC expansion and multi-potency capabilities are well-established, attributed to contrasting telomere profiles and susceptibilities to replicative senescence. As DPSCs possess negligible human telomerase (hTERT) expression, we examined whether intrinsic differences in the susceptibilities of DPSC sub-populations to oxidative stress-induced biomolecular damage and premature senescence further contributed to this heterogeneity, via differential enzymic antioxidant capabilities between DPSCs.MethodsDPSCs were isolated from human third molars by differential fibronectin adhesion, and positive mesenchymal (CD73/CD90/CD105) and negative hematopoietic (CD45) stem cell marker expression confirmed. Isolated sub-populations were expanded in H2O2 (0-200 μM) and established as high or low proliferative DPSCs, based on population doublings (PDs) and senescence (telomere lengths, SA-β-galactosidase, p53/p16INK4a/p21waf1/hTERT) marker detection. The impact of DPSC expansion on mesenchymal, embryonic, and neural crest marker expression was assessed, as were the susceptibilities of high and low proliferative DPSCs to oxidative DNA and protein damage by immunocytochemistry. Expression profiles for superoxide dismutases (SODs), catalase, and glutathione-related antioxidants were further compared between DPSC sub-populations by qRT-PCR, Western blotting and activity assays.ResultsHigh proliferative DPSCs underwent > 80PDs in culture and resisted H2O2-induced senescence (50-76PDs). In contrast, low proliferative sub-populations exhibited accelerated senescence (4-32PDs), even in untreated controls (11-34PDs). While telomere lengths were largely unaffected, certain stem cell marker expression declined with H2O2 treatment and expansion. Elevated senescence susceptibilities in low proliferative DPSC (2-10PDs) were accompanied by increased oxidative damage, absent in high proliferative DPSCs until 45-60PDs. Increased SOD2/glutathione S-transferase ζ1 (GSTZ1) expression and SOD activities were identified in high proliferative DPSCs (10-25PDs), which declined during expansion. Low proliferative DPSCs (2-10PDs) exhibited inferior SOD, catalase and glutathione-related antioxidant expression/activities.ConclusionsSignificant variations exist in the susceptibilities of DPSC sub-populations to oxidative damage and premature senescence, contributed to by differential SOD2 and GSTZ1 profiles which maintain senescence-resistance/stemness properties in high proliferative DPSCs. Identification of superior antioxidant properties in high proliferative DPSCs enhances our understanding of DPSC biology and senescence, which may be exploited for selective sub-population screening/isolation from dental pulp tissues for regenerative medicine-based applications.

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The function of CUX1 in oxidative DNA damage repair is needed to prevent premature senescence of mouse embryo fibroblasts.

Publications

The function of CUX1 in oxidative DNA damage repair is needed to prevent premature senescence of mouse embryo fibroblasts.

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