Metabolic Stress and Mitochondrial Dysfunction in Ataxia-Telangiectasia.

The ataxia-telangiectasia mutated (ATM) protein kinase is, as the name implies, mutated in the human genetic disorder ataxia-telangiectasia (A-T). This protein has its "finger in many pies", being responsible for the phosphorylation of many thousands of proteins in different signaling pathways in its role in protecting the cell against a variety of different forms of stress that threaten to perturb cellular homeostasis. The classical role of ATM is the protection against DNA damage, but it is evident that it also plays a key role in maintaining cell homeostasis in the face of oxidative and other forms of non-DNA damaging stress. The presence of ATM is not only in the nucleus to cope with damage to DNA, but also in association with other organelles in the cytoplasm, which suggests a greater protective role. This review attempts to address this greater role of ATM in protecting the cell against both external and endogenous damage.

Isolation and in vitro Culture of Mouse Oocytes.

Females are endowed at birth with a fixed reserve of oocytes, which declines both in quantity and quality with advancing age. Understanding the molecular mechanisms regulating oocyte quality is crucial for improving the chances of pregnancy success in fertility clinics. In vitro culture systems enable researchers to analyse important molecular and genetic regulators of oocyte maturation and fertilisation. Here, we describe in detail a highly reproducible technique for the isolation and culture of fully grown mouse oocytes. We include the considerations and precautionary measures required for minimising the detrimental effects of in vitro culture conditions. This technique forms the starting point for a wide range of experimental approaches such as post-transcriptional gene silencing, immunocytochemistry, Western blotting, high-resolution 4D time-lapse imaging, and in vitro fertilization, which are instrumental in dissecting the molecular determinants of oocyte quality. Hence, this protocol serves as a useful, practical guide for any oocyte researcher beginning experiments aimed at investigating important oocyte molecular factors. Graphic abstract: A step-by-step protocol for the isolation and in vitro culture of oocytes from mice.

Oocytes mount a noncanonical DNA damage response involving APC-Cdh1-mediated proteolysis.

In mitotic cells, DNA damage induces temporary G2 arrest via inhibitory Cdk1 phosphorylation. In contrast, fully grown G2-stage oocytes readily enter M phase immediately following chemical induction of DNA damage in vitro, indicating that the canonical immediate-response G2/M DNA damage response (DDR) may be deficient. Senataxin (Setx) is involved in RNA/DNA processing and maintaining genome integrity. Here we find that mouse oocytes deleted of Setx accumulate DNA damage when exposed to oxidative stress in vitro and during aging in vivo, after which, surprisingly, they undergo G2 arrest. Moreover, fully grown wild-type oocytes undergo G2 arrest after chemotherapy-induced in vitro damage if an overnight delay is imposed following damage induction. Unexpectedly, this slow-evolving DDR is not mediated by inhibitory Cdk1 phosphorylation but by APC-Cdh1-mediated proteolysis of the Cdk1 activator, cyclin B1, secondary to increased Cdc14B-dependent APC-Cdh1 activation and reduced Emi1-dependent inhibition. Thus, oocytes are unable to respond immediately to DNA damage, but instead mount a G2/M DDR that evolves slowly and involves a phosphorylation-independent proteolytic pathway.

The Presence of Immature GV- Stage Oocytes during IVF/ICSI Is a Marker of Poor Oocyte Quality: A Pilot Study.

Here we investigate whether the presence of germinal vesicle-stage oocytes (GV- oocytes) reflects poor oocyte developmental competence (or quality). This was a prospective, non-randomised, cohort pilot-study involving 60 patients undergoing in vitro fertilization/ intracytoplasmic sperm injection for whom complete pregnancy outcome data were available. Patients in whom GV- oocytes were retrieved (GV+) at transvaginal oocyte retrieval (TVOR) were compared with those from whom no GVs were retrieved (GV-). We found that GV+ (n = 29) and GV- (n = 31) patients were similarly aged (35.4 vs. 36.4 years; p = 0.446). GV+ patients had a mean of 2.41 ± 2.03 GVs and comparable yields of MII oocytes to GV- patients (11 ± 6.88 vs. 8.26 ± 4.84; p = 0.077). Compared with GV- patients, GV+ patients had markedly lower implantation rates (11.8% vs. 30.2%; p = 0.022) as well as oocyte utilisation rates for clinical pregnancy (2.3% vs. 6.8%; p = 0.018) and live-birth (1.9% vs. 5.7%; p = 0.029). DNA damage levels measured using γH2AX immunostaining were not different in oocytes from women <36 years versus those ≥36 years (p = 0.606). Thus, patients who have GV- stage oocytes at TVOR exhibit poor oocyte quality reflected in reduced per-oocyte pregnancy success rates and uniformly high levels of oocyte DNA damage.

Cep55 overexpression promotes genomic instability and tumorigenesis in mice.

High expression of centrosomal protein CEP55 has been correlated with clinico-pathological parameters across multiple human cancers. Despite significant in vitro studies and association of aberrantly overexpressed CEP55 with worse prognosis, its causal role in vivo tumorigenesis remains elusive. Here, using a ubiquitously overexpressing transgenic mouse model, we show that Cep55 overexpression causes spontaneous tumorigenesis and accelerates Trp53+/- induced tumours in vivo. At the cellular level, using mouse embryonic fibroblasts (MEFs), we demonstrate that Cep55 overexpression induces proliferation advantage by modulating multiple cellular signalling networks including the hyperactivation of the Pi3k/Akt pathway. Notably, Cep55 overexpressing MEFs have a compromised Chk1-dependent S-phase checkpoint, causing increased replication speed and DNA damage, resulting in a prolonged aberrant mitotic division. Importantly, this phenotype was rescued by pharmacological inhibition of Pi3k/Akt or expression of mutant Chk1 (S280A) protein, which is insensitive to regulation by active Akt, in Cep55 overexpressing MEFs. Moreover, we report that Cep55 overexpression causes stabilized microtubules. Collectively, our data demonstrates causative effects of deregulated Cep55 on genome stability and tumorigenesis which have potential implications for tumour initiation and therapy development.

Enhanced dependency of KRAS-mutant colorectal cancer cells on RAD51-dependent homologous recombination repair identified from genetic interactions in Saccharomyces cerevisiae.

Activating KRAS mutations drive colorectal cancer tumorigenesis and influence response to anti-EGFR-targeted therapy. Despite recent advances in understanding Ras signaling biology and the revolution in therapies for melanoma using BRAF inhibitors, no targeted agents have been effective in KRAS-mutant cancers, mainly due to activation of compensatory pathways. Here, by leveraging the largest synthetic lethal genetic interactome in yeast, we identify that KRAS-mutated colorectal cancer cells have augmented homologous recombination repair (HRR) signaling. We found that KRAS mutation resulted in slowing and stalling of the replication fork and accumulation of DNA damage. Moreover, we found that KRAS-mutant HCT116 cells have an increase in MYC-mediated RAD51 expression with a corresponding increase in RAD51 recruitment to irradiation-induced DNA double-strand breaks (DSBs) compared to genetically complemented isogenic cells. MYC depletion using RNA interference significantly reduced IR-induced RAD51 foci formation and HRR. On the contrary, overexpression of either HA-tagged wild-type (WT) MYC or phospho-mutant S62A increased RAD51 protein levels and hence IR-induced RAD51 foci. Likewise, depletion of RAD51 selectively induced apoptosis in HCT116-mutant cells by increasing DSBs. Pharmacological inhibition targeting HRR signaling combined with PARP inhibition selectivity killed KRAS-mutant cells. Interestingly, these differences were not seen in a second isogenic pair of KRAS WT and mutant cells (DLD-1), likely due to their nondependency on the KRAS mutation for survival. Our data thus highlight a possible mechanism by which KRAS-mutant-dependent cells drive HRR in vitro by upregulating MYC-RAD51 expression. These data may offer a promising therapeutic vulnerability in colorectal cancer cells harboring otherwise nondruggable KRAS mutations, which warrants further investigation in vivo.