DNA fiber assay for the analysis of DNA replication progression in human pluripotent stem cells

Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabilities during prolonged in vitro culture that threaten to preclude their use in cell‐based regenerative medicine. The rapid proliferation of pluripotent cells leads to constitutive replication stress, hindering the progression of DNA replication forks and in some cases leading to replication‐fork collapse. Failure to overcome replication stress can result in incomplete genome duplication, which, if left to persist into the subsequent mitosis, can result in structural and numerical chromosomal instability. We have recently applied the DNA fiber assay to the study of replication stress in human PSC and found that, in comparison to somatic cells states, these cells display features of DNA replication stress that include slower replication fork speeds, evidence of stalled forks, and replication initiation from dormant replication origins. These findings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA fiber assay has enabled the development of an advanced nucleoside‐enriched culture medium that increases replication fork progression and decreases DNA damage and mitotic errors in human PSC cultures. The DNA fiber assay allows for the study of replication fork dynamics at single‐molecule resolution. The assay relies on cells incorporating nucleotide analogs into nascent DNA during replication, which are then measured to monitor several replication parameters. Here we provide an optimized protocol for the fiber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters

Poly(ADP-Ribose) glycohydrolase (PARG) vs. poly(ADP-Ribose) polymerase (PARP) – function in genome maintenance and relevance of inhibitors for anti-cancer therapy

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that catalyze the addition of poly(ADP-ribose) (PAR) subunits onto themselves and other acceptor proteins. PARPs are known to function in a large range of cellular processes including DNA repair, DNA replication, transcription and modulation of chromatin structure. Inhibition of PARP holds great potential for therapy, especially in cancer. Several PARP1/2/3 inhibitors (PARPi) have had success in treating ovarian, breast and prostate tumors harboring defects in the homologous recombination (HR) DNA repair pathway, especially BRCA1/2 mutated tumors. However, treatment is limited to specific sub-groups of patients and resistance can occur, limiting the use of PARPi. Poly(ADP-ribose) glycohydrolase (PARG) reverses the action of PARP enzymes, hydrolysing the ribose-ribose bonds present in poly(ADP-ribose). Like PARPs, PARG is involved in DNA replication and repair and PARG depleted/inhibited cells show increased sensitivity to DNA damaging agents. They also display an accumulation of perturbed replication intermediates which can lead to synthetic lethality in certain contexts. In addition, PARG is thought to play an important role in preventing the accumulation of cytoplasmic PAR and therefore parthanatos, a caspase-independent PAR-mediated type of cell death. In contrast to PARP, the therapeutic potential of PARG has been largely ignored. However, several recent papers have demonstrated the exciting possibilities that inhibitors of this enzyme may have for cancer treatment, both as single agents and in combination with cytotoxic drugs and radiotherapy. This article discusses what is known about the functions of PARP and PARG and the potential future implications of pharmacological inhibition in anti-cancer therapy

Chemical genetic analyses of quantitative changes in Cdk1 activity during the human cell cycle

Cyclin-dependent kinase 1 (Cdk1) controls cell proliferation and is inhibited by promising anticancer agents, but its mode of action and the consequences of its inhibition are incompletely understood. Cdk1 promotes S- and M-phases during the cell-cycle but also suppresses endoreduplication, which is associated with polyploidy and genome instability. The complexity of Cdk1 regulation has made it difficult to determine whether these different roles require different thresholds of kinase activity and whether the surge of activity as inhibitory phosphates are removed at mitotic onset is essential for cell proliferation. Here, we have used chemical genetics in a human cell line to address these issues. We rescued cells lethally depleted of endogenous Cdk1 with an exogenous Cdk1 conferring sensitivity to one ATP analogue inhibitor (1NMPP1) and resistance to another (RO3306). At no 1NMPP1 concentration was mitosis in rescued clones prevented without also inducing endoreduplication, suggesting that these two key roles for Cdk1 are not simply controlled by different Cdk1 activity thresholds. We also rescued RO3306-resistant clones using exogenous Cdk1 without inhibitory phosphorylation sites, indicating that the mitotic surge of Cdk1 activity is dispensable for cell proliferation. These results suggest that the basic mammalian cycle requires at least some qualitative changes in Cdk1 activity and that quantitative increases in activity need not be rapid. Furthermore, the viability of cells that are unable to undergo rapid Cdk1 activation, and the strong association between endoreduplication and impaired proliferation, may place restrictions on the therapeutic use of a Cdk1 inhibitors

Resistance of uveal melanoma to the interstrand cross-linking agent mitomycin C is associated with reduced expression of CYP450R

background: Uveal melanoma (UM) is the most common primary intraocular tumour of adults, frequently metastasising to the liver. Hepatic metastases are difficult to treat and are mainly unresponsive to chemotherapy. To investigate why UM are so chemo-resistant we explored the effect of interstrand cross-linking agents mitomycin C (MMC) and cisplatin in comparison with hydroxyurea (HU). methods: Sensitivity to MMC, cisplatin and HU was tested in established UM cell lines using clonogenic assays. The response of UM to MMC was confirmed in MTT assays using short-term cultures of primary UM. The expression of cytochrome P450 reductase (CYP450R) was analysed by western blotting, and DNA cross-linking was assessed using COMET analysis supported by γ-H2AX foci formation. results: Both established cell lines and primary cultures of UM were resistant to the cross-linking agent MMC (in each case P<0.001 in Student's t-test compared with controls). In two established UM cell lines, DNA cross-link damage was not induced by MMC (in both cases P<0.05 in Students's t-test compared with damage induced in controls). In all, 6 out of 6 UMs tested displayed reduced expression of the metabolising enzyme CYP450R and transient expression of CYP450R increased MMC sensitivity of UM. conclusion: We suggest that reduced expression of CYP450R is responsible for MMC resistance of UM, through a lack of bioactivation, which can be reversed by complementing UM cell lines with CYP450R

MYCN expression induces replication stress and sensitivity to PARP inhibition in neuroblastoma

This study investigates the influence expression of the MYCN oncogene has on the DNA damage response, replication fork progression and sensitivity to PARP inhibition in neuroblastoma. In a panel of neuroblastoma cell lines, MYCN amplification or MYCN expression resulted in increased cell death in response to a range of PARP inhibitors (niraparib, veliparib, talazoparib and olaparib) compared to the response seen in non-expressing/amplified cells. MYCN expression slowed replication fork speed and increased replication fork stalling, an effect that was amplified by PARP inhibition or PARP1 depletion. Increased DNA damage seen was specifically induced in S-phase cells. Importantly, PARP inhibition caused a significant increase in the survival of mice bearing MYCN expressing tumours in a transgenic murine model of MYCN expressing neuroblastoma. Olaparib also sensitized MYCN expressing cells to camptothecin- and temozolomide-induced cell death to a greater degree than non-expressing cells. In summary, MYCN expression leads to increased replication stress in neuroblastoma cells. This effect is exaggerated by inhibition of PARP, resulting in S-phase specific DNA damage and ultimately increased tumour cell death. PARP inhibition alone or in combination with classical chemotherapeutics is therefore a potential therapeutic strategy for neuroblastoma and may be more effective in MYCN expressing tumours

Increased replication stress determines ATR inhibitor sensitivity in neuroblastoma cells

Despite intensive high-dose multimodal therapy, high-risk neuroblastoma (NB) confers a less than 50% survival rate. This study investigates the role of replication stress in sensitivity to inhibition of Ataxia telangiectasia and Rad3-related (ATR) in pre-clinical models of high-risk NB. Amplification of the oncogene MYCN always imparts high-risk disease and occurs in 25% of all NB. Here, we show that MYCN-induced replication stress directly increases sensitivity to the ATR inhibitors VE-821 and AZD6738. PARP inhibition with Olaparib also results in replication stress and ATR activation, and sensitises NB cells to ATR inhibition independently of MYCN status, with synergistic levels of cell death seen in MYCN expressing ATR- and PARP-inhibited cells. Mechanistically, we demonstrate that ATR inhibition increases the number of persistent stalled and collapsed replication forks, exacerbating replication stress. It also abrogates S and G2 cell cycle checkpoints leading to death during mitosis in cells treated with an ATR inhibitor combined with PARP inhibition. In summary, increased replication stress through high MYCN expression, PARP inhibition or chemotherapeutic agents results in sensitivity to ATR inhibition. Our findings provide a mechanistic rationale for the inclusion of ATR and PARP inhibitors as a potential treatment strategy for high-risk NB