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High grade serous ovarian cancer (HGSOC) is a major cause of female cancer mortality. The approval of poly (ADP-ribose) polymerase (PARP) inhibitors for clinical use has greatly improved treatment options for patients with homologous recombination repair (HRR)-deficient HGSOC, although the development of PARP inhibitor resistance in some patients is revealing limitations to outcome. A proportion of patients with HRR-proficient cancers also benefit from PARP inhibitor therapy. Our aim is to compare mechanisms of resistance to the PARP inhibitor olaparib in these two main molecular categories of HGSOC and investigate a way to overcome resistance that we considered particularly suited to a cancer like HGSOC, where there is a very high incidence of TP53 gene mutation, making HGSOC cells heavily reliant on the G2 checkpoint for repair of DNA damage and survival. We identified alterations in multiple factors involved in resistance to PARP inhibition in both HRR-proficient and -deficient cancers. The most frequent change was a major reduction in levels of poly (ADP-ribose) glycohydrolase (PARG), which would be expected to preserve a residual PARP1-initiated DNA damage response to DNA single-strand breaks. Other changes seen would be expected to boost levels of HRR of DNA double-strand breaks. Growth of all olaparib-resistant clones isolated could be controlled by WEE1 kinase inhibitor AZD1775, which inactivates the G2 checkpoint. Our work suggests that use of the WEE1 kinase inhibitor could be a realistic therapeutic option for patients that develop resistance to olaparib.

// Lanlan Yang 1 , Ann-Marie Ritchie 1 and David W. Melton 1 1 Edinburgh Cancer Research Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, UK Correspondence to: David W. Melton, email: David.Melton@ed.ac.uk Keywords: nucleotide excision repair, ERCC1-XPF, ERCC1 homodimerization, ubiquitination, destabilisation Received: April 04, 2017 Accepted: July 11, 2017 Published: July 21, 2017 ABSTRACT DNA repair pathways present in all cells serve to preserve genome stability, but in cancer cells they also act reduce the efficacy of chemotherapy. The endonuclease ERCC1-XPF has an important role in the repair of DNA damage caused by a variety of chemotherapeutic agents and there has been intense interest in the use of ERCC1 as a predictive marker of therapeutic response in non-small cell lung carcinoma, squamous cell carcinoma and ovarian cancer. We have previously validated ERCC1 as a therapeutic target in melanoma, but all small molecule ERCC1-XPF inhibitors reported to date have lacked sufficient potency and specificity for clinical use. In an alternative approach to prevent the repair activity of ERCC1-XPF, we investigated the mechanism of ERCC1 ubiquitination and found that the key region was the C-terminal (HhH) 2 domain which heterodimerizes with XPF. This ERCC1 region was modified by non-conventional lysine-independent, but proteasome-dependent polyubiquitination, involving Lys33 of ubiquitin and a linear ubiquitin chain. XPF was not polyubiquitinated and its expression was dependent on presence of ERCC1, but not vice versa. To our surprise we found that ERCC1 can also homodimerize through its C-terminal (HhH) 2 domain. We exploited the ability of a peptide containing this C-terminal domain to destabilise both endogenous ERCC1 and XPF in human melanoma cells and fibroblasts, resulting in reductions of up to 85% in nucleotide excision repair and near two-fold increased sensitivity to DNA damaging agents. We suggest that the ERCC1 (HhH) 2 domain could be used in an alternative strategy to treat cancer.

BackgroundMacrophages show versatile functions in innate immunity, infectious diseases, and progression of cancers and cardiovascular diseases. These versatile functions of macrophages are conducted by different macrophage phenotypes classified as classically activated macrophages and alternatively activated macrophages due to different stimuli in the complex in vivo cytokine environment. Dissecting the regulation of macrophage activations will have a significant impact on disease progression and therapeutic strategy. Mathematical modeling of macrophage activation can improve the understanding of this biological process through quantitative analysis and provide guidance to facilitate future experimental design. However, few results have been reported for a complete model of macrophage activation patterns.ResultsWe globally searched and reviewed literature for macrophage activation from PubMed databases and screened the published experimental results. Temporal in vitro macrophage cytokine expression profiles from published results were selected to establish Boolean network models for macrophage activation patterns in response to three different stimuli. A combination of modeling methods including clustering, binarization, linear programming (LP), Boolean function determination, and semi-tensor product was applied to establish Boolean networks to quantify three macrophage activation patterns. The structure of the networks was confirmed based on protein-protein-interaction databases, pathway databases, and published experimental results. Computational predictions of the network evolution were compared against real experimental results to validate the effectiveness of the Boolean network models.ConclusionThree macrophage activation core evolution maps were established based on the Boolean networks using Matlab. Cytokine signatures of macrophage activation patterns were identified, providing a possible determination of macrophage activations using extracellular cytokine measurements.

A high throughput screen allowed the identification of N-hydroxyimide inhibitors of ERCC1-XPF endonuclease activity with micromolar potency, but they showed undesirable selectivity profiles against FEN-1. A scaffold hop to a hydroxypyrimidinone template gave compounds with similar potency but allowed selectivity to be switched in favour of ERCC1-XPF over FEN-1. Further exploration of the structure-activity relationships around this chemotype gave sub-micromolar inhibitors with >10-fold selectivity for ERCC1-XPF over FEN-1.

ERCC1-XPF is a structure-specific endonuclease that is required for the repair of DNA lesions, generated by the widely used platinum-containing cancer chemotherapeutics such as cisplatin, through the Nucleotide Excision Repair and Interstrand Crosslink Repair pathways. Based on mouse xenograft experiments, where ERCC1-deficient melanomas were cured by cisplatin therapy, we proposed that inhibition of ERCC1-XPF could enhance the effectiveness of platinum-based chemotherapy. Here we report the identification and properties of inhibitors against two key targets on ERCC1-XPF. By targeting the ERCC1-XPF interaction domain we proposed that inhibition would disrupt the ERCC1-XPF heterodimer resulting in destabilisation of both proteins. Using in silica screening, we identified an inhibitor that bound to ERCC1-XPF in a biophysical assay, reduced the level of ERCC1-XPF complexes in ovarian cancer cells, inhibited Nucleotide Excision Repair and sensitised melanoma cells to cisplatin. We also utilised high throughput and in silica screening to identify the first reported inhibitors of the other key target, the XPF endonuclease domain. We demonstrate that two of these compounds display specificity in vitro for ERCC1-XPF over two other endonucleases, bind to ERCC1-XPF, inhibit Nucleotide Excision Repair in two independent assays and specifically sensitise Nucleotide Excision Repair-proficient, but not Nucleotide Excision Repair-deficient human and mouse cells to cisplatin. (C) 2015 Elsevier BA/. All rights reserved.

Levels of the actin bundling protein fascin correlate with invasion and metastasis and reveal prognostic value in many epithelial carcinomas. However, we know very little about the potential role of fascin in melanoma. The purpose of this study is to compare fascin expression in primary melanomas and melanoma metastasis. Fascin expression was examined through the immunohistochemistry of paraffin embedded tissue microarrays including 560 cores of primary tumour and metastasis. Fascin expression was significantly elevated in 48 metastases compared with 254 primary tumours (P=0.034). In 187 patients with primary melanomas, fascin was not correlated with survival (P=0.067), whereas low fascin was significantly correlated with the presence of ulceration (P=0.005). Our results indicate that fascin status does not correlate with progression in melanoma. Upregulated fascin expression was detected in melanoma metastases, but was not correlated to patient outcome.

Nitrofurans are commonly used for the treatment of trypanosomal diseases including Chagas disease. More recently, following the fortuitous discovery that nifurtimox was clinically active against neuroblastoma, nitrofuran compounds are being investigated for activity against cancer. Herein, we show that nitrofuran compounds are similarly potent to human malignant melanoma and neuroblastoma cells. Furthermore, a recently discovered nitrofuran compound, NFN1, was 50- to 175-fold more potent than nifurtimox against human melanoma and neuroblastoma cell lines. As nitrofuran compounds are known to act as pro-drugs, producing DNA-damaging reactive intermediates upon activation, we investigated the DNA repair pathways involved. We show that, contrary to research in Escherichia coli, the Nucleotide Excision Repair pathway is not required to repair nitrofuran-induced DNA damage in mammalian cells. Instead, we show that inhibiting repair of single-strand DNA breaks with the poly(ADP-ribose) polymerase (PARP) inhibitor, Olaparib, enhances nitrofuran toxicity in melanoma and neuroblastoma cells. We propose that this is due to mammalian cells utilising Type 2 nitroreductases for nitrofuran activation producing Reactive Oxygen Species which cause DNA damage that is repaired by the Single Strand Break Repair and/or Base Excision Repair pathways, whereas in bacteria and trypanosomes, Type 1 nitroreductases are also utilised resulting in different DNA lesions. In addition we show that, consistent with Reactive Oxygen Species being formed upon nitrofuran activation and the ability of melanin to absorb Reactive Oxygen Species, production of melanin in melanoma cells offers some protection from NFN1- and hydrogen peroxide-induced toxicity. Our data suggest that combinations of Olaparib and nitrofuran compounds may be advantageous for the treatment of melanoma and neuroblastoma, but that the protection offered to melanoma cells by their melanin pigment must be taken into account.

The DNA in the cells in our skin is damaged by UV irradiation. DNA repair pathways protect us against the carcinogenic consequences of unrepaired DNA damage, but the same DNA repair pathways can also protect life-threatening melanoma skin cancers from DNA-damaging chemotherapeutics.

Skeletal muscle regeneration requires coordination between dynamic cellular populations and tissue microenvironments. Macrophages, recruited via CCR2, are essential for regeneration; however, the contribution of macrophages and the role of CCR2 on nonhematopoietic cells has not been defined. In addition, aging and sex interactions in regeneration and sarcopenia are unclear. Muscle regeneration was measured in young (3–6 mo), middle (11–15 mo), old (24–32 mo) male and female CCR2−/− mice. Whereas age-related muscle atrophy/sarcopenia was present, regenerated myofiber cross-sectional area (CSA) in CCR2−/− mice was comparably impaired across all ages and sexes, with increased adipocyte area compared with wild-type (WT) mice. CCR2−/− mice myofibers achieved approximately one third of baseline CSA even 84 d after injury. Regenerated CSA and clearance of necrotic tissue were dependent on bone marrow–derived cellular expression of CCR2. Myogenic progenitor cells isolated from WT and CCR2−/− mice exhibited comparable proliferation and differentiation capacity. The most striking cellular anomaly in injured muscle of CCR2−/− mice was markedly decreased macrophages, with a predominance of Ly6C− anti-inflammatory monocytes/macrophages. Ablation of proinflammatory TLR signaling did not affect muscle regeneration or resolution of necrosis. Of interest, many proinflammatory, proangiogenic, and chemotactic cytokines were markedly elevated in injured muscle of CCR2−/− relative to WT mice despite impairments in macrophage recruitment. Collectively, these results suggest that CCR2 on bone marrow–derived cells, likely macrophages, were essential to muscle regeneration independent of TLR signaling, aging, and sex. Decreased proinflammatory monocytes/macrophages actually promoted a proinflammatory microenvironment, which suggests that inflammaging was present in young CCR2−/− mice.

MicroRNAs (miRNAs) regulate many biological processes including muscle development. However, little is known regarding miRNA regulation of muscle regeneration. Murine tibialis anterior muscle was evaluated after cardiotoxin-induced injury and used for global miRNA expression analysis. From day 1 through day 21 following injury, 298 miRNAs were significantly changed at least at one time point, including 86 miRNAs that were altered >10-fold compared with uninjured skeletal muscle. Temporal miRNA expression patterns included inflammation-related miRNAs (miR-223 and -147) that increased immediately after injury; this pattern contrasted to that of mature muscle-specific miRNAs (miR-1, -133a, and -499) that abruptly decreased following injury followed by upregulation in later regenerative events. Another cluster of miRNAs were transiently increased in the early days of muscle regeneration including miR-351, a miRNA that was also transiently expressed during myogenic progenitor cell (MPC) differentiation in vitro. Based on computational predictions, further studies demonstrated that E2f3 was a target of miR-351 in myoblasts. Moreover, knockdown of miR-351 expression inhibited MPC proliferation and promoted apoptosis during MPC differentiation, whereas miR-351 overexpression protected MPC from apoptosis during differentiation. Collectively, these observations suggest that miR-351 is involved in both the maintenance of MPC proliferation and the transition into differentiated myotubes. Thus, a novel, time-dependent sequence of molecular events during muscle regeneration has been identified; miR-351 inhibits E2f3 expression, a key regulator of cell cycle progression and proliferation, and promotes MPC proliferation and protects early differentiating MPC from apoptosis, important events in the hostile tissue environment after acute muscle injury.

Aims: Elevated expression of DNA repair and replicationgenes has been reported in thick, non-fixed primarymelanomas that subsequently went on to metastasize,when compared to non-recurrent primary tumours.This increased expression could contribute to theextreme resistance shown by melanoma to DNA-dam-aging chemotherapeutics. We have investigated thehypothesis that levels of key DNA repair and replicationproteins are prognostic biomarkers in melanoma.Methods and results: We used a tissue microarraycontaining samples from all stages of melanomagenesisto investigate the hypothesis that levels of key DNArepair and replication proteins are prognostic biomar-kers in a larger, more representative and readilyavailable set of fixed primary melanomas. High expres-sion of topoisomerase IIa (TOP2A), that relievestorsional stress during DNA replication, and XRCC5(Ku80), required for DNA double-strand break repair,were associated with significantly worse survival.Conclusions: Two (XRCC5 and TOP2A) of seven DNArepair and replication proteins studied were prognosticfor melanoma.Keywords: chemotherapy, ERCC1, melanomagenesis, topoisomerase IIa, XRCC5

The ERCC1-XPF complex is a structure-specific endonuclease essential for the repair of DNA damage by the nucleotide excision repair pathway. It is also involved in other key cellular processes, including DNA interstrand crosslink (ICL) repair and DNA double-strand break (DSB) repair. New evidence has recently emerged, increasing our understanding of its requirement in these additional roles. In this review, we focus on the protein-protein and protein-DNA interactions made by the ERCC1 and XPF proteins and discuss how these coordinate ERCC1-XPF in its various roles. In a number of different cancers, high expression of ERCC1 has been linked to a poor response to platinum-based chemotherapy. We discuss prospects for the development of DNA repair inhibitors that target the activity, stability or protein interactions of the ERCC1-XPF complex as a novel therapeutic strategy to overcome chemoresistance.

Galectin-3 plays an important role in adhesion, proliferation, differentiation, angiogenesis and metastasis in multiple tumours. To investigate the role of galectin-3 in melanoma pathogenesis we examined the expression of galectin-3 in melanocytic lesions and analysed the correlation between galectin-3 expression and clinicopathologic factors including patient survival and BRAF mutation status.We evaluated the expression of galectin-3 in 53 cases of benign naevi, 31 cases of dysplastic naevi, 59 in-situ melanomas, 314 cases of primary melanoma and 69 metastatic melanomas using tissue microarray and immunohistochemistry.Marked differences in expression of galectin-3 were seen between different categories of melanocytic lesions (ANOVA p0.0001). An increase in expression of galectin-3 between benign naevi and thin primary melanomas and a progressive decrease in expression between thin primary melanomas and thicker melanomas or metastatic melanomas was seen. Strong galectin-3 expression was associated with improved overall survival (p=0.002 and p=0.0002 for cytoplasmic and nuclear expression, respectively) and melanoma-specific survival (p=0.017 and p=0.003 for cytoplasmic and nuclear expression, respectively). A multifactorial Cox regression analysis suggested that galectin-3 expression was an independent prognostic marker for overall survival in melanoma (risk ratio 0.73, 95% CI 0.547-0.970, p=0.031 for cytoplasmic expression and risk ratio 0.76, 95% CI 0.587-0.985, p=0.036 for nuclear expression). No association between galectin-3 expression and BRAF mutation status was observed.This study suggests that galectin-3 is a marker of progression in melanocytic lesions and a novel prognostic marker in primary melanoma.

The mitogen-activated protein kinase (MAPK) pathway is important in melanoma. In this pathway, DUSP6 phosphatase negatively controls the activation of extracellular signal-regulated (ERK) kinase. Through comparison of melanoma signalling pathways between immortal mouse melanocytes and their tumourigenic derivatives, retrieved from mouse xenografts, we identified a molecularly distinct subtype of melanoma, characterized by reduced ERK activity and increased DUSP6 expression. Overexpression of DUSP6 enhanced anchorage-independent growth and invasive ability of immortal mouse melanocytes, suggesting that increased DUSP6 expression contributes to melanoma formation in the mouse xenografts. In contrast, reduced tumourigenicity was observed after DUSP6 overexpression in human melanoma cells. A minority of thick human primary melanomas had high DUSP6 expression and the same poor melanoma-specific survival as the majority of thick primaries with low DUSP6 levels. We have demonstrated that DUSP6 is important in melanoma and that it plays a different role in our distinct subtype of mouse melanoma compared with that in classic human melanoma.

Background: The incidence of malignant melanoma is increasing faster than that for any other cancer. Histological examination of skin excision biopsies remains the standard method for melanoma diagnosis and prognosis. Significant morphological overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis. Methods: To identify improved molecular markers to support histological examination, we used microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis to identify differentially expressed microRNAs (miRNAs). Differential expression was validated by qRT–PCR, and functional studies were carried out after transfection of miRNA precursors or inhibitors into melanoma cells to modulate miRNA expression. Results: In all, 20 miRNAs showed highly significant differential expression between benign naevi and either primary or metastatic melanomas, the majority being downregulated in melanoma, whereas only 2 miRNAs, namely miR-203 and miR-205, were differentially expressed between primary and metastatic melanomas. In functional in vitro assays, overexpression of miR-200c and miR-205 inhibited anchorage-independent colony formation and overexpression of miR-211 inhibited both anchorage-independent colony formation and invasion. Conclusion: We have identified a series of differentially expressed miRNAs that could be useful as diagnostic or prognostic markers for melanoma and have shown that three miRNAs (namely miR-200c, miR-205 and miR-211) act as tumour suppressors.

Summary Increased expression of DNA repair genes contributes to the extreme resistance shown by melanoma to conventional DNA-damaging chemotherapeutics. One such chemotherapeutic effective against a range of other cancers, but not melanoma, is cisplatin. The DNA repair protein, ERCC1, is needed to remove cisplatin-induced DNA damage. We have shown that ERCC1 is essential for melanoma growth and resistance to cisplatin in a mouse xenograft model. Untreated xenografts of our transformed Ercc1-proficient melanocyte cell line grew very rapidly as malignant melanoma. Cisplatin treatment caused initial shrinkage of xenografts, but cisplatin-resistant regrowth soon followed. Cells reisolated into culture had twofold elevated levels of ERCC1 compared to both input cells and cells reisolated from untreated xenografts. An isogenic Ercc1-deficient derivative grew equally well in vitro as the Ercc1-proficient melanocyte cell line. However, in xenografts, the Ercc1-deficient melanomas were much slower to establish and were completely cured by just two cisplatin treatments.

Age-related cognitive decline and neurodegenerative diseases are a growing challenge for our societies with their aging populations. Accumulation of DNA damage has been proposed to contribute to these impairments, but direct proof that DNA damage results in impaired neuronal plasticity and memory is lacking. Here we take advantage ofErcc1Δ/−mutant mice, which are impaired in DNA nucleotide excision repair, interstrand crosslink repair, and double-strand break repair. We show that these mice exhibit an age-dependent decrease in neuronal plasticity and progressive neuronal pathology, suggestive of neurodegenerative processes. A similar phenotype is observed in mice where the mutation is restricted to excitatory forebrain neurons. Moreover, these neuron-specific mutants develop a learning impairment. Together, these results suggest a causal relationship between unrepaired, accumulating DNA damage, and age-dependent cognitive decline and neurodegeneration. Hence, accumulated DNA damage could therefore be an important factor in the onset and progression of age-related cognitive decline and neurodegenerative diseases.

The Ercc1 gene is essential for nucleotide excision repair and is also important in recombination repair and the repair of interstrand crosslinks. We have previously used a floxed Ercc1 allele with a keratinocyte-specific Cre recombinase transgene to inactivate Ercc1 in the epidermal layer of the skin and so generate a mouse model for UV-induced non-melanoma skin cancer. Now, in an attempt to generate a model for UV-induced melanoma, we have used the floxed Ercc1 allele in combination with a Cre transgene under the control of the tyrosinase gene promoter to produce mice with Ercc1-deficient melanocytes that are hypersensitive to UV irradiation. These animals developed normally, but died when 4-6 months old with severe colonic obstruction. Melanocytes are derived from the neural crest and the tyrosinase promoter is also expressed in additional neural crest-derived lineages, including the progenitors of the parasympathetic nervous system that innervates the gastrointestinal tract and controls gut peristalsis. A functional enteric nervous system developed in floxed Ercc1 mice with the tyrosinase Cre transgene, but was found to have degenerated in the colons of affected mice. We suggest that accumulating unrepaired endogenous DNA damage in the Ercc1-deficient colonic parasympathetic ganglia leads to the degeneration of this network and results in a colonic obstructive disorder that resembles late-onset Hirschsprung disease in man.

In Europe up to one in four couples experience difficulty conceiving and in half of these cases the problem has been attributed to sub or infertility in the male partner. The development of assisted reproductive technologies (ART) such as in vitro fertilization and intra-cytoplasmic spermatozoa injection has allowed some such couples to achieve a pregnancy. Concerns have been raised over the increasing use of ART not least because of the discovery of elevated levels of DNA damage in sperm from subfertile men. The impact of damaged DNA originating in the male germ line is poorly understood, but is thought to contribute to early pregnancy loss (recurrent miscarriage), placental problems and have a long-term impact on the health of the offspring. DNA repair is essential for meiotic recombination and correction of DNA damage in germ cells and proteins involved in all the major repair pathways are expressed in the testis. In this review, we will consider evidence that the production of sperm containing damaged DNA can be the result of suboptimal DNA repair and/or a mild environmental insult, such as heat stress, and how studies in mice may give us insight into the origins and consequences of DNA damage in human sperm.

The ERCC1/XPF complex is responsible for incision at the 5' side of the lesion during nucleotide excision repair and is also involved in homologous recombination and interstrand cross-link repair. The aim of the current study was to set up a better model for examination of Ercc1 deficiency in the murine liver and to determine the DNA lesions responsible for the premature polyploidy observed. We used the Cre/lox system with an adenovirus carrying Cre recombinase to conditionally induce Ercc1 deficiency in murine hepatocytes in vitro. Increased levels of apoptosis were apparent in our Ercc1-deficient cultures, both spontaneously and after UV irradiation and oxidative DNA damage. Increased apoptosis was also observed in simple Ercc1-deficient livers and the time course of the development of polyploidy was characterised. Livers from simple Ercc1 knockout mice contained mitochondria with disrupted outer membranes. Lipid accumulation was observed in older Ercc1-deficient hepatocyte cultures and in young Ercc1-deficient and wild-type livers. Lipids disappeared from the wild-type livers with age, but persisted in Ercc1-deficient livers, suggesting that a reduced ability to repair oxidative DNA damage and a malfunction of oxidative pathways could be responsible for the Ercc1-deficient liver phenotype. Real-time RT-PCR was used to determine differences in expression of cell cycle regulation and survival genes between Ercc1-deficient and control livers. Higher mRNA levels of Igfbp2, a possible marker for polyploidy, and p21 were detected in Ercc1-deficient livers. The pro-apoptotic factor, Bax, showed increased levels of mRNA expression in young Ercc1-deficient livers. However, no elevation in the levels of reactive oxygen species, or of malondialdehyde DNA adducts, a product of oxidative DNA damage, were found in Ercc1-deficient liver and no elevated levels of genes involved in the oxidative damage response were seen.

Dynamic, epigenetic mechanisms can regulate macrophage phenotypes following exposure to different stimulating conditions and environments. However, temporal patterns of microRNAs (miRNAs or miRs) across multiple macrophage polarization phenotypes have not been defined. We determined miRNA expression in bone marrow-derived murine macrophages over multiple time points (0.5, 1, 3, 24 h) following exposure to cytokines and/or LPS. We hypothesized that dynamic changes in miRNAs regulate macrophage phenotypes. Changes in macrophage polarization markers were detected as early as 0.5 and as late as 24 h; however, robust responses for most markers occurred within 3 h. In parallel, many polarization-specific miRNAs were also changed by 3 h and expressed divergent patterns between M1 and M2a conditions, with increased expression in M1 (miR-155, 199a-3p, 214-3p, 455-3p, and 125a) or M2a (miR-511 and 449a). Specifically, miR-125a-5p exhibited divergent patterns: increased at 12–24 h in M1 macrophages and decreasing trend in M2a. VEGF in the culture media of macrophages was dependent upon the polarization state, with greatly diminished VEGF in M2a compared with M1 macrophage culture media despite similar VEGF in cell lysates. Inhibition of miR-125a-5p in media-only controls (MO) and M1 macrophages greatly increased expression and secretion of soluble VEGF receptor-1 (sVEGFR1) leading to diminished VEGF in the culture media, partially converting MO and M1 into an M2a phenotype. Thus, the divergent expression patterns of polarization-specific miRNAs led to the identification and demonstrated the regulation of a specific macrophage polarization phenotype, sVEGFR1 by inhibition of miR-125a-5p.

Catechol-based inhibitors of ERCC1-XPF endonuclease activity were identified from a high-throughput screen. Exploration of the structure-activity relationships within this series yielded compound 13, which displayed an ERCC1-XPF IC50 of 0.6 μM, high selectivity against FEN-1 and DNase I and activity in nucleotide excision repair, cisplatin enhancement and γH2AX assays in A375 melanoma cells. Screening of fragments as potential alternatives to the catechol group revealed that 3-hydroxypyridones are able to inhibit ERCC1-XPF with high ligand efficiency, and elaboration of the hit gave compounds 36 and 37 which showed promising ERCC1-XPF IC50 values of

Macrophages are important in vascular inflammation and environmental factors influence macrophage plasticity. Macrophage transitions into pro-inflammatory (M1) or anti-inflammatory (M2) states have been defined predominately by measuring cytokines in culture media (CM). However, temporal relationships between cellular and secreted cytokines have not been established. We measured phenotypic markers and cytokines in cellular and CM of murine bone marrow-derived macrophages at multiple time points following stimulation with IFN-γ+LPS (M1), IL-4 (M2a), or IL-10 (M2c). Cytokines/proteins in M1-polarized macrophages exhibited two distinct temporal patterns; an early (0.5–3 hr), transient increase in cellular cytokines (GM-CSF, KC-GRO, MIP-2, IP-10 and MIP-1β) and a delayed (3–6 hrs) response that was more sustained [IL-3, regulated on activation normal T cell expressed and secreted (RANTES), and tissue inhibitor of metalloproteinases 1 (TIMP-1)]. M2a-related cytokine/cell markers (IGF-1, Fizz1, and Ym1) were progressively (3–24 hrs) increased post-stimulation. Additionally, novel patterns were observed. First, and unexpectedly, cellular pro-inflammatory chemokines, MCP-1 and MCP-3 but not MCP-5, were comparably increased in M1 and M2a macrophages. Second, Vegfr1 mRNA was decreased in M1 and increased in M2a macrophages. Finally, VEGF-A was increased in the CM of M1 cultures and strikingly reduced in M2a coinciding with increased Vegfr1 expression, suggesting decreased VEGF-A in M2a CM was secondary to increased soluble VEGFR1. In conclusion, macrophage cytokine production and marker expression were temporally regulated and relative levels compared across polarizing conditions were highly dependent upon the timing and location (cellular vs. CM) of the sample collection. For most cytokines, cellular production preceded increases in the CM suggesting that cellular regulatory pathways should be studied within 6 hours of stimulation. The divergent polarization-dependent expression of Vegfr1 may be essential to controlling VEGF potentially regulating angiogenesis and inflammatory cell infiltration in the vascular niche. The current study expands the repertoire of cytokines produced by polarized macrophages and provides insights into the dynamic regulation of macrophage polarization and resulting cytokines, proteins, and gene expression that influence vascular inflammation.

53BP1 protein is re-localized to the sites of DNA damage after ionizing radiation (IR) and is involved in DNA-damage-checkpoint signal transduction. We examined the dynamics of GFP-53BP1 in living cells. The protein starts to accumulate at the sites of DNA damage 2-3 minutes after damage induction. Fluorescence recovery after photobleaching experiments showed that GFP-53BP1 is highly mobile in non-irradiated cells. Upon binding to the IR-induced nuclear foci, the mobility of 53BP1 reduces greatly. The minimum (M) domain of 53BP1 essential for targeting to IR induced foci consists of residues 1220-1703. GFP-M protein forms foci in mouse embryonic fibroblast cells lacking functional endogenous 53BP1. The M domain contains a tandem repeat of Tudor motifs and an arginine- and glycine-rich domain (RG stretch), which are often found in proteins involved in RNA metabolism, the former being essential for targeting. RNase A treatment dissociates 53BP1 from IR-induced foci. In HeLa cells, dissociation of the M domain without the RG stretch by RNase A treatment can be restored by re-addition of nuclear RNA in the early stages of post-irradiation. 53BP1 immunoprecipitates contain some RNA molecules. Our results suggest a possible involvement of RNA in the binding of 53BP1 to chromatin damaged by IR.

ERCC1-XPF is the endonuclease that cuts 5′ of the damage in nucleotide excision repair (NER). Unlike other NER proteins, ERCC1-XPF is also involved in recombination and the repair of DNA interstrand cross-links. Unique among the NER gene knockouts, Ercc1 null mice are severely runted with high levels of hepatocyte polyploidy. To understand the link between DNA repair deficiency and polyploidy we have compared the premature polyploidy in Ercc1 null liver with the normal development of polyploidy in aging control mice. Polyploidy was accelerated dramatically in Ercc1 null hepatocytes, such that ploidy levels were equivalent in 3-week-old Ercc1 null and 1- to 2-year-old wild-type mouse liver. Levels of the cyclin-dependent kinase inhibitor, p21, were increased in the nuclei of Ercc1 null hepatocytes, and this increase was concentrated in, but not confined to, the polyploid hepatocytes. Much lower levels of p21 messenger RNA (mRNA) were found in old wild-type liver with equivalent levels of ploidy. We suggest that the more rapid accumulation of DNA damage in Ercc1 null liver leads to an increase in p21 levels, but that there is not a simple direct link between p21 levels and premature polyploidy. The failure to observe any link between p21 levels and polyploidy in aged wild-type liver may be attributable to the much lower levels of accumulated DNA damage, the much greater timescale involved, or the existence of a p21-independent mechanism for polyploidy. In conclusion, the premature polyploidy in Ercc1-deficient liver differs from the normal aging-related process. (Hepatology 2003;38:958-966.)

DNA repair has an essential role in protecting the genome from damage by endogenous and environmental agents. Polymorphisms in DNA repair genes and differences in repair capacity between individuals have been widely documented. For colorectal cancer, the loss of mismatch repair gene activity is a key genetic determinant. Nucleotide excision repair (NER), recombination repair (RR) and base excision repair (BER) pathways have critical roles in protection against other cancers, and we wished to investigate their role in colorectal cancer. We have compared the frequency of polymorphisms in the NER genes, XPD, XPF, XPG, ERCC1; in the BER gene, XRCC1; and in the RR gene, XRCC3; in colorectal cancer patients and in a control group. No significant associations were found for any of the NER gene polymorphisms or for the XRCC1 polymorphism. The C allele (position 18067) of the XRCC3 gene was weakly but significantly associated with colorectal cancer (odds ratio 1.52, 95% confidence interval 1.04-2.22, P=0.03). For all patients who were heterozygous for any of the repair genes studied, tumour tissue was investigated for loss of heterozygosity (LOH). Only one example of LOH was found for all the genes examined. From the association and LOH data, we conclude that these genes do not have an important role in protection against colorectal carcinogenesis.

Class switch recombination (CSR) is a programmed gene rearrangement in which a B cell which is producing IgM and IgD antibody develops into an IgG-, IgA- or IgE-expressing cell. This is achieved by recombination between switch regions located 5′ of each of the immunoglobulin heavy chain constant regions, except Cδ. The mechanism of CSR has not been resolved but it is thought to involve a double-strand break followed by end joining. It has previously been suggested that the nucleotide excision repair protein ERCC1 may be involved in CSR due to its known roles in removal of 3′ single-stranded tails in various types of recombination. In this study, we examined class switching in cultured splenocytes from ERCC1-deficient mice and found no evidence of any deficiency.

Ercc1 is essential for nucleotide excision repair (NER) but, unlike other NER proteins, Ercc1 and Xpf are also involved in recombination repair pathways. Ercc1 knockout mice have profound cell cycle abnormalities in the liver and die before weaning. Subsequently Xpa and Xpc knockouts have proved to be good models for the human NER deficiency disease, xeroderma pigmentosum, leading to speculation that the recombination, rather than the NER deficit is the key to the Ercc1 knockout phenotype. To investigate the importance of the recombination repair functions of Ercc1 we studied spermatogenesis and oogenesis in Ercc1 -deficient mice. Male and female Ercc1 -deficient mice were both infertile. Ercc1 was expressed at a high level in the testis and the highest levels of Ercc1 protein occurred in germ cells following meiotic crossing over. However, in Ercc1 null males some germ cell loss occurred prior to meiotic entry and there was no evidence that Ercc1 was essential for meiotic crossing over. An increased level of DNA strand breaks and oxidative DNA damage was found in Ercc1 -deficient testis and increased apoptosis was noted in male germ cells. We conclude that the repair functions of Ercc1 are required in both male and female germ cells at all stages of their maturation. The role of endogenous oxidative DNA damage and the reason for the sensitivity of the germ cells to Ercc1 deficiency are discussed.

ERCC1-XPF, through its role in nucleotide excision repair (NER), is essential for the repair of DNA damage caused by UV light. ERCC1-XPF is also involved in recombinational repair processes distinct from NER. In rodent cells chromosome aberrations are a common consequence of UV irradiation. We have previously shown that ERCC1-deficient cells have a lower ratio of chromatid exchanges to breaks than wild type cells. We have now confirmed this result and have shown that XPF-deficient cells also have a lower ratio than wild type. However, cells deficient in the other NER genes, XPD, XPB and XPG, all have the same ratio of exchanges to breaks as wild type. This implies that ERCC1-XPF, but not other NER proteins, is involved in the formation of UV-induced chromosome aberrations, presumably through the role of ERCC1-XPF in recombinational repair pathways rather than NER. We suggest that ERCC1-XPF may be involved in the bypass/repair of DNA damage in replicating DNA by an exchange mechanism involving single strand annealing between non-homologous chromosomes. This mechanism would rely on the ability of ERCC1-XPF to trim non-homologous 3' tails.

Late onset ataxia reported in three independently derived PrP null lines of mice has been attributed to the overexpression of the doppel protein in the CNS of these mice rather than to the loss of PrP. The central role of PrP in the transmissible spongiform encephalopathies (TSEs), the proximity of the gene which encodes doppel (Prnd) to the PrP gene (Prnp) and the structural similarity shared by PrP and doppel have led to the proposition that ataxia which develops during TSE disease could, in part, be due to doppel. In order to address this hypothesis, we have crossed our two inbred lines of PrP null mice, which either express (RCM) or do not express (NPU) thePrndgene in the CNS, with mice expressing twoPrnpa[108F189V]alleles of the PrP gene. We have found that the TSE infection does not influence the level of expression ofPrndin the CNS at the terminal stages of disease. Moreover, we have demonstrated that the level of expression ofPrndin the CNS has no influence on the incubation period, vacuolar pathology nor amount or distribution of PrPScdeposition in the brains of the TSE-infected mice. Doppel has therefore no apparent influence on the outcome of TSE disease in transgenic mice, suggesting it is unlikely to be involved in the naturally occurring TSE diseases in other species.

We demonstrated that young male and female mice similarly regenerated injured skeletal muscle; however, female mice transiently increased adipocyte area within regenerated muscle in a sex hormone-dependent manner. We extended these observations to investigate the effect of aging and sex on sarcopenia and muscle regeneration. Cardiotoxin injury to the tibialis anterior muscle of young, middle, and old-aged C57Bl/6J male and female mice was used to measure regenerated myofiber cross-sectional area (CSA), adipocyte area, residual necrosis, and inflammatory cell recruitment. Baseline (uninjured) myofiber CSA was decreased in old mice of both sexes compared to young and middle-aged mice. Regenerated CSA was similar in male mice in all age groups until baseline CSA was attained but decreased in middle and old age female mice compared to young females. Furthermore, adipocyte area within regenerated muscle was transiently increased in young females compared to young males and these sex-dependent increases persisted in middle and old age female mice and were associated with increased Pparg Young female mice had more pro-inflammatory monocytes/macrophages in regenerating muscle than young male mice and increased Sca-1(+)CD45(-)cells. In conclusion, sex and age influence pro-inflammatory cell recruitment, muscle regeneration, and adipocyte area following skeletal muscle injury.

The ERCC1 gene is essential for the repair of UV-induced DNA damage. Unlike most genes in the nucleotide excision repair (NER) pathway, ERCC1 is also involved in recombinational repair. Perhaps for this reason, ERCC1 knockout mice are not a model for the human NER deficiency disorder, xeroderma pigmentosum. Instead, ERCC1 null mice are severely runted and die before weaning from liver failure with accelerated hepatocyte polyploidy that is more reminiscent of a premature ageing disorder. To permit study of the role of ERCC1 in other tissues we have corrected the liver ERCC1 deficiency with a transgene under the control of a liver-specific promoter. The transgene alleviated runting and extended the lifespan. The elevated level of oxidative DNA damage and premature liver polyploidy were reversed and liver function was corrected. A widespread mitochondrial dysfunction was identified and an essential role for ERCC1 in the kidney was also revealed with transgene-containing ERCC1-deficient animals going on to die of renal failure. The nuclei of kidney proximal tubule cells became polyploid in a similar way to the premature liver polyploidy observed in younger ERCC1-deficient animals. We believe that this is a response to the accumulation of endogenous DNA damage in these particularly susceptible tissues which cannot be repaired in ERCC1-deficient animals.

The PrP gene of the host exerts a major influence over the outcome of transmissible spongiform encephalopathy (TSE) disease, but the mechanism by which this is achieved is not understood. We have introduced a specific mutation into the endogenous murine PrP gene using gene targeting to produce transgenic mice with a single amino acid alteration (proline to leucine) at amino acid position 101 in their PrP protein (P101L). The effect of this alteration on incubation time, targeting and PrP(Sc) formation has been studied in TSE-infected animals. Transgenic mice carrying the P101L mutation in PrP have remarkable differences in incubation time and targeting of central nervous system pathology compared with wild-type littermates, following inoculation with infectivity from human, hamster, sheep and murine sources. This single mutation can alter incubation time across three species barriers in a strain-dependent manner. These findings suggest a critical role for the structurally 'flexible' region of PrP in agent replication and targeting of TSE pathology.

Gene amplification is widely used for the production of pharmaceuticals and therapeutics in situations where a mammalian system is essential to synthesise a fully active product. Current gene amplification systems require multiple rounds of selection, often with high concentrations of toxic chemicals, to achieve the highest levels of gene amplification. The use of these systems has not been demonstrated in specialised mammalian cells, such as embryonic-stem cells, which can be used to generate transgenic animals. Thus, it has not yet proved possible to produce transgenic animals containing amplified copies of a gene of interest, with the potential to synthesise large amounts of a valuable gene product. We have developed a new amplification system, based around vectors encoding a partially disabled hypoxanthine phosphoribosyltransferase (HPRT) minigene, which can achieve greater than 1000-fold amplification of HPRT and the human growth hormone gene in a single step in Chinese hamster-lung cells. The amplification system also works in mouse embryonic-stem cells and we have used it to produce mice which express 30-fold higher levels of human protein C in milk than obtained with conventional transgenesis using the same protein C construct. This system should also be applicable to large animal transgenics produced by nuclear transfer from cultured cell lines.

A wide range of DNA lesions, both UV and chemically induced, are dealt with by the nucleotide excision repair (NER) pathway. Defects in NER result in human syndromes such as xeroderma pigmentosum (XP), where there is a 1000-fold increased incidence of skin cancer. The ERCC1 protein is essential for NER, but ERCC1 knockout mice are not a model for XP. In the absence of exogenous DNA-damaging agents, these mice are runted and die before weaning, with dramatically accelerated liver polyploidy and elevated levels of p53. Here we present a morphological, immunological, and molecular study to understand the mechanism for the unusual liver pathology in ERCC1-deficient mice. We show that the enlarged ERCC1-deficient hepatocytes are arrested in G2 and that DNA replication and the normal process of binucleation are both reduced. This is associated with a p53-independent increase in expression of the cyclin-dependent kinase inhibitor p21. The most dramatic feature of the ERCC1-deficient liver phenotype, the accelerated polyploidy, is not rescued by p53 deficiency, but we show that p53 is responsible for the reduced DNA replication and binucleation. We consider that the liver phenotype is a response to unrepaired endogenous DNA damage, which may reflect an additional non-NER-related function for the ERCC1 protein.—Nunez, F., Chipchase, M. D., Clarke, A. R., Melton, D. W. Nucleotide excision repair gene (ERCC1) deficiency causes G2 arrest in hepatocytes and a reduction in liver binucleation: the role of p53 and p21.

Classical genetic analysis has identified Sinc/Prni as the major gene controlling mouse scrapie incubation time. Sinc/Prni is linked to Prnp, the gene encoding the prion protein (PrP). Prnp alleles express distinct PrP protein variants, PrP A and PrP B, which arise from codon 108L/F and 189 T/V dimorphisms. Prnp genotype segregates with incubation time length which suggests, but does not prove, that incubation time is controlled by PrP dimorphisms, and that the Sinc/Prni and Prnp loci are congruent. We have used gene targetting to construct mice in which the endogenous Prnp allele has been modified to express PrP B instead of PrP A. Challenge with a mouse-adapted BSE strain results in dramatically shortened incubation times and demonstrates that PrP dimorphisms at codon 108 and/or 189 control incubation time, and that Sinc/Prni and Prnp are congruent.

The ERCC1 protein is essential for nucleotide excision repair in mammalian cells and is also believed to be involved in mitotic recombination. ERCC1-deficient mice, with their extreme runting and polyploid hepatocyte nuclei, have a phenotype that is more reminiscent of a cell cycle arrest/premature ageing disorder than the classic DNA repair deficiency disease, xeroderma pigmentosum. To understand the role of ERCC1 and the link between ERCC1-deficiency and cell cycle arrest, we have studied primary and immortalised embryonic fibroblast cultures from ERCC1-deficient mice and a Chinese hamster ovary ERCC1 mutant cell line. Mutant cells from both species showed the expected nucleotide excision repair deficiency, but the mouse mutant was only moderately sensitive to mitomycin C, indicating that ERCC1 is not essential for the recombination-mediated repair of interstrand cross links in the mouse. Mutant cells from both species had a high mutation frequency and the level of genomic instability was elevated in ERCC1-deficient mouse cells, both in vivo and in vitro. There was no evidence for an homologous recombination deficit in ERCC1 mutant cells from either species. However, the frequency of S-phase-dependent illegitimate chromatid exchange, induced by ultra violet light, was dramatically reduced in both mutants. In rodent cells the G1 arrest induced by ultra violet light is less extensive than in human cells, with the result that replication proceeds on an incompletely repaired template. Illegitimate recombination, resulting in a high frequency of chromatid exchange, is a response adopted by rodent cells to prevent the accumulation of DNA double strand breaks adjacent to unrepaired lesion sites on replicating DNA and allow replication to proceed. Our results indicate an additional role for ERCC1 in this process and we propose the following model to explain the growth arrest and early senescence seen in ERCC1-deficient mice. In the absence of ERCC1, spontaneously occurring DNA lesions accumulate and the failure of the illegitimate recombination process leads to the accumulation of double strand breaks following replication. This triggers the p53 response and the G2 cell cycle arrest, mediated by increased expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1). The increased levels of unrepaired lesions and double strand breaks lead to an increased mutation frequency and genome instability.

Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta- lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele. We show that the BLG-Cre construct specifies mammary specific gene deletion, and furthermore that it is temporally regulated, predominantly occurring during lactation. We fully characterised the extent of gene deletion in one line (line 74). In this strain the virgin gland is characterised by low levels (7%) of Cre mediated deletion, whereas 70–80% of cells within the lactating mammary gland have undergone recombination. Immunohisto-chemistry and indirect in situ PCR were used respectively to demonstrate that both Cre protein and Cre activity were evenly distributed throughout the population of secretory epithelial cells. The level of background recombination in non-mammary tissues was found to be ≤1.1%, irrespective of mammary gland developmental status. Crossing the transgenic BLG-Cre strain described here to mice harbouring other floxed alleles will facilitate the functional analysis of those genes during differentiation and development of the mammary gland.

Prion diseases are fatal transmissible neurological disorders afflicting a range of mammalian species. Although still controversial, a large body of data suggests that the causative agent may be composed entirely of a small glycoprotein. The brains of infected animals have accumulations of a pathogenic protease-resistant isoform (PrPsc) of a normal host-encoded glycoprotein, PrPc or prion protein. A number of lines of biochemical evidence implicate the disease-specific isoform, PrPsc, as the transmissible agent and genetic analysis has shown tight linkage between PrP gene mutations and polymorphisms and differential susceptibility to prion diseases, Perhaps the strongest evidence for a protein-only model of the agent is that PrP gene-ablated mice are resistant to scrapie and that mice with PrP mutation, corresponding to those found in a human familial prion disease, spontaneously develop a transmissible prion disease. This review describes the critical role that transgenic technology has played in the study of the biology of prion diseases and considers the issues raised by this work.

DNA mismatch repair (MMR) deficiency is associated with increased risk of developing several types of cancer and is the most common cause of hereditary ovarian cancer after BRCA1 and BRCA2 mutations. While there has been extensive investigation of MMR deficiency in colorectal cancer, MMR in ovarian cancer is relatively under-investigated. This review summarizes the mechanism of MMR, the ways in which MMR deficiency can promote carcinogenesis in general and then assesses the available studies regarding MMR deficiency in ovarian cancers with specific emphasis on implications for disease incidence and therapy. The incidence of germline MMR gene mutations in ovarian cancer is only 2% but other mechanisms of gene inactivation mean that loss of expression of one of the seven main genes (MSH2, MSH3, MSH6, MLH1, MLH3, PMS1 and PMS2) occurs in up to 29% of cases. Both mutational and expression data suggest that MMR deficiency is more common in non-serous ovarian cancer. Some studies suggest an improved survival for patients with MMR deficiency compared to historical controls but these do not account for the preponderance of non-serous tumors. A number of in vitro studies have suggested that MMR deficiency is a cause of platinum resistance. To date this has not been categorically demonstrated in the clinic. Larger studies that account for stage of presentation and immunohistochemical subtype are required to assess the effect of MMR deficiency on survival and chemosensitivity. Investigation of MMR related synthetic lethality in colorectal cancer has identified dihydrofolate reductase, DNA polymerase β and DNA polymerase γ and PTEN-induced putative kinase 1 as synthetic lethal to certain MMR defects by causing accumulation of oxidative DNA damage. These synthetic lethal targets require tested and others should be sought within the context of MMR deficient ovarian cancer in an attempt to provide novel therapeutic strategies for these patients.

Currently, mammalian cell technology has become the focus of biopharmaceutical production, with strict regulatory scrutiny of the techniques employed. Major concerns about the presence of animal-derived components in the culture media led to the development of serum-free (SF) culture processes. However, cell adaptation to SF conditions is still a major challenge and limiting step of process development. Thus, this study aims to assess the impact of SF adaptation on monoclonal antibody production (mAb), identify the most critical steps of cell adaptation to the SF EX-CELL medium, and create basic process guidelines.. The success of SF adaptation was dependent on critical steps that included: accentuated cell sensitivity to common culture procedures (centrifugation, trypsinization); initial cell concentration; time given at each step of serum-reduction; and, most importantly, medium supplements used to support adaptation. Indeed, only one of the five supplement combinations assessed (rhinsulin, ammonium metavanadate, nickel chloride and stannous chloride) succeeded for the CHO-K1 cell line used. This work also revealed that the chemically-defined EX-CELL medium benefits mAb production in comparison with the general purpose Dulbecco's Modified Eagle's Medium, but the complete removal of serum attenuates these positive effects., The authors acknowledge funding and support from the Portuguese Foundation for Science and Technology (FCT), namely grant ref SFRH/BD/46661/2008 for Maria Elisa Rodrigues and SFRH/BD/46660/2008 for Ana Rita Costa.

Deficiencies in different steps of purine metabolism give rise to a number of human inherited disorders. Lesch-Nyhan syndrome is a severe neurological disorder, caused by a deficiency in the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). HPRT-deficient mice have been generated, but have proved to be an unsuccessful model of the human disease. We have suggested that this may be due to a greater dependency in rodents on the other purine salvage enzyme, adenine phosphoribosyltransferase (APRT). We have generated an APRT-deficient mouse line by gene targeting, with a phenotype that closely resembled the symptoms of APRT deficiency in man. APRT null mice were viable, but 90% died prematurely before 6 months of age, displaying highly abnormal kidney morphology, with pathology characteristic of tubule obstruction. These mice have elevated urinary levels of adenine and 2,8-dihydroxyadenine, a highly insoluble adenine derivative, plus birefringent crystalline deposits and calculi within tubules throughout the kidney. A standard therapy for APRT-deficient human patients is the administration of the xanthine oxidase inhibitor, allopurinol. This has proved an effective therapy for APRT null mice, preventing accumulation of 2,8-dihydroxyadenine and much of the resultant renal obstruction, allowing us to establish a breeding line. We believe that these mice should provide a useful model for further study of APRT deficiency in humans. Furthermore, by generating APRT and HPRT double mutants, we will be able to test our hypothesis that both genes must be inactivated in mice before a model for Lesch-Nyhan syndrome can be obtained.

Bullous congenital ichthyosiform erythroderma (BCIE) is a dominantly inherited blistering skin disorder caused by point mutations in the suprabasal cytokeratins 1 or 10. Targeting the murine cytokeratin 10 gene in ES cells resulted in mice with different phenotypes in the homozygotes and heterozygotes; both of which exhibit similarities to specific clinical characteristics of BCIE. Homozygotes suffered from severe skin fragility and died shortly after birth. Heterozygotes were apparently unaffected at birth, but developed hyperkeratosis with age. In both genotypes, aggregation of cytokeratin intermediate filaments, changes in cytokeratin expression, and alterations in the program of epidermal differentiation were observed. In addition we demonstrate, for the first time, the existence of the murine equivalent of human cytokeratin 16.

We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies.

Summary Understanding how drugs work in vivo is critical for drug design and for maximizing the potential of currently available drugs. 5-nitrofurans are a class of prodrugs widely used to treat bacterial and trypanosome infections, but despite relative specificity, 5-nitrofurans often cause serious toxic side effects in people. Here, we use yeast and zebrafish, as well as human in vitro systems, to assess the biological activity of 5-nitrofurans, and we identify a conserved interaction between aldehyde dehydrogenase (ALDH) 2 and 5-nitrofurans across these species. In addition, we show that the activity of nifurtimox, a 5-nitrofuran anti-trypanosome prodrug, is dependent on zebrafish Aldh2 and is a substrate for human ALDH2. This study reveals a conserved and biologically relevant ALDH2-5-nitrofuran interaction that may have important implications for managing the toxicity of 5-nitrofuran treatment., Graphical Abstract Highlights ► Zebrafish provide a viable assay for the biological toxicity of 5-nitrofurans ► ALDH2 inhibitors prevent 5-nitrofuran toxicity in zebrafish and yeast ► Genetic dependence on ALDH2 for 5-nitrofuran toxicity in zebrafish and yeast systems ► 5-Nitrofurans bind to and are substrates of human ALDH2, 5-nitrofurans are antibiotics activated by pathogen specific enzymes, however, less is known about what happens in the host. Zhou et al. identify aldehyde dehydrogenase 2 as a 5-nitrofuran activating enzyme that has implications for managing some of the toxicity associated with 5-nitrofuran treatment.