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The mechanisms that drive leukocyte recruitment to the kidney are incompletely understood. Dipeptidase-1 (DPEP1) is a major neutrophil adhesion receptor highly expressed on proximal tubular cells and peritubular capillaries of the kidney. Renal ischemia reperfusion injury (IRI) induces robust neutrophil and monocyte recruitment and causes acute kidney injury (AKI). Renal inflammation and the AKI phenotype were attenuated in Dpep1 −/− mice or mice pretreated with DPEP1 antagonists, including the LSALT peptide, a nonenzymatic DPEP1 inhibitor. DPEP1 deficiency or inhibition primarily blocked neutrophil adhesion to peritubular capillaries and reduced inflammatory monocyte recruitment to the kidney after IRI. CD44 but not ICAM-1 blockade also decreased neutrophil recruitment to the kidney during IRI and was additive to DPEP1 effects. DPEP1, CD44, and ICAM-1 all contributed to the recruitment of monocyte/macrophages to the kidney following IRI. These results identify DPEP1 as a major leukocyte adhesion receptor in the kidney and potential therapeutic target for AKI.

Significance Inherited pathogenic mutations in genes required for copper delivery to cytochrome c oxidase (CcO) perturb mitochondrial energy metabolism and result in fatal mitochondrial disease. A prior attempt to treat human patients with these mutations by direct copper supplementation was not successful, possibly because of inefficient copper delivery to the mitochondria. We performed a targeted search to identify compounds that can efficiently transport copper across biological membranes and identified elesclomol (ES), an investigational anticancer drug, as the most efficient copper delivery agent. ES rescues CcO function in yeast, zebrafish, and mammalian models of copper deficiency by increasing cellular and mitochondrial copper content. Thus, our study offers a possibility of repurposing this anticancer drug for the treatment of disorders of copper metabolism., Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.

Despite extensive molecular characterization, human glioblastoma remains a fatal disease with survival rates measured in months. Little improvement is seen with standard surgery, radiotherapy and chemotherapy. Clinical progress is hampered by the inability to detect and target glioblastoma disease reservoirs based on a diffuse invasive pattern and the presence of molecular and phenotypic heterogeneity. The goal of this study was to target the invasive and stem-like glioblastoma cells that evade first-line treatments using agents capable of delivering imaging enhancers or biotherapeutic cargo. To accomplish this, a combinatorial phage display library was biopanned against glioblastoma cell model systems that accurately recapitulate the intra- and inter-tumor heterogeneity and infiltrative nature of the disease. Candidate peptides were screened for specificity and ability to target glioblastoma cells in vivo. Cargo-conjugated peptides delivered contrast-enhancing agents to highly infiltrative tumor populations in intracranial xenograft models without the obvious need for blood brain barrier disruption. Simultaneous use of five independent targeting peptides provided greater coverage of this complex tumor and selected peptides have the capacity to deliver a therapeutic cargo (oncolytic virus VSVΔM51) to the tumor cells in vivo. Herein, we have identified a series of peptides with utility as an innovative platform to assist in targeting glioblastoma for the purpose of diagnostic or prognostic imaging, image-guided surgery, and/or improved delivery of therapeutic agents to glioblastoma cells implicated in disease relapse.

DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg(+/-) offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg(-/-) mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg(+/-) and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg(-/-) animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg(-/-) animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg(-/-) mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.

Recent studies show the importance of mitochondrial dysfunction in the initiation and progression of acute kidney injury (AKI). However, no biomarkers exist linking renal injury to mitochondrial function and integrity. To this end, we evaluated urinary mitochondrial DNA (UmtDNA) as a biomarker of renal injury and function in humans with AKI following cardiac surgery. mtDNA was isolated from the urine of patients following cardiac surgery and quantified by quantitative PCR. Patients were stratified into no AKI, stable AKI, and progressive AKI groups based on Acute Kidney Injury Network (AKIN) staging. UmtDNA was elevated in progressive AKI patients and was associated with progression of patients with AKI at collection to higher AKIN stages. To evaluate the relationship of UmtDNA to measures of renal mitochondrial integrity in AKI, mice were subjected to sham surgery or varying degrees of ischemia followed by 24 h of reperfusion. UmtDNA increased in mice after 10-15 min of ischemia and positively correlated with ischemia time. Furthermore, UmtDNA was predictive of AKI in the mouse model. Finally, UmtDNA levels were negatively correlated with renal cortical mtDNA and mitochondrial gene expression. These translational studies demonstrate that UmtDNA is associated with recovery from AKI following cardiac surgery by serving as an indicator of mitochondrial integrity. Thus UmtDNA may serve as valuable biomarker for the development of mitochondrial-targeted therapies in AKI.

The invasive nature of glioblastoma renders them incurable by current therapeutic interventions. Using a novel invasive human glioma model, we previously identified the neurotrophin receptor p75(NTR) (aka CD271) as a mediator of glioma invasion. Herein, we provide evidence that preventing phosphorylation of p75(NTR) on S303 by pharmacological inhibition of PKA, or by a mutational strategy (S303G), cripples p75(NTR)-mediated glioma invasion resulting in serine phosphorylation within the C-terminal PDZ-binding motif (SPV) of p75(NTR). Consistent with this, deletion (ΔSPV) or mutation (SPM) of the PDZ motif results in abrogation of p75(NTR)-mediated invasion. Using a peptide-based strategy, we identified PDLIM1 as a novel signaling adaptor for p75(NTR) and provide the first evidence for a regulated interaction via S425 phosphorylation. Importantly, PDLIM1 was shown to interact with p75(NTR) in highly invasive patient-derived glioma stem cells/tumor-initiating cells and shRNA knockdown of PDLIM1 in vitro and in vivo results in complete ablation of p75(NTR)-mediated invasion. Collectively, these data demonstrate a requirement for a regulated interaction of p75(NTR) with PDLIM1 and suggest that targeting either the PDZ domain interactions and/or the phosphorylation of p75(NTR) by PKA could provide therapeutic strategies for patients with glioblastoma.

The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx₉CxnCx₁₀C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6Δ cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx₉CxnCx₁₀C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6Δ cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient.

Epilepsy is a debilitating disease affecting 1-2% of the world's population. Despite this high prevalence, 30% of patients suffering from epilepsy are not successfully managed by current medication suggesting a critical need for new anti-epileptic drugs (AEDs). In an effort to discover new therapeutics for the management of epilepsy, we began our study by screening drugs that, like some currently used AEDs, inhibit histone deacetylases (HDACs) using a well-established larval zebrafish model. In this model, 7-day post fertilization (dpf) larvae are treated with the widely used seizure-inducing compound pentylenetetrazol (PTZ) which stimulates a rapid increase in swimming behavior previously determined to be a measurable manifestation of seizures. In our first screen, we tested a number of different HDAC inhibitors and found that one, 2-benzamido-1 4-naphthoquinone (NQN1), significantly decreased swim activity to levels equal to that of valproic acid, 2-n-propylpentanoic acid (VPA). We continued to screen structurally related compounds including Vitamin K3 (VK3) and a number of novel Vitamin K (VK) analogs. We found that VK3 was a robust inhibitor of the PTZ-induced swim activity, as were several of our novel compounds. Three of these compounds were subsequently tested on mouse seizure models at the National Institute of Neurological Disorders and Stroke (NINDS) Anticonvulsant Screening Program. Compound 2h reduced seizures particularly well in the minimal clonic seizure (6Hz) and corneal-kindled mouse models of epilepsy, with no observable toxicity. As VK3 affects mitochondrial function, we tested the effects of our compounds on mitochondrial respiration and ATP production in a mouse hippocampal cell line. We demonstrate that these compounds affect ATP metabolism and increase total cellular ATP. Our data indicate the potential utility of these and other VK analogs for the prevention of seizures and suggest the potential mechanism for this protection may lie in the ability of these compounds to affect energy production.

The ERCC1-XPF structure-specific endonuclease is necessary for correct processing of homologous recombination intermediates requiring the removal of end-blocking nonhomologies. We previously showed that targeting the endogenous CHO APRT locus with plasmids designed to generate such intermediates revealed defective recombination phenotypes in ERCC1 deficient cells, including suppression of targeted insertion and vector correction recombinants and the generation of a novel class of aberrant recombinants through a deletogenic mechanism. In the present study, we examined some of the mechanistic features of ERCC1-XPF in processing recombination intermediates by varying gene targeting parameters. These included altering the distance between the double-strand break (DSB) in the targeting vector and the inactivating mutation in the APRT target gene, and changing the position of the target gene mutation relative to the DSB to result in target mutations that were either upstream or downstream from the DSB. Increasing the distance from the DSB in the targeting vector to the chromosomal target gene mutation resulted in an ERCC1 dependent decrease in the efficiency of gene targeting from intermediates presenting lengthy end-blocking nonhomologies. This decrease was accompanied by a shift in the distribution of recombinant classes away from target gene conversions to targeted insertions in both wild-type and ERCC1 deficient cells, and a dramatic increase in the proportion of aberrant recombinants in ERCC1 deficient cells. Changing the position of the target gene mutation relative to the DSB in the plasmid also altered the distribution of targeted insertion subclasses recovered in wild-type cells, consistent with two-ended strand invasion followed by resolution into crossover-type products and vector integration. Our results confirm expectations from studies of Rad10-Rad1 in budding yeast that ERCC1-XPF activity affects conversion tract length, and provide evidence for the mechanism of generation of the novel, aberrant recombinant class first described in our previous study.

Genetic hybrids of the genus Xiphophorus have historically been useful models for study of the genetic aspects of tumor formation. In the most studied Xiphophorus tumor model, two-gene loci, XMRK and DIFF, are implicated as critical both to UV-induced and spontaneous melanoma formation in BC(1) hybrids of crosses between X. maculatus and X. helleri, with X. helleri as the recurrent backcross parent. In addition to UV, the direct-acting carcinogen N-methyl-N-nitrosourea (MNU) has been used to induce tumors in Xiphophorus BC(1) hybrids from several cross types. In the present study, we address the hypothesis that excess melanomas in MNU-treated BC(1) hybrids may have been generated by direct mutation of CDKN2AB, a candidate gene for DIFF. MNU treatment of F(1) and BC(1) hybrid fish significantly increased tumor incidence at 6 months; however, no association was found between MNU-induced tumor formation and zygosity of the candidate tumor tumor-suppressor CDKN2AB in BC(1) hybrids, consistent with previously reported results. Sequence analysis of the X. maculatus CDKN2AB locus of heterozygous individuals (both BC(1) and F(1) hybrids) did not reveal any mutations caused by MNU, suggesting that the mechanism of MNU-induced melanoma formation in this Xiphophorus model does not involve direct mutation of CDKN2AB but may result from mutation of other critical genes.

Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism – intramitochondrial quality control (IMQC) – is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma1 is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments in cultured Oma1-deficient mouse embryonic fibroblasts link together impeded supercomplex stability and inability to maintain proper respiration under conditions that require maximal bioenergetic output. Finally, transient knockdown of OMA1 in zebrafish leads to impeded bioenergetics and morphological defects of the heart and eyes. Together, our biochemical and genetic studies in yeast, zebrafish and mammalian cells identify a novel and conserved physiological role for Oma1 protease in fine-tuning of respiratory function. We suggest that this unexpected physiological role is important for cellular bioenergetic plasticity and may contribute to Oma1-associated disease phenotypes in humans.

MUC1 is a transmembrane glycoprotein expressed by normal breast epithelium and virtually all breast cancers. MUC1 is normally restricted to the apical surface of epithelia and loss of this polarized distribution in breast carcinomas is associated with lymph node metastasis. Our previous work found that MUC1 can bind intercellular adhesion molecule-1 (ICAM-1), mediating adhesion of breast cancer cells to a simulated blood vessel wall, and also triggering a calcium-based signal in the MUC1-bearing cells. It is possible that the depolarized membrane distribution of MUC1 in breast carcinomas may facilitate interactions with stromal/endothelial ICAM-1 leading to adhesion and subsequent migration through the vessel wall. In the current study, we provide evidence that ICAM-1 can influence the migration of cells that express endogenous or transfected MUC1. Migration across a gelatin-coated Transwell membrane could be increased in a step-wise manner by the sequential addition of ICAM-1-expressing cells (endothelial cells and fibroblasts), and ICAM-1-inducing inflammatory cytokines (tumour necrosis factor-alpha and interleukin-1 beta). Antibodies against MUC1 or ICAM-1, but not a control antibody, could abrogate migratory increases. Cells that did not express MUC1 were unresponsive to ICAM-1. Our current findings build on our earlier work, by suggesting that the end result of the MUC1/ICAM-1-mediated cell-cell adhesion and calcium-based signal is migration. This has implications for the exit of circulating tumour cells from the vasculature, as well as tumour cell migration through fibroblast-containing stroma underlying the endothelial wall.

The MUC1 mucin is normally restricted to the apical surface of breast epithelial cells. In tumors, it is frequently overexpressed and underglycosylated. The MUC1 peptide core mediates firm adhesion of tumor cells to adjacent cells via binding to intercellular adhesion molecule-1 (ICAM-1). There is increasing evidence that MUC1 is involved in signaling, with current reports focusing on phosphorylation of the MUC1 cytoplasmic tail after indirect or artificial modes of stimulation. ICAM-1 is the only known direct ligand of the MUC1 extracellular domain. The data presented herein show that MUC1 expressed on the surface of breast cancer cell lines or transfected 293T cells can initiate a calcium-based oscillatory signal on contact with ICAM-1-transfected NIH 3T3 cells, and we present a novel method of quantifying and comparing calcium oscillations. The MUC1-induced signal appears to be distinct from those previously described, and may involve a Src family kinase, phosphoinositol 3-kinase, phospholipase C, and lipid rafts, but not mitogen-activated protein kinase. As calcium signaling has been associated with cytoskeletal change and motility, it is possible that the functions of MUC1 include heterotypic cell-cell adhesion followed by a calcium-based promigratory signal within tumor cells, thus facilitating metastasis.

Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. ...

BACKGROUND The MUC1 mucin is present on the apical surface of normal secretory epithelia. In breast carcinoma, MUC1 expression is variable in amount and cellular localization, the significance of which is controversial. The authors undertook a detailed analysis of staining pattern combined with a comprehensive literature review to better understand the role of MUC1 in breast carcinoma. METHODS Seventy-one patients with breast carcinoma were examined for MUC1, β-catenin, and E-cadherin staining patterns. These data were compared with data from 25 articles from the literature examining the expression of MUC1 in breast carcinoma. RESULTS All invasive carcinomas showed some MUC1 staining. In invasive ductal carcinomas, MUC1 was detected in the apical membrane (15%), cytoplasm (93%), or circumferential membrane (13%), with 81% of tumors showing a mixture of patterns. Tumors with low overall MUC1 expression (≤ 50% positive tumor cells) had a higher nuclear grade than tumors with high overall MUC1 expression (> 50%; P = 0.01). Tumors with high and low cytoplasmic expression had no difference in nuclear grade (P > 0.3). Circumferential membrane staining was correlated with positive lymph node status (P = 0.011). CONCLUSIONS In the literature, similar findings prevailed in which overall MUC1 expression was increased in lower grade (10 of 14 studies), estrogen receptor positive (8 of 13 studies) tumors and was associated with a better prognosis (8 of 13 studies). High cytoplasmic staining was associated with a worse prognosis, an association that was not explained by differences in histologic grade. Thus, the presence of MUC1 in the majority of tumor cells is associated with better differentiated tumors and with an improved prognosis. However, aberrantly localized MUC1 in the tumor cell cytoplasm or nonapical membrane is associated with a worse prognosis. Cancer 2001;91:1973–82. © 2001 American Cancer Society.

Opa1 catalyzes fusion of inner mitochondrial membranes and formation of the cristae. OPA1 mutations in humans lead to autosomal dominant optic atrophy. OPA1 knockout mice lose viability around embryonic day 9 from unknown reasons, indicating that OPA1 is essential for embryonic development. Zebrafish are an attractive model for studying vertebrate development and have been used for many years to describe developmental events that are difficult or impractical to view in mammalian models. In this study, Opa1 was successfully depleted in zebrafish embryos using antisense morpholinos, which resulted in disrupted mitochondrial morphology. Phenotypically, these embryos exhibited abnormal blood circulation and heart defects, as well as small eyes and small pectoral fin buds. Additionally, startle response was reduced and locomotor activity was impaired. Furthermore, Opa1 depletion caused bioenergetic defects, without impairing mitochondrial efficiency. In response to mitochondrial dysfunction, a transient upregulation of the master regulator of mitochondrial biogenesis, pgc1a, was observed. These results not only reveal a new Opa1-associated phenotype in a vertebrate model system, but also further elucidates the absolute requirement of Opa1 for successful vertebrate development.

The study of gene function has been greatly facilitated by the development of strategies to modify genomic DNA. Gene targeting is one of the most successfully applied techniques used to examine the roles of specific genes in a wide variety of model systems from yeast to mammals. Our laboratory has pioneered the use of the Chinese hamster ovary (CHO) cell culture model system to study pathways of DNA repair and recombination at the hemizygous CHO APRT locus. By using a simple and effective gene targeting method, we have generated a number of DNA repair-deficient cell lines that have been used in targeted recombination experiments to investigate pathways of recombinational repair in somatic mammalian cells. These methods can be readily customized to generate a variety of cell lines deficient in specific genes of interest and can be applied to study the roles of other DNA repair proteins in pathways of mammalian recombinational repair.

Gene targeting by homologous recombination in mammalian cells is an important tool for generating genetically modified mice used for modeling human diseases. Gene targeting approaches are also useful for studying the mechanisms of homologous recombination. We have developed gene targeting methods that we have specifically used to investigate the mechanisms of recombination in cultured mammalian cells. In this chapter, we describe the generation of Chinese hamster ovary (CHO) cell gene disruption ("knockout") mutants in the repair/recombination gene ERCC1. Using this approach, we have constructed pairs of isogenic ERCC1-proficient and -deficient (null) CHO cell lines and used them as recipients for gene targeting assays in which a hemizygous mutant hamster adenine phosphoribosyltransferase (APRT) locus is corrected by homologous recombination with plasmid vectors containing hamster APRT DNA sequence homologous to the target gene in each cell line. The configuration of the targeting vector leads to experimental outcomes in which certain classes of APRTrecombinants are over- or under-represented depending on the repair gene status of the transfection recipient. We describe methods both for targeted gene knockout of ERCC1, and for APRT targeted gene correction by homologous recombination, and some of our experimental results using these approaches.

Werner syndrome (WS) is a rare, segmental progeroid syndrome caused by defects in the WRN gene, which encodes a RecQ helicase. WRN has roles in many aspects of DNA metabolism including DNA repair and recombination. In this study, we exploited two different recombination assays previously used to describe a role for the structure-specific endonuclease ERCC1-XPF in mitotic and targeted homologous recombination. We constructed Chinese hamster ovary (CHO) cell lines isogenic with the cell lines used in these previous studies by depleting WRN using shRNA vectors. When intrachromosomal, mitotic recombination was assayed in WRN-depleted CHO cells, a hyperrecombination phenotype was observed, and a small number of aberrant recombinants were generated. Targeted homologous recombination was also examined in WRN-depleted CHO cells using a plasmid-chromosome targeting assay. In these experiments, loss of WRN resulted in a significant decrease in nonhomologous integration events and ablation of recombinants that required random integration of the corrected targeting vector. Aberrant recombinants were also recovered, but only from WRN-depleted cells. The pleiotropic recombination phenotypes conferred by WRN depletion, reflected in distinct homologous and nonhomologous recombination pathways, suggest a role for WRN in processing specific types of homologous recombination intermediates as well as an important function in nonhomologous recombination.

DNA interstrand crosslinks (ICLs) are among the most deleterious cytotoxic lesions encountered by cells, mainly due to the covalent linkage these lesions create between the two strands of DNA which effectively blocks replication and transcription. Although ICL repair in mammalian cells is not fully understood, processing of these lesions is thought to begin by “unhooking” at the site of the damaged base accompanied by the generation of a double strand break and ultimately repair through translesion synthesis and homologous recombination. A key player in this repair process is the heterodimeric protein complex ERCC1-XPF. Although some models of ICL repair restrict ERCC1-XPF activity to the unhooking step, recent data suggest that this protein complex acts in additional downstream steps. Here, we review the evidence implicating ERCC1-XPF in multiple steps of ICL repair. Environ. Mol. Mutagen., 2010. © 2010 Wiley-Liss, Inc.

The invasive nature of cancers in general, and malignant gliomas in particular, is a major clinical problem rendering tumors incurable by conventional therapies. Using a novel invasive glioma mouse model established by serial in vivo selection, we identified the p75 neurotrophin receptor (p75NTR) as a critical regulator of glioma invasion. Through a series of functional, biochemical, and clinical studies, we found that p75NTR dramatically enhanced migration and invasion of genetically distinct glioma and frequently exhibited robust expression in highly invasive glioblastoma patient specimens. Moreover, we found that p75NTR-mediated invasion was neurotrophin dependent, resulting in the activation of downstream pathways and producing striking cytoskeletal changes of the invading cells. These results provide the first evidence for p75NTR as a major contributor to the highly invasive nature of malignant gliomas and identify a novel therapeutic target., Author Summary Gliomas are highly malignant and invasive tumors with tendrils that extend far from the primary tumor site, rendering conventional therapies ineffective and leading to an invariably poor prognosis. To understand the molecular mechanisms underlying this invasive behavior, we injected immunocompromised mice with human gliomas and compared invasive cells, which left the primary tumor site, to noninvasive cells, which remained at the site of injection. We identified the neurotrophin receptor p75NTR—which normally functions during development to induce neurite outgrowth and promote neuronal cell death—as an important regulator of glioma invasion. We present the first evidence that this neurotrophin receptor can also be a potent mediator of glioma invasion, and we show that the expression of this receptor is sufficient to impart a dramatic invasive behavior on genetically distinct tumors. These data highlight a previously unknown function of this receptor and suggest it may be a novel therapeutic target in the treatment of this devastating cancer., By in vivo selection of a human glioma, the authors identify the p75NTR neuotrophin receptor as a critical molecule regulating increased invasiveness.

Damselfish neurofibromatosis (DNF) is a transmissible disease characterized by peripheral nerve sheath and pigment cell tumors which occurs in bicolor damselfish ( Stegastes partitus ) on Florida reefs. The damselfish virus-like agent (DVLA) is associated with the development of DNF and contains a 2.4 kb DNA genome which was found at high levels in tumors and tumor-derived cell lines and at lower levels in non-tumor tissues of both spontaneously diseased fish (TF) and fish with experimentally induced tumors (EF). An analysis of transcription patterns revealed up to five DVLA derived RNAs ranging in size from 300 to 1400 bp in these cell types. DNA was the most commonly distributed DVLA component in TF and EF followed by RNA. Prevalence of transcripts varied by tissue type. The smallest transcripts were the most common in all cell types and the most complete patterns, which included the larger transcripts, were observed primarily in tumors. The presence of viral RNAs in addition to DNA in non-tumor tissues suggested these tissues were infected by DVLA and indicated a wide tissue tropism for this agent. The high levels of DVLA nucleic acids found in tumors suggest that replication is occurring there. However, the potential for DVLA replication in other tissues where only a limited range of transcripts were present is not known. The mechanism of tumorigenesis by this agent is unknown. However, the association of the larger transcripts with most tumor tissues and their absence in most non-tumor tissues suggests that these RNAs may be involved in tumor formation.

Background Cancer is a leading cause of death globally and the majority of patients will die from the formation of metastases rather than from their primary tumour as most metastases are resistant to conventional therapies. Based on numerous clinical observations it is clear that different cancers have a propensity to metastasize to specific organs. The central hypothesis for our current study is that an organs vasculature is intimately involved in mediating the interaction between specific cancer cells and the target organ for which they will metastasize to. In this respect we have focused on how melanoma cancer cells metastasize to the liver and lungs, two major sites of metastatic disease within the body, in order to provide insight for the development of new treatments for metastatic cancer. Materials and methods Herein we used an unbiased combinatorial phage in vivo biopanning strategy to isolate a library of peptidedisplaying-phage that home to the liver and lungs of animals treated with a pro-inflammatory stimulus, lipopolysccharaide (LPS). Using a functional screen we isolated a single phage that prevented the recruitment of leukocytes into the liver and lungs of animals following LPS treatment. We investigated if the lung/liver inhibitory phage were also able to inhibit the formation of lung metastasis using a human melanoma metastasis xenograft model. Results Using intravital microscopy, we identified a peptide-displaying-phage, and its corresponding displayed-peptide, that has the ability to inhibit adhesion and recruitment of neutrophils in the liver sinusoids in response to LPS. Excitingly, we also found that animals injected (i.v.) with human melanoma cells (70W) in the presence of the phage, or its corresponding displayed-peptide, showed reduced metastatic lung burden. As the isolated peptide was capable of inhibiting both leukocyte recruitment and melanoma lung metastasis, we asked if leukocytes played a functional role in the establishment of melanoma metastasis. We found that in animals that received Lys6G (Anti Gr-1) antibody to deplete circulating leukocytes there was a dramatic increase in 70W melanoma metastasis to the lungs and that this increase in metastatic state was still blocked by the addition of the peptide. Conclusion