An initial characterisation of the Unfolded Protein Response pathway in haematopoietic canine cancer cell lines - a necessary step for the future development of new therapies in dogs with neoplasia.

Introduction: New and more effective therapies for canine cancer patients are urgently required and this necessitates advanced experimental research. Dogs are good models for studies in comparative oncology; however, canine cancer cell biology research is currently limited by low availability of validated antibody reagents and techniques. This study characterises the expression of key components of the unfolded protein response (UPR) in a panel of haematopoietic canine cancer cell lines using commercially available antibodies, and validates the methods used to study this pathway. Material and Methods: The CLBL-1 canine lymphoma cell line and the GL-1 canine leukaemia cell line sourced externally and two counterparts established in house (CNK-89 and CLB70) were used as models of different lymphoma and leukaemia canine cell lines for the study. The human U2OS cell line served as the control. Antibodies were selected for identifying UPR proteins according to known canine cell reactivity and canine-murine and canine-human homology. Endoplasmic reticulum stress was induced with thapsigargin and MG132 in the cell lines. Etoposide was used to induce DNA damage in the cells. The techniques used for this validation analysis were RNA sequencing to observe the expression of UPR components in canine cell lines, Western blot to observe changes of protein expression levels after inducing ER stress in the cells, and flow cytometry in order to study cell death. Results: Substantial variations in both the basic expression and agonist-induced activation of the UPR pathway were observed in canine cancer cell lines, although the biological significance of these differences requires further investigation. Conclusion: These findings will be a starting point for future studies on cancer biology in dogs. They will also contribute to developing novel anticancer therapies for canine patients and may provide new insights into human oncology.

Studying the DNA damage response pathway in hematopoietic canine cancer cell lines, a necessary step for finding targets to generate new therapies to treat cancer in dogs.

Background: Dogs present a significant opportunity for studies in comparative oncology. However, the study of cancer biology phenomena in canine cells is currently limited by restricted availability of validated antibody reagents and techniques. Here, we provide an initial characterization of the expression and activity of key components of the DNA Damage Response (DDR) in a panel of hematopoietic canine cancer cell lines, with the use of commercially available antibody reagents. Materials and methods: The techniques used for this validation analysis were western blot, qPCR, and DNA combing assay. Results: Substantial variations in both the basal expression (ATR, Claspin, Chk1, and Rad51) and agonist-induced activation (p-Chk1) of DDR components were observed in canine cancer cell lines. The expression was stronger in the CLBL-1 (B-cell lymphoma) and CLB70 (B-cell chronic lymphocytic leukemia) cell lines than in the GL-1 (B-cell leukemia) cell line, but the biological significance of these differences requires further investigation. We also validated methodologies for quantifying DNA replication dynamics in hematopoietic canine cancer cell lines, and found that the GL-1 cell line presented a higher replication fork speed than the CLBL-1 cell line, but that both showed a tendency to replication fork asymmetry. Conclusion: These findings will inform future studies on cancer biology, which will facilitate progress in developing novel anticancer therapies for canine patients. They can also provide new knowledge in human oncology.

When more is less: heritable gain-of-function chk1 mutations impair human fertility.

Heritable loss-of-function mutations in genes encoding key regulators of DNA repair and genome stability can result in degenerative progeroid and/or cancer predisposition syndromes; however, such mutations have never been found to affect the Chk1 protein kinase, despite its central role in DNA damage signalling and checkpoint activation. Remarkably, two recent reports now demonstrate that heritable, gain-of-function mutations within the Chk1 C-terminal regulatory domain can cause female infertility in humans. In vitro, oocytes from individuals heterozygous for such mutant Chk1 alleles fail to undergo the first mitotic division after fertilization owing to arrest in G2 phase of the cell cycle. This arrest results from inhibition of the master regulator of mitosis, the cyclin-dependent kinase CDK1, through the same molecular mechanisms that are engaged by activated Chk1 to impose G2 checkpoint arrest in somatic cells bearing DNA damage. Remarkably, the failure of this first zygotic division in heterozygotes in vitro can be rescued through treatment with selective Chk1 inhibitor drugs, allowing development of apparently normal blastocysts and offering hope that a pharmacological solution to this cause of infertility may be possible.

DNA damage response proteins in canine cancer as potential research targets in comparative oncology.

The DNA damage response (DDR) is a complex signal transduction network that is activated when endogenous or exogenous genotoxins damage or interfere with the replication of genomic DNA. Under such conditions, the DDR promotes DNA repair and ensures accurate replication and division of the genome. High levels of genomic instability are frequently observed in cancers and can stem from germline loss-of-function mutations in certain DDR genes, such as BRCA1, BRCA2, and p53, that form the basis of human cancer predisposition syndromes. In addition, mutation and/or aberrant expression of multiple DDR genes are frequently observed in sporadic human cancers. As a result, the DDR is considered to represent a viable target for cancer therapy in humans and a variety of strategies are under investigation. Cancer is also a significant cause of mortality in dogs, a species that offers certain advantages for experimental oncology. Domestic dogs present numerous inbred lines, many of which display predisposition to specific forms of cancer and the study of which may provide insight into the biological basis of this susceptibility. In addition, clinical trials are possible in dogs and may lead to therapeutic insights that could ultimately be extended to humans. Here we review what is known specifically about the DDR in dogs and discuss how this knowledge could be extended and exploited to advance experimental oncology in this species.

The Nucleocapsid protein triggers the main humoral immune response in COVID-19 patients.

In order to control the COVID-19 pandemic caused by SARS-CoV-2 infection, serious progress has been made to identify infected patients and to detect patients with a positive immune response against the virus. Currently, attempts to generate a vaccine against the coronavirus are ongoing. To understand SARS-CoV-2 immunoreactivity, we compared the IgG antibody response against SARS-CoV-2 in infected versus control patients by dot blot using recombinant viral particle proteins: N (Nucleocapsid), M (Membrane) and S (Spike). In addition, we used different protein fragments of the N and S protein to map immune epitopes. Most of the COVID-19 patients presented a specific immune response against the full length and fragments of the N protein and, to lesser extent, against a fragment containing amino acids 300-685 of the S protein. In contrast, immunoreactivity against other S protein fragments or the M protein was low. This response is specific for COVID-19 patients as very few of the control patients displayed immunoreactivity, likely reflecting an immune response against other coronaviruses. Altogether, our results may help develop method(s) for measuring COVID-19 antibody response, selectivity of methods detecting such SARS-CoV-2 antibodies and vaccine development.

Claspin - checkpoint adaptor and DNA replication factor.

Claspin was discovered as a Chk1-interacting protein necessary for Chk1 phosphorylation and activation by the upstream kinase, ATR, in response to DNA synthesis inhibition in Xenopus oocyte extracts. Subsequent investigations have defined a molecular model in which Claspin acts as an adaptor or scaffold protein to facilitate activation of Chk1 by ATR within a multiprotein complex that forms on single-stranded DNA at stalled replication forks and sites of DNA damage. Interestingly, Claspin is an unstable protein whose degradation via the proteasome is tightly regulated via ubiquitination and controlled by multiple ubiquitin ligases and deubiquitinases. As a result, Claspin levels fluctuate during the cell cycle, contributing to the regulation of checkpoint proficiency and playing a key role in terminating checkpoint-mediated cell cycle arrest. In addition to its role in signalling genotoxic stress, Claspin is required to maintain normal rates of replication fork progression during unperturbed DNA replication and may contribute to the regulation of replication origin firing. Consistent with this, Claspin can bind directly to DNA, with particular affinity for branched or forked molecules, and it interacts with multiple protein components of the replisome. As expected for a protein with key roles in checkpoint signalling and genome duplication, aberrations of Claspin expression and structure have been observed in cancer. Claspin is furthermore targeted to facilitate viral replication and plays a role in suppressing cellular DNA synthesis in response to nongenotoxic endoplasmic reticulum stress. Here, we review the functions and regulation of Claspin with a focus on areas of active research.

Chk1 KA1 domain auto-phosphorylation stimulates biological activity and is linked to rapid proteasomal degradation.

The DNA damage-activated protein kinase Chk1 is known to undergo auto-phosphorylation, however the sites and functional significance of this modification remain poorly understood. We have identified two novel Chk1 auto-phosphorylation sites, threonines 378 and 382 (T378/382), located in a highly conserved motif within the C-terminal Kinase Associated 1 (KA1) domain. T378/382 occur within optimal consensus Chk1 phosphorylation motifs and substitution with phospho-mimetic aspartic acid residues results in a constitutively active mutant Chk1 kinase (Chk1-DD) that arrests cell cycle progression in G2 phase of the cell cycle in the absence of DNA damage. Remarkably, the mutant Chk1-DD protein is also subject to very rapid proteasomal degradation, with a half-life approximately one tenth that of wild-type Chk1. Consistent with this, T378/T382 auto-phosphorylation also accelerates the proteasomal degradation of constitutively active Chk1 KA1 domain structural mutants. T378/382 auto-phosphorylation and accelerated degradation of wild-type Chk1 occurs at low levels during unperturbed growth, but surprisingly, is not augmented in response to genotoxic stress. Taken together, these observations demonstrate that Chk1 T378/T382 auto-phosphorylation within the KA1 domain is linked to kinase activation and rapid proteasomal degradation, and suggest a non-canonical mechanism of regulation.

Charge regulation of nonpolar colloids.

Individual colloids often carry a charge as a result of the dissociation (or adsorption) of weakly-ionized surface groups. The magnitude depends on the precise chemical environment surrounding a particle, which in a concentrated dispersion is a function of the colloid packing fraction η. Theoretical studies have suggested that the effective charge Zeff in regulated systems could, in general, decrease with increasing η. We test this hypothesis for nonpolar dispersions by determining Zeff(η) over a wide range of packing fractions (10-5 ≤ η ≤ 0.3) using a combination of small-angle X-ray scattering and electrophoretic mobility measurements. All dispersions remain entirely in the fluid phase regime. We find a complex dependence of the particle charge as a function of the packing fraction, with Zeff initially decreasing at low concentrations before finally increasing at high η. We attribute the non-monotonic density dependence to a crossover from concentration-independent screening at low η, to a high packing fraction regime in which counterions outnumber salt ions and electrostatic screening becomes η-dependent. The efficiency of charge stabilization at high concentrations may explain the unusually high stability of concentrated nanoparticle dispersions which has been reported.

Charging Poly(methyl Methacrylate) Latexes in Nonpolar Solvents: Effect of Particle Concentration.

The electrophoresis of a well-established model system of charged colloids in nonpolar solvents has been studied as a function of particle volume fraction at constant surfactant concentration. Dispersions of poly(12-hydroxystearic acid)-stabilized poly(methyl methacrylate) (PMMA) latexes in dodecane were prepared with added Aerosol OT surfactant as the charging agent. The electrophoretic mobility (µ) of the PMMA latexes is found to decrease with particle concentration. The particles are charged by a small molecule charging agent (AOT) at finite concentration, and this makes the origin of this decrease in µ unclear. There are two suggested explanations. The decrease could either be due to the reservoir of available surfactant being exhausted at high particle concentrations or the interactions between the charged particles at high particle number concentrations. Contrast-variation small-angle neutron scattering measurements of PMMA latexes and deuterated AOT-d34 surfactant in latex core contrast-matched solvent were used to study the former, and electrokinetic modeling was used to study the latter. As the same amount of AOT-d34 is found to be incorporated with the latexes at all volume fractions, the solvodynamic and electrical interactions between particles are determined to be the explanation for the decrease in mobility. These measurements show that, for small latexes, there are interactions between the charged particles at all accessible particle volume fractions and that it is necessary to account for this to accurately determine the electrokinetic ζ potential.

The internal structure of poly(methyl methacrylate) latexes in nonpolar solvents.

HYPOTHESIS: Poly(methyl methacrylate) (PMMA) latexes in nonpolar solvents are an excellent model system to understand phenomena in low dielectric media, and understanding their internal structure is critical to characterizing their performance in both fundamental studies of colloidal interactions and in potential industrial applications. Both the PMMA cores and the poly(12-hydroxystearic acid) (PHSA) shells of the latexes are known to be penetrable by solvent and small molecules, but the relevance of this for the properties of these particles is unknown. EXPERIMENTS: These particles can be prepared in a broad range of sizes, and two PMMA latexes dispersed in n-dodecane (76 and 685nm in diameter) were studied using techniques appropriate to their size. Small-angle scattering (using both neutrons and X-rays) was used to study the small latexes, and analytical centrifugation was used to study the large latexes. These studies enabled the calculation of the core densities and the amount of solvent in the stabilizer shells for both latexes. Both have consequences on interpreting measurements using these latexes. FINDINGS: The PHSA shells are highly solvated (∼85% solvent by volume), as expected for effective steric stabilizers. However, the PHSA chains do contribute to the intensity of neutron scattering measurements on concentrated dispersions and cannot be ignored. The PMMA cores have a slightly lower density than PMMA homopolymer, which shows that only a small free volume is required to allow small molecules to penetrate into the cores. Interestingly, the observations are essentially the same, regardless of the size of the particle; these are general features of these polymer latexes. Despite the latexes being used as a model physical system, the internal chemical structure is complex and must be fully considered when characterizing them.

Autophagy is critically required for DNA repair by homologous recombination.

Autophagy delivers damaged cytoplasmic constituents to lysosomes for degradation, thus preserving cellular integrity and protecting against disease. Remarkably, autophagy-deficient cells also exhibit aberrant DNA damage responses with therapeutic implications, such as suppression of checkpoint kinase-1 function, impaired DNA double-strand break repair by homologous recombination, and increased reliance on error-prone non-homologous end-joining for survival.

DNA damage control: regulation and functions of checkpoint kinase 1.

Checkpoint kinase 1 (Chk1) is a master regulator of the DNA damage and replication checkpoints in vertebrate cells. When activated via phosphorylation by its upstream regulatory kinase, ATR, Chk1 prevents cells with damaged or incompletely replicated DNA from entering mitosis, and acts to stabilize stalled replication forks and suppress replication origin firing when DNA synthesis is inhibited. Chk1 blocks mitosis by maintaining high levels of inhibitory tyrosine phosphorylation of the mitotic cyclin-dependent kinase 1; however, the mechanisms that underlie replication fork stabilization and suppression of origin firing are less well defined. Although Chk1 function is evidently acutely regulated during these responses, how this occurs at the molecular level is incompletely understood. Recent evidence that Chk1 contains a 'kinase-associated 1' domain within its regulatory C-terminal region promises new insights. Additional modifications catalysed by other protein kinases, such as cyclin-dependent kinase 1, Akt, and RSK, can combine with ubiquitylation to regulate Chk1 subcellular localization and protein stability. Interestingly, it is clear that Chk1 has less well-defined functions in homologous recombination, chromatin modification, gene expression, spindle checkpoint proficiency, and cytokinesis. Here, we provide an overview of Chk1 regulation and functions, with an emphasis on unresolved questions that merit further research.

KA1-targeted regulatory domain mutations activate Chk1 in the absence of DNA damage.

The Chk1 protein kinase is activated in response to DNA damage through ATR-mediated phosphorylation at multiple serine-glutamine (SQ) residues within the C-terminal regulatory domain, however the molecular mechanism is not understood. Modelling indicates a high probability that this region of Chk1 contains a kinase-associated 1 (KA1) domain, a small, compact protein fold found in multiple protein kinases including SOS2, AMPK and MARK3. We introduced mutations into Chk1 designed to disrupt specific structural elements of the predicted KA1 domain. Remarkably, six of seven Chk1 KA1 mutants exhibit constitutive biological activity (Chk1-CA) in the absence of DNA damage, profoundly arresting cells in G2 phase of the cell cycle. Cell cycle arrest induced by selected Chk1-CA mutants depends on kinase catalytic activity, which is increased several-fold compared to wild-type, however phosphorylation of the key ATR regulatory site serine 345 (S345) is not required. Thus, mutations targeting the putative Chk1 KA1 domain confer constitutive biological activity by circumventing the need for ATR-mediated positive regulatory phosphorylation.

Loss of autophagy causes a synthetic lethal deficiency in DNA repair.

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.

Interaction between surfactants and colloidal latexes in nonpolar solvents studied using contrast-variation small-angle neutron scattering.

The interaction between deuterium-labeled Aerosol OT surfactant (AOT-d34) and sterically stabilized poly(methyl methacrylate) (PMMA) latex particles dispersed in nonpolar solvents has been studied using contrast-variation small-angle neutron scattering (CV-SANS). The electrophoretic mobilities (µ) of the latexes have been measured by phase-analysis light scattering, indicating that µ is negative. Two analogues of the stabilizers for the particles have been studied as free polymers in the absence of PMMA latexes: poly(12-hydroxystearic acid) (PHSA) polyester and poly(methyl methacrylate)-graft-poly(12-hydroxystearic acid) (PMMA-graft-PHSA) stabilizer copolymer. The scattering from both PHSA in dodecane and PMMA-graft-PHSA in toluene is consistent with extended polymer chains in good solvents. In dodecane, PMMA-graft-PHSA forms polymer micelles, and SANS is consistent with ellipsoidal aggregates formed of around 50 polymer chains. CV-SANS measurements were performed by measuring SANS from systems of PHSA, PMMA-graft-PHSA, and PMMA latexes with 10 and 100 mM surfactant solutions of AOT-d34 in both polymer/particle and AOT contrast-matched solvent. No excess scattering above the polymer or surfactant was found for PHSA in dodecane or PMMA-graft-PHSA in dodecane and toluene. This indicates that AOT does not significantly interact with the free polymers. Excess scattering was observed for systems with AOT-d34 and PMMA latexes dispersed in particle contrast-matched dodecane, consistent with the penetration of AOT into the PMMA latexes. This indicates that AOT does not interact preferentially with the stabilizing layers but, rather, is present throughout the colloids. Previous research ( Langmuir 2010, 26, 6967-6976 ) suggests that AOT surfactant is located in the latex PHSA-stabilizer layer, but all the results in this study are consistent with AOT poorly interacting with alkyl-stabilizer polymers.

Counterion condensation on spheres in the salt-free limit.

A highly-charged spherical colloid in a salt-free environment exerts such a powerful attraction on its counterions that a certain fraction condenses onto the surface of a particle. The degree of condensation depends on the curvature of the surface. So, for instance, condensation is triggered on a highly-charged sphere only if the radius exceeds a certain critical radius R*. R* is expected to be a simple function of the volume fraction of particles. To test these predictions, we prepare spherical particles which contain a covalently-bound ionic liquid, which is engineered to dissociate efficiently in a low-dielectric medium. By varying the proportion of ionic liquid to monomer we synthesise nonpolar dispersions of highly-charged spheres which contain essentially no free co-ions. The only ions in the system are counterions generated by the dissociation of surface-bound groups. We study the electrophoretic mobility of this salt-free system as a function of the colloid volume fraction, the particle radius, and the bare charge density and find evidence for extensive counterion condensation. At low electric fields, we observe excellent agreement with Poisson-Boltzmann predictions for counterion condensation on spheres. At high electric fields however, where ion advection is dominant, the electrophoretic mobility is enhanced significantly which we attribute to hydrodynamic stripping of the condensed layer of counterions from the surface of the particle.

Fascin 1 is transiently expressed in mouse melanoblasts during development and promotes migration and proliferation.

Fascins, a family of actin-bundling proteins, are expressed in a spatially and temporally restricted manner during development and often in cancer. Fascin 1 has a clear role in cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of fascin 1 in the melanocyte lineage and in melanoma cells. Fascin 1 knockout causes hypopigmentation in adult mice owing to migration and cell cycle progression defects in melanoblasts, the melanocyte precursor cell. Study of live embryo skin explants reveals that E14.5 fascin 1-null melanoblasts migrate slower, and generate fewer and thinner pseudopods. By contrast, fascin 1 expression drives faster migration and lamellipodia protrusion in melanocytes in vitro. In addition, fascin 1 depletion retards melanoblast proliferation in vivo and melanoma cell growth in vitro. These data indicate that fascin 1 not only promotes cell migration in mouse melanocytes but it also has a role in growth and cell cycle progression.

Chk1 is essential for the development of murine epidermal melanocytes.

Embryonic deletion of mouse Chk1 is lethal; however, whether Chk1 is essential in all individual tissues is unknown. By breeding C57Bl/ 6 mice homozygous for a conditional allele of Chk1 (Chk1fl/fl) and bearing melanocyte-specific Tyr::Cre and DCT:: LacZ transgenes, we investigated the consequences of Chk1 deletion in the melanocytic lineage. We show that adult Tyr::Cre; Chk1fl/fl mice lack coat pigmentation and epidermal melanocytes in the hair follicles, but retain eye pigmentation in the retinal pigmented epithelium (RPE). Melanoblasts formed normally during embryogenesis in Tyr::Cre; Chk1fl/fl mice at early times (embryonic day 10.5; E10.5) but were completely absent by stage E13.5, most probably as a consequence of spontaneous DNA damage and apoptosis. By contrast, melanoblast numbers were only slightly reduced in heterozygous Tyr::Cre; Chk1fl/ + embryos, and these mice exhibited normal coat pigmentation as adults. Thus, Chk1 is essential for the developmental formation of murine epidermal melanocytes but hemizygosity has little, if any, permanent developmental consequence in this cell type.