Recent advances in reactive oxygen species measurement in biological systems.

Reactive oxygen species (ROS) play an essential role in facilitating signal transduction processes within the cell. However, the precise details of the redox dynamics involved are not well understood. The generation of ROS is tightly controlled both spatially and temporally within the cell, making the study of ROS dynamics particularly difficult. In order to measure these dynamics, precise tools that can specifically examine the relevant ROS are required. Recent advancements in methodologies for ROS measurement have allowed the study of ROS biology at a level of precision previously unachievable. Here, we discuss improvements to fluorescent ROS dye technologies, genetically encoded ROS reporters, nanoparticle delivery systems, and nanotube ROS probes. These technologies improve specificity, localization and sensitivity over previously available ROS probes.

Ellipticine derivative induces potent cytostatic effect in acute myeloid leukaemia cells.

A panel of novel ellipticine isomers were designed and synthesised with the aim of evaluating their anti-cancer effects on selected leukaemia cell lines. A preliminary NCI 60-cell screen demonstrated that these compounds display promising anti-tumour activity across a number of different cell types. We have consequently examined the effect of these derivatives in detail on the Acute Myeloid Leukaemia (AML) cell line, MV4-11. Cell cycle analyses revealed that the compounds had a range of distinctive cell cycle effects. 7-Hydroxyisoellipticine showed the most promise with respect to cytostatic activity. We demonstrated that this compound inhibited proliferation of leukaemia cells by preventing cells from progressing from G2 phase into mitosis over a period of 24 h at a concentration of 5 µM. Our research suggests that this is mediated by an induction of reactive oxygen species (ROS), which in turn activates the DNA damage response pathway. As a result of the activation of p53, cyclin B1 is inhibited. The induction of this pathway leads to apoptosis which is seen at 48 h using the same dose of 7-hydroxyisoellipticine. This study provides for the first time detailed cellular information on the potential use of isoellipticines as chemotherapeutic agents.

Less stress, more success? Oncological implications of surgery-induced oxidative stress.

Reactive oxygen species (ROS) possess important cell signalling properties. This contradicts traditional thought which associated ROS activity with cell death. Emerging evidence clearly demonstrates that ROS signalling acts as a key regulator in tumour cell survival and in the cellular processes required for tumour cells to successfully metastasise and proliferate. The discovery of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) family of enzymes in the last decade has unravelled much of the mystery surrounding how ROS are generated. Tumour cells are now known to express Nox enzymes which produce ROS required for cellular signalling. Activation of Nox enzymes occurs via interaction with proinflammatory cytokines and growth factors, all of which are released following surgical trauma. As our understanding of the signalling capabilities of ROS grows, the oncological implications of ROS activity are gradually being revealed. Nox-derived ROS are known to play a central role in each step of the metastatic cascade including invasion, adhesion, angiogenesis and proliferation. This article describes how surgery creates a ROS-rich environment, which facilitates redox signalling, and also examines the role played by Nox enzymes in this process. The authors then explore current knowledge of the oncological implications of surgery-induced redox signalling, and discuss current and future therapeutic strategies targeted at ROS and Nox enzymes in cancer patients.

Redox survival signalling in retina-derived 661W cells.

Reactive oxygen species have been implicated in processes involving cellular damage and subsequent cell death, especially in organs such as the eye that are constantly exposed to excitatory signals. However, recent studies have shown that oxidant species can also act as intracellular signalling molecules promoting cell survival, but little is known about this mechanism in the retina. The present study demonstrates for the first time that hydrogen peroxide (H2O2) is generated rapidly and acts as a pro-survival signal in response to a variety of apoptotic stimuli in retina-derived 661W cells and in the retinal ganglion cell line RGC-5. Focussing on 661Ws and serum deprivation, we systematically investigated pro-survival and pro-death pathways and discovered that the rapid and transient burst of H2O2 activates the AKT survival pathway. Activation of the apoptotic machinery takes place following the decline of H2O2 to basal levels. To substantiate this proposed pro-survival role of H2O2, we inhibited the oxidant burst, which exacerbated cell death. Conversely, maintenance of the oxidant signal using exogenous H2O2 enhanced cell survival. Overall, the results presented in this study provide evidence for a novel role of H2O2 in mediating survival of retinal cells in response to apoptotic stimuli.

Analysis of differential gene expression in colorectal cancer and stroma using fluorescence-activated cell sorting purification.

Tumour stroma gene expression in biopsy specimens may obscure the expression of tumour parenchyma, hampering the predictive power of microarrays. We aimed to assess the utility of fluorescence-activated cell sorting (FACS) for generating cell populations for gene expression analysis and to compare the gene expression of FACS-purified tumour parenchyma to that of whole tumour biopsies. Single cell suspensions were generated from colorectal tumour biopsies and tumour parenchyma was separated using FACS. Fluorescence-activated cell sorting allowed reliable estimation and purification of cell populations, generating parenchymal purity above 90%. RNA from FACS-purified and corresponding whole tumour biopsies was hybridised to Affymetrix oligonucleotide microarrays. Whole tumour and parenchymal samples demonstrated differential gene expression, with 289 genes significantly overexpressed in the whole tumour, many of which were consistent with stromal gene expression (e.g., COL6A3, COL1A2, POSTN, TIMP2). Genes characteristic of colorectal carcinoma were overexpressed in the FACS-purified cells (e.g., HOX2D and RHOB). We found FACS to be a robust method for generating samples for gene expression analysis, allowing simultaneous assessment of parenchymal and stromal compartments. Gross stromal contamination may affect the interpretation of cancer gene expression microarray experiments, with implications for hypotheses generation and the stability of expression signatures used for predicting clinical outcomes.

Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas.

Immature T cells and some T cell hybridomas undergo apoptotic cell death when activated through the T cell receptor complex, a phenomenon that is probably related to antigen induced negative selection of developing T cells. This activation-induced apoptosis depends on active protein and RNA synthesis in the dying cells, although none of the genes required for this process have previously been identified. Antisense oligonucleotides corresponding to c-myc block the constitutive expression of c-Myc protein in T cell hybridomas and interfere with all aspects of activation-induced apoptosis without affecting lymphokine production in these cells. These data indicate that c-myc expression is a necessary component of activation-induced apoptosis.

Dicing with death: dissecting the components of the apoptosis machinery.

Apoptosis, a mode of cell death commonly observed when death is a desirable or programmed event, has several characteristic structural features. These features appear to be induced by a range of gene products which, together, supervise and participate in the controlled dismantling of the cell. In this article the molecular components of the apoptotic machinery and the proteins implicated in the regulation of this mechanism of cell death are discussed.

Mechanisms of ROS modulated cell survival during carcinogenesis.

There is increasing evidence within the literature that the decreased susceptibility of tumour cells to stimuli that induce apoptosis can be linked to their inherently increased redox potential. The review primarily focuses on the PI3-kinase/Akt pathway, and the multiple points along this signalling pathway that may be redox regulated. The PI3-kinase/Akt pathway can influence a cells' sensitivity to death inducing signals, through direct manipulation of apoptosis regulating molecules or by regulating the activity of key transcription factors. Proteins involved in the control of apoptosis that are directly regulated by the PI3-kinase/Akt pathway include caspase-9, Bad and the transcription factor GSK-3beta. Lately, it is becoming increasingly obvious that phosphatases are a major counter balance to the PI3-kinase/Akt pathway. Phosphatases such as PP2A and PP1alpha can dephosphorylate signalling molecules within the PI3-kinase/Akt pathway, blocking their activity. It is the balance between the kinase activity and the phosphatase activity that determines the presence and strength of the PI3-kinase/Akt signal. This is why any protein modifications that hinder dephosphorylation can increase the tumours survival advantage. One such modification is the oxidation of the sulphydryl group in key cysteine residues present within the active site of the phosphatases. This highlights the link between the increased redox stress in tumours with the PI3-kinase/Akt pathway. This review will discuss the various sources of reactive oxygen species within a tumour and the effect of these radicals on the PI3-kinase/Akt pathway.

A pilot study on the identification of human papillomavirus genotypes in tongue cancer samples from a single institution in Ecuador.

The relationship between human papillomavirus (HPV) and oropharyngeal squamous cell carcinoma has been established. However, data from Ecuador is limited. The objective of this study was to characterize HPV infection in Ecuadorian patients with tongue cancer. Fifty-three patients with tongue cancer treated at the tertiary referral center Sociedad de Lucha Contra el Cancer (SOLCA), Guayaquil, between 2006 and 2011 were identified. Linear Array® HPV genotyping was used to identify the presence and types of HPV on formalin-fixed paraffin-embedded biopsy samples from these patients with tongue cancer. HPV was identified in 42% (n=22) and high-risk (HR) HPV in 17% (n=9), with 18 different HPV types identified. The most common types were the HR HPV 33 (14%) and low-risk HPV 67 (14%), followed by the HR HPV 58. More than one HPV type was identified in 27.3% of cases. HPV 33 was frequently associated with other HPV types. No statistically significant differences in gender (P=0.58) and age (P=0.12) were observed between HPV-positive and HPV-negative cases. HPV was identified in almost half of the tongue cancer samples, with subtypes 33 and 67 being the most common. This suggested that HPV played an important role in this disease in the population studied. Given these results, current HPV vaccines may not be as effective in reducing tongue cancer rates in this population.

Immune modulator therapy for microscopic colitis in a case series of 73 patients.

BACKGROUND: Microscopic colitis (MC) is a common cause of chronic diarrhoea. Various treatment options have been described, but there are limited data describing outcomes of corticosteroid-sparing treatments. AIM: To evaluate the outcomes of patients with active MC treated with immune modulators. METHODS: All patients seen at Mayo Clinic, Rochester between January 1, 1997 and November 30, 2016 with a histological diagnosis of MC were identified. Patients treated with an immune modulator of interest were selected and clinical outcomes recorded. RESULTS: Seventy-three MC patients (50 collagenous colitis and 23 lymphocytic colitis) with a median disease duration of 24 months (range, 7-60) were included. The indications for treatment were budesonide-refractoriness in 66%, budesonide dependence in 29%, and budesonide intolerance in 5%. Median age was 51.8 years (range, 43.4-63.1) and 61 (84%) were female. Thiopurines were used in 49 patients (67%) for a median of 4 months (range, 1.5-15). Complete and partial response occurred in 43% and 22% respectively. Adverse effects resulting in therapy cessation occurred in 17 patients (35%). Twelve patients (16%) were treated with methotrexate for a median of 14 months (3-18.8). Complete and partial response occurred in 58% and 17%, respectively. Anti-TNF therapy was used in 10 patients (14%) for a median of 4 months (range, 2.3-5.5). Complete response occurred in four patients and partial response in four patients. CONCLUSIONS: The majority of patients with active MC responded to thiopurines, methotrexate, or anti-TNF therapy. Larger controlled studies are required to confirm the efficacy and safety of these medications in MC.

Multiple death pathways in retina-derived 661W cells following growth factor deprivation: crosstalk between caspases and calpains.

During development of the mammalian retina, neurons that do not succeed in establishing functional synaptic connections are eliminated by apoptosis, allowing the formation of a finely tuned network. Growth factors play a crucial role in controlling the balance between apoptosis and survival signals not only at developmental stages but also in long-term preservation of retinal functions. In the present work, we explore the apoptotic mechanisms triggered by growth factor deprivation of retina-derived 661W cells. Under serum starvation conditions, these cone photoreceptors underwent cell death with participation of caspase-9, -3 and -12. Interestingly, inhibition of caspases did not prevent apoptosis but only resulted in a temporary delay. We show m-calpain activation in parallel with caspases, indicating that more than one execution pathway is available to cone photoreceptors. Moreover, crosstalk of the caspase and calpain pathways was detected, suggesting a loop that may act to amplify the apoptotic cascade.

Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis.

Apoptosis is a form of cell death in which the cell "participates," such that metabolic energy and often protein synthesis are required for the death to occur. Once begun, the process of apoptosis proceeds in an ordered fashion. In the earliest phase DNA fragmentation occurs, accompanied by cell shrinkage and dilation of the endoplasmic reticulum. This is followed by cell fragmentation with the formation of sealed membrane vesicles, termed apoptotic bodies. In the present study we have demonstrated that the fungal metabolite cytochalasin B inhibits cell fragmentation and the formation of apoptotic bodies, probably by its ability to interfere with actin polymerization. This effect was seen when HL-60 cells were pretreated with cytochalasin B and then exposed to one of a number of apoptosis-inducing agents, including UV irradiation, camptothecin, aphidocholin, or PMA plus ionomycin. The observed effect was not peculiar to HL-60 cells, inasmuch as it was also seen for both Molt-4 and U-937 cell lines. Cytochalasin B had no effect on DNA fragmentation occurring in the earliest stage of apoptosis, and it appeared to have no inhibitory effects on nuclear fragmentation. Staurosporin had an effect similar to that seen with cytochalasin B, probably due to its ability to inhibit protein kinase C, which is a known potentiator of microfilament assembly. These data demonstrate that microfilament assembly is necessary for the formation of apoptotic bodies in the later stages of the apoptotic process.

Effects of surgery on the cancer stem cell niche.

Recent identification of a cancer stem cell (CSC) phenotype in solid tumors has greatly enhanced the understanding of the mechanisms responsible for cancer cell metastasis. In keeping with Pagets 'seed and soil' theory, CSCs display dependence upon stromal derived factors found within the niche in which they reside. Inflammatory mediators act as a 'fertilizer' within this niche when interacting with CSCs at the tumor-stromal interface and can potentiate the metastatic ability of CSCs. Interestingly, the same components of the pro-inflammatory milieu experienced by cancer patients perioperatively are known to promote the metastagenic potential of CSCs. On the basis of this observation we discuss how surgery-induced inflammation potentiates colon CSC involvement in the metastatic process. We hypothesize that the high rates of recurrence and metastasis associated with tumor resection are potentiated by the effects of surgery-induced inflammation on CSCs. Finally we discuss potential therapeutic strategies for use in the perioperative window to protect cancer patients from the oncological effects of the pro-inflammatory milieu.

Functional aspects of apoptosis in hematopoiesis and consequences of failure.

Apoptosis is an internally directed, physiological method of cell destruction. Cellular components are dismantled within the confines of an intact cell membrane, and rapid ingestion by phagocytic cells prevents local inflammation. A variety of genes have now been identified as positive or negative regulators of apoptosis. Transfection experiments and studies of gene cooperation in viral transformation suggest that full cellular transformation requires not only the deregulation of proliferation, but also the inhibition of concomitant apoptosis programs. The regulation of apoptosis is fundamental to hematopoietic homeostasis. Stem cell renewal is continuously counterbalanced by apoptosis in functionally inactive or terminally differentiated cells. Extensive cell death in developing lymphocyte populations ensures that only cells recognizing non-self antigens are released into the periphery, and the finite lifespan of terminally differentiated cells enables the extensive cell turnover demanded by functional aspects of the hematopoietic system. The requirement of each hematopoietic sub-population for a specific sub-set of survival factors, provides a flexible mechanism for dictating the cellular composition of the mature population and for controlling population size. Surplus cell production and apoptosis are therefore normal features of hematopoiesis. The consequences of deregulated apoptosis are severe. Excessive apoptosis in lymphocyte populations plays a major role in the pathogenesis of acquired immunodeficiency syndrome (AIDS), whereas ineffective apoptosis has been associated with the development of inflammation, autoimmunity and hematological malignancies. The identification of various genetic abnormalities which influence apoptosis in leukaemic cells (e.g., mutant p53, Bcr-Abl and over-expression of Bcl-2), suggests that the acquisition of an anti-apoptotic lesions is an important event in the multi-step evolution of hematological malignancies. In addition, the nature of some leukaemias particularly the chronic leukemias, in which the leukemic cells are nonproliferative and long lived, suggests that anti-apoptotic lesions are early events in the pathogenesis of these diseases. It is likely that the utilization of mechanisms to evade apoptosis would facilitate disease progression in all leukemias and contribute to the development of multi-drug resistance. A better understanding of apoptosis mechanisms in hematopoietic cells, and their exploitation by leukemic cells should be useful in the development of improved cytotoxic regimes.

Studies on the filtration properties of isolated renal basement membranes.

1. The filtration properties of films of renal basement membrane were studied in vitro using pressure filtration chambers. 2. Retention of cytochrome c by the films was found to be dependent upon the filtration pressure indicating that it was transferred across the films by convective as well as diffusive flow. In contrast, serum albumin was transferred by diffusive movement only. 3. When solutions containing both cytochrome c and IgG were filtered it was found that increasing the filtration pressure reduced the flux of cytochrome c across the films. A similar phenomenon occurred when serum was filtered, less protein passed through the films at high filtration pressures. These phenomena are explained by concentration-polarisation effects. 4. The flux of cytochrome c through the films was found to decrease in a non-linear manner as the films thickness was increased. With thin films, the flux of cytochrome c increased in a non-linear manner as the concentration of the protein in the overstanding solution was increased. With thicker films the flux was linearly dependent on concentration. These findings are interpreted as supporting the view that movement of cytochrome c occurs, at least in part, by convective flow.

Purification and characterisation of an 'elastase-like' enzyme from rabbit polymorphonuclear leucocytes.

An enzyme with proteolytic activity has been isolated from the subcellular granules of rabbit polymorphonuclear leucocytes. Purification of the enzyme involved extraction of the granule membranes with 0.01 M sodium phosphate buffer, pH 7.4, containing 1 M NaCl and 0.1% Triton X-100, followed by gel exclusion chromatography on Sephadex G-75. The enzyme hydrolysed p-nitrophenol-N-tert-butyloxylcarbonyl-L-alaninate, a synthetic substrate for elastase, but failed to hydrolyse elastin. The enzyme also hydrolysed azo-albumin with a pH optimum between 7.5 and 8.5. Inhibition studies indicated that the enzyme was a serine proteinase (EC 3.4.21.-) and it was found to have an apparent molecular weight of 25 000 by polyacrylamide gel electrophoresis. The purified enzyme behaved as a single on SDS-polyacrylamide gel electrophoresis, had a single isolectric point at pH 5.9, yet showed multiple components on centrifugation.

Oxidative stress induces caspase-independent retinal apoptosis in vitro.

Apoptosis is the mode of cell death in retinitis pigmentosa (RP), a heterogeneous group of retinal degenerations. The activation of the caspase proteases forms a pivotal step in the initiation and execution phase of apoptosis in many cells. Inhibition of caspases has been reported to prevent apoptosis in many model systems. However, we demonstrate the absence of caspase activation during retinal cell apoptosis in vitro which involves phosphatidylserine (PS) externalisation, DNA nicking and cell shrinkage. In addition, zVAD-fmk, DEVD-CHO and BD-fmk, inhibitors of the caspases, were unable to alter the characteristics or kinetics of apoptosis, implying that retinal cell death in vitro follows a caspase-independent pathway. We have previously demonstrated the ability of reactive oxygen species (ROS) to act as mediators of retinal cell apoptosis in vitro as well as the ability of antioxidants to prevent retinal cell apoptosis. Here we demonstrate the oxidative inactivation of caspases in this model of retinal apoptosis and provide evidence for an oxidative stress driven cell death pathway that does not involve caspase activity and which retains key features of apoptotic cell death. Furthermore, our data indicates that apoptotic events such as PS exposure, DNA nicking and cell shrinkage may occur independently of caspase activity.

Cell shrinkage and apoptosis: a role for potassium and sodium ion efflux.

In this study we have shown that redistribution of the lipid composition of the external and internal leaflets of the PM during apoptosis results in two main alterations in the cell surface, externalisation of PS, and a looser packing of the lipid hydrophobic head groups in the external leaflet. Significantly, neither of these alterations can be directly implicated in the mechanism of apoptotic cell shrinkage, however they do have functions in other phases of the apoptotic process. Progressional studies involving morphological and flow cytometric evaluation, and DNA gel electrophoresis revealed that apoptotic cell shrinkage is associated with a decrease in [Na+]i and [K+]i which occurs after visualisation of chromatin condensation and internucleosomal DNA fragmentation, and prior to apoptotic body formation. When apoptotic cultures were supplemented with inhibitors of the Na+, K+-ATPase pump or the Ca2+-dependent K+ channel, essentially all of the respective Na+ or K+ efflux during apoptosis can be inhibited, suggesting that essentially all of the Na+ and K+ efflux can be ascribed to active pumping via the Na+, K+-ATPase pump and the Ca2+-dependent K+ channel.

Caspase-independent photoreceptor apoptosis in vivo and differential expression of apoptotic protease activating factor-1 and caspase-3 during retinal development.

Apoptosis is the mode of photoreceptor cell death in many retinal dystrophies. Exposure of Balb/c mice to excessive levels of light induces photoreceptor apoptosis and represents an animal model for the study of retinal degenerations. Caspases have emerged as central regulators of apoptosis, executing this tightly controlled death pathway in many cells. Previously we have reported that light-induced photoreceptor apoptosis occurs independently of one the key executioners of apoptosis, caspase-3. This present study extends these results reporting on the lack of activation of other caspases in this model including caspases-8, -9, -7, and -1. Furthermore, photoreceptor apoptosis cannot be inhibited with the broad range caspase inhibitor zVAD-fmk indicating that light-induced retinal degeneration is caspase-independent. We demonstrate that cytochrome c does not translocate from mitochondria to the cytosol during photoreceptor apoptosis. We also show that during retinal development apoptotic protease activating factor (Apaf-1) protein levels are markedly decreased and this is associated with the inability to activate the mitochondrial caspase cascade in the mature retina. In addition, there is also a significant reduction in expression of caspases-3 and -9 during retinal maturation and these levels do not increase following light exposure. Finally, we show that the calcium-dependent proteases calpains are active during light-induced retinal degeneration and establish that the calcium channel blocker D-cis-diltiazem completely inhibits photoreceptor apoptosis.

Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis.

Recent work has highlighted the importance of protein post-translational modifications such as phosphorylation (enzymatic) and nitrosylation (nonenzymatic) in the early stages of apoptosis. In this study, we have investigated the levels of protein carbonylation, a nonenzymatic protein modification that occurs in conditions of cellular oxidative stress, during etopside-induced apoptosis of HL60 cells. Within 1 h of VP16 treatment, a number of proteins underwent carbonylation due to oxidative stress. This was inhibited by the antioxidant N-acetyl-L-cysteine. Among the proteins found to be carbonylated were glycolytic enzymes. Subsequently, we found that the rate of glycolysis was significantly reduced, probably due to a carbonylation mediated reduction in enzymatic activity of glycolytic enzymes. Our work demonstrates that protein carbonylation can be rapidly induced through cytotoxic drug treatment and may specifically inhibit the glycolytic pathway. Given the importance of glycolysis as a source of cellular ATP, this has severe implications for cell function.