Dynamic interaction between breast cancer cells and osteoblastic tissue: comparison of two- and three-dimensional cultures

Breast cancer cell colonization of osteoblast monolayers grown in standard tissue culture (2D) is compared to colonization of a multi-cell-layer osteoblastic tissue (3D) grown in a specialized bioreactor. Colonization of 3D tissue recapitulates events observed in clinical samples including cancer penetration of tissue, growth of microcolonies, and formation of "Single cell file" commonly observed in end-stage pathological bone tissue. By contrast, adherent cancer cell colonies did not penetrate 2D tissue and did not form cell files. Thus, it appears that 3D tissue is a more biologically (clinically) relevant model than 2D monolayers in which to study cancer cell interactions with osteoblastic tissue. This direct comparison of 2D and 3D formats is implemented using MC3T3-E1 murine osteoblasts and MDA-MB-231 human metastatic breast cancer cells, or the metastasis-suppressed line, MDA-MB-231BRMS1, for comparison. When osteoblasts were co-cultured with metastatic cells, production of osteocalcin (a mineralization marker) decreased and secretion of the pro-inflammatory cytokine IL-6 increased in both 2D and 3D formats. Cancer cell penetration of the 3D tissue coincided with a changed osteoblast morphology from cuboidal to spindle-shaped, and with osteoblasts alignment parallel to the cancer cells. Metastasis-suppressed cells did not penetrate 3D tissue, did not cause a change in osteoblast morphology or align in rows. Moreover, they proliferated much less in the 3D culture than in the 2D culture in a manner similar to their growth in bone. In both systems, the cancer cells proliferated to a greater extent with immature osteoblasts compared to more mature osteoblasts.     

Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia model

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara‐C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara‐C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara‐C were elucidated by whole‐exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara‐C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.     

Disulfide stress: a novel type of oxidative stress in acute pancreatitis

Glutathione oxidation and protein glutathionylation are considered hallmarks of oxidative stress in cells because they reflect thiol redox status in proteins. Our aims were to analyze the redox status of thiols and to identify mixed disulfides and targets of redox signaling in pancreas in experimental acute pancreatitis as a model of acute inflammation associated with glutathione depletion. Glutathione depletion in pancreas in acute pancreatitis is not associated with any increase in oxidized glutathione levels or protein glutathionylation. Cystine and homocystine levels as well as protein cysteinylation and γ-glutamyl cysteinylation markedly rose in pancreas after induction of pancreatitis. Protein cysteinylation was undetectable in pancreas under basal conditions. Targets of disulfide stress were identified by Western blotting, diagonal electrophoresis, and proteomic methods. Cysteinylated albumin was detected. Redox-sensitive PP2A and tyrosine protein phosphatase activities diminished in pancreatitis and this loss was abrogated by N-acetylcysteine. According to our findings, disulfide stress may be considered a specific type of oxidative stress in acute inflammation associated with protein cysteinylation and γ-glutamylcysteinylation and oxidation of the pair cysteine/cystine, but without glutathione oxidation or changes in protein glutathionylation. Two types of targets of disulfide stress were identified: redox buffers, such as ribonuclease inhibitor or albumin, and redox-signaling thiols, which include thioredoxin 1, APE1/Ref1, Keap1, tyrosine and serine/threonine phosphatases, and protein disulfide isomerase. These targets exhibit great relevance in DNA repair, cell proliferation, apoptosis, endoplasmic reticulum stress, and inflammatory response. Disulfide stress would be a specific mechanism of redox signaling independent of glutathione redox status involved in inflammation.     

An immune edited tumour versus a tumour edited immune system: prospects for immune therapy of acute myeloid leukaemia

Cell based therapies for acute myeloid leukaemia (AML) have made significant progress in the last decade benefiting the prognosis and survival of patients with this aggressive form of leukaemia. Due to advances in haematopoietic stem cell transplantation (HSCT) and particularly the advent of reduced intensity conditioning (RIC), the scope of transplantation has now extended to those patients previously ineligible due to age and health restrictions and has been associated with a decrease in transplant related mortality. The apparent graft versus leukaemia (GvL) effect observed following HSCT demonstrates the potential of the immune system to target and eradicate AML cells. Building on previously published pre-clinical studies by ourselves and others, we are now initiating a Phase I clinical study in which lentiviral vectors are used to genetically modify AML cells to express B7.1 (CD80) and IL-2. By combining allogeneic HSCT with immunisation, using the autologous AML cells expressing B7.1 and IL-2, we hope to stimulate immune eradication of residual AML cells in poor prognosis patients that have achieved donor chimerism. In this report we describe the background to cell therapy based approaches for AML, and discuss difficulties associated with the deployment of a chronically stimulated, hence exhausted/depleted immune system to eradicate tumour cells that have already escaped immune surveillance.This article is a symposium paper from the “Robert Baldwin Symposium: 50 years of Cancer Immunotherapy”, held in Nottingham, Great Britain, on 30th June 2005.     

Discrimination between apoptosis and necrosis of neurons under oxidative stress

Flow cytometric studies of rat cerebellum neurons are described under conditions inducing cell death. Using a double labeling technique, discrimination between apoptotic and necrotic cell transformations is demonstrated. Histidine containing neuropeptides were found to regulate cell stability, taking part in selection of the preferable way of neuronal death under oxidative stress.     

Mathematical model of acute myeloblastic leukaemia: an investigation of the relevant kinetic parameters

A simple model of acute myeloblastic leukaemia (AML) development is introduced, explicitly including cell growth, cell differentiation and cell-cell interaction. Each of these processes is described by a single model parameter. It is hypothesized that the leukaemic cell is characterized by an alteration of only one of these processes. The kinetic behaviour of the AML system is examined separately for possible alterations of each of the three parameters describing the three processes involved. It is shown that, on the basis of the existing data on AML kinetics, the alteration of the growth and cell-cell interaction parameters can be eliminated as a possible source of AML. Thus kinetic data support the modification of the differentiation process as the origin of the AML state. Further, the growth characteristics of normal and leukaemic cells in the presence of each other are analysed. It is shown how the initial growth of leukaemic cells depends on the difference in the differentiation of normal and leukaemic cells and how the same difference determines the decay of normal cells in the presence of the predominantly leukaemic population. Correlations between the kinetic parameters of the normal and leukaemic populations are suggested to characterize the leukaemic state.     

MicroRNA control of myelopoiesis and the differentiation block in acute myeloid leukaemia

In the relatively short period of time since their discovery, microRNAs have been shown to control many important cellular functions such as cell differentiation, growth, proliferation and apoptosis. In addition, microRNAs have been demonstrated as key drivers of many malignancies and can function as either tumour suppressors or oncogenes. The haematopoietic system is not outside the realm of microRNA control with microRNAs controlling aspects of stem cell and progenitor self-renewal and differentiation, with many, if not all, haematological disorders associated with aberrant microRNA expression and function. In this review, we focus on the current understanding of microRNA control of haematopoiesis and detail the evidence for the contribution and clinical relevance of aberrant microRNA function to the characteristic block of differentiation in acute myeloid leukaemia.     

Neuroprotection by tempol in a model of iron-induced oxidative stress in acute ischemic stroke

Studies suggest iron exacerbates the damage caused by ischemic stroke. Our aim was to elucidate the effect of iron overload on infarct size after middle cerebral artery occlusion (MCAO) and to evaluate the efficacy of tempol, a superoxide dismutase mimetic, as a neuroprotective agent. Rats were administered iron +/- tempol before MCAO; control rats received saline. The middle cerebral artery was occluded for 24 h, and the size of the resultant infarct was assessed and expressed as the percentage of the hemisphere infracted (%HI). Iron treatment increased infarct size compared with control (51.83 +/- 3.55 vs. 27.56 +/- 3.28%HI iron treated vs. control, P = 0.01); pretreatment with tempol reversed this (51.83 +/- 3.55 vs. 26.09 +/- 9.57%HI iron treated vs. iron + tempol treated, P = 0.02). We hypothesized that reactive oxygen species (ROS) were responsible for the iron-induced damage. We measured ROS generated by exogenous iron in brain and peripheral vasculature from rats that had not undergone MCAO. There was no increase in ROS production in the brain of iron-treated rats or in brain slices incubated with iron citrate. However, ROS generation in carotid arteries incubated with iron citrate was significantly increased. ROS generation from the brain was assessed after MCAO by dihydroethidine staining; there was a dramatic increase in the ROS generation by the brain in the iron-treated rats compared with control 30 min after MCAO. We propose that iron-induced ROS generation in the cerebral vasculature adds to oxidative stress during an ischemic episode after the disruption of the blood-brain barrier.     

Survival and antioxidant defence of the yeast Saccharomyces cerevisiae during starvation and oxidative stress

The role of catalase in response of the yeast Saccharomyces cerevisiae to oxidative stress induced by hydrogen peroxide under starvation was investigated. It was shown that under conditions used in this study 0.5 mM H2O2 did not change the number of viable cells in the wild strain YPH250, but this parameter was decreased by 15% in the acatalsaemic strain YWT1. Cells treatment with 0.5 mM H2O2 for 30 min did not modify the levels of carbonyl proteins in the parental strain, but caused its 1.4-fold increase in the defective strain. The observed 1.5-fold activation of catalase in the wild strain cells in response to H2O2-stress suggests that under starvation conditions catalase can be involved in the yeast cell protection, particularly they can prevent oxidative modification of some antioxidant and associated enzymes.     

The relationship between excitotoxicity and oxidative stress in the central nervous system

Excitotoxicity and oxidative stress are two phenomena that have been repeatedly described as being implicated in a wide range of disorders of the nervous system. Such disorders include several common idiopathic neurological diseases, traumatic brain injury, and the consequences of exposure to certain neurotoxic agents. While there is evidence that metabolic derangements can laed to these adverse processes, and that these processes may synergize in their damaging effects, the degree of interdependence, and the causal relation between them is not clear. The intent of this review is to delineate potential mechanisms which may unit hyperexcitation to the excessive generation of reactive oxygen species. The degree of linkage between these events appears rather strong. It is likely that excitoxicity frequency leads to a pro-oxidant condition but that high rates of generation of reactive oxygen species are not invariably accompanied by a hyperexcited neuronal state Both excitoxic and ‘oxidotoxic’ states result from the failure of normal compensatory anti excitatory and antioxidant mechanisms to maintain cellular homeostatis.     

Reversible and irreversible modifications of skeletal muscle proteins in a rat model of acute oxidative stress

Oxidative stress caused by an imbalance of the production of “reactive oxygen species” (ROS) and cellular scavenging systems is known to a play a key role in the development of various diseases and aging processes. Such elevated ROS levels can damage all components of cells, including proteins, lipids and DNA. Here, we study the influence of highly reactive ROS species on skeletal muscle proteins in a rat model of acute oxidative stress caused by X-ray irradiation at different time points. Protein preparations depleted for functional actin by polymerization were separated by gel electrophoresis in two dimensions by applying first non-reductive and then reductive conditions in SDS-PAGE. This diagonal redox SDS-PAGE revealed significant alterations to intra- and inter-molecular disulfide bridges for several proteins, but especially actin, creatine kinase and different isoforms of the myosin light chain. Though the levels of these reversible modifications were increased by oxidative stress, all proteins followed different kinetics. Moreover, a significant degree of protein was irreversibly oxidized (carbonylated), as revealed by western blot analyses performed at different time points.     

Crystalline inclusions in the cytoplasm and nuclei of cells of acute myeloid leukaemia

In a survey by electron microscopy of peripheral blood and/or bone marrow from 230 adult patients with acute myeloid leukaemia, five were observed to contain crystalline inclusions in the cytoplasm of the leukaemic cells and a sixth contained crystals in the nuclei. In four cases, two of FAB type M2 and two of M4, the cytoplasmic crystals were hexagonal in section and 1-2 micron long. Two examples showed internal periodicities in the range 3.3-4.0 nm when the electronmicrographs were analysed by optical diffractometry. A single case of M1 contained smaller trapezoidal crystals with a 4.9nm periodicity. The sixth patient, with unusual cytological abnormalities and a rare t(3; 6) chromosomal translocation, contained six-sided crystals in the nuclei of some relatively undifferentiated cells. To the best of our knowledge such intranuclear crystals have not previously been reported in leukaemia. The relevance of the crystals to the leukaemic process is discussed.     

Relationship between respiratory enzymes and survival of Escherichia coli under starvation stress in lake water

Survival, electron transport system (ETS) activity and the activity of NADH and succinate dehydrogenase of Escherichia coli ML30 were studied under starvation stress at different temperatures in a filtered-autoclaved lake water microcosm. ETS activity in E. coli declined rapidly at 30 degrees C but more slowly at 4 degrees and 15 degrees C over a 20 d starvation period. The decrease in ETS activity in E. coli only started after 6 d of incubation at 4 degrees C and 15 degrees C. Viability of E. coli, as determined by plate counts, declined faster at 37 degrees C than at the other temperatures and remained highest at 4 degrees C in filtered-autoclaved lake water. There was also a significant cell size reduction at 37 degrees C in filtered-autoclaved lake water but not at 4 degrees C. ETS activity after up to 16 d of starvation increased after the addition of nutrient broth to the filtered-autoclaved lake water at 15 degrees C and 30 degrees C suggesting that cells were still able to respond to nutrients, even after prolonged starvation. The response to the addition of nutrient broth, however, declined with the length of the starvation period. The activity of both succinate and NADH dehydrogenase declined over a 13 d starvation period. The loss of activity was fastest at 37 degrees C compared to lower incubation temperatures but even at 4 degrees C, a significant proportion of the activity was lost over the 13 d period.     

Topological changes in DNA as a reflection of adaptation of Escherichia coli to oxidative stress under glucose starvation and transition to growth

Changes in the topological state of DNA occur in a starving Escherichia coli culture under oxidative stress caused by the addition of hydrogen peroxide. The addition of a carbon and energy source to this culture results in a second stress reaction. This supports previous data indicating that different mechanisms are responsible for the cell defense against oxidative stress in exponential and starving E. coli cultures. Polyamine synthesis is involved in the cell adaptation to the stress. Putrescine binding to DNA and its dissociation seem to modulate the DNA topological state, which regulates the expression of the adaptive genes. An increase in the activity of the polyamine-synthesizing system in response to oxidative stress leads to a putrescine flux across the cytoplasmic membrane, due to which the antioxidant activity of putrescine protects the membrane phospholipids and contributes to the restoration of the cell energy-generating function.     

Changes in the topological state of DNA duringEscherichia coli adaptation to oxidative stress under glucose starvation and after the transition to growth

Changes in the topological state of DNA occur in a starvingEscherichia coli culture under oxidative stress caused by the addition of hydrogen peroxide. The addition of a carbon and energy source to this culture results in a second stress reaction. This supports previous data indicating that different mechanisms are responsible for the cell defense against oxidative stress in exponential and starvingE. coli cultures. Polyamine synthesis is involved in the cell adaptation to the stress. Putrescine binding to DNA and its dissociation seem to modulate the DNA topological state, which regulates the expression of the adaptive genes. An increase in the activity of the polyamine-synthesizing system in response to oxidative stress leads to a putrescine flux across the cytoplasmic membrane, due to which the antioxidant activity of putrescine protects the membrane phospholipids and contributes to the restoration of the cell energy-generating function     

Hematological indices and oxidative stress biomarkers response to the starvation of Clarias gariepinus

Starvation effects for five weeks on energy reserves, oxidative stress and hematological indices in Nile catfish Clarias gariepinus was studied. The low protein level in starved fish may result from the lowering effect of prolonged starvation on protein synthesis rather than due to its degenerating protein. Moreover, the elevated level of serum amino acids may promote gluconeogenesis in liver. In addition, the lipid depletion in starved fish may be related to the preferential uses of lipids as an energy to starve fish. Also, unchanged glycemic level may introduce a potent evidence for the presence of active gluconeogenesis, depending on both amino and fatty acids precursors. Also, kidney and liver showed disturbances in metabolites associated with oxidative damage such as elevations in total peroxide, carbonyl protein and DNA fragmentation; these may cause dysfunction to these organs after five weeks of starvation. Total peroxide, carbonyl protein and DNA fragmentation were significantly increased in gills, liver and kidney by 29.9, 30.9 and 30.5; 83.6, 84.6 and 53.7; 82.4, 43.3 and 75.7%, respectively. Starvation induced severe anemia and loss of body weight in the fish. However, white muscle did not show any oxidative damage after five weeks of starvation.