Electrokinetic determinations of enzymatic susceptibilities of cell surface-associated RNA

Earlier investigations suggested, using electrokinetic evidence, that RNA is present at the surfaces of some types of cultured and freshly isolated cells. In this report, further investigations of the nature of cell surface RNA of cultured Ehrlich ascites (EAT) cells are reported. These experiments were carried out by determining the changes in electrophoretic mobility of EAT cells after treatment with several highly purified nucleases, neuraminidase, and hyaluronidase. The results suggested that cell surface RNA is located at surface sites separate from those susceptible to neuraminidase and hyaluronidase, that alpha and omega termini of RNA are absent from the electrokinetic surface, and that the RNA present at the cell surface might exist predominantly in a double-stranded form. A model is proposed in which cell surface RNA strand termini are buried out of the electrokinetic surface, but where RNA extends from these buried termini into the electrokinetic surface in loops.     

Electrokinetic determinations of enzymatic susceptibilities of cell surface-associated RNA

Earlier investigations suggested, using electrokinetic evidence, that RNA is present at the surfaces of some types of cultured and freshly isolated cells. In this report, further investigations of the nature of cell surface RNA of cultured Ehrlich ascites (EAT) cells are reported. These experiments were carried out by determining the changes in electrophoretic mobility of EAT cells after treatment with several highly purified nucleases, neuraminidase, and hyaluronidase. The results suggested that cell surface RNA is located at surface sites separate from those susceptible to neuraminidase and hyaluronidase, that α and ω termini of RNA are absent from the electrokinetic surface, and that the RNA present at the cell surface might exist predominantly in a double-stranded form. A model is proposed in which cell surface RNA strand termini are buried out of the electrokinetic surface, but where RNA extends from these buried termini into the electrokinetic surface in loops.     

Effects of Novel Dinuclear Cisplatinum(II) Complexes on the Electric Properties of Human Breast Cancer Cells

The aim of this study was to determine the influence of cisplatin and novel dinuclear platinum(II) complexes on the electrical properties of the membrane and the level of lipid peroxidation in the human breast cancer cell lines MDA-MB-231 and MCF-7. The basal electrical surface properties of cells are known. Changes in cell function may affect these surface properties, and those changes can be detected by electrokinetic measurements. The surface charge density of the breast cancer cell lines MDA-MB-231 and MCF-7 were measured as a function of pH. A four-component equilibrium model was used to describe the interaction between the solution ions and the breast cancer cell surface. The experimental and the theoretical charge variation curves of the breast cancer cells at pH 2.5–9 were in agreement. Measurements of the cellular malondialdehyde levels with high performance liquid chromatography were used to determine the extent of lipid peroxidation. The acid and base functional group concentrations and average association constants with hydroxyl ions were smaller in breast cancer cell membranes treated with cisplatin or novel dinuclear platinum(II) complexes compared with untreated cancer cells, and the average association constants with hydrogen ions were higher. The levels of lipid peroxidation products in breast cancer cells treated with cisplatin or novel dinuclear platinum(II) complexes were also higher than in untreated cancer cells.     

Altered electrophysiologic and pharmacologic response of smooth muscle cells on exposure to electrical fields generated by blood flow.

The flow of the blood past the vascular wall gives rise to an electrical potential. This field is calculated to have a periodic waveform with a transluminal peak-to-peak amplitude of approximately 1.35 V/m-1. Digital imaging fluorescent microscopy was used to measure changes in the membrane potentials of smooth muscle cells by following changes in the fluorescence of the potential sensitive dye, 3,3'-dipropyloxacarbocyanine iodide (di-O-C5[3]). The effect of the low level electrical field on the membrane potentials of cultured smooth muscle vascular cells was shown to cause a steady-state depolarization of approximately 10 mV. The degree of steady-state depolarization was shown to directly vary with the frequency of the applied field and the effect was not dependent on the presence of extracellular Ca+2 or Mg+2. These effects are though to be most consistent with an electroconformational coupling mechanism. The presence of this electrokinetic field was also shown to alter the electrophysiological response of smooth muscle cells treated with 5-hydroxytryptamine. Cells exposed concurrently to both 5-HT and the electrical field showed an increased membrane depolarization thus implying that the electrokinetic field may be important in both normal and pathologic cellular responses.     

Changes in the cytoskeletons during the differentiation of a myeloid leukemia cell line]

Cytoskeletons are supposed to play important roles in the cell growth, metastasis and invasion as well as the reduction of the adhesion to the matrix, which are characteristic of cancer cells. The dynamic changes of cytoskeletons were observed during the differentiation of mouse myelomonocytic leukemic cells, M1. We discuss an increase of the vimentin expression, which is correlated with the changes in the cell morphology, and a reduction of the vimentin degradation activity during the differentiation of M1 cells. And we also discuss about an increase of alpha-actinin synthesis, which is presumed to correlate with the cell adhesion and mobility.     

Involvement of leukocytes in the regulation of erythrocyte electrokinetic properties

Factor analysis was used to study the interrelations between the electrophoretic mobility of erythrocytes and some characteristics of blood leukocytes of healthy subjects and patients with arterial hypertension. In health, the stabilization of the mean mobility of erythrocytes in the electric field was shown to be independent of the changes in the blood leukogram (mainly, owing to the redistribution in the population of the cells with different properties). In disease, the pattern of this redistribution is altered, and white blood cells are involved in the regulation of the electrokinetic parameters of erythrocytes: the latter became sensitive to the total count of leukocytes and to the counts of stab neutrophils, monocytes, and eosinophils. The mechanisms of the involvement of leukocytes in the regulation of the electrokinetic properties of erythrocytes are discussed.     

Emerging role of exosome signalling in maintaining cancer stem cell dynamic equilibrium

Cancer stem cells (CSCs) are a small subset of heterogeneous cells existed in tumour tissues or cancer cell lines with self‐renewal and differentiation potentials. CSCs were considered to be responsible for the failure of conventional therapy and tumour recurrence. However, CSCs are not a static cell population, CSCs and non‐CSCs are maintained in dynamic interconversion state by their self‐differentiation and dedifferentiation. Therefore, targeting CSCs for cancer therapy is still not enough,exploring the mechanism of dynamic interconversion between CSCs and non‐CSCs and blocking the interconversion seems to be imperative. Exosomes are 30‐100 nm size in diameter extracellular vesicles (EVs) secreted by multiple living cells into the extracellular space. They contain cell‐state‐specific bioactive materials, including DNA, mRNA, ncRNA, proteins, lipids, etc. with their specific surface markers, such as, CD63, CD81, Alix, Tsg101, etc. Exosomes have been considered as information carriers in cell communication between cancer cells and non‐cancer cells, which affect gene expressions and cellular signalling pathways of recipient cells by delivering their contents. Now that exosomes acted as information carriers, whether they played role in maintaining dynamic equilibrium state between CSCs and non‐CSCs and their mechanism of activity are unknown. This review summarized the current research advance of exosomes’ role in maintaining CSC dynamic interconversion state and their possible mechanism of action, which will provide a better understanding the contribution of exosomes to dedifferentiation and stemness acquisition of non‐CSCs, and highlight that exosomes might be taken as the attractive target approaches for cancer therapeutics.     

Blood-based DNA methylation signatures in cancer: A systematic review

DNA methylation profiles are in dynamic equilibrium via the initiation of methylation, maintenance of methylation and demethylation, which control gene expression and chromosome stability. Changes in DNA methylation patterns play important roles in carcinogenesis and primarily manifests as hypomethylation of the entire genome and the hypermethylation of individual loci. These changes may be reflected in blood-based DNA, which provides a non-invasive means for cancer monitoring. Previous blood-based DNA detection objects primarily included circulating tumor DNA/cell-free DNA (ctDNA/cfDNA), circulating tumor cells (CTCs) and exosomes. Researchers gradually found that methylation changes in peripheral blood mononuclear cells (PBMCs) also reflected the presence of tumors. Blood-based DNA methylation is widely used in early diagnosis, prognosis prediction, dynamic monitoring after treatment and other fields of clinical research on cancer. The reversible methylation of genes also makes them important therapeutic targets. The present paper summarizes the changes in DNA methylation in cancer based on existing research and focuses on the characteristics of the detection objects of blood-based DNA, including ctDNA/cfDNA, CTCs, exosomes and PBMCs, and their application in clinical research.     

Dynamic enhancements of sample loading and analyte concentration in capillary isoelectric focusing for proteome studies

Capillary isoelectric focusing (CIEF) involves the use of the entire capillary filled with a mixture containing protein/peptide analytes and carrier ampholytes. Thus, the preparative capabilities of CIEF are inherently greater than most capillary-based electrokinetic separation techniques. To further increase sample loading and, therefore, the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from a solution reservoir, is demonstrated using a low p/ protein calibration kit and tryptic peptides from Saccharomyces cerevisiae. The proteins/peptides continuously migrate into the capillary and encounter a pH gradient established by carrier ampholytes originally present in the capillary for focusing and separation. Dynamic introduction and focusing in CIEF can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Depending on the mobilities of yeast peptides, the loading capacity of each peptide is measured to be around 8 to 45-fold of that obtained in conventional CIEF. By comparing with the concentrations of dilute yeast peptides originally present in the reservoir, an overall concentration factor of 1400-7700 together with excellent separation resolution is achieved using dynamic introduction and focusing. This concentration effect is further illustrated by detecting 10 pg/microL of bradykinin peptide spiked in yeast protein digest using only ultraviolet absorption.     

Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype

Tumor-associated macrophages (TAM) may have tumor-promoting activity, but it is not clear how their phenotype is achieved. In this study, we demonstrate that ovarian cancer cells switch cocultured macrophages to a phenotype similar to that found in ovarian tumors. Tumor cells caused dynamic changes in macrophage cytokine, chemokine, and matrix metalloprotease mRNA, and protein-inducing mediators that are found in human cancer. Macrophage mannose, mannose receptor, and scavenger receptors (SR-As) were also up-regulated by coculture, but not by conditioned medium. To further validate the model, we studied SR-A regulation on TAM in vitro and in vivo. Coculture of murine macrophages from mice deficient in TNF-alpha or its receptors revealed that TNF-alpha was key to SR-A induction via its p75 receptor. SR-A expression was also reduced in TAM from ovarian cancers treated with anti-TNF-alpha Abs or grown in TNF-alpha(-/-) mice. Chemical communication between tumor cells and macrophages may be important in regulating the cancer cytokine microenvironment.     

Invasiveness-triggered state transition in malignant melanoma cells

Cancer cells are considered to have high morphological heterogeneity in human melanoma tissue. Here, we report that epithelial cancer cells are dominant in different development stages of human melanoma tissues. The cellular and molecular mechanisms that maintain melanoma cells in the epithelial state are further investigated in the A2058 cell line. We find that micropore (8 µm) transwell invasion, but not superficial migration in the scratch assay, can induce remarkable morphological changes between epithelial and mesenchymal melanoma cells within 4 days. The morphological switch is associated with dynamic changes of epithelial–mesenchymal transition (EMT) hallmarks E-cadherin and vimentin. Further immunoflurencent staining and co-immunoprecipitation assay showed the uncoupling of the M3 muscarinic acetylcholine receptor (mAChR) and the p75 neurotrophin receptor (p75NTR) in epithelial melanoma cells. Specific knockdown of M3 mAChR by small interfering RNA (siRNA) significantly abrogates the transition of spindle-shaped mesenchymal cells to epithelial cells. Collectively, we report a cellular model of invasiveness-triggered state transition (ITST) in which melanoma cell invasion can induce morphological changes between epithelial and mesenchymal cells. ITST is one of the biological basis for maintaining metastatic melanoma cells in the epithelial state. Furthermore, M3 mAChR receptor-mediated ITST provides a novel therapeutic strategy to inhibit the development of malignant melanoma.     

Dynamic and electrokinetic behavior of erythrocyte membrane in diabetes mellitus and diabetic cardiovascular disease

The dynamic and electrokinetic properties of erythrocyte membrane are explored as significant indices involved in the association of diabetes and diabetic cardiovascular disease. Lipid peroxidation studies reveal malondialdehyde concentration to reach a maximum in diabetic cardiovascular patients. Lower fluidity of erythrocyte membrane implies declined ability of erythrocyte to deform in pathogenic state, which is supported by decreased osmotic resistance. Membrane protein profile modification detected by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicates a significant reduction in the quantity of ankyrin protein band 2.1 in diabetic subjects. In addition the reduction in an immunoreactive band against polyclonal anti-ankyrin antibody during Western blot analysis confirms the modification of ankyrin protein in diseased erythrocyte (reported for the first time). The electrokinetic behavior of erythrocyte membrane is monitored by laser Doppler velocimetry mode of the Nano-ZS. Changes in zeta potential values of the red blood cell membrane are consistent with decreased membrane fluidity in diseased erythrocytes (reported for the first time). Membrane potential values of control, diabetic and diabetic cardiovascular erythrocytes are -37.24+/-1.5 mV, -28.44+/-1.34 mV, and -22.21+/-1.21 mV respectively indicating a gradual lowering of zeta potential when erythrocyte membrane undergoes progressive changes - from simple agglomeration to fluid gel formation - and finally to a rigid gel.     

Hydrolysis of nucleoside triphosphate in plasma membranes of the hepatocytes of normal, regenerating and foetal livers and in cancer cells of hepatomas.

Electron histochemical studies show that changes in the nucleoside triphosphatase activity in plasma membranes of cancer cells can proceed in different directions. Some cells show a high activity of magnesium-dependent NTPase over the whole membrane surface (perimeter), while others have a low enzymic activity which is present only in certain regions of the membranes, the remaining cells possessing no enzyme activity at all. These changes are not strictly characteristic of cancer cells alone.     

Nuclear-cytoskeletal interactions: evidence for physical connections between the nucleus and cell periphery and their alteration by transformation.

The overall coordination of cell structure and function that results in gene expression requires a spatial and temporal precision that would be unobtainable in the absence of structural order within the cell. Cells contain extensive and elaborate three-dimensional skeletal networks that form integral structural components of the plasma membrane, cytoplasm, and nucleus. These skeletal networks form a dynamic tissue matrix are composed of the nuclear matrix, cytoskeleton, and extracellular matrix. The tissue matrix is an interactive network which undergoes dynamic changes as cells move and change shape. Pathologists have long recognized cancer in pathologic specimens based on the altered morphology of tumor cells compared to their normal counterparts. The structural order of cells appears to be altered in transformed cells. This structural order is reflected in the altered morphology and motility observed in transformed cells compared to their normal counterparts, however, it is unclear whether the structural changes observed in cancer cells have any functional significance. We report here on the nature of the physical connections between the nucleus and cell periphery in nontransformed cells and demonstrate that the nucleus is dynamically coupled to the cell periphery via actin microfilaments. We also demonstrate that the dynamic coupling of the nucleus to the cell periphery via actin microfilaments is altered in Kirsten-ras transformed rat kidney epithelial cells. This loss of structure-function relationship may be an important factor in the process of cell transformation.     

Influence of mono- and multivalent cations on the electrokinetic properties of Rana pipiens kidney cells.

Changes in the cation composition of the ambient cell environment, at constant ionic strengths, altered the electrokinetic properties of the surface of normal adult Rana pipiens kidney cells in a manner which reflected its valence. the effect was expressed in the changes of the magnitude of the net surface charge, at any pH, and in the shift of the isoelectric point of the cell surface.     

Change in electrokinetic potential in rat brain synaptosomes exposed to estradiol

The effect of different estradiol concentrations on electrokinetic potential of rat brain synaptosomes has been investigated. Changes in synaptosome electrokinetic potential at early stages of estradiol action are shown. The data obtained show that synaptosomes may be used as a model system to study the correlation between electrokinetic potential of cells and their physiological and biochemical status.     

A phenotypic switch in the dispersal strategy of breast cancer cells selected for metastatic colonization

An important question in cancer evolution concerns which traits make a cell likely to successfully metastasize. Cell motility phenotypes, mediated by cell shape change, are strong candidates. We experimentally evolved breast cancer cells in vitro for metastatic capability, using selective regimes designed to simulate stages of metastasis, then quantified their motility behaviours using computer vision. All evolved lines showed changes to motility phenotypes, and we have identified a previously unknown density-dependent motility phenotype only seen in cells selected for colonization of decellularized lung tissue. These cells increase their rate of morphological change with an increase in migration speed when local cell density is high. However, when the local cell density is low, we find the opposite relationship: the rate of morphological change decreases with an increase in migration speed. Neither the ancestral population, nor cells selected for their ability to escape or invade extracellular matrix-like environments, displays this dynamic behavioural switch. Our results suggest that cells capable of distant-site colonization may be characterized by dynamic morphological phenotypes and the capacity to respond to the local social environment.     

Tissue transglutaminase, inflammation, and cancer: how intimate is the relationship?

Despite significant advances in surgery and biology, cancer remains a major health problem. It is now well accepted that metastasis and cancer cells’ acquired or inherent resistance to conventional therapies are major roadblocks to successful treatment. Chronic inflammation is an important driving force that provides a favorable platform for cancer’s progression and development and suggests a link between inflammation and metastatic transformation. However, how chronic inflammation contributes to metastatic cell transformation is not well understood. According to the current theory of cancer progression, a small subpopulation of cancer stem cells (CSCs) in tumors is responsible for their metastasis, resistance, and sustenance. Whether CSCs originate from normal stem cells or from dedifferentiation of terminally differentiated cells remains unknown. Recent evidence indicates that stem cells are not unique; malignant or nonmalignant cells can reprogram and de-differentiate to acquire a stemness phenotype. Thus, phenotypic plasticity may exist between stem cells and non-stem cells, and a dynamic equilibrium may exist between the two phenotypes. Moreover, this equilibrium may shift in one direction or another on the basis of contextual signals in the microenvironment that influence the interconversion between stem and non-stem cell compartments. Whether the inflammatory microenvironment influences this interconversion and shifts the dynamic equilibrium towards stem cell compartments remains unknown. We recently found that aberrant tissue transglutaminase (TG2) expression induces the mesenchymal transition (EMT) and stem cell characteristics in epithelial cells. This finding, in conjunction with the observation that inflammatory signals (e.g., TGFβ, TNFα, and NF-κB) which induce EMT, also induce TG2 expression, suggests a possible link between TG2, inflammation, and cancer progression. In this review, we summarize TG2-driven processes in inflammation and their implications in cancer progression.     

Cancer metabolism and tumor microenvironment: fostering each other?

Science China Life Sciences - The changes associated with malignancy are not only in cancer cells but also in environment in which cancer cells live. Metabolic reprogramming supports tumor cell...