The effect of pyrophosphate and diphosphonates on calcium transport in red cells.

The effect of 0.5 mM pyrophosphate (PPi), disodium ethane-4-hydroxy-1,1-diphosphonate (EHDP) and disodium dichloromethane diphosphonate (Cl2MDP) on the ATP-dependent Ca2+ extrusion from the human red cell ghosts was studied. PPi and Cl2MDP had no effect, when introduced into the cells or added outside to the medium. EHDP slightly increased the calcium concentration in the released cells and slightly decreased the rate constant of the calcium transport, having opposite effects when it was inside or outside the cells. PPi and the 2 diphosphonates were not found to move easily across the red cell membrane.     

Presence of lectin-like substances in the basement membrane of the human kidney glomerulus]

Crude extracts from the human glomerular basement membranes solubilized by pepsin or bacterial collagenase agglutinate normal or transformed human cells. Cytoagglutination is inhibited by N-acetyl-osamines. These properties are reminiscent of lectins. When agglutinated cells are incubated for an additional 20 hrs. period in minimal serum free medium but in presence of these basement membrane extracts, they attach to the glass and spread out.     

Impact of interactions between normal and transformed epithelial cells and the relevance to cancer

The majority of human cancers are initiated when a single cell in an epithelial sheet becomes transformed. Cell transformation arises from the activation of oncoproteins and/or inactivation of tumor suppressor proteins. Recent studies have independently revealed that interaction and communication between transformed cells and their normal neighbors have a significant impact on the fate of the transformed cell. Several reports have shown that various phenomena occur at the interface between normal and transformed epithelial cells following the initial transformation event. In epithelia of Drosophila melanogaster, transformed and normal cells compete for survival in a process termed cell competition. This review will summarize current research and discuss the impact of these studies on our understanding of how primary tumors emerge and develop within a normal epithelium.     

Ginkgo biloba extract inhibits oxygen species production generated by phorbol myristate acetate stimulated human leukocytes

A Ginkgo biloba extract (Gbe) containing flavonoids, among other compounds, was tested for the release of activated oxygen species (O-2, H2O2, OH.) during the stimulation of human neutrophils (PMNs) by a soluble agonist. The extract slows down O2 consumption (respiratory burst) of stimulated cells by its inhibitory action on NADPH-oxidase, the enzyme responsible for the reduction of O2 to O-2. Consequently, superoxide anion (O-.2) and hydrogen peroxide (H2O2) production is significantly decreased when the PMNs stimulation is done in the presence of the extract at concentrations of 500, 250 and 125 micrograms/ml. Moreover, the hydroxyl radical generation (OH.) is very much decreased at concentrations as low as 15.6 micrograms Gbe/ml, which indicates that the extract also has free radical scavenging activity. Gbe is able at least to reduce very severely the activity of myeloperoxidase contained in neutrophils. This enzyme, secreted into the intra and extracellular medium, catalyzes the oxidation of chloride (Cl-) by H2O2 to yield strong oxidants (HOCl, chloramines) which are implicated in inflammatory processes.     

Establishment of normal diploid and malignant heteroploid cell lines from non-treated and benzo(a)pyrene treated hamster embryo cell cultures.

Two normal diploid control cell lines and a heteroploid malignant transformed cell line from B(a)P treated hamster embryo cell cultures were established. The 14-month-old B(a)P transformed cell line grew 8-times faster than the 20-month-old control cell line. The control cell line showed normal diploid chromosome complement in 93% cells and heteroploidy in 7% cells while B(a)P treated line showed 83% heteroploid cells and only 17% diploid cells. This is the first report on the establishment of diploid hamster cell cultures grown for extended period.     

Tumor specific surface antigen in the culture medium of a rat cell line transformed by Rous sarcoma virus]

TSSA is detected on transformed cells by a mixed hemadsorption reaction. The medium of cultures of Rat cells transformed by Rous sarcoma virus (Prague strain, sub-group C) contains a soluble factor which specifically inhibits this reaction. This factor thus possesses the antigenic activity of TSSA which is associated with the presence of a component of molecular weight 42,000 by polyacrylamide gel electrophoresis.     

Characterization and relative abundance of maxi-chloride channels in Epstein-Barr virus (EBV) producer: B95-8 cells

Several Epstein-Barr virus (EBV)-transformed cell lines were used to investigate the pathogenesis of lymphoproliferative diseases and nasopharyngeal carcinoma. The studies focus on the events occurring inside the membrane. On only one occasion, the cell membrane of EBV-transformed B lymphocytes from a cystic fibrosis patient was found to express defective Cl channels (CFTR; Cystic Fibrosis Transmembrane conductance Regulator), as in the airway epithelial cell. No other type of channel in EBV-transformed cells has so far been investigated. In this study, the cell membrane of the B95-8 cell was examined by the patch-clamp technique and compared to the non-EBV-infected BJAB cell. The high conductance (300 pS) maxi-chloride (Cl) channel activity was the most frequently observed event in inside-out configurations. Under similar experimental conditions, we have found a significantly higher probability of detecting maxi-Cl channel activity on the cell membrane of B95-8 cells (69%) than on BJAB cells (27%), or as previously reported on resting murine B lymphocytes (38%) or intact human T lymphocytes (37%). The relative abundance of the maxi-Cl channel on B95-8 cells may be linked to EBV infection and/or secretory ability.     

Hypergravity promotes cell proliferation

When HeLa cells, chicken embryo fibroblasts, sarcoma Galliera cells, Friend leukemia virus transformed cells and human lymphocytes are cultured in a hypergravitational field (e.g. 10 X g) proliferation rate is increased by 20-30%, whereas glucose consumption per cell is lower than at 1 X g. Tracking of cell movements on gold-coated substrates reveals that cell migration is hindered at high-g. These findings suggest that under gravitational stress the cell is either capable of shifting to other metabolic pathways and/or consumes less energy at high-g than at 1 X g. This work describes ground-based investigations related to experiments to be performed on future Spacelab missions.     

Characterization of the partial inhibition, by deficiency in serum factors, of multiplication of SV3T3 transformed fibroblasts]

Partial inhibition, by serum starvation, of multiplication of SV3T3 transformed fibroblasts was studied by cultivating cells in a medium containing 1% of serum. Two different and successive states are described: a first step, with a rate of multiplication for 24 hrs, which is lowered (2 instead of 3 in a normal medium containing 10% of serum) but which remains constant (i.e. exponential multiplication), the observed inhibition being reversed by normal medium; a second step, where the rate of multiplication for 24 hrs, decreases very fast, and which is not reversible.     

A haemagglutinin in the tissue fluid of the Pacific oyster,Crassostrea gigas,with specificity for sialic acid residues in glycoproteins

Summary An agglutinin for human red cells has a specificity for sialic acid and a high affinity for bovine salivary glycoprotein. Digestion of the glycoprotein with Pronase or neuraminidase indicated that binding of sialic acid to receptors in the agglutinin is the first tepp in the mechanism of formation of a stable complex between ligand and receptor.We wish to thank Dr. P. Walne, Conwy Laboratory, MAFF, for generous supplies of Crassostrea gigas. One of us (SWH) wishes to thank the Natural Environment Research Council for financial support.     

Detection of human antigens on the surface of mouse cells transformed by chromosome transfer]

Mouse embryo cells, transformed in vitro by the transfer of chromosomes from HeLa human tumour cells, express a surface antigen (s) also found on HeLa cells. This antigen(s), which has been detected both by indirect immunofluoresence and by a 125I-protein A binding assay, is not an antigen(s) shared by both Human and Mouse cells.     

Detection of the H-RAS oncogene in human thyroid anaplastic carcinomas

We have transfected high-molecular-weight DNA from human thyroid carcinomas into murine 3T3 cells. As a result we identified several foci of morphologically distinct transformed cells in each of the tumour DNA transfected cultures. After a total of three rounds of transfection, the transformed cells were shown to form tumours in nude mice. Southern blot analysis of DNA prepared from third-round transfectants demonstrated the presence of human Alu repetitive sequences and, after hybridization with probes for known oncogenes, indicated the presence of the human H-RAS oncogene in 3T3 cells transfected with three out of four anaplastic carcinoma DNA samples. It appears therefore that activation of RAS genes may be an important event in the development of the anaplastic thyroid tumours.     

Silencing of RB1 but not of RB2/P130 induces cellular senescence and impairs the differentiation potential of human mesenchymal stem cells

Stem cell senescence is considered deleterious because it may impair tissue renewal and function. On the other hand, senescence may arrest the uncontrolled growth of transformed stem cells and protect organisms from cancer. This double function of senescence is strictly linked to the activity of genes that the control cell cycle such as the retinoblastoma proteins RB1, RB2/P130, and P107. We took advantage of the RNA interference technique to analyze the role of these proteins in the biology of mesenchymal stem cells (MSC). Cells lacking RB1 were prone to DNA damage. They showed elevated levels of p53 and p21^cip1 and increased regulation of RB2/P130 and P107 expression. These cells gradually adopted a senescent phenotype with impairment of self-renewal properties. No significant modification of cell growth was observed as it occurs in other cell types or systems. In cells with silenced RB2/P130, we detected a reduction of DNA damage along with a higher proliferation rate, an increase in clonogenic ability, and the diminution of apoptosis and senescence. Cells with silenced RB2/P130 were cultivated for extended periods of time without adopting a transformed phenotype. Of note, acute lowering of P107 did not induce relevant changes in the in vitro behavior of MSC. We also analyzed cell commitment and the osteo-chondro-adipogenic differentiation process of clones derived by MSC cultures. In all clones obtained from cells with silenced retinoblastoma genes, we observed a reduction in the ability to differentiate compared with the control clones. In summary, our data show evidence that the silencing of the expression of RB1 or RB2/P130 is not compensated by other gene family members, and this profoundly affects MSC functions.     

VIP receptors and control of short circuit current in the human intestinal clonal cell line Cl.19A

At the maximally effective concentration of 10 nM, VIP induced a marked (12.5-fold stimulation above basal), and sustained increase in short circuit current in the human intestinal epithelial cell line Cl.19A grown on permeable filters and placed in Ussing chambers. Half-maximal increase of Isc was observed for 0.1 nM VIP. This was well correlated with the VIP-stimulated adenylate cyclase activity (ED50:0.07 nM). Binding studies using 125I-VIP indicated that Cl.19A cells express a peptide-specific VIP receptor with a dissociation constant of 0.07 nM. Covalent labeling of receptors followed by SDS-PAGE analysis of membrane proteins resulted in the identification of a 63,000 dalton binding protein in Cl.19A cells.     

Effects of mistletoe (Viscum album L.) extracts on cultured tumor cells

Bacterially fermented mistletoe preparations (BFMP) were tested on rat hepatoma tissue culture (HTC) cells and human leukemia Molt 4 cells. A dose-dependent inhibition of the growth rate of the cells was observed. For both cell lines, cytostatic concentrations, expressed in weight of fresh plant, were 0.5 mg/ml culture medium for oak BFMP and 1 mg/ml for apple tree BFMP. However, the action of the two preparations was markedly different on each cell line. Non-viable HTC cells were not stained by trypan blue while non-viable Molt 4 cells were fully colored by this reagent. A lysis of cellular membranes of HTC cells was observed by electron microscopy. Furthermore, oak BFMP inhibited the growth of virus transformed 3T3-SV40 cells more than that of non-transformed 3T3 cells. In contrast to BFMP, non-fermented extracts and a purified mistletoe lectin showed a greater inhibition of the growth of Molt 4 cells than of HTC cells. Samples withdrawn at different times during fermentation gradually lost their inhibitory effect on the growth of Molt 4 cells while their action on HTC cells increased up to the 4th day of fermentation. These results are discussed in relation to the cytotoxic substances of mistletoe already characterized.     

T-antigen regulated expression reduces apoptosis of Tag-transformed human myoblasts

The generation of human myogenic cell lines could potentially provide a valuable source for cell transplantation in myopathies. The dysregulation of proliferative-differentiative signals by viral oncogenes can result in the induction of apoptosis. Whether apoptosis occurred in myogenic cells expressing large T antigen (Tag) from SV40 upon differentiation was unknown. Human muscle satellite cells were transfected with two different constructs, containing either an origin-defective SV40 genome or Tag under vimentin promoter control. When differentiation was triggered, Tag expression reduced the formation of myotubes and dead cells showing apoptotic features were present. However, the cells expressing SV40 Tag under vimentin promoter control retained their capacity to form myotubes and expressed the myofibrillar proteins as myosin heavy chain and dystrophin when Tag expression was silent. Their apoptotic rate was similar to that of untransfected cells. The observation that apoptosis can be prevented by the down-regulation of Tag suggests that the programmed cell death induced in transformed cells can be reversed, and confirms the regulatory efficiency of the human vimentin promoter.     

Interferon-beta can induce the production of plasminogen activator by cultured human cancer cells

Three cultured human cell lines, renal cancer cells (ACHN), bladder cancer cells (EJ), and fibroblasts transformed in culture by Co-60 gamma rays (KMST-6), when treated with interferon-beta, produced 1.5 to 4 times as much plasminogen activator as the untreated control cultures. This enhanced production of PA was inhibited by cycloheximide or actinomycin D.     

Membrane transport of Na and K and cell dehydration in sickle erythrocytes

The cellular concentration of Hb S plays a central role in the kinetic of Hb S polymerization and cell sickling. Blood of patients with homozygous sickle cell (SS) anemia contains a variable fraction of cells which are markedly dehydrated and have increased Hb S concentration. Since a decrease in cellular Hb S concentration reduces Hb S polymerization and sickling, the study of the processes leading to sickle cell dehydration has important pathophysiological and therapeutic implications. Sickle cell dehydration is due to cellular loss of K and Cl. K loss in sickle cells can take place via either the Ca²⁺-activated K⁺ channel, or the K?Cl cotransport, or the combined effect of oxidative damage and deformation of the red cell membrane. Inhibitors of K transport through these pathways could be used to prevent dehydration of sickle cells in vivo, provided that they can be administered safely.     

Structure and function of the type 1 insulin-like growth factor receptor

The type 1 insulin-like growth factor receptor (IGF-1R), a transmembrane tyrosine kinase, is widely expressed across many cell types in foetal and postnatal tissues. Activation of the receptor following binding of the secreted growth factor ligands IGF-1 and IGF-2 elicits a repertoire of cellular responses including proliferation, and the protection of cells from programmed cell death or apoptosis. As a result, signalling through the IGF-1R is the principal pathway responsible for somatic growth in foetal mammals, whereas somatic growth in postnatal animals is achieved through the synergistic interaction of growth hormone and the IGFs. Forced overexpression of the IGF-1R results in the malignant transformation of cultured cells: conversely, downregulation of IGF-1R levels can reverse the transformed phenotype of tumour cells, and may render them sensitive to apoptosis in vivo. Elevated levels of IGF-IR are observed in a variety of human tumour types, whereas epidemiological studies implicate the IGF-1 axis as a predisposing factor in the pathogenesis of human breast and prostate cancer. The IGF-1R has thus emerged as a therapeutic target for the development of antitumour agents. Recent progress towards the elucidation of the three-dimensional structure of the extracellular domain of the IGF-1R represents an opportunity for the rational assembly of small molecule antagonists of receptor function for clinical use.