Cell junction and cyclic AMP: III. Promotion of junctional membrane permeability and junctional membrane particles in a junction-deficient cell type

Summary The cyclic nucleotide effect on junction was studied in C1-1D cells, a mouse cancer cell type that fails to make permeable junctions in ordinary confluent culture. Upon administration of cyclic AMP, dibutyryl cyclic AMP, dibutyryl cyclic AMP plus caffeine (db-cAMP-caffeine), or cholera toxin (an adenylate cyclase activator), the cells acquired permeable junctions; they became electrically coupled and transferred fluorescent tracer molecules among each other—a transfer exhibiting the molecular size limit of permeation of normal cell-to-cell channels. The effect took several hours to develop. With the db-cAMP-caffeine treatment, junctional permeability emerged within two hours in one-fifth of the cell opopulation, and within the next few hours in the entire population. This development was not prevented by the cytokinesis inhibitor cytochalasin B. Permeable junctions formed also in two other conditions where the cell-endogenous cyclic AMP level may be expected to increase: serum starvation and low cell density. After three weeks of starving the cells of serum, a junctional permeability arose in confluent cultures, which on feeding with serum disappeared within two to three days. At low cell density, namely below confluency, the cells made permeable junctions, unstarved. In cultures of rather uniform density, the frequency of permeable junctions was inversely related to the average density, over the subconfluent range; at densities of about 1×10⁴ cells/cm², where the cells had few mutual contacts, 80% of the pairs presumed to be in contact were electrically coupled. In cultures with adjoining territories of high (confluent) and low cell density, there was coupling only in the last, and in this low-density state the cells were also capable of coupling with other mammalian cell types (mouse 3T3-BalbC and human Lesch-Nyhan cells).Correlated electron microscopy of freeze-fractured cell junctions showed no membrane differentiation in confluent C1-1D cultures. The junctions acquired differentiations, namely particle clusters of gap junction and strands of tight junction, upon cyclic nucleotide application or serum starvation and in the lowdensity condition. With db-cAMP-caffeine, these differentiations appeared within 4 hr of the treatment (confluent cultures), growing in size over the next hours. Treatment with cycloheximide, but not with cytochalasin B, prevented the development of recognizable gap junction and tight junction in cultures supplied with db-cAMP-caffeine.     

Changes in intercellular exchange intensity in an L cell culture]

Intercellular junctions permeable to fluorescein Na were studied with the aid of intracellular glass microelectrodes in the cultures of transformed mouse-embryo cells (L-strain). The degree of coupling was correlated with the cell culture density. In addition, the degree of coupling increased after short-time incubated of the cells with 1mM lanthanum. Such an incubation did not influence other features of cells: the value of the membrane potential, the character of growth or the inclusion of the vital dye neutral red. Some other factors stimulated cellular aggregation (concanavalin A, protamine, decreasing the pH) but did not influence the degree of coupling.     

Highly permeable contacts between epithelial cells in culture]

Cultured epithelial cells producing monolayered sheets were used to study intercellular contacts permeable to sodium fluorescein. Cells in dense cultures were more capable of forming permeable junctions than cells of sparse cultures. In addition, the standard microelectrode technique revealed some differences in cellular membrane potentials in dense and sparse cultures. A possible correlation is discussed between intercellular contact formation and other features of cells depending on cell culture density.     

The use of lead as an ionic tracer for investigating routes of passive fluid transfer

Several inorganic ions, including lead, barium, silver, and thallium, have been tested as possible tracers for demonstrating fluid-accessible channels in functional epithelia at the ultrastructural level. The most useful of the ionic tracers examined was the lead (plumbous) ion, administered for short time intervals (less than 2 min) and "captured" with phosphate used as the buffer in the fixative. Passive fluid and ion-accessible channels of rat parotid salivary gland have been examined with this method. At short tracer infusion times (0.5-1.0 min), localization of the tracer was primarily extracellular, although intracellular deposits were observed in the following sites: smooth membrane-delimited endocytic vesicles of both epithelial and connective tissue cells, inner Golgi cisternae, and occasional cisternae of rough endoplastic reticulum. The lead tracer readily penetrated tight junctions between parotid acinar cells but rarely passed through the tight junctions between intercalated duct cells and did not penetrate junctions of striated duct cells. Fat cells observed in the stroma of this gland were the only cells that exhibited lead tracer in the cytosol, suggesting that the plasmalemma of this cell type is more permeable to exogenous ions than the plasmalemma of other cell types present in this gland.     

Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium

Summary Studies of brain microvessel endothelial cell physiology and blood-brain barrier properties are often hampered by the requirement of repeatedly producing and characterizing primary endothelial cell cultures. The use of viral oncogenes to produce several immortalized brain microvessel cell lines has been reported. The resulting cell lines express many properties of the blood-brain barrier phenotype but do not completely mimic primary endothelial cells in culture. As immortalized brain microvessel endothelial cell lines have not yet been produced from mice, we transformed mouse brain endothelial cells with the adenovirus E1A gene using a retroviral vector (DOL). Eight of 11 clones produced exhibited an endothelial-like cobblestone morphology and were characterized as endothelial with a panel of antibodies, lectins, and ultrastructural criteria. These cells are endothelial in origin and share ultrastructural features with primary cultures of endothelial cells. Examination of freeze fracture and transmission electron micrographs show adherens junctions exist between the transformed cells, and culture in astrocyte-conditioned medium induces the formation of gap junctions. This is one indication that responses to astrocyte-derived factors are retained by the transformed cell lines.     

LOW-RESISTANCE JUNCTIONS BETWEEN CANCER CELLS IN VARIOUS SOLID TUMORS

Electrical coupling, which reveals the presence of specialized low-resistance intercellular junctions, has now been found in four types of tumors, Sarcoma 180, Novikoff hepatoma, and Morris hepatomas 3924-A and 7777. Coupled cancer cells were distinguished from coupled normal cells by intracellular marking techniques. Although the evidence suggests that coupling may be extensive in some cases, it is not possible to say that the coupling was normal. In particular, the results do not exclude less obvious defects in the specialized junctions, such as abnormal distribution or decreased permeability to molecules other than small inorganic ions. The results are discussed in relation to previous studies of coupling in Novikoff hepatomas and in cultures of S1801 and II cell lines.     

Low resistance junctions between normal and between virus transformed fibroblasts in tissue culture

The occurrence of low resistance junctions between normal chick embryo fibroblasts and between fibroblasts transformed with Rous sarcoma virus in tissue culture was studied with intracellular microelectrodes. The results showed that these junctions were present between normal chick fibroblasts in proliferating cultures as well as between cells in ‘density dependent inhibited cultures’. Mechanical injury to a fibroblast within a small group of coupled cells caused the injured fibroblast to uncouple immediately from its neighboring cells without interrupting coupling between the healthy uninjured cells. In the case of fibroblasts transformed with a Rous sarcoma virus, the results further showed that low resistance junctions were present when the transformation appeared in the infected cells and remained present thereafter.     

Cell permeabilization by poliovirus 2B viroporin triggers bystander permeabilization in neighbouring cells through a mechanism involving gap junctions

Poliovirus 2B protein is a well‐known viroporin implicated in plasma membrane permeabilization to ions and low‐molecular‐weight compounds during infection. Translation in mammalian cells expressing 2B protein is inhibited by hygromycin B (HB) but remains unaffected in mock cells, which are not permeable to the inhibitor. Here we describe a previously unreported bystander effect in which healthy baby hamster kidney (BHK) cells become sensitive to HB when co‐cultured with a low proportion of cells expressing poliovirus 2B. Viroporins E from mouse hepatitis virus, 6K from Sindbis virus and NS4A protein from hepatitis C virus were also able to permeabilize neighbouring cells to different extents. Expression of 2B induced permeabilization of neighbouring cell lines other than BHK. We found that gap junctions are responsible mediating the observed bystander permeabilization. Gap junctional communication was confirmed in 2B‐expressing co‐cultures by fluorescent dye transfer. Moreover, the presence of connexin 43 was confirmed in both mock and 2B‐transfected cells. Finally, inhibition of HB entry to neighbouring cells was observed with 18α‐glycyrrhethinic acid, an inhibitor of gap junctions. Taken together, these findings support a mechanism involving gap junctional intercellular communication in the bystander permeabilization effect observed in healthy cells co‐cultured with poliovirus 2B‐expressing cells.     

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.     

Inhibition of chicken embryo lens differentiation and lens junction formation in culture by pp60v-src.

A culture system was developed which permitted the differentiation of chicken lens epithelial cells to lentoid bodies which contained several cell layers, accumulated high levels of delta-crystallin, and produced extensive gap junctions. This differentiation process was prevented when the cells were infected with a temperature-sensitive src mutant of Rous sarcoma virus and maintained at the permissive temperature. These transformed cells continued to proliferate and also synthesized the major lens gap junction protein, MP28, at near-normal rates. However, this MP28 was not assembled to produce gap junctions. Cultures shifted to the nonpermissive temperature formed lentoid bodies similar to those in uninfected lens cultures, including the establishment of gap junctions containing MP28.     

The chick retinal pigment epithelium grown on permeable support demonstrates functional polarity

The retinal pigment epithelium (RPE) from the chick embryo was cultured on permeable support. Using confluent cultures and analysis of the incubation medium, the present study demonstrates that RPE cells cultured on permeable membrane retain functional polarity, a characteristic of the RPE in vivo. The degree of intercellular permeability in the confluent RPE cultures was estimated by following [3H]inulin movement from the apical side to the basal side of the cultures. Twenty-four hours after exposure of the apical side of the culture to [3H]inulin, the 3H concentration in the apical medium remained at 3.4 to 4.4 times of that in the basal medium. The barrier function of RPE disappears in the presence of EDTA. Net unidirectional fluid movement from the apical side of the cultures to the basal side of the cultures is regularly observed in confluent RPE cultures. The rate varies among different preparations of cultures and the highest is 1.60-1.84 microliters/cm2/h. When cultures are given 26 h of [35S]methionine, more than 20 bands with molecular weights ranging from 20,000 to greater than 250,000 Da can be detected in the medium as assessed by autoradiography of SDS-polyacrylamide gels. While six macromolecules appear to be equally concentrated in the basal medium and the apical medium, the majority are in higher concentration in the basal medium. Analysis of the 10% TCA-precipitable fraction of the medium showed that the specific activities in the apical medium and basal medium were 24.0 +/- 0.4 X 10(6) and 46.4 +/- 0.2 X 10(6) (mean +/- SEM, N = 8) cpm/ml/mg RPE protein, respectively. When cultures react with VIP (vasoactive intestinal peptide), the elevated intracellular cyclic AMP is extruded into the medium bathing the cells. However, the rate of extrusion into the basal medium is twice as fast as that into the apical medium. Electron microscopy of the confluent RPE cultures shows morphological polarization of the cells. The intercellular spaces appear to be closed at the apical side of the cells by junctional complexes consisting of tight junctions, zonular adherens junctions, and gap junctions.     

Morphology, cell-cell interactions, and migratory activity of IAR-2 epithelial cells transformed with the RAS oncogene: contribution of cell adhesion protein E-cadherin

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphologic and mi gration characteristics of epithelial cells of Iar1162 and IAR1170 clones derived from a mixed culture of on cogene N-RasV12-transformed cell line IAR-2. It was found that the mutant oncogene RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, formed E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the mutant oncogene RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR 1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of locomotor activity in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the locomotor activity of transformed epithelial cells and plays an important role in their collective migration.     

Effects of extracellular calcium and magnesium on junctional intercellular communication in human fibroblasts

The role of extracellular calcium and magnesium in intercellular communication via permeable junctions was studied by measuring metabolic co-operation between ASS- and ASL- human fibroblasts. Communication through pre-existing junctions was not affected by the removal of extracellular calcium and magnesium from the medium. On the other hand, the establishment of communication through new permeable junctions, when ASS- and ASL- cells were mixed, was dependent on the presence of extracellular calcium and magnesium.     

Morphology,cell-cell interactions,and migratory activity of IAR-2 epithelial cells transformed with the <Emphasis Type="Italic">RAS</Emphasis> oncogene: Contribution of cell adhesion protein E-Cadherin

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphological and migration characteristics of epithelial cells of IAR1162 and IAR1170 clones derived from a mixed culture of N-RasV12 oncogene-transformed IAR-2 cell line. It was found that the oncogenic RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, assembled E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the oncogenic RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of cell motility in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the motility of transformed epithelial cells and plays an important role in their collective migration.     

Selectivity in metabolic cooperation between cultured mammalian cells

Selective communication between cultured mammalian cells was detected as selectivity in metabolic cooperation. Whilst the majority of the cell types examined (human skin fibroblast, PC13, G3, Don, PyY) showed metabolic cooperation at almost all (>95%) of their homotypic cell-to-cell contacts, they did not necessarily show cooperation at such a high proportion of their heterotypic contacts. Less than 10% of G3/human fibroblast contacts, and usually less than 30% of G3/PC13 contacts were observed to be positive for metabolic cooperation. L cells differed from these other cell types in that they formed permeable junctions at a greater proportion of their heterotypic cell-to-cell contacts (contacts between L and PyY cells) than their homotypic contacts. We question why it was that the contacts between any two poorly-compatible cell types were positive for metabolic cooperation in only a small proportion of cases. We could find no indication that this phenomenon was attributable to heterogeneity within the cell stocks. Time course studies upon PC13 and G3 cells showed that the proportion of cooperation-positive contacts was not constant but that it continued to increase over many hours of co-culture. In comparing the homotypic and heterotypic interactions of these cell types, selectivity manifested as a difference in the rate of appearance of permeable junctions. We discuss possible explanations for these findings.     

The ultrastructure of the rat primary decidual zone

The rat primary decidual zone (PDZ) is a transitory, avascular region of transformed fibroblasts surrounding the implanting embryo. Tracer studies have indicated that the PDZ is selectively permeable to macromolecules, permeability decreasing with increasing molecular weight of the tracer. To clarify the morphological basis of the permeability barrier, we have studied the ultrastructure of the PDZ with particular emphasis on the intercellular features and cellular junctions. The cells of the PDZ were large and tightly packed; their apposed membranes showed extensive interdigitations in some regions, but elsewhere they were relatively straight. Tight junctions, gap junctions, and desmosomelike junctions were observed between decidual cells. The tight junctions usually consisted of one or two points of membrane fusion, and they were oriented both parallel and perpendicular to the long axis of the PDZ. These junctions were frequently associated with gap junctions. Scattered pockets of dilated extracellular space between decidual cells contained collagen fibrils and an amorphous, dense material. These extracellular components were also sequestered by the decidual cells in deep invaginations of the cell surface that were continuous with the extracellular space. Decidual cells also exhibited flangelike processes that penetrated the basal laminae of the adjacent epithelium and capillary endothelium. Our present observations indicate that decidual cells are connected by tight junctions, and a previous study demonstrated that macromolecules up to 40 kDa readily cross the PDZ; hence, the tight junctions appear to be discontinuous. We suggest that the structures restricting the movement of large macromolecules (66 kDa and larger) across the PDZ from blood vessels to the embryo may include discontinuous tight junctions, membrane interdigitations, and amorphous intercellular material.     

Intercellular communication and tissue growth

Summary Three cancer cell strains that fail to make permeable membrane junctions were tested for ability to transfer an endogenous hypoxanthine derivative from cell to cell. The cells of these strains, loaded with³H-hypoxanthine, were grown in contact with cells from a mutant line incapable of incorporating exogenous hypoxanthine. The transfer of the³H-hypoxanthine derivative to the mutant cells was determined by radio-autography and, in the same preparations, the presence of permeable membrane junctions was determined by intercellular fluorescein tracer diffusion and electrical measurement. The cells of the three strains showed no transfer of hypoxanthine derivative to contiguous mutant cells; the cells that make permeable junctions did show such transfer, under the same conditions.In contrast to this contact-requiring mode of transfer, a contact-independent transfer phenomenon was observed with these three cancer cell strains.     

The lesion in a metabolic cooperation-defective embryonal carcinoma variant is of broad specificity with regard to metabolite and to contiguous cell type

Homotypic and heterotypic permeable junction formation between a metabolic cooperationdefective embryonal carcinoma variant R5/3 and its parent PC13TG8 have been studied by uridine prelabelling. The results indicate that the lesion results not in a modified specificity of cell recognition but in a deficiency in forming permeable junctions irrespective of contiguous cell type. New techniques have been developed for the study of intercellular transfer of sodium ions and of amino acids of the urea cycle. These have been used to demonstrate that the R5/3 lesion reduces the capacity of the cells to participate in intercellular transfer of three unrelated categories of small molecule.     

Changes in surface relief of suspended cells are morphological signs of the initial stage of neoplastic transformation in fibroblastic monolayer cultures.

The percentages of cells with different types of cell surface relief were determined in cell suspensions derived from monolayer cultures. Primary cultures of rat embryo fibroblasts (REF) and cell lines REF (LT) and REF-1, immortalized cells of which preserved normal phenotypic characteristics of the initial primary culture REF, as well as morphologically transformed tumorigenic lines REF (LT) ras and REF-2EJ were studied. In REF suspensions the cells with the blebbed type of surface relief were shown to be predominant as compared with those with microvillus relief whereas cell suspensions derived from both immortalized and fully transformed cultures display the reverse ratio of cells with those types of surface relief. Therefore, the pattern of cell surface relief in cell suspensions derived from fibroblastic monolayer cultures may serve as a morphological marker of the initial stage of neoplastic transformation-immortalization when typical morphological signs of cell transformation are not yet manifested in monolayer cultures.     

Characterization of accelerated iron-ion-induced damage in gap junction-competent and -incompetent thyroid follicular cells

Early- and late-passage cultures of Fischer rat thyroid cells differ in their growth properties and gap junction competency. Previous studies comparing early- and late-passage cultures exposed to gamma rays and proton beams revealed that differences in growth rate did not influence their responses; however, the presence of connexin 32 gap junctions conferred resistance to gamma radiation. To further assess differences in radiation quality, suspension cultures of early- and late-passage cells were exposed to accelerated iron ions, and their comparative biological responses were measured. The iron-ion-irradiated cells displayed sustained levels of incorporated dUTP, reflecting persistent DNA damage. These results were supported by the frequency of chromosomal damage measured by micronucleus formation. Iron-ion irradiation induced micronuclei at a rate of eight per gray per 100 binucleated cells scored in early-passage cells and nine per gray per 100 binucleated cells scored in late-passage cells. Relative to photons, the calculated radiobiological effectiveness for frequency of micronuclei was 5.7 and 6.4 for the early- and late-passage cultures, respectively (P > 0.05). Levels of apoptosis fluctuated as a function of dose, and modest increases above basal levels persisted throughout the 48-h period. The comparison of retained follicular structures revealed differences in the alpha components of the linear-quadratic dose-response curves (0.60 Gy(-1) for early-passage and 0.71 Gy(-1) for late-passage cultures, P < 0.014). Cell cycle phase redistribution resulted in a G2 arrest (P < 0.001) for both early- and late-passage cultures. In conclusion, the response of thyroid follicular cells to high-LET radiation was not influenced by the presence of gap junctions or the proliferative status of the target cells.