Contact inhibition in tissue culture

Conclusions Contact inhibition of movement is here defined simply as the stopping of the continued locomotion of a cell in the direction which has produced a collision with another cell; so that one cell does not use another as a substratum. Amongst fibroblasts and epithelial cells this inhibition seems to be brought about by a mechanism which it is suggested consists essentially of a spasm of contraction in the region of the contact, set off by some signal from the cell contacted. Many other kinds of cells show the general phenomenon of contact inhibition; but there is no certainty that they have the same contractile mechanism. The survey of the literature which this review has entailed suggests that it might be useful to end with four somewhat negative points: (1) Contact inhibition as originally defined is not concerned with mitosis. It may of course become so. (2) Contact inhibition of movement is difficult to analyse reliably without quantitative estimations and deliberate experiments. Anecdotes are not enough. (3) Malignant cells are not properly described as being devoid of contact inhibition. It is suggested that they are defective as compared with their cells of origin. (4) From the point of view invasion interest. It is the heterologous inhibition of tumour cells by normal cells that is relevant.     

Ontogeny of the mouse liver studied with hepatocyte membrane antigens and a comparison with alpha-fetoprotein synthesis

The mouse liver was studied during embryogenesis and postnatal development using indirect immunoperoxidase method with rat monoclonal antibodies to two membrane antigens of the hepatocytes. B10 antigen was first detected in the liver of 16 day old embryos on the hepatocyte membrane in the area of biliary capillary in the center of an acinar structure, thus reflecting the onset of formation of acinar structure in the liver. Formation of the liver beams after birth is accompanied by changes in the pattern of the antigen distribution--it is present on the lateral side of the hepatocyte membrane. D2 antigen is equally distributed over the hepatocyte membrane. When the acinar organization of the liver is replaced by the beam-like structure after birth, the antigen is redistributed--the part of the hepatocyte membrane facing circulatory sinus contains this antigen in a much greater quantity.     

A protein associated with the mouse and rat hepatocyte junctional complex

Summary In this study we have examined a protein associated with bile canaliculi of mouse and rat hepatocytes that is detected by monoclonal antibody BG9.1. The protein is seen by indirect-immunofluorescence microscopy as 2 discrete parallel lines at the lateral borders of adjacent hepatocytes. This pattern is present during development in the day 13 fetal mouse liver. Electron microscopy with immuno-gold labeling indicated that the protein is associated with the cytoplasmic surface of junctional complexes located on either side of bile canaliculi. BG9.1 reacts with a protein of 192000 apparent molecular weight on immunoblots of plasma membrane isolated from mouse and rat hepatocytes. It has been reported that unlike most cellular components, tight junctions are not soluble in sodium deoxycholate. Extraction of isolated hepatic plasma membrane sheets with deoxycholate and other reagents did not eliminate the pattern seen by indirect-immunofluorescence microscopy and enhanced the intensity of reactions on immunoblots. BG9.1 also binds to the junctional-complex region in other epithelial cell types. These results indicate that BG9.1 detects a deoxycholate-insoluble protein associated with junctional complexes and suggests that the protein is a component of tight junctions.     

Gap junctional communication in the preimplantation mouse embryo.

In this study, we examined cell-to-cell communication via gap junctional channels between the cells of the early mouse embryo from the 2-cell stage to the preimplantation blastocyst stage. The extent of communication was examined by monitoring for the presence of ionic coupling, the transfer of injected fluorescein (molecular weight 330) and the transfer of injected horseradish peroxidase (molecular weight 40,000). In the 2-cell, 4-cell and precompaction 8-cell embryos, cytoplasmic bridges between sister blastomeres were responsible for ionic coupling and the transfer of injected fluorescein as well as the transfer of injected horseradish peroxidase.In contrast, no communication was observed between blastomeres from different sister pairs. Junction-mediated intercellular communication was unequivocably detected for the first time in the embryo at the early compaction stage (late 8-cell embryo). At that stage, ionic coupling was present and fluorescein injected into one cell spread to all eight cells of the embryo. Injected horseradish peroxidase was passed to only one other cell, however, again indicating the presence of cytoplasmic bridges between sister blastomeres. Junctional communication with respect to both ionic coupling and dye transfer was retained between all the cells throughout compaction. At the blastocyst stage, trophoblast cells of the blastocyst were linked by junctional channels to other trophoblast cells as well as to cells of the inner cell mass, as indicated by the spread of injected fluorescein. In addition, the extent of communication between the cells of the inner cell mass was examined in inner cell masses isolated by immunosurgery; both ionic coupling and the complete spread of injected fluorescein were observed.     

Gap junctional communication in the post-implantation mouse embryo.

We studied the extent of cell-to-cell communication via junctional channels in in vitro-implanted mouse blastocysts by monitoring ionic coupling and the spread of two injected low molecular weight dyes, fluorescein and Lucifer yellow. In the early attached embryos, both trophoblasts and cells of the inner cell mass (ICM) were ionically coupled to one another. Dye injections in either trophoblasts or ICM cells resulted in spread to the entire embryo. As older and more developed embryos were examined, the spread of injected dye was progressively more limited. In the most developed embryos examined, dye injected into a cell in the ICM region resulted in spread throughout the ICM but not into the surrounding trophoblast cells, while dye injected into a trophoblast cell did not spread to any other cell in the embryo. Simultaneous monitoring of ionic coupling and dye injections in embryos of intermediate stages in this transition revealed that the trophoblast and ICM cells were ionically coupled, even across the apparent boundary where no dye was observed to pass. In the latest stage embryos examined in which no injected dye was observed to move out of the ICM, ionic coupling was still observed between the cells of the ICM and the trophoblasts. Furthermore, in the more developed embryos, dye injected into the ICM region frequently was not transferred to all the cells of the ICM, thus suggesting a further compartmentalization of due spread within the ICM. Our observations that ionic coupling is more extensive than the detectable spread of injected dyes may perhaps reflect a reduced number of junctional channels. With fewer channels less dye would pass between cells, so that, together with continuous quenching, the transfer of injected dye would not be detectable. This partial segregation of cell-to-cell communication as indicated by the limited dye spread may parallel specific differentiation processes, in particular that of giant trophoblast, embryonic ectoderm and extraembryonic endoderm differentiation.     

Functional analysis of the mouse alpha-fetoprotein enhancers and their subfragments in primary mouse hepatocyte cultures.

We have compared the activities of mouse alpha-fetoprotein (AFP) enhancers I, II, and III with their minimal enhancer fragments (Mers) I, II, and III and with the entire 7-kilobase pair enhancer domain by transient expression assay in primary fetal mouse liver cells. The level of expression directed by the AFP promoter [p(-1009)AFPcat] alone is stimulated at least 10-fold by the entire AFP enhancer domain (-1009 to -6983). Enhancer I can drive the level of chloramphenicol acetyltransferase activity equivalent to that of the entire enhancer domain, whereas the increase in activity by enhancers II and III is significantly lower (1.5-fold). MersI, II, and III all mediate a greater increase in activity than their corresponding enhancer regions. The increase with MerI is 16-fold. Using DNase I protection analyses we identified 3 protein-binding regions in MerI; site Ia binds liver and brain nuclear proteins; site Ib binds liver, kidney, and brain nuclear proteins as well as purified C/EBP; site Ic binds liver and kidney nuclear proteins. Site-specific mutation of Ia, Ib, or Ic showed a 10-25% reduction in chloramphenicol acetyltransferase expression; deletion of the C/EBP-binding site in Ib showed a 45% reduction in activity and mutation of all 3 sites (Ia, Ib, and Ic) resulted in a 75% reduction in activity. Our studies indicate no single trans-acting factor is absolutely essential for enhancer activity, and that the enhancer activity of MerI is mediated via a combinatorial and additive mechanism.     

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.     

Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid

Intercellular gap-junctional communication was measured using metabolic co-operation in co-cultures of argininosuccinate synthetase-deficient and argininosuccinate lyase-deficient human fibroblasts. 18-alpha-glycyrrhetinic acid (AGA) was found to inhibit communication by more than 95% at concentrations as low as 2 microM. Concentrations up to 100 microM were not cytotoxic over a period of 2 hours. Communication inhibition was of rapid onset and was readily reversible. Communication remained continuously yet reversibly blocked in cells cultured in the presence of AGA for 20 days. The related compounds 18-beta-glycyrrhetinic acid and carbenoxolone also caused communication inhibition. The effect is probably not mediated via mineralocorticoid or glucocorticoid receptors since aldosterone and glucocorticoids had no effect on communication. AGA thus has properties of a useful inhibitor in the study of intercellular junctional communication.     

4-Hydroxynonenal reduces junctional communication between endothelial cells in culture

The effect of 4-hydroxynonenal (HNE), a lipid peroxidation product, on junctional communication (JC) among cultured vascular endothelial cells was assessed by both study of the transfer of microinjected 6-carboxyfluorescein between neighboring cells and measurement by a "cut-loading and dye transfer" technique. Both methods indicated that at concentrations higher than 10(-9) M and testing times between 6 and 8 h HNE reduces endothelial cell junctional communication. At 10(-8) M, a gradual development of HNE effect appears during 6-8 h of exposure but is followed by a slow recovery completed at 20 h. The reduction in junctional communication is not produced by the inhibition of protein synthesis, as tested by radiolabeled leucine incorporation. The HNE effect might be relevant to pathological processes in which lipid peroxidation is associated with uncontrolled cell proliferation, as in atherogenesis and promotion of carcinogenesis by chronic inflammation.     

Gap junctional communication and compaction during preimplantation stages of mouse development

The ability of gap junction antibodies to block dye transfer and electrical coupling was examined in the compacted 8-cell mouse zygote. In control zygotes, Lucifer yellow injected into 1 cell transferred to the rest of the embryo. When antibodies raised against the major protein extracted from gap junctions were co-injected with Lucifer yellow, dye transfer failed in 86% of the zygotes tested and electrical coupling was almost completely inhibited. Subsequently, the antibody-containing cells were extruded. When the antibodies were injected into 1 cell at the 2-cell stage, 82% of the zygotes divided normally to the 8-cell stage. Cells containing gap junction antibodies were uncompacted, but continued to divide. We conclude that these antibodies inhibit gap junctional communication in the early mouse zygote and that communication through gap junctions may be involved in the maintenance of compaction.     

Synthesis of DNA and alpha-fetoprotein in the monolayer culture of mouse hepatocytes

Relation between processes of proliferation and synthesis of the embryonal serum protein alpha-fetoprotein (AFP), the influence on these processes of polyelectrolyte dextran sulfate (DS) and dimethyl-sulfoxide (DMSO) has been studied in the monolayer culture of mouse hepatocytes. In control cultures the correlations between the time of appearance and the level of DNA and AFP synthesis were observed. DS and DMSO were found to inhibit both processes. Cell proliferation could be reestablished by addition of epidermal growth factor. In case of the influence of DMSO, it wasn't followed by the induction of AFP synthesis. This the processes of DNA and AFP synthesis in monolayer cultures of mouse hepatocytes can be separated. The elongated incubation of hepatocytes with collagenase during their obtaining, abolished the effects of DS. This shows that surface components of hepatocytes, lost upon enzyme degradation, may be involved in the mechanism of DS effect.     

Regulation of alpha-fetoprotein and transferrin synthesis and secretion in mouse hepatoma cells

Significant differences in the glucocorticoid- and cyclic nucleotide-mediated regulation of the secretory glycoproteins, α-fetoprotein and transferrin, have been observed to develop in a mouse hepatoma cell line, Hepa-2, after many passages in culture. Treatment of low-passage cells with hydrocortisone (10^?6m), N⁶,O²-dibutyryl cyclic AMP (10^?3m), or 8-bromo-cyclic AMP (10^?3m) results in 1.5-, 2- to 4-, and 5.5- to 6-fold increases, respectively, in the rates of synthesis and secretion of α-fetoprotein. As expected of secretory proteins, the ratio of synthesis to secretion is 1 and remains unaltered when treatment with hydrocoritsone, N⁶,O²-dibutyryl cyclic AMP, and 8-bromo-cyclic AMP causes a stimulation of synthesis and secretion. Similar studies showing that albumin and transferrin synthesis and secretion are also balanced in these low-passage cells have been published and indicate that the regulation of synthesis and secretion remains coupled in these low-passage cells. In high-passage Hepa-2 cells, however, we have shown that the relative rate of α-fetoprotein synthesis is higher than its rate of secretion and that the ratio of synthesis to secretion is 4. Similarly, the ratio of transferrin synthesis to secretion is 3.6, whereas it remains unaltered for albumin. When the high-passage cells are treated with N⁶,O²-dibutyryl cyclic AMP, there is a greater increase in the rate of secretion for both glycoproteins, resulting in a reduction of the ratio of synthesis to secretion from 4 to 1.63 for α-fetoprotein and from 3.6 to 2.3 for transferrin. This effect on the secretion of α-fetoprotein and transferrin is specific for the cyclic nucleotides and occurs only in high-passage cells. Hydrocortisone treatment causes an increase in α-fetoprotein synthesis and secretion. However, the ratio of synthesis to secretion increases from 3.96 in control to 5.5 in treated cells. Our studies show, therefore, that there is an increase in this ratio because of a slightly greater effect on synthesis which is not reflected in secretion. Similarly, hydrocortisone exerts a greater increase in transferrin synthesis than secretion and causes the ratio of synthesis to secretion to increase from 3.6 to 6.2. We propose that during continued subculturing a Hepa-2 variant is selected in which the regulation of serum glycoprotein synthesis and secretion is uncoupled. Furthermore, this effect is specific for secretory glycoproteins since the regulation of albumin synthesis and secretion by hydrocortisone and cyclic nucleotides remained unaltered.     

Gap junctional communication in the extraembryonic tissues of the gastrulating mouse embryo

We characterized gap junctional communication in the extraembryonic tissues of the 7.5-d gastrulating mouse embryo. At this stage of development, the extraembryonic tissues form a large part of the conceptus, and link the embryo proper to the maternal tissue. Using Lucifer yellow injections, cells in most extraembryonic tissues were observed to be very well dye coupled, the only exception being the peripheral regions of the ectoplacental cone. Of particular interest was the fact that no dye coupling was detected between the three major extraembryonic tissues. Thus, the extraembryonic ectoderm (EEC), the extraembryonic endoderm (EEN), and the ectoplacental cone (EPC) corresponded to separate communication compartments, with the EPC being further subdivided into three compartments. Interestingly, the EEN was observed to exhibit a very low level of dye coupling with the adjacent visceral embryonic endoderm (EN), and consistent with the latter dye coupling results was the finding that the EEN was ionically coupled to the EN, but not with any other extraembryonic tissues. However, in the EPC, ionic coupling studies show that the central region was well coupled ionically to the EEC, but only weakly coupled to the peripheral EPC. These findings, in conjunction with our previous study (1988. J. Cell Biol. 107:241-255), demonstrate that the 7.5-d mouse conceptus is subdivided into at least nine major Lucifer yellow-delineated communication compartments, with ionic coupling across some of these compartments effectively unifying the embryo into two large domains corresponding to the embryo proper and the major extraembryonic tissues.     

Prevention of organochlorine-induced inhibition of gap junctional communication by chaetoglobosin K in astrocytes

Innumerable toxic substances present in the environment inhibit gap junctions, intercellular membrane channels that play fundamental roles in coordinated function of cells and tissues. Included are persistent organochlorine compounds, which pose health risks to humans and animals owing to their widespread use, bioaccumulation, and ability to inhibit gap junction channel-mediated intercellular communication in liver, lung, skin, heart, and brain cells. In this study, the organochlorine xenobiotics dieldrin and endosulfan, at micromolar concentrations, were found to inhibit gap junction-mediated intercellular communication and induce hypophosphorylation of connexin 43 in cultured rat astrocytes, the predominant cell type in the brain coupled through gap junctions. This inhibition of gap junctional communication was substantially reduced by preincubation with chaetoglobosin K (ChK), a bioactive natural produce previously shown to have ras tumor suppressor activity. Chaetoglobosin K also prevented dieldrin and endosulfan-induced hypophosphorylation of connexin 43 and prevented dieldrin-induced connexin 43 plaque dissolution in both astrocytes and cultured liver epithelial cells. The results suggest that stabilization of the native, phosphorylated form of connexin 43 by ChK may contribute to its ability to prevent organochlorine-induced inhibition of gap junction-mediated communication and dissolution of gap junction plaques within the plasma membrane. This revised version was published online in July 2006 with corrections to the Cover Date.     

Organ culture of adult mouse intestine

Summary Explants of adult mouse jejunum have been maintained in organ culture with or without fructose added to the medium in order to stimulate the intestinal glucose-6-phosphatase (G-6-Pase). When the fructose is added, at the beginning of the culture, a three-fold increase of G-6-Pase is measured during the first 24h. If the fructose is added after 24 h of culture, no significant increase of the G-6-Pase is registered in comparison with the controls. Proteins, DNA content and dissacharidase activities are not modified during the culture. Alkaline phosphatase activity presents a twofold increase in the controls and stimulated explants. The ultrastructural localization of the G-6-Pase is not altered during the culture.This work was supported by research grants from the Medical Research Council of Canada (J.S.H.) (D.M.)Mr. Chabot is a recipient of a studentship from the Medical Research Council of CanadaD. Ménard, Ph.D. is chercheur-boursier from the CRSQ     

Mechanism of inhibition of junctional communication between animal cells by phorbol ester

Abstract. The tumour promotor 4 β -phorbol 12-myristate 13-acetate (TPA) reduces the rate of junction formation between V79 Chinese hamster lung cells in culture but not between BHK21/13 Syrian hamster fibroblasts. TPA may act on all cells but only affect junction formation in those situations where the rate of junction formation is already low.     

Organ culture of adult mouse intestine

Summary Explants of adult mouse intestine have been maintained in organ culture for 24 to 48 hr. The best results have been obtained with a mixture of DMEM-HEPES medium and NCTC-135 enriched with 10% fetal bovine serum. The morphology of the mucosa is well preserved at the light electron microscopic level: absorbing cells exhibit an increase in secondary lysosomes; goblet cells and Paneth cells remain active; numerous mitoses are observed in the crypts; and vigorous re-epithelization takes place on the margin of the explants. This work was supported by a research grant from the Medical Research Council of Canada (to J. S. H.) and partly by the F.C.A.C. Québec (to J. S. H.).     

Mechanism of inhibition of junctional communication between animal cells by phorbol ester

The tumour promotor 4 beta-phorbol 12-myristate 13-acetate (TPA) reduces the rate of junction formation between V79 Chinese hamster lung cells in culture but not between BHK21/13 Syrian hamster fibroblasts. TPA may act on all cells but only affect junction formation in those situations where the rate of junction formation is already low.