Hybrids between normal cells,communication-deficient and communication-competent tumor cells

Summary Communication competent rat cells, communication incompetent (i.e., gap junction defective) rat tumor cells and communication competent rat tumor cells were fused in different combinations and the resultant hybrid cells were tested with regard to their growth properties and communication-competence. The ability to communicate via gap junctions was strictly inherited in a dominant fashion. Hybrids of normal cells and non-communicating tumor cells exhibited normal growth properties,i.e., the transformed phenotype was not expressed. This adds evidence to the hypothesis that the transformed growth of communication-incompetent tumor cells may be due to the loss or closure of their gap junctions.Communication-competent tumor cells behaved different when fused with normal cells in that their transformed growth was observed in each of the hybrids examined. No complementation was observed when different communication-incompetent cells or different tumor cells were fused.     

Differential regulation of communication by retinoic acid in homologous and heterologous junctions between normal and transformed cells

The permeability of junctions between cells of the same type (homologous junctions) is greatly increased by retinoic acid (10(-9)-10(-8) M), a probable morphogen, and this responsiveness is shared by a variety of normal and transformed cell types (Mehta, P.P., J.S. Bertram, and W.R. Loewenstein. 1989. J. Cell Biol. 108:1053-1065). Here we report that the heterologous junctions between the normal and transformed cells respond in the opposite direction; their permeability is reduced by retinoic acid (greater than or equal to 10(-9) M) and its benzoic acid derivative tetrahydrotetramethylnaphthalenylpropenylbenzoic acid (greater than or equal to 10(-11) M). The opposite responses of the two classes of junction are shown to be concurrent; in cocultures of normal 10T1/2 cells and their methylcholanthrene-transformed counterparts, the permeability of the heterologous junctions, which is lower than that of the homologous junctions to start with, falls (within 20 h of retinoid application), at the same time that the permeability of the homologous junctions rises in both cell types. Such a counter-regulation requires a minimum of three degrees of cellular differentiation. A model is proposed in which the differentiations reside in a trio of junctional channel protein. The principle of the model may have wide applications in the regulation of intercellular communication at tissue boundaries, including embryonic ones.     

Correlation of (Na---K)-ATPase activity with growth of normal and transformed cells

The (Na⁺---K⁺)-stimulated Mg²⁺-dependent ATPase activities of 3T3 and SV40 transformed 3T3 cells were compared as a function of cell population density. For normal cells the enzyme activity remained relatively constant during exponential growth, but decreased sharply coincident with contact inhibition of growth at confluence. This decrease in activity could be reversed by stimulating contact-inhibited cultures to undertake renewed short-term growth either by adding fetal calf serum or changing the medium completely. Transformed cells did not experience a decrease in (Na⁺---K⁺)-ATPase activity upon reaching confluence, but this is consistent with the fact that they were still growing exponentially at this stage. However, non-confluent cultures of both normal and transformed cells incurred a marked decrease in levels of the enzyme when growth was inhibited by serum depletion. The results have been interpreted as indicating that levels of (Na⁺---K⁺)-ATPase in both normal and transformed cells are correlated with growth. Hence the different patterns of ATPase activity displayed by malignant cells and their normal counterparts with increase in cell number appear to be a reflection of their dissimilar growth behaviours rather than of any innate difference between them.     

Glutaraldehyde-fixed transformed and non-transformed cells induce contact-dependent inhibition of growth in non-transformed C3H/10T1/2 mouse fibroblasts, but not in 3-methylcholanthrene-transformed cells

C3H/10T1/2 mouse fibroblasts showed a pronounced inhibition of growth when reaching a critical cell density. The situation of high cell density could be mimicked by the addition of glutaraldehyde-fixed cells to sparsely seeded proliferating cells. Treatment of the C3H/10T1/2 cells with 3-methylcholanthrene led to a high frequency of piled up foci (118 type II and type III foci in 78 cultures). Cells of a type III focus of a treated culture were cloned. These cells grew in soft-agar and reached 10 times higher cell densities when grown in culture dishes, than did their non-transformed counterparts. Glutaraldehyde-fixed transformed cells did not differ from fixed non-transformed cells in the ability to inhibit the growth of sparsely seeded non-transformed cells. On the other hand, both the addition of fixed normal or transformed C3H/10T1/2 cells did not affect the growth rate of transformed cells. In a concept explaining the density-dependent inhibition of growth of non-transformed cells by a specific interaction of plasma membrane-localized effectors with plasma membrane-localized receptors, the present findings would indicate that the transformed cells used express active effectors but are functionally defective in the receptors or in the signal transmission.     

A study of communication specificity between cells in culture

We have examined the specificity of communication between cells in culture by co-culturing cells derived from mammalian, avian, and arthropod organisms. Both mammalian and avian culture cells have similar gap junctional phenotypes, while the insect (arthropod) cell lines have a significantly different gap junctional structure. Electrophysiological and ultrastructural methods were used to examine ionic coupling and junctional interactions between homologous and heterologous cell types. In homologous cell systems, gap junctions and ionic coupling are present at a high incidence. Also, heterologous vertebrate cells in co-culture can communicate readily. By contrast, practically no coupling (0-8%) is detectable between heterologous insect cell lines (Homopteran or Lepidopteran) and vertebrate cells (mammalian myocardial or 3T3 cells). No gap junctions have been observed between arthropod and vertebrate cell types, even though the heterologous cells may be separated by less than 10 nm. In additional studies, a low incidence of coupling was found between heterologous insect cell lines derived from different arthropod orders. However, extensive coupling was detected between insect cell lines that are derived from the same order (Homoptera). These observations suggest that there is little or no apparent specificity for communication between vertebrate cells in culture that express the same gap junctional phenotype, while there is a definite communication specificity that exists between arthropod cells in culture.     

Nonsensitized lymphocytes produce leukocyte interferon when cultured with foreign cells.

Nonsensitized human lymphocytes produce interferon when cultured with either transformed or normal heterologous cells. De novo RNA and protein synthesis was not required in a foreign cell for it to act as the inducer. The interferon produced has been characterized as being leukocyte type rather than immune or fibroblast type.     

Inhibition of neoplastic cell growth by quiescent cells is mediated by serum concentration and cAMP phosphodiesterase inhibitors

We have demonstrated that confluent monolayers of the mouse fibroblast cell line C3H/10T1/2 (10T1/2) have the ability to cause reversible growth inhibition of cocultured transformed cells. This was first demonstrated for de novo transformed cells and later extended to established cell lines of proven oncogenicity in vivo. This growth inhibition could be increased by growing the 10T1/2 cells to high density in increasing concentrations of serum or by elevating intracellular concentrations of cAMP using inhibitors of phosphodiesterase (PDE). These manipulations, which in cocultures of nontransformed and transformed cells caused complete inhibition of tumor cell growth, had no effect on growth rate or saturation density of either ceil type when cultured alone, demonstrating the cooperative nature of this phenomenon. This cooperation could not be produced by transfer of culture medium, demonstrating the requirement for intimate cell contact. Inhibition of the formation of transformed foci of cells in these mixed cultures was accompanied by a decrease in the incorporation of labeled thymidine into these cultures; the kinetics of this inhibition and recovery suggested a rapidly reversible effect on cell cycle transit times. The potent inhibitor of cAMP PDE, Ro 20-1724 induced dose dependent increases in intracellular cAMP in both nontransformed and in transformed cells. However, at a concentration of 10^?4 M Ro 20-1724, which inhibited tumor cell growth in mixed cultures, cAMP was elevated 30-fold in nontransformed versus only 3-fold in transformed cells. The inhibitory effects of PDE inhibitors on tumor growth have been extended to an in vivo model system, utilizing Lewis lung carcinoma cells growing as metastases in the lungs of C57B1 mice. In these mice, inoculated intravenously with a single cell suspension of Lewis lung cells, the formation of lung metastases was dramatically decreased by the twice daily administration of either isobutylmethylxanthine or Ro 20-1724; PDE inhibitors were shown to be active in vitro. The latter compound, which showed highest activity in vitro, was also substantially more potent in vivo as an inhibitor of lung tumor colony formation and doubled the life span of the tumor bearing animals. Cell cycle analysis of lung tumor colonies by the labeled mitosis method showed that both phosphodiesterase inhibitors caused a prolonged G₁ phase in the cell cycle but failed to influence other phases. Although detailed analysis of host tissues is not complete, prolonged treatment with these drugs caused no statistically significant weight loss or changes in counts of red or white blood cells indicating a selective growth inhibition of transformed cells at these doses. Studies to determine the mechanism of the cellular communication and the nature of the signal are in progress.     

The impact of homologous and heterologous nurse cells on the division capability of sparsely plated cells

The growth-promoting effects of nurse cells of carrot, tomato, patato, maize, bean, carnation and two species of tobacco were studied on carrot, tomato, tobacco and potato cells plated at low densities. In an area immediately below the nurse cells the plating efficiency was very high and found to be independent of cell density. In an area outside the nurse cells, in some cases, the plating efficiency tended to be much higher in combinations with cells from a heterologous source as compared with those from a homologous source. Moreover, in the same area with some combinations the plating efficiency decreased when cell density was lowered, while with other combinations this phenomenon did not occur. This decrease was independent of the absolute value of plating efficiency. In experiments in which the concentration of conditioning factors was presumably changed, no significant difference in the plating efficiency was noticed. We therefore suggest that different plating efficiencies observed with heterologous nurse cells were not due to a higher level of conditioning factors, but rather to the production of different types of conditioning factors that are presumably degraded with different efficiency.     

Dependence of intracellular alkali-ion concentrations of 3T3 and SV 40-3T3 cells on growth density.

Intracellular contents of potassium and of sodium are determined for 3T3 and SV 40-3T3 cells in dependence of growth density. In parallel, total cell volume and volume of intracellular water is determined for these cells suspended in physiological buffer. Intracellular potassium concentration thus evaluated for suspended 3T3 cells exhibits a sharp decrease at cellular growth densities which lead to density dependent inhibition of cell proliferation. In the case of SV 40-3T3 cells, this drop of potassium concentration with increasing cellular growth density is not observed, which correlates well with the absence of cell density dependent inhibition of cell growth in the transformed cell line. These results support the notion that processes of stimulation of quiescent 3T3 cells or of cell density dependent inhibition of their proliferation are mediated by processes including changes of potassium transport characteristics leading to increase or decrease respectively of their intracellular potassium concentration. Furthermore, these and other results suggest, that a difference between normal and transformed cells most relevant to their different proliferation behaviour might reside in different transport characteristics for potassium of the plasma membranes of these cells.     

Connexins and cancer

The hypothesis, that gap junctional intercellular communication plays a key role in carcinogenesis and more generally in growth control was formulated nearly 40 years ago. From this time, data accumulated, showing that this type of communication is frequently decreased or absent in cells treated with tumor promoting agents, among transformed cells or between transformed/tumor cells and normal cells. This observation has been made on various cell types and whatever their tissue and species origins, by using in vitro and in vivo models. It led to the general assumption that the inhibition of gap junctional intercellular communication may play a role in carcinogenesis at two levels: (1) during tumor promotion by favoring the clonal expansion of initiated cells and (2) after the phenotypic transformation of cells by preventing the diffusion of putative "normalizing" factors between tumor cells and surrounding normal cells. During the past decade, the discovery that gap junction proteins, the connexins (Cx), may act as tumour suppressors, by reverting the phenotype of transformed cells confirmed the idea that their lack of function would be actively involved in carcinogenesis. However, we still do not know precisely what are the molecular processes that gap junctional intercellular communication may regulate and still do have very few data concerning the gap junction situation in human cancers. All these aspects are presented from an historical point of view and discussed below.     

Nontransformed cells can normalize gap junctional communication with transformed cells

We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.     

A G2/M cell cycle block in transformed cells by contact with normal neighbors

Neighbour suppression of growth of tumour cells by stationary normal cells might be important in early stages of cancer. We have studied this using suppressor and non-suppressor lines of 3T3 fibroblasts and SV40 transformed derivatives. Growth suppression of transformed cells depended on direct contact with stationary confluent cultures of 3T3 cells but not on gap junction communication. It was not caused by apoptosis nor through the normal G0/G1 block present in the confluent normal cells. Instead, there was a progressive elongation of the cell cycle leading to arrest in G2/M in the transformed cells. This indicates an unusual type of growth arrest not previously involved in social control of cell growth.     

Transmembrane ferricyanide reduction in tobacco callus cells

Transmembrane ferricyanide reduction in whole cells of normal and of transformed tobacco (Nicotiana tabacum) callus tissue was compared. It was found that low concentrations of indoleacetic acid (IAA, 0.1 μM), gibberellic acid (GA, 0.3 μM), and benzyl adenine (BA, 0.03 μM) stimulate external ferricyanide reduction in normal tobacco callus cells, but inhibit this reaction up to 67% in transformed cells when hormones are applied to cells 10 min prior to assay. Higher concentrations of these growth regulators (1 μM or greater) inhibit transmembrane ferricyanide reduction in both types of cells, with the exception of IAA, giving an initial stimulation of the rate (12%), followed by 24% inhibition after 2 min. The observed external ferricyanide reduction by whole tobacco callus cells may be explained on the basis of a transplasmalemma redox system, which may be associated with the iron metabolism of these cells.     

PCC4azal teratocarcinoma stem cell differentiation in culture: III. Cell-to-cell communication properties

The cell-to-cell communication properties of the PCC4azal embryonal carcinoma cells and their differentiated endoderm-like cells and giant cells were characterized by ionic coupling, metabolic coupling, and transfer of an injected fluorescein dye. All PCC4azal cell types communicate well with one another: stem cells with stem cells, endoderm-like cells with endoderm-like cells, and giant cells with giant cells. In addition, the stem cells communicate well with giant cells as assayed by metabolic coupling. The ability of the undifferentiated teratocarcinoma cells to metabolically couple with a variety of heterologous cell types was examined. The stem cells were always efficient donors but very poor recipients in almost all combinations with cells of fibroblast or epithelial morphology that were derived from several different species. In contrast, these same heterologous cell types were both efficient donors and efficient recipients with each other.     

Interferons and bone. A comparison of the effects of interferon-alpha and interferon-gamma in cultures of human bone-derived cells and an osteosarcoma cell line

Recombinant human interferon-alpha 2C and recombinant human interferon-gamma (5-1000 U/ml) inhibit the proliferation of normal human bone-derived cells and a human osteosarcoma cell line. In the bone-derived cells the inhibitory effect of interferon-gamma was significantly greater than that of interferon-alpha, whereas in the osteosarcoma cell line the inhibitory effects of both interferons were quantitatively similar. Interferon-alpha did not affect the alkaline phosphatase activity of either type of cells. In contrast, interferon-gamma affected the activity of the enzyme in both cell types: in the bone-derived cells the effect of interferon-gamma was stimulatory whereas in the osteosarcoma cells the effect was inhibitory. In both cell types interferon-gamma selectively inhibited the incorporation of radiolabelled proline into type I collagen. In the osteosarcoma cells, the effects of both interferons on collagen synthesis were quantitatively similar. In the bone-derived cells, however, interferon-alpha decreased proline incorporation into collagen and non-collagen proteins to a similar extent and thus did not affect collagen synthesis when expressed as a percentage of total protein synthesis. Two-dimensional polyacrylamide gel electrophoresis of the radiolabelled proteins of the cell layer synthesised by both cell types in the presence of either interferon demonstrated that this treatment enhanced or induced the synthesis of a total of 21 individual proteins (19 in bone cells, 14 in osteosarcoma), ranging in apparent molecular mass over 14-87 kDa. The set of proteins induced was different in all four combinations of cells and interferon. A tentative identification of several of the proteins was possible based upon estimation of molecular mass, preferential induction by interferon-alpha or interferon-gamma and differential induction in normal and transformed bone-derived cells. The results of this study demonstrate that interferons have complex effects upon the proliferative and biosynthetic activities of human bone-derived cells and demonstrate significant differences between the responses of normal cells and transformed bone-derived cell line. Further investigations will be required in order to determine whether or not these differences are unique to the osteosarcoma cell line or are a characteristic of the effects of interferons on bone-derived cells in general.     

Regulation of mouse oocyte growth: probable nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth

Intercellular communication, as determined by two different assay procedures, was established in vitro between mouse oocytes free of adhering follicle cells and monolayers of either follicle or 3T3 cells. Both of these cell types are known to be able to form homologous gap junctions, and follicle cells naturally form heterologous gap junctions with oocytes in vivo. Monolayers of L cells that are communication deficient did not establish intercellular communication with oocytes as determined by the two different assays for intercellular communication. The diameter of oocytes cultured for 4 days in medium or on monolayers of L cells decreased markedly, 9.7 and 13.1 micron, respectively. In contrast, oocytes cultured for 4 days on follicle cell monolayers increased on the average about 4.7 micron in diameter. Oocytes cultured for 4 days on monolayers of 3T3 cells decreased slightly in diameter, i.e., 2.1 micron. Results from these experiments support a nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth.     

Maintaining connexin43 gap junctional communication in v-Src cells does not alter growth properties associated with the transformed phenotype

Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine "normal" cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.     

Effect of proteolytic inhibitors on growth and surface architecture of normal and transformed cells

Protease inhibitors were tested for their effect on the growth of normal and SV40-transformed mouse fibroblasts. The protease inhibitors TAME¹ and EWTI¹, which act competitively on proteases, reduce the growth of transformed cells more than that of untransformed parent cells. However, transformed cells grown in medium containing these drugs do not show contact inhibition of cell division or decreased agglutinability with Concanavalin A. The inhibition of growth is due to an extended duration of all phases of the cell cycle. The protease inhibitor TLCK¹, an active site titrant reacting irreversibly with trypsin, blocks transformed cells in the premitotic stage of the cell cycle. This effect does not occur in the untransformed parent cells. The decrease in agglutinability of transformed cells treated with TLCK is correlated with a partial synchronisation in the G2 stage of the cell cycle. Our results do not support the hypothesis that protease inhibitors induce transformed cells to assume a normal growth pattern and that this is accompanied by a decreased agglutinability with plant lectins.     

Control of the uptake of amino acids by serum in chick embryo cells,untransformed or transformed with rous sarcoma virus

Summary Forty to fifty minutes after removal of serum, the net total uptake of amino acids in growing secondary cultures of normal or virus-transformed chick embryo cells, stopped or proceeded only at a highly reduced rate. In both normal and transformed cells, theinitial (0–40 min) rate of the above uptake was the same in the absence of serum as in its presence. The initial rate of the total uptake of amino acids in growing transformed cells was about the same as in growing normal cells. Neither in the normal nor in the transformed cells was the rate of the total uptake of amino acids reduced by cell confluence alone. In highly dense, hyperconfluent cultures of normal cells in which cell growth was arrested, the rate of uptake in the absence or in the presence of serum was four- to fivefold lower than the rate obtained in growing normal cells under similar conditions; in the absence of serum, the net uptake stopped after 40 min in the hyperconfluent cultures as well. It appears that cells growing in tissue culture require a serum factor for maintenance of the required high rates of uptake of amino acids and that the inhibition of growth at high cell densities is a result of depletion of this factor from serum, or the inability of the cells in a dense culture to respond to the factor. A serum factor is apparently also required for maintenance of the reduced rates of uptake of amino acids observed in hyperconfluent cultures.     

Contact with basement membrane heparan sulfate enhances the growth of transformed vascular endothelial cells, but suppresses normal cells.

Modulation of vascular endothelial cell growth by basement membrane heparan sulfate was investigated using four lines of normal and transformed cells. The growth of transformed endothelial cells, but not normal cells, on reconstituted basement membrane was severely suppressed when heparan sulfate, one of the components of the membrane, was specifically degraded by an enzyme, heparitinase. Similarly, when cells were grown on surfaces coated with heparan sulfate, as little as 60 pg/cm2 of heparan sulfate caused growth enhancement of transformed cells, but suppression of normal cells. These results together with our previous observations (IMAMURA, T and MITSUI, Y. (1987) Exp. Cell Res., 172: 92-100) argue that transformed cells have reversed a mechanism by which basement membrane heparan sulfate functions as a physiological suppressor for the growth of normal endothelial cells.