Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions

Chinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., mec-). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The isolated Chinese hamster CI-4 cells which expressed the mec- phenotype most stringently showed the following characteristics: 1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the mec+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (mec+). Confluent CI-4 cells grown in the presence of 1 mM db-cAMP showed 9% coupled cells. 2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The mec+ parental cells showed small gap junction plaques (0.013% of the total cell surface analyzed). 3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92% positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic fibroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68%. Other cells of spontaneous mec- phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix. 4. The mec- defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of mec-, as well as mec+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the mec- defect.     

Reactivation and Dedifferentiation of Differentiated Murine Erythroleukemic Cell Nuclei

A murine erythroleukemic cell line, 745 A4-TG, deficient in hypoxanthine-guanine-phosphoribosyl transferase, can be induced with 3 mM hexamethylene bisacetamide to yield at least 50% of cells undergoing irreversible erythroid differentiation and finally losing capacity for cell divisions. The effects of such induced differentiation of 745 A4-TG on its ability to form viable and proliferating hybrids when fused with 3T3 1T22 fibroblasts were investigated. We found that when the induced 745 A4-TG cells were used, more continuously proliferating hybrids were obtained than could be accounted for by the residual uninduced cells which remained in these induced preparations. This suggests that some of the induced 745 A4-TG cells, when fused with 3T3 1T22 reverted from the induced phenotype of a limited capacity for cell proliferation to an uninduced state of continuous proliferation. This observation was further confirmed with the use of fully differentiated 745 A4-TG cells, which were obtained after selection with a bromodeoxyuridine suicide treatment to eliminate the uninduced and the partially differentiated cells in the preparations. When these selected, fully differentiated cells, as characterized by their lack of proliferation capacity and thymidine kinase activity, were fused with 3T3 1T22 (also deficient in thymidine kinase), it was found that not only were viable hybrid colonies obtained in a selection medium, which precluded the proliferation of either parental cells, but these hybrids continued to proliferate for more than two months in selection medium. These data thus confirmed that some fully differentiated erythroleukemic nucleus components in the hybrids were reactivated to regain capacity for cell proliferation and to dedifferentiate to synthesize thymidine kinase for survival in the selection medium. The lack of hemoglobin synthesis by these hybrids also indicates dedifferention of these murine erythroleukemic components in the hybrids.     

Antigenic immunostaining patterns in somatic hybrids of human HeLa cells and mouse fibroblasts 3T3.4E propagated in conventional medium and delipidized serum

Somatic cell hybrids were obtained with electric pulse by fusion of human epithelial HeLa cells derived from a carcinoma of the uterine cervix and mouse fibroblasts 3T3.4E, deficient in thymidine kinase. Hybrids were selected and propagated in HAT media; some experiments were carried out in medium with delipidized serum. The hybrid cells were characterized by indirect immunofluorescence with a biotin-streptavidin system using a panel of nine monoclonal antibodies specific for membrane and cytoplasmic antigens of parental cells: intermediate filaments (keratins and vimentin), HLA class 1 (beta 2-microglobulin), cell activation (EGF and transferrin receptors) and cellular adhesion (fibronectin and laminin). All of these antigens were expressed in HeLa cells cultured in conventional medium or with delipidized serum. Conversely mouse fibroblasts contained only vimentin, fibronectin and laminin. All the parental antigens were present in first passage hybrid cells cultured in conventional medium. Vimentin, fibronectin and laminin were maintained in fourth passage hybrids whereas keratins, beta 2-microglobulin, EGF and transferrin receptors were no longer detected. When propagated in medium with delipidized serum, hybrid cells re-expressed these antigens after 5 days of culture. These findings suggest that the reexpression of HeLa cell antigens in hybrid cells was related to deficiency in vitamin A.     

Electrical excitability and chemosensitivity of mouse neuroblastoma X mouse or human fibroblast hybrids

Intracellular microelectrode recording techniques were used to measure passive membrane properties, electrical excitability and chemosensitivity of mouse neuroblastoma cells and somatic cell hybrids formed between these cells and either L cells or human diploid fibroblasts. Different clones of the hybrid cells showed varying degrees of neuronal or fibroblastic membrane-differentiated function; a selection technique involving incubation of the cells with aminopterin gave quite homogeneous non-dividing populations of cells within a given clone of the neuroblastoma x L cell hybrids. Despite relatively uniform chromosomal numbers within a given clone, the neuroblastoma x human fibroblast hybrids were morphologically and electrophysiologically heterogeneous. The possibility is considered that this may represent the effect of variable segregation of the human chromosomal complement.     

Enucleation of mammalian cells by cytochalasin B. II. Formation of hybrid cells and heterokaryons by fusion of anucleate and nucleated cells

The development of methods for the formation of hybrid cells and heterokaryons by virus-induced fusion of chemically-enucleated cells and nucleated cells has been described. Heterokaryons and hybrid cells formed by fusion of anucleate mouse peritoneal macrophages (MPM) and nucleated mouse L and human HEp-2 cells were identified by mixed haemadsorption, by their sensitivity to trypsin and by their capacity to ingest antibody-coated sheep red blood cells. The expression of macrophage markers in these cells declined rapidly after fusion. Hybrid cell and heterokaryon formation was identified in mixed cultures of anucleate L cells and nucleated MPM, and was accompanied by the reactivation of DNA synthesis in the macrophage nuclei. Other hybrids and heterokaryons were formed by virus-induced fusion of anucleate MPM and nucleated chick embryo erythrocytes and anucleate L cells and nucleated HEp-2 cells. The value of anucleate-nucleate cell hybrids in the study of metabolic and genetic regulation in mammalian cells is discussed.     

Correction of the biochemical defect in porphobilinogen deaminase deficient cells by non-viral gene delivery

Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in acute intermittent porphyria (AIP). AIP is a genetic disease characterized by neurovisceral and psychiatric disturbances. Despite a palliative treatment, it may still be lethal. An initial step towards gene therapy was recently taken by showing that PBGD could be expressed to correct the enzyme deficiency in AIP fibroblasts. The aim of the present study was to investigate whether the biochemical defect can be corrected by using non-viral gene delivery. The biochemical defect in human and mouse PBGD deficient fibroblasts was demonstrated by analyzing synthesis of the heme precursor, protoporphyrin (PP), after addition of 5-aminolevulinic acid (ALA). Human AIP fibroblasts synthesized 21% and mouse PBGD deficient fibroblasts only 11% of the PP amount synthesized in respective control cells. Gene delivery increased the PBGD activity 88–200 fold in human AIP fibroblasts and synthesis of PP was increased from 21–152% of normal after ALA incubation. Similar results were obtained in mouse PBGD deficient cells, although the PP levels were several-fold lower as compared to human cells. HPLC analysis confirmed that PP was the main porphyrin intermediate that was formed. Addition of porphobilinogen (PBG) resulted in 3–7 fold lower synthesis of PP as compared to ALA addition. These results show that non-viral gene delivery of plasmids encoding PBGD results in a high expression of functional PBGD shown by induced synthesis of PP in PBGD deficient cells after supplementation of ALA and PBG.     

UV-Irradiation of related mouse hybrid cells: Similar increase in capacity to replicate intact minute-virus-of-mice but differential enhancement of survival of UV-irradiated virus

3 hybrid cell lines between mouse fibroblasts (A9) and mouse lymphoma cells (L5178YS) were compared with regard to the ability of UV-pre-irradiated cells to replicated intact (unirradiated) Minute-Virus-of-Mice (MVM) and to reactivate UV-irradiated MVM. UV irradiation of cells before virus infection enhanced their ability to plaque intact virus (Enhanced Capacity) to a similar extent in the 3 hybrid cell lines. However, pretreatment of cells with UV radiation enhanced the survival of UV-damaged virus (Enhanced Reactivation) in only 1 of these hybrids. The lack of detectable Enhanced Reactivation in the other hybrids without concomitant change in their Enhanced Capacity, suggets that these processes are at least partly independent. Virus survival in unirradiated cells was similar for all 3 hybrid cell lines, indicating that the absence of detectable Enhanced Reactivation in 2 of the hybrids was not due to the constitutive expression of this process, but might rather result from its loss or extinction. The expression of both Enhanced Capacity and Enhanced Reactivation requires synthesis of protein de novo shortly after cell irradiation.     

Transfer of purine metabolites between cells through the medium and via cell contacts in cocultures of HGPRT+ and HGPRT- cells

Cells with and without hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity were used to examine the transfer of purine metabolites through the medium and via cell contacts. HGPRT- Chinese hamster and human fibroblasts were able to incorporate 3H-labeled purine metabolite(s) from medium in which mouse HGPRT+ B82 cells had been grown for 24 h with [3H]hypoxanthine, but mouse A9 fibroblasts that were deficient in HGPRT, adenine phosphoribosyltransferase (APRT), and methylthioadenosine phosphorylase (MTAP) were unable to incorporate these metabolites. This suggests that in recipient cells incorporation is due to [3H]MTA, which has been shown previously to be the major 3H-labeled purine metabolite to accumulate in B82 medium, being cleaved by MTAP to [3H]adenine, which is phosphoribosylated by APRT to [3H]AMP. Incorporation by recipient cells of metabolites from the medium is referred to as contact-independent metabolite transfer (CIMT). In autoradiograms of B82/A9 cocultures that were labeled with [3H]hypoxanthine, grains were found over A9 that were not in contact with B82, although A9 did not act as recipients of CIMT. This is termed proximity-dependent metabolite transfer (PDMT). Both CIMT and PDMT interfered with the assessment of nucleotide exchange between HGPRT+ and HGPRT- cells through cell contacts, which is referred to as contact-dependent metabolite transfer (CDMT). These problems were unique to HGPRT+ mouse L cells. However, HGPRT- mouse L cells, A9, could be used as potential recipients. A9 were positive recipients of CDMT with only one of five cell lines tested, which suggested that these cells were selective communicators. CDMT could not be studied with [3H]guanine because the nuclei of HGPRT- cells became labeled.     

Antigenic immunostaining patterns in somatic hybrids of human HeLa cells and mouse fibroblasts 3T3.4E propagated in conventional medium and delipidized serum

Somatic cell hybrids were obtained with electric pulse by fusion of human epithelial HeLa cells derived from a carcinoma of the uterine cervix and mouse fibroblasts 3T3.4E, deficient in thymidine kinase. Hybrids were selected and propagated in HAT media; some experiments were carried out in medium with delipidized serum. The hybrid cells were characterized by indirect immunofluorescence with a biotin-streptavidin system using a panel of nine monoclonal antibodies specific for membrane and cytoplasmic antigens of parental cells: intermediate filaments (keratins and vimentin), HLA class 1 (β₂-microglobulin), cell activation (EGF and transferrin receptors) and cellular adhesion (fibronectin and laminin).     

The plasmacytoma J558L lacks constitutively active NF-kappa B and is deficient in early response gene activation.

In mature B cells the nuclear factor NF-kappa B which binds within the kappa enhancer is constitutively present in the nucleus. However, the lambda light chain producing myeloma J558L has been found to lack constitutively functional NF-kappa B. Deoxycholate released functional NF-kappa B from cytoplasmic extracts and functional NF-kappa B was present in J558L following cycloheximide but not phorbol ester treatment. J558L was also unable to respond to phorbol ester stimulation with synthesis of mRNA from the early response gene TIS11. J558L differs from S107, another myeloma which was found to be deficient in the synthesis of NF-kappa B but not in the activation of TIS11. Somatic cell hybrids were used to further define the defect in J558L; hybrids were made with the myelomas S107 and S194 and the pre-B cell line 70Z/3. In general, complementation of the defect in J558L was observed; however there was not a direct correlation between the levels of TIS11 mRNA and NF-kappa B expression in the somatic cell hybrids, suggesting that the pathways of activation of these genes, while possibly sharing common elements, are not identical. The defect in J558L was surprising given that it has frequently been used for the expression of transfected light chain genes.     

Galactocerebrosidase activity in somatic cell hybrids derived from twitcher mouse/control human fibroblasts is associated with human chromosome 17.

Somatic cell hybrids derived from twitcher mouse cells and from control human fibroblasts were selected by two different methods. One method utilized 6-thioguanine-resistant twitcher cells as a parental line and the other used neomycin-resistant control human fibroblasts as a parental line so that hybrid lines could be selected in either HAT or in G-418 medium, respectively. The hybrid lines were analyzed for galactocerebrosidase activity. Since the twitcher cell lines are deficient in galactocerebrosidase activity, the presence of this activity in these hybrid lines depends upon the presence of human chromosome contents. Both galactocerebrosidase-positive and -deficient hybrid lines were analyzed for their human chromosome contents by the use of isozyme markers. In hybrids derived from both selection methods the expression of galactocerebrosidase activity was associated with the presence of human chromosome 17 marker isozymes. This was confirmed cytogenetically by means of trypsin-banded Giemsa staining of intact human chromosome 17 in three galactocerebrosidase-positive hybrid lines.     

Induction of alkaline phosphatase in cultured human fibroblasts. Comparison of normal cells and those from patients with cystic fibrosis.

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP. Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

Analysis of myogenesis by somatic cell hybridization: III. Myogenic competence of hybrids derived from rat L6 myoblasts and mouse primary fibroblasts and myoblasts

Hybrid cells derived from rat L6 myoblasts and mouse primary fibroblasts (M x F hybrids), as well as those derived from rat L6 myoblasts and mouse primary myoblasts (M x M hybrids), were examined for their ability to engage in myogenesis as judged by muscle fiber formation plus the expression of skeletal muscle myosin and creatine kinase (CK). Of 172 primary hybrid colonies scored, 59% were myogenic in the M x F fusion and 97% exhibited muscle fiber formation in the M x M fusion. Individual hybrid clones from each cross were isolated, expanded and analyzed for myogenic capabilities as well. All three M x M and all ten M x F isolated clones exhibited preferential elimination of mouse chromosomes. Nonetheless, all were capable of fusing spontaneously and of elaborating skeletal muscle myosin and CK. The three M x M hybrids expressed only MM-CK whereas nine out of ten M x F hybrids produced all three CK isoenzymes (MM, MB, BB). These results suggest that M X M hybrids express CK patterns reminiscent of the rat L6 parental cells while M X F hybrids apparently mimic mouse muscle fiber CK patterns. Various models are discussed which address these phenomena.     

Plasmalogen content and beta-adrenoceptor signalling in fibroblasts from patients with Zellweger syndrome. Effects of hexadecylglycerol

In Zellweger or cerebro-hepato-renal syndrome (CHRS), the assembly of peroxisomes is defective, resulting in deficient plasmalogen formation. Plasmalogens are part of the membrane lipid composition. In fibroblasts of CHRS patients, the plasmalogen fraction of phosphatidylethanolamine (PPE) was about half of that in control cells while total phospholipid (PL) content, individual PL and plasma membrane fluidity were normal. CHRS cell strains had higher beta-adrenoceptor numbers and isoproterenol-stimulated cAMP responses. Receptors were more efficiently coupled to adenylate cyclase than in control cells. Stimulations of cAMP with NaF or forskolin were the same as in control cells. Restoring synthesis of plasmalogens with hexadecylglycerol (HDG), a plasmalogen precursor, resulted in a proportionate increase in PPE of about 40% in both control and CHRS fibroblasts. Exposure to HDG reduced surface beta-adrenoceptor sites and cAMP-responses to isoproterenol in CHRS cells only, while post-receptor stimulations of cAMP were reduced in both cell types. Plasmalogen contents inversely correlated with isoproterenol-stimulated cAMP levels. The increased numbers of functional beta-adrenoceptors in CHRS fibroblasts may be the result of a higher expression and/or of a prolonged functional half-life of the receptor protein. In vivo, this may contribute to the clinical manifestations of the disease.     

The normalization of tumor cell morphology related to an increase in their size: research on giant cells produced by mitomycin C treatment]

This study shows that artificial increase in cell site leads to morphological normalization of transformed fibroblasts. Mouse L cells (clone 171/5) were used. As most transformed cells, they were poorly spread on the substratum, made only dot-like focal contacts with it, rounded quickly at room temperature and did not contain prominent actin cables. Giant cells were obtained by incubation of these cells in the medium supplemented with mitomycin C (0.15-0.20 mcg/ml). DNA synthesis and mitosis were blocked by this treatment, while protein synthesis was changing very slightly. As a consequence, the cell size increased dramatically from 3 to 11 days of the cell incubation in the mitomycin containing medium. The degree of cell spreading per mcg of protein increased significantly in the giant cells. These cells do not round after moderate cooling, and well developed system of actin cables and matured streak-like focal contacts associated with these cables are formed in them. These results, along with our previous data on the restoration of cell spreading and cytoskeleton structure in giant multinucleated cells, provide strong evidences that the increase in cell size per se can induce qualitative changes in cell morphology. It can be suggested that there are some scaling-dependent factors regulating the processes of cytoskeleton assembly and formation of cell-substrate contacts.     

Induction of alkaline phosphatase in cultured human fibroblasts: Comparison of normal cells and those from patients with cystic fibrosis

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP.Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and myelin-associated glycoprotein) in regenerating adult mouse sciatic nerve

The localization of the neural cell adhesion molecules L1, N-CAM, and the myelin-associated glycoprotein was studied by pre- and postembedding staining procedures at the light and electron microscopic levels in transected and crushed adult mouse sciatic nerve. During the first 2-6 d after transection, myelinated and nonmyelinated axons degenerated in the distal part of the proximal stump close to the transection site and over the entire length of the distal part of the transected nerve. During this time, regrowing axons were seen only in the proximal, but not in the distal nerve stump. In most cases L1 and N-CAM remained detectable at cell contacts between nonmyelinating Schwann cells and degenerating axons as long as these were still morphologically intact. Similarly, myelin-associated glycoprotein remained detectable in the periaxonal area of the degenerating myelinated axons. During and after degeneration of axons, nonmyelinating Schwann cells formed slender processes which were L1 and N-CAM positive. They resembled small-diameter axons but could be unequivocally identified as Schwann cells by chronical denervation. Unlike the nonmyelinating Schwann cells, only few myelinating ones expressed L1 and N-CAM. At the cut ends of the nerve stumps a cap developed (more at the proximal than at the distal stump) that contained S-100-negative and fibronectin-positive fibroblast-like cells. Most of these cells were N-CAM positive but always L1 negative. Growth cones and regrowing axons expressed N-CAM and L1 at contact sites with these cells. Regrowing axons of small diameter were L1 and N-CAM positive where they made contact with each other or with Schwann cells, while large-diameter axons were only poorly antigen positive or completely negative. 14 d after transection, when regrowing axons were seen in the distal part of the transected nerve, regrowing axons made L1- and N-CAM-positive contacts with Schwann cells. When contacting basement membrane, axons were rarely found to express L1 and N-CAM. Most, if not all, Schwann cells associated with degenerating myelin expressed L1 and N-CAM. In crushed nerves, the immunostaining pattern was essentially the same as in the cut nerve. During formation of myelin, the sequence of adhesion molecule expression was the same as during development: L1 disappeared and N-CAM was reduced on myelinating Schwann cells and axons after the Schwann cell process had turned approximately 1.5 loops around the axon. Myelin-associated glycoprotein then appeared both periaxonally and on the turning loops of Schwann cells in the uncompacted myelin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in Expression of Fibroblast Surface Antigen (SFA) during Cytodifferentiation and Heterokaryon Formation

Fibroblast surface antigen (SF antigen, SFA) is a major glycoprotein antigen detected in connective tissue cells (primitive mesenchymal cells, fibroblasts, and astroglial cells). In this study the expression of SFA was followed during differentiation of the mesenchymal cells of the mouse metanephros and during heterokaryon formation produced by Sendai-virus induced fusion of human fibroblasts and chick red blood cells. It was demonstrated by immunofluorescence that SFA was lost from the kidney mesenchymal cells when they differentiate into epithelial cells of the secretory tubuli. During this process SFA became detectable in the basement membrane formed around the tubuli. In cell fusion experiments human SFA which was present as fibrillar network on the surface of cultured fibroblasts, was gradually lost from the heterokaryons when the incorporated chick nuclei became activated. These two sets of experiments indicate that SFA can be used as a phenotypic marker of Cytodifferentiation.     

Regulation of expression of tyrosine aminotransferase in somatic cell hybrids between rat hepatoma cells and mouse fibroblasts

Somatic cell hybrids between rat hepatoma cells and mouse 3T3 fibroblasts fail to produce the liver-specific enzyme tyrosine aminotransferase. A novel approach using gamma-irradiation to induce chromosome loss from the non-expressing parent cell was applied to dissect genetically the factors in 3T3 cells that interact with the regulation of expression of tyrosine aminotransferase in these hybrids. Suppression of basal and steroid-inducible tyrosine aminotransferase activities was progressively relieved with increasing dose of radiation. The wide range in degree of reexpression suggests a complex of regulatory mechanisms. Suppression of steroid-inducibility was not linked to the mouse X-chromosome. Nor did the mouse genome affect the modulation of enzyme activity induced by insulin and by serum.     

Contact inhibition of growth of human diploid fibroblasts by immobilized plasma membrane glycoproteins

The human embryonic fibroblasts used in this study show pronounced inhibition of growth when reaching a critical cell density. High cell density and growth inhibition has previously been mimicked by the addition of glutaraldehyde-fixed cells or of isolated plasma membranes to sparsely seeded proliferating fibroblasts (Wieser, R. J., R. Heck, and F. Oesch, 1985, Exp. Cell Res., 158:493-499). In this report, we describe the successful solubilization of the growth-inhibiting glycoproteins and their covalent coupling to silicabeads (10 microns), which had been derivatized with 3-isothiocyanatopropyltriethoxysilane. The beads, bearing the plasma membrane proteins, were added to sparsely seeded, actively proliferating fibroblasts, and growth was measured by the determination of cell number or of incorporation of [3H]thymidine into DNA. The growth was inhibited in a concentration-dependent manner, whereby 50% inhibition was achieved with 0.3 micrograms of immobilized protein added to 5 X 10(3) cells. Terminal galactose residues of plasma membrane glycoproteins with N-glycosydically bound carbohydrates were responsible for the inhibition of growth. Dense cultures of human fibroblasts are characterized by an accelerated synthesis of procollagen type III. We have found that this cellular response can also be induced by the addition of immobilized plasma membrane glycoproteins to sparsely seeded cells. These observations support the conclusion that the addition of immobilized plasma membrane glycoproteins to sparsely seeded fibroblasts mimics the situation occurring at high cell density. These results show that cell-cell contacts via plasma membrane glycoproteins carrying terminal galactose residues are important for the regulation of the proliferation of cultured human fibroblasts and presumably of the accelerated synthesis of collagen type III.