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).     

Expression of epidermal differentiation antigens in cultures of interspecific somatic hybrids (human keratinocytes X mouse fibroblasts 3T3-4E)

Interspecific somatic hybrids have been prepared by fusion of human epidermal cells with mouse fibroblasts 3T3-4E using PEG 4000. Expression of epidermal differentiation antigens (bullous pemphigoid antigens, BP, keratin subsets 55-57 k and 67 k), markers of basal and suprabasal cells, were studied by immunocytochemistry for 10 passages. These markers were detected in the hybrids early after fusion, indicating that cells from both compartments were able to fuse with 3T3-4E cells. However, the hybrids expressing high molecular weight keratins were no longer detected after 7 days in primary cultures and serial passages, whereas those expressing BP antigens and vimentin persisted. Low molecular weight keratins 52 K and 50 K were detected by SDS-PAGE at the second passage in precipitates formed between labeled hybrid lysates and total keratin rabbit antiserum. Karyotype analysis showed mainly murine chromosomes and a submetacentric human chromosome between the 6th and the 10th passage.     

Correlation of the inability to sustain growth in defined serum-free medium with the suppression of tumorigenicity in Wilms' nephroblastoma.

We report the investigation of the growth properties of tumorigenic and reverted nontumorigenic Wilms' nephroblastoma cells when cultured in serum-free medium. Wilms' tumor, a pediatric nephroblastoma, has been associated with deletions encompassing the p13 band of chromosome 11 and an independent loss of heterozygosity at 11p15. Weissman et al. (Science 236:175-180, 1987) transferred a human der(11) chromosome into the G401.6TG.6 Wilms' tumor cell line via the microcell-mediated chromosome transfer technique. The resulting microcell hybrids were nontumorigenic when assayed in nude mice; however these cells retained all of the in vitro growth and morphological characteristics of the tumorigenic parental cells in 10% fetal calf serum (FCS). Segregation of the der(11) chromosome from the nontumorigenic microcell hybrid cells resulted in the reappearance of the tumorigenic phenotype in vivo. In vitro culture of these cell lines in serum-free medium supplemented with 0.1% bovine serum albumin (BSA) and 10 ng/ml Na2O3Se resulted in sustained growth of both the tumorigenic parent and the tumorigenic segregant while the nontumorigenic microcell hybrids were unable to divide. The separate addition of either 10 ng/ml of epidermal growth factor (EGF) or 5 micrograms/ml of insulin did not alter this effect. However, the addition of 5 micrograms/ml of transferrin stimulated the nontumorigenic microcell hybrid cells to grow at a rate comparable to the tumorigenic cells. In addition, conditioned serum-free medium from the tumorigenic parental or tumorigenic segregant cell lines was able to stimulate the growth of the nontumorigenic microcell hybrid cells, whereas the reciprocal experiment had no effect on the growth of the tumorigenic cells. These data suggest that the inability of the microcell hybrid cells to grow in serum-free conditions is correlated with their genetic nontumorigenic phenotype and that a specific growth factor, transferrin, can bypass or alter this negative growth regulatory pathway(s) in vitro.     

Integrin-mediated migration of murine B82L fibroblasts is dependent on the expression of an intact epidermal growth factor receptor

To evaluate the mechanisms by which epidermal growth factor (EGF) regulates actin-based cellular processes such as cell migration, we first examined the effects of EGF on cell adhesion, which is essential for cell migration. In mouse B82L fibroblasts transfected with the full-length EGF receptor, EGF promotes cell rounding and attenuates cell spreading on fibronectin, laminin, and vitronectin, and thus appears to reduce the strength of cell adhesion. Moreover, EGF synergizes with multiple extracellular matrix (ECM) components in the promotion of integrin-mediated cell migration of several different cell types, including fibroblasts and various carcinoma and osteosarcoma cell lines. Interestingly, co-presentation (co-positioning) of EGF with laminin or fibronectin is essential for EGF-stimulated migration. When EGF is mixed with the cells instead of the ECM components, it has little effect on cell migration. These results suggest that co-presentation of EGF with ECM components can enhance the polarization events required for directional cell movement. To identify the EGF receptor elements critical for the EGF stimulation of cell migration, B82L fibroblasts were transfected with either mutated or wild-type EGF receptors. Surprisingly, we found that B82L-Parental cells that lack the EGF receptor are not able to migrate to fibronectin, even though they can adhere to fibronectin. However, the introduction of wild-type EGF receptors into these fibroblasts enables them to migrate toward fibronectin even in the absence of EGF. The requirement of the EGF receptor for cell migration does not appear to result from the secretion of EGF or TGF-alpha by the cells transfected with the EGF receptor. Furthermore, cells expressing EGF receptors that are kinase-inactive, or C-terminally truncated, exhibit little migration toward fibronectin, indicating that an intact EGF receptor kinase is required for fibronectin-induced cell migration. In addition, neutralizing anti-EGF receptor antibodies attenuate cell migration in the presence of EGF, and inhibit migration to fibronectin or laminin alone. These results further suggest that the EGF receptor is downstream of integrin activation in the signal transduction pathways leading to fibroblast migration.     

Expression of human hepatic genes in somatic cell hybrids

Four diploid human cell types (lymphocytes, fibroblasts, amniotic fluid cells, and hepatocytes) were fused to mouse hepatoma cells, HH. HH synthesized and secreted several liver-specific gene products including albumin, transferrin, and alpha-fetoprotein. The resulting interspecific hybrids were compared to determine whether or not the pattern of human hepatic gene expression was similar when these various cells were fused with the mouse hepatoma line. The expression of six human hepatic genes was examined, including albumin, alpha-fetoprotein, ceruloplasmin, transferrin, alpha-1-antitrypsin, and haptoglobin. Albumin was most frequently expressed while alpha-fetoprotein was not detected in any of the hybrids studied. The patterns of expression of human serum proteins differed between the hybrid series. Hybrids derived from human fibroblasts produced primarily albumin, while those derived from lymphoblastoid cells and amniocytes had a higher frequency of clones secreting alpha-1-antitrypsin. The findings reported here suggest that the frequency of hybrid clones expressing human hepatic gene products and the array of proteins produced are influenced by the histogenetic state of the human parental cell type.     

Lack of expression of differentiation in mouse teratoma-fibroblast somatic cell hybrids

Somatic cell hybrids between multipotent mouse teratoma cells and mouse fibroblasts were established and isolated. The hybrid cells possess one chromosome set of each parental type and express major histocompatibility antigens of both strains of mice from which the parental cells were derived. Upon inoculation into F1 mice, hybrid cells produced tumors which were typical fibrosarcomas as were the tumors produced by the fibroblastic parental cell line. None of the well differentiated tissue types characteristic of the teratoma parent cell tumors were expressed in the hybrid tumors. The hybrid tumors possessed the majority of the chromosomes of the two parental cells; however, the modal numbers were slightly reduced in comparison with those of the cell populations inoculated. The possible role of ‘genic balance’ in phenotypic expression of cell hybrids is discussed.     

The serum-free growth of cultured cells in bovine colostrum and in milk obtained later in the lactation period

Seven established cell lines, including both epithelial cells and fibroblasts (MDCK, Vero, CV-1, NRK, 3T3, F2408, and NIL8) and four early passage cell strains (bovine articular chondrocytes, bovine smooth muscle cells, human foreskin fibroblasts, and rat embryo cells) were cultured in serum-free medium supplemented with milk obtained 1 day after birth (colostrum) or 80 days after birth (older milk). MDCK, Vero, CV-1, NRK, and 3T3 grew readily in colostrum and attained saturation densities ranging from 22% to 63% of that in serum. There was no growth of F2408, NIL8, or the early passage strains in bovine colostrum. None of the 11 cell cultures grew in older milk. The temporal dependence of growth in milk was examined in detail using MDCK cells. Growth equivalent to that in serum occurred in 3% colostrum and in 15% milk obtained 2 days after birth. Milk obtained 3 days and 10 days after birth was not effective as a growth supplement for MDCK cells at any concentration. Those cells, unable to grow in colostrum or in older milk, could be induced to grow if culture dishes were precoated with fibronectin. In addition to fibronectin, it was necessary in some cultures to supplement colostrum or older milk with insulin and/or transferrin in order to achieve growth. In the presence of fibronectin and appropriate factors, the final saturation density attained in colostrum or older milk ranged from 25% to 100% of that in serum. The fibronectin contents of bovine colostrum and milk were determined. The fibronectin level of colostrum was found to be approximately 5% of bovine serum. There was no detectable fibronectin in the 80-day-old milk.     

Behavior of transforming growth factors in serum-free media: an improved assay for transforming growth factors

Transforming growth factors (TGFs) are a relatively new category of factors that induce the anchorage-independent growth of non-transformed cells. These factors are usually detected by their ability to induce normal rat kidney (NRK) fibroblasts to grow in soft agar. Until now, this assay has been performed in serum-containing medium (SCM). Unfortunately, the background activity of this assay is variable and dependent on several factors, including passage number of the cells and the serum lot used. Furthermore, the addition of either EGF or TGF-beta alone results in the appearance of additional colonies, which decreases the sensitivity of the assay. To circumvent these problems, serum-free media have been developed that support the growth of the NRK cells at low density in both monolayer culture and soft agar. Long-term growth in monolayer cultures occurs in serum-free medium supplemented with laminin, insulin, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and high density lipoprotein (HDL). Growth in soft agar occurs when TGFs are added to a serum-free medium, AIG medium, that contains insulin, transferrin, FGF and HDL. In contrast to the background activity observed when the assay is performed in SCM, no colonies form in the AIG medium unless TGFs are added and few, if any, colonies form if EGF or TGF-beta are added alone. Thus, the AIG medium provides an improved assay for TGFs. In addition, the AIG medium should prove useful for examining other factors, including serum factors, for TGF activity.     

Regulation of lipid synthesis from acetate in diploid fibroblast cultures —variation with passage level and stage of cell growth

Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [¹⁴C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein-free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultures to change in exogenous lipid is dependent on passage level and state of growth.     

Cell fusion of human and mouse cells as a source for new cells retaining human markers. Analysis of DNA content, membrane and cytoplasmic antigen expression.

Flow cytometry and ultrastructural morphometry were used to study some characteristics of cells obtained by fusion with polyethylene-glycol 4000 between mouse fibroblasts 3T3.4E and normal keratinocytes (3T3.4E x NHK) or hand wart human keratinocytes (3T3.4E x HWK), at late passages. The cell cycle and the expression of human beta 2-microglobulin, human EGF-receptors (EGF-r), vimentin were simultaneously studied by flow cytometry. Epithelial CaSki cells, derived from a human uterine carcinoma, expressing high levels of beta 2-microglobulin, EGF-r and vimentin, were used as a positive control. In mouse fibroblasts 3T3.4E only vimentin was expressed whereas in cells derived from fusion, human beta 2-microglobulin, human EGF-r and vimentin were detected. The cell cycle analysis revealed that the peak position of G0/G1 differed with the cells (channel 11 for 3T3.4E cells, 13 for 3T3.4E x HWK and 15 for 3T3.4E x NHK). The area of the cell compartments from each cell type was also different by quantitative ultrastructural morphometry. The hybrid phenotype was maintained in late passages in cells (3T3.4E x NHK) and (3T3.4E x HWK), as shown by the expression of human antigens, differences in DNA contents and nuclear area. Flow cytometry may be a very accurate and precise tool for studying low antigenic expression. The combination of different methods including analysis of DNA content, antigenic expression and ultrastructural morphometry confirmed that 3T3.4E, 3T3.4E x NHK and 3T3.4E x HWK cells are different cell types. These techniques are complementary to cell phenotype analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of cell attachment and cell number by fibronectin and laminin

We have examined the effect of laminin and fibronectin on the attachment and growth on type IV collagen of a line of mouse epithelial cells and a strain of adult human fibroblasts. Laminin stimulated attachment of the epidermal cells and fibronectin stimulated fibroblast attachment. At high concentrations (100 micrograms/ml), the attachment proteins altered the growth of cells in culture. The epidermal cells grew better in media containing fibronectin-free serum supplemented with laminin. Fibroblasts, on the other hand, grew best in media containing serum supplemented with fibronectin. These data suggest that laminin promotes epithelial cell growth whereas fibronectin promotes fibroblast growth. This observation was confirmed when these cells were cocultured in the presence of the attachment proteins or of their respective antibodies. The mouse epidermal cells grew best when laminin was added to cocultures of fibroblasts and epithelial cells. Fibroblasts grew best in the presence of antibody to laminin and poorly in the presence of antibody to fibronectin. Thus, fibronectin and laminin may participate in the regulation of cell populations in vivo and may be involved in epithelial-mesenchymal interactions.     

Behavior of transforming growth factors in serum-free media: An improved assay for transforming growth factors

Summary Transforming growth factors (TGFs) are a relatively new category of factors that induce the anchorage-independent growth of non-transformed cells. These factors are usually detected by their ability to induce normal rat kidney (NRK) fibroblasts to grow in soft agar. Until now, this assay has been performed in serum-containing medium (SCM). Unfortunately, the background activity of this assay is variable and dependent on several factors, including passage number of the cells and the serum lot used. Furthermore, the addition of either EGF or TGF-β alone results in the appearance of additional colonies, which decreases the sensitivity of the assay. To circumvent these problems, serum-free media have been developed that support the growth of the NRK cells at low density in both monolayer culture and soft agar. Long-term growth in monolayer cultures occurs in serum-free medium supplemented with laminin, insulin, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and high density lipoprotein (HDL). Growth in soft agar occurs when TGFs are added to a serum-free medium, AIG medium, that contains insulin, transferrin, FGF and HDL. In contrast to the background activity observed when the assay is performed in SCM, no colonies form in the AIG medium unless TGFs are added and few, if any, colonies form if EGF or TGF-β are added alone. Thus, the AIG medium provides an improved assay for TGFs. In addition, the AIG medium should prove useful for examining other factors, including serum factors, for TGF activity. Editor's Statement This communication describes a modification of the standard assay for transforming growth factors. The techniques employed make use of advantages provided by recent advances in serum-free cell culture to provide a well-defined detection system that is more sensitive than conventional procedures. Experimental approaches described in this article also should be helpful in unraveling differences in cellular behavior encountered under anchorage-dependent vs. anchorage-independent conditions. D. W. Barnes     

Improved clonal and nonclonal growth of human,rat and bovine adrenocortical cells in culture

Summary This report describes the development of a culture system for long-term growth and cloning of human fetal adrenocortical cells. Optimal conditions for stimulating clonal growth were determned by testing the efficacy of horse serum (HS), fetal bovine serum (FBS), fibroblast growth factor (FGF), epidermal growth factor (EGF), fibronectin, and a combination of growth factors, UltroSer G, in stimulating growth from low density. Optimal conditions for clonal growth were achieved using fibronectin-coated dishes and DME/F12 medium with 10% FEBS, 10% HS, 2% UltroSer G, and 100 ng/ml FGF or 100 pM EGF. Conditions for growth at clonal density were found to be optimal for growth of early passage, nonclonal cultures at higher densities. The improved growth conditions used for cloning were shown to allow continued long-term growth of nonclonal human adrenocortical cells without fibroblasts overgrowth. All cells in cultures grown in HS, FBS, and UltroSer G had morphologic characteristics of adrenocortical cells, whereas cells grown in FBS only rapidly became overgrown with fibroblasts. Clonal and nonclonal early passage human adrenocortical cells had smilar mitogenic responses to FGF and EGF. Whereas FGF, EGF, and UltroSer G showed similar stimulation of DNA synthesis and clonal growth in human adrenocortical cells and human adrenal gland fibroblasts, the tumor promoter 12-O-teradecanoylphorbol-13-acetate stimulated growth only in adrenocortical cells and was strongly inhibitory to growth in fibroblasts. In both cell types, forskolin inhibited DNA synthesis. Human adrenocortical cell cultures were functional and synthesized cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. The improved growth conditions for clonal growth of human adrenocortial cells also provided optimal conditions for long-term growth of cultured rat adrenocortical cells and ncreased the cloning efficiency of cultured bovine adrenocortical cells. This work was supported by Research grants AG-00936 and AG-06108 from the National Institute on Aging, Bethesda, MD.     

The enhanced expression of cellular fibronectin in mouse-mouse somatic cell hybrids

The expression of cellular fibronectin in mouse-mouse somatic cell hybrids was compared with that in the parental cells by indirect immunofluorescence and enzyme-linked sandwich immunoassay. The hybrid clones were all found to express much larger amounts of cellular fibronectin than the parental cells. This indicates that somatic cell hybridization does not necessarily suppress the expression of cellular fibronectin.     

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.     

Effect of serum,fibronectin, and laminin on adhesion of rabbit intestinal epithelial cells in culture

Rabbit intestinal epithelial cells, obtained after a limited hyaluronidase digestion, were incubated in medium with or without calf serum, on bacteriological plastic dishes. The dishes, either plain or coated with an air-dried type I collagen film, were pretreated with medium alone or with medium containing purified laminin or purified fibronectin. Cells did not attach in significant numbers to untreated bacteriological plastic, even in the presence of serum. Cells did attach to collagen-coated dishes, and were judged viable on the basis of their incorporation of radiolabeled leucine into cell protein. Cell adhesion to the collagen substrate increased in proportion to the concentration of serum in the medium, with maximal attachment at 5% serum or greater. Pretreatment of plain or collagen-coated dishes with increasing amounts of fibronectin enhanced cell adhesion in a concentration-dependent manner. Either serum, or fibronectin-free serum in the medium enhanced cell attachment to substrates pretreated with cither fibronectin or laminin. Thus, intestinal epithelial cells appear to possess surface receptors for both laminin and fibronectin. The evidence further suggests that calf serum may contain factors, other than fibronectin, capable of enhancing intestinal epithelial cell attachment to collagen substrates.     

Primary culture of adult mouse lung fibroblasts in serum-free medium: responses to growth factors

We report a completely serum-free system for primary culture of fibroblasts from explants of adult mouse lung tissue which permits bioassays for cytokine activity to be performed using unselected populations of cells at low passage number, without interference by serum binding proteins or interacting growth factors. Cultures were established on collagen-coated surfaces in medium MCDB 201 containing albumin, transferrin, epidermal growth factor, lipids, prostaglandin E1, vitamin E, and reducing agents. The cells were morphologically and ultrastructurally typical of fibroblasts in culture and demonstrated expression of vimentin and induction of expression of desmin in culture. Proliferation of the cells was reproducible between different primary cultures and was growth factor dependent. Both cycling and growth-arrested cells exhibited increased DNA synthesis when stimulated with epidermal growth factor, platelet-derived growth factor, or basic fibroblast growth factor, which functioned as complete mitogens, but did not respond to insulin, tumor necrosis factor or interleukin-1 beta. Maximal induction of DNA synthesis by epidermal growth factor required the continued presence of the mitogen in the culture medium. These results cannot be satisfactorily explained by the competence-progression model of responses to mitogenic stimuli but support and extend the findings of other studies using diploid fibroblasts.     

Regulation of growth and gene activity in euploid hybrids between human neonatal fibroblasts and epithelioid amniotic fluid cells.

Pure populations of proliferating synkaryons were obtained from polyethylene glycol-mediated crosses between diploid human foreskin fibroblasts and epithelioid amniotic fluid cells. These hybrids proved to be chromosomally stable tetraploids. They continuously produced heteropolymeric G6PD and showed strictly additive patterns of silver staining of both parental sets of nucleolar organizing chromosomes. Collagenous proteins characteristic of the fibroblast parent were synthesized, while fibronectin production appeared to be directed by the epithelioid portion of the genome. Even though these heterotypic hybrids proliferated at a reduced rate and achieved fewer population doublings relative to homotypic (fibroblast X fibroblast) crosses, they survived passage by trypsinization better than pure populations of epithelioid cells. These observations suggest a concerted action of both parental genomes with respect to proteins responsible for "household" functions, but complementation and possibly modulation of gene action with respect to "luxury" protein synthesis and cell growth.     

Induction of homokaryocyte, heterokaryocyte and hybrid formation by lysolecithin

Lysolecithin has been utilized to induce homo- and heterokaryocyte formation in CV-1, F5-1 and WI-38 cells and hybrid formation in 1R and mKSBu100 cells cultured in vitro. A new fusion technique utilizing solutions of albumin or delipidized serum as vehicles for lysolecithin has been introduced. Optimal concentrations of lysolecithin for inducing cell fusion and reducing cell damage for various cell lines have been evaluated. Lysolecithin produced 15–25% homokaryocytes and about 5–7% heterokaryocytes. Two different mutant cell lines fused in the presence of lysolecithin produced hybrids which survived in selective medium.     

Regulation of terminal differentiation of cultured human keratinocytes by vitamin A

Vitamin A is known to exert an important influence on epithelial differentiation. The fetal calf serum supplement of cell-culture medium contains enough of the vitamin to affect the differentiation of cultured keratinocytes derived from epidermis and from other stratified squamous epithelia. The cellular and molecular properties of the cultures are altered when the medium is supplemented with serum from which the vitamin A has been removed by solvent extraction (delipidized serum). Cell motility is reduced, the adhesiveness of cells increases and pattern formation is prevented. In both epidermal and conjunctival keratinocytes, removal of vitamin A leads to the synthesis of a 67 kd keratin characteristic of terminally differentiating epidermis and to much reduced synthesis of the 52 kd and 40 kd keratins typical of conjunctiva. These changes, both cellular and molecular, are reversed by the addition of retinyl acetate to the medium containing delipidized serum. Cell motility and pattern formation are restored, and detachment of the most mature cells from the surface of the stratified epithelium is promoted. Synthesis of the 67 kd keratin is prevented and the synthesis of the 40 and 52 kd keratins is stimulated. The nature of the keratins synthesized is regulated by the concentration of vitamin A, and each cell type adjusts its synthesis differently at a given vitamin concentration.