Cardiomyocytes cultured in serum-free medium: Growth and creatine kinase activity

Primary cultures of newborn rat heart cells were grown for up to 3 weeks in serum-free medium supplemented by insulin, hydrocortisone, transferrin and fetuin. The cells resumed spontaneous beating at 20 h post plating. Mean rates of beating on the second and third day were 79.5 and 94 beats per min, respectively. Cell proliferation occurred during the first 3 days of culture with maximal rates of DNA and protein synthesis on the second day. The highest values of creatine kinase activity were observed on days 2–5 and the three cytoplasmic isozymes, MM, MB and BB, were present in the cultures in proportions similar to those of the newborn heart, indicating stability of the differentiated state of the cells. The relative amount of each isozyme remained unchanged throughout the experiments, MM constituted 70–90% of enzyme activity, MB contributed up to 30% and BB did not exceed 15% of activity. The very low proportion of BB and the lack of increase in this isozyme with age of culture support our earlier morphological observations that non-myocytes do not overgrow the culture.     

Proliferation and differentiation of chick skeletal muscle cells cultured in a chemically defined medium

By using the technique of nuclear transplantation in Paramecium [1], amicronucleate and renucleate clones were prepared in P. caudatum. The major differences between amicronucleate and micronucleate cells in the vegetative stage are elongation of cell cycle time, decrease in food vacuole formation, and shortening of the buccal cavity in the amicronucleate cells. These characteristics of amicronucleate cells are closely related with the absence of micronucleus, because all of these abnormalities were cured when the micronucleus was transplanted again into the amicronucleate. It is evident that the germinal micronucleus plays an important role not only during the sexual cycle but also in vegetative growth. Elongation of the cell cycle time in amicronucleates was also observed in P. bursaria and P. jenningsi.     

Specific alterations in the pattern of histone-3 synthesis during conversion of human leukemic cells to terminally differentiated cells in culture

The presence of nano- to micromolar concentrations of 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in suspension cultures of human promyelocytic leukemia cells, HL-60, or human monocytic leukemia cells, THP-1, resulted in the appearance of macrophage-like cells attached to the substratum. The terminally TPA-differentiated cells continued to synthesize histones at a low rate even though DNA replication had ceased. The pattern of synthesis of histone variants in differentiated cells differed from that in undifferentiated cells and resembled that of quiescent or density-arrested cells. In undifferentiated cells, all three histone-H3 variants are synthesized, while in quiescent cells, only the H3.3 variant is synthesized. When TPA-differentiated macrophages were placed in normal medium, the pattern of histone synthesis was not altered, thus substantiating previous findings that the differentiation is irreversible. Further, TPA-differentiated macrophages and macrophages isolated from a normal human donor exhibited identical pattern of histone synthesis. Altogether, the results indicate that changes in the synthetic rates of histones during the TPA-induced maturation of human leukemic cells is not directly due to TPA or terminal cell differentiation per se but is due to the cessation of cell proliferation and DNA replication.     

Detection of melanogenic proteins in cultured chick-embryo melanocytes

The phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), was used as a reversible inhibitor of melanogenesis. Chick-melanocyte cultures of the black genotype, E/E, were grown in conditioned medium plus TPA. After growth in TPA and after its removal, the cells were pulse labeled with 3H-leucine. The membrane fraction, which included all tyrosinase activity as well as both mature and immature melanosomes, was solubilized with Triton X-100. The proteins were separated using two-dimensional electrophoresis and visualized by fluorography. One defined melanogenic protein, tyrosinase, was isolated, and its location was determined in the two-dimensional protein pattern. The protein patterns for both the TPA-inhibited cells and the cells in which the TPA effects were reversed after removal were compared. In addition to tyrosinase, at least nine TPA-sensitive proteins were found. These were designated as being putative melanogenic proteins which, along with tyrosinase, may be responsible for melanin-granule synthesis.     

Ultrastructural organization of nucleoproteins during aging of cultured human embryonic fibroblasts

Analysis of nucleoproteins in resting human embryonic fibroblasts in vitro at different population doubling levels (PDL) using electron microscopy revealed the disappearance of non-nucleolar ribonucleoprotein structures at high PDL, the nucleoli became larger and the filamentous masses containing the nascent nucleolar RNA displayed a fibrillo-granular pattern which has never been described previously. In addition, conventional fixation revealed the disappearance of most of the stainable chromatin whose threads were unusually spaced and shortened specially at the nuclear surface after loosening. We interpret these changes in chromatin organization as the consequence of the alkali-sensitive sites that accumulate during senescence.     

Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lactation [11]. By measuring the incorporation of glucose carbon from [U-¹⁴C]glucose into intermediary metabolites and metabolic products in mammary epithelial cells from virgin, pregnant, and lactating mice, we demonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counterparts. When isolated from lactating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.     

Binding uptake and degradation of antithrombin III X protease complexes by cultured corneal endothelial cells

Interaction of 125I-labeled human antithrombin III (125I-AT III) X protease complexes with bovine corneal endothelial cells has been studied in tissue culture. 125I-AT III does not bind to endothelial cells, but its complexes with either thrombin or trypsin bind specifically to the cultures. The binding of 125I-AT III X protease complexes is not via the moiety of the free antithrombin III (AT III) or the free protease, since neither AT III nor thrombin compete on the binding of 125I-AT III X thrombin complexes. Only unlabeled AT III X thrombin complexes compete on the binding of the iodinated ligand. 125I-AT III X trypsin complexes bind with a KD of 1.4 X 10(-7) M to high affinity-binding sites present on the cell surface of corneal endothelial cells. Saturation of binding to the cell surface is observed at a concentration of 2.5 X 10(-7) M 125I-AT III X trypsin complexes and the number of binding sites per cell is about 4 X 10(4). The cell surface binding reaches a maximum by 15 min and then decreases with time. The cells, when incubated at 37 degrees C, appear to internalize the bound complexes by adsorptive endocytosis which proceeds at a rate of 0.5-0.8 pmole/1 X 10(6) cells/h. The internalization process of 125I-AT III X protease complexes is saturated at a concentration of 2.5 X 10(-7) M. Since the cells release 125I-labeled material into the extracellular media which cannot be precipitated by trichloroacetic acid (TCA), it probably represents degradation of 125I-AT III X protease complexes into small fragments at a linear rate of about 0.5 pmole/1 X 10(6) cells/h. The described process of AT III X protease complexes binding, internalization and subsequent degradation by corneal endothelial cells may represent a clearing mechanism for extracellular AT III X protease complexes formed under pathological conditions.     

Biosynthesis of EGF receptor, transferrin receptor and colligin by cultured human keratinocytes and the effect of retinoic acid

The biosynthesis of EGF and transferrin receptor by human keratinocytes in culture has been followed using specific monoclonal antibodies. In addition, keratinocytes are shown to synthesise a Mr 47 000 protein that binds to gelatin-Sepharose. Peptide mapping confirms the identity of this protein with colligin, a newly described cell surface-associated glycoprotein that also binds to native collagens (Kurkinen et al., J biol chem 259 (1984) 5915) [9]. Vitamin A and its analogues have profound effects on the differentiation, morphology and motility of human keratinocytes in culture. We show here that retinoic acid (RA) has no effect on the growth rate of the cells or the synthesis of EGF receptor and colligin, but stimulates the synthesis of transferrin receptor.     

Large macromolecules can be introduced into cultured mammalian cells using erythrocyte membrane vesicles

Plasmid 6.4 kbp DNA, 14 kbp DNA, lambda phage particles, all of which contained herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene, or IgM molecules, were mixed with erythrocyte membranes and treated with neutral detergent. The transparent mixture was diluted with phosphate-buffered saline (PBS), followed by centrifugation to collect membrane vesicles containing the large macromolecules. 10-15% of 6.4 kbp, 3% of 14 kbp, 4-7% of the lambda phage particles and 14.5% of IgM were trapped within erythrocyte membrane vesicles. The membrane vesicles containing these molecules were fused with L cells, or rat F2408#20 cells, both of which are deficient in thymidine kinase activity. In each case, transformants were obtained. 2 X 10(5) - 7 X 10(5) phage PFU or 1.5 X 10(6) - 8 X 10(7) DNA molecules were required to obtain one transformant from L cells, but 2-3 X 10(7) phage PFU or 2 X 10(9) - 1 X 10(10) DNA molecules were required for one transformant from rat cells. Number of colonies which transiently expressed TK genes in L cells was also determined by autoradiography. The ratio of stable transformants to colonies positive for transient expression in cells treated with low doses of DNA or lambda phage was 46-68%. The transformation efficiency of human fibroblast cells by pSV2-gpt DNA trapped in erythrocyte membrane vesicles was less than that of L cells by HSV-TK DNA, but almost the same as that of rat cells by HSV-TK DNA.     

Stimulation of sialyltransferase activity of melanoma cells by retinoic acid

Retinoic acid (RA) treatment of murine S91-C2 melanoma cells has been found to augment the activity of glycoprotein: sialyltransferase in a dose-dependent and time-dependent process. The enzymatic activity in cells treated with 10 microM RA reached a maximal level, 3-fold higher than in untreated cells, 72 h after initiation of treatment. In contrast, the addition of RA directly into the reaction mixture had no stimulatory effect on sialyltransferase. The endogenous glycoproteins to which sialic acid is transferred from cytidine monophosphate (CMP)-[14C] sialic acid by the action of sialyltransferase have been identified by fluorography after polyacrylamide gel electrophoresis. One of these acceptors, a glycoprotein of Mr 160 000, comigrated in gel electrophoresis with a cell surface sialoglycoprotein that can be labeled by the periodate-tritiated borohydrate procedure more intensely on intact RA-treated than on untreated cells. Removal of sialic acid residues exposed on the surface of either control or RA-treated cells enhanced 2- to 3-fold the transfer of sialic acid to endogenous acceptors. These results suggest that the increased sialyltransferase activity in RA-treated melanoma cells may be responsible for the enhanced sialylation of certain cell surface glycoproteins. RA treatment of several other tumor cell lines also resulted in stimulation of sialyltransferase activity indicating that this effect of RA is not limited to the S91-C2 melanoma cells.     

Progress through G1 and S in relation to net protein accumulation in human NHIK 3025 cells

We have investigated whether human NHIK 3025 cells are dependent upon a net increase in cellular protein content in order to traverse G1 and S. The increase in DNA and protein content was studied by means of two-parameter flow cytometry using populations of cells synchronized by mitotic selection. By adding 1 μM cycloheximide to the medium protein synthesis was partially inhibited, resulting in negligible net accumulation of protein. The cells were able to enter S and progress through S under such conditions. The latter was the case whether the cells had been accumulating protein during G1 or not. The results further indicate that the larger cells enter S earlier and traverse S at a higher rate than the smaller cells. Our conclusion is that net accumulation of protein does not seem to be a prerequisite for traverse through G1 and S, i.e. DNA replication may be dissociated from the general growth of cell mass.     

The synthesis and intracellular localization of adenovirus hexon protein studied by microinjection of mRNA into human cells

Polyadenylated RNA isolated 18 h after infection of HeLa cells with adenovirus type 2 was both translated in vitro and microinjected into the cytoplasm of human transformed amnion cells. The hexon polypeptide could be specifically immunoprecipitated from the products of cell-free translation with a rabbit-anti-hexon serum. The same serum when used in immunofluorescence assays of microinjected cells revealed hexon protein synthesized 6 h after microinjection. The intensity of the staining persisted up to 16 h after injection of messenger RNA (mRNA). Newly-synthesized hexon protein was characteristically located mainly in the nucleus. Essentially the same results were obtained when normal amnion cells were microinjected.     

Decreased longevity of human diploid cells after incorporation of latex spheres within their cytoplasm

The effects of cytoplasmic incorporation of latex spheres (a cytoplasmic marker) on the growth potential of human diploid cells was examined. After incorporation of latex spheres within their cytoplasm, GM2290 (diploid, Lesch-Nyhan) cells showed a reduced replicative potential. A lower percentage of cells exposed to latex particles incorporated [³H]thymidme during any subsequent 24-h test period when compared with comparable, untreated cells. The overall life expectancy of the cultures treated with spheres was reduced approx. 25%. A similarly treated and examined transformed cell line (HeLa) showed no similar adverse effects after incorporation of latex spheres. The results suggest that latex spheres should be used with caution in experiments on in vitro cellular senescence.     

Reinitiation of host DNA synthesis in senescent human diploid cells by infection with simian virus 40

Human diploid fibroblasts, TIG-1, cease to proliferate at about 60-62 population doubling level. In their senescent state used in this study, the percentage of nuclei labeled by [3H]thymidine for 48 h was around 1-2% in fresh medium containing 5-40% fetal bovine serum. The percentage of labelled nuclei increased up to 10-fold after infection with SV40. This increase reflects stimulation of cell DNA synthesis because: 1. The increase also occurred when ts A900 was used for infection at the non-permissive temperature, under these conditions viral DNA synthesis is inhibited; 2, the increase paralleled the stimulation of [3H]thymidine incorporation into DNA in a Hirt-precipitate fraction from SV40-infected cells. UV-irradiated SV40 had reduced ability to induce DNA synthesis. A viable deletion mutant of SV40, d1940, had almost the same activity to induce cell DNA synthesis as did wild-type SV40. Equilibrium density gradient centrifugation analysis of DNA labelled with 5-bromodeoxyuridine (BrdU) supported semiconservative replication rather than repair synthesis. We conclude that a considerable fraction of human diploid cells in a senescent population initiate host DNA replication by infection with SV40, although these cells cannot be stimulated with fetal bovine serum.     

Expression of c-fos in parietal endoderm, amnion and differentiating F9 teratocarcinoma cells

The expression of the cellular proto-oncogene, c-fos, in extra-embryonic tissues of the mouse was investigated using a v-fos DNA probe and an affinity-purified antiserum raised against a C-terminal synthetic peptide. At 13.5 days of development, parietal endoderm--a tissue not previously studied using these methods--was found to express c-fos RNA at a higher level than the amnion or placenta. The previously reported dramatic increase in c-fos RNA levels in extra-embryonic membranes during gestation was found to be confined to the amnion. The antipeptide serum specifically recovered proteins with Mr values of 46,000 and 39,000 from extracts of parietal endoderm and amnion cells labelled for 15 min with 35S-methionine. On sodium-dodecyl-sulphate/polyacrylamide gel electrophoresis these proteins co-migrated with proteins immunoprecipitated using serum from rats inoculated with FBJ-MuSV-transformed cells (tumour-bearing rat serum). Pulse-chasing and 32P-labelling experiments showed that the protein with an Mr of 46,000 was rapidly converted into higher-molecular-weight phosphorylated derivatives. F9 teratocarcinoma stem cells differentiated into parietal-endoderm-like cells in response to treatment with retinoic acid and dibutyryl cyclic AMP. However, this differentiation was not accompanied by any large transient increase in c-fos RNA expression.     

Cytokeratin patterns of human oral epithelia: Differences in cytokeratin synthesis in gingival epithelium and the adjacent alveolar mucosa

Human oral mucosa includes various epithelia that are commonly classified as lining, masticatory, and specialized epithelia. Although adjacent tissues, the gingiva and alveolar mucosa represent two different types of epithelia: the gingiva is cornified and exhibits high rate ridges, whereas the mucosa does not normally cornify and exhibits a relatively smooth-contoured borderline between the epithelium and the underlying connective tissue. We examined the cytokeratin patterns of both epithelia using one- and two-dimensional gel electrophoresis. The gingiva expresses a great complexity of cytokeratins, including significant amounts of components nos. 1, 2, 5, 6, 10, 11, 13, 14, 16, and 17, as well as traces of cytokeratins nos. 4 and 15, i.e., a pattern similar to those of vaginal mucosa and epidermis containing proliferative keratinocytes. In contrast, the alveolar mucosa contains only two major cytokeratins, i.e., nos. 4 and 13, together with two minor amounts of cytokeratins nos. 5, 6, 14, and 17, thus resembling the patterns of certain other stratified, noncornified epithelia, such as the esophagus. Immunofluorescence microscopy using monoclonal antibodies to cytokeratins nos. 4 and 13 revealed the presence of these proteins in the suprabasal layers of alveolar mucosa, whereas in the gingiva, only certain small, suprabasal clusters of cells appeared to contain these cytokeratins. The cytoskeletal differences between gingival and alveolar mucosa are discussed in relation to the differences in their morphology and function, and with respect to pathological processes characteristic of these epithelia.     

Induction of glycophorin gene expression in cultured murine erythroleukemia cells

In order to identify the mRNA for mouse glycophorin, mRNA was isolated from immature erythroid cells obtained from the spleens of anemic mice, translated in vitro in mRNA-dependent rabbit reticulocyte lysate, and then immunoprecipitated with a specific antiserum. Glycophorin mRNA was shown to be present only in erythroid cells. In immunofluorescent and in vitro translation studies, it was shown that glycophorin mRNA is absent in uninduced murine erythroleukemia (MEL) cells, but is induced in dimethylsulfoxide-treated differentiating cells.     

Adhesion of human amnion epithelial cells to extracellular matrix : Evidence for multiple mechanisms

Human amnion epithelial cells attach and flatten slowly (approximately 65 min) onto plastic in the presence of serum but much more rapidly (20-30 min) onto subcellular matrix (SCM) deposited by the same cells. This matrix contains both fibronectin and laminin, but neither molecule on its own can reproduce its adhesive properties. Cells attach on surfaces containing fibronectin and laminin and extend filopodial and lamellipodial areas of cytoplasm without extensive flattening in the perinuclear region. Matrix deposited onto plastic by amnion epithelial cells has trypsin-sensitive and trypsin-resistant, papain-sensitive adhesion-promoting components. Cell spreading triggered by the latter but not the former can be inhibited by pretreating the adhering cells with heparin. Other GAGs are without effect. The results are discussed in terms of multiple interactions between epithelial cells and basal laminae.     

Fibroblast feeder layers inhibit differentiation of retinoic acid-treated embryonal carcinoma cells by increasing the probability of stem cell renewal

The appearance of differentiated cells in embryonal carcinoma (EC) cultures can be inhibited by culturing the cells on fibroblast feeder layers. To determine whether or not feeder layers act by increasing the probability of stem cell renewal, growth and differentiation were monitored in cultures of F9 (subclone OTF9 -63) EC cells exposed to retinoic acid (RA) in either the presence or absence of feeder layers. By measuring the fraction of laminin-positive TROMA 1-positive or alkaline phosphatase-negative cells, it was determined that the frequency of differentiated cells in RA-treated F9 cultures was reduced by 70-80% when cells were cultured on fibroblast feeder layers instead of gelatin-coated dishes. Experiments in which EC cells were cultured in close proximity to a feeder layer demonstrated that cell-cell contact was required for maximal inhibition of differentiation. The probability of stem cell renewal was determined by measuring the number of colony-forming cells in RA-treated cultures as a function of time. Analysis of the data demonstrated that the probabilities of stem cell renewal were 0.5 and 0.25 during the first and second 48 h periods, respectively, following addition of RA for cells cultured without feeder layers. Cultures maintained on feeder layers exhibited a stem cell renewal probability of 0.72. Thus, feeder layers reduce the frequency of differentiated cells in RA-treated cultures by increasing the probability of stem cell renewal. Determining the mechanism by which feeder layers counteract the effect of a chemically defined differentiation inducer should help to uncover the processes that regulate the probability of stem cell renewal.     

Characteristics of a human epidermal squamous carcinoma cell line at different extracellular calcium concentratrions

A continuous line derived from a human skin squamous cell carcinoma has been grown in media of high, normal and low Ca²⁺ concentrations. The growth rate was unaffected by the Ca²⁺ levels even though morphological changes were observed. Desmosomes were absent at low Ca²⁺ and areas of cell piling were observed at high Ca²⁺. Cell protein staining patterns on polyacrylamide gels were identical for cells grown at the three Ca²⁺ levels. The variations were minor for the glycoproteins reacted with ¹²⁵I-conA. Lactoper-oxidase iodination revealed changes in cell surface proteins, most markedly in the emergence of new proteins at high Ca²⁺.