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.     

Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells

Growth of SCC-13 squamous carcinoma cultures in the presence of retinoids considerably reduced the expression of two differentiation markers, the cellular capability to form cross-linked envelopes, and the enzyme transglutaminase required for cross-linking. A limited survey of retinoids showed that all-trans retinoic acid, 13-cis retinoic acid, and arotinoid Ro 13-6298 were highly effective in the absence of hydrocortisone and were only slightly antagonized by its presence in the medium. In contrast, retinyl acetate, retinol, and retinol bound to its plasma binding protein were quite active in the absence of hydrocortisone but were essentially inactive in its presence. Dexamethasone was also highly effective in antagonizing the suppressive action of retinyl acetate on envelope formation, while the corticosteroid antagonists cortexolone and progesterone were inactive. These results suggest that there are separate pathways, which are differentially regulated by hydrocortisone, for either the metabolism or action of retinol and retinoic acid in SCC-13 cells.     

Changes in cell-substratum adhesion and nuclear-cytoskeletal anchorage during cytodifferentiation of BeWo choriocarcinoma cells

Changes in the substratum anchorage of cells and nuclei were examined during methotrexate (MTX)-induced cytodifferentiation of BeWo human choriocarcinoma cells. During this process cytotrophoblast-like cells (CTLs) transform into giant mono- and multinucleated syncytiotrophoblast-like cells (STLs). Cells treated with MTX for 24 h exhibited significantly faster rates of substratum detachment by EDTA, trypsin-EDTA, EDTA-glycine, and DMSO than did uninduced controls. The decrease in cell-substratum adhesiveness occurred prior to the onset of morphological transformation. By 48 h, when morphological transformation was first observed, there had occurred a marked change in nuclear-cytoskeletal anchorage to the substratum, as evidenced by a difference in sensitivity of Triton-extracted STL and CTL monolayers to detachment by KI. STL monolayers were completely detached within 5 min of exposure to 0.3 M KI, while CTL monolayers remained firmly attached to the substratum for at least 3 h. KI-extracted residues were examined by electron microscopy and found to consist of nuclear shells attached to intermediate filaments. When cytoskeletal residues and KI-extracted proteins of STL and CTL cells were compared by two-dimensional polyacrylamide gel electrophoresis (PAGE), qualitative and quantitative differences were seen in a number of minor components. Thus the sensitivity of STL nuclear-cytoskeletal monolayers to removal by KI, an effective actin depolymerizing agent, may involve changes in the organization, stability, or interactions of actin with other components of the cytoskeletal framework.     

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.     

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.     

Mammary serum factor initiates cell growth in 3T3 cell variants which are non-responsive to EGF

The structural organization of the histone-containing spermatozoa of the goldfish C. auratus is studied. Chemical fractionation shows that the five standard histones are present. Histone H1 subfractions are also similar to those found in other organisms. This is the first organism studied in which no peculiar basic protein is found in spermatozoa, since in all the organisms described up to now, unique protein components are always present, either alone or accompanied by somatic-like histones. Ultrastructural studies show that this chromatin is organized as a bundle of fibers of about 25 nm diameter, which upon spreading give the typical ‘beads-on-a-string’ appearance. Nuclease digestion demonstrates a repeat length of 205 base pairs (bp), slightly longer than in somatic tissues, but shorter than in echinoderm spermatozoa.     

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.     

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.     

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.     

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.     

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.     

Structural and biochemical aspects of the effects of various media used for isolation and fractionation of mouse liver nuclei

We have carried out an electronmicroscopic study of replicas of the underside of culture cells. The techniques developed for the preparation of such replicas are described in detail. We find that the cells do not rest on the surface of the culture dish itself, but rather are attached to a protein film adsorbed from the culture medium. The underside of cells is usually rather smooth looking. It is difficult to clearly define the site of attachment of cells to their substrate. The features interpreted as attachment sites are often bored and diffuse. Heavy bundles of micro-filaments can be recognized in the sole plate of BHK cells. These bundles are usually parallel to the long axes of the cells, and often terminate in cell projections. They are only rarely encountered in the sole plate of transformed BHK cells.     

Reversible pinocytosis of horseradish peroxidase in lymphoid cells

A detailed study of fluid phase endocytosis of horseradish peroxidase (HRP) in rat lymph node cells (LNC) is presented in this paper. Preliminary experiments have shown that HRP was internalized by non-receptor-mediated endocytosis and interacted minimally or not at all with plasma membrane of LNC, and can then be considered as a true fluid phase marker for these cells. Kinetics of uptake of HRP was found not to be linear with incubation time at 37 degrees C and deviation from linearity can be attributed to constant exocytosis of HRP. The kinetics of exocytosis cannot be described by a single exponential process. Rather, a minimum of two exponentials is required to account for exocytosis. This suggests that at least two intracellular compartments are involved in this process. The first turns over very rapidly with a t 1/2 release of about 3 min and is saturated after 10 min of exposure with HRP. The second, which turns over very slowly, is characterized by a t 1/2 release of about 500 min and accounts for the intracellular accumulation of HRP. Similar biphasic kinetics of exocytosis were observed with unfractionated LNC, with T lymphocyte-enriched LNC and with lymphocytes purified according to their density. This suggests that most, if not all, LNC are able to release HRP and that each cell type is endowed with the two intracellular compartments. Kinetics of uptake of HRP in these two compartments indicated that they are probably filled by two endocytic pathways, at least partially independent. Taken together, these results seem to indicate that a rapid membrane recycling occurs in lymphocytes. Furthermore, the weak base ammonium chloride and the carboxylic ionophore monensin were shown in our study to inhibit fluid phase endocytosis of HRP. The inhibition was time-dependent and required a preincubation of the cells with the drugs to be observed. Our results suggest that a perturbation of the vesicular traffic or a sequestration of membranes involved in HRP uptake is induced by these drugs. Under these conditions the release of cell-associated HRP was also reduced and to the same extent as the inhibition of uptake. Distribution of HRP between the two compartments and the t 1/2 release of HRP from either compartment were not perturbed. Taken together these results seem to indicate that exocytosis is not specifically affected by these drugs. Inhibition of uptake in drug-treated cells could result from a general decrease of membrane recycling or to the formation of smaller pinocytic vesicles with a different surface to volume ratio.     

The distribution of calmodulin in living mitotic cells

Calmodulin has been labeled with rhodamine isothiocyanate (CaM-RITC) and used as a probe for the location of calmodulin in vivo. CaM-RITC retains its capacity to regulate the activity of brain phosphodiesterase in a Ca²⁺-dependent manner in vitro, indicating that the labeled protein is still active. After injection into living mammalian cells CaM-RITC incorporates rapidly into the mitotic spindle; the details of its localization there mimic closely the distribution of Calmodulin seen by immunofluorescence. In interphase cells the CaM-RITC is excluded from the nucleus, but shows no region of specific concentration within the cytoplasm. Neither a 2-fold increase in cellular CaM nor the injection of anti CaM has any observable effect on the progress of mitosis.     

Factor VIII-related antigen : A pericellular matrix component of cultured human endothelial cells

We studied the extracellular localization of factor VIII-related antigen (VIIIR: Ag) in cultures of human endothelial cells. The cells deposited both VIIIR: Ag and fibronectin already during their initial adhesion phase and in immunofluorescence microscopy of spread cells extracellular VIIIR: Ag was localized to fibrils coaligning with pericellular fibronectin. When human fibroblasts, which do not synthesize VIIIR: Ag, were cultured in endothelial cell post-culture medium, a fibrillar matrix localization of VIIIR: Ag was seen, comparable to that of endothelial cell cultures. A fibrillar VIIIR: Ag-specific staining was also seen in cell-free pericellular matrices of endothelial cells, produced by deoxycholate treatment. In immunoelectron microscopy, VIIIR: Ag was seen in fibrillar extracellular material between and underneath the cells and in cell-free matrices of endothelial cells as well.In immunofluorescence microscopy of cell-free matrices, VIIIR: Ag codistributed with both fibronectin and type III procollagen. Digestion of the matrices with purified bacterial collagenase abolished the type III procollagen-specific fluorescence, whereas the fibrillar VIIIR: Ag-specific staining, codistributing with fibronectin, remained unaffected. In electrophoresis of isolated, metabolically labelled endothelial cell matrices, major polypeptides with M_r 220–240; 180; 160; 80 and 45 kD and some minor polypeptides were resolved. In addition, immunoblotting revealed fibronectin, VIIIR: Ag and type III procollagen as components of cell-free matrices of endothelial cells. Direct overlay of iodinated cellular fibronectin on electrophoretically separated polypeptides of cultured endothelial cells, transferred to nitrocellulose, suggested that fibronectin binds directly to VIIIR: Ag. Our results indicate that VIIIR: Ag produced by human endothelial cells is a component of the pericellular matrix and is not bound to collagen but may directly associate with fibronectin.     

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.     

High resolution autoradiographical detection of RNA in the interchromatin granules of DRB-treated cells

Isolated rat liver cells were pulse-labelled with tritiated uridine and post-incubated in the presence of an excess of unlabelled uridine and of adenosine analog DRB (5-6-dichloro-1-beta-D-ribofuranosyl benzimidazole). Nuclear radioactivity was detected with high resolution autoradiography. A significant labelling of the interchromatin granules was revealed in these conditions. Pretreatments of cells with low doses of actinomycin D in order to preferentially inhibit ribosomal RNA (rRNA) synthesis prevented the labelling of the interchromatin granules during subsequent DRB treatments. These observations indicate that in DRB-treated cells, the interchromatin granules are sites of transfer or of accumulation of nucleolar RNA. Our results are discussed in connection with our knowledge of the action of DRB on RNA metabolism in mammalian cells and with recent data concerning the still enigmatic interchromatin granules which are present in the nuclei of most cells.     

Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells

These studies examined the effect of exogenous pyruvate on the growth and differentiation of primary cell cultures of rat tracheal epithelial cells. The cell cultures were derived from outgrowths of tracheal explants, and require pyruvate for survival and growth in the presence of 10% FBS. In pyruvate-supplemented (2 mM) medium, the number of cells attached to the dish increased rapidly, while exfoliation of cells into the medium as well as formation of cornified envelopes were relatively low. The growth response to pyruvate was concentration-dependent in these cell cultures. In the absence of pyruvate, the extent of terminal differentiation to keratinization gradually increased. This was characterized by a cessation of growth after one week, and an increase in exfoliation until all cells had sloughed from the dish. Accompanying these changes was a marked increase in the formation of cornified envelopes. Cells undergoing DNA synthesis were present throughout 2 weeks of culture in pyruvate-deprived medium, even as the total number of cells was diminishing. Several compounds, including other 2-oxocarboxylic acids, were ineffective growth substitutes for pyruvate. These results indicate that the requirement for pyruvate is quite stringent in these cultures and that one way pyruvate promotes the growth of tracheal epithelial cells is by inhibiting terminal differentiation.     

Adhesive and migratory behavior of normal and sulfate-deficient sea urchin cells in vitro

Dissociated cells from different stage embryos of the sea urchin Lytechinus pictus were compared in their adhesion to various substrates. Micromeres from 16-cell stage embryos bind to tissue culture and Petri dishes but not to Petri dishes coated with human plasma fibronectin. Other cell types did not adhere to any of the substrates tested. By hatched blastula stage, about 28% of the cells adhered to fibronectin as well as to tissue culture dishes. By the mesenchyme blastula stage, there was a further increase in the proportion of cells adhering to these substrates. At no stage did cells adhere to native rat tail collagen. Primary mesenchymal cells were isolated by their selective adhesion to tissue culture dishes in the presence of horse serum. These cells were then examined for their migratory capacity. Cell spreading and migration followed adhesion and occurred on fibronectin but not on the other substrates tested. Based on analysis of video tapes, greater than 60% of these cells moved faster than 1 micron/min. On the other hand, cells from sulfate-deprived embryos, in which primary mesenchyme migration is blocked in situ, failed to spread and migrated little on the same substratum. This defect was reversed by a 6 h pretreatment of the cells in normal sea water. Thus, the in vitro migratory behavior parallels that observed in vivo. These results support the hypothesis that the primary mesenchymal cells produce a sulfate-dependent component that is required for cell spreading and migration.