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.     

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.     

Multiplication of Swiss 3T3 cells in a serum-free medium

Gently trypsinized Swiss 3T3 cells inoculated into medium MCDB 402 attach readily to polylysine-coated surfaces and remain viable for several days in the absence of exogenously added protein. Short-term multiplication under defined conditions can be obtained by supplementing the MCDB 402 with fibroblast growth factor (FGF), insulin (INS), and dexamethasone (DEX). Addition of bovine plasma fibronectin further improves attachment and viability. This system does not require initial plating in serum or the addition of poorly defined extracts for cellular attachment or for multiplication. In the complete system minus FGF, cells plated at a low density attach to the culture surface and become quiescent. The addition of FGF or PDGF 48–72 h after plating stimulates a high level of DNA synthesis during the following 24 h. EGF also stimulates DNA synthesis in these cells, but to a lesser extent. Insulin and dexamethasone are not needed for the initial DNA synthesis response to FGF, but are needed for continuing multiplication over a period of several days. This system provides a means for studying the effects of specific mitogens on Swiss 3T3 cells in the absence of undefined supplements, and without complications due to density-dependent inhibition.     

Guanylate cyclase activators hemin and sodium nitroprusside stimulate cell growth in serum-free medium

Hemin and sodium nitroprusside, which strongly activate purified rat brain guanylate cyclase in vitro, were also found to stimulate glioma C6 and neuroblastoma M1 and N1E-115 cells to divide in serum-free medium. Hemin and sodium nitroprusside each stimulate C6 cell growth to a comparable extent. Sodium nitroprusside was less potent than hemin for inducing growth of neuroblastoma cells. Moreover, both agents when added together caused a synergic cell growth enhancement which is comparable to the synergism observed in their guanylate cyclase stimulation in vitro. These results suggest that activation of guanylate cyclase may play a role in the proliferative response to these compounds.     

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.     

Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: Relationship to fetal age

Explants of small intestinal tissue have been cultured from fetal and young rats (from 13-day fetuses to 3-week-old rats). Growth of morphologically typical epithelial cells was obtained from explants of tissue from 14–20 day fetuses. Optimal growth was obtained using tissue from 17-day fetuses with outgrowth from the explant being observed 1-day after explant. Eighty per cent of explants developed epithelial growth by 11 days in culture. Initially, the epithelial outgrowth showed no morphological evidence of differentiation but after 5–10 days in culture differentiation into goblet or elongated cells with alkaline phosphatase activity occurred. Cells with brush borders and goblet cells were identified using electron microscopy. No differentiation occurred if the explant was removed even though growth continued.It was very difficult to culture tissue from fetuses older than 20 days' gestation, and when small intestine of 18–20-day fetuses was divided into two parts (proximal and distal) and cultured separately, growth of epithelial cells from explants of the proximal segment was less successful than that of the distal segment, indicating that the growth ability of these epithelial cells in vitro was closely related to tissue maturation in vivo. In contrast to the apparent relationship between fetal age and successful growth of intestinal epithelial cells, squamous epithelial cells of the esophagus could be grown from explants of 14-day fetus through newborn and 3-week-old rats.     

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.     

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.     

Albumin secretion and protein synthesis by cultured diploid and tetraploid rat hepatocytes separated by elutriation

Diploid and tetraploid rat hepatocyte subpopulations were isolated by elutriation and cultured for 24 h. Albumin secretion and protein synthesis rates were two-fold lower in 2n than in 4n hepatocytes. [35S]methionine-labelled proteins analysed by acrylamide gel electrophoresis showed a strikingly similar pattern in the two cell subpopulations. No differences in cellular proteins or in the intensity of labelling were observed. These results show (1) that viable diploid and tetraploid hepatocyte subpopulations can be separated by elutriation under sterile conditions and then cultured; and (2) strongly suggest that the same genes are transcribed and further translated at the same rate in both hepatocyte subpopulations.     

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.     

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.     

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.     

Ploidy class-dependent variations during 24 h of glucose-6-phosphate and succinate dehydrogenase activity and single-stranded RNA content in isolated rat hepatocytes

The time-dependent variations over 24 h of glucose-6-phosphate dehydrogenase (G6PDH) activity, succinate dehydrogenase (SDH) activity and single-stranded RNA (ssRNA) content have been investigated by cytophotometric analysis of cytochemically stained isolated hepatocytes of different ploidy classes from adult male rats. A marked variation of 48 % over the day in G6PDH activity of the mononuclear diploid cells was revealed, but no significant variation in the binuclear tetraploid cells. The cells of the inbetween ploidy classes showed an amplitude of variation of 38 % (binuclear diploid cells) and 24% (mononuclear tetraploid cells), respectively. All cells showed a maximum activity of the enzyme at the middle of the day and a minimum during the night. The relative enzyme activity per mononuclear diploid cell was significantly higher than the relative activity in the other cells, especially at its maximum. The variation of the SDH activity in hepatocytes isolated from the same rats was similar in all cells, irrespective of their ploidy class. The activity was highest at the end of the activity phase of the animals. The SDH activity per cell was directly proportional to the quantity of genome copies. The ssRNA content of the hepatocytes showed a time-dependent variation with a maximum during the resting phase of the animals and a minimum during their activity phase. The variation was larger in the mononuclear diploid cells than in the cells of other ploidy classes and the ssRNA content was also significantly higher in these cells than in the hepatocytes of other ploidy classes when calculated on the basis of genome copies. It is concluded that the large amplitude of variation over the day and the high relative amount of G6PDH activity and ssRNA content in mononuclear diploid cells is related to the function of these cells as stem cells of the liver parenchyma.     

Replacement of serum with a defined medium increases beta-adrenergic receptor number in cultured glioma cells

Lymphocyte chromosomes from a cercopithecoid species, Macaca mulatta, were studied for the occurrence of lateral asymmetry in constitutive heterochromatin. The technique consisted of growing the lymphocytes for one cell cycle in BrdUrd, staining with 33258 Hoechst, exposing them to UV light, treating them with 2 SSC and staining with Giemsa. This procedure revealed asymmetric staining in the region of constitutive heterochromatin of the nucleolar organizer marker chromosome (no. 13 of the complement). In these chromosomes, the darkly staining region was confined at any given point to a single chromatid, while the corresponding region on the sister chromatid was lightly stained. This pattern of asymmetric staining in the constitutive heterochromatic region was not observed in any other chromosome of Macaca mulatta. The lateral asymmetry of constitutive heterochromatin in this species is presumed to reflect the strand bias in the distribution of thymine in the alphoid DNA fractions.     

dCMP-aminohydrolase activity during early sea urchin development. An example of negative enzyme control during embryogenesis

In sea urchin, unfertilized eggs have a very high level of dCMP-aminohydrolase (dCMPase) activity, which decreases gradually and at the pluteus stage it is only about a quarter of that found in the unfertilized egg. But in abnormal embryos and in disaggregated cells from embryos, no decrease in the dCMPase activity takes place. To understand the control mechanism involved in this enzyme activity during development, we have analyzed the effect of various drugs which interfere with information transfer, such as actinomycin C, puromycin, 5-azacytidine, 2-thio-uracil and p-fluoro-DL-phenylalanine on dCMPase activity in embryos of Paracentrotus lividus and Sphaerechinus granularis. Among these drugs only actinomycin induces a remarkable increase of the dCMPase activity in embryos with respect to unfertilized eggs. Puromycin has a differential and dose-dependent effect. Other drugs, although they affect normal development and macromolecular synthesis, do not significantly alter the dCMPase activity. On the basis of these results we suggest the presence of a repressor mechanism in the control of dCMP-aminohydrolase level during early embryogenesis of sea urchin.     

Z-DNA immunoreactivity of Drosophila polytene chromosomes : Effects of the fixatives 45% acetic acid and 95% ethanol and of DNase I nicking

Drosophila salivary chromosomes have been isolated at neutral pH and physiological ionic strength. They display only background level binding of antibodies against Z-DNA. Following exposure to the commonly used fixative 45% acetic acid all of the polytene chromosomes, X and autosomes, show a massive increase in anti-Z-DNA antibody binding. The enhancement from background to intense fluorescence occurs whether the chromosomes are stabilised by two orders of magnitude lower concentration of formaldehyde than that used to minimise protein extraction in classical acid squash preparations, or by physiological concentrations of spermine and spermidine. Nicking of acetic acid-treated chromosomes by DNase I dramatically reduces their Z-DNA immunoreactivity. The histones and non-histones extracted by 45% acetic acid from unfixed and formaldehyde-fixed Drosophila chromatin have been analysed. Exposure of isolated salivary chromosomes to the non-protein-extracting fixative 95% ethanol also enhances Z-DNA immunoreactivity. All of these phenomena must be taken into account in the search for the Z-DNA conformation in cells by cytological techniques.     

Cell division cycle in mammalian cells : VIII. Mapping of G1 into six segments using temperature-sensitive cell cycle mutants

The G1 blocks in three temperature-sensitive (ts) Syrian hamster cell-cycle mutants have been mapped in relation to other G1 landmarks. Two mutants reported here, ts-559 and ts-694, show defective progression only in G1. When shifted from the permissive temperature of 33 degrees C to the non-permissive temperature of 39 degrees C, G1 cells of these two mutants show no further cell cycle progression, while cells in S, G2 and mitosis progress through the cell cycle but become blocked after entering G1. The two mutants complement each other, and also complement the previously reported mutant ts-550C with blocks in both G1 and G2 of the cell cycle. The locations of the G1 blocks in both ts-559 and ts-694 are before the hydroxyurea arrest point. The G1 ts point in ts-694 is prior to the isoleucine deprivation and serum starvation points, while the G1 block in ts-559 is after the serum starvation point but before the isoleucine block. Other G1 block points which have been reported are in mutants of different species and isolated in different laboratories, causing difficulties for relative positioning of the blocks in G1. The mutants for mapping in this study have been isolated from the same cell line. The G1 ts arrest points of ts-559 and ts-694, and that found in ts-550C, together with nutritional deprivations and metabolic inhibitors, provide seven reference points which divide G1 into six segments, each of which is bracketed by two adjacent points: mitosis, ts-694 block, serum starvation arrest point, ts-559 block, isoleucine deprivation arrest point, ts-550C block, hydroxyurea or excess-thymidine arrest segment.     

High hydrostatic pressure and the dissection of fertilization responses : I. The relationship between cortical granule exocytosis and proton efflux during fertilization of the sea urchin egg

High hydrostatic pressure applied between sperm attachment and the onset of cortical granule exocytosis will inhibit this exocytotic event in sea urchin eggs. Such pressure-treated zygotes, nevertheless, are activated and capable of development. Thus, this technique can be used as a tool to study the relationship between cortical granule breakdown and other fertilization-related responses. We have studied whether the exocytosis of cortical granules is necessary for proton efflux (acid release) to occur. Our results indicate that although Ca²⁺ is released while the eggs are under pressure (a prerequisite for the following events to take place), cortical granule exocytosis and acid release are pressure-sensitive and completely inhibited at pressures above 400 atm (6000 psi) and 275 atm (4000 psi), respectively. However, upon decompression, acid release is initiated which amounts to 65–70% of that seen in the unpressurized controls, suggesting that the efflux mechanism does not require cortical granule exocytosis and must result from some modification of the original plasma membrane of the egg. The remaining 30–35% of the acid release is related to cortical granule exocytosis, since it can be obtained upon induction of the cortical granule fusion 30 min later under atmospheric pressure. The initiation of acid release after decompression indicates that the efflux mechanism is not transiently turned on at fertilization, but undergoing long-term modification; the recovery of the ability to induce cortical granule fusion after fertilization under pressure suggests a refilling of cytoplasmic Ca²⁺ stores within this time course.     

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.