Structure and genomic organization of the mouse dihydrofolate reductase gene

The genomic organization of the mouse dihydrofolate reductase gene has been determined by hybridization of specific cDNA sequences to restriction endonuclease-generated fragments of DNA from methotrexate-resistant S-180 cells. The dihydrofolate reductase gene contains a minimum of five intervening sequences (one in the 5′ untranslated region and four in the protein-coding region) and spans a minimum of 42 kilobase pairs on the genome. Genomic sequences at the junction of the intervening sequence and mRNA-coding sequence and at the polyadenylation site have been determined. A similar organization is found in independently isolated methotrexate-resistant cell lines, in the parental sensitive cell line and in several inbred mouse strains, indicating that this organization represents that of the natural gene.     

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.     

Gene expression in normal and virally transformed mouse 3T3B and hamster BHK21 cells

Cell lines 3T3B (mouse), 3T3B-SV40, BHK21 (hamster) and BHK21 polyoma virus (PyY) were labelled with [³⁵S]methionine under conditions in which 500–600 cpm were incorporated per cell during a 20 h incubation period. Two-dimensional gel electrophoresis analysis of the total [³⁵S]methionine-labelled polypeptides from 200–300 cells followed by fluorography revealed about 500 acidic (isoelectric focusing, IEF) and 150 basic polypeptides (non-equilibrium pH gradient electrophoresis, NEPHGE) whose position could be reproducibly assessed. Counting of 33 abundant acidic polypeptides present in both 3T3B and 3T3B-SV40 revealed significant changes in the relative proportion of ten of them. Seven, including the subunit of the 100 Å filaments ‘fibroblast type’ (55K) (1.1% in 3T3B; 0.6% in 3T3B-SV40), three cytoarchitectural proteins and three soluble proteins, corresponded to a decrease of 40% or more in the radioactivity of the spots in transformed cells, and only in three cases was there a significant increase in radioactivity of polypeptides in 3T3B-SV40 cells. Among the polypeptides that show less than 40% variation we have identified total actin (42K) (13% of total label in 3T3B; 10% in 3T3B-SV40), α- and β-tubulin (55K) (1.6% of total label in 3T3B; 2% in 3T3B-SV40), eleven polypeptides present in Triton skeletons, and nine soluble proteins. We have also observed 25 obvious changes in polypeptide intensities (16 acidic and 9 basic) but these were not quantitated. Only three polypeptides were found in transformed cells that were not detected in normal cells. One of these corresponded to the large T antigen and the other two to Triton-soluble proteins of a molecular weight in the range of 52–54K. Similar quantitative studies on the hamster BHK21/BHK21PyY pair confirmed at least the major observations made in 3T3B and 3T3B-SV40.     

The effect of serum on the calcium content of BALB/c 3T3 mouse cells

The 5'-termini of purified rat liver nucleolar and cytoplasmic 28S ribosomal RNA (rRNA) are precisely located within the homologous rDNA sequence by S1 nuclease protection mapping using an appropriate rDNA restriction fragment. The 5'-termini of nucleolar 28S rRNA are heterogeneous in length. The bulk of the nucleolar 28S rRNA map within two CTC motifs in rDNA located in the internal transcribed spacer 2 at the 50-60 and 5-15 bp upstream from the site of the homogeneous 5'-terminus of the cytoplasmic 28S rRNA. These results provide direct proof that nucleolar 28S rRNA molecules contain excess sequences at their 5'-termini and require further processing to generate the mature cytoplasmic 28S rRNA.     

Cytochalasin B-induced alterations of insulin binding and microfilament organization in cultured fibroblasts

The effect of cytochalasin B (CB) on insulin binding has been investigated in confluent cultures of chick embryo fibroblasts. Time- and dose-dependent increases in binding of [¹²⁵I]insulin was observed after incubation of fibroblasts with CB. At 10 μg/ml, CB caused a 2-fold increase in binding, due to an increase in the number of binding sites from 9.3 × 10³ to 2.0 × 10⁴ per cell. Removal of CB from the growth medium was accompanied by a decrease in [¹²⁵I]insulin binding to control values in 24 h. Increase in the binding of insulin in CB-treated CEF was also accompanied by enhancement of insulin to stimulation of [³H]thymidine incorporation into acid-insoluble material. CB treatment also caused disorganization and disappearance of microfilament bundles and changes in cell shape from flat, with a few blebs and folds on the cell surface, to round with numerous blebs and folds. The data from this study suggest that changes in the number of surface insulin-binding sites may be related to the state of organization of cytoskeletal structures in chick embryo fibroblasts.     

Demonstration of competence and progression activities for human fibroblasts

Human embryonic lung fibroblasts (HEL) completed 4.5 population doublings in 6 days when maintained in DMEM supplemented with 10% human whole blood serum (WBS), plasma-derived serum (PDS) or defibrinogenated plasma containing 10 mM CaCl₂. Plasma in the absence of additional calcium promoted less growth. Sera and plasma chromatographed through carboxymethyl Sephadex (CMS) supported only one population doubling. Increased growth resulting in three doublings was observed in CMS-treated WBS or PDS supplemented with commercially prepared platelet-derived growth factor (PDGF). The magnitude of this PDGF response was dependent on serum concentration. A significant increase in the proportion of cells incorporating [³H]thymidine was observed in confluent cultures exposed to PDGF prior to incubation in WBS-CMS or PDS-CMS indicating competence and progression activities for human fibroblasts. In contrast, cells maintained in the presence of plasma-CMS failed to grow in response to PDGF. Factors bound to CMS columns restored growth-promoting activity to PDGF-supplemented WBS-CMS, PDS-CMS and plasma-CMS. However, growth-promoting CMS-bound components from plasma were lost during dialysis through membranes excluding materials above 12000 MW.     

Fertilization-induced changes in concanavalin A binding to mouse eggs

The binding of concanavalin A (ConA) to zona-free unfertilized and fertilized mouse eggs has been investigated using tritiated ConA. At low lectin concentrations (1–5 μg ml^?1) the fertilized egg shows a higher affinity for [³H]ConA than does the unfertilized egg. In saturation conditions, however, unfertilized and fertilized eggs show the same binding capacity (1.55 × 10⁸ ConA molecules/egg). The results indicate that ConA-binding sites change qualitatively following fertilization; possible connections between this change and other fertilization-induced changes in the egg surface are discussed.     

Two sites for calcium action in compaction of the mouse embryo

Compact mouse morulae were decompacted in calcium-free medium and allowed to recompact in standard embryo culture medium. When the recompaction medium contained trifluoperazine (TFP)(0.05 mM), an inhibitor of the calcium-dependent protein calmodulin, the embryos failed to recompact. This effect could not be overcome by either db-cAMP (1.0 mM) or theophylline (0.75 mg/ml). When the recompaction medium contained less than standard calcium (0.085 or 0.17 mM) the embryos recompacted at a slower rate than in control medium (1.7 mM). The calcium ionophore A23187, at concentrations up to 1.5 X 10(-3) mM, had no significant stimulatory effect upon the recompaction rte of embryos in the reduced calcium medium. In addition, the calcium antagonist Verapamil (0.3 mM), which blocks calcium uptake by cells, significantly inhibited recompaction in standard culture medium. Large doses of diazepam inhibited recompaction only slightly in standard culture medium. Large doses of diazepam inhibited recompaction only slightly in standard culture medium. We conclude that calcium uptake into the cytoplasm is required for recompaction, but that cell surface calcium is also required and is rate-limiting under these experimental conditions.     

Protease activity associated with loss of adhesiveness in mouse teratocarcinoma

Actin-binding proteins were assayed in various tissues using an 125I-actin overlay procedure. Four major G actin-binding proteins of 90000, 65000, 58000 and 40000 Mr have been identified. The 90K protein is present in all tissues and binds labelled actin in a calcium-sensitive manner with binding increasing 3-4-fold in the presence of Ca2+. The distribution of the 58K and 65K protein which are not Ca2+-sensitive was more variable. These proteins were present in different ratios in different tissues. 125I-actin binding to all four actin-binding proteins is specific and can be displaced by preincubation of the gels with unlabelled actin. The interaction of actin with these proteins does not appear to involve ionic forces, since binding is not diminished by varying the salt concentration. Skeletal muscle glycolytic enzymes, the lens crystallins and the histones also bind 125I-actin. This binding cannot be displaced by preincubation with unlabelled actin and is presumably non-specific. The calcium sensitivity of two highly purified actin-binding proteins, the 90K human platelet protein and villin was compared using 125I-actin. The platelet 90K protein binds actin at less than 10(-7) M free calcium, but detectable binding to villin does not occur below 10(-6) M free calcium. The ubiquity of these actin-binding proteins is clear and we conclude that the calcium-sensitive 90K actin-binding protein in all of these tissues is the same as the platelet protein.     

Uptake and stability of human and bovine acid alpha-glucosidase in cultured fibroblasts and skeletal muscle cells from glycogenosis type II patients

Acid alpha-glucosidase (EC 3.2.1.20) was purified from human placenta and bovine testis by affinity chromatography using concanavalin A (conA) and Sephadex G 200. When added to the culture medium of human fibroblasts, the enzyme purified from bovine testis is taken up with a 200-fold higher efficiency than the enzyme from human placenta. Uptake of acid alpha-glucosidase from bovine testis is mediated by the mannose-6-phosphate receptor, whereas only a minor fraction of placental enzyme appears to be equipped with the mannose-6-phosphate recognition marker. Once internalized, both human and bovine acid alpha-glucosidase demonstrate a half-life of about 10 days in fibroblasts from control individuals and patients with different clinical forms of glycogenosis type II (Pompe's disease, acid alpha-glucosidase deficiency). Evidence is presented that the mannose-6-phosphate receptor is also present on the plasma membrane of the clonal myogenic skeletal muscle cell lines G8-1 and L6J1 (respectively from mouse and rat origin) and on cultured human skeletal muscle cells derived from a muscle biopsy. Addition of bovine testis acid alpha-glucosidase to skeletal muscle cell cultures from an adult patient with glycogenosis type II leads to complete correction of the enzyme deficiency.     

Isolation of an aggregation inhibitory factor from non-adhesive mouse teratoma cells

An aggregation inhibitory factor (AIF) has been extracted from mouse ascites teratoma cells (that do not aggregate in culture) that retards adhesion of cultured teratoma cells of the same cell line (that do aggregate). Preliminary characterization of AIF on polyacrylamide gels suggests that AIF is a protein composed of four subunits. Extraction of AIF from ascites teratoma cells was accomplished without significant loss of viability by a technique involving the application of an electric field to large numbers of whole cells suspended in a hypertonic electrode buffer. In tests of adhesion, AIF consistently and immediately inhibited aggregation of cultured teratoma cells after 5, 10, 15, and 30 min of incubation. Furthermore, a reduced concentration of AIF resulted in a corresponding decrease in inhibition, suggesting a concentration-dependent action. AIF may help explain how cultured teratoma cells adhere, whereas ascites teratoma cells of the same subline do not adhere.     

Ultrastructural and biochemical characterization of a cloned mouse keratinocyte cell line

A cloned population of mouse C3H/He keratinocytes was obtained from the 14th passage of an epidermal cell line. A two-step cloning procedure using Petriperm dishes was performed. The cloned population, grown at 34 °C, was subcultured more than 30 times over a one year period. By day 14, three cell layers were formed; the ultrastructural morphology and immunofluorescence characterization of these layers showed numerous tonofilament bundles and well organized desmosome tonofilament structures. They thereby resemble the proliferative compartment of the epidermis. High resolution acrylamide gel electrophoresis of the keratins extracted from the cloned cells showed the presence of many keratin subunits. The tonofilaments extracted from the cell layers, as well as from the supernatant cells, contained a small quantity of high MW keratins (rel. MW 63 000; apparent isoelectric point 5.5–6.2). These results indicate that the cloned keratinocyte cell line had retained a certain maturation capacity in culture.     

Detection of a fetal antigen on mouse erythroleukemic Friend cells

A fetal antigen, expressed on all fetal erythrocytes during normal ontogeny, was detected on Friend erythroleukemic cells but not on circulating erythrocytes from leukemic mice. Friend cells were shown to express the fetal antigen both by immunofluorescence and antiserum absorption. The fetal antigen thus allows a clearcut distinction between the tumoral step from which emerge the Friend and erythro-proliferative cells.     

Density-dependent regulation of growth in somatic hybrids between normal Chinese hamster fibroblasts and V79-8 (G1) cells

The purpose of this work was to determine the relationship between the presence of a G1 period in the mitotic cycle and a cell's ability to respond to density-dependent regulation of growth (DDR). Somatic hybrids were obtained between normal fibroblasts from newborn Chinese hamsters, which show a strong response to DDR, and V79-8 Chinese hamster cells, which are insensitive to DDR. Two variant V79-8 sublines were used, one reported to lack a G1 period (G1-) and the other with a G1 period (G1+). Fourteen hybrid clones were isolated in selective medium and analysed for growth properties and cell cycle parameters; their hybrid nature was supported by chromosome counts. All hybrid clones, irrespective of whether a V79-8 G1- or G1+ cell was one of the parents, showed pronounced DDR and had G1 periods of various lengths. Previous experiments had shown the absence of G1 to be dominant in somatic hybrids between V79-8 G1- and G1+ cell lines. Our results may mean that the G1- property provided by V79-8 is unable to overcome the very long G1 of normal fibroblasts, or in cells that can be arrested in G1 in response to DDR, some function prevents the dominant effect of the G1- cell on at least part of the G1 period.     

Proliferation, differentiation and maturation of a mouse epidermal keratinocyte cell line

The spontaneous development of a cell line of neonatal mouse C3H/He epidermal cells is described. The culture has been serially passaged at 29 °C over 18 months in the absence of any dermal support. The cell morphology of the 18th passage is reported. During early growth phase, the morphology of the cell layers was similar to that observed in the basal and differentiating strata of the epidermis: numerous tonofilament bundles and desmosome-filament complexes were observed. During late growth phase, maturation and vertical stratification occurred: demonstrated by the tonofilament accumulation, cell organelle degradation, nuclear pyknosis, presence of keratohyalin granules and horny cell layers with thickened membranes. Hemidesmosome-like structures were shown. No basal lamina or membrane coating granules were detectable. The 18th passage cultured cells did not induce tumors in nude mice. This keratinocyte cell line is not permanent, however: a malignant transformation occurred after 25 subcultures which resulted in an undifferentiated cell population.     

Adult human retinal cells in culture. Identification of cell types and expression of differentiated properties

A method for culturing adult mammalian retinal neurons in serum-free N2 medium supplemented with nerve growth factor (NGF) is described. Identification of neurons in cultures of dispersed human retina was based upon morphology, immunocytochemical localization of bound tetanus toxin, and autoradiographic localization of 3H-neurotransmitter candidates (gamma-aminobutyric acid, glycine, dopamine) accumulated by high-affinity uptake mechanisms. Neurons would not attach to glass or plastic substrates, consequently the present studies were performed using neurons plated upon a feeder layer. Serum was required for the initial phase of attachment. The feeder layer was derived from retinal cells that had been plated on glass or plastic in the presence of serum and had later been passaged. Since these cells exhibited glial fibrillary acidic protein (GFAP) immunoreactivity, they were tentatively identified as being glial in origin. Under these conditions, neuron- and glia-specific properties were retained up to 28 days. The presence of interstitial retinol-binding protein (IRBP) in medium of cultures of neuronal cells on feeder layers was demonstrated by an immunoblot technique using rabbit antibovine IRBP antibodies. No IRBP was detected in medium in which the feeder layers alone had been cultured. IRBP biosynthesis was demonstrated by incubation of the cultures with [35S]methionine. Immunoprecipitable [35S]IRBP was detected only in medium from cultures containing neurons; cells of the feeder layer did not synthesize and secrete this glycoprotein. These findings are consistent with the hypothesis that IRBP, a 135K constituent of the interphotoreceptor matrix, is synthesized in vivo by a neuronal cell, specifically, the photoreceptors.     

Effect of transferrin on alkaline phosphatase activity and collagen synthesis in osteoblastic cells derived from newborn mouse calvaria

The effect of transferrin was tested on osteoblastic cells (clone MC3T3-E1) cultured in serum-free medium containing 1% bovine serum albumin (BSA). Transferrin (Tf) stimulated increases of protein content and protein synthesis, but not of DNA content and cell number, in the cells. This protein also increased alkaline phosphatase activity and collagen synthesis in combination with 1% BSA. Actinomycin D and cycloheximide inhibited alkaline phosphatase activity induced by Tf, suggesting that Tf may enhance de novo synthesis of the enzyme. These results indicate that Tf may be involved in differentiation of osteoblastic cells, but not in their proliferation, in vitro.