Relationship of adhesiveness of cells in culture with specific enzyme activity.

l-Glutamine is required by mouse teratoma cells and other mouse ascites tumor cells in the synthesis of complex carbohydrates involved in intercellular adhesion. Since l-glutamine is synthesized by the enzyme glutamine synthetase (GS) (EC 6.3.1.2), these studies were undertaken to determine if a relationship exists between cellular adhesiveness and GS specific activity. Two types of experiment were performed to examine this relationship. Actinomycin D enhanced both teratoma cell GS specific activity and cellular adhesiveness over controls in batch cultures at confluency. Also, the relationship between cell adhesiveness and GS specific activity during the cell cycle was studied using cell populations synchronized with thymidine plus Colcemid. In these synchronized cultures, cellular adhesiveness displayed an oscillatory pattern with peaks of GS specific activity occurring just prior to peaks of adhesiveness. The levels of GS specific activity and intercellular adhesiveness were enhanced by the addition of hydrocortisone, a steroid known to induce GS specific activity in mouse teratoma cells. These results demonstrate a correlation between GS specific activity and cellular adhesiveness. Based upon previous work which implicates l-glutamine in intercellular adhesion, it is not unreasonable to speculate that GS specific activity and cellular adhesiveness may be causally related.     

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.     

X-chromosome activity in heterokaryons and hybrids between mouse fibroblasts and teratocarcinoma stem cells

To determine whether 2X-active cells contain factors capable of reactivating the inactive mammalian X chromosome, fibroblast lines, having a cytologically or genetically marked inactive X, were fused with 2X-active mouse embryos or ovarian teratocarcinoma stem cells. Fusions with 2–16 cell embryos were uninformative because no mitosis occurred in heterokaryons. Fusions with 2X-active teratocarcinoma cells, and screening for re-expression of alleles on the inactive X showed that reactivation did not occur with detectable frequency in heterokaryon. Hybridization of HPRT^?M. musculus × M. caroli cells with XO HPRT^? teratocarcinoma cells yielded hybrids with a frequency of >10^?6; these hybrids all expressed the Hpt allele on the inactive M. caroli X, but not the M. caroliGpd or Pgk. Late replication-banding studies of hybrids and 6-thioguanine-resistant revertants showed that the reactivated Hp⁺ allele was still located on the late replicating X. Similar results were obtained with hybridization of this line to 1X-active (male-derived) fibroblast lines, indicating that hybridization per se, rather than a specific factor contributed by the teratocarcinoma cell partner, was reponsible for the frequent localized derepression of the Hpt⁺ allele on the inactive X.     

Phosphorylation of non-histone proteins associated with mitosis in HeLa cells

Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with ³²P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8–10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation—dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis.     

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.     

Hl histones from mammalian testes : Hlt is associated with spermatogenesis in humans

Hlt is a testis-specific Hl histone variant associated with meiosis and post-meiotic stages of male germ cell development. We have now made tentative identification of Hlt in humans by a variety of criteria including electrophoretic and extractive properties. While Hlt was readily identified in extracts from normal testes, it was not detectable in extracts from aspermatogenic testes or from placenta. Identification of Hlt in humans confirms the widespread association of this unusual Hl variant with spermatogenesis among mammals ranging from rodents to primates.     

Thymidine triphosphate synthesis in senescent WI38 cells : Relationship to loss of replicative capacity

The effect of in vitro age on thymidine triphosphate (TTP) synthesis was assessed in WI38 cultures according to the following measurements: (1) thymidine kinase activity of broken cell preparations; (2) in situ incorporation of [3H]thymidine into acid-soluble material; and (3) total intracellular TTP content as determined by an enzymatic assay. All three parameters were maximal in exponentially proliferating populations and minimal in quiescent monolayers; no significant differences between young and old cultures were observed despite the reduced replicative capacity of the latter. The addition of serum to density-arrested cultures induced both TTP synthesis and DNA replication after a lag of approx. 12 h; although a greater percentage of young cells initiated replication as compared with old, pool sizes expanded to a similar extent in both populations. Pool expansion did not require entry into S phase; the pool sizes of control and cytosyl arabinoside-treated cultures were comparable. These findings suggest that senescent cells retain the ability to synthesize TTP, even though they are incapable of replicating DNA. Because TTP synthesis is a cell cycle-dependent event that normally begins in late G1, senescent cells might be blocked in the latter portion of the prereplicative phase and not in G0 as are quiescent cells.     

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.     

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.     

Embryonal carcinoma cell growth and differentiation. Production of and response to molecules with transforming growth factor activity

Transforming growth factors are known to induce anchorage-independent growth of non-transformed cells, and are released by a variety of cells, including MSV-transformed cells. This study demonstrates that the differentiated cells derived from F9 and PC-13 embryonal carcinoma cells, but not the parental cells themselves, respond by increased growth to several factors released by MSV-transformed cells, including partially purified sarcoma growth factor. The chemical properties of the growth-promoting activity are shown to match the chemical properties of the transforming growth factors released by MSV-transformed cells. Furthermore, F9 and PC-13 embryonal carcinoma cells, which do not respond to factors released by MSV-transformed cells, are shown to release factors with transforming growth factor activity. Based on the close relationship between mouse embryonal carcinoma cells and cells of early mouse embryos, it is suggested that molecules with transforming growth factor activity may play a role during the early stages of mammalian development.     

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.     

Modification of the surface ATPase activity in cultured hepatoma cells by lipid-depleted media

Several compounds have been described which elute fibronectin from a gelatin-Sepharose affinity support. In the present study, it has been found that the potent chaotrophic agent, lithium di-iodosalicylic acid, is 20-fold more effective in eluting fibronectin from collagen than any other presently described fibronectin elution agent. Lithium di-iodosalicylic acid and certain other fibronectin elution agents have been characterized in regard to several parameters involved in the elution of fibronectin from collagen and plastic substrata. By assaying for retention of the cell adhesive activity of fibronectin, it has been demonstrated that 8 M urea + 0.1 M citric acid, pH 4.7, is the most effective condition for preservation of biological activity following elution of fibronectin from the gelatin-Sepharose affinity support.     

Changes in endogenous cell surface galactosyltransferase activity during in vitro limb bud chondrogenesis

When the subridge mesoderm of the embryonic chick limb bud is cultured in the absence of the apical ectodermal ridge and adjacent ectoderm, the cells rapidly progress through the various stages of chondrogenesis. During the first day of culture, the cells initiate condensation, and during subsequent days, deposit a cartilage matrix. In the present study, we show that early in the first day there is a progressive 2-fold increase in cell surface galactosyltransferase activity towards endogenous acceptors. Later in the first day, although the cells are still in condensation, endogenous galactosyltransferase activity has decreased, suggesting in situ galactosylation of surface acceptors. During subsequent development, when cartilage matrix is being deposited, surface galactosyltransferase activity remains low. All controls have been performed to insure cell surface localization of enzyme activity. Two other surface glycosyltransferases show very low levels of activity, which do not change significantly during culture. We suggest that during cellular condensation, an interaction between surface galactosyltransferases and acceptors on adjacent cells occurs, and this interaction may be causally related to subsequent chondrogenic differentiation.     

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.     

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.     

Inhibition of dihydrofolate reductase gene expression following serum withdrawal or db-cAMP addition in methotrexate-resistant mouse fibroblasts

We have used a methotrexate (MTX)-resistant mouse 3T6 cell line (M50L3), that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of 300, to study the mechanism for turning off DHFR gene expression following withdrawal of serum factors or elevation of the intracellular level of cAMP. When resting (G0) M50L3 cells are serum-stimulated to reenter the cell cycle, the level of DHFR activity begins to increase at about the same time the cells begin synthesizing DNA. The increase in enzyme activity is preceded by increases in the synthesis rate of the enzyme, and the content and production rate of DHFR mRNA. These increases, as well as entry into S phase, are blocked when the cells are serum-stimulated in the presence of dibutyryl cyclic AMP (db-cAMP) and theophylline. In this study, we found that when these drugs were added, or the serum stimulus was withdrawn during S phase (20 h following stimulation), the subsequent increase in DHFR level was blocked. Immunoprecipitation of DHFR from pulse-labelled cells showed that both treatments led to a rapid decrease in synthesis rate of the enzyme. The effect on total protein synthesis was much less than on DHFR synthesis. In DNA-excess filter hybridization experiments, we found that the content of cytoplasmic DHFR mRNA decreased in parallel with the synthesis rate of the enzyme. This was due in part to a decrease in the production rate of DHFR mRNA relative to total mRNA. In addition, drug addition or serum withdrawal led to a significant destabilization of DHFR (as well as total) mRNA. About 85% of poly(A)(+) DHFR mRNA was associated with polysomes in resting, growing, or cAMP-treated cells, suggesting that DHFR gene expression was not controlled at the translational level under these conditions.     

Karyotypic normalcy and quasi-normalcy of developmentally totipotent mouse teratocarcinoma cells

Malignant mouse teratocarcinoma cells are, in some cases, able to undergo normal, complete differentiation after injection into blastocysts. Thus far, only three lines—of unrelated origin—have been found (all in this laboratory) to be developmentally totipotent in blastocyst tests. The karyotypes of these lines, and their somatic- and germ-cell derivatives, were investigated by G-banding methods, as a possible clue to their developmental superiority. The first, OTT 6050 (129 strain), is an embryo-derived induced tumor maintained as an ascites transplant line. Its stem cells (from embryoid body “cores”) have 40 chromosomes in the modal class, which comprises two subclasses: one all normal and one with a metacentric chromosome (isochromosome-8). However, mosaic animals from injected blastocysts have only the normal subclass in their teratocarcinomaderived cells; all are of $\text{X}\text{Y}$ male sex chromosome type. Presence of the Y chromosome was verified after transmission through the germ line of two fertile mosaic males, in their F₁ male progeny. The second teratocarcinoma line, 72484-395 (LT strain), is a spontaneous ovarian solid tumor maintained by subcutaneous transplantation. Karyotypes of cells from the tumor, and also of teratocarcinoma-derived cells in mosaic animals, were normal and of $\text{X}\text{X}$ female sex chromosome type. Karyotypes of the F₁ progeny, from tumor-strain germ cells of a fertile mosaic female, were also normal. The third line, NG 2 (129 strain), is a mutant clonal in vitro line deficient in hypoxanthine phosphoribosyltransferase. It originated from an embryo-derived experimental tumor (OTT 5568) that was established in culture (PSA1 line); the culture was then mutagenized and selected for 6-thioguanine resistance. The NG 2 line proved to be quasi-normal, with only two karyotypic anomalies: trisomy of chromosome 6 and $\text{X}\text{O}$ female sex chromosome constitution. Thus, developmental totipotency in all three lines, including one maintained in vitro, is accompanied by karyotypic normalcy or near-normalcy. Other culture lines reported to be aneuploid have not yet given evidence of totipotency. Karyotypic normalcy may therefore have predictive value useful in choosing teratocarcinoma lines with relatively high developmental prospects. This is of importance in identifying those mutant lines that would be promising candidates for introduction, via blastocyst injection, of specific mutant genes into mice.     

Glycopeptides prepared from mouse cerebrum inhibit protein synthesis and cell division in baby hamster kidney cells, but not in their polyoma virus-transformed analogs

Glycopeptides isolated from mouse cerebral cortex cell surfaces (BCSG) were shown to inhibit cell growth and protein synthesis in baby hamster kidney (BHK)-21 cells, whereas polyoma virus-transformed BHK-21 cells (pyBHK-21) were refractory to the inhibitory activity of the glycopeptides. Growth inhibition was shown to be reversible and non-lethal to BHK-21 cells. Despite that difference in sensitivity to the action of the glycopeptides, both cell lines could bind the inhibitor in a saturable fashion and in similar quantities. After trypsinization, BHK-21 cells appeared refractory to the inhibitor, whereas pyBHK-21 cells became sensitive. The data suggested the presence of a receptor for BCSG on the cell surface of both cell lines. Incubating BCSG with conditioned medium from pyBHK-21 cells resulted in loss of the glycopeptide's inhibitory activity. In contrast, medium conditioned by BHK-21 cells had no effect on the inhibitory activity of BCSG. We hypothesize that the refractoriness of pyBHK-21 cells to BCSG is related to their autonomous growth characteristics and failure to respond to topo-inhibitory growth control. BCSG may be a naturally occurring growth regulator whose function can be explored by use of the BHK-21/ pyBHK-21 model system.