Electrokinetic determinations of enzymatic susceptibilities of cell surface-associated RNA

Earlier investigations suggested, using electrokinetic evidence, that RNA is present at the surfaces of some types of cultured and freshly isolated cells. In this report, further investigations of the nature of cell surface RNA of cultured Ehrlich ascites (EAT) cells are reported. These experiments were carried out by determining the changes in electrophoretic mobility of EAT cells after treatment with several highly purified nucleases, neuraminidase, and hyaluronidase. The results suggested that cell surface RNA is located at surface sites separate from those susceptible to neuraminidase and hyaluronidase, that α and ω termini of RNA are absent from the electrokinetic surface, and that the RNA present at the cell surface might exist predominantly in a double-stranded form. A model is proposed in which cell surface RNA strand termini are buried out of the electrokinetic surface, but where RNA extends from these buried termini into the electrokinetic surface in loops.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

Restricted amino acid nutrition induces attachment to glass and spreading of Ehrlich ascites tumour cells

Ehrlich ascites tumour (EAT) cells were cultured in vitro in Eagle's MEM and Medium 199 with a lowered amino-acid content. Under these conditions EAT cells lose their rounded shape typical of highly malignant cancer cells, and begin to spread on the substratum. The changes in EAT cell morphology are preceded by a decrease in the rate of protein synthesis. These changes were maintained for three days after returning the cells to Eagle's MEM with a normal amino-acid content, but the return to control media did not cause reasumption of growth in the once spread cells. The increase in glucose content (up to five-fold) or the presence of inhibitors of DNA synthesis did not prevent the attachment and flattening of EAT cells in media with a lowered amino acid content. Several possible mechanisms of the influence of restricted amino-acid availability on the changes in EAT cell surface properties are pointed out and the need for study of cancer cell responses to restricted nutrition is discussed.     

Cell-substratum attachment and cell surface hyaluronate of Rous sarcoma virus-transformed chondrocytes

Hyaluronate is associated with the cell surface of cultured Rous sarcoma virus-transformed chondrocytes. Detachment of these cells from their substratum by a variety of reagents is accompanied by release of 75-100% of this hyaluronate into solution. Treatment of the cells with 200 U/ml protease-free Streptomyces hyaluronidase at 37 degrees C cause release of greater than 90% of the cell surface hyaluronate and complete cell detachment. Treatment with a lower concentration of Streptomyces hyaluronidase (30 U/ml) at 25 degrees C or a corresponding activity of testicular hyaluronidase gives similar results, but only in the presence of mM EGTA. Treatment with the lower activities of either hyaluronidase or with 1 mM EGTA alone release only approximately 45% of the cell surface hyaluronate and does not cause significant cell detachment. It is concluded that there are two populations of cell surface hyaluronate differing in their accessibility or their resistance to dissociation from other components of the cell surface. It is proposed that the less readily released fraction is located between the transformed chondrocyte surface and substratum and is necessary for their interaction.     

Electrokinetic properties of isolated cerebral-cortex synaptic vesicles

Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of -3.55mum.s(-1).V(-1).cm in saline-sorbitol, pH7.2, at 25 degrees C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pK(a) in the range 3.0-3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn(2+) or Ca(2+) was more effective in decreasing vesicle mobility than was Mg(2+), Sr(2+) or Ba(2+). The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.     

Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near-confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37 degrees C) stimulated a significant increase in receptor-mediated 125I-LDL internalization and degradation relative to PBS-treated control cells. Binding studies at 4 degrees C revealed an increased affinity of LDL receptor sites on neuraminidase-treated cells compared to control BAEC (6.9 vs. 16.2 nM/10(6) BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase-treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125I-LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor-upregulated cultured normal human fibroblasts resulted in an inhibition of 125I-LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125I-LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor-mediated 125I-LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor-mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.     

Induction of granulomatous experimental autoimmune thyroiditis in mice with in vitro activated effector T cells and anti-IFN-gamma antibody.

Experimental autoimmune thyroiditis (EAT) can be induced in mice after the transfer of mouse thyroglobulin (MTg)-sensitized donor spleen cells that have been activated in vitro with MTg. CD4+ T cells are required for the transfer of EAT in this model. Because CD4+ T cells produce various lymphokines, such as IFN-gamma, that may be involved in the activation or regulation of the immune response to MTg and the development of EAT, the present study was undertaken to determine whether a neutralizing mAb to IFN-gamma could modulate the induction or expression of EAT. The anti-IFN-gamma mAb XMG-1.2 had no effect on sensitization of donor cells. However, addition of XMG-1.2 mAb during in vitro activation of MTg-primed spleen cells resulted in more severe EAT in recipient mice. The thyroid lesions in recipients of cells cultured with MTg and XMG-1.2 mAb also exhibited granulomatous changes, which differed qualitatively from the predominantly lymphocytic cell infiltrates in recipients of cells cultured with MTg alone. Recipients of MTg-activated spleen cells also developed severe granulomatous EAT when they were given injections of XMG-1.2 mAb. The effects of XMG-1.2 could be neutralized by IFN-gamma. Recipients of cells cultured in the presence of XMG-1.2 mAb had augmented autoantibody responses, although there were no apparent differences in the IgG subclass distribution of the anti-MTg autoantibody responses. These studies suggest that neutralization of endogenous IFN-gamma results in increased activity of cells capable of inducing granulomatous EAT in mice.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Summary Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothyocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.This study was supported by a grant from the Cancer Society of Saint Joseph County, Indiana and from the Phi Beta Psi Sorority     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothiocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.     

Host-Dependent Restriction of Mengovirus Replication

Mengovirus infection of a restrictive cell line, Maden's bovine kidney (MDBK), results in a virus yield 1,000-fold less than that obtained from productively infected cell lines such as L cells or Ehrlich ascites tumor cells (EAT). Cells of both types of host systems are infected with comparable efficiencies and are completely killed as a consequence of infection. Infective center assays, coupled with the observation of total cell killing, suggest that comparable numbers of cells synthesize viral antigen and release virus in both types of host system. Viral-specific ribonucleic acid (RNA) synthesis is initiated and proceeds in an identical fashion for approximately 4 hr after the infection of MDBK, EAT, or L-cells. At this time, viral RNA synthesis in MDBK ceases, whereas viral RNA synthesis in EAT and L-cells continues at a linear rate. These results indicate that none of the early viral events leading to the initiation of viral-specific RNA synthesis constitutes the primary site of mengovirus restriction in MDBK. Rather it appears that the cessation of viral RNA synthesis in restrictive cells constitutes the primary limiting event. Based on its delayed interaction with mengovirus RNA synthesis, it appears that the host-related restrictive agent is initially compartmentalized and then released as a consequence of infection subsequent to those early events in mengovirus infection leading to the initiation and continued synthesis of viral RNA.     

The role of hyaluronic acid in intercellular adhesion of cultured mouse cells

Aggregation of cultured mouse cells was measured by the rate of disappearance of particles from a suspension of single cells. Treatment with several enzymes which degrade hyaluronic acid (testicular hyaluronidase, streptomyces hyaluronidase, streptococcal hyaluronidase and chondroitinase ABC) inhibited the aggregation of SV-3T3 and several other cell types. Since streptomyces and streptococcal hyaluronidases are specific for hyaluronic acid, it is suggested that hyaluronic acid is involved in the observed aggregation. Hyaluronidase-induced inhibition of aggregation was complete in the absence of divalent cations, but only partial in their presence. This finding is consistent with the hypothesis that two separate mechanisms are responsible for aggregation; one dependent upon and the other independent of calcium and magnesium. Aggregation was also inhibited by high levels of hyaluronic acid. A similar effect was obtained with fragments of hyaluronic acid consisting of six sugar residues or more. Chondroitin (desulfated chondroitin 6-sulfate) and to a lesser extent desulfated dermatan sulfate also inhibited aggregation. Other glycosaminoglycans (chondroitin 4-sulfate, chondroitin 6-sulfate, heparin and heparan sulfate) had little or no effect on aggregation. It is suggested that the hyaluronic acid inhibits aggregation by competing with endogenous hyaluronic acid for cell surface binding sites.     

Intracellular movement of cell surface receptors after endocytosis: resialylation of asialo-transferrin receptor in human erythroleukemia cells

The intracellular movement of cell surface transferrin receptor (TfR) after internalization was studied in K562 cultured human erythroleukemia cells. The sialic acid residues of the TfR glycoprotein were used to monitor transport to the Golgi complex, the site of sialyltransferases. Surface-labeled cells were treated with neuraminidase, and readdition of sialic acid residues, monitored by isoelectric focusing of immunoprecipitated TfR, was used to assess the movement of receptor to sialyltransferase-containing compartments. Asialo-TfR was resialylated by the cells with a half-time of 2-3 h. Resialylation occurred in an intracellular organelle, since it was inhibited by treatments that allow internalization of surface components but block transfer out of the endosomal compartment. Moreover, roughly half of the resialylated molecules were cleaved when cells were retreated with neuraminidase after culturing, indicating that this fraction of the molecules had returned to the cell surface. These results suggest that TfR is transported from the cell surface to the Golgi complex, the intracellular site of sialyltransferases, and then returns to the cell surface. This pathway, which has not been previously described for a cell surface receptor, may be different from the route followed by TfR in iron uptake, since reported rates of transferrin uptake and release are significantly more rapid than the resialylation of asialo-TfR.     

Prevention by retinoic acid of anionic site redistribution on the surface of cultured human sarcoma cells

The distribution of cell surface negatively-charged macromolecules was determined electron microscopically on untreated and on retinoic acid (RA)-treated cultured human osteosarcoma Hs791 and chondrosarcoma Hs705 cells using cationized ferritin (CF), an electron-dense marker of anionic sites. Labeling on the surface of prefixed cells was continuous and uniform whether they were grown in the absence or presence of RA. In contrast, CF distribution on unfixed cells was markedly affected by RA; CF labeling of untreated cells occurred in patches and clusters whereas the label on RA-treated cells was continuous, as on prefixed cells. CF labeling of unfixed cells decreased considerably after incubation of the cells either with hyaluronidase or neuraminidase. There was also a reduction in patching and clustering. Changes induced by RA in the apparent membrane microviscosity, in neuraminidase-releasable sialic acid, or in transglutaminase activity could not be related to the effect of RA on CF-induced anionic site redistribution since these characteristics were modulated differently in the two cell lines. In contrast, RA increased the sialylation of specific cell surface membrane glycoproteins on both cell types. These results suggest that RA prevents redistribution of cell surface sialoglycoconjugates and glycosaminoglycans by CF. This effect may be the result of increased sialylation of specific surface components and may be related causally to the suppression of the transformed phenotype in the sarcoma cells.     

Ultrastructure of the Surfaces of Cells Infected with Avian Leukosis-Sarcoma Viruses

When stained with ruthenium red (RR), chick embryo cells infected with various strains of Rous sarcoma virus (RSV) and with avian leukosis viruses RAV-1 and RAV-3 showed an increase in the layer of acid mucopolysaccharides (AMPS) at their surfaces as compared with uninfected cells. This increase was most prominent in cells infected with the Fujinami strain of RSV. The layer was resistant to digestion with neuraminidase or trypsin but was readily removed by exposure to hyaluronidase. The thickness of this AMPS layer was not correlated with the varying degree of loss of contact inhibition exhibited by cells infected with the different strains of virus. The staining of the cell envelope with a solution of phosphotungstic and chromic acids (PTA-CR) suggested the presence of glycoproteins. The outer surface of the virions showed the same staining as the cell surface with RR and PTA-CR, and the budding virus particle was seen to incorporate the RR layer of the cell into its structure. The RR layers of cells and virions appeared to fuse, as did those between virus particles, suggesting that these layers play a role in the aggregation of virus particles and in their adherence to the surface of the cell.     

A cytokinetic approach to determine the range of O2-dependence of pyrimidine(deoxy)nucleotide biosynthesis relevant for cell proliferation

In vitro cultured Ehrlich ascites tumour (EAT) cells were used because of the ease of their manipulation under different levels of hypoxia. They were used to clarify further the complex mechanism of oxygen-dependent cell proliferation. On reducing the oxygen concentration from 20% to lower levels (1-7%) an increase in the length of the population doubling time with concomitant reductions in protein, RNA and DNA content of cultures were observed. The incorporation of [14C]HCO3- into the RNA fraction of cells by de novo biosynthesis of uridine monophosphate (UMP) was reduced proportionally to the microenvironmental O2 tension. Uptake of this labelled precursor by cells in the presence of N-phosphonoacetyl-L-aspartate was found to be similarly inhibited. To correlate the reduction of cell growth under hypoxia with the functional pyrimidine supply, hypoxic cells were cultured in the presence of a balanced mixture of deoxynucleosides and/or uridine (100 microM deoxycytidine, 10 microM deoxyadenosine, 10 microM deoxyguanosine, 100 microM uridine). Above 3% O2 in the protective atmosphere, no improvement of growth parameters by the exogenous pyrimidinenucleotide precursors was obtained, whereas these compounds had a positive influence below this level. The increase in cell number was raised to about 60% of that of control cultures (20% O2) irrespective of the oxygen tension. In addition, when above 3% O2 the incorporation of HCO3- into RNA was comparable to that of controls, indicating that the pyrimidine de novo pathway is not a limiting factor in RNA biosynthesis. In conclusion, whereas at suboptimal O2 levels (5-7%) no correlation between pyrimidine metabolism and reduction of proliferation rate appears to exist, at low O2 concentrations (less than 3%) the rate of orotate/UMP production seems to be an important factor in the growth cessation of EAT cells; at critical O2 tensions (less than 1%) the lack of pyrimidine-deoxynucleosides substantially reduces cell cycle progression.     

Transcription termination and RNA processing in the 3''-end spacer of mouse ribosomal RNA genes.

The 3' termini of ribosomal RNA precursors from mouse FM3A cultured cells are mapped to eight sites within 625 bp downstream from the 3' terminus of 28 S rRNA. Three additional sites are mapped in liver RNA from C3H/He strain mice. Two of them, the sites at 570 bp and 625 bp are assumed to be termination sites in vivo, because they correspond to in vitro termination sites of RNA polymerase I, and 45 S RNAs having these 3' termini decay with kinetics distinct from others. The amount of 45 S RNA having the 3' terminus at other sites is variable among several mouse strains, despite their having the same DNA sequence in these regions. The ability to produce 3' termini in these sites seems to follow Mendel's law of inheritance. Therefore, we postulate that these nine sites are RNA processing sites which are controlled genetically.     

Stimulation of density-inhibited cell cultures by insulin

Cell proliferation in density-inhibited chick embryo cell cultures was induced by microgram quantities of insulin, neuraminidase, trypsin or papain. Other proteins tested, including albumin, fetuin, ribonuclease and hyaluronidase were inactive except in very high concentrations (> 100 μg/ml). The insulin chick embryo model was selected for detailed analysis of the initiation of proliferation. Insulin insolubilized by conjugation with Sepharose particles was also active, but only in so far as it was released in soluble form from the particles. This was measured by a radioimmunoassay. Under the conditions giving maximal cell proliferation less than 0.002-0.2% of insulin was taken up by the cells. This suggests that an interaction of insulin with the cell surface only is sufficient to stimulate the cells. Insulin released the density-inhibited cells from G₁ phase to produce an almost synchronous wave of proliferation. The following sequence of events was characteristic of the cells after stimulation by insulin: an early increase in sugar uptake and decrease in leucine uptake, increase in cell volume, stimulation of RNA and protein synthesis, increase in thymidine uptake, DNA synthesis, mitosis and cell division.     

Cell surface glycosaminoglycans: Identification and organization in cultured human embryo fibroblasts

A morphologically detectable cell coat, composed of glycoprotein, glycolipid, and glycosaminoglycan, is present on the external surface of most vertebrate cells. We have invetigated the composition and organization of glycosaminoglycans in the cell coat of cultured human embryo fibroblasts by labeling cells with ³H-glucosamine and Na₂³⁵SO₄ and subsequently treating cultures with specific enzymes. Components released were identified by chromatography and specific enzymatic digestion. In situ incubation with leech hyaluronidase (4 μg/ml) removed only hyaluronic acid from the cell surface whereas testicular hyaluronidase (0.5 mg/ml) removed both hyaluronic acid and chondroitin sulfate. Trypsin (0.1 mg/ml) released a large mass of glycopeptides in addition to hyaluronic acid, chondroitin sulfate, and heparan sulfate. The affinity of the cell coat for the cationic dye, ruthenium red, was reduced by leech hyaluronidase treatment. Sequential enzyme digestions of the cell surface showed that hyaluronic acid could be removed without the concomitant or subsequent release of sulfated glycosaminoglycans, suggesting that the hyaluronic acid is not a structural backbone for glycosaminoglycan complexes of the external cell surface.     

Phytohemagglutinin-induced change in the distribution of acidic sugars in surface membrane of lymphoid cells and blocking of the radiation effect.

Cell electrophoretic mobilities (EPM) of cultured lymphoblastoid cells were measured after removal of acidic sugars to investigate whether the localization of these acidic sugars was altered by the action of phytohaemagglutinin (PHA). After treatment with neuraminidase or hyaluronidase, the EPM of control cells decreased 50.1 and 0.3%, while that of PHA-treated cells decreased 25.2 and 39.0%, respectively. These results suggest that hyaluronic acid appeared at the periphery of the cell surface in place of some sialic acid after incubation with PHA. The change became evident after 10 min incubation with PHA and reached its maximum after 20 min at 37 °C, but no change was observed at 4 °C. The EPM decreased with time after X-irradiation, and reached a minimum value after 4 h. The addition of PHA to culture before irradiation completely blocked the X-ray-mediated reduction in EPM. PHA administration after irradiation stopped further EPM reduction. These results seem to suggest a rapid rearrangement of membrane molecules linking with the receptors and acidic sugars induced by PHA, and blocking of further conformation change by X-irradiation.