Transformed BHK cells exhibiting normal or subnormal sugar uptake

The uptake of deoxyglucose was compared in BHK cells and in DMN4B cells, a conditionally transformed line of BHK cells which exhibits transformed behavior at 38.5° but not at 32°. At 32°, DMN4B cells took up deoxyglucose more slowly than BHK cells, reflecting a higher Km for uptake of this sugar. When both cell lines were grown at 38.5°, the Km for DMN4B cells was reduced to a level only slightly greater than for BHK cells, and deoxyglucose uptake became similar in the two cell lines. Growth in glucose-free medium for 22 hours stimulated deoxyglucose uptake in both BHK and DMN4B cells; under these conditions, uptake was equal in the two cells lines, both at 32° and 38.5°. Glycolysis, as measured by lactic acid production, was slower in DMN4B than BHK cells, but in contrast to deoxyglucose uptake, this difference was observed at 38.5° rather than 32°. The observation that the subnormal deoxyglucose uptake of DMN4B cells in the untransformed state (32°) can be normalized by growth at 38.5°, a temperature permissive for transformation, suggests that membrane changes facilitating sugar uptake, which have been found in other transformed cells, are associated with transformation in DMN4B cells as well. However, the failure of uptake to exceed normal in these cells indicates that their transformed behavior is not attributable to excessive sugar uptake per se.     

Abnormal response of glycolipid synthesis to temperature in transformed BHK cells thermosensitive for growth control.

In DMN4B cells, a line of chemically mutagenized BHK hamster cells which exhibit transformed behavior at 38.5°C but not at 32°C, [¹⁴C]-palmitate incorporation into mono-, di-, and trihexosylceramides was unimpaired at 32°C when compared with incorporation rates in untransformed BHK cells. At 38.5°C, labeling of these glycolipids increased greatly in the BHK cells, but failed to increase comparably in the DMN4B cells. Assay of cell-free preparations of the galactosyltransferase which catalyzes trihexosylceramide synthesis revealed a stimulatory effect of increased temperature on activity of the BHK enzyme, but not the DMN4B enzyme. The results suggest that transformation can result from a mutation affecting glycolipid synthesis.     

Changes in RNA polymerase II in a cell cycle-specific temperature-sensitive mutant of hamster cells

tsAF8 cells are a temperature-sensitive mutant of BHK cells that arrest at the nonpermissive temperature in the G₁ phase of the cell cycle. The activity of solubilized RNA polymerase II and its ability to bind [³H]-γ-amanitin decrease in tsAF8 cells at 40.6°, with a half-life of ～ 10 hr. No appreciable changes occur in these two parameters in tsAF8 cells at 34° or in BHK cells at either 34° or 40.6°. Protein synthesis is not appreciably affected for at least 24 hr after tsAF8 cells are shifted to 40.6°. These results indicate that in tsAF8 cells at the nonpermissive temperature, there is a defect in either the synthesis, the assembly, or the stability of RNA polymerase II, and that the loss of RNA polymerase II molecules is not due to widespread cellular damage.     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.     

The response of normal and polyoma virus-transformed BHK-21 cells to exogenous purines

Adenosine (10 μM) stimulates the initial growth rate of BHK/21 cells seeded at low but not high density in monolayer culture; it does not affect final cell density or permit growth in agar. In labelling experiments With tritiated thymidine, adenosine also increases the response of quiescent cells to low concentrations of serum. Dialysis of serum to remove oxypurines only marginally reduces its effect on quiescent cell labelling or growth, indicating that BHK/21 cells are able to synthesise purines. The response of quiescent cells to 5% serum is inhibited by high MW (2 × 10⁶) dextran sulphate at 2 μg per milliliter. Low MW dextran sulphate (30,000) and heparin at 20 μg per milliliter produce the same effect. Exogenous adenosine (10 μM) prevents this inhibition. Many other purine derivatives replace adenosine for all the above activities but xanthine is completely inactive in all. It, therefore, appears that nucleotide synthesis is a necessary function of these compounds. The growth of cells of a polyoma-virus-transformed BHK/21 line in monolayer is not stimulated by exogenous purine, though their colony-forming ability in agar is increased five-fold. The stimulating effects of exogenous purines on normal BHK/21 cells and the absolute requirement for them in the presence of polyanions is discussed in relation to possible mechanisms of growth control.     

Production of non-defective and defective oligomers of viral DNA in mouse 3T3 cells transformed by a thermosensitive mutant of polyoma virus

Cultures of three lines of mouse 3T3 cells transformed independently by the thermosensitive ts-a mutant of polyoma virus yield virus upon lowering their incubation temperature to 31°C. At 31°C, the internal pools of DNA of all three lines contain not only superhelical viral monomers, but also a small proportion of viral oligomers.From one of these three cell lines, several sublines of different clonal morphology were isolated at 38.5°C. The viral DNA synthesized at 31°C by each different subline displayed a unique oligomer pattern which has been stable through many cell passages and further reclonings. In contrast to the parental line, the monomer in most of these sublines is a minor component of the viral DNA pool. In one subline, more than 80% of the viral DNA consists of superhelical molecules about 1.6-times the size of a monomer. The specific infectivity of these molecules is only about one-tenth that of monomers, whereas the efficiency in transforming hamster (BHK21) cells is about twice that of monomers.     

Complement-dependent cytotoxic activity of serum mannan-binding protein towards mammalian cells with surface-exposed high-mannose type glycans

Serum mannan-binding protein (S-MBP), a lectin specific for mannose andN-acetylglucosamine, activates complement through the classical pathway. With the help of complement, S-MBP lyses red blood cells which have been coated with yeast mannan and kills bacteria which haveN-acetylglucosamine and/orl-glycero-d-manno-heptose on their core oligosaccharide. In this study, we examined whether mammalian cells, on which S-MBP could bound, are killed by a complement-dependent mechanism. When baby hamster kidney (BHK) cells were treated with an -mannosidase inhibitor, 1-deoxymannojirimycin (dMM), most of the cellular oligosaccharides were transformed from the complex-type to the high mannose-type. S-MBP bound to the dMM-treated BHK cells in the presence of Ca²⁺, and this binding was eliminated by mannose. When dMM-treated cells, labelled with⁵¹Cr, were incubated with complement, radioactivity was released in a dose-dependent manner by S-MBP and complement. This release was not observed with heat-inactivated complement. These observations suggest that S-MBP is able, with the help of complement, to kill not only exogenous microorganisms but also mammalian cells which have high mannose-type oligosaccharides exposed on their surfaces. Abbreviations: S-MBP, serum mannan-binding protein; BHK, baby hamster kidney; dMM, 1-deoxymannojirimycin; FITC, fluorescein-isothiocyanate; TBS, Tris buffered saline; HIV, human immunodeficiency virus; CH₅₀, 50% haemolytic units. The complement nomenclature used was recommended by the World Health Organization (1986).This paper is dedicated to Nathan Sharon.     

Thymidine nucleotide synthesis and catabolism by CHO cells and their changes during the cell cycle

At 0°C, CHO cells efficiently incorporated [³H]thymidine into the nucleotide fraction, but not into DNA. Upon reincubation of asynchronous cultures at 37°C, 15–25% of the radioactivity contained in the cellular nucleotide fraction was released, in the form of thymidine, into the culture medium. At 0°C, however, radioactivity of the nucleotide fraction was retained within the cells. Similarly, dTMP phosphatase (EC 3.1.3.35) in cell extracts was active at 37°C, but not at 0°C, whereas thymidine kinase (EC 2.7.1.21) was active at both temperatures. If synchronous cultures in Gl phase were prelabeled at 0°C and reincubated at 37°C, almost all radioactivity in the nucleotide fraction was released into the medium, whereas in S-phase cultures nearly all radioactivity of the nucleotide fraction was incorporated into DNA. In synchronous S-phase cultures treated with hydroxyurea, radioactivity in the nucleotide fraction was released into the medium at a rate considerably lower than that observed for Gl-phase cells. Rates of endogenous synthesis of thymidine nucleotides were calculated from changes of cellular thymidine nucleotide content, incorporation of thymidine nucleotides into DNA and release of thymidine into the medium during reincubation of prelabeled cultures in thymidine-free medium. The results obtained (see Table III) reveal marked differences between Gl and S phases with respect to the determinants of thymidine nucleotide metabolism.     

Hypothermic preconditioning of endothelial cells attenuates cold-induced injury by a ferritin-dependent process

Hypothermia for myocardial protection or storage of vascular grafts may damage the endothelium and impair vascular function upon reperfusion/rewarming. Catalytic iron pools and oxidative stress are important mediators of cold-induced endothelial injury. Because endothelial cells are highly adaptive, we hypothesized that hypothermic preconditioning (HPC) protects cells at 0°C by a heme oxygenase-1 (HO-1) and ferritin-dependent mechanism. Storage of human coronary artery endothelial cells at 0°C caused the release of lactate dehydrogenase, increases in bleomycin-detectible iron (BDI), and increases in the ratio of oxidized/reduced glutathione, signifying oxidative stress. Hypoxia increased injury at 0°C but did not increase BDI or oxidative stress further. HPC at 25°C for 15–72 h attenuated these changes by an amount achievable by pretreating cells with 10–20 μM deferoxamine, an iron chelator, and protected cell viability. Treating cells with hemin chloride at 37°C transiently increased intracellular heme, HO-1, BDI, and ferritin. Elevated heme/iron sensitized cells to 0°C but ferritin was protective. HPC increased iron maximally after 2 h at 25°C and ferritin levels peaked after 15 h. HO-1 was not induced. When HPC-mediated increases in ferritin were blocked by deferoxamine, protection at 0°C was diminished. We conclude that HPC-mediated endothelial protection from hypothermic injury is an iron- and ferritin-dependent process.     

Conditionally lethal mutations in Chinese hamster cells. I. Isolation of a temperature-sensitive line and its investigation by cell cycle studies

The isolation of a temperature sensitive cell line from the Chinese hamster line CCL39 of the American Type Culture Collection is described. At the nonpermissive temperature (39°C) the cells become attached to the surface of tissue culture dishes, but no microscopically observable colonies are formed upon prolonged incubation. Exposure to the high temperature for more than 24 hours leads to an almost complete loss in viability. A karyotypic analysis showed that this new line has lost one of the medium-sized metacentric chromosomes, although no proof is available so far to show that this loss is not simply coincidental. In nonsynchronized cultures transferred to 39°C DNA synthesis stops first, RNA synthesis shortly thereafter, while protein synthesis (turnover) continues for a longer time. After such a shift the cell number increases by less than 15% as measured with the Coulter counter. Studies with synchronized cultures give the following results: (1) one round of DNA synthesis can occur at 39°C when the cells are released from serum starvation or a hydroxyurea block, or when mitotic cells are placed at 39°C; (2) the entry of cells into metaphase of mitosis at 39°C is almost normal when the preceding time interval at 39°C is only eight hours (release of cells from G₁/S boundary), but considerably reduced when the cells spend an additional 12 to 15 hours at 39°C in G₁ (release from serum starvation). Infection by SV40 virus temporarily induces DNA synthesis after it has come to a stop at the nonpermissive temperature, but cells permanently transformed by SV40 still exhibit the temperature-sensitive phenotype.     

Studies on the nature of protease-induced growth stimulation in normal and transformed BHK cells.

In the presence of growth-limiting serum concentrations trypsin displays mitogenic activity on actively-growing but not quiescent BHK cells. These results suggest that BHK cells arrested in G1 (G0) are not sensitive to protease-induced growth stimulation. Previous work strongly suggested that the trypsin active-site is not directly involved in its mitogenic activity on BHK cells. Additional studies on denatured trypsin fragments further indicate that the molecular conformation and size of native trypsin may not be absolutely required for mitogenic activity. Cellular multiplication induced by the addition of fresh serum to quiescent BHK cultures is not inhibited by high concentrations of soybean trypsin inhibitor. Similar to our previous findings with trypsin, it has been further observed that plasmin is not sufficient to initiate the growth of BHK cells in soft agar. Trypsin also fails to enhance the growth of a thermosensitive polyoma-transformed BHK line in soft agar at the restrictive temperature. Finally, the growth of transformed BHK cells in soft agar does not display a requirement for plasminogen and is not inhibited by soybean trypsin inhibitor. These studies argue against the involvement of plasmin or other exogenous trypsin-like enzymes in the growth and transformation of BHK cells.     

N-glycosylation of recombinant human fucosyltransferase III is required for its in vivo folding in mammalian and insect cells

Human alpha3/4fucosyltransferase (FT3) catalyses the synthesis of fucosylated glycoconjugates involved in cell-cell interactions. FT3 has two potential N-glycosylation sites at Asn(154) and Asn(185). Soluble secretory forms of the enzyme (SFT3) and mutant forms with the first, second and both glycosylation sites (SFT3DN1, SFT3DN2, SFT3DN) mutated have been expressed in baby hamster kidney (BHK) and Spodoptera frugiperda (Sf9) cells. Deletion of the first or both sites caused total enzyme inactivation. Deletion of the second site caused 99% and 75% decrease of secretory enzyme expression in BHK and Sf9 cells, respectively. Sf9 cells produced 1 mg/l SFT3 and 0.3 mg/l SFT3DN2; these values were 175- and 3750-fold higher, respectively, than those observed for BHK cells. A significant amount of protein was accumulated intracellularly in Sf9 cells which for SFT3 was active and for SFT3DN2 was inactive, indicating the importance of the glycans from the second glycosylation site for protein folding. The corresponding full-length forms FT3, FT3DN1 and FT3DN2 associated with calnexin as observed by immunoprecipitation studies, which indicated the possible role of this chaperon in the folding of glycosylated glycosyltransferases.     

Release of the glucose‐regulated protein 94 by baby hamster kidney cells

Glucose‐regulated protein 94 (grp94) is a major component of the endoplasmic reticulum (ER) lumen of eukaryotic cells. We showed that grp94 is released from baby hamster kidney (BHK‐21) cells into a serum‐free medium. The exit of grp94 into the medium was not related to the protein discharge due to cell death and was independent of de novo protein synthesis. The treatment of cells with brefeldin A and monensin, the inhibitors of the classical pathway of protein secretion, did not decrease the extracellular level of grp94, indicating that the discharge of grp94 from cells does not occur through the ER/Golgi–dependent pathway. Exosomes, membrane vesicles secreted by several cell types, were not involved in the release of grp94 from cells. Methyl‐β‐cyclodextrin, a substance that disrupts the lipid raft organization, considerably reduced the extracellular level of grp94, indicating that lipid rafts are involved in the liberation of grp94 from BHK‐21 cells. The results suggest that BHK‐21 cells release grp94 into the serum‐free medium via the nonclassical secretory pathway in which lipid rafts play an important role. Copyright © 2012 John Wiley & Sons, Ltd.     

Premature chromosome condensation in a ts DNA-mutant of BHK cells

A temperature-sensitive mutant of BHK, designated is BN-2, shows a rapid drop in ³H-thymidine incorporation along with accumulation of the cells in the G1 phase of the cycle when asynchronous cultures are shifted from 33.5°C to the nonpermissive temperature of 39.5°C. Synchronized cultures of ts BN-2 cells did not enter DNA synthesis when shifted up in G1. Shift-up of cultures at the beginning of the S phase resulted in an approximately normal rate of DNA synthesis for about 2 hr. The rate of DNA synthesis then quickly declined, and the cells became arrested in mid-S after completion of approximately 0.5 rounds of DNA replication. At the same time, the majority of the cells were observed to lose the nuclear membrane and displayed premature chromosome condensation. These events were followed by the appearance of cells containing several micronuclei and eventual cell disruption and death. The nonpermissive temperature appeared to have no effect on either the elongation of short fragments of DNA or the execution of mitosis after the completion of the S phase under permissive conditions. The ts defect in this mutant may directly limit the initiation of DNA synthesis or alter the regulation of chromatin condensation.     

Characterization of freeze-thaw induced ultrastructural damage to endothelial cells in vitro

Summary The pathophysiology of endothelial cells is important to a variety of vascular conditions including coagulation and hemostasis resulting from clinical frostbite. Use of an in vitro model system demonstrated that when bovine endothelial cells were frozen at 1°C or 20°C/min and thawed immediately (20°C/min), a variety of ultrastructural alterations occurred. Membraneous structures were most extensively damaged, with mitochondria the most sensitive organelle. Low amplitude mitochondrial swelling, first evident at 0°C, progressed to high amplitude swelling by −10°C (frozen). In addition, the rough endoplasmic reticulum was dilated and formed large vesicles with a homogeneous matrix. Nuclear changes first occurred at −15°C. These included separation and distortion of the nuclear membrane, changes in chromatin distribution, and disruption of the nucleolus. Scanning electron microscopy revealed perforated plasma membranes in some cells at −10°C (frozen) and in most cells by −20°C. Cultures frozen at 20°C/min revealed mostly the same ultrastructural damage noted at 1°C/min except a higher percentage of cells exhibited alterations. Data from the recovery index and lactic dehydrogenase (LDH) release correlated well with observed ultrastructural changes. Early swelling of mitochondria and dilation of rough endoplasmic reticulum was not lethal in the absence of freezing. Increased swelling in cytoplasmic organelles coupled with nuclear alterations at −15°C resulted in a decreased survival rate and release of significant quantities of LDH by −20°C. No unique morphological changes were temperature specific, but the total number of cells that displayed alterations increased as temperature decreased. The views, opinions or findings, or both, contained in this report are those of the authros and should not be construed as indicative of an official Department of the Army position, policy, or decision unless so designated by other official documentation.     

Cytolytic T lymphocyte mediated chromium-51 release versus spontaneous release from blast and spleen cell targets at low temperatures

The rate of spontaneous ⁵¹Cr release from spleen cell and LPS blast target cells is strongly temperature dependent. Between 32 and 37 °C the rate of spontaneous release increases dramatically with temperature. Cytolytic T-lymphocyte-mediated lysis of these target cells is also temperature dependent, but lysis does not increase greatly above 32 °C. The ratio of specific ⁵¹Cr release to spontaneous release can be significantly improved by doing ⁵¹Cr-release assays at temperatures below 37 °C.     

The release of cytosolic proteins from cells treated with concanavalin A during scraping from plastic culture dishes

When rat hepatoma cells (HTC and R117-21B), treated with concanavalin A (conA) at 37 °C, were scraped from plastic culture dishes with a silicone-rubber policeman, the cell membranes were broken and the cytoplasm was released. This phenomenon was also observed in cells treated with conA at 4 °C, even though it took a longer time to show the same effect. The effect of 10 μg/ml of conA on the release of the cellular proteins reached a plateau when the treatment was carried out at 37 °C. Ninety percent of this effect was abolished by 10 mM of α-methyl-d-mannoside. The effect was completely nullified by 100 mM. At 4 °C, however, even 100 mM of this sugar could not abolish this effect. The apparent decrease in the cellular proteins with conA after scraping was observed not only in the logarithmic phase, but also in the stationary phase of cell growth. The breakdown of plasma membranes with conA eventually caused decrease in tyrosine aminotransferase activity, even though the lectin induced the enzyme activity in cultured cells.     

Effects of temperature,metabolic and cytoskeletal inhibitors on the rate of BHK cell adhesion to polystyrene

Adhesion of baby hamster kidney fibroblasts (BHK cells) to a Falcon tissue culture flask was measured under various physiological conditions. While 75–80% of the fibroblasts adhere at temperatures from 19–50°, cellular adhesion decreased dramatically below 19°. Less than 10% of the cells adhere to the substratum even after prolonged incubations at temperatures of 8° or below. This lack of adhesion at low temperatures cannot be overcome by the application of increased gravitational force to the cells. No correlation exists between cellular ATP concentrations or respiration rates and the rate of cell adhesion to the substratum. One millimolar Na F and 1 mM 2,4 dinitrophenol together lower cellular ATP concentration by 95% but adhesion is reduced by only 50%. NaN₃ and KCN greatly lower cellular ATP concentrations without a corresponding inhibition of adhesion. Inhibition of cellular respiration by these compounds occurs at lower concentrations than does the inhibition of adhesion. Two micrograms/milliliters of cytochalasin B inhibits adhesion by 90%, 0.1 mM vinblastine sulphate or colchicine by less than 50% and 50 μg/ml colcemid by less than 30%. Fixing the cells with formaldehyde, hardening their membranes with ZnCl₂ or treating the cells with toluene, all cause an inhibition in adhesion. Again, application of increased gravitational force cannot overcome these latter inhibitions of BHK cell adhesion to the surface of the flasks.     

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity. I. General requirements of the reaction and temporal relationship between lethal hits and cytolysis.

The effect of various physical and chemical parameters on the cytotoxic reaction was studied in a ⁵¹Cr-release assay in order to analyze the mechanism by which human blood mononuclear cells (MC) damage antibody-sensitized target cells infected with herpes simplex virus. Centrifugation of the target cell-MC mixture consistently increased the velocity of the reaction. In addition, uncentrifuged target cell-MC cultures showed a sigmoidal kinetic curve of ⁵¹Cr release with an initial lag phase of at least 10 min, whereas ⁵¹Cr release in centrifuged cultures followed a linear pattern with time without an initial lag. These findings indicate that direct contact between target and effector cells is necessary for cytotoxicity to occur. The reaction as a whole was temperature dependent, proceeding well at 37 °C and not at all at 4 °C. Incubation of the MC at 46 °C for 10 min abolished their cytotoxic potential without affecting their viability; similar heating of the target cells did not affect their background isotope release or sensitivity to the lytic process. Heating target cell-MC mixtures at 46 °C for 10 min thus provided a tool by which the temporal relationship between the mounting of “lethal hits” and specific isotope release, or cell lysis, could be studied. Using this technique, we observed virtually simultaneous occurrence of lethal hits and cell lysis, measured at various intervals between 10 and 360 min postincubation. Likewise, we were unable to demonstrate a transient period of increased osmotic fragility in target cells after contact with MC but before actual cell lysis. Taken together, these findings imply either that cell lysis, as indicated by ⁵¹Cr release, results from a sudden nonosmotic injury to the target cell membrane or, alternatively, osmotic damage leading to ⁵¹Cr release occurs too rapidly to be detected by the methods employed in this study. These findings imply either a qualitative or a quantitative difference between antibody-dependent cellular cytotoxicity (ADCC) mediated by K cells and cytotoxicity mediated by sensitized T cells.The cytotoxic reaction was completely inhibited by 10 mM EDTA and did not occur in a Ca²⁺- and Mg²⁺-free medium. Neither Ca²⁺ nor Mg²⁺ alone produced as much cytotoxicity as the two cations in tandem; in addition, when added to the culture medium in suboptimal amounts, the two cations were either additive or synergistic. These observations suggest that both cations are necessary in ADCC and also that there may be separate Ca²⁺- and Mg²⁺-dependent events in the lytic pathway.