Fibroblast receptor for cell-substratum adhesion: studies on the interaction of baby hamster kidney cells with latex beads coated by cold insoluble globulin (plasma fibronectin)

Studies were carried out on the interactions of uncharged latex beads (0.76 micrometer) with baby hamster kidney cells. Binding of beads to the cells occurred if the beads were coated by cold insoluble globulin (CIG) (plasma fibronectin) but not if the beads were coated by bovine albumin. Bovine albumin-coated beads did not bind to the cells even in the presence of excess CIG in the incubation medium. Binding of beads occurred randomly over the entire surfaces of cells in suspension. However, cell receptors for CIG beads were no longer detectable on the upper surface of cells spread onCIG-coated tissue culture dishes. Binding of CIG beads to cells occurred at all temperatures tested from 4 degrees to 37 degrees C but the rate was lowest at 4 degrees C. At 37 degrees C, binding was accompanied by endocytosis and the beads were found inside vesicles which appeared to be lysosomes. There was also release of radioactivity from radiolabeled CIG beads during incubation with the cells at 37 degrees C. Binding of CIG beads to cells did not require divalent cations. Finally, the cell receptor for CIG beads was lost after cell trypsinization. The data are discussed in terms of current ideas about the basis for cell adhesion.     

Fibroblasts maintain a complete endocytic pathway in the presence of lysosomotropic amines

We have investigated the effects of the lysosomotropic amines, ammonium chloride and chloroquine, on the delivery of fluid-phase pinocytic tracers to lysosomes in Chinese hamster ovary (CHO) cells. In preliminary experiments, 15 mM ammonium chloride and 0.1 mM chloroquine were found to be sufficient to give maximal protection of endocytosed material from digestion in a lysosome. In the presence of either amine at these concentrations, the generation time of CHO cells was depressed by less than 30% even though selective depletion of lysosomal hydrolases was observed. For cells treated with either amine for 1 or 6 days the amount of horseradish peroxidase (HRP) internalized in a 1-h pulse was approximately 50-70% of that of control. By cell fractionation, cells treated with amine for 2 or 6 days were found to accumulate fluorescein-dextran or HRP in lysosomes. HRP accumulation in lysosomes in amine-treated cells was also observed by electron microscopy. Little exocytosis of lysosomal HRP into the media was observed under any condition. We conclude that in long-term amine-treated CHO cells endocytic vesicle traffic is maintained.     

Identification and purification of NK cells with lysosomotropic vital stains: correlation of lysosome content with NK activity

The lysosome content of lymphocytes has been analyzed with lysosomotropic vital stains and the fluorescence-activated cell sorter (FACS). Large granular lymphocytes (LGL), which account for virtually all natural killing activity in peripheral blood, are quantitatively different from small lymphocytes (SL) in this respect. LGL obtained by Percoll gradient density centrifugation accumulate more of the lysosomotropic vital dyes than SL do, staining with either neutral red or mepacrine (quinacrine). Furthermore among the LGL-rich, low density lymphocyte population highly, granulated cells can be separated from less granulated ones by mepacrine staining and FACS. Thus, separated highly granulated LGL express very high natural killing, whereas the less granulated low density large lymphocytes do not kill.     

The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement

Lysosomotropic amines, such as ammonium chloride, are known to protect cells from the cytotoxic effects of diphtheria toxin. These drugs are believed to inhibit the transport of the toxin from a receptor at the cell exterior into the cytoplasm where a fragment of the toxin arrests protein synthesis. We studied the effects of lysosomotropic agents on the cytotoxic process to better understand how the toxin enters the cytoplasm. The cytotoxic effects of diphtheria toxin were not inhibited by antitoxin when cells were preincubated at 37 degrees C with toxin and ammonium chloride, exposed to antitoxin at 4 degrees C, washed to relieve the ammonium chloride inhibition, and finally warmed to 37 degrees C. The antigenic determinants of the toxin were, therefore, either altered or sheltered. It is likely that the combination of ammonium chloride and a low temperature trapped the toxin in an intracellular vesicle from which the toxin could proceed to the cytoplasm. Because lysosomotropic amines raise the pH within acidic intracellular vesicles, such as lysosomes, they could trap the toxin within such a vesicle if an acidic environment were necessary for the toxin to penetrate into the cytoplasm. We simulated acidic conditions which the toxin might encounter by exposing cells with toxin bound to their surface to acidic medium. We then measured the effects of lysosomotropic amines on the activity of the toxin to see if the acidic environment substituted for the function normally inhibited by the drugs. The drugs no longer protected the cells. This suggests that exposing the toxin to an acidic environment, such as that found within lysosomes, is an important step in the penetration of diphtheria toxin into the cytoplasm.     

Immunochemical purification of probe-labeled plasma membrane proteins: An approach to the molecular anatomy of the cell surface

The probe 2,4,6-trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labelling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate-polyacrylamide gels of probe-labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus-transformed cells. Moreover, the transformed cells display a nonvirion component of 80–100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37°C but not at 41°C, the expression of this component is related to the expression of the transformed phenotype.     

The effect of lysosomotropic detergents on the permeability properties of the lysosome membrane

Compounds such as N-dodecylimidazole and N-dodecylmorpholine kill cells in culture. Their cytotoxicity has been attributed to accumulation in lysosomes where protonation confers detergent properties resulting in membrane destabilization. This hypothesis has been tested by examining the ability of N-dodecylimidazole and N-dodecylmorpholine to decrease the latency of alpha-glucosidase in isolated rat liver lysosomes. No effect was observed. Nor was N-dodecylimidazole apparently able to increase the permeability of isolated rat liver lysosomes to L-alanine, as no diminution of the disruptive effect of L-alanine methyl ester was seen. N-Dodecylimidazole (10-20 micrograms per ml) caused lactate dehydrogenase release from cystinotic fibroblasts, but marginally toxic concentrations failed to induce cystine release, as might have been expected if lysosome membrane damage had occurred. It is concluded that the cytotoxic effects of lysosomotropic detergents may be mediated by a non-lysosomal mechanism.     

Antibody-induced modulation of proteins in vesicular stomatitis virus-infected fibroblasts.

When vesicular stomatitis virus-infected baby hamster kidney cells were treated with rabbit anti-vesicular stomatitis virus serum, there was a loss of the viral glycoprotein G into acid-soluble products. This degradation occurred within minutes at 37 degrees C and required the presence of G protein at the cell surface. The degree of degradation depended on antiserum concentration. The antiserum, also, prevented maturation of extracellular virions and induced partial degradation of the intracellular viral proteins, without affecting host proteins. The degradation could not be prevented by the presence of lysosomotropic agents, protease inhibitors, colchicine, or cytochalasin B. Similar kinetics and specificity of degradation was obtained with cells infected with vesicular stomatitis virus mutants that were less cytopathic. These results characterize a model system for studying the parameters and consequences of antigenic modulation as well as for studying the fate of viral antigens during persistent infections.     

Growth characteristics in cell culture and pathogenicity in mice of two terrestrial rabies strains indigenous to Canada

Two strains of street rabies virus from striped skunks (Mephitis mephitis) were used to infect either a murine neuroblastoma (NA 1300) or a baby hamster kidney (BHK-21/C13) cell culture and the cell infection rates were noted during 4 days postinfection. These cultures were then passaged for four consecutive passages, and the viruses obtained in the supernatant fluids of passage 4 were then treated as original isolates and used to infect both neuroblastoma and baby hamster kidney cells. The mortality period in Swiss white mice caused by the various virus suspensions was noted. The virus strain from the brain of skunks from Saskatchewan infected neuroblastoma and baby hamster kidney cells equally well, produced similar virus titres in supernatant fluids after four subcultures in both cell types, and appeared to produce similar mortality periods in mice from either the original brain tissue or from cell culture supernatant fluids. On the other hand, the virus from the brains of skunks from Ontario readily infected neuroblastoma but poorly infected baby hamster kidney cell cultures. Passage of this strain through four subcultures in both cell types produced virus titres in the supernatant fluids of equal magnitude. However, reisolation of the virus from the supernatant fluid of passage 4 in neuroblastoma cell cultures showed a similar pattern to that from the original brain, while the virus from baby hamster kidney cell passage supernatant fluid was considerably altered. Although the mortality period in mice was similar with virus from the brain and neuroblastoma cell cultures, this period was shortened when mice were inoculated with baby hamster kidney culture supernatant virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic amines are selective cytotoxic agents for pigmented cells

We have shown that morpholine, a cyclic amine, exerts a selective inhibition of growth on melanocytic pigmented cell lines compared to nonpigmented cells. The ID50 of morpholine for the pigmented B-16 cell line HFH was 1200 micrograms/ml, compared to values greater than 2400 micrograms/ml for baby hamster kidney, Chinese hamster ovary and NP, an unpigmented primate cell line. Two other cyclic amines piperazine and piperidine, were similarly found to be selectively toxic to melanocytes. This selective toxicity could be synergistically enhanced by pretreatment of the cells with theophylline, a stimulator of tyrosinase activity, which indicates that the selective toxicity may be associated with melanin synthesis. Low passage HFH, high passage HFH and Syrian hamster melanoma RPMI 1846 cells that were pretreated with theophylline showed between 13 and 29% greater toxicity compared to controls treated with theophylline or morpholine alone. Unpigmented NP primate cells, Chinese hamster ovary and mouse fibroblast L929 remained unaffected. These cyclic amines join a list of other amines that have also been shown to be melanocytotoxic.     

The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole

The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (C12-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15% of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C12-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to approximately 125% of control values within 24 h. Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. Treatment with concanavalin A (50 micrograms/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by C12-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 micrograms/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to less than 20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CH-loaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by C12-Im. These observations also confirm the previous conclusions that C12-Im kills cells as a consequence of lysosomal disruption.     

Resistance of the human beta1-adrenergic receptor to agonist-induced ubiquitination: a mechanism for impaired receptor degradation

Down-regulation is a classic response of most G protein-coupled receptors to prolonged agonist stimulation. We recently showed that when expressed in baby hamster kidney cells, the human beta1-but not the beta2-adrenergic receptor (AR) is totally resistant to agonist-mediated down-regulation, whereas both have similar rates of basal degradation (Liang, W., Austin, S., Hoang, Q., and Fishman, P. H. (2003) J. Biol. Chem. 278, 39773-39781). To identify the underlying mechanism(s) for this resistance, we investigated the role of proteasomes, lysosomes, and ubiquitination in the degradation of beta1AR expressed in baby hamster kidney and human embryonic kidney 293 cells. Both lysosomal and proteasomal inhibitors reduced beta1AR degradation in agonist-stimulated cells but were less effective on basal degradation. To determine whether beta1AR trafficked to lysosomes we used confocal fluorescence microscopy. We observed some colocalization of beta1AR and lysosomal markers in agonist-treated cells but much less than that of beta2AR even in cells co-transfected with arrestin-2, which increases beta1AR internalization. Ubiquitination of beta2AR readily occurred in agonist-stimulated cells, whereas ubiquitination of beta1AR was not detectable even under conditions optimal for that of beta2AR. Moreover, in cells expressing betaAR chimeras in which the C termini have been switched, the chimeric beta1AR with beta2AR C-tail underwent ubiquitination and down-regulation, but the chimeric beta2AR with beta1AR C-tail did not. Our results demonstrate for the first time that beta1AR and beta2AR differ in the ability to be ubiquitinated. Because ubiquitin serves as a signal for sorting membrane receptors to lysosomes, the lack of agonist-mediated ubiquitination of beta1AR may prevent its extensive trafficking to lysosomes and, thus, account for its resistance to down-regulation.     

Density-dependent inhibition of cell growth is correlated with the activity of a cell surface phosphatidylinositol-specific phospholipase C.

We previously identified a novel phospatidylinositol-specific phospholipase C (PI-PLC) present at the surface of Swiss 3T3 cells using a fluorescent analog of PI and showed that this cell surface PI-PLC (csPI-PLC) activity increases with increasing cell density (J. Biol. Chem. 265, 5337-5340 (1990)). Here, we find that csPI-PLC activity also increased in cultures in which growth was inhibited due to serum deprivation. csPI-PLC was inhibited by agents that inhibit other mammalian PI-PLCs, but not by treatments which inhibit glycosyl PI-PLCs (GPI-PLCs). We also extended our studies using fluorescent PI to other cell types and found that csPI-PLC activity was present only in cell lines that exhibit growth inhibition upon reaching confluency (Swiss 3T3, 3T3-L1, BALB/c 3T3, and normal rat kidney (NRK) fibroblasts), but not in cell lines that are tumorigenic and/or do not exhibit growth inhibition in a density-dependent manner (Chinese hamster ovary (CHO), mouse L, SV-40 transformed BALB/c 3T3 (SV-T2), baby hamster kidney (BHK), and Chinese hamster lung (V79) fibroblasts). These results support the hypothesis that csPI-PLC plays a role in the density-dependent inhibition of cell growth.     

Comparative analysis of caffeine and 3-aminobenzamide as DNA repair inhibitors in Syrian baby hamster kidney cells

The effects of caffeine and 3-aminobenzamide (3-AB) on Syrian baby hamster kidney cells treated with DNA-alkylating agents and ultraviolet-light suggest that two different DNA-repair mechanisms are involved. Both these agents enhanced the cell kill after methyl methanesulfonate (MMS) treatment. However, enhanced lethality was observed only with caffeine post-treatment when cells were exposed to nitrogen mustard (HN2) or ultraviolet light (UV); 3-AB did not appreciably change cell killing by these agents. With MMS-treated cultures, the effect of caffeine was maximal about 16 h later. The effect of 3-AB on the other hand, was exerted during the first 4 h after exposure to MMS. Caffeine's effect on cell survival could be abolished by low concentrations of cycloheximide, whereas 3-AB's effect could not. Furthermore, the G2 block in cell cycle progression, after MMS treatment, was not observed if the cells were post-treated with caffeine. In the presence of 3-AB, MMS-treated cells were arrested in G2 phase at a much earlier time compared to cells not treated with 3-AB. Finally caffeine post-treatment produced a 10-fold increase in nuclear fragmentation in MMS-treated cells. 3-AB did not cause nuclear fragmentation by itself but further enhanced the nuclear fragmenting effect of caffeine when both agents were present during the posttreatment. Therefore, we propose that 3-AB and caffeine each prevent a different repair mechanism from being effective.     

Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C.

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are rapidly deactivated by a membrane-bound phosphatase activity. The efficiency of this regulation suggests CFTR and protein phosphatases may be associated within a regulatory complex. In this paper we test that possibility using co-immunoprecipitation and cross-linking experiments. A monoclonal anti-CFTR antibody co-precipitated type 2C protein phosphatase (PP2C) from baby hamster kidney cells stably expressing CFTR but did not co-precipitate PP1, PP2A, or PP2B. Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from baby hamster kidney membrane extracts. Exposing baby hamster kidney cell lysates to dithiobis (sulfosuccinimidyl propionate) caused the cross-linking of histidine-tagged CFTR (CFTR(His10)) and PP2C into high molecular weight complexes that were isolated by chromatography on Ni(2+)-nitrilotriacetic acid-agarose. Chemical cross-linking was specific for PP2C, because PP1, PP2A, and PP2B did not co-purify with CFTR(His10) after dithiobis (sulfosuccinimidyl propionate) exposure. These results suggest CFTR and PP2C exist in a stable complex that facilitates regulation of the channel.     

Materials released into culture medium by normal and oncogenic virus-transformed mammalian cells.

Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polyoma virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.     

Materials released into culture medium by normal and oncogenic virus-transformed mammalian cells

Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polymo virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.     

Lysosomal origin of the ferric iron required for cell killing by hydrogen peroxide

The lysosomotropic amines methylamine (40 mM) and chloroquine (125 mM) prevented the killing of cultured hepatocytes by hydrogen peroxide generated in the medium by glucose oxidase. Maximum protection required several hours preincubation with either amine. Sensitivity of the hepatocytes to H2O2 was restored either by the addition of ferrous or ferric iron to the culture medium, or by incubating the cells for 4 hours in the absence of either amine prior to treatment with H2O2. Neither methylamine nor chloroquine had any effect on the cell killing by t-butyl hydroperoxide, a hepatotoxin that does not require iron. The protective effect of the lysosomotropic amines was distinguished from that of the ferric iron chelator deferoxamine in two ways: 1) deferoxamine protected hepatocytes from H2O2 toxicity but did not require a pretreatment period; and 2) in contrast to methylamine or chloroquine, deferoxamine had no effect on lysosomal pH as assessed by the fluorescent probe acridine orange. The data suggest that a lysosomal pool is the source of the ferric iron necessary for the killing of hepatocytes by H2O2.     

Effect of cytolytic infection on maintenance of resistance to HVJ (Sendai virus) in an altered BHK cell culture

Altered baby hamster kidney (BHK-R) cells were serially cultured in the continuous presence of hemagglutinating virus of Japan (HVJ). These cells showed a distinct resistance to superinfection with the homologous HVJ. This resistance of BHK-R cells gradually disappeared after serial passages in the presence of ultraviolet-irradiated HVJ particles which lost infectivity but still preserved hemagglutinating and neuraminidase activities. When BHK-R cells were serially cultured in the presence of a temperature-sensitive mutant of HVJ at non-permissive temperature, the cells also lost the resistance. The resistance of BHK-R cells remained unchanged, even after prolonged incubation in virus-free maintenance medium under the conditions of no cell division. It was suggested that killing of virus-sensitive cells, which were generated during cell proliferation, was required for maintenance of the resistance.     

A growth hormone-vesicular stomatitis virus G hybrid protein is rapidly degraded in lysosomes following transport to the cell surface

We have expressed the hybrid protein, GHG3, in baby hamster kidney cells to study protein turnover. GHG3 contains the cytoplasmic and transmembrane domains of vesicular stomatitis virus G protein linked to the C-terminus rat growth hormone. Turnover of GHG3 was prevented by lysosomal inhibitors (leupeptin, chloroquine, primaquine or monensin), while the accumulated GHG3 was localized to intracellular vesicles, results indicating that degradation occurred in lysosomes. The kinetics of degradation at 34 degrees C were determined in pulse-chase studies of metabolically labeled cells. After a lag period of 1 h, degradation was rapid (t1/2 = 1.25 h). The fate of GHG3 during the lag period was determined by immunofluorescence. We detected GHG3 on the cell surface when growth hormone antiserum was added to the growth medium 90 min prior to fixation and staining. No staining was observed if protein synthesis was inhibited with cycloheximide 90 min prior to the addition of growth hormone antiserum, a result indicating that GHG3 was rapidly removed from the cell surface. Unless the cells were pretreated with cycloheximide, antiserum was also detected in intracellular vesicles, which showed that GHG3 was endocytosed. These data indicate that a pool of GHG3 is transported rapidly to the cell surface, endocytosed and with little or no recycling directed to lysosomes for degradation.     

The leader polypeptide of Theiler's virus is essential for neurovirulence but not for virus growth in BHK cells.

A leader polypeptide of unknown function is encoded by cardioviruses, such as Theiler's murine encephalomyelitis virus. Although the deletion of this polypeptide has little effect on the growth of parental GDVII virus in baby hamster kidney (BHK) cells, the mutant virus is completely attenuated and fails to kill mice receiving intracerebral inoculations of high doses of the virus.