Isolation and characterization of Daucus carota L. cell lines resistant to 2-deoxy-D-glucose

Variant carrot (Daucus carota L.) cell lines resistant to the growth inhibitory effects of the glucose analogue 2-deoxy-D-glucose (dGlc) were isolated utilizing a feeder plate technique. Growth of sensitive cells was less than 7.5% of controls on medium supplemented with 3.0 mM dGlc, whereas resistant variants achieved growth ranging from 15% to 70% of that in controls. Increased levels of acid invertase activity in variant cell lines in response to dGlc in the culture medium, together with decreased sensitivity of the acid invertase enzyme (EC 3.2.1.26) to dGlc, is proposed as one of several potential mechanisms contributing to the observed dGlc resistance.     

Properties and action mechanism of the toxic lectin modeccin: Interaction with cell lines resistant to modeccin,abrin, and ricin

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2-mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell-free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits. Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice-versa. The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possesss different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.     

Isolation and detailed characterization of human cell lines resistant to -threo-chloramphenicol

The isolation and characterization of chloramphenicol resistant derivatives of the human cell line HeLa B is described. Growth of resistant lines was unaffected in the presence of 100 μg/ml -threo-chloramphenicol, whereas growth of the parental cells was inhibited at 12.5 μg/ml. The incorporation of [³⁵S]methionine into mitochondrial protein of intact resistant cells continued normally in the presence of 100 μg/ml chloramphenicol (cytoplasmic protein synthesis was blocked by addition of 50 μg/ml emetine). Under these conditions the electrophoretic profile of labelled, presumptive mitochondrially-made proteins was similar to that of the parental cell line labelled in the absence of chloramphenicol. The cell lines selected in the presence of chloramphenicol also showed increased resistance to some other inhibitors of mitochondrial protein synthesis, e.g. carbomycin and mikamycin. [¹⁴C]Chloramphenicol was found to have normal access to the interior of resistant cells and it is therefore unlikely that resistance results from altered cell permeability. No modification of the drug by acetylation or glucuronide conjugation mechanisms was observed. The possibilities remain that resistance is mediated by altered permeability of the mitochondrial membrane, or from modification to a component of the mitochondrial protein synthetic system.     

Conformation-dependent antigenic determinants in the toxic lectin ricin.

The major part of the ricin-precipitable antibodies in sera produced by immunizing rabbits with formaldehyde-treated ricin is precipitated also by the isolated ricin A and B chains. In contrast, in antisera produced by immunizing with formaldehyde-treated ricinus agglutinin only a small part of the antibodies cross-reacting with ricin can be precipitated by the isolated A and B chains, or bound to immunoabsorbents containing the isolated ricin chains. In immunodiffusion studies with anti-ricinus agglutinin sera, a star-shaped precipitate was formed when isolated A and B chains recombined to form intact ricin. Both anti-ricin and anti-ricinus agglutinin sera neutralized effectively the ability of ricin to inhibit protein synthesis in HeLa cells. Anti-ricin serum also neutralized the inhibitory effect of the isolated A chain on protein synthesis in a cell-free system and the ability of the isolated B chain to induce indirect hemagglutination. In contrast, antiricinus agglutinin serum did not neutralize the biologic activities of the isolated ricin A and B chains. Anti-ricinus agglutinin serum formed a precipitate with the hybrid ricin A chain/abrin B chain, and protected against the toxic effect on HeLa cells of this hybrid, indicating conformational changes of ricin A chain upon binding to the B chain. It is concluded that the anti-ricinus agglutinin serum contains antibodies directed against conformational determinants present on intact ricin, but not present or exposed in the isolated A and B chains. At least part of these conformational determinants appears to be carried by the A chain.     

Isolation of L-ethionine resistant cell lines in Vigna sinensis L. after mutagen treatment

Chemical mutagenesis was employed for the isolation of variant cell lines resistant to L-Ethionine (L-Eth) in Vigna sinensis L. We have measured cell survival after treatment of Vigna sinensis cell suspensions with different mutagens: Ethyl methanesulfonate (EMS), N-methyl-N-nitrosoguanidine (NTG) and Acridine Orange (AO). NTG was more toxic than EMS and AO.L-Eth resistant colonies were isolated by plating on selective medium after NTG treatment. The frequencies of appearance of resistant cells of MS-3 media supplemented with 20 g/ml L-Eth were 1.3 × 10^-6 to 1.8 × 10^-5. The highest number of L-Eth resistant calli were recorded in cells treated with 10 g/ml NTG. Few resistant colonies also appeared spontaneously from non-mutagenized cultures with a frequency of 2.9 × 10^-7 to 5.7 × 10^-7. However, a number of isolated colonies were discarded after successive retesting. The number of resistant calli dropped from 204 to 22 during successive retests. The significance of these observations has also been discussed.     

Mechanism of resistance to ricin toxin in selected mouse lymphoma cell lines

The role of impaired toxin uptake in conferring cellular resistance to the plant toxin RCA_II (ricin) has been examined using a murine BW5 147 lymphoma line and a toxin-resistant variant (BW5 147 Ric^R.3) selected by repeated exposure to RCA_II. The toxin-resistant variant is 250 times more resistant to RCA_II in long-term growth experiments and 1,000 times more resistant in short-term protein synthesis assays. Experiments with ferritin-conjugated ¹²⁵I-labeled RCA_II (ferritin-¹²⁵I-RCA_II) indicated that toxin binding to sensitive and resistant cells is similar at low toxin concentrations where maximum differential cytotoxicity occurs but that major difference exist with respect to toxin uptake. In sensitive cells toxin is internalized via endocytosis, and as seen previously in other systems subsequent rupture of some of the toxin-containing endocytotic vesicles releases toxin into the cytoplasm, where it inhibits protein synthesis. The process of toxin transfer to the cytoplasm is presumed to account for the one-hour lag before toxin-induced inhibition of protein synthesis can be detected. Endocytotic uptake of toxin is impaired in resistant BW5147Ric^R.3 cells, and they are unaffected by toxin concentrations that inhibit protein synthesis and kill sensitive parental cells. Killing of resistant cells at low toxin concentrations was accomplished by encapsulating RCA_II into lipid vesicles capable of fusing with the plasma membrane. Direct introduction of toxin into resistant cells using lipid vesicles as carriers produced rapid inhibition (< 15 min) of protein synthesis and eliminated the lag in toxin action seen in sensitive cells exposed to free toxin. These findings are discussed in relation to the mechanism of toxin action and proposals that toxin activity requires structural modification of the toxin molecule at the cell surface before transport into the cell.     

RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes

The modification reaction of 28 S rRNA in eukaryotic ribosomes by ricin A-chain was characterized. To examine whether ricin A-chain release any bases from 28 S rRNA, rat liver ribosomes were incubated with a catalytic amount of the toxin, and a fraction containing free bases and nucleosides was prepared from the postribosomal fraction of the reaction mixture by means of ion-exchange column chromatography. Thin-layer chromatographic analysis of this fraction revealed a release of 1 mol of adenine from 1 mol of ribosome. When the ribosomes or naked total RNAs were treated with ricin A-chain in the presence of [32P] phosphate, little incorporation of the radioactivity into 28 S rRNA was observed, indicating that the release is not mediated by phosphorolysis. Thus, considering together with the previous result (Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K. (1987) J. Biol. Chem. 262, 5908-5912), the results in the present experiments demonstrated that ricin A-chain inactivates the ribosomes by cleaving the N-glycosidic bond of A4324 of 28 S rRNA in a hydrolytic fashion.     

Isolation and characterization of high endothelial cell lines derived from mouse lymph nodes

Summary Two long-term cultured cell lines were established from BALB/c mouse axillary and cervical lymph nodes that exhibited a combination of functional, morphological, and phenotypic characteristics consistent only with high endothelial venule cells. Spleen lymphocytes selectively bound and migrated across the cell lines. On Matrigel, these cell lines formed tubules with lumens, a characteristic unique to endothelial cells. Morphologically the cells were 20–30 μm in diameter and exhibited contact inhibition. The cells were not myeloid in origin because they lacked sodium fluoride-inhibitable nonspecific esterase activity, myeloperoxidase activity, and F4/80 antigen. The cell line phenotypes were compared to high endothelial venule (HEV) cells in tissue sections. HEV cells in lymph node tissue sections expressed endoglin, PECAM-1, ICAM-1, VCAM-1, laminin, fibronectin, collagen IV, H2K^d, MECA 79, MECA 325, and vWF. The cell lines expressed endoglin, VCAM-1, fibronectin, and H2K^d. The cell line derived from cervical lymph nodes also expressed laminin and H2D^d. Neither cell line expressed collagen IV, IA^d, ICAM-1, ICAM-2, dendritic cell antigen, or PECAM-1. They also did not express MECA antigens or intracellular vWF, consistent with reports of many cultured endothelial cells. To further substantiate cell line identification, antiserum generated against the cell lines bound specifically to HEV cells in frozen lymph node tissue sections and to both of the lymph node-derived cell lines but not control cell lines. Thus, the lymph node derived-cell lines expressed molecules found on HEV cellsin vivo and most importantly retained the functions of tubule formation, lymphocyte adhesion, and promotion of lymphocyte migration.     

Mouse lymphoma cell lines resistant to pea lectin are defective in fucose metabolism

Two mutants of the BW5147 mouse lymphoma cell line have been selected for their resistance to the toxic effects of pea lectin. These cell lines, termed PLR1.3 and PHAR1.8 PLR7.2, have a decreased number of high affinity pea lectin-binding sites (Trowbridge, I.S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Intact cell labeling experiments using [2-3H]mannose indicated that PLR1.3 cells have a block in the conversion of GDP-[3H]mannose to GDP-[3H]fucose whereas PHAR1.8 PLR7.2 cells appear to be blocked in the transfer of fucose from GDP-[3H]fucose to glycoprotein acceptors. In vitro experiments with extracts of PLR1.3 cells confirmed the failure to convert GDP-mannose to GDP-fucose and indicated that the defect is in GDP-mannose 4,6-dehydratase (EC 4.2.1.47), the first enzyme in the conversion of GDP-mannose to GDP-fucose. The block in the PLR1.3 cells could be bypassed by growing the cells in the presence of fucose, demonstrating that an alternate pathway for the production of GDP-fucose presumably via fucose 1-phosphate is functional in this line. PLR1.3 cells grown in 10 mM fucose showed normal high affinity pea lectin binding. PHRA1.8 PLR7.2 cells synthesize GDP-fucose and have normal or increased levels of GDP-fucose:glycoprotein fucosyltransferase when assayed in vitro. The fucosyltransferases of this clone can utilize its own glycoproteins as fucose acceptors in in vitro assays. These findings indicate that this cell line fails to carry out the fucosyltransferase reaction in vivo despite the fact that it possesses the appropriate nucleotide sugar, glycoprotein acceptors, and fucosyltransferase. The finding of decreased glycoprotein fucose in two independent isolates of pea lectin-resistant cell lines and the restoration of high affinity pea lectin binding to PLR1.3 cells following fucose feeding strongly implicates fucose as a major determinant of pea lectin binding.     

Isolation and characterization of lectin-resistant mouse cell lines defective in their ability to yield hybrid cells

Lectin-resistant variants of the mouse mammary tumor cell line FM3A in suspension were isolated after multiple-step selection using toxic concentrations of phytohemagglutinin (PHA) and concanavalin A (Con A). In fusion experiments, both PHA- and Con A-resistant lines were found to have lost the ability to yield hybrids with suspension-grown cells, although they were capable of yielding hybrids with monolayer-grown cells. This phenotype of PHA-resistant lines was stable during a long period of culture in the absence of PHA.     

Isolation and characterization of two novel colon cancer cell lines from Chinese patients

Incidence of colon cancer has increased rapidly in China. Although many colon cancer cell lines have been established previously, most of them were derived from patients from western countries. Epidemiological, clinical, cytogenetic, and molecular biological studies showed that there are considerable differences between Chinese and western countries colon cancer patients. Therefore, establishment of novel colon cancer cell line from Chinese is useful for studying the racial difference of this disease and can be important for studying the pathogenesis of colon cancer in China. In our laboratory, two novel continuous human colon cancer cell lines, SHT-1 and SHH-1, have been established in vitro from Chinese patients, and both cell lines have been passaged for 4 yr, and they have been continuously subcultured with more than 800 population doubling and without signs of senescence. Both cell lines were obtained from primary tumor tissues during colon cancer surgery. Cells grew rapidly with a doubling time of 36–39 h and a plating efficiency of 26–28%. These cells exhibited an epithelial morphology and expressed cytokeratin. Tumor developed in severe combined immunodeficient (SCID) mice 4–6 wk after inoculated subcutaneously with the cultured cancer cells. Karyotypic analysis and comparative genomic hybridization (CGH) analysis in SHT-1 cells revealed a hypertriploid modal number of 76 with numerous numerical and structural abnormalities previously linked to colon cancer. In another cell line (SHH-1), CGH analysis revealed that −1p13 was the only cytogenetic anomaly.     

Isolation and characterization of aDatura innoxia cell line resistant to ethanol

A cell line ofDatura innoxia was selected in suspension culture to be resistant to 1% (vol/vol) ethanol (EtOH^R). EtOH^R cells were cross-resistant to 1% (vol/vol) methanol and 1% (vol/vol) 2-propanol but not 1% (vol/vol)n-propanol orn-butanol, whereas wild type (WT) cells were resistant only to methanol. Resistance in EtOH^R cells is probably a result of a very low level of alcohol dehydrogenase (ADH) activity which was only 9 to 10% of that in WT cells and was undetectable during much of the EtOH^R growth cycle. In the absence of ethanol, EtOH^R cells have a I₅₀ for the toxic ethanol analog allyl alcohol, which is nearly 3 times higher than that in WT cells. In the presence of ethanol, EtOH^R cells have an I₅₀ for allyl alcohol which is 12 times more than WT cells. This difference correlated well with the decrease in ADH activity found in EtOH^R cells grown on ethanol. When ethanol was removed from the suspension medium, ADH activity in EtOH^R cells gradually increased to WT levels. When re-exposed to ethanol after 200 cell generations, ADH activity quickly decreased and growth resumed after a 4- to 6-day lag period. Lipid analysis showed a 37% increase in total lipid in EtOH^R cells, mostly in polar lipids, di- and triglycerides. The fatty acid composition of these lipid classes was shifted toward the more polyunsaturated. These lipid changes were probably a reflection of the increased plastid number in the EtOH^R cells and may be a result of growth in ethanol rather than a reason for resistance. EtOH^R cells seem to be regulatory mutants able to quickly lower ADH activity in the presence of ethanol.     

Navy (haricot)-bean (Phaseolus vulgaris) lectin. Isolation and characterization of two components from a toxic agglutinating extract

Two protein components were isolated in a highly purified state from a toxic fraction from the navy (haricot) bean (Phaseolus vulgaris). One, a lectin, strongly agglutinated horse erythrocytes and leucocytes, agglutination being readily observable with both types of cell at a lectin concentration of 4mug/ml. The other component (component 1), although possessing some similarity in composition, was thought to be non-agglutinating or, at most, only very weakly so. Component 1 had a mol.wt. of about 143000 and a subunit mol.wt. of about 37000, suggesting a tetrameric structure probably with identical subunits. Alanine was the only N-terminal amino acid identified and the molecule was notable in being devoid of tryptophan and cysteine, low in methionine and high in leucine, glutamic acid and aspartic acid. The lectin was somewhat smaller (mol.wt. about 114000) and apparently also composed of four identical subunits of mol.wt. about 30000. Dansylation showed that arginine occupied the N-terminus of the polypeptide chain. Aspartic acid, serine, threonine and leucine were the predominant amino acids of the lectin, and the sulphur-containing amino acids were entirely absent. Both constituents were glycoproteins and the compositions of the carbohydrate portions (4.9% for component 1 and 8.1% for the lectin) were generally similar, consisting of mannose and glucosamine with smaller amounts of glucose and traces of xylose and arabinose.     

Chinese hamster ovary cell variants resistant to monensin, an ionophoric antibiotic. I. Isolation and altered endocytosis of ricin

Chinese hamster ovary (CHO) cell variants resistant to a carboxylic ionophore, monensin, have been isolated. Two monensin-resistant variants (MonR-31 and MonR-32) showed a three- to fourfold higher resistance to monensin than did CHO. These MonR clones also showed fourfold higher resistance to another carboxylic ionophore, nigericin, and twofold higher resistance to valinomycin. They were also slightly more resistant to other unrelated drugs such as adriamycin, colchicine, bleomycin, and chloroquine, and in particular, they showed about threefold higher resistance to ricin, a toxin of Ricinus communis. MonR clones were found to retain a normal level of [125I]ricin binding, but internalization of [125I]ricin into the MonR clones was one-half or less than with CHO. Present data suggest that drug-resistant clones selected in culture may provide a way to isolate cells with altered response to various bioactive molecules.     

Binding diversity of the two binding sites of ricin B lectin

Ricin B is a galactose-binding protein, which contains two binding sites. We have compared the binding properties of the two binding sites of ricin B chain toward different mono- and disaccharide ligands. The free energies of binding are calculated using the free energy perturbation simulation (thermodynamic integration method) and linear interaction energy approach using CHARMM force field. The second binding site of the protein was found to be weaker compared to the first. The details of the hydrogen-bonding scheme suggested the origin of the epimeric specificity of the protein. The reason for the weaker binding capacity of the second binding site has been addressed.