A variant form of beta-actin in a mutant of KB cells resistant to cytochalasin B

Lines of KB cells resistant to cytochalasin B have been isolated and characterized. The mutant Cyt 1 exhibits increased cross-resistance to cytochalasin E. Cyt 1 cells bind less cytochalasin B than parental KB cells. Two-dimensional gel analysis shows that Cyt 1 cells carry an alteration in beta-actin. This is further confirmed by one-dimensional peptide analysis. The altered beta-actin (beta'-actin) is synthesized when poly(A)+ RNA from Cyt 1 cells is translated in a reticulocyte-cell-free translation system. These results suggest that the primary site of action of cytochalasin B on cellular motility processes is beta-actin.     

Freeze-fracture study of adenovirus-induced KB cell surface alterations.

A technique of destabilization of KB cell plasma membrane by treatment with hypotonic citrate buffer was employed. The technique did not alter the viability or ability of the cells to synthesize macromolecules or produce virus, but did permit the visualization, by the freeze-fracture technique, of plasma membrane modifications occurring in response to adenovirus adsorption. The modifications consisted of a rearrangement of the membrane particles (MPs) on both protoplasmic and external leaflets of the plasma membrane. The rearrangement delineated bare areas, 139 nm in mean diameter, devoid of MPs and protruding outwards. The membrane changes were transient and were only observed when KB cells were maintained with adenovirus particles at 0 °C. The changes disappeared rapidly upon warming to 37 °C, reforming the ‘random pattern’ of MPs, normally visible on the cell plasma membrane. The same type of study was carried out with purified adenovirus capsid components (hexon, penton, penton base and fiber), with smaller virus particles (poliovirus) and with larger ‘artificial’ adenovirus particles made of latex beads coated with adenovirus pentons. The dimensions of the bare regions devoid of MPs appeared to be related to the size of the particles used, suggesting the existence of a ‘recognition pattern’ specific for virus particles.     

Functional studies on B cell hybridomas with B cell surface antigens. IV. Direct effects of cytochalasin B on differentiation

We previously established B cell hybridomas between M12.4.1 B lymphoma of BALB/c mice and normal B cell of C57BL/6 (B6) mice. These hybridomas express Iab, Iad, and IgM molecules on the cell membrane, and can induce the generation of IgM secretion when treated with purified goat anti-mouse-mu antibody (anti-mu) without T cell factors. In this study, TH2.54, a subclone of a B cell hybridoma, was treated with cytochalasin B (CB), a fungal product that disrupts microfilaments, and the direct effect of CB on the proliferation and differentiation of TH2.54 was examined. CB considerably suppressed the spontaneous proliferation of hybrid cells. This product, however, did not inhibit the generation of IgM secretion by TH2.54 treated with anti-mu. Surprisingly, CB could directly induce the development of IgM-secreting cells by TH2.54 at a relatively high frequency. Among cytochalasins, dihydrocytochalasin B (H2CB), cytochalasin C (CC), and cytochalasin D (CD) showed marked effects on the induction of IgM secretion as well as CB. In addition, the differentiative effect of CB was greatly inhibited by N6, O2-dibutyryladenosine 3':5'-cyclic monophosphate (dbc-AMP), but not by N2, O2-dibutyrylguanosine 3':5'-cyclic monophosphate (dbc-GMP). Analysis by flow microfluorometry (FMF), cytotoxicity assays, and quantitative absorption tests demonstrated that CB treatment of TH2.54 resulted in a significant decrease in the expression of Iab, Iad, and IgM molecules on the cell membrane. In contrast, parental M12.4.1 neither generated any IgM secretion nor changed Iad expression on the cell membrane under the same conditions. The present study suggests very strongly that microfilament-microtubule systems are not involved in the differentiative process of TH2.54 induced by anti-mu. The results also indicate that CB can provide the initiative signal for differentiation of TH2.54 into the maturation lineage; this is followed by a significant change in the expression of Ia and IgM molecules on the cell membrane.     

A Chinese hamster ovary cell mutant resistant to aphidicolin

A triple (aphr ara-Ar and araCr) mutant (AP7) of Chinese hamster ovary cells resistant to DNA polymerase inhibitors is described. The aphidicolin-resistance of the mutant was stable and inherited as a dominant genetic trait. The DNA polymerase alpha from the wild type (aphs) and the mutant (aphr) cells differed in their elution profiles on DEAE-cellulose chromatography and in their molecular weights which were 192,000 for the wild type (CHO-K-1, AC6a) and 165,000 for the mutant (AP7) enzymes.     

Inhibition of mycoplasma cell division by cytochalasin B

Mycoplasma gallisepticum has subcellular organelles which may function as a primitive "mitotic-like" apparatus. To investigate these further, we have studied the effects of cytochalasin B (CB) on M. gallisepticum. We found that CB inhibits cell division; this is the only procaryote thus far reported to be inhibited by CB. CB does not inhibit glucose or macromolecule precursor uptake. It stops cellular DNA synthesis, however, although RNA and protein synthesis continue (at a reduced rate). CB removal results in a resumption of DNA synthesis, followed by cell division. There appears to be some degree of cell synchrony in this first division after CB removal. These results, together with morphological data, indicate that CB blocks at two points in the cell cycle: at the time "mitotic-like" structures are formed and at the time of cell division. It is suggested that the CB blocks may result from a disruption of actin-like protein structures required at these points in the cell cycle.     

Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells

Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.     

Binding of cytochalasin B to platelets

The binding of cytochalasin B (CB) to human platelets and to isolated platelet cytosol and membranes has been analyzed with [3H]CB. High- and low affinity classes of saturable binding sites were associated with intact platelets. Binding at very low concentrations of CB (i.e., high-affinity binding) was partially prevented by 100 mM D-galactose or D-glucose and to a much lesser extent by L-glucose. Binding to platelet cytosol also involved two classes of sites with affinities and capacities similar to those observed with the whole cells. None of this binding, however, was affected by 100 mM D-galactose. Saturable binding to platelet membranes occurred at sites with a uniform binding affinity. Approximately 52% of this binding was prevented by 1 M D-galactose and another 15% by cytochalasin E (CE). We hypothesize that binding in the cytosol is to monomeric (low-affinity) and polymerized (high-affinity) actin, whereas membrane binding (high-affinity only) occurs primarily at sites involved with galactose transport.     

Characterization of a cytochalasin D-resistant mutant of Entamoeba histolytica

Characterization of a cytochalasin D-resistant mutant of the human parasite Entamoeba histolytica capable of growing at 10 microM cytochalasin is described. The mutant cells also show resistance to 5 mM colchicine and 100 microM cytochalasin B, drugs proved deleterious for wild type trophozoites. The mutants show increased osmotic fragility and electric mobility but reduced phagocytic activity, and agglutination by Concanavalin A. On the other hand pinocytic activity remains unaltered when compared with the wild type cells. Polymerized actin, seen by staining with phalloidin, often appears polarized to one end of the trophozoites and forms few of the endocytic invaginations found in wild type amebas. An altered distribution of part of the actin could explain the differences in surface properties and motility observed in the mutant amebas.     

Chronic B cell deficiency from birth prevents age-related alterations in the B lineage

Aging is accompanied by a decline in B lymphopoiesis in the bone marrow and accumulation of long-lived B cells in the periphery. The mechanisms underlying these changes are unclear. To explore whether aging in the B lineage is subjected to homeostatic regulation, we used mutant mice bearing chronic B cell deficiency from birth. We show that chronic B cell deficiency from birth, resulting from impaired maturation (CD19(-/-) and CD74(-/-)) or reduced survival (baff-r(-/-)), prevents age-related changes in the B lineage. Thus, frequencies of early and late hematopoietic stem cells, B lymphopoiesis, and the rate of B cell production do not substantially change with age in these mice, as opposed to wild-type mice where kinetic experiments indicate that the output from the bone marrow is impaired. Further, we found that long-lived B cells did not accumulate and peripheral repertoire was not altered with age in these mice. Collectively, our results suggest that aging in the B lineage is not autonomously progressing but subjected to homeostatic regulation.     

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallel-aligned intermediate filaments. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41 degrees C). At the permissive temperature for transformation (36 degrees C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41 degrees C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.     

Genetic characterization of human KB cell lines resistant to epidermal growth factor: Pseudomonas exotoxin conjugates

Pleiotropic human KB cell mutants, selected for resistance to a conjugate of epidermal growth factor with Pseudomonas exotoxin (PE-EGF), were characterized genetically. These mutants have a pleiotropic phenotype, which includes reduced number of EGF receptors and reduced growth rate. Hybrid cells between HeLa D98 and four out of five of these resistant cell lines were more resistant to PE-EGF than hybrids formed between HeLa D98 and parental KB cells. This result indicates that the phenotype of PE-EGF resistance is incompletely dominant in four out of five cases and recessive in one out of five variants. In three separate experiments, transfection of DNA from two of the dominant resistant cell lines resulted in transformation of wild-type KB cells to PE-EGF resistance, confirming the dominant nature of these mutations, which affect levels of EGF receptor in KB cells.     

Chinese hamster cell mutant resistant to ML236B (Compactin) is defective in endocytosis of low-density lipo-protein.

A fungal metabolite, ML236B (Compactin), isolated from Penicillium citrinum, is a specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (EC 1.1.1.34). Three ML236B-resistant (ML236Br) mutants, MF-1, MF-2, and MF-3, were isolated from V79 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The fluctuation test showed 2.2 X 10(-6) mutants per cell per generation of a spontaneous mutation frequency of ML236Br clones. These ML236Br clones showed a four- to fivefold-higher resistance to the drug than did their parental V79. Radioactive acetate, but not mevalonate, incorporation into the sterol fraction increased about 10-fold in ML236Br clones in comparison with that in V79. The cellular level of HMG-coenzyme A reductase in three ML236Br mutants was found to be a few-fold higher than that of V79 when cultured in the presence of lipoproteins. The 125I-labeled low-density lipoprotein-binding assay showed binding activity in three ML236Br clones comparable to that of the parental V79 cells. By contrast, an internalization assay of 125I-labeled low-density lipoprotein into the cells showed significantly reduced activity in three ML236Br clones in comparison with V79.     

Proline accumulation in a barley mutant resistant to trans-4-hydroxy-L-proline

Five proline analogues were tested for inhibition of the growth of mature barley (Hordeum vulgare L.) embryos in sterile culture. Inhibition by all analogues was relieved by proline. Inhibition by trans-4-hydroxy-L-proline was relieved by low amounts of proline. Twenty thousand mature embryos were dissected from M₂ seeds after sodium azide mutagenesis. Four plants (Rothamsted 5201, 6102, 6901, 6902) were selected with good growth on 4 mM trans-4-hydroxyproline. Properties of mutant R5201 were studied in detail. Selfed progeny of R5201 were all resistant to trans-4-hydroxyproline and also to L-thiazolidine-4-carboxylic acid and trans-3-hydroxy-L-proline but not L-azetidine-2-carboxylic acid. The content of soluble proline in progeny of R5201 was higher in leaves by a factor of up to six-fold. Proline content was measured in the soluble fraction of the terminal 20 mm of 4 d old plants subjected to severe water stress in 40% w/v polyethylene glycol. Leaves of the mutant contained more proline initially and accumulated proline morer rapidly than the parental leaves. As mutant leaves were larger and lost water more rapidly the greater increase in proline may have been caused by more severe water stress. Resistance to trans-4-hydroxyproline in R5201 was due to a single partially dominant nuclear gene.Abbreviations AZC L-azetidine-2-carboxylic acid - HYP trans-4-hydroxy-L-proline - ORN L-ornithine - CIT L-citrulline     

Mutant cell lines resistant to azetidine-2-carboxylic acid: alterations in the synthesis of proline from glutamic acid

Two mutant Chinese hamster lung fibroblast lines have been isolated that are resistant to the toxic proline analog L-azetidine-2-carboxylic acid. The line designated AZCA-1 has 30-fold elevated activity of pyrroline-5-carboxylate synthase and a large increase in the rate of proline production and release compared to controls. Pyrroline-5-carboxylate synthase activity is not elevated in the resistant line designated AZCA-4, but the enzyme is less sensitive to inhibition by ornithine and proline than control enzyme. Intracellular proline is elevated in AZCA-4 cells, with no change in the rate of release of proline synthesized from glutamate. Resistance to azetidine carboxylic acid in both mutant lines is attributed to the expanded intracellular proline pool that results from alterations in pyrroline-5-carboxylate synthase. These results indicate that intracellular proline levels are determined at least in part by the regulated activity of pyrroline-5-carboxylate synthase.