Surface Of CHO Cells Research Articles

Surface presentation of LPS is sufficient for initiation of signaling events

The activation of phagocytic leukocytes by LPS has been implicated in the pathogenesis of Gram-negative sepsis. The identification of CD14 as a mediator of LPS-inducible signal transduction was a crucial event in understanding the mechanism by which LPS-induced cellular activation occurs. CD14, a 55 kDa glycosyl phosphatidylinositol (GPI)-linked protein present on the surface of phagocytic leukocytes, has been shown to bind LPS and to mediate cellular activation. While it is generally agreed that the interaction between lipid A and CD14 is central to cellular activation by LPS, details of the downstream events which follow remain obscure. The CD11/CD18 integrins are a second group of LPS receptors. The observation that CD11/CD18 could enable LPS responsiveness independent of CD14 when expressed on the surface of CHO cells suggested that the ability of CD14 to participate in the LPS signaling cascade was not unique. We hypothesized that the common function shared by these two LPS receptors was their ability to bind LPS and bring it in close proximity to the plasma membrane, where it could then interact with a signal transducer. To further investigate the role of LPS binding in cellular signaling, we decided to exploit the activity of two other proteins known to bind LPS: lipopolysaccharide binding protein (LBP) and bactericidal/permeability-increasing protein (BPI). We found that when these normally soluble proteins were expressed in a CHO-K1 fibroblast as a GPI-anchored receptor, both could substitute for CD14 in initiating the nuclear translocation of NF-κB in response to LPS stimulation. Pharmacological studies with an LPS antagonist demonstrated that these artificial LPS receptors had characteristics that were qualitatively identical to CD14. These data support the hypothesis that a receptor distinct from CD14 functions as the actual signal transducer, and suggests that surface binding of LPS to the cell membrane is the crucial first step in initiation of the downstream signaling events. We conclude that any LPS binding protein that can focus LPS on the cell surface is sufficient to activate cells.

Characterization of membrane translocation by anthrax protective antigen.

Solving the crystallographic structure of the ring-shaped heptamer formed by protective antigen (PA), the B moiety of anthrax toxin, has focused attention on understanding how this oligomer mediates membrane translocation of the toxin's A moieties. We have developed an assay for translocation in which radiolabeled ligands are bound to proteolytically activated PA (PA63) at the surface of CHO or L6 cells, and translocation across the plasma membrane is induced by lowering the pH. The cells are then treated with Pronase E to degrade residual surface-bound material, and protected ligands are quantified after fractionation by SDS-PAGE. Translocation was most efficient (35%-50%) with LFN, the N-terminal PA binding domain of the anthrax lethal factor (LF). Intact LF, edema factor (EF), or fusion proteins containing LFN fused to certain heterologous proteins [the diphtheria toxin A chain (DTA) or dihydrofolate reductase (DHFR)] were less efficiently translocated (15%-20%); and LFN fusions to several other proteins were not translocated at all. LFN with different N-terminal residues was found to be degraded according to the N-end rule by the proteasome, and translocation of LFN fused to a mutant form of DHFR with a low affinity for methotrexate (MTX) protected cells from the effects of MTX. Both results are consistent with a cytosolic location of protected proteins. Evidence that a protein must unfold to be translocated was obtained in experiments showing that (i) translocation of LFNDTA was blocked by introduction of an artificial disulfide into the DTA moiety, and (ii) translocation of LFNDHFR and LFNDTA was blocked by their ligands (MTX and adenine, respectively). These results demonstrate that the acid-induced translocation by anthrax toxin closely resembles that of diphtheria toxin, despite the fact that these two toxins are unrelated and form pores by different mechanisms.

Functional role of glycosylation on the recombinant CD38/ADP-ribosyl cyclase in CHO cells

Current evidence suggests that CD38, a lymphocyte differentiation antigen, has been found to be functionally indistinguishable from the native form when it was expressed as a soluble and non-glycosylated protein in yeast. Studies were conducted to evaluate the functional role of glycosylation on the membrane-bound CD38 in mammalian cells. The stable transformants of CHO cells were established with pXJ41–CD38 construct and the recombinant CD38 was detected at the surface of CHO cells using a polyclonal antibody to rat CD38 by immunocytochemistry. The recombinant protein displaying ADP-ribosyl cyclase activity was purified from CHO cells, which appeared as 42 and 46 kDa bands on immunoblot under non-reducing and reducing conditions, respectively. The recombinant CD38 was then subjected to deglycosylation with N-glycosidase F that resulted in a 33 kDa band on immunoblot under reducing condition. Further the partial deglycosylation of the recombinant CD38, performed at various time intervals, resulted in a series of bands (33–46 kDa) on immunoblot. Kinetic analysis indicated that deglycosylation of the recombinant CD38 showed a considerable decrease in V max and an increase in K m of ADP-ribosyl cyclase activity. These observations clearly suggest that glycosylation plays an important role to maintain the enzymatic activity and substrate affinity of CD38/ADP-ribosyl cyclase for mediating the signalling events in mammalian cells.

Cellular distribution of a GPI-anchored complement regulatory protein CD59: homodimerization on the surface of HeLa and CD59-transfected CHO cells.

Human glycosyl phosphatidylinositol-anchored protein CD59 was solubilized in detergent-insoluble complexes (DICs) and in post-nuclear pellets by a two-step solubilization procedure using Triton X-100 and octylglucoside. CD59 molecules are recovered in both fractions, the amount being greater in the latter fraction in all cell types tested. Specific labeling of surface CD59 molecules revealed that the CD59 detected in DICs originated from intracellular compartments, whereas that in post-nuclear pellets was in part derived from the cell surface. Cross-linking of surface proteins with chemical cross-linker followed by Western blotting with anti-CD59 antibody revealed cross-linked products with molecular masses of 28-36 kDa on HeLa and human CD59 cDNA-transfected CHO cells; the CD59-associating molecules were estimated to be 13-18 kDa in size. The cross-linked products were extracted in the post nuclear pellets, and CD59 existed mainly as a cross-linked form on the cell surface. Two-dimensional electrophoresis of the cross-linked products revealed no trace of molecules other than CD59. The cross-linked products showed the same N-terminal sequences as CD59 and a strikingly similar amino acid composition to that of CD59. Thus, most likely, the cross-linked products are CD59 dimers. The finding that CD59 localized on outer membranes is all in the form of dimers suggests the importance of dimerization for CD59 functioning.

Thyrotropin Receptor Cleavage at Site 1 Does Not Involve a Specific Amino Acid Motif but Instead Depends on the Presence of the Unique, 50 Amino Acid Insertion

Thyrotropin (TSH) receptor (TSHR) A and B subunits are formed by intramolecular cleavage of the single chain receptor at two separate sites. The region involved in cleavage at Site 2 has been identified, but previous mutagenesis studies failed to identify Site 1. We now report fortuitous observations on the effect of trypsin on the TSHR that localizes a small region harboring Site 1. Thus, as detected by immunoblotting and by 125I-TSH cross-linking to TSHR expressed on the surface of intact CHO cells, trypsin clipped a small polypeptide fragment bearing a glycan moiety from the C terminus of the A subunit. Based on the TSHR primary structure, this small fragment (1-2 kDa) contains Asn-302. This information, together with estimation of the size of the deglycosylated A subunit relative to a series of C-terminal truncated TSHR ectodomain variants, places cleavage Site 1 in the vicinity of, or closely upstream to, residue 317. Remarkably, mutagenesis of every amino acid residue between residues 298-316 (present study) and 317-362 (previous data) did not prevent cleavage at Site 1. However, cleavage at this site was abrogated by deletion of a 50-amino acid segment (residues 317-366) unique to the TSHR in the glycoprotein hormone receptor family. In summary, these data provide novel insight into TSHR intramolecular cleavage. Cleavage at Site 1 does not depend on a specific amino acid motif and differs from cleavage at Site 2 by involvement of a mechanism requiring the presence of the enigmatic TSHR 50-amino acid "insertion."

Myelin-associated glycoprotein interacts with neurons via a sialic acid binding site at ARG118 and a distinct neurite inhibition site.

Inhibitory components in myelin are largely responsible for the lack of regeneration in the mammalian CNS. Myelin-associated glycoprotein (MAG), a sialic acid binding protein and a component of myelin, is a potent inhibitor of neurite outgrowth from a variety of neurons both in vitro and in vivo. Here, we show that MAG's sialic acid binding site is distinct from its neurite inhibitory activity. Alone, sialic acid-dependent binding of MAG to neurons is insufficient to effect inhibition of axonal growth. Thus, while soluble MAG-Fc (MAG extracellular domain fused to Fc), a truncated form of MAG-Fc missing Ig-domains 4 and 5, MAG(d1-3)-Fc, and another sialic acid binding protein, sialoadhesin, each bind to neurons in a sialic acid- dependent manner, only full-length MAG-Fc inhibits neurite outgrowth. These results suggest that a second site must exist on MAG which elicits this response. Consistent with this model, mutation of arginine 118 (R118) in MAG to either alanine or aspartate abolishes its sialic acid-dependent binding. However, when expressed at the surface of either CHO or Schwann cells, R118-mutated MAG retains the ability to inhibit axonal outgrowth. Hence, MAG has two recognition sites for neurons, the sialic acid binding site at R118 and a distinct inhibition site which is absent from the first three Ig domains.

Insight into Screening Immunoglobulin Gene Combinatorial Libraries in a Phage Display Vector: A Tale of Two Antibodies

Combinatorial libraries of immunoglobulin genes in “phage display” vectors are a powerful tool for obtaining antigen-specific antibody fragments. To date, this approach has been used to isolate abundant, but not rare, human autoantibodies of IgC class. We have compared the relative efficiencies of panning pComb3 libraries made from intrathyroidal plasma cells for abundant human autoantibodies to thyroid peroxidase (TPO) and rare autoantibodies to the thyrotropin receptor (TSHR). TPO-specific Fab were readily obtained from a library using three different forms of recombinant antigen, (i) purified TPO, (ii) impure TPO in culture medium and, (iii) TPO expressed on the surface of CHO cells. In contrast, TSHR-specific Fab were not isolated. This was the case despite repeated pannings of six libraries from three optimal patients (IgG/kappa and IgG/lambda libraries for each patient). Both purified recombinant TSHR and CHO cells expressing TSHR on their surface were used. Library enrichment was observed on some screenings. However, Fab expressed by individual clones or from enriched libraries were not specific as determined by (i) binding to purified, radio-labeled antigen, (ii) FACS analysis of TSHR on intact CHO cells and, (iii) inhibition of radiolabeled TSH binding. Remarkably, in screening for both TPO- and TSHR-specific Fab, neither library enrichment nor the retention of cDNA inserts of the correct size correlated with obtaining Fab with the antigenic specificity sought. Indeed, excellent enrichment could be observed with conditioned medium from untransfected cells. Our data suggest that the key to isolating rare antibodies from phage display libraries is not the creation of vast libraries of greater diversity or even the development of more stable vectors. Rather, success in this endeavor appears to require reducing the “noise” of non-specific clones in a moderately sized library.

Transport of sphingomyelin to the cell surface is inhibited by brefeldin A and in mitosis, where C6-NBD-sphingomyelin is translocated across the plasma membrane by a multidrug transporter activity.

Sphingomyelin is a major lipid of the mammalian cell surface. The view that sphingomyelin, after synthesis in the Golgi lumen, reaches the outer leaflet of the plasma membrane on the inside of carrier vesicles has been challenged by inconsistencies in the results of transport studies. To investigate whether an alternative pathway to the cell surface exists for sphingomyelin, brefeldin A and mitotic cells were used to block vesicular traffic between the Golgi complex and the plasma membrane. Exogenous sphingomyelinase was applied in the cold to assay for the presence of sphingomyelin on the surface of CHO cells. Newly synthesized radiolabeled sphingomyelin was found to equilibrate with cell surface sphingomyelin within 1.5 hours at 37 degrees C. Brefeldin A and mitosis inhibited this transport but, surprisingly, not the surface appearance of the short-chain sphingomyelin analog N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl(C6-NBD)-sphingo myelin as assayed by depletion of this lipid in the medium by the scavenger albumin. Transport of C6-NBD-sphingomyelin in the presence of brefeldin A was blocked by cyclosporin A and PSC 833, inhibitors of the multidrug resistance P-glycoprotein. The same was observed in HepG2 and HeLa cells, and for short-chain glucosylceramide, which demonstrates the general nature of the transporter-dependent sphingolipid translocation across the plasma membrane.

A deletion in the alpha subunit locks platelet integrin alpha IIb beta 3 into a high affinity state.

The integrin alpha IIb beta 3 (GPIIb/IIIa) mediates platelet aggregation by a change in affinity for the ligand fibrinogen. The amino acids 991-995 (GFFKR) at the NH2-terminus of the cytoplasmic domain are highly conserved in all known integrin alpha subunits. We postulated that the GFFKR-region is important for the inside-out signal transduction and has an influence on the affinity state of integrins. To test this hypothesis, a mutant with a deletion in the GFFKR region was designed. The DNA-constructs were constructed by PCR, sequenced, cotransfected with the beta 3 subunit into CHO cells and cell surface expression was proven with immunoprecipitation and flow cytometry. The GFFKR-deletion mutant demonstrated a high affinity binding of the mAb PAC-1 and I125-labeled fibrinogen. The metabolic inhibitors 2-deoxyglucose and NaN3 did not change the affinity state of the deleted receptor. Neither did the truncation of the cytoplasmic domain of the beta 3 subunit. Additionally, expression of the deleted integrin in the erythropoetic cell line K562 revealed a high affinity state. A deletion of the GFFKR-region in the cytoplasmic domain of the alpha subunit locks integrin alpha IIb beta 3 in a high affinity state. This is an intrinsic property of the deleted receptor since there is no energy dependence and no cell type specifity. Thus, the GFFKR-region is involved in inside-out signaling in alpha IIb beta 3. Furthermore, cell lines expressing this activated alpha IIb beta 3 integrin may be used as models for activated platelets.

Temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1.

We investigated cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 strain IIIB expressed on the surface of CHO cells. These cells formed syncytia when incubated together with CD4-positive human lymphoblastoid SupT1 cells or HeLa-CD4 cells but not when incubated with CD4-negative cell lines. A new assay for binding and fusion was developed by using fluorescent phospholipid analogs that were produced in SupT1 cells by metabolic incorporation of BODIPY-labeled fatty acids. Fusion occurred as early as 10 min after mixing of labeled SupT1 cells with unlabeled CHO-gp160 cells at 37 degrees C. When both the fluorescence assay and formation of syncytia were used, fusion of SupT1 and HeLa-CD4 cells with CHO-gp160 cells was observed only at temperatures above 25 degrees C, confirming recent observations (Y.-K. Fu, T.K. Hart, Z.L. Jonak, and P.J. Bugelski, J. Virol. 67:3818-3825, 1993). This temperature dependence was not observed with influenza virus-induced cell-cell fusion, which was quantitatively similar at both 20 and 37 degrees C, indicating that cell-cell fusion in general is not temperature dependent in this range. gp120-CD4-specific cell-cell binding was found over the entire 0 to 37 degrees C range but increased markedly above 25 degrees C. The enhanced binding and fusion were reduced by cytochalasins B and D. Binding of soluble gp120 to CD4-expressing cells was equivalent at 37 and 16 degrees C. Together, these data indicate that during gp120-gp41-induced syncytium formation, initial cell-cell binding is followed by a cytoskeleton-dependent increase in the number of gp120-CD4 complexes, leading to an increase in the avidity of cell-cell binding. The increased number of gp120-CD4 complexes is required for fusion, which suggests that the formation of a fusion complex consisting of multiple CD4 and gp120-gp41 molecules is a step in the fusion mechanism.

Immunochemical and molecular analysis of antigen binding to lipid anchored and soluble forms of an mhc independent human α/β T cell receptor

We have constructed antigen-specific chimeric human T cell receptor (TCR) molecules deleted of the transmembrane domain and containing the signal sequence for the biosynthesis of the phosphatidyl inositol glycan (GPI) linkage. These membrane-anchored forms of the TCR α and β chains have been expressed in non-T cells, and they are recognized by α or β TCR specific monoclonal antibodies. We have utilized both immunochemical methods and flow cytometry to prove that the enzyme phosphatidylinositol phospholipase C (PI/PLC) is able to cleave the GPI anchored TCR as a heterodimer from the CHO cell surface. We have demonstrated that the α/β TCR heterodimer on the surface of CHO cells will recognize and bind polymers containing fluorescein (FL-polymer), and the binding activity is completely eliminated by the enzyme, PI/PLC. Moreover, soluble forms of the α/β heterodimer will bind tightly to FL substituted sepharose, which demonstrates the retention of biological activity by the TCR after solubilization. Molecular modelling of the putative antigen binding site of the α FLβ FL TCR was derived from the known atomic coordinates of eight different hapten or peptide specific antibodies. Mutagenesis of several residues predicted from the model to be important in FL binding gave results consistent with involvement of Ig equivalent CDR2 and CDR3 domains in the antigen binding pocket. Therefore, using a model hapten system in studying recognition of the TCR independent of MHC interactions, we conclude that amino acid residues located in similar positions within CDR domains as compared to the case of MHC restricted TCR recognition are used in the binding of either hapten or peptide antigens.

Homotypic and heterotypic Ca ++-independent cell adhesion activities of biliary glycoprotein, a member of carcinoembryonic antigen family, expressed on CHO cell surface

Homotypic and heterotypic cell adhesion activities of a carcinoembryonic antigen (CEA) family member, biliary glycoprotein a (BGPa), have been examined. CHO cells transfected with the cDNA for BGPa, CEA, non-specific cross-reacting antigen (NCA) and CGM6 have been used. The BGPa producers showed both homotypic and heterotypic adhesion to CEA and NCA producers. However, they hardly adhered to CGM6 producers. Calcium ion was not required for BGPa-mediated homotypic and heterotypic cell adhesion as well as for the adhesions of other members of CEA family. The results strongly suggested that BGPa may play some important roles through Ca ++-independent cell adhesion activities.

Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells.

Bone is a dynamic tissue that responds to many factors including vitamin D, parathyroid hormone, estrogen, calcitonin, and bone morphogenetic proteins (BMPs). The ability to stimulate new bone growth would permit novel therapies for situations where bone mass has been lost due to accident or disease. Purified BMP-2, in conjunction with a suitable matrix, is sufficient to stimulate the synthesis of new bone (Wang et al., 1990). We have expressed recombinant human BMP-2 at high levels in Chinese hamster ovary cells using methotrexate-mediated gene amplification. Several forms of BMP-2 are secreted from CHO cells: (1) an amino-terminal propeptide of 40-45 kDa, (23) a mature active 30 kDa homodimer consisting of 18-22 kDa subunits, and (3) a small amount of uncleaved 60 kDa precursor protein. The mature, active protein is predominantly a 30 kDa homodimer consisting of subspecies of 18 and 22 kDa which differ by proteolytic processing at their amino termini. Mature BMP-2 and propeptide contain high mannose and complex N-linked oligosaccharides, respectively. The molar amount of secreted, processed propeptide is approximately 5-fold higher than mature BMP-2 in conditioned medium. BMP-2 associates with both the extracellular matrix and the surface of CHO cells, which may in part account for the unequal levels of extracellular propeptide and mature forms of the molecule in the conditioned medium. Recombinant BMP-2 can be expressed in sufficient quantities to assess its therapeutic potential for bone regeneration.

An approach to imaging of living cell surface topography by scanning tunneling microscopy

Since a scanning tunneling microscope (STM) was developed, an observation of living cell surface has been one of the final aims in the biological application of STM. By developing a new style of STM which was combined with an optical microscope and with a novel system for the centering of both images, we successfully got the STM images of living cell surface of T24 cells (human bladder cancer cell line) and CHO cells (Chinese hamster ovary fibroblast) cultured on highly oriented pyrolytic graphite under the appropriate condition (V > 8.0 V, I < 0.2 nA). Unexpectedly, the living T24 cell showed a slightly uneven surface with a steep foot slope. The CHO cell showed more rough surface with steeper slope of cell foot. Although the STM system had a fine spatial resolution less than 3 nm, the profile of living cell surface covered with electrolyte was clear only when the scanning area was more than 10 μm square.

Binding of polycation DEAE-dextran to chinese hamster ovary cells induces reversible Ca 2+ influx and inhibits capping of concanavalin A acceptor proteins

Binding of the polycation DEAE-dextran to the cell surface of HA-1 CHO cells caused a marked increase in 45Ca 2+ exchange influx. The effect was fairly selective for Ca 2+, undirectional (efflux was not increased) and was rapidly reversed by treatment with polyanion dextran sulfate. 45Ca 2+ influx could not be stimulated by treatment with multivalent lectins or fibronectin. In addition to stimulating 45Ca 2+ flux, DEAE-dextran inhibited the capping of concanavalin-A acceptor proteins. Inhibition of capping occurred over the same DEAE-dextran concentration range (20–200 μg/ml) which stimulated 45Ca 2+ uptake, possibly implicating increased cellular [Ca 2+] in the inhibition of concanavalin A acceptor protein capping in this cell type. The profound effect of DEAE-dextran on cellular Ca 2+ uptake and the rapid reversal of the effect by dextran sulfate might make the polycation a useful agent for the induction of transient increases in cellular [Ca 2+].

The polycation deae dextran binds to the CHO cell surface causing marked modulation of treatment response and surface properties

The polycation deae dextran binds to the CHO cell surface causing marked modulation of treatment response and surface properties

Interactions of lectins with CHO cell surface membranes. II. Differential effects of local anesthetics on endocytosis of Con A and WGA binding sites.

Using fibroblastic CHO cells, we have examined 1) the internalization and redistribution of surface binding sites for the lectins Concanavalin A and wheat germ agglutinin and 2) the sensitivity of these processes to putative inhibitors of cytoskeletal activity. Following initial exposure to fluorescein conjugated Con A (CAF) or WGA (WGAF) at 4 degrees C, kinetic analysis of internalization and intracellular aggregation of lectin at 37 degrees C indicated more rapid aggregate formation in the case of WGA than in the case of Con A. Treatment with tertiary amine local anesthetics (tetracaine, dibucaine, and xylocaine) or with a lysosomatrophic amine, m-dansyl cadaverine, blocked internalization of Con A but not of WGA. Similar differential effects on redistribution of Con A and WGA were not however observed with the antimicrotubule agents colchicine and nocodazole. Simultaneous treatment of cells with WGAF and with rhodamine labeled Con A (CAR) resulted in the accumulation of both labels in a central perinuclear aggregate; a similar experiment in the presence of local anesthetic resulted in a diffuse peripheral distribution of CAR and a central aggregate of WGAF. These results suggest 1) CHO cells possess at least two distinct pathways for lectin internalization and redistribution, which can be discriminated in terms of drug sensitivity; 2) CHO cells can sort out and independently internalize different populations of lectin binding sites; and 3) different pathways may be a manifestation of biochemically distinct linkages between cytoskeletal elements and various groups of surface glycoproteins. Present findings concur with our previous results concerning the mutual independence of the surface binding sites for Con A and WGA (Emerson and Juliano, 1982).

Interactions of lectins with CHO cell surface membranes. I. Competition studies indicate concanavalin a and WGA bind to discrete populations of sites.

The binding of radioiodinated lectins to the CHO cell surface was measured for the following affinity purified plant agglutinins; concanavalin A, wheat germ agglutinin, Ricinus agglutinins (I, II) pea agglutinin, peanut agglutinin, and Bandeiria simpliciafolia agglutinin (BSLI). The number of binding sites at saturation ranged from 6 x 10(5) for BSL I to 5 x 10(7) for pea and Ricinus. Affinity constants calculated by the Steck-Wallach procedure ranged form 2 x 10(5) m(-1) for pea lectin to 4.5 x 10(5) M(-1) L for peanut lectin. Competition studies between homologous and heterologous radiolabeled and unlabeled lectins indicated that homologous unlabeled lectin could fully block binding of radiolabeled lectin. For heterologous pairs, a variety of results were observed. Of particular interest is the finding that concanavalin A and wheat germ agglutinin mutually failed to compete for binding indicating that these two lectins bind to distinct, nonoverlapping populations of surface sites. This finding suggests that the binding sites for WGA and Con A reside, for the most part, on discrete populations of membrane molecules; this concept is further validated in a companion paper in the upcoming issue of this journal (Schwartz et al., 1982).

Chinese hamster ovary chromosomes and antigens in tobacco/hamster heterokaryons.

Tobacco GGLL/hamster CHO hamster heterokaryon frequency following cell fusion has been raised to 4-10 percent with a modified PEG procedure. In heterokaryons, GGLL nuclei become activated, adhere to and appear to fuse with CHO nucleo. Metaphase CHO chromosomes can be identified 24 hours after fusion and immunofluorescent CHO cell surface antigens diffuse into the plant cell membrane. Diffusion coefficients, D, are estimated to be between 0.6 X 10(-11) and 1.6 X 10(-11) cm2sec-1.

The adhension of Chinese hamster cells. I. Effects of temperature, metabolic inhibitors and proteolytic dissection of cell surface macromolecules.

AbstractWe have investigated factors controlling the rate of adhesion of suspension culture CHO cells to serum coated glass surfaces. Thus, we have examined (1) the effect of metabolic depletion via KCN treatment; (2) effects of colchicine and cytochalasin‐B, two drugs which are presumptive inhibitors of microtubule and microfilament activities, respectively; (3) effects of temperature on adhesion; (4) the correlation between changes in cell adhesion rate and alterations of lacteroperoxidase labelled cell surface proteins consequent to treatment with proteases.We have found that colchicine is completely without effect on the initial adhesion process in CHO cells, indicating that microtubules are not a determinant of adhesion kinetics. Cytochalasin‐B and depletion of ATP stores by KCN both diminish CHO cell adhesion, but seem to act in different ways. Treatment with cytochalasin‐B results in a dose dependent reduction in the initial slope of adhesion versus time curves, while KCN treatment produces a pronounced lag period during which little cell attachment takes place, followed by a relatively rapid rise to control levels of cell attachment.Analysis of CHO cell adhesion kinetics as a function of temperature reveals that the inhibition produced by low temperature seems similar to that produced by KCN. Thus at elevated (25°C) temperature, CHO cells adhere rapidly, while at 4°C adhesion is completely inhibited. However, at intermediate temperatures (10°C) a pronounced lag period followed by a rapid rise is noted, a result similar to that seen in KCN treated cells.Treatment of CHO cells with low doses (10 μg/ml) of crystalline trypsin results in the loss of several major cell surface proteins, without any appreciable effect on adhesion kinetics. High doses (100–1,000 μg/ml) of trypsin result in the cleavage of other proteins whose loss may be related to a parallel loss in adhesive ability. Some of the CHO cell surface proteins seem resistant to doses of trypsin (1,000 μg/ml) which completely abolish the ability of cells to adhere.Our observations on the effects of inhibitors, temperature, and surface proteolysis on CHO cell adhesion kinetics suggest the involvement of (a) microfilaments, (b) plasma membrane fluidity, and (c) a subclass of the surface proteins as important determinants of the adhesion process.