Wild-type Chinese Hamster Ovary Cells Research Articles

Fibroblast Growth Factor-2 Antagonist Activity and Angiostatic Capacity of Sulfated Escherichia coli K5 Polysaccharide Derivatives

The angiogenic basic fibroblast growth factor (FGF2) interacts with tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Here, we report the FGF2 antagonist and antiangiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in N- and/or O-position after N-deacetylation. O-Sulfated and N,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to 125I-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG.FGF2.FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wild-type CHO cells. They also inhibited 125I-FGF2 binding to FGFR1-overexpressing HSPG-bearing CHO cells and adult bovine aortic endothelial cells. K5 derivatives also inhibited FGF2-mediated cell proliferation in endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective. Also, highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected endothelial FGF2-T-MAE cells in fibrin gel and spontaneous angiogenesis in vitro on Matrigel of FGF2-T-MAE and HUVE cells. Finally, the highly N,O-sulfated K5 derivative exerted a potent antiangiogenic activity on the chick embryo chorioallantoic membrane. These data demonstrate the possibility of generating FGF2 antagonists endowed with antiangiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds.

The Effect of Precursor Structures on the Action of Glucosaminyl 3-O-Sulfotransferase-1 and the Biosynthesis of Anticoagulant Heparan Sulfate

To understand how 2-O-sulfation of uronic acid residues influences the biosynthesis of anticoagulant heparan sulfate, the cDNA encoding glucosaminyl 3-O-sulfotransferase-1 (3-OST-1) was introduced into wild-type Chinese hamster ovary cells and mutant pgsF-17 cells, which are defective in 2-O-sulfation. 3-OST-1-transduced cells gained the ability to bind to antithrombin. Structural analysis of the heparan sulfate chains showed that 3-OST-1 generates sequences containing GlcUA-GlcN(SO(3))3(SO(3)) and GlcUA-GlcN(SO(3))3(SO(3))6(SO(3)) in both wild-type and mutant cells. In addition, IdoUA-GlcN(SO(3))3(SO(3)) and IdoUA-GlcN(SO(3))3(SO(3))6(SO(3)) accumulate in the mutant chain. These disaccharides were also observed by tagging [6-(3)H]GlcN-labeled pgsF-17 heparan sulfate in vitro with [(35)S]PAPs and purified 3-OST-1. Heparan sulfate derived from the transduced mutant also had approximately 2-fold higher affinity for antithrombin than heparan sulfate derived from the transduced wild-type cells, and it inactivated factor Xa more efficiently. This study demonstrates for the first time that (i) 3-O-sulfation by 3-OST-1 can occur independently of the 2-O-sulfation of uronic acids, (ii) 2-O-sulfation usually occurs before 3-O-sulfation, (iii) 2-O-sulfation blocks the action of 3-OST-1 at glucosamine residues located to the reducing side of IdoUA units, and (iv) that alternative antithrombin-binding structures can be made in the absence of 2-O-sulfation.

Further evidence for the involvement of insulin receptor substrates in epidermal growth factor-induced activation of phosphatidylinositol 3-kinase.

In accordance with our recent results obtained with cultured rat hepatocytes [Fujioka, T. & Ui, M. (2001) Eur. J. Biochem. 268, 25-34], epidermal growth factor (EGF) gave rise to transient tyrosine phosphorylation of insulin receptor substrates (IRS-1 and IRS-2), thereby activating the bound phosphatidylinositol 3-kinase in human epidermoid carcinoma A431 cells normally abundant in EGF receptors (EGFR) and Chinese hamster ovary (CHO) cells transfected with full-length EGFR. These actions of EGF, although much smaller in magnitude than those of insulin or IGF-I in the same cells, were accompanied by tyrosine phosphorylation of EGFR rather than insulin or IGF-I receptors, never observed in wild-type CHO cells expressing no EGFR, and totally inhibited by an inhibitor of EGFR kinase, AG1478, that was without effect on insulin or IGF-I actions. Recombinant IRS-1 was phosphorylated on tyrosines upon incubation with purified EGFR from A431 cells and 32P-labeled ATP. When CHO cells were transfected with C-terminal truncated EGFR lacking three NPXY motifs responsible for direct binding to phosphotyrosine-binding domains of IRSs, no effect of EGF could be observed. We suggest that tyrosine phosphorylation of IRS-1 or IRS-2 could mediate EGFR-induced activation of phosphatidylinositol 3-kinase in mammalian cells.

Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite.

Glycosylphosphatidylinositol (GPI) anchors and glycoinositolphospholipids (GIPLs) from parasitic protozoa have been shown to exert a wide variety of effects on cells of the host innate immune system. However, the receptor(s) that are triggered by these protozoan glycolipids has not been identified. Here we present evidence that Trypanosoma cruzi-derived GPI anchors and GIPLs trigger CD25 expression on Chinese hamster ovary-K1 cells transfected with CD14 and Toll-like receptor-2 (TLR-2), but not wild-type (TLR-2-deficient) Chinese hamster ovary cells. The protozoan-derived GPI anchors and GIPLs containing alkylacylglycerol and saturated fatty acid chains or ceramide were found to be active in a concentration range of 100 nM to 1 microM. More importantly, the GPI anchors purified from T. cruzi trypomastigotes, which contain a longer glycan core and unsaturated fatty acids in the sn-2 position of the alkylacylglycerolipid component, triggered TLR-2 at subnanomolar concentrations. We performed experiments with macrophages from TLR-2 knockout and TLR-4 knockout mice, and found that TLR-2 expression appears to be essential for induction of IL-12, TNF-alpha, and NO by GPI anchors derived from T. cruzi trypomastigotes. Thus, highly purified GPI anchors from T. cruzi parasites are potent activators of TLR-2 from both mouse and human origin. The activation of TLR-2 may initiate host innate defense mechanisms and inflammatory response during protozoan infection, and may provide new strategies for immune intervention during protozoan infections.

A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins.

Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5)) and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.

The DRAG test: an assay for detection of genotoxic damage.

A high throughput assay (the DRAG test) is described, which could be a useful tool for the detection of repairable DNA adducts, and which is based on the inhibition of the growth of DNA repair-deficient Chinese hamster ovary (CHO) cells. The cytotoxicity of a test substance towards DNA repair-deficient CHO cell lines is compared with the corresponding cytotoxicity in the parental wild-type CHO cell line (AA8). A more pronounced toxicity toward a DNA repair-deficient cell line is interpreted as being the consequence of its inability to repair the DNA adduct induced by the compound. (+)-7beta,8alpha-Dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, camptothecin, ethyl methanesulphonate and mitomycin C were used as reference substances, and the overall results indicate that the DRAG test could be useful in the screening of compounds for the production of repairable DNA adducts. The main advantages with the DRAG test are that it provides a relevant endpoint, it is rapid, it requires small amounts of the test item, and it permits a large number of compounds to be tested.

Repair of sulfur mustard-induced DNA damage in mammalian cells measured by a host cell reactivation assay.

DNA damage is thought to be the initial event that causes sulfur mustard (SM) toxicity, while the ability of cells to repair this damage is thought to provide a degree of natural protection. To investigate the repair process, we have damaged plasmids containing the firefly luciferase gene with either SM or its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES). Damaged plasmids were transfected into wild-type and nucleotide excision repair (NER) deficient Chinese hamster ovary cells; these cells were also transfected with a second reporter plasmid containing RENILLA: luciferase as an internal control on the efficiency of transfection. Transfected cells were incubated at 37 degrees C for 27 h and then both firefly and RENILLA: luciferase intensities were measured on the same samples with the dual luciferase reporter assay. Bioluminescence in lysates from cells transfected with damaged plasmid, expressed as a percentage of the bioluminescence from cells transfected with undamaged plasmid, is increased by host cell repair activity. The results show that NER-competent cells have a higher reactivation capacity than NER-deficient cells for plasmids damaged by either SM or CEES. Significantly, NER-competent cells are also more resistant to the toxic effects of SM and CEES, indicating that NER is not only proficient in repairing DNA damage caused by either agent but also in decreasing their toxicity. This host cell repair assay can now be used to determine what other cellular mechanisms protect cells from mustard toxicity and under what conditions these mechanisms are most effective.

Repair bias of large loop mismatches during recombination in mammalian cells depends on loop length and structure

Repair of loop mismatches was investigated in wild-type and mismatch binding-defective Chinese hamster ovary (CHO) cells. Loop mismatches were formed in vivo during extrachromosomal recombination between heteroallelic plasmid substrates. Recombination was expected to occur primarily by single-strand annealing (SSA), yielding 12- or 26-base nonpalindromic loop mismatches, and 12-, 26-, or 40-base palindromic loop mismatches. Nonpalindromic loops were repaired efficiently and with bias toward loop loss. In contrast, the 12-base palindromic loop was repaired with bias toward loop retention, indicating that repair bias depends on loop structure. Among the palindromic loops, repair bias was dependent on loop length, with bias shifting from loop retention to loop loss with increasing loop size. For both palindromic and nonpalindromic loops, repair efficiencies and biases were independent of the general (MSH/MLH) mismatch repair pathway. These results are discussed with respect to the maintenance of large nonpalindromic insertions, and of small and large palindromes, in eukaryotic genomes.

CD36, a Member of the Class B Scavenger Receptor Family, as a Receptor for Advanced Glycation End Products

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. Five AGE receptors identified so far are RAGE (receptor for AGE), galectin-3, 80K-H, OST-48, and SRA (macrophage scavenger receptor class A types I and II). Since SRA is known to belong to the class A scavenger receptor family, and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36, although belonging to the class B scavenger receptor family, can recognize AGE proteins as ligands. This was tested at the cellular level in this study using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CD36-CHO cells). Cellular expression of CD36 was confirmed by immunoblotting and immunofluorescent microscopy using anti-CD36 antibody. Upon incubation at 37 degrees C, (125)I-AGE-bovine serum albumin (AGE-BSA) and (125)I-oxidized low density lipoprotein (LDL), an authentic ligand for CD36, were endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CD36-CHO cells, but not wild-type CHO cells. In binding experiments at 4 degrees C, (125)I-AGE-BSA exhibited specific and saturable binding to CD36-CHO cells (K(d) = 5.6 microg/ml). The endocytic uptake of (125)I-AGE-BSA by these cells was inhibited by 50% by oxidized LDL and by 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of oxidized LDL. Our results indicate that CD36 expressed by these cells mediates the endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major oxidized LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, these results suggest that, like oxidized LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

Infectivity ofChlamydia trachomatisSerovar LGV but Not E Is Dependent on Host Cell Heparan Sulfate

The ability of heparan sulfate, heparin, and other glycosaminoglycans to inhibit the infectivity of Chlamydia trachomatis serovars E and LGV was examined using a simple competitive inhibition assay with three cell types from the human female reproductive tract, including primary human endosalpingeal cells. With the majority of the glycosaminoglycans tested, LGV was more significantly inhibited than serovar E. We have compared chlamydial infectivity between a wild-type Chinese hamster ovary cell line and two glycosaminoglycan-deficient cell lines. LGV was shown to be unable to infect heparan sulfate-deficient and GAG-deficient Chinese hamster ovary cell lines, whereas the E serovar infected these cells as efficiently as the control (nondeficient) cells. These two sets of experiments confirmed that serovar LGV is more dependent on a heparan sulfate-related mechanism of infectivity than is serovar E. This is further supported by the fact that attempts to purify a heparan sulfate-like molecule from either serovar cultured in glycosaminoglycan-deficient cell lines were nonproductive. Previous reports have suggested that chlamydia are able to produce a heparan sulfate-like molecule that is important for attachment and infectivity. We have attempted to detect possible binding of a specific heparan sulfate antibody to C. trachomatis by flow cytometry. Results showed no binding of the heparan sulfate antibody to C. trachomatis serovar LGV or E. Our results strongly indicate that chlamydiae do not produce a heparan sulfate-like molecule but rather use host cell heparan sulfate in order to infect cells.

Biogenesis of Nonspecific Lipid Transfer Protein and Sterol Carrier Protein x: STUDIES USING PEROXISOME ASSEMBLY-DEFECTIVE pex CELL MUTANTS

Nonspecific lipid transfer protein (nsLTP; also called sterol carrier protein 2) with a molecular mass of 13 kDa is synthesized as a larger 15-kDa precursor (pre-nsLTP) with an N-terminal 20-amino acid extension presequence, as well as with the peroxisome targeting signal type 1 (PTS1), Ala-Lys-Leu, at the C terminus. The precursor pre-nsLTP is processed to mature nsLTP by proteolytic removal of the presequence, most likely after being imported into peroxisomes. Sterol carrier protein x (SCPx), a 59-kDa branched-chain fatty acid thiolase of peroxisomes, contains the entire pre-nsLTP moiety at the C-terminal part and is converted to the 46-kDa form and nsLTP after the transport to peroxisomes. We investigated which of these two potential topogenic sequences functions in biogenesis of nsLTP and SCPx. Morphological and biochemical analyses, making use of Chinese hamster ovary cell pex mutants such as the PTS1 receptor-impaired pex5 and PTS2 import-defective pex7, as well as green fluorescent protein chimeras, revealed that both pre-nsLTP and SCPx are imported into peroxisomes by the Pex5p-mediated PTS1 pathway. Nearly half of the pre-nsLTP remains in the cytosol, as assessed by subcellular fractionation of the wild-type Chinese hamster ovary cells. In an in vitro binding assay, only mature nsLTP, but not pre-nsLTP, from the cell lysates interacted with the Pex5p. It is likely, therefore, that modulation of the C-terminal PTS1 by the presequence gives rise to cytoplasmic localization of pre-nsLTP.

Thyroid-stimulating Hormone and Cyclic AMP Activate p38 Mitogen-activated Protein Kinase Cascade: INVOLVEMENT OF PROTEIN KINASE A, Rac1, AND REACTIVE OXYGEN SPECIES

p38 mitogen-activated protein kinases (p38-MAPKs) are activated by cytokines, cellular stresses, growth factors, and hormones. We show here that p38-MAPKs are activated upon stimulation by thyroid-stimulating hormone (TSH) or cAMP. TSH caused the phosphorylation of p38-MAPK in Chinese hamster ovary cells stably transfected with the human TSH receptor but not in wild-type Chinese hamster ovary cells. The effect of TSH was fully mimicked by the adenylyl cyclase activator, forskolin, and by a permeant analog of cAMP. The effect of forskolin was reproduced in FRTL5 rat thyroid cells. TSH also stimulated the phosphorylation of MAPK kinase 3 or 6, over the same time scale as that of p38-MAPKs. TSH and forskolin stimulated the activity of the alpha-isoform of p38-MAPK assayed by phosphorylation of the transcription factor ATF2. The activity of MAPK-activated protein kinase-2 was stimulated by TSH and forskolin. This stimulation was abolished by SB203580, a specific inhibitor of p38-MAPKs. The protein kinase A inhibitor H89 inhibited the stimulation of phosphorylation of p38-MAPKs by forskolin, whereas inhibitors of protein kinase C, p70(S6k), and phosphatidylinositol 3-kinase were ineffective. Expression of the dominant negative form of Rac1, but not that of Ras, blocked forskolin-induced p38-MAPK activation. Diphenylene iodonium, a potent inhibitor of NADPH oxidase(s), and ascorbic acid, an effective free radical scavenger, suppressed TSH- or forskolin-stimulated p38-MAPK phosphorylation, indicating that the generation of reactive oxygen species plays a key role in signaling from cAMP to p38-MAPKs. Inhibition of the p38-MAPK pathway with SB203580 partially but significantly, attenuates cAMP- and TSH-induced expression of the sodium iodide symporter in FRTL-5 cells. These results point to a new signaling pathway for the G(s)-coupled TSH receptor, involving cAMP, protein kinase A, Rac1, and reactive oxygen species and resulting in the activation of a signaling kinase cascade that includes MAPK kinase 3 or 6, p38-MAPK, and MAPK-activated protein kinase-2.

In vivo trafficking and catabolism of IgG1 antibodies with Fc associated carbohydrates of differing structure

We have now produced mouse-human chimeric IgG1 in wild-type Chinese hamster ovary (CHO) cell lines Pro-5 as well as in the glycosylation mutants Lec 2, Lec 8, and Lec 1. Analysis of the attached carbohydrates shows those present on IgG1-Lec 1 were mannose terminated. Carbohydrate present on IgG1-Lec8 was uniformly biantennary terminating in N-acetylglucosamine. The glycosylation profiles of IgG1-Lec 2 and IgG1-Pro-5 were heterogeneous. Only IgG1-Pro-5 was sialylated with sialic acid present on only a small percentage of the carbohydrate structures. When the in vivo fate of antibodies labeled with (125)I-lactotyramine was determined, it was found that the majority of all of the antibodies, irrespective of the structure of their attached carbohydrate, is catabolized in the skin and muscle. However, the attached carbohydrate structure does influence the amount that is catabolized in the liver and the liver serves as a major site for the catabolism of proteins bearing carbohydrate with the Lec2 (with terminal galactose) or Lec1(with terminal mannose) structure.

Exploring the outcome of genetic modifications of glycosylation in cultured cell lines by concurrent isolation of the major classes of vertebrate glycans.

In the preceding article (Manzi,A.E., Norgard-Sumnicht,K., Argade,S., Marth,J.D., van Halbeek,H. and Varki.A. [2000] GLYCOBIOLOGY:, 10, 669-688), we reported a comprehensive approach for the extraction, fractionation, and isolation of all of the major classes of sugar chains (glycans) from vertebrate tissues. Here we apply this "Glycan Isolation Protocol" to a variety of cultured mammalian cell lines, including two wild-type Chinese hamster ovary (CHO) cell lines and some of their genetically modified variants that were predicted or known to have defined abnormalities in the biosynthesis of one or more classes of glycans. We also use this approach to characterize clone 489, a new derivative of the GAG-deficient CHO clone pgsA-745, in which sulfation has been restored by transfection of a wild-type CHO cDNA library. By metabolically labeling the cell lines with [6-(3)H]glucosamine we were able to monitor the recovery of all major classes of glycans. The results allow us to reach several conclusions: first, the protocol described in the preceding paper is further validated by finding good recovery of total radioactivity and appropriate distribution of label in the correct glycan classes in the fractions from a variety of cell lines; second, the amount of radioactivity recovered in free glycosylphosphatidylinositol (GPI) lipids is remarkably high when compared to that found in GPI anchors, with the former being the dominant form in some cells; third, cells with known genetic mutations in specific glycosylation pathways are shown to have the expected changes in the distribution of recovered radioactivity in the appropriate fractions; fourth, the N- and O- glycans recovered via the protocol are of adequate quality to demonstrate marked differences in their structural profiles and/or content; fifth, the protocol can pick up unexpected differences of glycan classes not predicted to be affected by the primary defect; finally, the reappearance of sulfation in the novel clone 489 is not due to restoration of GAG sulfation, but rather due to the new expression of sulfation in the fraction enriched in N- and O-linked glycopeptides. These results demonstrate the power of this comprehensive approach for the concurrent exploration and profiling of the different major classes of glycans in cells.

Plasmodium falciparum: Cloned and Expressed CIDR Domains of PfEMP1 Bind to Chondroitin Sulfate A

Degen, R., Weiss, N., and Beck, H.-P. 2000. Plasmodium falciparum: Cloned and expressed CIDR domains of PfEMP1 bind to chondroitin sulfate A. Experimental Parasitology95, 113–121. Adherence of erythrocytes infected with mature asexual Plasmodium falciparum parasites (iRBC) to microvascular endothelial cells contributes to the pathology of P. falciparum malaria. It has been shown that the variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) confers adhesion to a wide range of cell surface receptors. Previously, the cysteine-rich interdomain region (CIDR) of PfEMP1 has been identified as binding site to CD36. We provide evidence that the same region can also mediate binding to chondroitin sulfate A (CSA). CIDR domains of two different parasite strains were expressed in Escherichia coli as a 6xHis-tagged protein. Purified recombinant protein bound to Chinese hamster ovary (CHO) cells which naturally express chondroitin sulfate A. Treatment of wild-type CHO cells with chondroitinase ABC reduced binding up to 94.4%. Competitive binding using soluble CSA inhibited binding to CHO cells by up to 100% at 2 mg/ml and by 62.4% at 0.5 mg/ml, whereas 1 mg/ml heparan sulfate had only a little effect (18.1%). In contrast, a recombinant 6xHis-tagged DBL1 domain showed no binding to wild-type CHO cells. Such an approach of analyzing various domains of PfEMP1 as recombinant proteins may elucidate their functions and may lead to novel anti-adherence therapeutics, especially for maternal malaria infections.

Sensitivity to nitrogen mustard relates to the ability of processing DNA damage in Chinese hamster ovary cells.

The hallmark of the excision repair pathways is the removal of DNA adducts by excision of the damaged nucleotides. In the course of repair, transient DNA strand breaks occur, which can be measured by the Comet assay. We have investigated the processing of DNA damage, mediated by nitrogen mustard, in wild-type AA8 Chinese hamster ovary cells, and in UV5, UV20 and UV41 DNA repair deficient cell lines. Whereas DNA repair could not be detected by unscheduled DNA synthesis at nitrogen mustard doses below 10 microM, processing of nitrogen mustard-mediated DNA damage was observed by the Comet assay at a 100-times lower concentration. Wild-type Chinese hamster ovary AA8 cells were able to process nitrogen mustard-mediated DNA damage within 4-24 hr depending on the dose of nitrogen mustard (0.1-10 microM). None of the repair-deficient cell lines was able to completely process the DNA damage induced by 10 microM nitrogen mustard. At nitrogen mustard doses that conferred 10% colony forming ability, the repair-deficient cells had an altered processing of nitrogen mustard-mediated DNA damage: In the AA8, UV20, and UV41 cells, the amplitude of strand breaks peaked early (within 4 hr), the level of strand breaks in the nitrogen mustard exposed UV20 and UV41 cells did not return to the baseline of the unexposed reference culture, and the peak in strand breaks in the UV5 cell line occurred after 4 hr. Our results indicate that the single cell gel electrophoresis (Comet) assay is suitable for assessing repair capability of DNA alkylations.

A unique cytosolic activity related but distinct from NQO1 catalyses metabolic activation of mitomycin C.

Mitomycin C (MMC) is a prototype bioreductive drug employed to treat a variety of cancers including head and neck cancer. Among the various enzymes, dicoumarol inhibitable cytosolic NAD(P)H:quinone oxidoreductase1 (NQO1) was shown to catalyse bioreductive activation of MMC leading to cross-linking of the DNA and cytotoxicity. However, the role of NQO1 in metabolic activation of MMC has been disputed. In this report, we present cellular and animal models to demonstrate that NQO1 may play only a minor role in metabolic activation of MMC. We further demonstrate that bioreductive activation of MMC is catalysed by a unique cytosolic activity which is related but distinct from NQO1. Chinese hamster ovary (CHO) cells were developed that permanently express higher levels of cDNA-derived NQO1. These cells showed significantly increased protection against menadione toxicity. However, they failed to demonstrate higher cytotoxicity due to exposure to MMC under oxygen (normal air) or hypoxia, as compared to the wild-type control CHO cells. Disruption of the NQO1 gene by homologous recombination generated NQO1–/– mice that do not express the NQO1 gene resulting in the loss of NQO1 protein and activity. The cytosolic fractions from liver and colon tissues of NQO1–/– mice showed similar amounts of DNA cross-linking upon exposure to MMC, as observed in NQO1+/+ mice. The unique cytosolic activity that activated MMC in cytosolic fractions of liver and colon tissues of NQO1–/– mice was designated as cytosolic MMC reductase. This activity, like NQO1, was inhibited by dicoumarol and immunologically related to NQO1. © 2000 Cancer Research Campaign

Purification of the Serine Palmitoyltransferase Complex Responsible for Sphingoid Base Synthesis by Using Affinity Peptide Chromatography Techniques

Serine palmitoyltransferase (SPT), a membrane-bound enzyme of the endoplasmic reticulum, catalyzes the condensation of palmitoyl coenzyme A (CoA) and L-serine to produce 3-ketodihydrosphingosine. This enzyme contains at least two different subunits, named the LCB1 and LCB2 proteins. In the present study, we expressed a FLAG- and His(6) peptide-tagged version of the hamster LCB1 protein in a Chinese hamster ovary cell mutant strain lacking the endogenous LCB1 subunit and purified SPT from the cells near to homogeneity by affinity peptide chromatography. The endogenous LCB2 protein was co-purified with the tagged LCB1 protein in purification of SPT. In various aspects, including optimum pH, acyl-CoA specificity, and sphingofungin sensitivity, the activity of purified SPT was consistent with the activity detected in lysates of wild-type Chinese hamster ovary cells. The optimum concentration of palmitoyl-CoA for 3-ketodihydrosphingosine formation by purified SPT was approximately 25 microM, and the apparent K(m) of L-serine was 0.28 mM. Competition analysis of the SPT reaction with various serine analogs showed that all of the amino, carboxyl, and hydroxyl groups of L-serine were responsible for the substrate recognition of the enzyme. SDS-polyacrylamide gel electrophoretic analysis of purified SPT, together with immunoprecipitation analysis of metabolically labeled LCB proteins, strongly suggested that the SPT enzyme consisted of the LCB1 and LCB2 proteins with a stoichiometry of 1:1.

Activation of extracellular signal-regulated kinases (ERK 44/42) by chlorpyrifos oxon in Chinese hamster ovary cells.

Acetylcholinesterase inhibition explains most but not all of the toxicological manifestations of exposure to the major organophosphorus insecticide chlorpyrifos (CP) and its metabolically activated form chlorpyrifos oxon (CPO); CPO is also reported to interact with muscarinic acetylcholine receptors and alter secondary messenger status. We find that CP and CPO activate extracellular signal-regulated kinases (ERK 44/42) in both wild-type (CHOK1) and human muscarinic receptor-expressing Chinese hamster ovary cells (CHO-M2). The degree of ERK 44/42 activation on treatment with 50 microM CPO for 40 minutes is 2- to 3-fold compared with control cells and is both concentration- and time-dependent. CP is at least 2-fold less potent than CPO as an activator of ERK 44/42 and the hydrolysis products 3,5,6-trichloropyridinol and diethyl phosphate are not activators. ERK 44/42 activation by CPO is insensitive to the protein kinase A inhibitor H-89, but is completely abolished by the phosphatidylinositol 3-kinase (P13-K) inhibitor wortmannin, the protein kinase C (PKC) inhibitor GF-109203X, and the mitogen-activated extracellular signal-regulated protein kinase kinase (MEK) inhibitor PD 098059. Therefore, CPO activates the ERK 44/42 signaling cascade in CHOK1 cells via a pathway dependent on P13-K, PKC, and MEK but not requiring PKA or the human M2 muscarinic receptor. In summary we find that CPO activates a mammalian signal transduction cascade involved in cell growth and differentiation. This occurs through a pathway common to growth factors and mitogens, consistent with a receptor-mediated event. However, CPO may also inhibit an enzyme involved in signal transduction. The specific target of CPO leading to the activation of ERK 44/42 and the potential effects of this activation on cell function remain to be determined.

Protective role for proteoglycans against cationic lipid cytotoxicity allowing optimal transfection efficiency in vitro

A dependence on proteoglycans for cationic lipid-mediated gene transfer has been suggested in previous studies [Mislick and Baldeschwieler (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12349-12354; Mounkes, Zhong, Cipres-Palacin, Heath and Debs (1998) J. Biol. Chem. 273, 26164-26170]. We have evaluated the mechanism of proteoglycan involvement in cationic lipid-mediated gene transfer. DNA plasmid uptake and gene expression were studied in wild-type Chinese hamster ovary (CHO) cells (CHO-K1), heparan sulphate-deficient CHO cells (pgsD-677) and proteoglycan-deficient CHO cells (pgsB-618). At an optimal ratio of cationic lipid to DNA, a substantial decrease in reporter gene expression was observed in proteoglycan-deficient cells compared with that in heparan sulphate-deficient and wild-type cells. However, there were no differences in reporter gene expression between the cell lines when transfected by electroporation. Moreover, all cell lines exhibited equal cationic-lipid-DNA complex uptake activities, as assessed by the measurement of intracellular 32P-labelled and rhodamine-labelled DNA plasmid. An analysis of reflected-light images of wild-type and proteoglycan-deficient cells suggested that cationic lipids were preferentially toxic to proteoglycan-deficient cells. Cell-growth assays confirmed this, showing that cationic lipids exhibited a greater anti-proliferative activity in proteoglycan-deficient cells and in chlorate-treated wild-type cells than in the other cell lines. The growth-inhibitory effect of cationic lipids was abrogated by the addition of exogenous sulphated glycosaminoglycans. We conclude that the glycosaminoglycan part of proteoglycans serves a protective role against cationic lipid cytotoxicity, allowing optimal transfection efficiency in vitro.