Characterization of cAMP-dependent proteolysis of GATA-6

Cyclic AMP-dependent proteolysis of GATA-6(Delta50) was characterized using inhibitors for intracellular signaling pathways. Among these kinase inhibitors, only H-89 and K252a inhibited the proteolysis induced by dbcAMP, a membrane permeable cAMP analogue, others such as PD98059, SB203580, calphostine C, PP1, and KN-93 did not do so. These results suggest that A-kinase, but not C-kinase, MEK, P38 MAP-kinases or Src kinase, could participate in the observed phenomenon. We further demonstrated that an inhibitor for ubiquitin isopeptidase (Delta12-PGJ2) inhibited the degradation of GATA-6(Delta50) in the presence of dbcAMP, suggesting that the cAMP-dependent proteolysis could be mediated through the ubiquitin-proteasome pathway, although proteasome activity did not change significantly during dbcAMP treatment. The full-length GATA-6 was also responsive to the induced degradation. Furthermore, mutation of a potential phosphorylation site (Ser-290-->Ala) for A- and C-kinases, and deletion of the PEST sequence of GATA-6 did not abolish the degradation. All these results suggest that cellular factor(s) may play a crucial role in mediating the activation of the cAMP-dependent process.     

Investigation of the bystander effect in CHO-K1 cells

AimInvestigation of the bystander effect in Chinese Hamster Ovary cells (CHO-K1) co-cultured with cells irradiated in the dose range of 0.1–4 Gy of high LET ¹²C ions and X-rays.BackgroundThe radiobiological effects of charged heavy particles on a cellular or molecular level are of fundamental importance in the field of biomedical applications, especially in hadron therapy and space radiation biology.Materials and methodsA heavy ion ¹²C beam from the Heavy Ion Laboratory of the University of Warsaw (HIL) was used to irradiate CHO-K1 cells. Cells were seeded in Petri dishes specially designed for irradiation purposes. Immediately after irradiation, cells were transferred into transwell culture insert dishes to enable co-culture of irradiated and non-irradiated cells. Cells from the membrane and well shared the medium but could not touch each other. To study bystander effects, a clonogenic survival assay was performed.ResultsThe survival fraction of cells co-cultured with cells irradiated with ¹²C ions and X-rays was not reduced.ConclusionsThe bystander effect was not observed in these studies.     

GD3 expression in CHO-K1 cells increases growth rate,induces morphological changes,and affects cell-substrate interactions

We have generated a panel of CHO-K1 cell clones with different glycolipid compositions by stable transfection of appropriate glycosyltransferases and studied the morphological and growth phenotype of a clone stably expressing Sial-T2. Compared with the GM3 expressing parental cells, Sial-T2 transfectants show low expression of GM3 and neo expression of GD3 and GT3. These cells show about 60% reduction of the mean cell area, and about 2-fold increase of the mean colony area and growth rate. Cells over expressing Sial-T2 showed a flattened appearance, and with time in culture they detached from the substrate leaving adhered material that was GD3 immunoreactive. No apoptotic or proteome differences could be detected in the Sial-T2 transfectants. Thus, increased expression of GD3 and GT3 influence parameters of growth and social behavior of CHO-K1 cells. However, the molecular and cellular basis underlying these influences requires further investigation.     

Metabolomics profiling of extracellular metabolites in CHO-K1 cells cultured in different types of growth media

An efficient mammalian cell system for producing bioproducts should retain high cell viability and efficient use of energy sources rendering the need to understand the effects of various variables on the cell system. In this study, global metabolite (metabolomics) analysis approach was used to try and understand the relationships between types of media used, culture growth behavior and productivity. CHO-KI cells producing IGF-1 were obtained from ATCC and grown in T-flask (37 °C, 5 % CO₂) until 70–80 % confluent in RPMI 1640 and Ham’s F12, respectively. Samples were taken at 8-hourly intervals for routine cell counting, biochemical responses, insulin like growth factor—1 (IGF-1) protein concentration and global metabolite analysis (gas chromatography mass spectrometry, GCMS). Conditioned media from each time point were spun down before injection into GCMS. Data from GCMS were then transferred to SIMCA-P + Version 12 for chemometric evaluation using principal component analysis. The results showed that while routine analysis gave only subtle differences between the media, global metabolite analysis was able to clearly separate the culture based on growth media with growth phases as confounding factor. Different types of media also appeared to affect IGF-1 production. Asparagine was found to be indicative of healthiness of cells and production of high IGF-1. Meanwhile identification of ornithine and lysine in death phase was found to be associated with apoptosis and oversupplied nutrient respectively. Using the biomarkers revealed in the study, several bioprocessing strategies including medium improvement and in-time downstream processing can be potentially implemented to achieve efficient CHO culture system.     

Hyperthermia-induced changes in the morphology of CHO-K1 and their refractile inclusions

1. 1.|Chinese hamster ovary cells (CHO-K1) were heated at temperatures of 42°C and above.

2. 2.|Cells were cultured in microcapillaries to eliminate handling stress, and morphological changes were observed by light microscopy.

3. 3.|Increased incidence of membrane blebbing was noted between 1 and 2 h and few cells were viable after 2 h at 43°C.

4. 4.|Morphological changes, including the appearance of potocytotic blebs, were recorded by cinemicroscopy of microcapillary cultures on a heated microscope stage.

5. 5.|Lipid-rich refractile cell inclusions changed shape before blebbing occurred.

6. 6.|Cell retraction and rearrangement of organelles seen at 1 h at 43°C are the reverse of those seen in spreading post-trypsinized cells and suggest a thermal effect on the cytoskeleton.

Protease inhibitors prevent the protein kinase A-dependent loss of Rap1 GTPase from the particulate fraction of COS1 cells

Immunoblotting with a monoclonal Rap1 antibody, we found that elevation of cyclic AMP, with forskolin and IBMX or CPT-cAMP, led to a rapid reduction in the levels of Rap1 protein associated with particulate, nuclear/perinuclear fractions from PC12 and COS1 cells. In contrast, cytoplasmic levels of Rap1 remained constant following cyclic AMP stimulation. To gain independent confirmation that cyclic AMP promoted loss of Rap1 in nuclear/perinuclear fractions we used a polyclonal Rap1 antibody, which gave similar results to the monoclonal antibody. This demonstrated that the loss in Rap1 immunoreactivity was not due to phosphorylation-dependent changes that alter immunorecognition. The reduction in Rap1 levels was blocked by PKA inhibitors and by a Rap1 serine to alanine PKA-phosphorylation site mutant (S180A). Peptide inhibitors of the proteasome, cathespin, and calpain II also inhibited the decrease in Rap1 levels, indicating that proteolytic degradation may contribute to maintaining Rap1 levels in the nuclear/perinuclear fraction of cells.     

Role of collagen tripeptide fragment GER on activation of adhesion and modification of fatty acid composition in membrane phospholipids of CHO-K1 cells

It has been found that the multiply repeated tripeptide fragment GER (Gly-Glu-Arg) from different collagen types stimulates the nonspecific adhesion of CHO-K1 cells. Activation of cell adhesion is accompanied by modifications to the fatty acid composition in the phospholipids of the cell membrane. Cell incubation with the synthetic GER peptide increases the unsaturation index of phosphatidylcholin (PC), phosphatidylethanolamine (PEA), and phosphatidylinositol (PI). Arachidonic (C20:4ω6) acid is mainly contributed to the increased index of PI. Not only arachidonic acid but other unsaturated fatty acids, such as docosatetraenoic (C22:4ω6), docosapentaenoic (C22:5ω3), and docosahexaenoic (C22:6ω3), are responsible for the increased index of PC and PEA. In addition, the elevation of the relative content of polyenoic fatty acids in PI is concomitant with a reduced amount of monoenoic fatty acids, mainly due to decline in the oleic (C18:1) acid level. The role of GER peptide in (1) the activation of cell adhesion as a regulator of active or inactive states of integrin receptors; (2) modification of fatty acid composition in major classes of phospholipids as a modulator of the fluidity in annular lipid zones surrounding to the adhesive molecules is discussed.     

Development of a fluorescence anisotropy-based assay for Dop,the first enzyme in the pupylation pathway

The Pup-proteasome system (PPS) carries out regulated tagging and degradation of proteins in bacterial species belonging to the phyla Actinobacteria and Nitrospira. In the pathogen Mycobacterium tuberculosis, where this proteolytic pathway was initially discovered, PPS enzymes are essential for full virulence and persistence in the mammalian host. As such, PPS enzymes are potential targets for development of antituberculosis therapeutics. Such development often requires sensitive and robust assays for measurements of enzymatic activities and the effect of examined inhibitors. Here, we describe the development of an in vitro activity assay for Dop, the first enzyme in the PPS. Based on fluorescence anisotropy measurements, this assay is simple, sensitive, and compatible with a high-throughput format for screening purposes. We demonstrate how this assay can also be reliably and conveniently used for detailed kinetic measurements of Dop activity. As such, this assay is of value for basic research into Dop and the PPS. Finally, we show that the assay developed here primarily for the mycobacterial Dop can be readily employed with other Dop enzymes, using the same simple protocol.     

Alterations of the glutathione-redox balance induced by metals in CHO-K1 cells

The effects of cadmium (Cd(2+)), mercury (Hg(2+)), lead (Pb(2+)), copper (Cu(2+)) and nickel (Ni(2+)) on the glutathione (GSH)-redox cycle were assessed in CHO-K1 by the neutral red uptake inhibition (NR) assay (NR(6.25), NR(12.5) and NR(25)). Mercury proved to be the most and lead the least toxic of the metals tested. The effects on GSH content and intracellular specific activities of enzymes involved in the GSH-redox balance were measured after a 24-h exposure. Total GSH content increased significantly in cultures exposed to the lowest metal concentration assayed (NR(6.25)), but fell to below control values when exposed to concentrations equivalent to NR(25). Oxidised glutathione content dropped significantly at NR(6.25), while somewhat higher values were obtained for cultures exposed to higher doses. Glutathione peroxidase (Gpx) activities were 1.2-, 1.5-, 1.6-, 2.0- and 2.5-fold higher than untreated controls for cadmium, copper, mercury, nickel and lead, respectively, at concentrations equivalent to NR(6.25). Gpx activity declined at metal concentrations equivalent to NR(12.5) and NR(25). Glutathione reductase activity remained almost unchanged except at low doses of mercury, nickel and lead. Glutathione-S-transferase activity decreased at rising metal concentrations. The results suggest that a homeostatic defence mechanism was activated when cells were exposed to doses equivalent to NR(6.25) while the ability of the cells to respond weakened as the dose increased. A close relationship was also observed between metal cytotoxicity, total GSH content and the dissociation energy of the sulphur-metal bonds. These facts confirm the involvement of antioxidant defence mechanisms in the toxic action of these ions.     

Evidence for the activation of 6-phosphofructo-1-kinase by cAMP-dependent protein kinase in Aspergillus niger

Abstract The change from pentose phosphate pathway to glycolysis plays a significant role in the physiology of Aspergillus niger during the induction of citric acid accumulation. Evidence is shown for the importance of 6-phophofructo-1-kinase in this process since it is activated by phosphorylation. By incubating a purified active form of enzyme together with commercially available alkaline phosphatase, 6-phosphofructo-1-kinase activity was lost after a certain time suggesting that the enzyme was dephosphorylated. Inactive 6-phosphofructo-1-kinase could be isolated from the cells in the early stage of growth in a high citric acid yielding medium. The enzyme was 'in vitro' activated by isolated protein kinase in the presence of cAMP, ATP and Mg²⁺ ions. Additional evidence for covalent phosphorylation of inactive 6-phosphofructo-1-kinase was obtained by incubating both enzymes together with labelled [ γ −³²P]ATP. The activating enzyme was partially purified from A. niger mycelium.     

Budding yeast Cdc6p induces re-replication in fission yeast by inhibition of SCF(Pop)-mediated proteolysis

In fission yeast, overexpression of the replication initiator protein Cdc18p induces re-replication, a phenotype characterized by continuous DNA synthesis in the absence of cell division. In contrast, overexpression of Cdc6p, the budding yeast homolog of Cdc18p, does not cause re-replication in S. cerevisiae. However, we have found that Cdc6p has the ability to induce re-replication in fission yeast. Cdc6p cannot functionally replace Cdc18p, but instead interferes with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein kinase Cdc2p. This activity of Cdc6p is entirely contained within a short N-terminal peptide, which forms a tight complex with Cdc2p and the F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF^Pop ubiquitin ligase in fission yeast. These interactions are mediated by two distinct regions within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p phosphorylation sites. The data suggest that disruption of re-replication control by overexpression of Cdc6p in fission yeast is a consequence of sequestration of Cdc2p and Pop2p, two factors involved in the negative regulation of Rum1p, Cdc18p and potentially other replication proteins. Received: 29 April 1999 / Accepted: 27 June 1999     

Rapid Swelling of a CHO-K1 Aspartate/Glutamate Transport Mutant in Hypo-osmotic Medium

Two Chinese hamster ovary cell (CHO-K1) mutants selected for defective glutamate transport via system X⁻ _AG are also highly permeable to small neutral molecules. Light microscopy demonstrated that exposure of one of these mutants, Ed-A1, to hypo-osmotic medium led to extremely rapid swelling, presumably due to increased water flux. When placed in 20% saline, Ed-A1 cells swelled to three times their original volume within 15 sec, a sixfold larger increase than parental CHO-K1. In spite of this rapid volume increase, mutant and wild-type cells remained viable for 20 min in dilute saline. A regulatory volume decrease in Ed-A1, and the continual swelling of CHO-K1, resulted in the two cells achieving equal size after 5 min in 20% saline. The time course of these volume changes permitted analysis of large numbers of cells by a hydrodynamic technique, steric field flow fractionation (FFF). Steric FFF demonstrated the expected inhibition of osmotic swelling of human erythrocytes by the mercurial, p-chloromercuribenzenesulfonic acid (PCMBS). However, PCMBS increased the apparent swelling rate of Ed-A1 and CHO-K1, suggesting that an aquaporin-like molecule is not responsible for any significant fraction of the water fluxes into either line. PCMBS also strongly inhibited aspartate transport by system X⁻ _AG. By taking advantage of their different swelling rates in hypotonic medium, steric FFF can separate mixtures of CHO-K1 and Ed-A1. Received: 2 August 1996/Revised: 25 October 1996     

The tumor suppressor LKB1 induces p21 expression in collaboration with LMO4, GATA-6, and Ldb1

The LKB1/STK11 serine/threonine kinase is mutated in Peutz-Jeghers syndrome and various sporadic cancers such as lung adenocarcinoma. We show here that LKB1 forms a complex with LMO4, GATA-6, and Ldb1, and enhances GATA-mediated transactivation in a kinase-dependent manner. We further demonstrate that LKB1 has the potential to induce p21 expression in collaboration with LMO4, GATA-6, and Ldb1 through the p53-independent mechanism. Our findings suggest that LKB1 regulates GATA-mediated gene expression and that this activity of LKB1 may be important for its tumor suppressor function.     

Localization of proteasomes and proteasomal proteolysis in the mammalian interphase cell nucleus by systematic application of immunocytochemistry

Proteasomes are ATP-driven, multisubunit proteolytic machines that degrade endogenous proteins into peptides and play a crucial role in cellular events such as the cell cycle, signal transduction, maintenance of proper protein folding and gene expression. Recent evidence indicates that the ubiquitin-proteasome system is an active component of the cell nucleus. A characteristic feature of the nucleus is its organization into distinct domains that have a unique composition of macromolecules and dynamically form as a response to the requirements of nuclear function. Here, we show by systematic application of different immunocytochemical procedures and comparison with signature proteins of nuclear domains that during interphase endogenous proteasomes are localized diffusely throughout the nucleoplasm, in speckles, in nuclear bodies, and in nucleoplasmic foci. Proteasomes do not occur in the nuclear envelope region or the nucleolus, unless nucleoplasmic invaginations expand into this nuclear body. Confirmedly, proteasomal proteolysis is detected in nucleoplasmic foci, but is absent from the nuclear envelope or nucleolus. The results underpin the idea that the ubiquitin-proteasome system is not only located, but also proteolytically active in distinct nuclear domains and thus may be directly involved in gene expression, and nuclear quality control.     

Differences in the solution structures of the parallel beta-helical pectate lyases as determined by limited proteolysis

The pectate lyase family of proteins has been shown to fold into a novel domain motif, the right-handed parallel beta-helix. As a means of gaining insight to the solution structure of the pectate lyases, the enzymes were subjected to limited proteolytic digestion by the endoproteases AspN, GluC and trypsin. The effects of proteolytic cleavage on enzymatic activity were determined, and the early products of proteolysis were identified by capillary electrophoresis, MALDI-TOF mass spectrometry and HPLC. A single peptide bond between Lys158 and Asp159 in pectate lyase B (PLb) was cleaved by both AspN and trypsin, with no detectable hydrolysis of PLb by GluC. Pectate lyase E (PLe) was hydrolyzed by trypsin between Lys164 and Asp165, a bond on an analogous loop structure found to be susceptible to proteolytic attack in PLb. AspN and GluC preferentially hydrolyzed peptide bonds (at Asp127 and Glu124, respectively) on another loop extending from the central beta-helical core of PLe. A single beta-strand of the central cylinder of the pectate lyase C (PLc) molecule was susceptible to all three proteases used. These data demonstrate that the most susceptible peptide bonds to proteolytic scission within the native enzymes lie on or near one of the three parallel beta-sheets that compose the core domain motif Despite the proximity of the proteolytic cleavages to the catalytic sites of the enzymes, significant retention of lyase activity was observed after partial proteolysis, indicating preservation of functional tertiary structure in the proteolytic products.     

Absence of Gup1p in Saccharomyces cerevisiae results in defective cell wall composition, assembly, stability and morphology

Saccharomyces cerevisiae Gup1p and its homologue Gup2p, members of the superfamily of membrane-bound O-acyl transferases, were previously associated with glycerol-mediated salt-stress recovery and glycerol symporter activity. Several other phenotypes suggested Gup1p involvement in processes connected with cell structure organization and biogenesis. The gup1Delta mutant is also thermosensitive and exhibits an altered plasma membrane lipid composition. The present work shows that the thermosensitivity is independent of glycerol production and retention. Furthermore, the mutant grows poorly on salt, ethanol and weak carboxylic acids, suggestive of a malfunctioning membrane potential. Additionally, gup1Delta is sensitive to cell wall-perturbing agents, such as Calcofluor white, Zymolyase, lyticase and sodium dodecyl sulphate and exhibits a sedimentation/aggregation phenotype. Quantitative analysis of cell wall components yielded increased contents of chitin and beta-1,3-glucans and lower amounts of mannoproteins. Consistently, scanning electron microscopy showed a strikingly rough surface morphology of the mutant cells. These results suggest that the gup1Delta is affected in cell wall assembly and stability, although the Slt2p/MAP kinase from the PKC pathway was phosphorylated during hypo-osmotic shock to a normal extent. Results emphasize the pleiotropic nature of gup1Delta, and are consistent with a role of Gulp1p in connection with several pathways for cell maintenance and construction/remodelling.     

Bioprocess development for the production of a recombinant MUC1 fusion protein expressed by CHO-K1 cells in protein-free medium

The mucin MUC1 is a candidate for use in specific immunotherapy against breast cancer, but this requires the large-scale production of a MUC1 antigen. In this study, a bioprocess for the expression of a recombinant MUC1 fusion protein with a cancer associated glycosylation in CHO-K1 cells has been developed. Cells permanently expressing parts of the extracellular portion of MUC1 fused to IgG Fc were directly transferred from adherent growth in serum-containing medium to suspension culture in the protein-free ProCHO4-CDM culture medium. Using the Cellferm-pro system, optimal culture parameter as pH and pO(2) were determined in parallel spinner flask batch cultures. A pH of 6.8-7.0 and a pO(2) of 40% of air saturation was found to give best cell growth and productivity of secreted recombinant protein. Specific productivity strongly depended the pO(2) and correlated with the online monitored oxygen uptake rate (OUR) of the cells, which indicates a positive influence of the rate of oxidative phosphorylation on productivity. The optimised conditions were applied to continuous perfusion culture which gave very high cell densities and space time yields of the recombinant MUC1 fusion protein, allowing production at gram scale. The product degradation was much lower in supernatants from continuous perfusion culture compared to batch mode. Antibodies reacting with cancer associated MUC1 glycoforms strongly bound to the fusion protein, indicating that the desired glycoforms were obtained and suggesting that the recombinant MUC1 protein could be tested for use in immunotherapy.