MT3 Cells Research Articles

Efficiency of Escherichia coli Jm109 and genetical engineering strains (E. coli MT2, E. coli MT3) in cadmium removal from aqueous solutions

Metallothioneins (MTs) are proteins with high metal binding affinity that exist in many organisms. Recently, this feature of MTs has been used in many areas, including environmental studies. In this study, the human MT2, and MT3 genes were cloned into Escherichia coli Jm109 to remove cadmium (Cd) from aqueous solutions. Minimum inhibition concentrations (MIC) of Cd for E. coli Jm109, E. coli MT2, and E. coli MT3 were determined as 154, 212, and 250 mg L −1 , respectively. 0.5, 5, and 25 mg L −1 Cd were applied to the strains. The maximum Cd removal was at the concentration of 0.5 mg L −1 . The optimal conditions for temperature, pH, and contact time were 37 °C, 7, and 24 h, respectively. E. coli MT3 was the most effective strain in Cd bioremoval. Scanning electron microscope (SEM) images showed that the morphological structures of the Cd treated cells were disrupted. The Energy dispersive spectroscopy (EDS) peaks of the Cd treated cells clearly showed Cd absorption into the cells. Scanning transmission electron microscope (STEM) images showed that there were clusters reflecting the accumulation of Cd in the MT3 cells. The appearance of attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) spectrum peaks of hydroxyl, carboxyl, amine, and sulfoxide groups in MT3 cells exposed to Cd confirmed that these groups bind Cd. As a result, it can be said that the MT3 gene can be used as an effective tool in Cd removal from waters. • The Cd removal capacities of genetically modified Escherichia coli were high. • E. coli MT3 was more effective in Cd removal than E. coli Jm109 and E. coli MT2. • SEM and STEM images showed that E. coli MT3 absorbs more Cd than other strains. • ATR-FTIR spectrum peaks confirmed that MT3 cells bind Cd. • MT3 can be used as an effective tool in Cd removal from water.

Metallothionein-3 modulates the amyloid β endocytosis of astrocytes through its effects on actin polymerization.

BackgroundAstrocytes may play important roles in the pathogenesis of Alzheimer’s disease (AD) by clearing extracellular amyloid beta (Aβ) through endocytosis and degradation. We recently showed that metallothionein 3 (Mt3), a zinc-binding metallothionein that is enriched in the central nervous system, contributes to actin polymerization in astrocytes. Because actin is likely involved in the endocytosis of Aβ, we investigated the possible role of Mt3 in Aβ endocytosis by cortical astrocytes in this study.ResultsTo assess the route of Aβ uptake, we exposed cultured astrocytes to fluorescently labeled Aβ1–40 or Aβ1–42 together with chloropromazine (CP) or methyl-beta-cyclodextrin (MβCD), inhibitors of clathrin- and caveolin-dependent endocytosis, respectively. CP treatment almost completely blocked Aβ1–40 and Aβ1–42 endocytosis, whereas exposure to MβCD had no significant effect. Actin disruption with cytochalasin D (CytD) or latrunculin B also completely blocked Aβ1–40 and Aβ1–42 endocytosis. Because the absence of Mt3 also results in actin disruption, we examined Aβ1–40 and Aβ1–42 uptake and expression in Mt3−/− astrocytes. Compared with wild-type (WT) cells, Mt3−/− cells exhibited markedly reduced Aβ1–40 and Aβ1–42 endocytosis and expression of Aβ1-42 monomers and oligomers. A similar reduction was observed in CytD-treated WT cells. Finally, actin disruption and Mt3 knockout each increased the overall levels of clathrin and the associated protein phosphatidylinositol-binding clathrin assembly protein (PICALM) in astrocytes.ConclusionsOur results suggest that the absence of Mt3 reduces Aβ uptake in astrocytes through an abnormality in actin polymerization. In light of evidence that Mt3 is downregulated in AD, our findings indicate that this mechanism may contribute to the extracellular accumulation of Aβ in this disease.

Abstract 5546: Coupling of hydroxyethyl starch to anticancer drugs for the improvement of tolerability and efficacy.

Abstract Background: Standard-of-care anti-cancer drugs exhibit anti-tumor activity but typically display dose limiting toxicities in patients. Poor solubility in water, a short half-life, and a narrow therapeutic window further limit their applicability. The aim of the current studies was to investigate whether coupling of hydroxyethyl starch (HES) to selected anti-cancer drugs with different cleavable linkers improves their efficacy and safety in human tumor xenograft models in mice. Materials and methods: Female NMRI nu/nu mice bearing subcutaneous xenografts of the human large cell lung cancer LXFL 529 were treated twice weekly i.v. for two to four weeks with either the vehicle only, etoposide, temsirolimus or their HES-conjugates. Dosing started at group median tumor volumes of 200-250 mm3. In another model, 5x106 tumor cells from the human breast carcinoma MT-3 cell line were inoculated s.c. followed by i.v. injection of vehicle, docetaxel, or HES-docetaxel conjugates starting on day 7 at palpable tumor size. Docetaxel was administered on 5 consecutive days [5 mg/kg]; the HES-docetaxel conjugates were given only once on day 7 [75 mg/kg]. Moreover, 1x107 cells of the human colon carcinoma HT-29 cell line were implanted s.c. to develop palpable tumors within 8 days. On day 8, the mice received a single i.v. dose of vehicle, irinotecan, or either HES-irinotecan or HES-SN38 conjugates. Tumor volume, body weight, and mortality were monitored during an observation period of up to 90 days. Results: The conjugates were well soluble in saline in contrast to the originator drugs. HES-temsirolimus exerted a higher anti-tumor activity, and HES-etoposide caused a substantially delayed progression of the large cell lung cancer xenograft LXFL 529 after the end of dosing in comparison to the respective originator drug. In the experiment with the breast carcinoma xenograft model, the HES-docetaxel conjugates were better tolerated than docetaxel, allowing the administration of higher doses. HES-docetaxel conjugates exerted a stronger anti-tumor activity compared to the originator drug. In the human colon carcinoma xenograft model, HES-SN38 at the same, or even at a lower dose level compared to irinotecan, caused a substantially stronger tumor growth inhibition dependent on the stability of the linker. Moreover, HES-irinotecan reduced tumor growth to a significantly greater degree than the irinotecan originator. Conclusion: We showed for the first time that the conjugation of HES to drugs is useful to improve their water solubility, making irritating excipients required to dissolve hydrophobic drugs, dispensable. In addition, the coupling of HES to anti-cancer drugs can improve their efficacy and tolerability in tumor xenograft models in mice. The extended release kinetics of HES-drug conjugates may improve the pharmacokinetic profile by reducing exposure peaks, which are typically related to unwanted drug effects. Citation Format: Silke Baasner, Corinna Lupp, Christoph Meyer, Dominik Heckmann, Norbert Zander, Steven Tucker, Martin Westphal. Coupling of hydroxyethyl starch to anticancer drugs for the improvement of tolerability and efficacy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5546. doi:10.1158/1538-7445.AM2013-5546

Influence of cancerostatic perifosine on membrane fluidity of liposomes and different cell lines as measured by electron paramagnetic resonance

AimTo test whether membrane fluidity and its changes are important for the sensitivity of cells to the action of perifosine (OPP), a new anticancer drug targeting cell membrane and not DNA.MethodInfluence of OPP on the membrane structure of OPP-resistant MCF7, and OPP-sensitive MT3 breast cancer cell lines, as well as of mouse fibroblasts (L929) cell lines, and model cells (liposomes) was investigated by electron paramagnetic resonance, using spin labeled derivative of OPP (P5) and 5-doxylpalmitoyl methylester (MeFASL(10,3)) as spin probes.ResultsOPP increased membrane fluidity of all cell lines at concentrations higher than 50 µM (on the level of P ≤ 0.05, t test). In cells, the differences were observed only by P5 and not by MeFASL(10,3). Average order parameter Seff decreased for about 12% in MCF7 and L929 and only for 8% in OPP-sensitive MT3 cells, showing that there was no correlation between membrane fluidity changes and sensitivity of cells to OPP. The only correlation we found was between OPP sensitivity and the cell growth rate. In liposomes, both spin probes were sensitive to the action of OPP. Seff decreased with increasing concentration of OPP. For MeFASL(10,3) a significant decrease was observed at 4 mol% OPP, while for P5 it was observed at 8 mol%.ConclusionInfluence of OPP on plasma membrane fluidity of breast cancer cells is not the determining factor in the sensitivity of cells to OPP.

Abstract 1726: Overcoming multi-drug resistance with Paclitaxel-loaded polymer nanoparticles

Abstract Multi-drug resistance against chemotherapeutic agents is a major problem in the treatment of cancer. It is primarily mediated by over-expression of P-glycoprotein (P-gp) encoded by the MDR-1 gene by the tumor cells. Most of the promising anticancer drugs such as doxorubicin, docetaxel, paclitaxel, and mitoxantrone are P-gp substrates. Thus, various ways for overcoming the multi-drug resistance, e.g. combination of an anticancer drug and a P-gp inhibitor or the formulation of nanoparticles in conjunction with anticancer drugs as well as the P-gp inhibitor, have been studied. Anticancer drug-loaded nanoparticles, such as paclitaxel-loaded polymer nanoparticles, are excellent candidates to overcome multi-drug resistance due to their enhanced therapeutic efficacy induced by increased cellular accumulation and sustained intracellular drug delivery. In this work, we developed an ampholytic copolymer that can encapsulate paclitaxel, and the resulting nanoparticles were found to have good water-solubility. In addition, in contrast to native paclitaxel, the paclitaxel-loaded nanoparticles were able to reverse multi-drug resistance in MCF7/ADR and MT-3/ADR cell lines, resulting in drug sensitivities comparable to the parental MCF7 and MT3 cell lines. P-gp expression was confirmed in both ADR cell lines by Western blotting, and its functionality was verified using a rhodamine accumulation and efflux assay. The nanoparticles were taken up by endocytosis in both cell lines and their enhanced therapeutic efficacy is probably due to the different mode of entry into the cell that may avoid the P-gp efflux pump. As a consequence higher concentrations of paclitaxel reach the nucleus and induce cell killing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1726. doi:10.1158/1538-7445.AM2011-1726

Cytotoxic Effect of the Pentacyclic Oxindole Alkaloid Mitraphylline Isolated fromUncaria tomentosaBark on Human Ewing's Sarcoma and Breast Cancer Cell Lines

Preparations from Uncaria tomentosa, a South American Rubiaceae, have been used in the Peruvian traditional medicine for the treatment of infective, inflammatory and tumoral processes. In this study, the pentacyclic oxindole alkaloid mitraphylline was isolated from the dried inner bark of this plant species, and its structure elucidated by analysis of NMR spectroscopic data. Mitraphylline was differentially identified from its stereoisomeric pair isomitraphylline by (15)N-NMR. Its antiproliferative and cytotoxic effects have been tested on human Ewing's sarcoma MHH-ES-1 and breast cancer MT-3 cell lines, using cyclophosphamide and vincristine as reference controls. A Coulter counter was used to determine viable cell numbers, followed by the application of the tetrazolium compound MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] an inner salt. A colorimetric method was employed to evaluate cell viability in this cytotoxic assay. Micromolar concentrations of mitraphylline (5 microM to 40 microM) inhibited the growth of both cell lines in a dose-dependent manner. The IC (50) +/- SE values were 17.15 +/- 0.82 microM for MHH-ES-1 and 11.80 +/- 1.03 microM for MT-3 for 30 hours, smaller than those obtained for the reference compounds. This action suggests that the pentacyclic oxindole alkaloid mitraphylline might be a new promising agent in the treatment of both human sarcoma and breast cancer.

Synthesis and Biophysical Characterization of Chlorambucil Anticancer Ether Lipid Prodrugs

The synthesis and biophysical characterization of four prodrug ether phospholipid conjugates are described. The lipids are prepared from the anticancer drug chlorambucil and have C16 and C18 ether chains with phosphatidylcholine or phosphatidylglycerol headgroups. All four prodrugs have the ability to form unilamellar liposomes (86-125 nm) and are hydrolyzed by phospholipase A(2), resulting in chlorambucil release. Liposomal formulations of prodrug lipids displayed cytotoxicity toward HT-29, MT-3, and ES-2 cancer cell lines in the presence of phospholipase A(2), with IC(50) values in the 8-36 microM range.

Bacterial consortium proteomics under 4‐chlorosalicylate carbon‐limiting conditions

In this study, the stable consortium composed by Pseudomonas reinekei strain MT1 and Achromobacter xylosoxidans strain MT3 (cell numbers in proportion 9:1) was under investigation to reveal bacterial interactions that take place under severe nutrient-limiting conditions. The analysis of steady states in continuous cultures was carried out at the proteome, metabolic profile, and population dynamic levels. Carbon-limiting studies showed a higher metabolic versatility in the community through upregulation of parallel catabolic enzymes (salicylate 5-hydroxylase and 17-fold on 2-keto-4-pentenoate hydratase) indicating a possible alternative carbon routing in the upper degradation pathway highlighting the effect of minor proportions of strain MT3 over the major consortia component strain MT1 with a significant change in the expression levels of the enzymes of the mainly induced biodegradation pathway such as salicylate 1-hydroxylase and catechol 1,2-dioxygenase together with important changes in the outer membrane composition of P. reinekei MT1 under different culture conditions. The study has demonstrated the importance of the outer membrane as a sensing/response protective barrier caused by interspecies interactions highlighting the role of the major outer membrane proteins OprF and porin D in P. reinekei sp. MT1 under the culture conditions tested.

Inhibition of breast cancer metastasis by dual liposomes to disturb complex formation

The interaction between tumour cells and blood components, mainly platelets, plays an important role in metastasis. In this study, the anti-metastatic effect of vesicles containing the cytotoxic drug perifosine (OPP) and the haemostatic inhibitor dipyridamole (DIP) was tested. These dual liposomes (DIP/OPP-L) encapsulating up to 400 μg DIP/ml and 6 mM OPP were prepared by extrusion technique. In vitro, DIP/OPP-L significantly inhibited the aggregation of platelets and reduced their adhesion to immobilized MT3 cells by up to 60% and 24.7%, respectively. Complex formation between platelets and tumour cells in vitro was completely prevented by DIP/OPP-vesicles. These combinatory liposomes also inhibited the metastatic capacity of circulating tumour cells by reducing the complex formation with platelets. Formation of lung and extrapulmonary metastases after intravenous administration of MT3 breast cancer cells was significantly reduced when mice were treated with a single intravenous dose of DIP/OPP-L containing 100 nmol lipid 6 h before tumour cell inoculation.

Interaction of alkylphospholipid liposomes with MT-3 breast-cancer cells depends critically on cholesterol concentration

We have investigated interaction of alkyphospholipid (APL) liposomes consisting of 1,1-dimethylpiperidin-1-ium-4-yl) octadecyl phosphate (OPP) and different concentrations of cholesterol (CH) with human MT-3 breast-cancer cells using electron paramagnetic resonance method (EPR) with advanced characterization of EPR spectra of spin labeled liposome membranes. After incubation of OPP liposomes with MT-3 cells, a reduction of liposome entrapped, water soluble spin-probe tempocholine (ASL) was observed, indicating that ASL is released from liposomes and is reduced by oxy-redoxy systems inside the cells. This process is fast if cholesterol content in the bilayer was 29 or 45 mol%, whereas at 56 mol% cholesterol the process is almost stopped. The rate of spin-probe reduction in first 10 min after incubation with cells is even faster as for the free ASL, indicating that liposomes with low amount of cholesterol accelerate penetration of ASL into the cells. A faster release of hydrophilic material from liposomes with low cholesterol content coincides with the presence of domains with highly disordered alkyl chain motion that disappears at 50 mol% of cholesterol. We propose that these highly fluid domains are responsible for interaction of OPP liposomes with cells and fast release of the entrapped material into the cells. These results suggest that micelles are not the only reason for cytotoxic effect of OPP liposom formulations, as it was suggested before. OPP in liposomes, containing 45 mol% cholesterol or less, also contributes to the cytotoxic effect, due to their fast interaction with breast-cancer cells.

Analysis of Biological Activities of Recombinant Rat Interleukin-5

We analyzed the biological activities of recombinant rat interleukin-5 (IL-5). Purified recombinant rat IL-5 promoted the proliferation of T88-M cells in a concentration-dependent manner, and its effect was inhibited by an anti-murine IL-5-neutralizing polyclonal antibody. When bone marrow cells from normal rats were incubated with recombinant rat IL-5 in medium containing methylcellulose, the colony formation by eosinophilic cells was induced. Furthermore, when rat peritoneal eosinophils were incubated with recombinant rat IL-5, the spontaneous decrease in eosinophil viability was inhibited in a time- and concentration-dependent manner. In addition, the recombinant rat IL-5-induced eosinophil survival was inhibited by an anti-murine IL-5-neutralizing polyclonal antibody. These findings suggest that rat IL-5 acts as B cell growth factor II, eosinophil differentiation factor, and eosinophil survival-enhancing factor.

Preparation of recombinant rat interleukin-5 by baculovirus expression system and analysis of its biological activities

Rat interleukin-5 (IL-5) cDNA was subcloned from peritoneal cells collected 4 h after intraperitoneal injection of Ascaris suum antigen solution into the immunized rats. Cysteine proteinase-deleted (CPd) rat IL-5 recombinant virus was constructed by inserting rat IL-5 cDNA into CPd virus having a deletion in the cysteine proteinase gene of the silkworm Bombyx mori nuclear polyhedrosis virus. On infection with the CPd rat IL-5 recombinant virus, the silkworm B. mori larvae produced rat IL-5 as a dimeric form in hemolymph. Recombinant rat IL-5 was purified more than 95.5% by anion-exchange chromatography and hydrophobic chromatography. The purified recombinant rat IL-5 promoted the proliferation of T88-M cells in a concentration-dependent manner, and its effect was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. When bone marrow cells from normal rats were incubated with recombinant rat IL-5 in medium containing methylcellulose, the colony formation by eosinophilic cells was induced. Furthermore, when rat peritoneal eosinophils were incubated with recombinant rat IL-5, the spontaneous decrease in the eosinophil viability was inhibited in time- and concentration-dependent manners. In addition, the recombinant rat IL-5-induced eosinophil survival was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. These findings suggest that rat IL-5 acts as B-cell growth factor II (BCGF-II), eosinophil differentiation factor (EDF), and eosinophil survival-enhancing factor.

C-kit ligand (SCF) in human multiple myeloma cells.

Here we review our recent experience addressing the role of SCF in multiple myeloma (MM). We first investigated the proliferation of MM cell lines and bone marrow samples from myeloma patients in response to rh-SCF alone and combined with Interleukin-6 (IL-6), IL-3, and IL-3/GM-CSF fusion protein PIXY 321. Neoplastic plasma cells were highly purified (>90%) by immunomagnetic depletion of T, myeloid, monocytoid and NK cells. The number of S-phase cells was evaluated after 3 days of liquid culture by the bromodeoxyuridine (BRDU) incorporation assay. The proliferation of RPMI 8226 and U266 cell lines was also assessed by a clonogenic assay. All the experiments were performed in serum-free conditions. RPMI 8226 cell line was not stimulated by SCF which also did not augment the proliferative activity of IL-6, IL-3 and PIXY-321. Conversely, SCF addition resulted in 2.4-fold increase of the number of U266 colonies and in a higher number of U266 and MT3 cells in S-phase. The c-kit ligand also enhanced the proliferation of MT3 and U266 cells mediated by the other cytokines. Anti-SCF polyclonal antibodies completely abrogated the proliferative response of MT3 cells to exogenous SCF and markedly reduced the spontaneous growth of the same cell line. Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) did detect SCF mRNA in MT3 and RPMI 8226 cells. Moreover, secreted SCF was found, in a biologically active form, in the supernatant of the two cell lines by the MO7e proliferation assay. These results suggest that an autocrine proliferative loop may be operative in MT3 cell line. When tested on fresh myeloma samples, SCF increased the number of S-phase plasma cells (4.7 +/- 1.6% vs 3.4 +/- 1.3% in control cultures; p = 0.02). Significant proliferation was also induced by IL6, IL-3 and PIXY-321. The addition of SCF significantly enhanced the proliferation of myeloma cells responsive to IL-6. Preliminary experiments performed on circulating plasma cells and myeloma precursors further supported the role of SCF on the proliferation of the neoplastic clone in MM.

Expression and functional role of c-kit ligand (SCF) in human multiple myeloma cells.

In this study we investigated the proliferation of three well-documented MM lines and 10 bone marrow samples from myeloma patients in response to rh-SCF alone and combined with Interleukin-6 (IL-6), IL-3 and IL-3/GM-CSF fusion protein PIXY 321. Neoplastic plasma cells were highly purified (> 90%) by immunomagnetic depletion of T, myeloid, monocytoid and NK cells. The number of S-phase cells was evaluated after 3 and 7 d of liquid culture by the bromodeoxyuridine (BRDU) incorporation assay. The proliferation of RPMI 8226 and U266 cell lines was also assessed by a clonogenic assay. All the experiments were performed in serum-free conditions. RPMI 8226 cell line was not stimulated by SCF which also did not augment the proliferative activity of IL-6, IL-3 and PIXY-321. Conversely, SCF addition resulted in 2.4-fold increase of the number of U266 colonies and in a higher number of U266 and MT3 cells in S-phase (24.5 +/- 2% SEM v 14.5 +/- 1% SEM and 32 +/- 3% SEM v 21 +/- 4% SEM, respectively; P < 0.05). The c-kit ligand also enhanced the proliferation of MT3 and U266 cells mediated by the other cytokines. Anti-SCF polyclonal antibodies completely abrogated the proliferative response of MT3 cells to exogenous SCF and markedly reduced the spontaneous growth of the same cell line. Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) did detect SCF mRNA in MT3 and RPMI 8226 cells. Moreover, secreted SCF was found, in a biologically active form, in the supernatant of the two cell lines by the MO7e proliferation assay. When tested on fresh myeloma samples, SCF increased the number of S-phase plasma cells (4.7 +/- 1.6% v 3.4 +/- 1.3% in control cultures: P = 0.02). Significant proliferation was also induced by IL-6 (7 +/- 2.3% of BRDU+ cells; P = 0.006), IL-3 (5.3 +/- 1.3%; P = 0.01) and PIXY-321 (5.4 +/- 1.6%; P = 0.02). The addition of SCF significantly enhanced the proliferation of myeloma cells responsive to IL-6. In summary, our results indicate that SCF is expressed in MM cells and stimulates the proliferation of neoplastic plasma cells.

Calcium oscillation induced by bradykinin in polyoma middle T antigen-transformed NIH3T3 fibroblasts: Evidence for dependence on protein kinase C

Bradykinin (BK) triggered long lasting intracellular free calcium ([Ca2+]i) oscillation in polyoma middle T-transformed cell line MT3 cells but not in the parental NIH3T3 cells. This periodic [Ca2+]i fluctuation was extracellular Ca(2+)-dependent and blocked by pretreatments with Ca2+ channel blockers, SK&F 96365 or CdCl2, suggesting a crucial role of Ca2+ entry across the plasma membrane possibly through a receptor-operated Ca2+ channel. Brief pretreatment with phorbol myristate acetate (PMA) completely abolished the BK-induced [Ca2+]i oscillation, and a protein kinase C (PKC) inhibitor, H-7, reversed the effect of PMA, indicating involvement of PKC. On the other hand, in some cells, oscillatory changes in [Ca2+]i were seen without agonist stimulation. The spontaneous oscillation was also dependent on extracellular Ca2+, but neither treatment with PMA nor H-7 had any effect under the same conditions.

Murine interleukin 5 receptor isolated by immunoaffinity chromatography: comparison of determined N-terminal sequence and deduced primary sequence from cDNA and implication of a role of the intracytoplasmic domain

The murine interleukin 5 receptor (IL-5R) was identified by utilizing an immobilized IL-5 and an immobilized monoclonal antibody against the murein IL-5R (designated H7 mAb). The H7 mAb immunoaffinity-purified materials from the extract of cell-surface radioiodinated T88-M cells (an IL-5-dependent early B cell line) using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) were reacted with an immobilized IL-5 matrix. SDS-PAGE of the adsorbed fraction revealed a single band at approximately 60 kDa. The binding of the 60 kDa protein to the immobilized IL-5 matrix was inhibited by the excess IL-5. The CHAPS-extract depleted of the 60 kDa protein by the absorption with H7 mAb did not contain any IL-5 binding proteins. Immunoaffinity procedure provided a final 7400-fold purification, based on an estimation of the content of the 60 kDa protein (approximate purity: 20%) from the silver-stained pattern of SDS-PAGE. Actin was copurified with the 60 kDa protein at an approximate ratio of 1:1, suggesting that the intracytoplasmic domain of the IL-5R may interact with actin. Furthermore, soluble IL-5R (molecular mass: 50 kDa) was purified by the H7 mAb-immunoaffinity chromatography. The purified soluble IL-5R was capable of inhibiting the binding of IL-5 to T88-M cells. Preparative SDS-PAGE followed by electroblotting onto a membrane permitted the determination of the N-terminal sequence of the IL-5R. The determined N-terminal sequence of the IL-5R and the deduced primary sequence from recently isolated cDNA were compared.

Interleukin 5 and interleukin 3 induce serine and tyrosine phosphorylations of several cellular proteins in an interleukin 5-dependent cell line

Murine interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and eosinophils. To elucidate the intracellular events via the IL-5 receptor, we have investigated IL-5-induced protein phosphorylation in an IL-5-dependent murine early B-lineage cell line (T88-M). The rapid phosphorylation of a 60-kDa protein at serine residues and of 140-, 92-, 53-, 48-, and 45-kDa proteins at tyrosine residues were induced by the stimulation of T88-M cells with IL-5. T88-M cells were found to proliferate in response to IL-3 as well as IL-5, and the phosphoproteins in T88-M cells could be induced by IL-3 stimulation in a similar manner of those induced by IL-5. Thus, these results suggest that the protein phosphorylation events in T88-M cells may be shared by both pathways via the IL-5 receptor and the IL-3 receptor.

Distribution of IL-5 receptor-positive B cells. Expression of IL-5 receptor on Ly-1(CD5)+ B cells.

mAb to murine IL-5R were prepared by means of fusion between mouse myeloma cells and spleen cells from a rat immunized with membrane-enriched fractions of IL-5-dependent early B cell line (T88-M). Two mAb (H7 and T21) were selected for their competitive inhibition of receptor binding by 35S-labeled IL-5 and of IL-5 biologic activities. The number of binding sites recognized by the mAb on different cell lines correlated with IL-5 responsiveness. Most surface IgM+ peritoneal B cells were H7+ and more than 70% were also Ly-1(CD5)dull+, and responded to IL-5 for polyclonal IgM production in a high frequency. A significant proportion of splenic B cells reacted with these mAb, although lower number (one-log less) than peritoneal B cells and a small proportion of H7dull+ splenic B cells seems to be Ly-1(CD5)dull+, 1 of 200 splenic B cells responded to IL-5 for IgM production. These results suggest that IL-5R+ B cells may consist of a subpopulation of B cells. Intriguingly, lymphoid populations of bone marrow cells were stained with H7 and T21, whereas myeloid populations were brightly stained with only T21. Finally, both H7 and T21 mAb specifically precipitated a protein of a Mr 60,000 from 125I-labeled cell lysates of IL-5R+ T88-M cells. The IL-5R with similar size (Mr 55,000 to 60,000) was precipitated from the cell lysates of peritoneal B cells. T21 mAb but not H7 mAb precipitated a protein of a Mr 110,000 from the cell lysates of bone marrow cells.

Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5.

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.

Characterization of the murine interleukin 5 receptor by using a monoclonal antibody

Murine interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. The receptor for IL-5 has been identified as two cross-linked complexes on T88-M cells (a murine IL-5-dependent early B cell line). In this study the IL-5 receptor was directly characterized by utilizing an immobilized IL-5 column and a rat monoclonal antibody, designated H7, directed against the IL-5 receptor. H7 completely inhibited specific binding of 35S-labeled IL-5 to T88-M cells, and bound to IL-5-responsive cells, e.g. T88-M, BCL1-B20 (a chronic B-cell leukemia), and MOPC104E (a myeloma), whereas H7 did not bind to IL-5-non-responsive cells, e.g. X5563 (a myeloma), FDC-P1 (an IL-3-dependent line), and MTH (an IL-2-dependent CTLL). H7 could barely bind to T88-M cells in the presence of IL-5, and immunoprecipitated a major band with an Mr of approximately 60 kd from the extract of surface-radioiodinated T88-M cells. The precipitation of this 60 kd molecule was inhibited by the addition of IL-5. Analysis with immobilized IL-5 also revealed that a 60 kd molecule bound specifically to IL-5-coupled beads compared with control beads. Furthermore, no additional molecule with a higher Mr that was recognized by H7 appeared under non-reducing, compared with reducing, conditions. The 60 kd molecule recognized by H7 could be digested with N-glycanase to yield a protein band of approximately 55 kd.(ABSTRACT TRUNCATED AT 250 WORDS)