Differential expression of mammalian or viral promoter-driven gene in adherent versus suspension cells

Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adherent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay), is as robust as when they are in adherent cells. The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play a critical role in maintaining colony formation or cell transformation. To compare promoter-driven expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 [SV40] and cytomegalovirus [CMV]) and mammalian (beta-actin) promoters fused to luciferase or beta-galactosidase reporter gene. Over-agar assay was used to suspend cells on top of agar, which allowed cell retrieval and analysis. We found that beta-actin and SV40 promoters exhibited suppressed gene expression of 70 and 56%, respectively, in cells suspended on agar compared with those attached on plates. The suppressed response by the exogenous beta-actin promoter in suspension was consistent with the response of the endogenous beta-actin promoter activity because the steady-state level of beta-actin messenger ribonucleic acid in suspended cells was significantly reduced by 50% relative to that expressed in attached cells. In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared with adherent cells. These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state.     

Mouse Fibroblast Cell Adhesion Studied by Neutron Reflectometry

Neutron reflectometry (NR) was used to examine live mouse fibroblast cells adherent on a quartz substrate in a deuterated phosphate-buffered saline environment at room temperature. These measurements represent the first, to our knowledge, successful visualization and quantization of the interface between live cells and a substrate with subnanometer resolution using NR. NR data, attributable to the adhesion of live cells, were observed and compared with data from pure growth medium. Independently of surface cell density, the average distance between the center of the cell membrane region and the quartz substrate was determined to be ∼180 Å. The membrane region (∼80 Å thick) contains the membranes of cells that are inhomogeneously distributed or undulating, likely conforming to the nonplanar geometry of the supporting adherence proteins. A second region of cell membranes at a greater distance from the substrate was not detectable by NR due to the resolution limits of the technique employed. Attachment of the live cell samples was confirmed by interaction with both distilled water and trypsin. Distinct changes in the NR data after exposure indicate the removal of cells from the substrate.     

A subpopulation of adherent accessory cells bearing both I-A and I-E or C subregion antigens is required for antigen-specific murine T lymphocyte proliferation.

A murine T lymphocyte proliferation assay that used antigen-primed lymph node T cells, was antigen specific, and required exogenous accessory cells was used to characterize the accessory cells that supported proliferation. These cells were Thy 1.2 negative, radioresistant, glass-adherent, and were functional only if alive. The accessory cell function of spleen adherent cells was much greater than that of peritoneal cells. Also, the accessory cell function of spleen adherent cells was proportional to the length of time such cells were incubated with antigen and very small numbers of such cells provided accessory cell function. Cytotoxic studies with subregion-restricted anti-Ia antibodies and complement indicated that accessory cell function resided in a subpopulation of spleen adherent cells that bore both I-A and I-E or C subregion antigens. The function of such cells was not related to a selective ability (vs other spleen adherent cells) to take up antigen. These data indicate that antigen-specific stimulation of T lymphocyte proliferation requires at least one specific subpopulation of spleen adherent cells that can be phenotypically identified by its expression of Ia antigens and are consistent with the possibility that Ia antigens may be Ir gene products.     

Development of pipette tip gap closure migration assay (s-ARU method) for studying semi-adherent cell lines

This work presents a pipette tip gap closure migration assay prototype tool (semi-adherent relative upsurge—s-ARU—method) to study cell migration or wound healing in semi-adherent cell lines, such as lymph node carcinoma of the prostate (LNCaP). Basically, it consists of a 6-well cover plate modification, where pipette tips with the filter are shortened and fixed vertically to the inner surface of the cover plate, with their heights adjusted to touch the bottom of the well center. This provides a barrier for the inoculated cells to grow on, creating a cell-free gap. Such a uniform gap formed can be used to study migration assay for both adherent as well as semi-adherent cells. After performing time studies, effective measurement of gap area can be carried out conveniently through image analysis software. Here, the prototype was tested for LNCaP cells, treated with testosterone and flutamide as well as with bacteriophages T4 and M13. A scratch assay using PC3 adherent cells was also performed for comparison. It was observed that s-ARU method is suitable for studying LNCaP cells migration assay, as observed from our results with testosterone, flutamide, and bacteriophages (T4 and M13). Our method is a low-cost handmade prototype, which can be an alternative to the other migration assay protocol(s) for both adherent and semi-adherent cell cultures in oncological research along with other biological research applications.     

Occupancy of the cancer cell urokinase receptor (uPAR): effects of acid elution and exogenous uPA on cell surface urokinase (uPA).

The development of a simple, sensitive fluorimetric assay for the measurement of cell surface-associated urokinase plasminogen activator (uPA) on viable, adherent HCT116 cells in microtitre plates, after a preincubation with purified human plasminogen is described. The assay determines plasmin activity by the cleavage of H-D-Val-Leu-Lys 4-aminomethyl coumarin under near physiological pH and ionic conditions with a sensitivity in the range of 5-100 mIU uPA/well at excitation 355 nm and emission 460 nm. Plasmin generated during the assay converted all cell-surface sc-uPA to tc-uPA, allowing the determination of total uPA activity. Inhibitor studies (PAI-2, amiloride or Glu-Gly-Arg chloromethylketone) confirmed the specificity of the uPA assay. Removal of these agents prior to assay allowed determination of the cell surface sc-uPA:tc-uPA ratio. Cell surface activity was only partially removed by acid elution. This corresponded with the loss of a number of proteins and uPA-containing species as detected by SDS-PAGE, gelatin enzymography and Western blotting. Although the major protein species eluted had a M(r) of 55 kDa, reacted with a commercial anti-human uPA mAb and correlated with the main lytic zone, other higher M(r) species were also eluted from HCT116 cells. Exogenous uPA increased cell-surface activity markedly on cells previously treated with acid. Following acid elution, cell surface uPA activity was restored after 30h in culture suggesting either de novo synthesis or release of pre-formed uPA with subsequent secretion and binding to uPAR. The assay has enabled studies on adherent cells to address questions about the regulation and expression of cell-surface uPA.     

Quantification of an in vitro cell-cell adhesion assay using interactive laser scanning cytometry.

We are interested in identifying cell-cell adhesion molecules on the surface of Sertoli cells that mediate Sertoli cell-spermatogenic cell adhesion. Numerous cell-cell adhesion assays employ microscopic observation, photomicroscopy or radioactive isotopes for quantification. Previously, we developed an in vitro assay for testicular cell interactions. This assay was, however, time consuming using photography for analysis. We have now modified this system using laser cytometry to quantify adherent cells. Rat testicular epithelial cells are cultured for approximately 6 days before labelling with fluorescein diactetate (FDA) to assess confluency by image scanning so that spermatogenic cell binding can be normalized to available epithelial cell surface area. Rat spermatogenic cells are labeled with FDA before addition to epithelial cell monolayers. In some studies, purified spermatogenic cell populations were isolated to determine average cell size. We found that spermatocyte area varied between 225-500 microns2, spermatids were 100-225 microns2 and residual bodies were less than 100 microns2. Using these parameters, scanning cytometry allows the differential analysis of adhesion by individual germ cell sub-classes from mixed cell suspensions, saving time, animals, and major expense. The scanning laser assisted assay is faster, more reproducible and less subjective than earlier cell-cell adhesion assays using light microscopy or isotopes. This experimental approach should facilitate any cell-cell adhesion assay in which one cell type is adherent to a substrate.     

Regulation of nitrate reductase activity in cultured spinach cells as studied by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay permitting the determination of nanogram quantities of nitrate reductase (NR) in cultured spinach cells has been developed and used for studies of the mechanism by which NR activity is regulated as a function of culture age. When 8-day old spinach cells were transferred to fresh medium, NR activity increased markedly in 2 days and thereafter decreased gradually until it became undetectable on the 10th day after the transfer. Determination of the amounts of NR by the immunosorbent assay indicated that the unique alteration of NR activity could be accounted for by the concomitant change in the amount of NR protein. Immunoblotting analysis of the subunit of NR also supported this result. It is concluded that the regulation of NR in spinach cells as a function of culture age is mediated by changes in the amount of the enzyme protein rather than by activation and inactivation of the preexisting proteins.     

Modulation of NMDA-mediated excitotoxicity by protein kinase C

Excessive activation of N-methyl-D-aspartate (NMDA) receptors leads to cell death in human embryonic kidney-293 (HEK) cells which have been transfected with recombinant NMDA receptors. To evaluate the role of protein kinase C (PKC) activation in NMDA-mediated toxicity, we have analyzed the survival of transfected HEK cells using trypan blue exclusion. We found that NMDA-mediated death of HEK cells transfected with NR1/NR2A subunits was increased by exposure to phorbol esters and reduced by inhibitors of PKC activation, or PKC down-regulation. The region of NR2A that provides the PKC-induced enhancement of cell death was localized to a discrete segment of the C-terminus. Use of isoform-specific PKC inhibitors showed that Ca(2+)- and lipid-dependent PKC isoforms (cPKCs), specifically PKCbeta1, was responsible for the increase in cell death when phorbol esters were applied prior to NMDA in these cells. PKC activity measured by an in vitro kinase assay was also increased in NR1A/NR2A-transfected HEK cells following NMDA stimulation. These results suggest that PKC acts on the C-terminus of NR2A to accentuate cell death in NR1/NR2A-transfected cells and demonstrate that this effect is mediated by cPKC isoforms. These data indicate that elevation of cellular PKC activity can increase neurotoxicity mediated by NMDA receptor activation.     

Spectrophotometric quantitation of anchorage-dependent cell numbers using the bicinchoninic acid protein assay reagent

A rapid and convenient method is described for the determination of the actual and relative number of adherent cells in tissue culture. The cell lines human melanoma C32, ATCC CRL 1585, mouse melanoma B16-F10, and pig epithelial LLC-PK1, suspended in Dulbecco's minimum essential medium containing no serum, were allowed to adhere to fibronectin adsorbed to wells of a 96-well microtiter plate. Nonadherent cells were removed by aspiration, wells were washed, and adherent cells were solubilized with 200 microliters of the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay reagent. Plates were heated to 60 degrees C for 30 min and absorbances read at 562 nm using a microtiter plate reader. A linear correlation was observed between the number of adherent cells in the range 2-8 X 10(5)/ml cells added and the protein content of the adherent cells as measured by the BCA protein reagent. The assay procedure gave absorbance values in the range of 0.100 to 1.30 making the method highly sensitive and reproducible. Blank wells containing only coupled protein and no cells gave little or no absorbance. Cell adhesion was fibronectin specific since little or no cell attachment was observed when microtiter plates were coupled with bovine serum albumin. Similar results were obtained with other cell types such as platelets. These results indicate that measurement of total cellular protein using the BCA protein reagent can be a rapid and sensitive assay for the detection and quantitation of adherent cells.     

Glutamatergic regulation of bone remodeling

L-glutamate (Glu) is the predominant neuromediator in the mammalian central nervous system (CNS). Bone is highly innervated and there is growing evidence of a neural control of bone cell metabolism. The recent discovery of Glu-containing nerve fibers in bone and Glu receptors (GluR) and transporters in bone cells suggest that this neuromediator may also act as a signaling molecule in bone and regulate bone cell function. Our previous studies have demonstrated that ionotropic N-Methyl-D-Aspartate (NMDA) GluR are highly expressed by mammalian osteoclasts. NMDA receptors (NMDAR) are heteromers associating the NR1 subunit and one of the four types of NR2 subunits (NR2A to D). We showed that osteoclasts express NR1, NR2B and NR2D subunits, suggesting a molecular diversity of NMDAR in these cells. Electrophysiological studies have confirmed that NMDAR are functional in mature osteoclasts, and features of Glu-induced current recorded in these cells indicate a major NR2D subunit composition. Using an in vitro assay of bone resorption, we showed that several antagonists of NMDAR binding to different sites of the receptor inhibit bone resorption. In particular, the specific NMDAR channel blocker MK801 had no effect on osteoclast attachment to bone and survival while it rapidly decreased the percentage of osteoclasts with actin ring structures that are associated with actively resorbing osteoclasts. NMDAR may thus be involved in adhesion-induced formation of the sealing zone required for bone resorption. NMDAR are also expressed by osteoclast precursors isolated from mouse bone marrow. We recently confirmed the presence of NR1, NR2B and NR2D in these cells and demonstrated their expression at all differentiation stages from osteoclast precursors to mature resorbing osteoclasts. No regulation of these subunits mRNA expression levels was observed throughout the osteoclastic differentiation sequence. Activation of NMDAR may therefore represent a new mechanism for regulating osteoclast formation and activity. While the origin of Glu in bone is still unknown, the possibility of a glutamatergic neurotransmission in this tissue is suggested by the detection of Glu in nerve fibers in close contact to bone cells. Furthermore, we recently demonstrated that sciatic neurectomy in growing rats induces a bone loss associated with a reduction of nerve profiles immunostained for Glu. These results suggest that Glu may be released from glutamatergic nerve profiles present in bone and therefore contribute to the local regulation of bone cell function.     

OKT3 monoclonal antibody induces production of colony-stimulating factor(s) for granulocytes and macrophages in cultures of human T lymphocytes and adherent cells

OKT3 monoclonal antibody (mab) recognizes a membrane antigen associated with the T cell antigen recognition receptor, and is known to be mitogenic and to induce lymphokine production. Our studies demonstrate the ability of OKT3 mab to induce from cultures of human T lymphocytes supplemented with adherent cells the production of colony-stimulating factor(s) for granulocytes and macrophages (GM-CSF) and interferon-gamma (IFN-gamma), an inhibitor of clonal growth of hematopoietic progenitor cells. As has been shown for the mitogenic and IFN-gamma-inducing activity of OKT3 mab, the induction of GM-CSF release in cultures of T cells is strictly dependent on the presence of adherent cells. However, the concentrations of OKT3 mab required for optimal GM-CSF production (50 ng/ml) were found to be 80-fold higher than those sufficient for maximal IFN-gamma production, proliferation, and interleukin 2 production. IFN-gamma activity induced by OKT3 mab partially inhibited colony and cluster formation from progenitor cells of granulocytes and macrophages in vitro. Therefore, neutralization of the IFN-gamma by monoclonal anti-human-IFN-gamma antibody before assay of conditioned medium in bone marrow cultures significantly enhanced the detection of GM-CSF. Kinetic studies demonstrated maximal cumulative GM-CSF production in response to optimal OKT3 mab concentrations on days 4 through 6 in cultures of T cells supplemented with 15% adherent cells. Highly enriched OKT4+ and OKT8+ T cell subsets co-cultured with adherent cells in the presence of OKT3 mab both produced GM-CSF and IFN-gamma and showed similar dose-response curves to OKT3 mab. The requirement for the presence of adherent cells could not be overcome by the addition of purified interleukin 1 or macrophage supernatants. Studies using irreversible inhibitors of DNA (mitomycin C) or protein biosynthesis (emetine-HCl) revealed the necessity of intact DNA synthesis and translation in mononuclear cells to produce GM-CSF in response to OKT3 mab. Loss of GM-CSF production was observed when either adherent cells or T lymphocytes were treated with emetine before co-culture with untreated cells of the other population in the presence of OKT3 mab. In contrast, mitomycin C reduced GM-CSF production significantly when T cells, but not adherent cells, were pretreated. These results suggest that T lymphocytes and adherent cells closely cooperate in the production of GM-CSF induced by OKT3 mab.     

Application of the chemiluminescent assay to cytotoxicity test: detection of menadione-catalyzed H2O2 production by viable cells.

Menadione-catalyzed H2O2 production by viable cells is proportional to viable cell number. The correlations between the viable cell number and the concentration of H2O2 produced are determined with the rapid chemiluminescent assay (S. Yamashoji, T. Ikeda, and K. Yamashoji, 1989, Anal. Biochem. 181, 149-152). This chemiluminescent assay of viable cells requires only 10 min and is much faster than NR (neutral red) inclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays, which require 3-5 h. When viable cells are incubated with antitumor drugs, detergents, mycotoxins, and glycoalkaloids for 24-48 h, a decrease in menadione-catalyzed H2O2 production in a dose- or incubation time-dependent manner is observed. In general, the 50% inhibition concentration determined by the chemiluminescent assay is lower than that determined by NR inclusion and MTT reduction assays, and the order of relative cytotoxic effects of agents is the same among these assays. Furthermore, clear cytotoxic effects are observed by the chemiluminescent assay after 1 h exposure of trypsinized cells to toxic compounds. Therefore, the chemiluminescent assay is expected to be more useful for the rapid detection of cytotoxic compounds than NR inclusion and MTT reduction assays.     

Heterogeneity of human natural killer cell populations.

Natural killing (NK) in human donors was determined by the ability of peripheral blood subpopulations to lyse the myeloid target, K562, in a 2 to 4 hr ⁵¹Cr release assay. The most active cell was a non-T cell which passed through nylon columns (representing 10 to 25% of column passed cells). A second column passed cell population, with characteristics of T lymphocytes (75 to 90% of column passed cells), was also capable of mediating natural killing. Non-T cells which were retained by the nylon columns (45 to 55% of adherent cells) lacked NK activity. However, nylon adherent T cells (45 to 55% of adherent cells) were consistently active in NK assays, illustrating an important subset of NK effector cell often overlooked. Both column passed and adherent T cells were further separated according to their ability to bind IgG or IgM immune complexes, showing that those mediating NK have receptors for IgG (Tγ+) but not for IgM (Tμ+).     

Inhibition of murine lymphokine-activated killer (LAK) cell activity by adherent cells

The effects of adherent cell depletion, indomethacin, and prostaglandin E2 (PGE2) on murine LAK cell activity were investigated. Removal of plastic adherent cells from splenocyte suspensions either prior to 5-day culture with 1000 U/ml of recombinant human IL-2 (rIL-2) or prior to assay resulted in an enhanced LAK cell cytotoxicity compared to that of whole spleen cell suspensions. Indomethacin enhanced LAK cell cytotoxicity of whole splenocyte suspensions if present during the culture period, but had no effect on whole splenocyte or adherent cell-depleted cell suspensions if added just prior to assay. PGE2 suppressed LAK cell activity of nonadherent splenocyte but not whole splenocyte suspensions when present during the culture period. In vivo treatment of mice with indomethacin enhanced cytotoxicity directed toward both LAK sensitive, natural killer (NK) resistant (P-815) and LAK, NK sensitive (YAC-1) tumor cell targets. Splenocytes from indomethacin-treated mice cultured with additional indomethacin and rIL-2 exhibited highest LAK cell activity. The results from this study indicate that LAK cells are regulated by adherent cells which suppress LAK cell activity. This suppression can be reversed both in vitro and in vivo by indomethacin. This study has important implications for the possible clinical use of indomethacin in the potentiation of in vivo and in vitro LAK cell activity for immunotherapeutic protocols.     

A novel time resolved fluorometric assay of anoikis using Europium-labelled Annexin V in cultured adherent cells

Background: Adherent cells undergo apoptosis when detached from their home ground, a process called anoikis (homelessness).Methods: We developed a new and sensitive method to analyse apoptosis and anoikis of adherent cell types using a time resolved fluorometric assay with Europium-labelled Annexin V. Anoikis was induced with tumor necrosis factor- /cycloheximide and three cell fractions of the cell cultures were prepared and analysed. Fraction 1 consisted of adherent cells, analysed while growing on their support (without detachment by trypsinisation). Fraction 2 contained detached cells due to anoikis (floating cells) and fraction 3 contained apoptotic bodies. Both fractions 2 and 3 were present in the culture medium and were isolated by differential centrifugation.Results: TNF- treatment of three different types of adherent cell cultures induced a significant increase of the amount of floating cells (anoikis) and apoptotic bodies compared to control cell cultures. Also in the adherent cell fractions a small amount of apoptosis was observed.Conclusions: The novel time resolved assay provides the ability to analyse the cell death cascade in adherent cell cultures of the same sample at the same time in a sensitive and reproducible way.     

Round versus flat: bone cell morphology, elasticity, and mechanosensing

There is increasing evidence that cell function and mechanical properties are closely related to morphology. However, most in vitro studies investigate flat adherent cells, which might not reflect physiological geometries in vivo. Osteocytes, the mechanosensors in bone, reside within ellipsoid containment, while osteoblasts adhere to flatter bone surfaces. It is unknown whether morphology difference, dictated by the geometry of attachment is important for cell rheology and mechanosensing. We developed a novel methodology for investigating the rheology and mechanosensitivity of bone cells under different morphologies using atomic force microscopy and our two-particle assay for optical tweezers. We found that the elastic constant of MLO-Y4 osteocytes when flat and adherent (>1 kPa) largely differed when round but partially adherent (<1 kPa). The elastic constant of round suspended MLO-Y4 osteocytes, MC3T3-E1 osteoblasts, and primary osteoblasts were similarly <1 kPa. The mechanosensitivity of round suspended MLO-Y4 osteocytes was investigated by monitoring nitric oxide (NO) release, an essential signaling molecule in bone. A preliminary observation of high NO release from round suspended MLO-Y4 osteocytes in response to 5 pN force is reported here, in contrast with previous studies where flat cells routinely release lesser NO while being stimulated with higher force. Our results suggest that a round cellular morphology supports a less stiff cytoskeleton configuration compared with flat cellular morphology. This implies that osteocytes take advantage of their ellipsoid morphology in vivo to sense small strains benefiting bone health. Our assay provides novel opportunities for in vitro studies under a controlled suspended morphology versus commonly studied adherent morphologies.     

Antigen-specific human T cell factors. I. T cell helper factor: biololgic properties

The in vitro induction and assay of an ovalbumin-specific human T cell helper factor are described. Peripheral blood T cells, cultured with ovalbumin in a Marbrook-Diener system, produce an antigen-specific factor(ThF120-OA), which can be purified by affinity chromatography. The in vitro studies with ThF120-OA pointed out that in the production of the factor as well as in the factor-B cell interaction the adherent cell determines the genetic restriction. The results of kinetic studies on T helper activities demonstrated that Thf120-OA provides an auxiliary activity at various moments during the differentiation of the human peripheral B cell into an antibody-secreting cell. The observed differences in the mode of action of Th cells and Th factor are discussed.     

Supravital exposure to propidium iodide identifies apoptosis on adherent cells

BACKGROUND: Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.     

Spontaneous helper factor production by nonadherent rabbit lymphoid cells and its feedback regulation by adherent cells

Normal, unstimulated rabbit lymphoid cells, when depleted of adherent cells, produced soluble helper factor activity that augmented antibody formation by rabbit spleen cells primed against sheep red blood cells (SRBC). Adherent cells inhibited the production of the helper factor by nonadherent cells via a soluble product. Thus unseparated (adherent cell-containing) appendix, lymph node, and spleen cell cultures did not produce the helper factor. On the other hand, the activity of the helper factor required the presence of adherent cells in the assay cultures. Peritoneal exudate cells, predominantly esterase positive, also inhibited the production of the helper factor if they were first exposed to the helper factor-containing culture supernatant. These results imply that a helper factor may participate in the feedback regulation of its own production via an adherent cell population.