Evaluation of particulate systems supporting tumor cell fractions in a preventive vaccination against intracranial rat glioma.

OBJECT: Irradiated autologous tumor cells are commonly used as a source of antigens in antiglioma vaccinations to activate the immune system. As cell number is often a limiting factor in these cells' preparation, the aim of the present study was to find a means that can lower the amount of cells required. Among strategies currently developed, adjuvant particulate systems offer a promising means to improve the antitumor immune response. In this study, the authors were interested in evaluating the role of particulate systems containing biodegradable microspheres that carry tumor cell fractions on their surfaces in the induction of a protective immunity in the 9L/Fischer 344 rat glioma model. The efficiency of these particulate systems was compared to that of irradiated 9L cells. METHODS: Particulate systems composed of poly(D,L-lactide-co-glycolide) (PLGA) microspheres that support 9L cell fractions on their surfaces (cell lysates or plasma membranes) or irradiated 9L cells alone were injected subcutaneously into the flanks of syngeneic Fischer 344 rats. Eighteen days later, the rats were intracranially injected with nonirradiated 9L cells. A study of survival in these animals and an analysis of the resulting immune response were then conducted. For the same amount of protein (50 microg) injected, irradiated 9L cells provided long-term survival in 30% of animals, whereas 9L plasma membranes adsorbed onto PLGA microspheres provided long-term survival in 10% of animals and cell lysates adsorbed onto microspheres provided long-term survival in 0%. Accordingly, particulate systems induced a lower T helper cell Type 1 (Th1) peripheral immune response than irradiated 9L cells. However, greater secretion of Th1 cytokines was observed when particulate systems were used than when cell fractions separated from microspheres were used, indicating the adjuvant property of these particulate systems. CONCLUSIONS: Particulate systems have adjuvant properties but are still less efficient than irradiated whole tumor cells for vaccinations. Encapsulation of an activating molecule in the microsphere will be the next developmental step in the search for efficient antiglioma vaccinations.

Enhancement of hyperthermic damage on M14 melanoma cells by liposome pretreatment.

Exponentially growing human melanoma cells (M14 cell line) were pretreated with various amounts of dipalmitoylphosphatidylcholine-containing multilamellar liposomes and then exposed to heat treatment (42.5 degrees C). Cell damage produced by the treatments, given separately or in combination, was evaluated in terms of cell survival. Our results demonstrate that the cell survival at 37 degrees C was not affected by liposome concentrations up to 1000 nmol of phospholipid/2.5 x 10(6) cells, while liposome treatment of cells before heat exposure determined a marked damaging effect even at 100 nmol of phospholipid/2.5 x 10(6) cells. The mechanisms of liposome-cell interaction have been investigated by electron microscopy or by electron spin resonance measurements of spin-labeled membranes of intact cells. Evidence has been obtained that liposomal lipids are either taken up by M14 cells or become incorporated in the cell membrane. The present data suggest the possibility that liposome treatments per se could be of potential value as a therapeutic approach, by increasing the effect of heat therapy.

Taccalonolides E and A: Plant-derived steroids with microtubule-stabilizing activity.

During the course of a mechanism-based screening program designed to identify new microtubule-disrupting agents from natural products, we identified a crude extract from Tacca chantrieri that initiated Taxol-like microtubule bundling. Bioassay-directed purification of the extract yielded the highly oxygenated steroids taccalonolides E and A. The taccalonolides caused an increased density of cellular microtubules in interphase cells and the formation of thick bundles of microtubules similar to the effects of Taxol. Mitotic cells exhibited abnormal mitotic spindles containing three or more spindle poles. The taccalonolides were evaluated for antiproliferative effects in drug-sensitive and multidrug-resistant cell lines. The data indicate that taccalonolide E is slightly more potent than taccalonolide A in drug-sensitive cell lines and that both taccalonolides are effective inhibitors of cell proliferation. Both taccalonolides are poorer substrates for transport by P-glycoprotein than Taxol. The ability of the taccalonolides to circumvent mutations in the Taxol-binding region of beta-tubulin was examined using the PTX 10, PTX 22, and 1A9/A8 cell lines. The data suggest little cross-resistance of taccalonolide A as compared with Taxol, however, the data from the PTX 22 cell line indicate a 12-fold resistance to taccalonolide E, suggesting a potential overlap of binding sites. Characteristic of agents that disrupt microtubules, the taccalonolides caused G(2)-M accumulation, Bcl-2 phosphorylation, and initiation of apoptosis. The taccalonolides represent a novel class of plant-derived microtubule-stabilizers that differ structurally and biologically from other classes of microtubule-stabilizers.

Transfer of proteins to membranes facilitates both cyanogen bromide cleavage and two-dimensional proteolytic mapping.

We have found that the transfer of gel-fractionated proteins to membranes facilitates phosphopeptide mapping. Nitrocellulose proves to be an excellent matrix for both cyanogen bromide cleavage and proteolytic digestion. Digestion of p56lck bound to a nitrocellulose membrane with cyanogen bromide or trypsin generated patterns of phosphopeptides indistinguishable from those produced by digestion of p56lck eluted from a gel. Immobilon-P and nylon membranes can also be used for proteolytic mapping, but not for cyanogen bromide cleavage. Since the use of membrane-bound protein eliminates the need for elution and precipitation of the protein, analysis is rapid. In addition, the recovery of the peptides from proteins digested on membranes is better and more consistent than it is from eluted and precipitated proteins.

A new technical approach for the ultrastructural analysis of hematopoietic cells using cell monolayers adherent to electron-transparent melamine resins.

The melamine resin is a polymer of a hexamethylol melamine ether which can coat glass slides with an electron-transparent foil (approximately 80 nm thick) after polymerization by p-toluene sulphonic acid and warming. Provided that the cells had been resuspended in a serum-free medium, normal peripheral blood, or bone marrow cells, blasts of different acute leukemias, cells of B-cell chronic lymphocytic leukemia, and of the cell-lines K562, KG1a, and HL-60 became adherent to the melamine-resin-covered glass slides. The optimal sedimentation time and cell concentration was 45 min and 10(7) cells/ml, respectively. Moreover, in serum-free culture medium the cells could be maintained adherent for up to 96 h without a great loss in cell number and viability. For transmission electron microscopical (TEM) analysis, the monolayers could be embedded in situ in epon after routine fixation and staining procedures. Alternatively, the foils could be removed from the glass and mounted on grids for whole mount electron microscopic analysis (WMEM). Both methods could be combined with immunogold labelling for the detection of surface antigens. This technique permits ultrastructural in situ analysis of morphological and/or immunological changes of cells induced by in vitro stimulation.

Liposome-associated retinoic acid. Increased in vitro antiproliferative effects on neoplastic cells.

The activity of liposome-associated retinoic acid was analyzed on in vitro cultured tumor cell lines and compared to the antiproliferative effects of free retinoic acid. It was found that liposome-associated retinoic acid is about 300 times more active than free retinoic acid in inhibiting in vitro cell growth of leukemic and melanoma cell lines. An increased activity of retinoic acid (10-20 times) was also obtained after premixing of this compound with empty liposomes, demonstrating that the retinoic acid efficiently interacts with liposomes which may facilitate solubility and cell uptake of retinoids.

Paramagnetic microspheres as immunological markers for light and electron microscopy.

M-450 Dynabeads are magnetizable polystyrene microspheres 4.5 micron in diameter to which antibodies of IgM isotype can be physically adsorbed. Antibody-coated Dynabeads can be used to label cell surfaces and then to separate the rosetted cells by application of an external magnetic field. We demonstrate here, using cell lines K562 and U937 and the previously undescribed monoclonal antibodies CH-F42 and CH-E25, that Dynabeads can also be used to label cells at the ultrastructural level. Dynabeads can therefore provide a useful bridge between light and electron microscopy. The preservation of specific rosettes at the ultrastructural level without the formation of artifactual aggregates requires rapid but gentle fixation in dilute suspension. We have achieved this by fixing briefly in a large volume of buffered glutaraldehyde followed by neutralization of excess glutaraldehyde with ethanolamine.

In vitro matrix assembly induced by critical assembly concentration (CAC).

L-2 cells are an immortalized cell line derived from yolk sac parietal endoderm cells, which are responsible for the production of Reichert's membrane, a thick basement membrane produced during rat gestation. Although the L-2 cells secrete all the major components of the basal lamina, they do not assemble a robust matrix in cell culture. We hypothesized that the reason L-2 cells fail to assemble a matrix in cell culture is because the concentrations of matrix components necessary for this matrix assembly do not reach a critical association concentration (CAC) under standard cell culture conditions. To limit the diffusion of secreted molecules while maintaining a nutrient-rich environment for the cells to thrive, we developed a technique that uses a dialysis membrane to limit protein diffusion in a 2-well plate format. This technique permits L-2 cells to assemble a robust matrix in as little as 24 hr that continues to be formed for at least 72 hr. This technique may address some of the physical limitations imposed by cell culture and could be readily applied to other cell types and medium conditions.

EB1 identifies sites of microtubule polymerisation during neurite development.

EB1 is a microtubule associated protein which interacts with the APC tumour suppressor protein and components of the cytoplasmic dynein/dynactin complex. EB1 is also a specific marker of growing microtubule tips. Here we demonstrate that EB1 protein levels are increased during axon but not dendrite formation in differentiated N2A neuroblastoma cells, and that EB1 localises to microtubule tips throughout extending neurites in these cells. In N2A axons, analysis of the ratio of EB1/beta-tubulin fluorescence demonstrated that the distal tip region contained the highest proportion of polymerising microtubules. Time-lapse confocal imaging of an EB1-GFP fusion protein in transfected N2A cells directly revealed the dynamics of microtubule extension in neurites, and demonstrated the existence of unusual, discrete knots of microtubule polymerisation at the periphery of non-process bearing cells which may represent an early event in neurite outgrowth. We conclude that EB1 localisation can be used to identify and analyse sites of microtubule polymerisation at a high resolution during neurite development, a process to which it may contribute.

A fibrous-bed bioreactor for continuous production of developmental endothelial locus-1 by osteosarcoma cells.

Genetically engineered human osteosarcoma cells containing developmental endothelial locus-1 (del-1) gene were studied for production of Del-1, a protein that has the properties of an extracellular matrix protein and can regulate vascular morphogenesis and remodeling. Del-1 has been studied as a potential anti-angiogenesis drug targeting solid tumors. In this study, osteosarcoma cells were cultured in a fibrous-bed bioreactor (FBB) to continuously produce Del-1. The FBB was constructed by packing a polyester fibrous matrix into a 1.5-l spinner flask. The effects of media composition, including the serum content in the medium, and dilution rate on cell growth, metabolism, and Del-1 production were studied. A gradual reduction of serum content from 10% (v/v) to 0.5% (v/v) caused no loss in Del-1 production. However, the production of Del-1 decreased significantly in a serum-free medium, suggesting some nutrients present in the serum were important to culture viability and Del-1 production. The continuous FBB culture was stable for long-term production of Del-1, with a higher Del-1 titer than that normally obtained in T-flask cultures and overall productivity similar to the total production from 300 25-cm(2) T-flasks. Reducing geneticin in the medium from 250 microg ml(-1) to zero at later culturing stages had no significant effect on Del-1 production. The FBB was operated for a period of more than 4 months without any notable degeneration, and reached a final cell density of 3 x 10(8) cells ml(-1) of packing volume with >90% cell viability. The good reactor performance can be attributed to the three-dimensional environment provided by the fibrous matrix that allows for efficient mass transfer and cell immobilization and growth. Scanning electron microscopic and confocal scanning laser microscopic studies of the cell-matrix showed that cells formed large aggregates in the fibrous matrix and cell density was relatively uniform in the matrix.

Effects of the lipophilic anticancer drug teniposide (VM-26) on membrane transport.

The epipodophyllotoxin glucopyranosides have previously been shown to interact with membrane lipids and to alter the activity of several lipid-embedded membrane proteins. To determine if these agents are acting as general membrane perturbants, we have further examined their effects on membrane processes in Ehrlich ascites tumor cells. [3H]VM-26 and [3H]VP-16 were taken up rapidly and concentrated within the cells in proportion to their lipophilicity. Neither agent was found to have any significant effect on the influx of L-[3H]leucine or alpha-[3H]aminoisobutyric acid. Likewise, these drugs had no significant effects on the hexose transporter. The nucleoside transporter, which is structurally and functionally similar to the hexose transporter, was dramatically affected, however. VM-26 was a non-competitive inhibitor of equilibrium-exchange influx of cytosine arabinoside in Ehrlich cells with a Ki of 15 microM. Equilibrium-exchange influx increased with temperature in control cells (Q10 = 2) but not in VM-26-treated cells; thus, VM-26 was a more potent inhibitor at higher temperatures. VM-26 also significantly reduced zero-trans influx in Ehrlich, P388, L5178Y, and ML-1 cells, and these effects were immediate in onset. VM-26 inhibited high-affinity binding of the nucleoside transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR), but VM-26 enhanced non-specific NBMPR binding to Ehrlich cells. The apparent specificity of the epipodophyllotoxins for the nucleoside transporter is discussed.

Human osteoblast-like cells phagocytose metal particles and express the macrophage marker CD68 in vitro.

Periprosthetic osteolysis is a major cause of aseptic loosening in artificial joint replacement. It is assumed to occur in conjunction with the activation of macrophages. We have shown in vitro that human osteoblast-like cells, isolated from bone specimens obtained from patients undergoing hip replacement, phagocytose fine particles of titanium alloy (TiAlV). The human osteoblast-like cells were identified immunocytochemically by the presence of bone-specific alkaline phosphatase (BAP). With increasing duration of culture, a variable number of the osteoblastic cells became positive for the macrophage marker CD68, independent of the phagocytosis of particles, with a fine granular cytoplasmic staining which was coexpressed with BAP as revealed by immunodoublestaining. The metal particles were not toxic to the osteoblastic cells since even in culture for up to four weeks massively laden cells were vital and had a characteristic morphology. Cells of the human osteosarcoma cell line (HOS 58) were also able to phagocytose metal particles but had only a low expression of the CD68 antigen. Fluorescence-activated cell scanning confirmed our immunocytochemical results. Additionally, the cells were found to be negative for the major histocompatibility complex-II (MHC-II) which is a marker for macrophages and other antigen-presenting cells. Negative results of histochemical tests for tartrate-resistant acid phosphatase excluded the contamination by osteoclasts or macrophages in culture. Our observations suggest that the osteoblast can either change to a phagocytosing cell or that the phagocytosis is an underestimated property of the osteoblast. The detection of the CD68 antigen is insufficient to prove the monocytic lineage. In order to discriminate between macrophages and osteoblasts additional markers should be used. To our knowledge, this is the first demonstration of cells of an osteoblastic origin which have acquired a mixed phenotype of both osteoblasts and macrophages.

Ultrastructural changes in tumor cells treated with liposomal forms of anticancer drugs.

AIM: To determine the main ultrastructural changes in MCF-7 sublines sensitive and resistant to cytotoxic action of anticancer drugs, resulting from the treatment with conventional and liposomal forms of cisplatin and doxorubicin. METHODS: Electron microscopy, light microscopy, MTT-test. RESULTS: It has been shown that the phenomenon of drug resistance is associated with complication of ultrastructural organization of cells and more high differentiation by the main cytomorphologic characteristics which promote their resistance to cytotoxic action of anticancer preparations. Cytoarchitectonics of all resistant cells possesses common patterns and doesn't depend on the particular drugs toward which the resistance has been developed. It has been shown that the cells of the parental form MCF-7 line are more sensitive to cytotoxic action of doxorubicin than to cisplatin. Liposomal forms of anticancer drugs used at the same concentrations that the conventional ones, especially that of doxorubicin, caused more expressed alterations in ultrastructural organization of cells of all studied sublines with dominance of apoptotic processes. CONCLUSION: Evaluating an effect of equal concentrations of cisplatin and doxorubicin in conventional and liposomal forms, one may conclude on higher cytotoxic action of doxorubicin vs. cisplatin that is expressed in a wider spectrum of ultrastructural changes of cell architectonics in different sublines of MCF-7 cells and higher rate of apoptosis.

[Basic studies on CaO-P2O5-MgO-SiO2-CaF system glass ceramics. 2. Ultrastructural study on interface between culture cells and glass ceramics].

The aim of this study was to determine biocompatibility of glass ceramics and adhesion of cultured cells to glass ceramics. Four established cultured cell lines, human fibrosarcoma cells (HT-1080), human gingival carcinoma cells (Ca9-22), human osteosarcoma cells (NY) and mouse osteoblasts (MC3T3-E1), were used. For phase-contrast and electron microscopic observation they were cultured on substrates of glass ceramics or polystyrene coverslips as a control. The results obtained were as follows. Glass ceramics caused neither cellular degeneration nor death, as revealed by phase-contrast microscopy. By transmission electron microscopy an amorphous structure similar to the basal lamina was observed at the interface between the substrates and Ca9-22, and between glass ceramics and NY. A similar structure sometimes existed between the substrates and MC3T3-E1. On the other hand HT-1080 showed no such structure. The findings suggest that the biocompatibility of glass ceramics was satisfactory. Furthermore, from the clinical point of view it seems to be possible to close the material-tissue interface with epithelial, fibrocytic and osteocytic cells.

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy.

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1-2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.

Imaging liposomes at electron microscopic level: encapsulated colloidal iron as an electrondense marker for liposome-cell interactions.

Colloidal iron was encapsulated into liposomes prepared by different methods to provide an electrondense marker for easy identification of liposomes in cell and tissue culture. Stable colloidal iron solution can be prepared at virtually any concentration. The diameters of more than 75 per cent of the iron particles measured between 1 and 5 nm. Liposomes with a distinct electrondense core were evident at a colloidal iron solution concentration of 0.6 g/l. The colloidal iron labelled liposomes were easily identified in cells after incubation by routine electron microscopic procedures. Liposomes could be found in lysosomes or endosomes of human M21 melanoma cells. Intact, as well as partially degraded liposomes were present after two hours of incubation.

Self-assembly of a high molecular weight basement membrane heparan sulfate proteoglycan into dimers and oligomers.

A high molecular weight basement membrane heparan sulfate proteoglycan, isolated from murine Englebreth-Holm-Swarm tumor, is seen in platinum replicas as an elongated flexible core (Mr = 450,000) consisting of a series of tandem globular domains from which extend, at one end, two to three heparan sulfate chains (average Mr = 80,000 each). This macromolecule will self-assemble into dimers and lesser amounts of oligomers when incubated in neutral isotonic buffer. These molecular species can be separated by zonal velocity sedimentation and assembly is seen to be time- and concentration-dependent. In rotary-shadowed platinum replicas the binding region is found at or near the end of the core at the pole opposite the origin of the heparan sulfate chains. Dimers are double-length structures and oligomers are seen as stellate clusters: in both, the heparan sulfate chains appear peripherally oriented. While isolated cores self-assemble, isolated heparan sulfate chains do not bind intact proteoglycans. Furthermore, proteolytic removal of a non-heparan sulfate containing core moiety destroys the ability of the proteoglycan monomer to form larger species or bind intact proteoglycan, further supporting the binding topography determined morphologically. These negatively charged macromolecular complexes may be important contributors to basement membrane structure and function.

Ultrastructural characterization of two new human endometrial carcinoma cell lines and normal human endometrial epithelial cells cultured on extracellular matrix.

Two new lines of human endometrial carcinoma (HEC) cells, one from an adenocarcinoma and one from a highly metastatic serous papillary carcinoma, were established in culture. Structural and morphologic properties of these cells at early passage were compared with those of cultured normal human endometrial epithelial (NHEE) cells. For these studies, cells were grown on a conventional plastic surface or on an extracellular matrix substrate (Matrigel), and examined by transmission electron microscopy and immunofluorescent light microscopy. The HEC cells appeared morphologically similar on plastic and Matrigel, whereas the NHEE cells showed significantly greater epithelial morphologic differentiation on Matrigel than on plastic. On extracellular matrix, the morphologic differences observed between HEC cells and NHEE cells were primarily of an architectural nature, which may be in part explained by differences between NHEE and HEC cells in the arrangement of actin microfilaments and cytokeratin intermediate filaments. Furthermore, HEC cells displayed extensive networks of vimentin intermediate filaments, which were absent from the NHEE cells. These observations support the hypothesis that architectural deregulation is a prominent feature of endometrial carcinoma, and that cytoskeletal alterations may uncouple HEC cell ultrastructural morphology from the influence of extracellular matrix.

Radioreceptor assay for detergent-solubilized rat insulinoma growth hormone receptors.

Growth hormone receptors from a rat insulinoma cell line, RIN-5AH were solubilized in the non-ionic detergent Triton X-100. A radioreceptor assay based on polyethylene glycol precipitation of the growth hormone: receptor complex showed time-dependent and saturable hormone binding. The affinity in detergent solution for biosynthetic human growth hormone of approx. 6 ng/ml was found similar to that of intact RIN cells. The solubilization and receptor assay conditions described are useful for further characterization and purification of RIN cell growth hormone receptors, which might provide an initial insight into the molecular mechanism of the growth hormone effects on islet beta-cells.

Regulation of cytoskeletal structure in human mammary carcinoma cells MCF-7 by culture substrata.

Three-dimensional cellular structures formed by MCF-7 human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7 cells from S-type to M-type does not occur in cells involved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.