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PubMed1995

Cytolytic effects of autologous lymphokine-activated killer cells on organotypic multicellular spheroids of gliomas in vitro.

Knowledge about lymphokine-activated killer (LAK) cell infiltration and LAK cell cytotoxicity is essential to improve the effectiveness of LAK cell therapy against gliomas. In the present study, organotypic multicellular spheroids (OMS) of glioma tissue were used as a culture model to study the effects of LAK cells on gliomas. Compared to tumour cell lines and spheroids derived from tumour cell lines, OMS have several advantages with respect to preservation of tumour cell heterogeneity and the maintenance of the tumour architecture, e.g. capillaries and extracellular matrix. Four glioma specimens, obtained at surgery, were cultured directly on agarose to form OMS, which were then co-cultured with either autologous LAK cells or autologous non-activated peripheral blood lymphocytes (PBLs). After various time periods of co-cultivation, the OMS were fixed and examined both histologically and immunocytochemically. The present results showed that LAK cells infiltrated the OMS completely within 24 h of co-cultivation and severe cellular damage was observed, whereas PBLs infiltrated the OMS poorly and there was only marginal cellular damage. The present study indicates that OMS of gliomas provide an experimental model to investigate the infiltration and cytotoxicity of LAK cells on glioma tissue in vitro.

Adult↗
PubMed1996

Cryopreservation of organotypic multicellular spheroids from human gliomas.

Fresh human glioma tissue can be cultured on agarose to form organotypic multicellular spheroids (OMS). The major advantage of OMS is the preservation of the cellular heterogeneity and the tumour architecture, which is lost in conventional monolayer cultures. The present study was undertaken to assess the possibilities of storing frozen OMS from seven gliomas which were frozen to determine the viability after thawing. OMS were frozen slowly to -196 degrees C using a programmable freezing machine in culture medium containing 45% serum and 10% of the cryopreservative agent dimethyl sulphoxide (DMSO). After 2 weeks storage at -196 degrees C, quick thawing, and culturing for another week, it appeared that the frozen-thawed OMS were viable and retained their histological characteristics. In addition, it is demonstrated that the cellular constituents of the OMS resumed metabolic and proliferative activities. It is concluded that it is possible to establish frozen stocks of viable glioma OMS. This will enable extensive studies on OMS, such as investigation of the biological behaviour of gliomas by using OMS obtained from primary and corresponding recurrent gliomas. In addition, cryopreservation of OMS makes it possible to correlate the results of in vitro tests on OMS with the patients' responses to similar therapeutic approaches.

Brain Neoplasms↗
PubMed2004

Co-expression of three MEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates during the biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites.

In higher plants, isopentenyl diphosphate (IPP) is synthesised both from the plastidic 2-C-methyl-d-erythritol 4-phosphate (MEP) and from the cytosolic mevalonate (MVA) pathways. Primary metabolites, such as phytol group of chlorophylls, carotenoids and the plant hormones abscisic acid (ABA) and gibberellins (GAs) are derived directly from the MEP pathway. Many secondary metabolites, such as monoterpene indole alkaloids (MIAs) in Catharanthus roseus, are also synthesised from this source of IPP. Using Northern blot and in situ hybridisation experiments, we show that three MEP pathway genes (1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MECS)) and the gene encoding geraniol 10-hydroxylase (G10H), a cytochrome P450 monooxygenase involved in the first committed step in the formation of iridoid monoterpenoids display identical cell-specific expression patterns. The co-localisation of these four transcripts to internal phloem parenchyma of young aerial organs of C. roseus adds a new level of complexity to the multicellular nature of MIA biosynthesis. We predict the translocation of pathway intermediates from the internal phloem parenchyma to the epidermis and, ultimately, to laticifers and idioblasts during MIA biosynthesis. Similarly, the translocation of intermediates from the phloem parenchyma is probably also required during the biosynthesis of hormones and photosynthetic primary metabolites derived from the MEP pathway.

Aldose-Ketose Isomerases↗
PubMed1991

Spatial variation of sequestered calcium in the multicellular stage of Dictyostelium discoideum as assayed by chlortetracycline fluorescence.

We have used chlortetracycline (CTC) as a fluorescent probe to detect the distribution of sequestered calcium in multicellular stages of Dictyostelium discoideum. Tips of late aggregates, slugs and early culminating masses fluoresce very strongly. Most of the fluorescence is intracellular in origin and emanates from a small number of intense punctate sources. The sources correspond in part to autophagic vacuoles vis. neutral-red staining, acidic digestive vesicles, and may also include intracellular organelles; cytoplasmic fluorescence is much weaker in comparison. The level of fluorescence drops in the middle portion of slugs and rises again in the posteriormost region, though not to as high a level as in the tip. This holds good irrespective of whether CTC is applied only in the neighbourhood of the aggregate centre, only in the aggregate periphery, or to the whole aggregate. We infer that there must be a good deal of mixing in the stages leading from aggregation to slug formation; thus the serial order in which cells enter an aggregate does not bear any relation to their ultimate fates. The other implication of our study is that calcium sequestration is much more extensive in prestalk and anterior-like cells than in prespore cells. These findings are discussed with regard to possible implications for pattern formation.

Animals↗
PubMed1979

Induction of cyclic AMP phosphodiesterase by disaggregation of the multicellular complexes of Dictyostelium discoideum.

When the multicellular complexes (slugs) of Dictyostelium discoideum were disaggregated, the activity of phosphodiesterase for adenosine 3':5'-monophosphate increased. The increase occurred first with the enzyme in the particulate fraction of the cell. Cytoplasmic and extracellular phosphodiesterase, as well as the macromolecular inhibitor for this enzyme, also increased at later times. The increase in these activities was blocked by inhibitors of protein and RNA synthesis. This indicates that the increase is not due to the activation of the enzyme, but due to its synthesis de novo, and that the synthesis is induced at the transcriptional level by the loss of cell contact. In the case of cellular phosphodiesterase, inhibitors of protein synthesis (cycloheximide and emetine) not only blocked the increase, but also caused a decrease in activity. The mechanism of such decrease was further investigated.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Rapid formation of multicellular spheroids of adult rat hepatocytes by rotation culture and their immobilization within calcium alginate.

Tyrosine aminotransferase (TAT) induction and albumin secretion abilities were examined in rat hepatocytes immobilized within calcium alginate; the immobilized hepatocytes lost these abilities within a week. An attempt was then made to immobilize multicellular spheroids of hepatocytes for the purpose of stabilizing the liver functions. Although it takes at least 4 days to form spheroids in the conventional method using monolayer-cultured cells, in this study we developed a new method for rapid spheroid formation. Isolated hepatocytes were seeded into a polystyrene dish and incubated on a rotary shaker. Hepatocytes started to aggregate after 6 h of the rotation culture, and spheroids approximately 100 microns in diameter formed within 24 h. The immobilized spheroids had higher TAT induction and albumin secretion abilities, which were maintained for a longer time, than the immobilized nonaggregated cells. Further stabilization was observed in immobilized heterospheroids formed in the presence of nonparenchymal liver cells. This method for the rapid formation of spheroids consisting of hepatocytes and nonparenchymal liver cells could be utilized in the construction of a bioartificial liver support system.

Albumins↗
PubMed1996

A Myxococcus xanthus cell density-sensing system required for multicellular development.

Progression through early Myxococcus xanthus multicellular fruiting body development requires the generation of and response to extracellular A signal. Extracellular A signal is a specific set of amino acids at an extracellular concentration greater than 10 muM. It functions as a cell density signal during starvation that allows the cells to sense that a minimal cell density has been reached and development can proceed. The generation of extracellular A signal requires the products of three asg genes. They have recently been identified as AsgA, a fused two-component histidine protein kinase and response regulator; AsgB, a putative DNA-binding protein; and AsgC, the M, xanthus major sigma factor. Other elements of the A signaling pathway map to the sasB locus and appear to be A signal transducers. These elements are regulators of the earliest A signal-dependent gene, whose promoter is a member of the sigma-54 family. Continued study of the A signaling pathway is expected to identify additional components of this network required for the complex behavioural response of fruiting body formation.

Cell Count↗
PubMed1998

Network thermodynamic model of coupled transport in a multicellular tissue--the islet of Langerhans.

Network thermodynamic modeling via bond graphs was used to describe the water and cryoprotectant additive (CPA) transport in a multicellular tissue. The model is presented as a tool to understand the osmotic behavior of the islets of Langerhans when exposed to ternary aqueous solutions containing an electrolyte and a CPA. It accounts for the effects of the location of cells within the tissue and an interstitial matrix, plus differential permeabilities to water and CPA. The interstitial matrix was assumed to be a porous medium able to store the chemical species being transported. Controlled osmotic stress experiments were conducted on isolated rat pancreas islets to measure the transient volumetric response to step-wise changes in dimethyl sulfoxide, Me2SO, concentration. The model provides a tool for predicting the transient volumetric response of peripheral and interior cells and of interstitial tissue, as well as the build up of solute concentration, during addition and removal of CPAs and freezing and thawing protocols. Inverse solution methods were applied to determine values for standard cell membrane permeability parameters Lp, omega and sigma as well as for the interstitial flow conductivities Kw and Kp'.

Animals↗
PubMed1989

Flow cytometric analysis of intracellular hematoporphyrin derivative in human tumor cells and multicellular spheroids.

Flow cytometry (FCM) has been used to investigate the intracellular fluorescence of hematoporphyrin derivative (HPD) in monolayer and spheroid cultures of WiDr cells. For exponentially-growing monolayer cultures mean cellular fluorescence was directly proportion to the external HPD levels in the range 5-100 micrograms ml-1 (r = 0.99). Heterogeneity of cellular fluorescence was quantified by determining the ratio of the fluorescence value below which were observed values for 98% of the cell population compared to the fluorescence value for 2%. In exponentially-growing cultures, decreasing levels of HPD in the medium led to an increase in the 98:2% ratio, i.e. an increase in heterogeneity of intracellular drug levels. The growth of cells as multicellular spheroids confers a spheroid-size-dependent resistance to photodynamic treatment. With increasing spheroid size (100, 250, 500, 750 and 1000 microns diam.) there was a decrease in mean intracellular HPD levels and a large linear increase in the 98:2% ratio (r = 0.94).

Adenocarcinoma↗
PubMed1998

Study of the effects of dispase in the chemotherapy of multicellular tumour spheroids of small-cell lung carcinoma in man.

Multicellular tumour spheroids (MTS), diameter 650 microm, from PC-6, SBC-1 and NCL-H60 small-cell lung carcinoma cell-lines in man were prepared by the liquid overlay culture method and used to study the influence of treatment with dispase (bacterial neutral protease from Bacillus polymyxa, 1000 units mL(-1)) on the effectiveness of carboplatin, as determined by colony-forming assay. When carboplatin alone was used on monolayers the curve of survival fraction against concentration was exponential in shape, indicating that the drug was active against the monolayer. When MTS were treated with medium concentrations (10(-5) and 10(-4) M) of carboplatin alone the survival fraction-concentration curve showed that the effectiveness of the treatment was less than that against the monolayer. On treatment of MTS with carboplatin and dispase the survival fraction-concentration curve was similar to that obtained for the monolayer and the survival fraction of the core of the MTS was also less than when carboplatin alone was used. These results imply that dispase dissolves the intercellular matrix of the MTS enabling enhanced infiltration of carboplatin into the core of the MTS. Dispase thus indirectly increases the effectiveness of carboplatin.

Antineoplastic Agents↗
PubMed2003

Analysis of nitric oxide consumption by erythrocytes in blood vessels using a distributed multicellular model.

Multiple sets of experimental data have shown that the red blood cell (RBC) consumes nitric oxide (NO) about 600-1000-fold slower than the equivalent concentration of cell-free hemoglobin (Hb). Diffusion barriers of various sources have been suggested to explain this observation. In this work, a multicellular, spatially distributed, two-dimensional model, that describes the production, transport, and consumption of NO in blood vessels and the surrounding tissue, is developed. The model is used to assess the relative significance of NO transport barriers that reduce the rate of NO consumption in the blood. Unlike previous models of this system, the model developed here accounts explicitly for the presence of, and interactions among, a population of RBCs inside the lumen of the blood vessel and is, therefore, better suited to analyze, quantitatively, the contribution of each transport barrier as NO diffuses from its site of synthesis to the interior of the RBCs where it interacts with Hb. The model, which uses experimentally derived parameters, shows that extracellular unstirred boundary layer diffusion alone cannot account for the reduced NO consumption by RBC compared to an equivalent concentration of cell-free Hb. Since this result is reached using a two-dimensional representation of the RBCs, which overestimates the importance of the boundary layer diffusion resistance, it would be expected that in the real three-dimensional case, diffusion through the extracellular boundary layer would contribute even less to the overall mass transfer resistance. Consistent with recent experimental findings, the results of our model suggest that, under physiological conditions, transmembrane (membrane and its associated cytoskeleton layer) diffusion limitations in RBCs represent a key source of resistance for NO uptake by RBCs.

Animals↗
PubMed2006

Crl activates transcription initiation of RpoS-regulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium.

In Salmonella enterica serovar Typhimurium, the stationary-phase sigma factor sigma(S) (RpoS) is required for virulence, stress resistance, biofilm formation, and development of the rdar morphotype. This morphotype is a multicellular behavior characterized by expression of the adhesive extracellular matrix components cellulose and curli fimbriae. The Crl protein of Escherichia coli interacts with sigma(S) and activates expression of sigma(S)-regulated genes, such as the csgBAC operon encoding the subunit of the curli proteins, by an unknown mechanism. Here, we showed using in vivo and in vitro experiments that the Crl protein of Salmonella serovar Typhimurium is required for development of a typical rdar morphotype and for maximal expression of the csgD, csgB, adrA, and bcsA genes, which are involved in curli and cellulose biosynthesis. In vitro transcription assays and potassium permanganate reactivity experiments with purified His(6)-Crl showed that Crl directly activated sigma(S)-dependent transcription initiation at the csgD and adrA promoters. We observed no effect of Crl on sigma(70)-dependent transcription. Crl protein levels increased during the late exponential and stationary growth phases in Luria-Beratani medium without NaCl at 28 degrees C. We obtained complementation of the crl mutation by increasing sigma(S) levels. This suggests that Crl has a major physiological impact at low concentrations of sigma(S).

Bacterial Proteins↗
PubMed1992

Lamina, a novel multicellular form of Methanosarcina mazei S-6.

A novel multicellular form of Methanosarcina mazei S-6 is described. It was termed lamina, and it formed during the exponential growth phase when packets or single cells were grown in 40 mM trimethylamine and a total concentration of 8.3 to 15.6 mM Ca2+ and/or Mg2+, in cultures that were not shaken. A distinct molecular event represented by the increment in expression and a spatial redistribution of an antigen during lamina formation is documented.

Antibodies, Monoclonal↗
PubMed1996

The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.

A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal-dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP-binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O-antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521.

ATP-Binding Cassette Transporters↗
PubMed2003

Novel developmental genes, fruCD, of Myxococcus xanthus: involvement of a cell division protein in multicellular development.

Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development upon nutrient starvation. In the present study, two novel developmental genes, fruC and fruD, of M. xanthus were identified and characterized. The FruD protein has significant amino acid sequence similarity to the DivIVA proteins of many bacteria including Bacillus subtilis. Vegetative cells of the fruD mutant exhibited a filamentous phenotype. The fruC and fruD mutants displayed similar delayed-development phenotypes. The formation of tightly aggregated mounds by fruC and fruD mutants was slower than that by the wild-type strain. Spore formation by the fruC and fruD mutants initiated after 30 h poststarvation, whereas wild-type M. xanthus initiated spore formation after 18 h. The fruCD genes were constitutively expressed as an operon during vegetative growth and development. S1 mapping revealed that transcription initiation sites of the fruCD operon were located 114 (P1) and 55 bp (P2) upstream of the fruC initiation codon. Only the P1 promoter was active during vegetative growth, while both the P1 and P2 promoters were active during development. The FruD protein was produced as a cytoplasmic protein and formed an oligomer during vegetative growth and development.

Amino Acid Sequence↗
PubMed2004

Behavior of Ca(2+) waves in multicellular preparations from guinea pig ventricle.

Ca(+) waves have been implicated in Ca(2+) overload-induced cardiac arrhythmias. To deepen understanding of the behavior of Ca(2+) waves in a multicellular system, consecutive two-dimensional Ca(2+) images were obtained with a confocal microscope from surface cells of guinea pig ventricular papillary muscles loaded with fluo 3 or rhod 2. In intact muscles, no Ca(2+) waves were detected under the resting condition, whereas they were frequently observed during the rest immediately after high-frequency stimulations where cytoplasmic Ca(2+) concentration and Ca(2+) stored in the sarcoplasmic reticulum (SR) were gradually decreasing. The intervals of Ca(2+) waves increased as they occurred later, their amplitudes and velocities remaining unchanged. A SERCA inhibitor reversibly prolonged the wave intervals. In Na(+)-free/Ca(2+)-free medium where neither Ca(2+) influx nor Na(+)/Ca(2+) exchange took place, recurrent Ca(2+) waves emerged at constant intervals in each cell. These results are consistent with the conclusion that the loading level of the SR is critical for induction of Ca(2+) waves. Each cell independently exhibited its own regular rhythm of Ca(2+) wave with a distinct interval. These waves propagated in either direction along the longitudinal axis within a muscle cell, but seldom beyond the cell boundary. In contrast, in partially damaged muscles that showed spontaneous Ca(2+) waves at rest in normal Krebs solution, their propagation often was unidirectional, decreasing in frequency. In these cases, however, Ca(2+) waves rarely moved beyond the cellular boundary. The gradient of the cytoplasmic Ca(2+) concentration was suggested to be the cause of the one-way propagation.

Animals↗
PubMed2002

Load-dependent induction of apoptosis in multicellular myocardial preparations.

Increased mechanical load has been proposed as an inductor of apoptosis, but it is unknown whether this can occur in the range of pre- and afterloads that prevail in the beating heart. We investigated apoptosis in cultured rabbit multicellular myocardial preparations over several days. Muscles contracted in absence of pre- and afterload (unloaded isotonic), in absence of preload but in presence of afterload (unloaded isometric), or in presence of both (loaded isometric). After up to 48 h of continuous contractions, apoptosis was assessed by TdT-mediated nick-end labeling (TUNEL) assay and DNA ladder analysis. In muscles that contracted loaded isometric, apoptosis was detected after 6-24 h. After 48 h, apoptosis was most prominent in this group, reflected by a high level of DNA ladder intensity (DLI; 27.8 +/- 11.5), whereas Bcl-xL (on RNA level) was significantly downregulated, and Fas remained unchanged. In unloaded isometric preparations, apoptosis was significantly less (6.9 +/- 5.9 DLI) and very similar to those contracting unloaded isotonic (6.1 +/- 5.1 DLI). We conclude that load-dependent apoptosis can occur at sarcomere lengths achievable in vivo and may mainly result from increased preload.

Animals↗
PubMed2004

Microenvironmental regulation of proliferation in multicellular spheroids is mediated through differential expression of cyclin-dependent kinase inhibitors.

Multicellular spheroids composed of transformed cells are known to mimic the growth characteristics of tumors and to develop gradients in proliferation with increasing size. This progressive accumulation of quiescent cells is presumably an active process that occurs in response to the microenvironmental stresses that develop within the three-dimensional structure, and, yet, little is known regarding either the signals that induce the cell cycle arrest or the molecular basis for the halt in proliferation. We have previously reported that regulation of cyclin-dependent kinase (CDK) inhibitors (CKIs) differs in monolayer versus spheroid cell culture. In this study, we have examined the expression of three CKIs in EMT6 mouse mammary carcinoma and MEL28 human melanoma spheroids, as a function both of spheroid size and of location within the spheroid. We report that expression of the CKIs p18(INK4c), p21(waf1/cip1), and p27(Kip1) all increase as the spheroid grows and develops a quiescent cell fraction. However, by examining protein expression in discrete regions of the spheroid, we have found that only p18(INK4c) and p27(Kip1) expression positively correlate with growth arrest, whereas p21(waf1/cip1) is expressed predominantly in proliferating cells. Further analysis indicated that, in the quiescent cells, p18(INK4c) is found in increasing association with CDK6, whereas p27(Kip1) associates predominantly with CDK2. In MEL28 cells, CDK2 activity is completely abrogated in the inner regions of the spheroid, whereas in EMT6 cells, CDK2 activity decreases in accordance with a decrease in expression. We also observed a decrease in all cell cycle regulatory proteins in the innermost spheroid fraction, including CDKs, CKIs, and cyclins. Induction of CKIs from separate families, as well as their association with distinct target CDKs, suggests that there may be multiple checkpoints activated to ensure cell cycle arrest in non-growth-conducive environments. Furthermore, because very similar observations were made in both a human melanoma cell line and a mouse mammary carcinoma cell line, our results indicate that these checkpoints, as well as the signal transduction pathways that activate them, are highly conserved.

Animals↗