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PubMed2002

Cellular function in multicellular system for hormone-secretion: electrophysiological aspect of studies on alpha-, beta- and delta-cells of the pancreatic islet.

We review a new method to explore the cellular functions in multicellular system by application of the perforated patch-clamp technique to intact pancreatic islet of Langerhans. Using this approach, the integrity of the islet is preserved and intercellular communication via gap junctions and paracrine processes are maintained. By using low-resistance patch electrodes, rapid current responses can be monitored under voltage-clamp control. We have applied this methodology to answer questions not resolved by patch-clamp experiments on isolated single insulin-secreting beta-cells. First, the role of a K(+)-current dependent on Ca(2+)-influx for the termination of burst of action potentials in beta-cells could be documented. Neither the current, nor the bursting pattern of electrical activity is preserved in isolated beta-cells. Second, the conductance of gap junctions (approximately 1 nS) between beta-cells was determined. Third, electrical properties of glucagon-producing alpha- and somatostatin-secreting delta-cells and the different mechanisms for glucose-sensing in these cells could be explored. The findings emanating from these experiments may have implications for neuroscience research such as the mechanism of oscillatory electrical activity in general and processes involved in the glucose-sensing in some neurons, which response to changes of blood glucose concentration.

Animals↗
PubMed1997

In vitro confirmation of newly established lung cancer cell lines using flow cytometry and multicellular tumor spheroids.

We report on a simplified method of cytomorphological in vitro confirmation of newly established lung cancer cell lines by using multicellular tumor spheroids (MTS) and flow cytometry (FCM). Eleven cell lines were established from 11 patients with lung cancer. The MTS were produced by culturing cells in agar-coated dish. Cytomorphological studies were made using smears of crushed MTS and frozen sections of MTS. The MTS were fixed doubly with paraformaldehyde and osmic acid for scanning and transmission electron microscopy. Bivariate fluorescence of fluorescein isothyocyanate (FITC, tumor associated antigen, TAA) and propidium iodide (DNA) were measured by FCM. The MTS grew anchorage-independently. Cytopathological and electron microscopic findings of MTS were similar to those of the original clinical specimens. The DNA index and TAA were useful in evaluating the presence or absence of contamination by cells of non-tumor origin. The new cell lines satisfied a minimum of four conditions to confirm their establishment: (a) they originated from humans, (b) they were cytomorphologically identified with specimens from primary lesions, (c) they showed tumorigenicity, and (d) they were free from contamination by cells of different origin. From these findings, the establishment of new cell lines can be confirmed in vitro by using MTS and FCM.

Antigens, Neoplasm↗
PubMed2001

Peptide pheromone-dependent regulation of antimicrobial peptide production in Gram-positive bacteria: a case of multicellular behavior.

Quorum sensing enables unicellular organisms to behave in a multicellular way by allowing population-wide synchronized adaptive responses that involve modulation of a wide range of physiological responses in a cell density-, cell proximity- or growth phase-dependent manner. Examples of processes modulated by quorum sensing are the development of genetic competence, conjugative plasmid transfer, sporulation and cell differentiation, biofilm formation, virulence response, production of antibiotics, antimicrobial peptides and toxins, and bioluminescence (for reviews see [38]). The cell-to-cell communication strategies involved in these processes are based on the utilization of small signal molecules produced and released into the environment by the microorganisms. These communication molecules are referred to as pheromones and act as chemical messengers that transmit information across space. The extracellular pheromones accumulate in the environment and trigger a response in the target cells when its concentration reaches a certain threshold value. Elucidation of the chemical nature of the pheromones modulating the processes mentioned above reveals that most of them are unmodified peptides, post-translationally modified peptides, N-acyl homoserine lactones, or butyrolactones. Lactone-based pheromones are the preferred communication signals in Gram-negative bacteria (for review see [47,48]), whereas peptide-based pheromones are the predominant extracellular signals among Gram-positive bacteria (for review see [37,61]). However, lactone-based pheromones are utilized as signals that modulate differentiation and secondary metabolism production in Streptomyces (for review see [20]). This review focuses on the major advances and current views of the peptide-pheromone dependent regulatory circuits involved in production of antimicrobial peptides in Gram-positive bacteria.

Anti-Bacterial Agents↗
PubMed1999

The role of gravity in the evolutionary emergence of multicellular complexity: microgravity effects on arthropod development and aging.

While experiments carried out in Space with isolated cells have shown that eucaryotic cells are able to sense and respond to the absence of gravity by modifying their reactions, experiments in which more complex processes have been investigated, such as Biological Systems undergoing development under Microgravity, have been surprisingly unaffected by the space environment. This can be considered a curious result since all organisms are evolutionarily adapted to the current level of the gravity force in our planet and should eventually change if this parameter will vary in a permanent manner. In fact, the small effects of the modifications in gravity on development in short term experiments may be equivalent to the difficulties in detecting the involvement of other basic physical processes such as diffusion-controled auto-organizative reactions in currently developing biological systems. An apparent exception to this lack of effect is experiments where brine shrimp dormant gastrulae directly exposed to the space environment accumulate developmental defects as a consequence of cosmic irradiation. In this article we discuss the idea that at a certain stage during the evolutionary emergence of multicellular organisms the cues laid by generic forces such as gravity were involved in the evolutionary organization of these primitive organisms. As evolution proceed, these early mechanisms may have been obscured and/or made redundant by the appearance of new internal, environment-independent biological regulatory mechanisms. On the other hand, behavioral responses that may be important, for example, in setting the life-spans of organisms may still be more readily susceptible to manipulation by external cues as experiments carried out by our group in Space and on the ground with Drosophila melanogaster indicate. Grant Numbers: ESP-1775.

Animals↗
PubMed2003

Bacterial multicellularity as a possible source of antibiotic resistance.

Knowledge about survival of micro-organisms in stressful situations not only influences the evolutionary theory in a fundamental way, but bears an extraordinary importance in finding a global solution to a very concrete urgent problem of mankind, namely bacterial resistance to antibiotics. Recent in vitro experiments demonstrate that the adaptive mutation process involving transient hypermutators could be one of the most important mechanisms whereby bacterial cells achieve the antibiotic resistance. An effective response of the mutation rates to specific selective conditions and an increasing number of conclusive evidence that bacterial cells are indeed communicative and co-operative organisms lead us to a hypothesis that the emergence of the antibiotic resistant mutants through the so-called adaptive mutation is deeply connected with the multicellular organisation of bacterial cells.

Anti-Bacterial Agents↗
PubMed1995

Volume estimation of multicellular colon carcinoma spheroids using Cavalieri's principle.

Multicellular tumour-spheroids are regarded as suitable models for cancer research, similar to avascular tumour parts. As a size parameter of the spheroids, usually their maximum diameter is used, estimated on a section presumed to be equatorial or near equatorial. Estimation of the volume of spheroids is of interest for the detection of subtle changes in different kinds of investigations. Since spheroids are often not truly spherical, and because model-based methods for volume determination may be biased, Cavalieri's principle, rediscovered recently for stereology, was used to determine the volume of the spheroids. Here we report the results of the volume estimation of colon carcinoma spheroids, together with an outline of the basic stereological principle and formulas used. The spheroids investigated had a volume between 1.4 and 92.3 mm3 (mean 33.9). The volume fraction of necrotic to viable tissue cells was between 0.6:1 and 2.2:1. The coefficient of error (CE) was remarkably low with 3.7% for the volumes. Both inter- and intraobserver-variability were extremely low with correlation coefficients (r2) of 99%. Thus, the high precision of the stereological method, combined with a low workload, make it ideally suitable for routine volume estimation.

Carcinoma↗
PubMed1998

Extravascular diffusion of tirapazamine: effect of metabolic consumption assessed using the multicellular layer model.

PURPOSE: Hypoxia-selective cytotoxic agents, like tirapazamine (TPZ), must diffuse considerable distances in tumors to reach their target cell population. This study uses a new three-dimensional tissue culture model, in which cells are grown as multicellular layers (MCL), to investigate whether metabolic consumption of TPZ is sufficiently rapid to compromise its extravascular diffusion in tumors. METHODS AND MATERIALS: V79-171b and MGH-U1 cells were grown as MCL to thicknesses of approximately 120 and 360 microm respectively. The extent of hypoxia in MCL, as assessed by EF5 binding, was modulated by altering gas-phase O2 content, and flux of TPZ through MCL was investigated by high-performance liquid chromatography (HPLC). Data were fitted to a diffusion-reaction mathematical model to determine the diffusion coefficient of TPZ in the MCL (DM) and the rate of its metabolic consumption under anoxia. These parameters were used to simulate TPZ transport in tumors. RESULTS: The flux of TPZ through well-oxygenated MCL (equilibrated with 95% O2) was well fitted as Fickian diffusion without reaction, with a D(M) of 7.4 x 10(-7) cm2s(-1) (12-fold lower than in culture medium) for V79 and 1.3 x 10(-6) cm2s(-1) for MGH-U1 MCL. Flux of TPZ was suppressed under anoxia, and fitting the data required inclusion of a reaction term with a rate constant for metabolic consumption of TPZ of 0.52 min(-1) for V79 and 0.31 min(-1) for MGH-U1 MCL. These transport parameters would translate into a 43% or 30% decrease respectively in TPZ exposure, as a result of drug metabolism, in the center of a slab of anoxic tissue 100 microm in thickness. CONCLUSIONS: MCL cultures provide an in vitro model for investigating the interaction between metabolic consumption and diffusion of bioreductive drugs. If rates of diffusion and metabolism similar to those measured in V79 and MGH-U1 MCL apply in tumors, then cells in large confluent regions of hypoxia would be partially protected by failure of TPZ penetration. Simulation of extravascular transport of TPZ-like bioreductive drugs demonstrates that the optimum metabolic rate constant is determined by two competing requirements: it should be high enough to ensure potent cytotoxicity under hypoxia, yet low enough that penetration is not severely compromised.

Animals↗
PubMed1997

Mechanotransduction of hair bundles arising from multicellular complexes in anemones.

Sea anemones are among the simplest animals to use hair bundles to detect vibrations. Although we previously found anemone bundles to be morphologically similar to vertebrate hair bundles, only indirect evidence implicated anemone bundles in mechanotransduction. Here, we test mechanotransduction of these bundles using loose-patch current recording from apical membranes of cells at the base of deflected bundles. Step bundle deflection results in graded membrane currents that are inward in some cells (positive) and outward in other cells (negative). Positive responses range from 5 to 30 pA, abruptly saturate with stronger stimuli, and increase in duration with prolonged deflections. Negative responses range from 10 to 150 pA, show a logarithmic relation to stimulus strength, and attenuate with prolonged deflections. Additionally, responses are reversibly inhibited by streptomycin. We present a model for anemone bundle mechanotransduction modified from the gating spring model for vertebrate mechanotransduction. Because anemone bundles comprise stereocilia arising from a multicellular complex, we propose that supporting cells on opposite sides of a bundle function as oppositely polarized hair cells. Thus, deflection induces ion channels to open in cells on one side of the complex, while allowing channels to close in cells on the opposite side of the complex.

Adaptation, Physiological↗
PubMed1993

Synergistic interaction of natural human tumor necrosis factor-alpha/natural human interferon-alpha mixture alone and in combination with cisplatin against head and neck laryngeal squamous carcinoma multicellular tumor spheroids.

We evaluated the effects of each component, natural human tumor necrosis factor-alpha (nHuTNF-alpha) and natural human interferon-alpha (nHu-IFN-alpha), and the combined nHuTNF-alpha/nHuIFN-alpha preparation with or without cisplatin (DDP) at tumor mass level. Multicellular tumor spheroids (MTS) of approximately 500 microns in diameter were produced from HEp-2 laryngeal squamous carcinoma cell line by liquid overlay culture technique. Cell kill effects of 72 hr exposure to nHuTNF-alpha, nHuIFN-alpha, or nHuTNF-alpha/nHuIFN-alpha against cells in MTS were 2-3-fold less than those in monolayer. In MTS, does response curves become progressively flat at high drug concentrations, indicative of poor drug penetration into the MTS core. Combination nHuTNF-alpha/nHuIFN-alpha produced synergistic cell kill for both MTS and monolayers. For monolayer cells exposure to nHuTNF-alpha/nHuIFN-alpha first (72 hr) followed by DDP (1 hr) after 1 hr rest period was synergistic with combination index (CI) of approximately 0.8 at LD50. Simultaneous (72 hr in nHuTNF-alpha/nHuIFN-alpha with DDP in last 1 hr) or reverse order of exposure were antagonistic. In contrast, for MTS, DDP followed by nHuTNF-alpha/nHuIFN-alpha was most synergistic with CI of approximately 0.2. Simultaneous exposure or nHuTNF-alpha/nHuIFN-alpha followed by DDP showed synergism with CI of approximately 0.5 and 0.8, respectively. The improved efficacy of DDP followed by nHuTNF-alpha/nHuIFN-alpha sequence for MTS cells appears to be due to increased nHuTNF-alpha/nHuIFN-alpha penetration into the MTS core aided by DDP.

Carcinoma, Squamous Cell↗
PubMed1999

Redox-regulation of intrinsic prion expression in multicellular prostate tumor spheroids.

The cellular function of the intrinsic prion protein (PrPc) remains largely unknown. In the present study PrPc expression was investigated in multicellular prostate tumor spheroids and was correlated to the intracellular redox state as evaluated using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In small tumor spheroids (diameter 100 +/- 20 microm) reactive oxygen species (ROS) levels were increased as compared with large (diameter 250 +/- 50 microm) spheroids. ROS generation was mediated by the mitochondrial respiratory chain and a NADPH oxidaselike enzyme, because carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, and diphenylene iodonium chloride (DPI) significantly reduced ROS levels. The elevated ROS were correlated to an increased expression of PrPc, Cu/Zn superoxide dismutase (SOD-1), and catalase in small as compared with large spheroids. In large tumor spheroids, PrPc was predominantly expressed in the peripheral cell layers and colocalized with SOD-1 and catalase. Raising intracellular ROS in large tumor spheroids by hydrogen peroxide, menadione, buthionine sulfoximine (BSO), and incubation in glutamine-reduced medium increased PrPc expression. In small spheroids PrPc was downregulated after incubation with the radical scavengers dehydroascorbate (DHA) and vitamin E. Our data indicate that PrPc expression in tumor spheroids is related to the intracellular redox state and may participate in antioxidative defense.

Antioxidants↗
PubMed2001

A multicellular spheroid-based sensor for anti-cancer therapeutics.

The progress in cellular engineering offers novel approaches for anti-cancer therapies. To investigate the effectiveness of potential therapies efficient screening methods are required. We propose an impedance measurement system which enables the use of multicellular spheroid models in bioelectronic screening systems either for non destructive life-time diagnostic or anti-cancer therapies. A biohybrid sensor system is created comprising gene-manipulated T47D clone 11 breast carcinoma spheroids positioned hydrodynamically in a capillary system with electrodes. A novel approach employing an antisense-5'butyrylcholinesterase expression system is probed on reaggregated tumor cells under simulated microgravity, inhibiting the gene transcription and translation of the embryonic proliferation marker butyrylcholinesterase expressed in different tumor types. Alterations in the morphology of cell aggregates e.g. apoptosis or necrosis can be detected by impedance spectroscopy monitoring the electric behavior of membranes and extracellular space with a high resolution and reproducibility. The hydrodynamic positioning of 3D in vitro cell aggregates and the short time for the measurements represent an innovative method for a synchronized multicapillary screening system. The combination of the measuring system with a bioreactor enables cyclic life time recordings of impedance spectra for monitoring the cell aggregate properties for a long period.

Antineoplastic Agents↗
PubMed1992

Genetic novelties in mitochondrial genomes of multicellular animals.

Mitochondrial genomes of multicellular animals are mostly small, circular molecules in which 13 protein genes, two ribosomal-RNA genes and 22 transfer-RNA genes are closely packed. Substantial rearrangements of genes have only occurred between phylogenetically distant organisms. However, a wealth of genetic novelties are found among these genomes that include modified genetic codes, unorthodox translation initiation codons, and structurally modified RNA components of the mitochondrion's translation system.

Animals↗
PubMed1996

Interacting signaling pathways controlling multicellular development in Dictyostelium.

cAMP functions as the key extracellular signaling molecule controlling Dictyostelium development acting through classic G-protein-coupled/serpentine receptors. Whereas aggregation is controlled by nanomolar pulses of cAMP, a more continuous micromolar signal controls multicellular differentiation by activating a transcriptional cascade via a receptor-mediated but non G-protein-coupled pathway. Potential mechanisms by which extracellular cAMP functions to differentially control aggregation followed by morphogenesis and cell-type differentiation are discussed. This review also summarizes new findings elucidating pathways controlling cell-type regulation in this organism, including signaling cascades mediated by glycogen synthase kinase 3 and cAMP-dependent protein kinase, key regulators of cell-type differentiation in metazoans, and newly identified transcription factors.

Animals↗
PubMed1997

Hypofractionated radiation induces a decrease in cell proliferation but no histological damage to organotypic multicellular spheroids of human glioblastomas.

The aim of this study was to examine the effect of radiation on glioblastoma, using an organotypic multicellular spheroid (OMS) model. Most glioblastoma cell lines are, in contrast to glioblastomas in vivo, relatively radiosensitive. This limits the value of using cell lines for studying the radiation effect of glioblastomas. The advantage of OMS is maintenance of the characteristics of the original tumour, which is lost in conventional cell cultures. OMS prepared from four glioblastomas were treated with hypofractionated radiation with a radiobiologically equivalent dose to standard radiation treatment for glioblastoma patients. After treatment, the histology as well as the cell proliferation of the OMS was examined. After radiation, a significant decrease in cell proliferation was found, although no histological damage to the OMS was observed. The modest effects of radiation on the OMS are in agreement with the limited therapeutic value of radiotherapy for glioblastoma patients. Therefore, OMS seems to be a good alternative for cell lines to study the radiobiological effect on glioblastomas.

Cell Division↗
PubMed1997

Influence of drug exposure parameters on the activity of paclitaxel in multicellular spheroids.

Paclitaxel is a chemotherapeutic drug which has clinical activity against several solid tumours including ovarian and metastatic breast cancers. Despite extensive preclinical evaluation in several experimental models, no studies have determined the effect of taxol on multicellular spheroids, a model which closely mimics the microregions of solid tumours. MCF-7 human breast carcinoma spheroids were significantly less sensitive than monolayers with IC50 values of 14.33 +/- 4.51 microM and 0.15 +/- 0.09 microM, respectively, following a 1 h drug exposure. Similarly, DLD-1 human colon carcinoma spheroids were also more resistant (IC50 = 33.0 +/- 8.89 microM) than monolayers (IC50 = 0.36 +/- 0.14 microM) following a 1 h drug exposure. Paclitaxel was unable to penetrate DLD-1 multicell layers (22 microns in thickness), suggesting that suboptimal drug exposures to paclitaxel occur in cells which reside some distance away from the surface of the spheroid. In the case of DLD-1 spheroids, extending the exposure time to 24 h whilst maintaining the same overall concentration x time (C x T) drug exposure parameters, resulted in greater cell kill (C x T required to kill 50% of cells = 13.67 +/- 3.21 microM/h) compared with 1 h drug exposures (C x T required to kill 50% of cells = 33.00 +/- 8.89 microM/h). Similar results were obtained with MCF-7 spheroids. In monolayers cultures, dose-response curves contained a marked plateau phase (a characteristic feature of cell cycle phase specific drug) and in the case of MCF-7 cells, cell kill was proportional to T as opposed to C x T. These results support the use of prolonged infusions of paclitaxel in the clinic, as extending the duration of drug exposure not only allows more cells to enter sensitive phases of the cell cycle, but would also allow paclitaxel more time to penetrate into avascular regions of solid tumours. It is likely that paclitaxel will only be effective against cells which reside close to tumour blood vessels and combination therapy with bioreductive drugs (such as tirapazamine) may produce synergistic effects in vivo.

Antineoplastic Agents, Phytogenic↗
PubMedDate not supplied

Multicellular resistance: a paradigm for clinical resistance?

Research on resistance to cancer treatment was mainly focused for 20 years on multidrug resistance (MDR). No useful method of reversing MDR, suitable for clinical use, has yet emerged from this large quantity of work. The reason could be an inadequate evaluation of the target. When grown in spheroids, cancer cells exhibit a phenomenon known as 'multicellular resistance' (MCR). Tumours in patients seem to present the same characteristics. The mechanisms underlying MCR can be classified into two forms: contact resistance and resistance inherent in the spheroid structure. Mechanisms of MCR include: inhibition of apoptosis, high proportion of quiescent cells, modulation of protein expression (including topoisomerases and repair enzymes), potential permeability problems, presence of a hypoxic and necrotic centre and other possible mechanisms that remain to be discovered. A new therapeutic class of drugs is required to overcome MCR. Compounds, which are able to disrupt communication and binding between tumour cells and their microenvironment, seem to be able to circumvent MCR. Interesting results are obtained in vitro and in vivo in mice with specific antibodies or peptides recognised by cell binding proteins. Interestingly, these compounds also appear to be able to inhibit metastasis. Hyaluronidase has already been used with anticancer drugs in patients and was shown to increase drug potency. The explanation given is that it improves drug penetration into spheroids. We now hypothesise that hyaluronidase, in fact, decreases MCR and thus could be the first member of a new therapeutic class.

Animals↗
PubMed2001

Identifying 'prime suspects': symbioses and the evolution of multicellularity.

The possible involvement of symbioses in the evolution of multicellularity is explored. Evidence is drawn principally from the biology of present day associations of plants and animals with prokaryotes. A particular emphasis is placed on future research opportunities in this area of biology that have been provided by the advent of specific molecular techniques and new model systems. With the application of new approaches that result from these advances, a more holistic understanding of the biology of the coevolved communities, composed of animals or plants and their associated prokaryotes, is within the reach of biologists over the next few decades.

Animals↗
PubMed1999

Evolution of multicellularity in the volvocine algae.

Recent studies reveal that relationships among the volvocine algae are more complex than was previously believed. Nevertheless, this group still appears to provide an unrivaled opportunity to analyze an evolutionary pathway leading from unicellularity (Chlamydomonas) to multicellularity with division of labor (Volvox). Significant progress in this regard was made in the past year when two genes playing key roles in Volvox cellular differentiation were cloned, and clues were uncovered regarding their mechanisms of action.

Animals↗