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PubMed2003

Multicellular dosimetry in voxel geometry for targeted radionuclide therapy.

A software package to investigate absorbed doses and dose-rates at the cellular and multicellular scale has been developed that considers two- and three-dimensional activity distributions and makes use of analytical representations of the point-dose kernels for (131)I, (32)P, and (90)Y. This software allows cell assemblies to be simulated by definition of the number, size, and geometry of cells and their nuclei, and radionuclide uptake can be specified to occur within the nucleus, the cytoplasm, at the membrane, or within the extracellular space. The software has been validated at a cellular scale by comparison with results obtained using spherical geometry, as found in the literature. At a multicellular scale, comparisons were made with a Monte Carlo simulation in voxel geometry. The software has been designed to work within a user-defined voxel geometry. This geometry is useful not only to simulate complex cell assemblies and realistic heterogeneous radionuclide distributions, but will also allow the use of histological and autoradiographic data. Absorbed dose distributions for a single cell calculated using this code varied significantly with activity localization within the cell, and to a lesser extent, with the cellular geometry. At a multicellular level, a two-dimensional heterogeneous activity distribution inferred from a two-dimensional image of a slice throughout a spheroid was used to calculate a dose-rate distribution. This resulted in a heterogeneous dose-rate delivery even for longer-range radionuclides such as (90)Y and (32)P.

Absorption↗
PubMed1994

Rapid acquisition of multicellular drug resistance after a single exposure of mammary tumor cells to antitumor alkylating agents.

BACKGROUND: Clinical drug resistance is either intrinsic (de novo) or often acquired rapidly in conjunction with chemotherapy. By contrast, the selection of drug-resistant mutant cell lines in monolayer culture systems is usually a more protracted process. Sublines of mouse EMT-6 mammary tumor cells selected for resistance to various alkylating agents in vivo after serial passage into syngeneic mice manifest their resistance in vitro only when cultured as three-dimensional multicellular aggregates or spheroids. PURPOSE: We examined whether a single exposure of mouse EMT-6 or human MDA-MB-231 breast cancer cells to alkylating agents in vitro is sufficient for the induction of a resistance phenotype, which may be detected by re-applying the drugs to cells grown as three-dimensional aggregates. METHODS: Mouse EMT-6 and human MDA-MB-231 breast cancer cells cultured as three-dimensional aggregates were exposed to a single dose of alkylating agent for 1-5 days. Aggregates were dispersed, and cells were plated as monolayer cultures for up to 8 weeks to allow for recovery. Colony-forming ability was assessed after a subsequent alkylating-agent exposure of cells cultured as monolayers or three-dimensional aggregates. RESULTS: A single in vitro exposure to 12.5-microM cisplatin (CDDP) for 5 days or 25 microM 4-hydroperoxycyclophosphamide (4-O2H CTX) for 1 or 3 days without changing the medium was sufficient to induce transient but substantial resistance in EMT-6 cells as determined by clonogenic assays. Such resistance was not detected when monolayer cell cultures were used. The concentration of 4-O2H-CTX and the length of time the cells remained in three-dimensional culture after initial exposure to this drug was associated with the degree of subsequent drug resistance of cells grown as three-dimensional cultures. Furthermore, this acquired resistance after a single drug exposure was accompanied by changes in the three-dimensional architecture of the cell aggregates, which now formed much more compact multicellular spheroids. Similarly, a single exposure to 4-O2H-CTX was enough to bring about resistance in MDA-MB-231 cells detectable only in three-dimensional cultures, as well as the change in three-dimensional architecture. CONCLUSIONS: Rapid acquisition of resistance likely represents a physiologic mechanism of adaptation operative at the multicellular level rather than a stable genetic change and may be one of the reasons for the rapid development of drug resistance acquired by tumors in vivo. IMPLICATIONS: In vivo drug exposure may result in transient and low levels of drug resistance that may nevertheless be clinically relevant.

Animals↗
PubMed1991

Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages.

After in-vitro fertilization, 2161 supernumerary embryos were frozen with 1,2-propanediol and sucrose as cryoprotectants at either pronucleate or multicellular (2-6 blastomeres) stages. By the end of March 1990, 494 pronucleate stage embryos and 492 multicellular stage embryos had been thawed and 54 and 47% of them, respectively were considered suitable for transfer. Ongoing pregnancy and implantation rates were 17.9 and 10.7%, respectively for embryos frozen at the pronucleate stage and 5.5 and 4.7% for embryos frozen at the multicellular stage. Ovarian stimulation with human menopausal gonadotrophin (HMG) after pharmacological hypophysectomy with a gonadotrophin releasing hormone agonistic analogue (GnRHa) using a long protocol permitted us to freeze significantly more embryos per cycle (7.2 +/- 4.1) than stimulation with HMG and GnRHa in a short protocol (4.7 +/- 3.4) or stimulation with clomiphene citrate (CC) and HMG (2.7 +/- 1.9). Ongoing pregnancy rates after transfer during the stimulated cycles were similar for the three types of treatment (27.1, 27.3 and 32.1%, respectively). However, ongoing pregnancy rates after frozen-thawed embryo transfers were significantly higher when originating from GnRHa + HMG treatments (14.3 and 14.8%, respectively for long and short protocols) than when originating from CC + HMG treatment (5.6%). Embryo cryopreservation has permitted the ongoing pregnancy rate to increase from 28.4 to 36.9% (P less than 0.01) even though more than half of the embryos have not been thawed. We conclude that embryos obtained after stimulation with GnRHa + HMG and frozen at the pronucleate stage are more likely to result in a pregnancy.

Cryopreservation↗
PubMed1991

Proteus mirabilis mutants defective in swarmer cell differentiation and multicellular behavior.

Proteus mirabilis is a dimorphic bacterium which exists in liquid cultures as a 1.5- to 2.0-microns motile swimmer cell possessing 6 to 10 peritrichous flagella. When swimmer cells are placed on a surface, they differentiate by a combination of events that ultimately produce a swarmer cell. Unlike the swimmer cell, the polyploid swarmer cell is 60 to 80 microns long and possesses hundreds to thousands of surface-induced flagella. These features, combined with multicellular behavior, allow the swarmer cells to move over a surface in a process called swarming. Transposon Tn5 was used to produce P. mirabilis mutants defective in wild-type swarming motility. Two general classes of mutants were found to be defective in swarming. The first class was composed of null mutants that were completely devoid of swarming motility. The majority of nonswarming mutations were the result of defects in the synthesis of flagella or in the ability to rotate the flagella. The remaining nonswarming mutants produced flagella but were defective in surface-induced elongation. Strains in the second general class of mutants, which made up more than 65% of all defects in swarming were motile but were defective in the control and coordination of multicellular swarming. Analysis of consolidation zones produced by such crippled mutants suggested that this pleiotropic phenotype was caused by a defect in the regulation of multicellular behavior. A possible mechanism controlling the cyclic process of differentiation and dediferentiation involved in the swarming behavior of P. mirabilis is discussed.

Cell Division↗
PubMed2004

Genes involved in formation of structured multicellular communities by Bacillus subtilis.

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.

Bacillus subtilis↗
PubMed2026

A conserved partner-switching system controls terminal differentiation in multicellular cyanobacteria.

UNLABELLED: Canonical partner-switching systems (PSSs) regulate sigma factor activity through reversible phosphorylation, but their established roles have been largely limited to stress responses and sporulation in Firmicutes. Whether this regulatory mechanism also controls developmental cell fate decisions in other bacterial phyla has remained unknown. Here, we identify a canonical PSS that governs heterocyst differentiation in the multicellular cyanobacterium Anabaena sp. PCC 7120. This system comprises the anti-sigma factor All2284 (NfsS) and the anti-anti-sigma factor All2283 (NfsR). Structural predictions and biochemical assays showed that NfsS phosphorylates NfsR on a conserved serine residue, whereas bacterial two-hybrid and co-purification assays demonstrated that NfsS binds the developmental sigma factors SigC and SigE. Deletion of nfsR abolished heterocyst formation and diazotrophic growth, and transcriptomic analysis revealed broad failure to induce late heterocyst genes, including nitrogen fixation functions such as nifHDK and fdxH. Phylum-wide comparative genomics further showed that PSS genes and putative functional clusters are strongly enriched in filamentous and heterocyst-forming taxa, indicating an association between the expansion of these signaling modules and the emergence of multicellularity and developmental specialization. Together, these findings establish a PSS as a direct regulator of terminal cell differentiation in a gram-negative bacterium and reveal partner switching as a conserved regulatory principle linking environmental signaling to developmental fate in a major bacterial phylum. IMPORTANCE: While partner-switching systems are classically associated with stress responses and sporulation control in Firmicutes, whether this regulatory logic governs developmental decisions in other bacterial phyla has remained unknown. Here, we establish that a related partner-switching mechanism operates in a distinct bacterial lineage, the cyanobacteria, where it controls a major developmental transition involving terminal cell differentiation. Specifically, we show that a phosphorylation-dependent checkpoint involving the anti-sigma factor NfsS and the anti-anti-sigma factor NfsR directly regulates heterocyst formation. Disruption of this switch abolishes cell differentiation and diazotrophic growth, revealing that this system is an obligate gatekeeper for terminal differentiation. Conceptually, these findings substantially extend the known functional repertoire of partner-switching circuits: rather than controlling stress adaptation or spore dormancy, this module has been co-opted to govern a complex, multicellular developmental program in an organism that underpins global carbon and nitrogen cycles. This work, therefore, establishes a new paradigm for phosphorylation-based control of developmental sigma factors and provides a tractable model for dissecting how conserved signaling modules are rewired to drive lineage-specific innovations across the bacterial domain.

cell differentiation↗
PubMed1987

Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures.

Madin-Darby canine kidney (MDCK) cells form polarized, multicellular epithelial structures in vitro. Low-level expression of pp60v-src in MDCK cells elicits plasticity in these multicellular structures. Plasticity was revealed by the displacement of cells from mechanically stressed regions of the epithelial monolayers; however, the two-dimensional relationship between the cells in the remainder of the monolayer was maintained. Electron microscopy of multicellular structures revealed abnormal separation of the lateral membranes of adjacent cells and selective uncoupling of the junctional complex; the zonula adherens was disrupted, but the zonula occludens and desmosomes were retained. Significantly, this result was not accompanied by transformation of the cells, as judged by the absence of anchorage-independent growth potential. These results demonstrate a nonmitogenic biological activity of pp60v-src which is experimentally dissociable from transformation. This morphoregulatory action on higher-order epithelial structures may reflect a function of related cellular tyrosine kinases.

Animals↗
PubMedDate not supplied

Cultured human nasal epithelial multicellular spheroids: polar cyst-like model tissues.

We report here a new readily cultured nonadherent hollow spheroidal epithelial tissue model: human nasal epithelial multicellular spheroids, prepared from brushings of human nasal epithelium in vivo. Although cultured cyst-like epithelial models developed from embryonic, transformed, or polypoid tissues have been reported previously, human nasal epithelial multicellular spheroids are derived from normal mature nontransformed human airway epithelial cells. In our studies, spheroids ranged in size from 50 to 700 microns diameter (averaging approximately 250 microns). Cells of the spheroid displayed morphological polarity and formed junctional complexes. Transcellular electrolyte transport may underlie the increase in spheroid size which occurred in culture. The ease and simplicity of the brushing and culture procedures reported here render normal and diseased human cell populations more readily accessible to investigation. We believe human nasal epithelial multicellular spheroids may have important applications in the study of electrolyte and fluid transport processes, ciliary motility, epithelial polarity, cellular metabolism, and drug cytotoxicity in normal and pathophysiological states of the human respiratory tract (e.g., cystic fibrosis).

Biological Transport, Active↗
PubMed1998

Amino acids stimulate phosphorylation of p70S6k and organization of rat adipocytes into multicellular clusters.

In previous studies we have shown that rat adipocytes suspended in Matrigel and placed in primary culture migrate through the gel to form multicellular clusters over a 5- to 6-day period. In the present study, phosphorylation of the insulin-regulated 70-kDa ribosomal protein S6 kinase (p70S6k) was observed within 30 min of establishment of adipocytes in primary culture. Two inhibitors of the p70S6k signaling pathway, rapamycin and LY-294002, greatly reduced phosphorylation of p70S6k and organization of adipocytes into multicellular clusters. Of all the components of the cell culture medium, amino acids, and in particular a subset of neutral amino acids, were found to promote both phosphorylation of p70S6k and cluster formation. Lowering the concentrations of amino acids in the medium to levels approximating those in plasma of fasted rats decreased both phosphorylation of p70S6k and cluster formation. Furthermore, stimulation of p70S6k phosphorylation by amino acids was prevented by either rapamycin or LY-294002. These findings demonstrate that amino acids stimulate the p70S6k signaling pathway in adipocytes and imply a role for this pathway in multicellular clustering.

Adipocytes↗
PubMed2003

Estimation of action potential changes from field potential recordings in multicellular mouse cardiac myocyte cultures.

BACKGROUND: Extracellular recordings of electrical activity with substrate-integrated microelectrode arrays (MEAs) enable non-invasive long-term monitoring of contracting multicellular cardiac preparations. However, to characterize not only the spread of excitation and the conduction velocity from field potential (FP) recordings, a more rigorous analysis of FPs is necessary. Therefore in this study we aim to characterize intrinsic action potential (AP) parameters by simultaneous recording of APs and FPs. METHODS: A MEA consisting of 60 substrate-integrated electrodes is used to record the FP-waveform from multicellular preparations of isolated embryonic mouse cardiomyocytes. Simultaneous current clamp recordings in the vicinity of individual microelectrodes and pharmacological interventions allowed us to correlate FP and AP components and their time course. RESULTS: The experiments revealed a linear relationship between AP rise time and FP rise time as well as a linear relationship between AP duration and FP duration. Furthermore a direct contribution of the voltage dependent Na(+)- and Ca(2+)-current to the FP could be identified. CONCLUSION: The characterization of the FP allows us for the first time to estimate AP changes and the contribution of individual current components to the AP by the help of non-invasive recording within a multicellular cardiac preparation during long-term culture.

Action Potentials↗
PubMed2002

The DC electrical-field-induced Ca(2+) response and growth stimulation of multicellular tumor spheroids are mediated by ATP release and purinergic receptor stimulation.

It has been demonstrated that adenosine 5'-triphosphate (ATP) is actively secreted by cells, thereby eliciting Ca(2+)-dependent signal transduction cascades in an autocrine and paracrine manner. In the present study the effects of direct current (DC) electrical fields on ATP release, the intracellular Ca(2+) concentration [Ca(2+)](i) and growth of multicellular prostate tumor spheroids were investigated. Treatment of multicellular tumor spheroids by a single DC electrical field pulse with a field strength of 750 Vm(-1) for 60 seconds resulted in a transient Ca(2+) response, activation of c-Fos and growth stimulation. The initial [Ca(2+)](i) signal was elicited at the anode-facing side of the spheroid and spread with a velocity of approximately 12 microm per second across the spheroid surface. The electrical-field-evoked Ca(2+) response as well as c-Fos activation and growth stimulation of tumor spheroids were inhibited by pretreatment with the anion channel blockers NPPB, niflumic acid and tamoxifen. Furthermore, the Ca(2+) response elicited by electrical field treatment was abolished following purinergic receptor desensitivation by repetitive treatment of tumor spheroids with ATP and pretreatment with the purinergic receptor antagonist suramin as well as with apyrase. Electrical field treatment of tumor spheroids resulted in release of ATP into the supernatant as evaluated by luciferin/luciferase bioluminescence. ATP release was efficiently inhibited in the presence of anion channel blockers. Our data suggest that electrical field treatment of multicellular tumor spheroids results in ATP release, which concomitantly activates purinergic receptors, elicits a Ca(2+) wave spreading through the tumor spheroid tissue and stimulates tumor growth.

Adenosine Triphosphate↗
PubMed2006

Identification of the proliferation/differentiation switch in the cellular network of multicellular organisms.

The protein-protein interaction networks, or interactome networks, have been shown to have dynamic modular structures, yet the functional connections between and among the modules are less well understood. Here, using a new pipeline to integrate the interactome and the transcriptome, we identified a pair of transcriptionally anticorrelated modules, each consisting of hundreds of genes in multicellular interactome networks across different individuals and populations. The two modules are associated with cellular proliferation and differentiation, respectively. The proliferation module is conserved among eukaryotic organisms, whereas the differentiation module is specific to multicellular organisms. Upon differentiation of various tissues and cell lines from different organisms, the expression of the proliferation module is more uniformly suppressed, while the differentiation module is upregulated in a tissue- and species-specific manner. Our results indicate that even at the tissue and organism levels, proliferation and differentiation modules may correspond to two alternative states of the molecular network and may reflect a universal symbiotic relationship in a multicellular organism. Our analyses further predict that the proteins mediating the interactions between these modules may serve as modulators at the proliferation/differentiation switch.

Animals↗
PubMed2005

Modeling multicellular response to nonuniform distributions of radioactivity: differences in cellular response to self-dose and cross-dose.

Radiopharmaceuticals are distributed nonuniformly in tissue. While distributions of radioactivity often appear uniform at the organ level, in fact, microscopic examination reveals that only a fraction of the cells in tissue are labeled. Labeled cells and unlabeled cells often receive different absorbed doses depending on the extent of the nonuniformity and the characteristics of the emitted radiations. The labeled cells receive an absorbed dose from radioactivity within the cell (self-dose) as well as an absorbed dose from radioactivity in surrounding labeled cells (cross-dose). Unlabeled cells receive only a cross-dose. In recent communications, a multicellular cluster model was used to investigate the lethality of microscopic nonuniform distributions of 131I iododeoxyuridine (131IdU). For a given mean absorbed dose to the tissue, the dose response depended on the percentage of cells that were labeled. Specifically, when 1, 10 and 100% of the cells were labeled, a D37 of 6.4, 5.7 and 4.5 Gy, respectively, was observed. The reason for these differences was recently traced to differences in the cellular response to the self- and cross-doses delivered by 131IdU. Systematic isolation of the effects of self-dose resulted in a D37 of 1.2 +/- 0.3 Gy. The cross-dose component yielded a D37 of 6.4 +/- 0.5 Gy. In the present work, the overall survival of multicellular clusters containing 1, 10 and 100% labeled cells is modeled using a semi-empirical approach that uses the mean lethal self- and cross-doses and the fraction of cells labeled. There is excellent agreement between the theoretical model and the experimental data when the surviving fraction is greater than 1%. Therefore, when the distribution of 131I in tissue is nonuniform at the microscopic level, and the cellular response to self- and cross-doses differs, multicellular dosimetry can be used successfully to predict biological response, whereas the mean absorbed dose fails in this regard.

Animals↗
PubMed1997

Reconstruction of trabecular tissue from human trabecular cells as a multicellular spheroid.

To understand the development of the trabecular meshwork of the eye, floating cellular aggregates (multicellular spheroids) were formed from human trabecular cells in a non-adherent environment of culture and incubated for up to one month. Dissociated trabecular cells formed multicellular spheroids within one day in the non-adherent environment, and apoptosis continued to occur in the spheroids which had been initially filled with cells. The final structure after one month appeared as a meshwork of cells with large extracellular spaces. Epidermal and basic fibroblast growth factor (EGF and bFGF) protected trabecular cells in the spheroids from apoptosis and, as a result, kept the spheroids filled with cells even after one month. In the absence of excess EGF or bFGF, the multicellular spheroids grown in vitro from human trabecular cells mimicked the mesh-like structure of normal trabecular tissue. In contrast, under an excess of these growth factors, spheroids of high cellularity, resembling the abnormal trabecular tissues of patients with congenital glaucoma, were formed.

Humans↗
PubMed1992

Combination of epidermal growth factor and insulin is required for multicellular spheroid formation of rat hepatocytes in primary culture.

We showed that the combination of epidermal growth factor (EGF) and insulin is an essential supplement to Williams' #E medium for the formation of floating multicellular spheroids in primary culture of rat hepatocytes. Isolated hepatocytes assembled to form floating multicellular spheroids within 96 h through transient assembly of monolayer islands within the initial 24 h in dishes coated with liver-derived proteoglycans. However, the assembly of multicellular spheroids was severely suppressed in the absence of either EGF or insulin. The reduction of spheroid assembly was correlated with decreased attachment and subsequent decreased formation of monolayer islands within 24 h. The minimum amounts of EGF and insulin required for the formation of floating spheroids were 1 ng/ml and 0.4 microgram/ml, respectively. These results suggest that the enhancement of hepatocyte attachment provided by the combination of EGF and insulin during the early phase of culture is required for the formation of floating spheroids.

Animals↗
PubMedDate not supplied

Differential expression of adhesion molecules (CD44, ICAM-1 and LFA-3) in cancer cells grown in monolayer or as multicellular spheroids.

Multicellular tumor spheroids have been used to examine numerous aspects of tumor biology since they often recreate the in vivo tumor environment much more closely than other models. Since the three-dimensional organization of cancer cells into spheroids is based upon cell-cell interactions which appear dramatically different in spheroids with respect to monolayer cultures, it can be hypothesized that a modulation in the expression of the molecules which are directly responsible for cell-cell and cell-matrix interactions, particularly the cell adhesion molecules (CAMs), may be involved. In order to test this postulate, the expression of three important CAMs involved in tumor processes (CD44, ICAM-1 and LFA-3) in the human cancer cell lines HT29 (colon adenocarcinoma), A431 (squamous epidermal carcinoma) and A2780 (ovarian carcinoma) grown in monolayer or as multicellular spheroids was compared. The results demonstrate that only two of the lines (HT29 and A431) formed spheroids after six days of gyratory culture while A2780 cells did not form such structures after up to 8 days of culture. In the two cell lines which did form early phase multicellular spheroids, flow cytometric analysis revealed that important differences exist between the same cells grown in monolayer and as spheroids in the quantity of expression of CAMs.

Adenocarcinoma↗
PubMed2000

Redox regulation of P-glycoprotein-mediated multidrug resistance in multicellular prostate tumor spheroids.

Multicellular prostate tumor spheroids develop intrinsic P-glycoprotein (Pgp)-mediated multidrug resistance with the appearance of quiescent cell areas. We have investigated the effect of intracellular reactive oxygen species (ROS) on Pgp expression in large, quiescent and drug-resistant multicellular spheroids (diameter 250 +/- 50microm). Using the ROS-sensitive fluorescence dye 2;7;-dichlorodihydrofluorescein diacetate (H(2)DCFDA), we demonstrated that these tumor spheroids are characterized by reduced intracellular ROS compared with drug-sensitive small spheroids (diameter 60 +/- 20microm) consisting predominantly of proliferating cells. The prooxidants hydrogen peroxide, menadione and glyceraldehyde raised ROS in large tumor spheroids and significantly down-regulated Pgp within 24 hr. Comparable effects were achieved with the known Pgp-reversing agents sodium orthovanadate, quinidine and cyclosporin A but not with verapamil. Consequently, the retention and toxicity of the anthracycline doxorubicin was increased in tumor spheroids treated with prooxidants. Co-administration of prooxidants and the free radical scavenger ebselen did not alter Pgp levels, indicating that down-regulation of Pgp is mediated via ROS. Down-regulation of Pgp by H(2)O(2) was abolished when either forskolin, 8-Br-cAMP or IBMX, which raise intracellular cAMP levels, was co-administered, indicating that Pgp expression is regulated by protein kinase A (PKA). Furthermore, Pgp was down-regulated by the PKA inhibitors Rp-cAMPs and H89. Since prooxidants stimulated the growth of multicellular spheroids and down-regulated the cyclin-dependent kinase inhibitor p27(kip1), we conclude that ROS-mediated Pgp down-regulation may be paralleled by recruitment of drug-resistant quiescent cells in the depth of the tumor tissue for cell-cycle activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Lack of multicellular drug resistance observed in human ovarian and prostate carcinoma treated with the proteasome inhibitor PS-341.

Almost all known conventional cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than in killing tumor cells grown as monolayer cell cultures. This "multicellular resistance" reflects the relative intrinsic drug-resistant phenotype of most solid tumors growing in vivo and is due to factors such as limited drug penetration or reduced fractions of proliferating cells. Proteasome inhibitors such as PS-341, a dipeptide boronic acid analogue, represent an interesting new class of potential anticancer drugs, which are entering early-phase clinical trials. PS-341 has been found to have good broad-spectrum cytotoxic activity in the 60-monolayer cell line National Cancer Institute screen. However, because its relative potency has not been tested in spheroid systems, we analyzed the activity of PS-341 in a spheroid/solid tumor context using four different human ovarian carcinoma cell lines and three prostate carcinoma cell lines, respectively. We found, with one exception, that PS-341 showed equal or greater activity in spheroids than in the respective monolayer cell cultures, even in a prostate cancer spheroid model with a very low growth fraction. PS-341 induced apoptotic cell death in carcinoma cells in both culture systems. We also noted a decrease in XIAP protein, a member of the inhibitor of apoptosis (IAP) family of apoptosis inhibitors, and phosphorylation of Bcl-XL in PS-341-treated ovarian carcinoma cells. Furthermore, DNA fragmentation, a hallmark of apoptosis (in this case, induced by PS-341), was completely inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD). Taken together, the results indicate that unlike most other known anticancer cytotoxic drugs, PS-341 appears to be as effective in killing tumor cells grown in the form of multicell spheroids as in killing tumor cells grown in monolayer cell culture. Hence, this compound has the potential to circumvent multicellular drug resistance and, as such, may show promising activity against solid tumors with low growth fractions in vivo, which are frequently intrinsically resistant to conventional cytotoxic anticancer drugs.

Antineoplastic Agents↗