Alter Cell Behavior Research Articles

Patterns of cell lineage, movement, and migration from germ layer specification to gastrulation in the amphipod crustacean Parhyale hawaiensis

The acquisition of specific cell fates throughout embryonic development is one of the core problems in developmental and evolutionary biology. In the amphipod Parhyale hawaiensis all three germ layers and the germ line are determined by the eight-cell stage. Despite this early fate determination, multiple cell types can be replaced following ablation of their founder cells, showing that this embryo also has significant regulative properties. Here we present a cellular-level resolution lineage analysis for P. hawaiensis embryos between fertilization and gastrulation, including analysis of cleavage patterns, division times, and clonal behaviors. We compare these cellular behaviors in wild type embryos with those in embryos where specific founder cells have been ablated, or where zygotic transcription has been inhibited. We observe that when germ line, endoderm or mesoderm founder cells are ablated, the remaining cells do not alter their cleavage or migration behaviors before the onset of gastrulation. In the absence of zygotic transcription, ingression movements proceed normally, but epibolic movements are disrupted. This indicates that the embryo's regulative response to germ layer founder loss, in the form of altered cell behavior, is realized in the ~32h between gastrulation and early germ band elongation, and is likely to require zygotic reprogramming rather than alternative deployment of maternally supplied determinants. Combining these data with the observations of previous studies, we propose a framework to elucidate the molecular mechanisms that regulate the determinative and regulative properties of the P. hawaiensis embryo.

Abstract 2156: c-Myb promotes growth, invasion and androgen-independence of prostate cancer cells

Abstract Most prostate cancer (PCa) patients treated with first line androgen-deprivation therapy (ADT) initially exhibit a dramatic regression of the androgen-dependent (AD) cancer cells; however, the tumors eventually progress to an androgen-independent (AI) stage. The resulting hormone-refractory tumors are highly aggressive and resistant to conventional therapies. The causal mechanisms for such a transition remain largely unknown; however, a number of genetic and epigenetic alterations are believed to be involved in the process of AI progression of PCa. In an earlier study, c-Myb was identified among the genes that are amplified at higher frequency in hormone-refractory PCa. The c-Myb proto-oncogene is a cellular progenitor of v-Myb oncogenes, which encodes for a transcription factor and confers its oncogenic activity by regulating the expression of several target genes. In the present study, we have investigated the functional role of c-Myb in prostate cancer cells. Our data showed that c-Myb was overexpressed in AI (C4-2) PCa cells as compared to AD (LNCaP) PCa cells of the same genotypic lineage. Furthermore, we found that c-Myb was regulated by androgens in a biphasic manner and its expression correlated with the effect of androgens on prostate cancer cell growth. Short-hairpin RNA (shRNA)-mediated silencing of c-Myb in C4-2 cells reduced their growth, clonogenicity, motility and invasion, whereas its ectopic expression in LNCaP cells had opposite effects. Moreover, we observed that the overexpression of c-Myb in LNCaP and C4-2 cells was associated with their growth in androgen-depleted media, suggesting its role in androgen-independence. Immunoblot analysis demonstrated loss of mesenchymal markers N-cadherin, vimentin, Slug, Snail, and Twist, and gain of epithelial marker, E-cadherin in c-Myb-silenced C4-2 cells. In contrast, c-Myb overexpressing LNCaP cells exhibited a loss of epithelial and gain of mesenchymal markers, thus indicating a role of c-Myb in epithelial to mesenchymal transition (EMT) of PCa cells. Altogether, our studies provide first experimental evidence for a functional role of c-Myb in altered growth, androgen-independence and malignant behavior of the prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2156. doi:10.1158/1538-7445.AM2011-2156

Stem Cell Dynamics in Response to Nutrient Availability

When nutrient availability becomes limited, animals must actively adjust their metabolism to allocate limited resources and maintain tissue homeostasis. However, it is poorly understood how tissues maintained by adult stem cells respond to chronic changes in metabolism. To begin to address this question, we fed flies a diet lacking protein (protein starvation) and assayed both germline and intestinal stem cells. Our results revealed a decrease in stem cell proliferation and a reduction in stem cell number; however, a small pool of active stem cells remained. Upon refeeding, stem cell number increased dramatically, indicating that the remaining stem cells are competent to respond quickly to changes in nutritional status. Stem cell maintenance is critically dependent upon intrinsic and extrinsic factors that act to regulate stem cell behavior. Activation of the insulin/IGF signaling pathway in stem cells and adjacent support cells in the germline was sufficient to suppress stem cell loss during starvation. Therefore, our data indicate that stem cells can directly sense changes in the systemic environment to coordinate their behavior with the nutritional status of the animal, providing a paradigm for maintaining tissue homeostasis under metabolic stress.

Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition

BackgroundEpithelial to mesenchymal transition (EMT), implicated as a mechanism for tumor dissemination, is marked by loss of E-cadherin, disruption of cell adhesion, and induction of cell motility and invasion. In most intraductal breast carcinomas E-cadherin is regulated epigenetically via methylation of the promoter. E-cadherin expression is therefore dynamic and open to modulation by the microenvironment. In addition, it has been observed that metastatic foci commonly appear more differentiated than the primary tumor, suggesting that cancer cells may further undergo a mesenchymal to epithelial reverting transition (MErT) in the secondary organ environment following the EMT that allows for escape.ResultsWe first examined E-cadherin expression in primary breast tumors and their corresponding metastases to liver, lung and brain and discovered that 62% (10/16) of cases showed increased E-cadherin expression in the metastases compared to the primaries. These observations led to the question of whether the positive metastatic foci arose from expansion of E-cadherin-positive cells or from MErT of originally E-cadherin-negative disseminated cells. Thus, we aimed to determine whether it was possible for the mesenchymal-like MDA-MB-231 breast cancer cells to undergo an MErT through the re-expression of E-cadherin, either through exogenous introduction or induction by the microenvironment. Ectopic expression of full-length E-cadherin in MDA-MB-231 cells resulted in a morphological and functional reversion of the epithelial phenotype, with even just the cytosolic domain of E-cadherin yielding a partial phenotype. Introduction of MDA-MB-231 cells or primary explants into a secondary organ environment simulated by a hepatocyte coculture system induced E-cadherin re-expression through passive loss of methylation of the promoter. Furthermore, detection of E-cadherin-positive metastatic foci following the spontaneous metastasis of MDA-MB-231 cells injected into the mammary fat pad of mice suggests that this re-expression is functional.ConclusionsOur clinical observations and experimental data indicate that the secondary organ microenvironment can induce the re-expression of E-cadherin and consequently MErT. This phenotypic change is reflected in altered cell behavior and thus may be a critical step in cell survival at metastatic sites.

T1191 Intra-Tumoral Variability in Prostaglandin E 2 Levels in Human Colorectal Cancer Liver Metastases is Associated With Differences in NAD+ Levels and Expression of NAD+-Dependent 15-Prostaglandin Dehydrogenase

Introduction Cyclooxygenase (COX)-2 (synthesis) and NAD+-dependent 15-prostaglandin (PG) dehydrogenase (15-PGDH; catabolism) are rate-limiting enzymes in control of tissue PGE2 levels. COX-2 and 15-PGDH function may be affected by the tumour micro-environment (eg, hypoxia) leading to changes in local PGE2 concentration and altered cancer cell behaviour. Epithelial-mesenchymal transition (EMT) is believed to be crucial for tumour cell invasion/metastasis. EMT is promoted by PGE2 in several cell types in vitro. We investigated the relationship between PGE2 levels, COX-2 and 15-PGDH expression, and NAD+ levels in human colorectal cancer liver metastases (CRCLM), and the role of hypoxia and PGE2 in EMT of CRC cells in vitro. Methods PGE2 and NAD+ levels, and immunoreactive 15-PGDH and COX-2 protein content, were measured in paired central and peripheral tissue from 20 CRCLM. 15-PGDH activity was measured by a [ 3 H]-PGE2 degradation assay in the presence or absence of exogenous NAD+ (1 μM). Parallel in vitro studies used a LIM1863 human CRC cell model of transforming growth factor (TGF) β-induced EMT and MCF-7 human breast cancer cells. Results PGE2 levels were increased by a mean (95% CI) of 26% (1% to 52%) in the centre of CRCLM relative to peripheral tissue (mean 762 vs 604 pg/mg). Counter-intuitively, immunoreactive 15-PGDH protein levels were also increased (14% (3% to 27%)) in tumour centres, as was 15-PGDH activity in the presence of added NAD+ (16% (3% to 29%)). However, NAD+ levels were 59% (25% to 72%) lower in the centre of CRCLM than the periphery (mean 192 vs 546 pmol/mg) suggesting inefficient 15-PGDH-dependent catabolism may explain raised PGE2 levels. By contrast, there were no significant regional differences in immunoreactive COX-2 protein levels. In central areas of CRCLM, cancer cells exhibiting low E-cadherin expression had higher 15-PGDH expression than neighbouring E-cadherin-positive cells. Our CRCLM tissue observations were mirrored by LIM1863 cells undergoing EMT as hypoxic (1% O2) LIM1863 cells contained significantly less NAD+ and exhibited higher 15-PGDH protein levels than normoxic cells. 15-PGDH activity was significantly decreased in hypoxic MCF-7 cells compared with normoxic counterparts when NAD+ was present at a limiting concentration (no exogenous added). Furthermore, PGE2 (0.1–10 μM) promoted TGF-β-induced EMT in LIM 1863 cells in a concentration-dependent manner. Conclusion Regional differences in PGE2 levels and 15-PGDH expression/activity are apparent in human CRCLM (possibly due to a hypoxic micro-environment). PGE2 promotes TGF-β-induced EMT in human CRC cells in vitro. Modulation of factors controlling PGE2 represents a novel therapeutic strategy against EMT in CRCLM.

Abstract 3189: Aggressive renal cancer cell behavior is partly mediated by CD98hc

Abstract CD98, a heteromeric transmembrane protein, consists of a heavy subunit (4F2hc, SLC3A2), extracellular linked to a light subunit. The light chains provide amino acid transport activity of CD98. Recently, we could show that CD98hc directly interacts with a conserved motif in integrin beta subunits, thereby mediating outside - in signaling. A genetic knockout of CD98hc in animals is embryonic lethal, whereas overexpression of CD98hc in somatic cells leads to malignant transformation. Recently, we reported that high CD98hc expression is a novel and reliable diagnostic marker for the more malignant subtype II papillary renal cell cancer marker (pRCC). Thereby, CD98hc expression directly correlated with grade of differentiation. In the present study, we identified the functional role of CD98hc in clear cell renal cell cancer (ccRCC) behavior. First, we generated stable low CD98hc ccRCCs by the use of a shRNA lentiviral construct and compared tumor cell behavior with high CD98hc expressing mock infected ccRCCs. We found that downregulation of CD98hc led to an altered tumor cell behavior including tumor cell survival, migration and proliferation. Expression of a silent mutation - lacking shRNA binding motif - reconstituted CD98hc expression in lowCD98hc ccRCC, thereby restoring aggressive tumor cell behavior. By introducing CD98hc mutations either lacking integrin binding capacities or light chain interactions, we could identify the functional role of leucine amino acid transport in tumor cell proliferation, as affected C14-leucine uptake was directly correlated with tumor cell proliferative activity. In contrast, mutants lacking interaction sites for integrins showed defects adhesion-induced FAK phosphorylation and c-src activation, leading to reduced tumor cell adhesion and spreading. A biological relevance of the in vitro finding was confirmed in in vivo tumor xenotransplant assays, whereby stable low CD98hc/ccRCCs either reconstituted with wild type CD98hc or with mutated CD98hc were used. Only wild type reconstitution was capable to restore full aggressiveness of tumor growth and metastasis formation. From our data we conclude that CD98 partially mediates functional activity of renal cancer cell behavior. Thus, CD98hc-mediated tumor cell behavior may offer a potential novel therapeutic target for the treatment of cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3189.

PWE-070 Intratumoural variability in prostaglandin E2 levels in human colorectal cancer liver metastases is associated with differences in NAD+ levels and expression of NAD+-dependent 15-prostaglandin dehydrogenase

<h3>Introduction</h3> Cyclooxygenase (COX)-2 (synthesis) and NAD+-dependent 15-prostaglandin (PG) dehydrogenase (15-PGDH; catabolism) are rate-limiting enzymes in control of tissue PGE2 levels. COX-2 and 15-PGDH function may be affected by the tumour micro-environment (eg, hypoxia) leading to changes in local PGE2 concentration and altered cancer cell behaviour. Epithelial-mesenchymal transition (EMT) is believed to be crucial for tumour cell invasion/metastasis. EMT is promoted by PGE2 in several cell types in vitro. We investigated the relationship between PGE2 levels, COX-2 and 15-PGDH expression, and NAD+ levels in human colorectal cancer liver metastases (CRCLM), and the role of hypoxia and PGE2 in EMT of CRC cells in vitro. <h3>Methods</h3> PGE2 and NAD+ levels, and immunoreactive 15-PGDH and COX-2 protein content, were measured in paired central and peripheral tissue from 20 CRCLM. 15-PGDH activity was measured by a [³H]-PGE2 degradation assay in the presence or absence of exogenous NAD+ (1 μM). Parallel in vitro studies used a LIM1863 human CRC cell model of transforming growth factor (TGF) β-induced EMT and MCF-7 human breast cancer cells. <h3>Results</h3> PGE2 levels were increased by a mean (95% CI) of 26% (1% to 52%) in the centre of CRCLM relative to peripheral tissue (mean 762 vs 604 pg/mg). Counter-intuitively, immunoreactive 15-PGDH protein levels were also increased (14% (3% to 27%)) in tumour centres, as was 15-PGDH activity in the presence of added NAD+ (16% (3% to 29%)). However, NAD+ levels were 59% (25% to 72%) lower in the centre of CRCLM than the periphery (mean 192 vs 546 pmol/mg) suggesting inefficient 15-PGDH-dependent catabolism may explain raised PGE2 levels. By contrast, there were no significant regional differences in immunoreactive COX-2 protein levels. In central areas of CRCLM, cancer cells exhibiting low E-cadherin expression had higher 15-PGDH expression than neighbouring E-cadherin-positive cells. Our CRCLM tissue observations were mirrored by LIM1863 cells undergoing EMT as hypoxic (1% O2) LIM1863 cells contained significantly less NAD+ and exhibited higher 15-PGDH protein levels than normoxic cells. 15-PGDH activity was significantly decreased in hypoxic MCF-7 cells compared with normoxic counterparts when NAD+ was present at a limiting concentration (no exogenous added). Furthermore, PGE2 (0.1–10 μM) promoted TGF-β-induced EMT in LIM 1863 cells in a concentration-dependent manner. <h3>Conclusion</h3> Regional differences in PGE2 levels and 15-PGDH expression/activity are apparent in human CRCLM (possibly due to a hypoxic micro-environment). PGE2 promotes TGF-β-induced EMT in human CRC cells in vitro. Modulation of factors controlling PGE2 represents a novel therapeutic strategy against EMT in CRCLM.

Age-Related Changes in Redox Signaling and VSMC Function

Epidemiological studies have shown that advancing age is associated with an increased prevalence of cardiovascular disease (CVD). Vascular smooth muscle cells (VSMC) comprise the major arterial cell population, and changes in VSMC behavior, function, and redox status with age contribute to alterations in vascular remodeling and cell signaling. Over two decades of work on aged animal models provide support for age-related changes in VSMC and/or arterial tissues. Enhanced production of reactive oxygen species (ROS) and insufficient removal by scavenging systems are hallmarks of vascular aging. VSMC proliferation and migration are core processes in vascular remodeling and influenced by growth factors and signaling networks. The intrinsic link between gene regulation and aging often relates directly to transcription factors and their regulatory actions. Modulation of growth factor signaling leads to up- or downregulation of transcription factors that control expression of genes associated with VSMC proliferation, inflammation, and ROS production. Four major signaling pathways related to the transcription factors, AP-1, NF-kappaB, FoxO, and Nrf2, will be reviewed. Knowledge of age-related changes in signaling pathways in VSMC that lead to alterations in cell behavior and function consistent with disease progression may help in efforts to attenuate age-related CVD, such as atherosclerosis.

Androgen Receptor Is a Potential Therapeutic Target for Bladder Cancer

To investigate whether androgen receptor (AR) could serve as a potential molecular target for the treatment of bladder cancer. Cell proliferation, apoptosis, and migration capacity were determined in human transitional carcinoma cell lines T24 and 253-J treated with small interfering RNA directed against AR, and expression levels of growth- and metastasis-related genes were assessed using quantitative reverse transcriptase-polymerase chain reaction. Tumor cell growth and apoptosis were also evaluated in vivo in T24 tumor-bearing nude mice receiving electroporation-assisted administration of anti-AR small interfering RNA. AR expression knockdown produced increased apoptosis, decreased proliferation, and migration of bladder cancer cells. Cyclin D1, Bcl-x(L), and matrix metallopeptidase-9 gene expression were also reduced with AR knockdown, which might have contributed to the altered biological behavior of cancer cells. In vivo experiments showed that silencing AR expression, by interference aided by electroporation, significantly suppressed AR-positive bladder tumor growth with decreased cell proliferation and increased apoptotic rates. Downregulation of AR expression inhibits bladder cancer cell growth in vitro and in vivo, implying that its use might be a potential therapeutic target for the treatment of bladder cancer.

Cancer systems biology: a network modeling perspective.

Cancer is now appreciated as not only a highly heterogenous pathology with respect to cell type and tissue origin but also as a disease involving dysregulation of multiple pathways governing fundamental cell processes such as death, proliferation, differentiation and migration. Thus, the activities of molecular networks that execute metabolic or cytoskeletal processes, or regulate these by signal transduction, are altered in a complex manner by diverse genetic mutations in concert with the environmental context. A major challenge therefore is how to develop actionable understanding of this multivariate dysregulation, with respect both to how it arises from diverse genetic mutations and to how it may be ameliorated by prospective treatments. While high-throughput experimental platform technologies ranging from genomic sequencing to transcriptomic, proteomic and metabolomic profiling are now commonly used for molecular-level characterization of tumor cells and surrounding tissues, the resulting data sets defy straightforward intuitive interpretation with respect to potential therapeutic targets or the effects of perturbation. In this review article, we will discuss how significant advances can be obtained by applying computational modeling approaches to elucidate the pathways most critically involved in tumor formation and progression, impact of particular mutations on pathway operation, consequences of altered cell behavior in tissue environments and effects of molecular therapeutics.

Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sites

Rather than maximizing toughness, as needed for silk and muscle titin fibers to withstand external impact, the much softer extracellular matrix fibers made from fibronectin (Fn) can be stretched by cell generated forces and display extraordinary extensibility. We show that Fn fibers can be extended more than 8-fold (>700% strain) before 50% of the fibers break. The Young's modulus of single fibers, given by the highly nonlinear slope of the stress-strain curve, changes orders of magnitude, up to MPa. Although many other materials plastically deform before they rupture, evidence is provided that the reversible breakage of force-bearing backbone hydrogen bonds enables the large strain. When tension is released, the nano-sized Fn domains first contract in the crowded environment of fibers within seconds into random coil conformations (molten globule states), before the force-bearing hydrogen bond networks that stabilize the domain's secondary structures are reestablished within minutes (double exponential). The exposure of cryptic binding sites on Fn type III modules increases steeply upon stretching. Thus fiber extension steadily up-regulates fiber rigidity and cryptic epitope exposure, both of which are known to differentially alter cell behavior. Finally, since stress-strain relationships cannot directly be measured in native extracellular matrix (ECM), the stress-strain curves were correlated with stretch-induced alterations of intramolecular fluorescence resonance energy transfer (FRET) obtained from trace amounts of Fn probes (mechanical strain sensors) that can be incorporated into native ECM. Physiological implications of the extraordinary extensibility of Fn fibers and contraction kinetics are discussed.

Contact inhibition revisited

Contact inhibition of cell movement was originally defined in the 1950s as a way of interpreting studies that were ethological and statistical in nature. Research done in succeeding decades provided a more detailed study of the initial contact and its consequences for the cell. The behavior called contact inhibition is characterized by the cessation of ruffling and forward movement in the lamellipodium of the cell making the contact. A new ruffling membrane then arises elsewhere on the cell perimeter. A comparison between the contact behavior described in the early literature and that of the nerve growth cone, described recently by Steketee and Tosney, suggests that filopodia mediate the sensing function in both cases. Since transformed cells have fewer filopodia than normal cells, the contact behavior may decline in direct response to the degraded function of filopodia. This new "filopodia focal signal transduction" hypothesis of contact inhibition elevates the filopodia sensing function and the cessation of lamellipodial advance to the highest importance as phenomena underlying the altered behavior of cancer cells.

Determination of the Salivary Anti-Phenolic Glycolipid-1 Antibody in Leprosy Patients as a Tool to Monitoring Multidrugtherapy

PROBLEM STATEMENT: Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) and its cognate targets, the Matrix Metalloproteinases (MMPs), were differentially expressed in human brain samples with or without HIV-1 infection or HIV-1 Encephalitis (HIVE). APPROACH: A through literature review demonstrated that cell culture models of Central Nervous System (CNS) cell types had been used to illustrate the intricate temporal patterns of TIMP-1/MMP expression, regulated by a variety of inflammatory cytokines. RESULTS: As MMPs and TIMP-1 can significantly altered the extracellular environment and cell signaling, the differential regulation of TIMP-1/MMP expression in neuroinflammation can impact neuronal function and survival in disease conditions. TIMP-1 pro-survival effects had been demonstrated in a variety of cell types including CNS neurons, protecting cells from a wide range of stress and insults. TIMP-1, also known to interact with non-MMP targets, altered cell behavior. In this review, we discussed the possibility that the upregulation of TIMP-1 by glia in acute neuroinflammation may be a neuroprotective response. CONCLUSION: It will be important to delineate the effects of TIMP-1 on neurons and identify receptors and downstream signaling pathways, in order to evaluate TIMP-1 as a therapeutic strategy for neuroinflammatory and neurodegenerative diseases.

Transcriptional Infidelity Promotes Heritable Phenotypic Change in a Bistable Gene Network

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.

67-kDa Laminin Receptor in Human Bile Duct Carcinoma

Background/Aim: Abnormal interaction of epithelial cells with laminin component of basement membrane may account for altered biological behavior of cells, influencing proliferation, adhesion, and motility. In the current study, we investigated the role of 67-kDa laminin receptor (67LR), a high affinity receptor for laminin, in aggressiveness of bile duct carcinoma. Methods: Fifty-two paraffin-embedded specimens and 22 fresh tissues of patients with bile duct carcinoma were analyzed using immunohistologic and real-time polymerase chain reaction techniques, respectively. Expression of 67LR on the bile duct carcinoma QBC939 cells was examined by flow cytometry. The effects of 67LR on the adhesive and invasive abilities of QBC939 cells were determined by adhesion and invasion assay in vitro. Results: Both at the mRNA and protein level, bile duct carcinoma cells expressed a higher level of 67LR than normal epithelial cells (p < 0.01). The expression of 67LR was correlated inversely with differentiation extent of tumor (p < 0.05). The 67LR level was significantly increased in patients with lymph node metastases than in patients without lymph node involvement (p < 0.01). Flow cytometry showed that 69.9 ± 1.1% of QBC939 cells expressed 67LR. Expression of 67LR increased the adhesion and invasion of QBC939 cells. 60 and 120 min of incubation of 67LR antibodies, MLuC5, induced 46.3 ± 2.2 and 61.3 ± 2.1% inhibition of adhesion, respectively. The invasive ability of QBC939 cells to Matrigel was also reduced by 67LR antibodies, MLuC5. Conclusions: Overexpressing 67LR on bile duct carcinoma was associated with invasion and metastasis of bile duct carcinoma. Blockage of 67LR suppressed adhesion and invasion of bile duct carcinoma.

Downregulation of CD147 expression alters cytoskeleton architecture and inhibits gelatinase production and SAPK pathway in human hepatocellular carcinoma cells

BackgroundCD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA) against CD147 (si-CD147) on hepatocellular carcinoma cells' (SMMC-7721) architecture and functions.MethodsFlow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK), vinculiln and mitogen-activated protein kinase (MAPK) expression in SMMC-7721 cells.ResultsDownregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression.ConclusionCD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.

Piezoelectricity and prostate cancer: Proposed interaction between electromagnetic field and prostatic crystalloids

There is evidence that electromagnetic fields (EMF) play some part in the pathogenesis of prostate cancer, but the pathogenic mechanism remains unknown. The normal prostate gland and both benign and malignant prostate lesions contain abundant calcium/phosphorus crystalloids with various morphologies, which seem to be heterogeneously and diffusely distributed within the gland. We hypothesize that an environmental EMF may result in simultaneous, multidirectional and diffuse compression or expansion of these crystalloids (a piezoelectric effect). This would result in a slight mechanical distortion of the prostate, potentially altering cell behavior and enhancing the expression of specific genes, particularly those involved in suppressing apoptosis. A mathematical model of the cell mechanical effect is presented, and the hypothesis is related to current clinical evidence and to potential validation by critical laboratory tests.

Fibronectin Fragmentation Is a Feature of Periodontal Disease Sites and Diabetic Foot and Leg Wounds and Modifies Cell Behavior

Fibronectin (FN) undergoes fragmentation in periodontal disease sites and in poorly healing diabetic wounds. The biologic effects of FN fragments on wound healing remain unresolved. This study characterized the pattern of FN fragmentation and its effects on cellular behavior compared to intact FN. Polyclonal antibodies were raised against FN and three defined recombinant segments of FN and used to analyze gingival crevicular fluid from periodontal disease sites in systemically healthy subjects and in subjects with diabetes, as well as chronic leg and foot wound exudates from subjects with diabetes. Subsequently, the behavior of human gingival fibroblasts (hGFs) and HT1080 reference cells were analyzed by measuring cell attachment, migration, and chemotaxis in the presence of intact FN or recombinant FN fragments. FN fragmentation was evident in fluids from periodontal disease sites and diabetic leg and foot wounds. However, no fragmentation pattern distinguished systemically healthy subjects from subjects with diabetes. hGFs and HT1080 cells required significantly higher concentrations of FN fragments to achieve attachment comparable to intact FN. Cells cultured on FN fragments also were morphologically different from cells cultured on full-length FN. Migration was reduced for hGFs cultured on FN fragments relative to full-length FN. In contrast, FN fragments increased HT1080 fibrosarcoma cell migration over intact FN. FN fragmentation is a prominent feature of periodontal and chronic leg and foot wounds in diabetes. Furthermore, cell culture assays confirmed the hypothesis that exposure to defined FN fragments significantly alters cell behavior.

Probing the bioelectrochemistry of living cells

Recent times have seen a rapidly expanding interest in the study of both single cell behaviour and populations of cells. This paper presents a concise review of current techniques employed for the transduction and processing of cellular signals. Among these, electrochemical methodology in the form of transistor and impedance methods has figured prominently. Indirectly connected to this approach has been the optical, light addressable potentiometric technique. In our research we are developing vibrational methods which are capable of examining populations of neurons, smooth muscle and human red blood cells on a substrate in a label-free fashion. These are based on transverse acoustic wave methodology and Kelvin nanoprobe physics. With respect to the former, synchronous oscillations of frequency are detected for neurons which are altered by the introduction of certain drugs. The same technique can be used to monitor chemical perturbation of the structure of smooth muscle cells from rat aorta. The Kelvin nanoprobe possesses sub-micron resolution and has been successfully employed in the characterization of both isolated, single neuron and red blood cells. Alterations in cell behaviour are reflected in apparent changes in work function, which in turn is associated with changes in cellular potential and dielectric properties.

Effect of different carbon nanotubes on cell viability and proliferation

Carbon nanotubes (CNTs) are a focus of intense research for their potentialapplications in multiple diverse applications, including innovative biomedicalapplications. Due to their very recent discovery, little information is availableabout the biocompatibility and toxicity of this new class of nanoparticle, and asystematic study on biological interference is lacking. Thus, we decided to explore thetoxicity of three different types of carbon nanotube, differing in preparation (arcdischarge versus catalysed chemical vapour deposition); size (10–50 versus 100–150 nmwide × 1–10 µm long); contaminants (amorphous C, graphite, fullerenes or iron) and morphological type(multi-walled, MW, or single-walled, SW) on human leukemic U937 cells. We found thatthese carbon nanotubes exert a strong effect on the proliferation of the reporterU937 monocytic cell. However, these CNTs did not significantly affect the cellviability. These results show that CNTs, though not directly exerting a directcytotoxic effect, are nonetheless able to deeply alter cell behaviour, and thus werecommend thorough analyses to limit health risk due to uncontrolled exposure.