Anchorage-independent Cells Research Articles

From anchorage dependent proliferation to survival: Lessons from redox signalling

Anchorage to extracellular matrix (ECM) is essential for the execution of the mitotic program of nontransformed cells as they need simultaneous signals starting from mitogenic molecules, as growth factors (GFs), and adhesive agents belonging to ECM. Reactive oxygen species play a key function during both GF and integrin receptor signalling and are therefore recognised to have a synergistic function with several others transducers for anchorage-dependent growth (ADG). Indeed, redox-regulated proteins include protein tyrosine phosphatases, protein tyrosine kinases, small GTPases, cytoskeleton proteins, as well as several transcription factors. In this review, we focus on the role of reactive oxygen species (ROS) as key second messengers granting a proper executed mitosis for anchorage-dependent cells through redox regulation of several downstream targets. Besides, redox signals elicited by ECM contact assure a protection from anoikis, a specific apoptosis induced by lack of anchorage. Cancer cells frequently show a deregulation of ROS production and a constitutive oxidative stress has been associated to the achievement of an invasive phenotype. Hence, in cancer cells, the constitutive deregulation of both mitogenic and survival pathways, likely mimicking autocrine/adhesive signals, helps to guide the transformed cells to escape the innate apoptotic response to abolish the signals started by cell/ECM contact, thus sustaining the spreading of anchorage-independent cancer cells and the metastases growth.

Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog

BackgroundGenomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays.ResultsTwo genes, cyclin-dependent kinase like 3 (cdkl3) and cytochrome c oxidase subunit (cox15), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15. Moreover, flow cytometric analysis indicated that cells with an insert of cdkl3 were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of cox15 was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than cdkl3. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either cdkl3 or cox15.ConclusionThese results are consistent with previous studies on the functionalities of cdkl3 and cox15. For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to cox15 expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.

E-Cadherin Cell-Cell Adhesion in Ewing Tumor Cells Mediates Suppression of Anoikis through Activation of the ErbB4 Tyrosine Kinase

Ability to grow under anchorage-independent conditions is one of the major hallmarks of transformed cells. Key to this is the capacity of cells to suppress anoikis, or programmed cell death induced by detachment from the extracellular matrix. To model this phenomenon in vitro, we plated Ewing tumor cells under anchorage-independent conditions by transferring them to dishes coated with agar to prevent attachment to underlying plastic. This resulted in marked up-regulation of E-cadherin and rapid formation of multicellular spheroids in suspension. Addition of calcium chelators, antibodies to E-cadherin (but not to other cadherins or beta(1)-integrin), or expression of dominant negative E-cadherin led to massive apoptosis of spheroid cultures whereas adherent cultures were unaffected. This correlated with reduced activation of the phosphatidylinositol 3-kinase-Akt pathway but not the Ras-extracellular signal-regulated kinase 1/2 cascade. Furthermore, spheroid cultures showed profound chemoresistance to multiple cytotoxic agents compared with adherent cultures, which could be reversed by alpha-E-cadherin antibodies or dominant negative E-cadherin. In a screen for potential downstream effectors of spheroid cell survival, we detected E-cadherin-dependent activation of the ErbB4 receptor tyrosine kinase but not of other ErbB family members. Reduction of ErbB4 levels by RNA interference blocked Akt activation and spheroid cell survival and restored chemosensitivity to Ewing sarcoma spheroids. Our results indicate that anchorage-independent Ewing sarcoma cells suppress anoikis through a pathway involving E-cadherin cell-cell adhesion, which leads to ErbB4 activation of the phosphatidylinositol 3-kinase-Akt pathway, and that this is associated with increased resistance of cells to cytotoxic agents.

Adenovirus-mediated PEDF expression inhibits prostate cancer cell growth and results in augmented expression of PAI-2

PEDF is one of the most potent inhibitor of angiogenesis. Loss of PEDF was found in human prostate tumors and associated with the progression toward a metastatic phenotype. To test the therapeutic potential of PEDF, we constructed a replication-defective adenoviral vector capable of efficient transduction and expression of PEDF (Ad-PEDF) in PC-3 prostate carcinoma cells. As controls, we used adenoviruses expressing beta-galactosidase (Ad-LacZ). We showed that overexpression of PEDF inhibited proliferation of cells and augmented apoptosis in serum-starved cells, in comparison with Ad-LacZ. Furthermore, Ad-PEDF suppresses anchorage-independent growth cells in soft agar and tumor formation in athymic nude mice associated with decreased microvessel density. Microarray analysis showed that 56 out of 8464 genes were found to be upregulated and downregulated in the PC-3 infected with Ad-PEDF as compared with Ad-LacZ. The differentially expressed genes cover a broad range of functional activities including catalytic activity, protein binding, signal transduction activity and cell invasion. Among them, PAI-2 and DRHC were confirmed to be upregulated with real-time PCR and Western blot, suggesting a possible association with PEDF-induced signaling for cell motility and metastasis. These results can contribute to our understanding of the molecular mechanisms of treatment strategies of PEDF for prostate cancer.

Application of microarrays to identify and characterize genes involved in attachment dependence in HeLa cells

The ability to modify cellular properties such as adhesion is of interest in the design and performance of biotechnology-related processes. The current study was undertaken in order to evaluate the effectiveness of modulating cellular adhesion in HeLa cells from a genomics perspective. Using DNA microarrays, differences in gene expression between two phenotypically distinct, anchorage-dependent and anchorage-independent, HeLa cell lines were identified. With the aid of several statistical methods and an extensive literature search, two genes were selected as potential targets for further study: siat7 e and lama4. Subsequently, experiments were carried out to investigate the effects of siat7 e and lama4 separately, on adhesion in HeLa cells by altering their expression in vivo. Decreasing the expression of siat7 e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells using short interfering RNA (siRNA) resulted in greater aggregation (i.e. clumping) and morphological changes as compared to untreated anchorage-independent HeLa cells. Similar effects were seen in anchorage-independent HeLa cells when the expression of lama4 which encodes laminin α4, a member of the laminin family of glycoproteins, was enhanced as compared to untreated anchorage-independent HeLa cells. Using a shear flow chamber, an attachment assay was developed; illustrating either increased expression of siat7 e or decreased expression of lama4 in anchorage-dependent HeLa cells reduced cellular adhesion. Collectively, the results of this study are consistent with the roles siat7 e and lama4 play in adhesion processes in vivo and indicate modifying the expression of either gene can influence adhesion in HeLa cells. The strategy of applying bioinformatics techniques to characterize and manipulate phenotypic behaviors is a powerful tool for altering the properties of various cell lines for desired biotechnology objectives.

Overexpression of integrin-linked kinase induces cardiac stem cell expansion

Recent evidence suggests that the adult heart contains stem cells that are capable of self-renewal as well as multilineage differentiation. However, their inherent capacity for self-renewal is limiting to cell replacement applications. Integrin-linked kinase is a multifunctional protein kinase that activates Wnt target genes implicated in the symmetric replication of embryonic stem cells. Primary cultures derived from human fetal cardiac tissue (19-22 weeks' gestation) were grown in serum-free media and evaluated for the presence of cardiac progenitor cells. The effect of integrin-linked kinase was ascertained by adenoviral overexpression. Cultures infected with wild-type integrin-linked kinase yielded a significant (P = .001), approximately 5-fold increase in both the absolute number and the frequency of c-Kit-positive, myosin-negative cells. Cardiospheres, comprised on morphologically homogeneous, anchorage-independent cells, were reproducibly present at days 7 to 10 and formed derivative cardiospheres in multiple passages. Integrin-linked kinase infection of primary cardiac cell cultures resulted in a greater number of primary spheres at each cell density tested, compared with untreated and virus controls (P = .001). Secondary spheres transferred to differentiation medium and 5-aza-deoxycytodine (10 micromol/L) generated cells exhibiting biochemical evidence of differentiation into cardiomyocytes, smooth muscle cells, and endothelial cells. This study demonstrates that self-renewing cardiospheres generated from human fetal cardiac cells are composed of cells exhibiting the properties of stem cells, including the capacity for self-renewal and multilineage differentiation. Our results suggest that integrin-linked kinase promotes stem cell amplification and can be applied therapeutically to overcome a major limitation in the field of cardiac regenerative medicine.

Recombinant mussel adhesive protein Mgfp-5 as cell adhesion biomaterial

Mytilus galloprovincialis foot protein type-5 (Mgfp-5) is one of the mussel adhesive proteins that participate in adhesion with the substratum. We previously reported the production of recombinant Mgfp-5 in Escherichia coli and showed that the recombinant protein had superior adhesion abilities versus those of Cell-Tak, a commercially available mussel adhesive protein mixture. In the present work, we investigated the feasibility of using recombinant Mgfp-5 as a cell adhesion agent. Purified and tyrosinase-modified recombinant Mgfp-5 was used to adhere living anchorage-independent cells such as insect Drosophila S2 cells and human MOLT-4 cells onto glass slides. Our results revealed that these cell lines efficiently attached to recombinant Mgfp-5-coated glass surfaces, and that surface-immobilized S2 cells were viable and able to undergo cell division for up to 1 week. Cytochemical studies with 4',6-diamidino-2-phenylindole (DAPI) staining of nuclei and immunofluorescence for secreted foreign human erythropoietin (hEPO) from recombinant S2 cells and quantitative comparative analyses of S2 cell binding ability with Cell-Tak and poly-L-lysine, the main cell adhesion agent, were performed to demonstrate successful usage of recombinant Mgfp-5 for cell biological applications. Collectively, these results indicate that recombinant Mgfp-5 may be a useful new cell adhesion biomaterial for anchorage-independent cells.

Proliferation, Viability, and Metabolism of Human Tumor and Normal Cells Cultured in Microcapsule

In this study, we investigated the effect of the microenvironment provided by alginate-poly-L-lysine-alginate (APA) microcapsule with liquefied or gelled core on the proliferation, viability, and metabolism of human cells, including anchorage-dependent MCF-7 breast cancer cells and primary fibroblasts, and anchorage-independent K-562 leukemia cells; cells in conventional culture were used as control. The growth pattern of cells in microcapsule was examined by phase-contrast micrography. The cell viability, proliferation, organization, and gene expression were evaluated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, hematoxylin and eosin staining, live/dead staining, 5-bromo-20-deoxyuridine labeling, and immunohistochemistry, respectively. Cell metabolism was determined by measuring glucose and lactate concentrations in medium. The results demonstrate that APA microcapsule with liquefied core provides a microenvironment for both anchorage-dependent and anchorage-independent cells to grow into a large cell aggregate and maintain cell viability at a constant level for a period of time. In conclusion, cells in APA microcapsule are alive and have proliferation potential with lower metabolism rate. APA microcapsule may be a useful tool for in vitro tumor cell modeling and anticancer drug screening as well as for cancer gene therapy. In addition, it lays a solid foundation for the use of microencapsulation in cell culture in vitro and cell implantation in vivo.

Characterization of Integrin and Anchorage Dependence in Mammary Epithelial Cells following c-erbB2-Induced Epithelial-Mesenchymal Transition

Signalling from the proto-oncogene c-erbB2 in mammary epithelial cells has earlier been shown to result in epithelial-mesenchymal transition (EMT) giving rise to fibroblast-like cells, and acquisition of anchorage-independent growth (AIG) usually determined by growth capacity in soft agar. In this study, we have analysed AIG associated with c-erbB2-induced EMT in a human mammary epithelial cell line. Intriguingly, cells capable of growth in soft agar were shown to be dependent on the function of β₁ integrin extracellular matrix receptors for growth in collagen. We therefore tested the hypothesis that apparent AIG was due to deposition of extracellular matrix in the agar. Although the fibroblastic cells had strongly upregulated expression of the fibronectin receptor subunit integrin α₅ andabundant fibronectin fibrils, these properties did not have a positive correlation with AIG. Furthermore, antibody blocking of integrin α₅ and β₁ failed to inhibit AIG. These results indicate that the anchorage-independent cells are not dependent on connection to extracellular matrix, but instead may be subject to a growth-inhibitory effect from the collagen in the absence of integrin signalling. This notion was supported by the finding that integrin blocking of the fibroblastic cells in fibrin was without effect on proliferation.

The postnatal rat aorta contains pericyte progenitor cells that form spheroidal colonies in suspension culture

Pericytes play an important role in modulating angiogenesis, but the origin of these cells is poorly understood. To evaluate whether the mature vessel wall contains pericyte progenitor cells, nonendothelial mesenchymal cells isolated from the rat aorta were cultured in a serum-free medium optimized for stem cells. This method led to the isolation of anchorage-independent cells that proliferated slowly in suspension, forming spheroidal colonies. This process required basic fibroblast growth factor (bFGF) in the culture medium, because bFGF withdrawal caused the cells to attach to the culture dish and irreversibly lose their capacity to grow in suspension. Immunocytochemistry and RT-PCR analysis revealed the expression of the precursor cell markers CD34 and Tie-2 and the absence of endothelial cell markers (CD31 and endothelial nitric oxide synthase, eNOS) and smooth muscle cell markers (alpha-smooth muscle actin, alpha-SMA). In addition, spheroid-forming cells were positive for NG2, nestin, PDGF receptor (PDGFR)-alpha, and PDGFR-beta. Upon exposure to serum, these cells lost CD34 expression, acquired alpha-SMA, and attached to the culture dish. Returning these cells to serum-free medium failed to restore their original spheroid phenotype, suggesting terminal differentiation. When embedded in collagen gels, spheroid-forming cells rapidly migrated in response to PDGF-BB and became dendritic. Spheroid-forming cells cocultured in collagen with angiogenic outgrowths of rat aorta or isolated endothelial cells transformed into pericytes. These results demonstrate that the rat aorta contains primitive mesenchymal cells capable of pericyte differentiation. These immature cells may represent an important source of pericytes during angiogenesis in physiological and pathological processes. They may also provide a convenient supply of mural cells for vascular bioengineering applications.

Cell Adhesion Status-dependent Histone Acetylation Is Regulated through Intracellular Contractility-related Signaling Activities

Although histone acetylation is important for epigenetic gene transcription, histone acetylation regulation by extracellular cues has rarely been evidenced. Here, we examined whether and how histone acetylation is regulated by cell adhesion-mediated signaling. Gastric carcinoma cells in suspension showed a higher histone acetylation, compared with fibronectin-adherent cells. This difference was supported by a decreased histone deacetylases activity. Furthermore, trichostatin A (TSA)-mediated histone acetylation was significantly increased only in suspended, but not in fibronectin-adherent, cells. Pharmacological inhibition of intracellular contractility-related myosin light chain kinase or RhoA-kinase (ROCK) or expression of ROCK1 small interfering RNA, dominant negative RhoA, or active Rac1 decreased basal and TSA-mediated histone H3 acetylations in suspended cells,whereas inhibition of calmodulin-dependent protein kinase II or transient overexpression of wild type myosin light chain kinase enhanced the acetylations. Meanwhile, chromatin immunoprecipitation showed higher basal and TSA-enhanced associations of ROCK1 promoter regions with Lys(9)-acetylated histone 3 in suspended cells than in fibronectin-adherent cells and expression of ROCK1 was higher and further increased by TSA treatment in suspension. In addition, phosphorylation of myosin light chain was further increased by TSA in suspension and higher in anchorage-independent cells over adherently growing cells, indicating an inverse relationship between ROCK1 expression-mediated contractility and cell adhesion abilities. Cell adhesion analysis showed that pharmacological activation of intracellular contractility-related signaling activities decreased cell adhesion abilities, whereas inhibition of them increased the adhesion. Taken together, these observations suggest that cell adhesion-related signal transduction regulates histone acetylation, presumably through a close functional linkage between intracellular contractility and histone deacetylases activity/histone acetylation.

Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes

Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly(ethylene glycol)(PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 microm x 20 microm and 15 microm x 15 microm in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell-surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 +/- 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.

TGF-beta1 (transforming growth factor-beta1)-mediated adhesion of gastric carcinoma cells involves a decrease in Ras/ERKs (extracellular-signal-regulated kinases) cascade activity dependent on c-Src activity.

Signalling by integrin-mediated cell anchorage to extracellular matrix proteins is co-operative with other receptor-mediated signalling pathways to regulate cell adhesion, spreading, proliferation, survival, migration, differentiation and gene expression. It was observed that an anchorage-independent gastric carcinoma cell line (SNU16) became adherent on TGF-beta1 (transforming growth factor beta1) treatment. To understand how a signal cross-talk between integrin and TGF-beta1 pathways forms the basis for TGF-beta1 effects, cell adhesion and signalling activities were studied using an adherent subline (SNU16Ad, an adherent variant cell line derived from SNU16) derived from the SNU16 cells. SNU16 and SNU16Ad cells, but not integrin alpha5-expressing SNU16 cells, showed an increase in adhesion on extracellular matrix proteins after TGF-beta1 treatment. This increase was shown to be mediated by an integrin alpha3 subunit, which was up-regulated in adherent SNU16Ad cells and in TGF-beta1-treated SNU16 cells, compared with the parental SNU16 cells. After TGF-beta1 treatment of SNU16Ad cells on fibronectin, Tyr-416 phosphorylation of c-Src was increased, but Ras-GTP loading and ERK1/ERK2 (extracellular-signal-regulated kinases 1 and 2) activity were decreased, which showed a dependence on c-Src family kinase activity. Studies on adhesion and signalling activities using pharmacological inhibitors or by transient-transfection approaches showed that inhibition of ERK1/ERK2 activity increased TGF-beta1-mediated cell adhesion slightly, but not the basal cell adhesion significantly, and that c-Src family kinase activity and decrease in Ras/ERKs cascade activity were required for the TGF-beta1 effects. Altogether, the present study indicates that TGF-beta1 treatment causes anchorage-independent gastric carcinoma cells to adhere by an increase in integrin alpha3 level and a c-Src family kinase activity-dependent decrease in Ras/ERKs cascade activity.

Functional characterization of adherent synovial fluid cells in rheumatoid arthritis: destructive potential in vitro and in vivo.

To characterize the morphologic and immunologic features of adherent synovial fluid cells derived from patients with rheumatoid arthritis (RA), and to explore their potential function in vitro and in vivo by focusing on cartilage destruction. Synovial fluid adherent cells obtained from patients with RA and from control subjects were characterized by immunohistochemistry, flow cytometry, and electron microscopy. In vitro, these cells were cultured in the presence of cartilage particles. Cartilage destruction was monitored by the release of sulfated glycosaminoglycans (sGAG) into the medium, and the level of matrix metalloproteinase 1 (MMP-1) in the cell culture supernatant was measured by enzyme-linked immunosorbent assay. To inhibit cartilage destruction in vitro, the MMP inhibitor marimastat was tested in this system. In vivo, in the SCID mouse coimplantation model, RA synovial fluid adherent cells and RA synovial fibroblasts (as positive controls) were coimplanted with human cartilage under the kidney capsule and maintained there for 60 days. In vitro, the synovial fluid adherent cells consisted of 2 subpopulations, large round-shaped macrophage-like cells (CD68+) and spindle-shaped fibroblast-like cells (Thy-1+). When passaged, the latter cells proliferated and organized themselves into 3-dimensional formations. This allowed them to reach collagen particles fixed with agarose. Fibroblasts derived from synovial tissues could not be used in this assay because they grew only in monolayers and not on agarose. The majority (>90%) of passaged RA synovial fluid adherent cells expressed the Thy-1+,CD45-,CD68-,CD86- phenotype. Electron microscopy did not reveal important morphologic differences between the 2 types of fibroblasts, those from synovial tissue or those from synovial fluid. However, synovial fluid adherent cells expressed lower levels of adhesion molecules, including CD54 and galectin 3, as well as the complement-regulatory molecule CD55. The in vitro release of sGAG associated with cell activity was 2.5-fold higher from RA synovial fluid adherent cells in comparison with that from negative control cells. The release of sGAG correlated with the concentration of MMP-1 and was inhibited by the broad-range MMP inhibitor marimastat in a dose-dependent manner. RA synovial fluid adherent cells coimplanted with cartilage in SCID mice showed the same invasive behavior as that displayed by tissue-derived RA synovial fibroblasts. Similar to tissue-derived RA synovial fibroblasts, RA synovial fluid adherent cells, which contain "floating" anchorage-independent fibroblast-like cells, mediate cartilage destruction independent of the hyperplastic synovial tissue.

Floating anchorage-independent fibroblast-like cells mediate cartilage destruction independently from the hyperplastic synovial tissue

We characterized the morphological and immunological features of adherent synovial fluid cells derived from patients with rheumatoid arthritis (RA), and explored their potential function in vitro and in vivo by focusing on cartilage destruction. Synovial fluid adherent cells were obtained from patients with RA and from controls, and were characterized by immunohistochemistry and flow cytometry. In vitro, these cells were cultured in the presence of cartilage particles. Cartilage destruction was monitored by the release of sulphated glycosaminoglycans (sGAG) into the medium, and the level of MMP-1 in the cell culture supernatant was measured by ELISA. To inhibit cartilage destruction in vitro, the matrix metalloproteinase (MMP) inhibitor Marimastat was tested in this system. In vivo, in the SCID mouse coimplantation model, RA synovial fluid adherent cells and RA synovial fibroblasts (as positive controls) were coimplanted with human cartilage under the kidney capsule, and were maintained there for 60 days. Synovial fluid adherent cells in vitro consist of two subpopulations, large round-shaped macrophage-like cells(CD68+) and spindle-shaped fibroblast-like cells (Thy-1+). When passaged, the latter cells proliferate and organize themselves in three-dimensional formations. The majority (>90%) of passaged RA synovial fluid adherent cells expressed the Thy-1+, CD68-, CD86-phenotype. Compared with synovial tissue fibroblasts, synovial fluid adherent cells expressed lower levels of adhesion molecules, including CD54 and galectin-3. Using RA synovial fluid adherent cells, the in vitro release of sGAG associated with cell activity was 2.5-fold higher compared with negative controls. The release of sGAG correlated with the concentration of MMP-1 and was inhibited by the broad range MMP inhibitor Marimastat in a dose-dependent manner. RA synovial fluid adherent cells coimplanted with cartilage in SCID mice showed the same invasive behaviour as tissue-derived RA synovial fibroblasts. Taken together, our data showed that RA synovial fluid adherent contains 'floating' anchorage-independent fibroblast-like cells similar to tissue-derived RA synovial fibroblasts, contributing to cartilage destruction independently of the process mediated by the hyperplastic synovial tissue.

Adhesion-mediated intracellular redistribution of c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein-long confers resistance to CD95-induced apoptosis in hematopoietic cancer cell lines.

Evasion of immune surveillance is a key step in malignant progression. Interactions between transformed hematopoietic cells and their environment may initiate events that confer resistance to apoptosis and facilitate immune evasion. In this report, we demonstrate that beta(1) integrin-mediated adhesion to fibronectin inhibits CD95-induced caspase-8 activation and apoptosis in hematologic tumor cell lines. This adhesion-dependent inhibition of CD95-mediated apoptosis correlated with enhanced c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein-long (c-FLIP(L)) cytosolic solubility compared with nonadhered cells. Cytosolic c-FLIP(L) protein preferentially associated with cytosolic Fas-associated death domain protein (FADD) and localized to the death-inducing signal complex after CD95 ligation in adherent cells. The incorporation of c-FLIP(L) in the death-inducing signal complex prevented procaspase-8 processing and activation of the effector phase of apoptosis. Adhesion to fibronectin increased c-FLIP(L) cytosolic solubility and availability for FADD binding by redistributing c-FLIP(L) from a preexisting membrane-associated fraction. Increased cytosolic availability of c-FLIP(L) for FADD binding was not related to increased levels of RNA or protein synthesis. These data show that adhesion of anchorage-independent cells to fibronectin provides a novel mechanism of resistance to CD95-mediated programmed cell death by regulating the cellular localization and availability of c-FLIP(L).

Transcriptional complementarity in breast cancer: application to detection of circulating tumor cells.

We used a combination of genetic subtraction, silicon DNA microarray analysis, and quantitative PCR to identify tissue- and tumor-specific genes as diagnostic targets for breast cancer. From a large number of candidate antigens, several specific subsets of genes were identified that showed concordant and complementary expression profiles. Whereas transcriptional profiling of mammaglobin resulted in the detection of 70% of tumors in a panel of 46 primary and metastatic breast cancers, the inclusion of three additional markers resulted in detection of all 46 specimens. Immunomagnetic epithelial cell enrichment of circulating tumor cells from the peripheral blood of patients with metastatic breast cancer, coupled with RT-PCR-based amplification of breast tumor-specific transcripts, resulted in the detection of anchorage-independent tumor cells in the majority of patients with breast cancer with known metastatic disease. Complementation of mammaglobin with three additional genes in RT-PCR increases the detection of breast cancers in tissue and circulating tumor cells.

Transcriptional complementarity in breast cancer: Application to detection of circulating tumor cells

Background: We used a combination of genetic subtraction, silicon DNA microarray analysis, and quantitative PCR to identify tissue- and tumor-specific genes as diagnostic targets for breast cancer. Methods and Results: From a large number of candidate antigens, several specific subsets of genes were identified that showed concordant and complementary expression profiles. Whereas transcriptional profiling of mammaglobin resulted in the detection of 70% of tumors in a panel of 46 primary and metastatic breast cancers, the inclusion of three additional markers resulted in detection of all 46 specimens. Immunomagnetic epithelial cell enrichment of circulating tumor cells from the peripheral blood of patients with metastatic breast cancer, coupled with RT-PCR-based amplification of breast tumor-specific transcripts, resulted in the detection of anchorage-independent tumor cells in the majority of patients with breast cancer with known metastatic disease. Conclusion: Complementation of mammaglobin with three additional genes in RT-PCR increases the detection of breast cancers in tissue and circulating tumor cells.

Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1.

It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G(1)/S checkpoint and maintain high levels of p27(KIP1) mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27(KIP1) is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin alpha(2)beta(1), which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.

Induction of protein phosphatase type 2A in response to disruption of cell-matrix interactions.

The proliferation of most cells is strictly dependent on cell-matrix interactions, a phenomenon called anchorage dependence. Because tumor cells are often independent of this regulation, it is important to characterize the molecular components that are involved in this control. We therefore investigated a possible role of serine/threonine protein phosphatases in the regulation of anchorage-dependent cell growth. We found that the activity of serine/threonine protein phosphatase type 2A (PP2A) and, to a lesser extent, that of type 1 (PP1), was upregulated in response to the disruption of cellular attachment. In the case of PP2A, this induction was due to the transcriptional activation of the gene and increased expression of its protein. The increase in phosphatase activity corresponded with a decrease in the phosphorylation of cellular proteins that occurred in anchorage-dependent cells, but to a much lesser degree in anchorage-independent cells. At the same time, the activity of cyclin-dependent kinases was downregulated in anchorage-dependent, but not in anchorage-independent cells. Thus, our results indicate that the balance of kinase and phosphatase activity in anchorage-dependent cells is tipped in favor of phosphatase activity, which seems to dominate the extent of reversible protein phosphorylations after cellular detachment. In contrast, anchorage-independent cells appear to neutralize elevated phosphatase activity through sustained, strong kinase activity.