B-Raf regulation of integrin α4ß1-mediated resistance to shear stress through changes in cell spreading and cytoskeletal association in T cells.

The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4ß1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4ß1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4ß1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4ß1 integrin and not other integrins, such as α5ß1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4ß1 integrin-mediated adhesion.

The PI3-kinase delta inhibitor idelalisib (GS-1101) targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL) cell to endothelial and marrow stromal cells.

CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

CD25 and CD69 induction by α4ß1 outside-in signalling requires TCR early signalling complex proteins.

Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4ß1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4ß1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4ß1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4ß1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation.

Small molecule agonist of very late antigen-4 (VLA-4) integrin induces progenitor cell adhesion.

Activation of the integrin family of cell adhesion receptors on progenitor cells may be a viable approach to enhance the effects of stem cell-based therapies by improving cell retention and engraftment. Here, we describe the synthesis and characterization of the first small molecule agonist identified for the integrin α4ß1 (also known as very late antigen-4 or VLA-4). The agonist, THI0019, was generated via two structural modifications to a previously identified α4ß1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to α4ß1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1α. Molecular modeling predicted that the compound binds at the α/ß subunit interface overlapping the ligand-binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin α4ß7 as well as α5ß1 and αLß2. When cross-linked to αLß2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the α/ß subunit interface and not at the ligand-binding site in the inserted ("I") domain of the αL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.

Targeting constitutively activated ß1 integrins inhibits prostate cancer metastasis.

Disseminated prostate cancer cells must survive in circulation for metastasis to occur. Mechanisms by which these cells survive are not well understood. By immunohistochemistry of human tissues, we found that levels of ß1 integrins and integrin-induced autophosphorylation of FAK (pFAK-Y397) are increased in prostate cancer cells in primary prostate cancer and lymph node metastases, suggesting that ß1 integrin activation occurs in metastatic progression of prostate cancer. A conformation-sensitive antibody, 9EG7, was used to examine ß1 integrin activation. We found that ß1 integrins are constitutively activated in highly metastatic PC3 and PC3-mm2 cells, with less activation in low metastatic LNCaP and C4-2B4 cells. Increased ß1 integrin activation as well as the anoikis resistance in prostate cancer cells correlated with metastatic potential in vivo. Knockdown of ß1 integrin abrogated anoikis resistance in PC3-mm2 cells. In agreement with ß1 integrin activation, PC3-mm2 cells strongly adhered to type I collagen and fibronectin, a process inhibited by the ß1 integrin-neutralizing antibody mAb 33B6. mAb 33B6 also inhibited the phosphorylation of ß1 integrin downstream effectors, focal adhesion kinase (FAK) and AKT, leading to a 3-fold increase in PC3-mm2 apoptosis. Systemic delivery of mAb 33B6 suppressed spontaneous metastasis of PC3-mm2 from the prostate to distant lymph nodes following intraprostatic injection and suppressed metastasis of PC3-mm2 to multiple organs following intracardiac injection. Thus, constitutively activated ß1 integrins play a role in survival of PC3-mm2 cells in circulation and represent a potential target for metastasis prevention.

Synthesis and evaluation of an anti-MLC1 × anti-CD90 bispecific antibody for targeting and retaining bone-marrow-derived multipotent stromal cells in infarcted myocardium.

A key issue regarding the use of stem cells in cardiovascular regenerative medicine is their retention in target tissues. Here, we have generated and assessed a bispecific antibody heterodimer designed to improve the retention of bone-marrow-derived multipotent stromal cells (BMMSC) in cardiac tissue damaged by myocardial infarction. The heterodimer comprises an anti-human CD90 monoclonal antibody (mAb) (clone 5E10) and an anti-myosin light chain 1 (MLC1) mAb (clone MLM508) covalently cross-linked by a bis-arylhydrazone. We modified the anti-CD90 antibody with a pegylated-4-formylbenzamide moiety to a molar substitution ratio (MSR) of 2.6 and the anti-MLC1 antibody with a 6-hydrazinonicotinamide moiety to a MSR of 0.9. The covalent modifications had no significant deleterious effect on mAb epitope binding. Furthermore, the binding of anti-CD90 antibody to BMMSCs did not prevent their differentiation into adipo-, chondro-, or osteogenic lineages. Modified antibodies were combined under mild conditions (room temperature, pH 6, 1 h) in the presence of a catalyst (aniline) to allow for rapid generation of the covalent bis-arylhydrazone, which was monitored at A(354). We evaluated epitope immunoreactivity for each mAb in the construct. Flow cytometry demonstrated binding of the bispecific construct to BMMSCs that was competed by free anti-CD90 mAb, verifying that modification and cross-linking were not detrimental to the anti-CD90 complementarity-determining region. Similarly, ELISA-based assays demonstrated bispecific antibody binding to plastic-immobilized recombinant MLC1. Excess anti-MLC1 mAb competed for bispecific antibody binding. Finally, the anti-CD90 × anti-MLC1 bispecific antibody construct induced BMMSC adhesion to plastic-immobilized MLC1 that was resistant to shear stress, as measured in parallel-plate flow chamber assays. We used mAbs that bind both human antigens and the respective pig homologues. Thus, the anti-CD90 × anti-MLC1 bispecific antibody may be used in large animal studies of acute myocardial infarction and may provide a starting point for clinical studies.

An anti-PR1/HLA-A2 T-cell receptor-like antibody mediates complement-dependent cytotoxicity against acute myeloid leukemia progenitor cells.

PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)-restricted leukemia-associated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)-like immunoglobulin G2a (IgG2a) antibody (8F4) with high specific binding affinity (dissociation constant [K(D)] = 9.9nM) for a combined epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes (P = .046). 8F4 mediated complement-dependent cytolysis of AML blasts and Lin(-)CD34(+)CD38(-) leukemia stem cells (LSCs) but not normal leukocytes (P < .005). Although PR1 expression was similar on LSCs and hematopoietic stem cells, 8F4 inhibited AML progenitor cell growth, but not normal colony-forming units from healthy donors (P < .05). This study shows that 8F4, a novel TCR-like antibody, binds to a conformational epitope of the PR1/HLA-A2 complex on the cell surface and mediates specific lysis of AML, including LSCs. Therefore, this antibody warrants further study as a novel approach to targeting leukemia-initiating cells in patients with AML.

Potent in vivo suppression of inflammation by selectively targeting the high affinity conformation of integrin α4ß1.

The development of antagonists to the α4 integrin family of cell adhesion molecules has been an active area of pharmaceutical research to treat inflammatory and autoimmune diseases. Presently being tested in human clinical trials are compounds selective for α4ß1 (VLA-4) as well as several dual antagonists that inhibit both α4ß1 and α4ß7. The value of a dual versus a selective small molecule antagonist as well as the consequences of inhibiting different affinity states of the α4 integrins have been debated in the literature. Here, we characterize TBC3486, a N,N-disubstituted amide, which represents a unique structural class of non-peptidic, small molecule VLA-4 antagonists. Using a variety of adhesion assay formats as well as flow cytometry experiments using mAbs specific for certain activation-dependent integrin epitopes we demonstrate that TBC3486 preferentially targets the high affinity conformation of α4ß1 and behaves as a ligand mimetic. The antagonist is capable of blocking integrin-dependent T-cell co-activation in vitro as well as proves to be efficacious in vivo at low doses in two animal models of allergic inflammation. These data suggest that a small molecule α4 integrin antagonist selective for α4ß1 over α4ß7 and, specifically, selective for the high affinity conformation of α4ß1 may prove to be an effective therapy for multiple inflammatory diseases in humans.

2E8 binds to the high affinity I-domain in a metal ion-dependent manner: a second generation monoclonal antibody selectively targeting activated LFA-1.

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α(L) subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K(D)) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4(+) and CD8(+) T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.

Adrenergic modulation of focal adhesion kinase protects human ovarian cancer cells from anoikis.

Chronic stress is associated with hormonal changes that are known to affect multiple systems, including the immune and endocrine systems, but the effects of stress on cancer growth and progression are not fully understood. Here, we demonstrate that human ovarian cancer cells exposed to either norepinephrine or epinephrine exhibit lower levels of anoikis, the process by which cells enter apoptosis when separated from ECM and neighboring cells. In an orthotopic mouse model of human ovarian cancer, restraint stress and the associated increases in norepinephrine and epinephrine protected the tumor cells from anoikis and promoted their growth by activating focal adhesion kinase (FAK). These effects involved phosphorylation of FAKY397, which was itself associated with actin-dependent Src interaction with membrane-associated FAK. Importantly, in human ovarian cancer patients, behavioral states related to greater adrenergic activity were associated with higher levels of pFAKY397, which was in turn linked to substantially accelerated mortality. These data suggest that FAK modulation by stress hormones, especially norepinephrine and epinephrine, can contribute to tumor progression in patients with ovarian cancer and may point to potential new therapeutic targets for cancer management.

Co-engagement of alpha(4)beta(1) integrin (VLA-4) and CD4 or CD8 is necessary to induce maximal Erk1/2 phosphorylation and cytokine production in human T cells.

The alpha(4)beta(1) integrin VLA-4 (very-late activation antigen-4) and the lineage-specific CD4 and CD8 receptors have been proposed as putative co-stimulatory receptors on T cells. To assess the relative contribution of signaling through the TCR, CD28 and these accessory molecules, we activated human T cells using soluble antibodies recognizing all four of these T-cell receptor classes (CD3, CD28, CD4/CD8, and VLA-4), and we assessed the degree of activation using higher-order flow cytometry detecting intracellular Erk1/2 phosphorylation and production of IL-2 and IFN-gamma. We found that: (1) co-stimulation via CD4/CD8, in addition to CD28, is required for optimal T-cell activation; (2) VLA-4 binding consistently potentiates CD4(+) and CD8(+) T-cell activation; (3) augmentation of T-cell activation through VLA-4 binding is most pronounced following engagement of CD4/CD8. These results confirm that multiple signals, including VLA-4 engagement, are necessary for maximal T-cell activation beyond that induced via the TCR and CD28.

LFA-1 affinity regulation is necessary for the activation and proliferation of naive T cells.

The activation of LFA-1 (lymphocyte function-associated antigen) is a critical event for T cell co-stimulation. The mechanism of LFA-1 activation involves both affinity and avidity regulation, but the role of each in T cell activation remains unclear. We have identified antibodies that recognize and block different affinity states of the mouse LFA-1 I-domain. Monoclonal antibody 2D7 preferentially binds to the low affinity conformation, and this specific binding is abolished when LFA-1 is locked in the high affinity conformation. In contrast, M17/4 can bind both the locked high and low affinity forms of LFA-1. Although both 2D7 and M17/4 are blocking antibodies, 2D7 is significantly less potent than M17/4 in blocking LFA-1-mediated adhesion; thus, blocking high affinity LFA-1 is critical for preventing LFA-1-mediated adhesion. Using these reagents, we investigated whether LFA-1 affinity regulation affects T cell activation. We found that blocking high affinity LFA-1 prevents interleukin-2 production and T cell proliferation, demonstrated by TCR cross-linking and antigen-specific stimulation. Furthermore, there is a differential requirement of high affinity LFA-1 in the activation of CD4(+) and CD8(+) T cells. Although CD4(+) T cell activation depends on both high and low affinity LFA-1, only high affinity LFA-1 provides co-stimulation for CD8(+) T cell activation. Together, our data demonstrated that the I-domain of LFA-1 changes to the high affinity state in primary T cells, and high affinity LFA-1 is critical for facilitating T cell activation. This implicates LFA-1 activation as a novel regulatory mechanism for the modulation of T cell activation and proliferation.

Clustering T-cell GM1 lipid rafts increases cellular resistance to shear on fibronectin through changes in integrin affinity and cytoskeletal dynamics.

Lipid rafts are small laterally mobile microdomains that are highly enriched in lymphocyte signaling molecules. GM1 gangliosides are a common lipid raft component and have been shown to be important in many T-cell functions. The aggregation of specific GM1 lipid rafts can control many T-cell activation events, including their novel association with T-cell integrins. We found that clustering GM1 lipid rafts can regulate beta1 integrin function. This was apparent through increased resistance to shear flow-dependent detachment of T cells adherent to the alpha4beta1 and alpha5beta1 integrin ligand fibronectin (FN). Adhesion strengthening as a result of clustering GM1 enriched lipid rafts correlated with increased cellular rigidity and morphology through the localization of cortical F-actin, the resistance to shear-induced cell stretching, and an increase in the surface area and symmetry of the contact area between the cell surface and adhesive substrate. Furthermore, clustering GM1 lipid rafts could initiate integrin 'inside-out' signaling mechanisms. This was seen through increased integrin-cytoskeleton associations and enhanced soluble binding of FN and VCAM-1, suggesting the induction of high-affinity integrin conformations. The activation of these adhesion-strengthening characteristics appears to be specific for the aggregation of GM1 lipid rafts as the aggregation of the heterogeneous raft-associated molecule CD59 failed to activate these functions. These findings indicate a novel mechanism to signal to beta1 integrins and to activate adhesion-strengthening processes.

CD45RA T-cell activation without proliferation by a partial agonist monoclonal antibody to beta1 integrin.

CD45RA T cells are fully co-activated by natural beta1 integrin ligands fibronectin (FN) and VCAM-1, as well as monoclonal antibody (mAb) 19H8, which binds a combinatorial epitope of the alpha4beta1 heterodimer. These integrin ligands stimulate CD3-dependent proliferation and the upregulation of early activation markers CD25 and CD69. However, beta1-specific antibody 33B6, which binds to a similar range of the predominant T-cell integrins as natural ligands FN (alpha4beta1 and alpha5beta1) and VCAM-1 (alpha4beta1), failed to costimulate proliferation in the CD45RA subset, while retaining the ability to costimulate early activation markers CD25 and CD69. After addition of exogenous human interleukin-2 to the culture media, 33B6 costimulation of proliferation is restored. These data provide evidence that a branch of the alpha4beta1 integrin-signaling pathway in CD45RA T cells can be independently regulated and exploited through the use of partial agonist ligands, including mAbs to the integrin heterodimer.

Maintenance and polarization of human TH2 central memory T cells by thymic stromal lymphopoietin-activated dendritic cells.

The identity of TH2 memory cells and the mechanism regulating their maintenance during allergic inflammation remain elusive. We report that circulated human CD4+ T cells expressing the prostaglandin D2 receptor (CRTH2) are TH2 central memory T cells, characterized by their phenotype, TH2 cytokine production, gene-expression profile, and the ability to respond to allergens. Only dendritic cells (DCs) activated by thymic stromal lymphopoietin (TSLP) can induce a robust expansion of CRTH2+CD4+ TH2 memory cells, while maintaining their central memory phenotype and TH2 commitments. CRTH2+CD4+ TH2 memory cells activated by TSLP-DCs undergo further TH2 polarization and express cystatin A, Charcot-Leydon crystal protein, and prostaglandin D2 synthase, implying their broader roles in allergic inflammation. Infiltrated CRTH2+CD4+ TH2 effector memory T cells in skin lesion of atopic dermatitis were associated with activated DCs, suggesting that TSLP-DCs play important roles not only in TH2 priming, but also in the maintenance and further polarization of TH2 central memory cells in allergic diseases.

PI3-K/Akt-mediated anoikis resistance of human osteosarcoma cells requires Src activation.

Considerable advances in understanding the mechanisms associated with anoikis resistance of normal and malignant epithelial cells have been made. However, little is still known about the pathways involved in anoikis resistance of non-epithelial cells such as fibroblasts and sarcomas. Our results show that Src activity contributes to anoikis resistance of human osteosarcoma SAOS-2 cells. Src was found to be upregulated in anoikis resistant SAOS cells, and pharmacological inhibition of its activity resulted in the restoration of anoikis sensitivity. A normal pattern of dephosphorylation of FAK was observed upon cell detachment of both anoikis sensitive and resistant SAOS-2 cells, suggesting that FAK activity during anoikis resistance is not essential. The activity of Akt was found to be upregulated in anoikis resistant SAOSar cells and the pharmacological inhibition of PI3-K activity restored sensitivity to anoikis resistant cells, reconfirming the critical role of PI3-K/Akt pathway in cell survival. Furthermore, pharmacological inhibition of Src resulted in a decrease of Akt phosphorylation at Ser473. Altogether, these studies indicated a survival pathway mediated by the Src-dependent activation of the PI3-K/Akt pathway in a manner independent of FAK activity.

Contrasting roles for domain 4 of VCAM-1 in the regulation of cell adhesion and soluble VCAM-1 binding to integrin alpha4beta1.

Cell adhesion mediated by the interaction between integrin alpha4beta1 and VCAM-1 is important in normal physiologic processes and in inflammatory and autoimmune disease. Numerous studies have mapped the alpha4beta1 binding sites in VCAM-1 that mediate cell adhesion; however, little is known about the regions in VCAM-1 important for regulating soluble binding. In the present study, we demonstrate that 6D VCAM-1 (an alternatively spliced isoform of VCAM-1 lacking Ig-like domain 4) binds alpha4beta1 with a higher relative affinity than does the full-length form of VCAM-1 containing 7 Ig-like extracellular domains (7D VCAM-1). In indirect binding assays, the EC50 of soluble 6D VCAM-1 binding to alpha4beta1 on Jurkat cells (in 1 mM MnCl2) was 2 x 10(-9) M, compared with 7D VCAM-1 at 11 x 10(-9) M. When used in solution to inhibit alpha4beta1 mediated cell adhesion, the IC50 of 6D VCAM-1 was 13 x 10(-9) M, compared with 7D VCAM-1 measured at 150 x 10(-9) M. Removal of Ig-like domains 4, 5, or 6, or simply substituting Asp328 in domain 4 of 7D VCAM-1 with alanine, caused increased binding of soluble 7D VCAM-1 to alpha4beta1. In contrast, cells adhered more avidly to 7D VCAM-1 under shear force, as it induced cell spreading at lower concentrations than did 6D VCAM-1. Finally, soluble 6D VCAM-1 acts as an agonist through alpha4beta1 by augmenting cell migration and inducing cell aggregation. These results indicate that the domain 4 of VCAM-1 plays a contrasting role when VCAM-1 is presented in solution or as a cell surface-expressed adhesive substrate.

A chondroitin sulfate chain attached to the bone dentin matrix protein 1 NH2-terminal fragment.

Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein shown by gene ablations to be critical for the proper mineralization of bone and dentin. In the extracellular matrix of these tissues DMP1 is present as fragments representing the NH2-terminal (37 kDa) and COOH-terminal (57 kDa) portions of the cDNA-deduced amino acid sequence. During our separation of bone noncollagenous proteins, we observed a high molecular weight, DMP1-related component (designated DMP1-PG). We purified DMP1-PG with a monoclonal anti-DMP1 antibody affinity column. Amino acid analysis and Edman degradation of tryptic peptides proved that the core protein for DMP1-PG is the 37-kDa fragment of DMP1. Chondroitinase treatments demonstrated that the slower migration rate of DMP1-PG is due to the presence of glycosaminoglycan. Quantitative disaccharide analysis indicated that the glycosaminoglycan is made predominantly of chondroitin 4-sulfate. Further analysis on tryptic peptides led us to conclude that a single glycosaminoglycan chain is linked to the core protein via Ser74, located in the Ser74-Gly75 dipeptide, an amino acid sequence specific for the attachment of glycosaminoglycans. Our findings show that in addition to its existence as a phosphoprotein, the NH2-terminal fragment from DMP1 occurs as a proteoglycan. Amino acid sequence alignment analysis showed that the Ser74-Gly75 dipeptide and its flanking regions are highly conserved among a wide range of species from caiman to the Homo sapiens, indicating that this glycosaminoglycan attachment domain has survived an extremely long period of evolution pressure, suggesting that the glycosaminoglycan may be critical for the basic biological functions of DMP1.

Acquisition of anoikis resistance in human osteosarcoma cells does not alter sensitivity to chemotherapeutic agents.

BACKGROUND: Chemotherapy-induced cell death can involve the induction of apoptosis. Thus, aberrant function of the pathways involved might result in chemoresistance. Since cell adhesion to the extracellular matrix acts as a survival factor that homeostatically maintains normal tissue architecture, it was tested whether acquisition of resistance to deadhesion-induced apoptosis (anoikis) in human osteosarcoma would result in resistance to chemotherapy. METHODS: Osteosarcoma cell lines (SAOS-2 and TE-85) obtained from ATCC and were maintained in complete Eagle's MEM medium. Suspension culture was established by placing cells in tissue culture wells coated with poly-HEMA. Cell cytotoxicity was determined using a live/dead cytotoxicity assay. Cell cycle/apoptosis analyses were performed using propidium iodide (PI) staining with subsequent FACS analysis. Apoptosis was also assayed by Annexin-FITC/PI staining. RESULTS: Etoposide, adriamycin, vinblastine, cisplatin and paclitaxel were able to induce apoptosis in human osteosarcoma cells SAOS-2 regardless of their anoikis resistance phenotype or the culture conditions (adhered vs. suspended). Moreover, suspended anoikis resistant TE-85 cells (TE-85ar) retained their sensitivity to chemotherapy as well. CONCLUSION: Acquisition of anoikis resistance in human osteosarcoma cells does not result in a generalized resistance to all apoptotic stimuli, including chemotherapy. Moreover, our results suggest that the pathways regulating anoikis resistance and chemotherapy resistance might involve the action of different mediators.