Decreased Integrin Expression Research Articles

Bone marrow mesenchymal stromal cell-derived extracellular matrix displays altered glycosaminoglycan structure and impaired functionality in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.

SMAD 3, Integrin and VEGF Expression in a Periodontal Ligament During Orthodontic Tooth Movement Induced by Hyperbaric Oxygen Therapy and Stichopus hermanii.

PurposeThe aim of this study is to analyze SMAD 3, integrin and VEGF expressions in the periodontal ligament during orthodontic tooth movement induced by hyperbaric oxygen therapy and Stichopus hermanii.Materials and MethodsThirty Cavia cobaya were divided into 5 groups, namely, a normal control group (KN) without installation of helical springs or administration of HBOT and Stichopus hermanii gel. The negative control K(-) had helical spring without administration of HBOT and Stichopus hermanii gel for 14 days, while P1 had helical spring for 14 days then on day 3–14, Stichopus hermanii gel was added. Also, the helical spring was installed in P2 for 14 days then on day 8–14, HBOT 2.4 ATA was added 3 × 30 minutes a day, while P3 had helical spring for 14 days then on day 3–14, the gel was applied, and on day 8–14, HBOT 2.4 ATA was administered 3 × 30 minutes a day. Furthermore, SMAD3, integrin, and VEGF expressions were examined using immunohistochemical staining.ResultsSMAD3, integrins, and VEGF expressions showed significant differences within the groups. The combination of HBOT and Stichopus hermanii increased the expression of SMAD3 and VEGF compared to the single administration of Stichopus hermanii. The combination treatment also decreased integrin expression compared to a single HBOT administration.ConclusionThe combination of HBOT and Stichopus hermanii increases the expression of SMAD3, integrins, and VEGF compared to control but did not show significant differences compared to single HBOT treatment.

Diagnostic and therapeutic options in recurrent implantation failure.

Recurrent implantation failure (RIF) is an uncommon, imprecisely defined clinical disorder characterized by failure to achieve pregnancy after repeated embryo transfers. The diverse etiologies and incomplete understanding of RIF provide significant diagnostic and therapeutic challenges to patients and providers. Careful clinical evaluation prior to assisted reproduction can uncover many treatable causes, including thyroid dysfunction, submucosal myomas, and tobacco use. The more-subtle causes often require a more-targeted assessment. Undetected, small polyps or small areas of intrauterine synechiae are relatively common and easily treated contributors to RIF. Molecular and cellular abnormalities pose a greater therapeutic challenge. Putative causes of RIF, including progesterone resistance, shifted window of receptivity, decreased integrin expression, and immunologic disturbances, should be considered in the evaluation of a patient with otherwise unexplained RIF. It may also be true that a more complex and standardized definition of RIF would be helpful in these cases. In this paper, we review the diagnostic and therapeutic approaches to RIF, with emphasis on disorders of endometrial receptivity.

MicroRNA-124 suppresses growth and aggressiveness of osteosarcoma and inhibits TGF-β-mediated AKT/GSK-3β/SNAIL-1 signaling.

Osteosarcoma is one of the most common malignant tumors in adolescent populations and the prognosis remains incompletely understand. Previous reports have demonstrated that microRNA‑124 (miR‑124) has inhibitory effects on various human malignancies and is associated with tumor progression. However, the clinical significance and potential mechanisms of miR‑124 in the progression of osteosarcoma is not clearly understood. In this study, the potential molecular mechanism of miR‑124 in osteosarcoma tumorigenesis, growth and aggressiveness was investigated. The growth, proliferation, apoptosis, migration and invasion of osteosarcoma cells were investigated following miR‑124 transfection were determined by colony formation assay, western blotting, immunofluorescence, migration/invasion assays and reverse transcription‑quantitative polymerase chain reaction. Invivo anti‑cancer effects of miR‑124 were analyzed by a tumor growth assay, immunohistochemistry and survival rate observations. The results demonstrated that miR‑124 transfection significantly decreased integrin expression in osteosarcoma cells, and further inhibited growth, proliferation, migration and invasion of osteosarcoma cells. Flow cytometry assays indicated that miR‑124 transfection attenuated apoptosis resistance of osteosarcoma to tunicamycin, potentially via the downregulation of P53 and Bcl‑2 apoptosis regulator expression. Mechanistic assays demonstrated that miR‑124 transfection suppressed TGF‑β expression in osteosarcoma. An animal study revealed that tumor growth was reduced in tumor cells transfected with miR‑124 compared with control cells, and the survival rate was prolonged in mice with miR‑124 transfected xenografts compared with control tumors. In conclusion, these results indicate that miR‑124 transection inhibits the growth and aggressive of osteosarcoma, potentially via suppression of TGF‑β‑mediated AKT/GSK‑3β/snail family transcriptional repressor 1 (SNAIL‑1) signaling, suggesting miR‑124 may be a potential anti‑cancer agent/target for osteosarcoma therapy.

The prostate metastasis suppressor gene NDRG1 differentially regulates cell motility and invasion.

Experimental and clinical evidence suggests that N‐myc downregulated gene 1 (NDRG1) functions as a suppressor of prostate cancer metastasis. Elucidating pathways that drive survival and invasiveness of NDRG1‐deficient prostate cancer cells can help in designing therapeutics to target metastatic prostate cancer cells. However, the molecular mechanisms that lead NDRG1‐deficient prostate cancer cells to increased invasiveness remain largely unknown. In this study, we demonstrate that NDRG1‐deficient prostate tumors have decreased integrin expression and reduced cell adhesion and motility. Our data indicate that loss of NDRG1 differentially affects Rho GTPases. Specifically, there is a downregulation of active RhoA and Rac1 GTPases with a concomitant upregulation of active Cdc42 in NDRG1‐deficient cells. Live cell imaging using a fluorescent sensor that binds to polymerized actin revealed that NDRG1‐deficient cells have restricted actin dynamics, thereby affecting cell migration. These cellular and molecular characteristics are in sharp contrast to what is expected after loss of a metastasis suppressor. We further demonstrate that NDRG1‐deficient cells have increased resistance to anoikis and increased invasiveness which is independent of its elevated Cdc42 activity. Furthermore, NDRG1 regulates expression and glycosylation of EMMPRIN, a master regulator of matrix metalloproteases. NDRG1 deficiency leads to an increase in EMMPRIN expression with a concomitant increase in matrix metalloproteases and thus invadopodial activity. Using a three‐dimensional invasion assay and an in vivo metastasis assay for human prostate xenografts, we demonstrate that NDRG1‐deficient prostate cancer cells exhibit a collective invasion phenotype and are highly invasive. Thus, our findings provide novel insights suggesting that loss of NDRG1 leads to a decrease in actin‐mediated cellular motility but an increase in cellular invasion, resulting in increased tumor dissemination which positively impacts metastatic outcome.

Stromal cells control the epithelial residence of DCs and memory T cells by regulated activation of TGF-β.

Cells of the immune system that reside in barrier epithelia provide a first line of defense against pathogens. Langerhans cells (LCs) and CD8(+) tissue-resident memory T cells (TRM cells) require active transforming growth factor-β1 (TGF-β) for epidermal residence. Here we found that integrins αvβ6 and αvβ8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-β. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required αvβ6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-β, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-β by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication.

Quantifying cell behaviors in negative-pressure induced monolayer cell movement

BackgroundNegative-pressure of 125 mmHg (NP) has been shown to accelerate wound healing. Effects of NP on human keratinocyte behaviors during wound healing process were highlighted in this study. MethodsAn NP incubator incorporating the electric cell–substrate impedance sensing (ECIS) technique has been built to quantify monolayer keratinocytes movement in serum-free media at the ambient pressure (AP) and NP for 12 h. Monolayer cell motions were continuously recorded by ECIS in the frequency range of 22.5–64 kHz. Membrane capacitance (Cm), cell–substratum resistance (α), and cell–cell junction resistance (Rb) were evaluated in cells at the different pressures. ResultsA greater monolayer cell migration distance was found in cells at NP. Decreased cell–substratum adhesion reflected in the significantly low α (AP:NP = ∼5 Ω0.5:∼3 Ω0.5⋅cm), decreased integrin expression, and increased cell–substratum distance were seen in cells at NP. A significantly increased Cm (AP:NP = ∼4:∼8 μF/cm2) in association with increased membrane ruffling and microtubule filaments were observed early in the monolayer cell movement at NP. A progressive drop in the Rb from 1.2 Ω·cm2 to 0.8 Ω·cm2 corresponding to the gradually decreased E-cadherin expressions were observed 6 h after wound closure after NP treatment. ConclusionA quick membrane ruffling formation, an early cell–substratum separation, and an ensuing decrease in the cellular interaction occur in cells at NP. These specific monolayer cell behaviors at NP have been quantified and possibly accelerate wound healing.

Carbon Ion Irradiation Inhibits Glioma Cell Migration Through Downregulation of Integrin Expression

To investigate the effect of carbon ion irradiation on glioma cell migration. U87 and Ln229 glioma cells were irradiated with photons and carbon ions. Migration was analyzed 24 h after irradiation. Fluorescence-activated cell sorting analysis was performed in order to quantify surface expression of integrins. Single photon doses of 2 Gy and 10 Gy enhanced α(ν)β(3) and α(ν)β(5) integrin expression and caused tumor cell hypermigration on both vitronectin (Vn) and fibronectin (Fn). Compared to integrin expression in unirradiated cells, carbon ion irradiation caused decreased integrin expression and inhibited cell migration on both Vn and Fn. Photon radiotherapy (RT) enhances the risk of tumor cell migration and subsequently promotes locoregional spread via photon induction of integrin expression. In contrast to photon RT, carbon ion RT causes decreased integrin expression and suppresses glioma cell migration on both Vn and Fn, thus promising improved local control.

Abstract 1547: Acquisition of resistance to the β1 integrin antagonist HYD1 correlates with decreased α4, α6, αV and β1 integrin expression and decreased adhesion to fibronectin, VCAM-1 and vitronectin

Abstract Our laboratory recently reported that the D amino acid-containing β1 integrin inhibitory peptide HYD1 causes caspase independent cell death in multiple myeloma (MM) cell lines as a single agent in vitro and in vivo (Nair et al Molecular Cancer Therapeutics, 2009). Exposure of MM cells to HYD1 induces autophagy, but autophagy was shown to contribute to survival following HYD1 treatment. HYD1 treatment induces cell death in MM cell lines indicative of necrosis. These characteristics included: (a) an increase in the levels of reactive oxygen species (ROS), (b) ATP depletion and (c) loss of mitochondrial membrane potential. To further characterize the mechanism of HYD1 induced cell death an isogenic drug resistant variant was developed. This variant, referred to as H929-60, was developed by chronically exposing parental H929 cells to increasing doses of HYD1. Acquired resistance correlated with decreased HYD1 induced TO-PRO-3 positivity, decreased levels of ROS, reduced ATP depletion and reduced mmp loss compared to the H929 cell line. Additionally, the drug resistant variant H929-60 cells displayed decreased cell surface binding of FAM-HYD1 compared to H929 cells. These data indicate that decreased expression of the binding target(s) of HYD1 may contribute to drug resistance. Gene expression profiling (GEP) was used as an unbiased approach to identify changes in gene expression that may contribute to HYD1 resistance. Differentially expressed genes were mined in silico using the pathway analysis programs Ingenuity and GeneGo. The integrin signaling hub showed substantial changes in gene expression in the drug resistant variant compared to the parental cell line. Specifically, α4, α6, αV and β1 integrins were all down regulated along with the integrin associated protein CD47 in the HYD1 resistant cell line. These changes were consistent with decreased protein expression of integrins on the cell membrane and decreased functional adhesion to fibronectin, vitronectin and VCAM-1. Reducing the expression of α4 integrin using shRNA in the parental H929 cell line conferred partial resistance to HYD1 induced cell death. Together our data show that the acquisition of resistance to HYD1 correlated with decreased integrin expression and functional binding to extracellular matrixes. Future studies aim to determine whether a decrease in α6, αV and β1 integrins on the cell surface contributes to HYD1 resistance. 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 1547.

Integrin and Matrix Metalloproteinase Expression in Human Carotid Plaque

Neointimal thickening is the major cause of restenosis after carotid endarterectomy (CEA) and carotid stenting. The biologic behavior of these lesions is regulated by the interaction between smooth muscle cells (SMCs), endothelial cells (ECs), and extracellular matrix (ECM) proteins. Although the contribution of the cellular components of neointimal lesions has been extensively studied, the role of the ECM proteins in lesion remodeling is less well defined. We examined primary and restenotic carotid endarterectomy specimens to determine their cellular morphology. Tissue was also preserved for protein extraction for Western immunoblotting and mRNA for RT-PCR and cDNA microarray analysis. All primary lesions demonstrated the features of complex atherosclerotic plaque. Restenotic lesions were composed of SMCs embedded in ECM. Microarray analysis demonstrated altered expression of 13 of 96 genes. Eight genes were increased more than 3-fold and five genes were decreased more than 3-fold in primary plaque compared with restenotic lesions. RT-PCR confirmed alpha2-, alpha6-, and beta3-integrin gene expression in reference tissue, primary plaque, and restenotic lesions, with the greatest expression in primary plaque. Primary plaque demonstrated increased protein expression of plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase (TIMP-1). By zymography, pro-MMP-2, pro-MMP-9 levels, and MMP-2 activity were also increased in primary plaque compared with reference and restenotic tissues. The decreased integrin expression and protease activity in restenotic lesions versus primary carotid plaques suggests that the neointimal lesions were in a quiescent phase. These alterations in protein expression and protease activity demonstrate the importance of proteinase/inhibitor imbalance in regulating plaque remodeling.

Protein Phosphatase1 gamma (PP1cγ) Is Required for Thrombin-Induced Integrin αIIbβ3 Activation.

Integrin αIIbβ3 activation is critical for platelet physiology and is controlled by agonists- induced signal transduction through kinases and phosphatases. Compared to kinases, a role of phosphatases in platelet integrin αIIbβ3 signaling and activation is less understood. We have previously demonstrated that the catalytic subunit of protein phosphatase 1 (PP1c) associates constitutively with the integrin αIIbβ3 and regulates myosin light chain phosphorylation. Platelets express three isoforms of PP1c namely PP1cα, PP1cβ/δ and PP1cγ1, however their role in platelet physiology is unclear. In this study, we explored a role for the PP1cγ in integrin αIIbβ3 activation using the PP1cγ null mice that lacks both PP1cγ1 and PP1cγ2 splice variants. Deletion of PP1cγ did not alter the expression of PP1cα and PP1cβ/δ in platelets. Compared to the wild type mice, PP1cγ null mice had significantly (P=0.03) more platelets but not other blood cells and increased megakaryocytes in an in vitro bone marrow culture system, suggesting that PP1cγ may regulate platelet production. Compared to the wild type platelets, PP1cγ null platelets exhibited a ∼50% decreased activation (P=0.007) of integrin αIIbβ3 in response to low doses of thrombin as measured by the binding of soluble Alexa-flour 488-conjugated fibrinogen or Jon/A, an antibody to high affinity integrin αIIbβ3. Consistent with this observation, PP1cγ null platelets showed decreased aggregation in response to thrombin or protease activated receptor activating peptide. Soluble fibrinogen binding and aggregation to ADP, collagen and cross linked collagen -related peptide was not different between the PP1cγ null and wild type platelets. Surface expression of integrin αIIbβ3 was comparable in the PP1cγ null platelets indicating that the decreased activation of αIIbβ3 in PP1cγ platelets was not due to decreased integrin expression. These studies suggest that PP1cγ is required for thrombin-induced αIIbβ3 inside-out signaling process. In contrast, αIIbβ3 outside-in signaling process was not altered in PP1cγ null mice as revealed by normal adhesion to immobilized fibrinogen. Intravital microscopy revealed that the thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly (P=0.03) delayed in PP1cγ null mice compared to wild type mice. These studies illustrates that PP1cγ is an essential component of thrombin signaling that is downstream of protease activated receptors and promotes thrombus formation in part by supporting soluble fibrinogen binding and platelet aggregation.

Induction of apoptosis by the calcium antagonist mibefradil correlates with depolarization of the membrane potential and decreased integrin expression in human lens epithelial cells.

Posterior capsule opacification is still the major complication in cataract surgery and is caused by migration and proliferation of residual lens epithelial cells. The challenge of a suitable therapy to inhibit capsule opacification is to specifically interfere with cellular mechanisms. Our approach using the T-calcium channel antagonist mibefradil is based on the hypothesis that this drug inhibits the signaling pathways mediated by cell adhesion. The influence of mibefradil dihydrochloride was investigated on primary human lens epithelial cells (hLEC) from cataract surgery and on the human lens cell line HLE-B3. Apoptosis was quantitatively analyzed by flow cytometry (% increase of the sub-G1 peak), and verified by confocal microscopy (annexin V-biotin, TUNEL reaction). The membrane potential was detected by a membrane potential-sensitive dye. Integrin expression and proliferation were measured by flow cytometry. T-calcium channels in hLEC were verified by the whole-cell configuration of the patch-clamp technique. Mibefradil induced apoptosis in hLEC. Early signs of apoptosis were observed after only 4 h of incubation with mibefradil, accompanied by a significantly reduced cell area. Apoptosis correlated with inhibited integrin expression, reduced proliferation and the depolarization of the membrane potential. We could identify calcium channels of the T-type in our primary hLEC. We suggest that depolarization of the membrane potential and the inhibition of integrin expression leads to the loss of cell adhesion, which is the reason for the induction of apoptosis. Thus, mibefradil seems to be a suitable drug to prevent cell adhesion, migration and proliferation.

Epidermal Ras blockade demonstrates spatially localized Ras promotion of proliferation and inhibition of differentiation.

While important in carcinogenesis, the role of Ras in normal self-renewing tissues such as epidermis is unclear. To address this, we altered Ras function in undifferentiated and differentiating epidermal layers. Ras blockade within undifferentiated basal epidermal cells leads to decreased integrin expression, diminished growth capacity and induction of differentiation. Ras blockade in post-mitotic suprabasal epidermis exerts no effect. In contrast, regulated Ras and Raf activation inhibits differentiation. These findings indicate that spatially restricted Ras/Raf signaling divides epidermis into an undifferentiated proliferative compartment and a differentiating post-mitotic compartment and suggest a new role for Ras in tissue homeostasis.

Regulation of alphaVbeta3 and alphaVbeta5 integrins by dexamethasone in normal human osteoblastic cells.

Long-term administration of pharmacological doses of glucocorticoids inhibits bone formation and results in osteoporosis. Since integrin-mediated cell-matrix interactions are essential for osteoblast function, we hypothesized that the detrimental effect of glucocorticoids on bone derived, at least in part, from decreased integrin-matrix interactions. Because alphavbeta3 and alphavbeta5 integrins can interact with several bone matrix proteins, we analyzed the effects of dexamethasone (Dex) on the expression of these integrins in normal human osteoblastic cells. We found adhesion of these cells to osteopontin and vitronectin to be dependent on alphavbeta3 and alphavbeta5, respectively; this ligand specificity was not altered by Dex. The effects of Dex on the adhesion of human osteoblastic cells to osteopontin and vitronectin were biphasic with an increase after 2 days, followed by a decrease after 8 days of treatment. Consistently, surface alphavbeta3 and alphavbeta5 integrins, which were increased after 2 days of Dex treatment, were decreased after 8 days. Similarly, total cellular alphav, beta3, and beta5 proteins, which were increased by Dex early in the culture, were diminished after 8 days. Metabolic labeling studies indicated that Dex exhibited biphasic regulation on the biosynthesis of alphavbeta5, with stimulation observed during the second day of treatment, followed by inhibition during the 8th day of exposure. By contrast, the biosynthesis of alphavbeta3 was inhibited by Dex on day 1 and remained inhibited on day 8. Analysis of the mRNA indicated that alphav and beta5 levels were increased by Dex during early exposure (1-3 days), followed by inhibition after prolonged exposure (>/=7 days). By contrast, Dex decreased beta3 mRNA level at all the time points analyzed. Consistently, Dex decreased beta3 promoter activity after 1 day and persisted over 8-day period. By contrast, Dex stimulated beta5 promoter activity after 1 or 2 days but had no effect after 8 days. To further evaluate mechanism(s) leading to the decreased integrin expression after prolonged Dex treatment, mRNA stability was analyzed. Dex was found to accelerate the degradation of alphav, beta3 and beta5 mRNA after an 8-day treatment. Thus, the regulation of alphavbeta3 was dependent on transcription and posttranscriptional events whereas the expression of alphavbeta5 was dependent mainly on posttranscriptional events after prolonged Dex treatment. In conclusion, Dex exhibited time-dependent regulation on the expression of alphavbeta3 and alphavbeta5 integrins in normal human osteoblastic cells. Short-term exposure to Dex increased the levels of alphavbeta3 and alphavbeta5 on the surface and cell adhesion to osteopontin and vitronectin whereas long-term exposure to Dex decreased the expression of both integrins and inhibited the cell adhesion to matrix proteins.

Induction of apoptosis in human neuroblastoma cells by abrogation of integrin‐mediated cell adhesion

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extra-cellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a “DNA ladder” on gel electrophoresis, by propidium iodine staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to β, integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interations by means of perturbing antibody against β, subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings incidate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death. Int. J. Cancer 70:688–698, 1997. © 1997 Wiley-Liss, Inc.

Induction of apoptosis in human neuroblastoma cells by abrogation of integrin-mediated cell adhesion.

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extracellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a "DNA ladder" on gel electrophoresis, by propidium iodide staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to beta 1 integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interactions by means of perturbing antibody against beta 1 subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings indicate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death.