Roser Pagan

RGD Peptides and monoclonal antibodies, antagonists of αv-Integrin, enter the cells by independent endocytic pathways

Cyclic synthetic peptides containing the arginine-glycine-aspartate motif (cRGD) and monoclonal antibodies (mAbs) targeted for individual integrins have been developed as potential therapeutic drugs for the treatment of several diseases. We showed that a cRGD peptide targeted for αvβ3 was internalized in αv-integrin expressing and nonexpressing melanoma cells by an integrin independent fluid-phase endocytosis pathway that does not alter the number of functional integrin receptors at the cell surface. In contrast, a blocking mAb directed to αv was internalized by an integrin-dependent endocytosis pathway that reduced the number of functional integrin receptors at the cell surface. We prove that melanoma cells pretreated with the mAb do not readhere to the substrate, whereas cells pretreated with cRGD peptide retain their readhesion capacity. Given the growing importance of RGD peptides, knowledge of these cellular mechanisms is required to improve the development of antiangiogenic and anti-inflammatory drugs.

Use of siRNAs and Antisense Oligonucleotides Against Survivin RNA to Inhibit Steps Leading to Tumor Angiogenesis

The antiapoptotic protein survivin is an attractive target in cancer therapy because it is expressed differently in tumors and normal tissues and it is potentially required for cancer cells to remain viable. Given that survivin is also overexpressed in endothelial cells (ECs) of newly formed blood vessels found in tumors, its RNA targeting might compromise EC viability and interfere with tumor angiogenesis. We used two antisense strategies against survivin expression, antisense oligonucleotides (aODN) and small interfering RNA (siRNA), to study in ECs the contribution of survivin in various steps leading to tumor angiogenesis. A 21-mer phosphorothioate aODN and two siRNA oligonucleotides against survivin mRNA were designed to downregulate survivin expression. Survivin targeting caused (1) a strong growth-inhibitory effect, (2) a 4-fold increase in apoptosis, (3) an accumulation of cells in the S phase and a decrease in G2/M phase, (4) a dose-dependent inhibition of EC migration on Vitronectin, and (5) a decrease in capillary formation. Control oligonucleotides, an unrelated oligonucleotide, and one with four mismatches, had no significant effect. All these results show that survivin is a suitable target in cancer therapy because its inhibition in EC causes both a proapoptotic effect and an interruption of tumor angiogenesis. The two strategies used, classic aODN and siRNA technology, were very effective. Moreover, the latter can be used in the low nanomolar range, thus increasing the sensitivity of the treatment.

Effects of growth and differentiation factors on the epithelial-mesenchymal transition in cultured neonatal rat hepatocytes

BACKGROUND/AIMS Loss of specific differentiation markers, adoption of a migrating morphology and progressive replacement of the cytokeratin network by vimentin intermediate filaments characterize the epithelial-mesenchymal transition of cultured neonatal rat hepatocytes. In a previous study (Hepatology 1997; 25: 598-606), we reported that this process can be differentially regulated by EGF and DMSO, two agents that affect hepatocyte growth and differentiation. The aim of the present study was to determine if growth activation or differential gene expression could explain the differences in EMT observed between these two factors. METHODS We compared the effects of EGF, HGF, TGF-beta1 and DMSO on growth, proto-oncogene expression, epithelial-mesenchymal transition markers and expression of liver transcription factors in cultured neonatal rat hepatocytes using thymidine incorporation, Northern blotting and Western blotting analysis. RESULTS When TGF-beta1 or DMSO was added to the cultures supplemented with EGF and HGF, the mitogenic activity induced by these factors was inhibited. DMSO down-regulated c-myc and c-fos expression. mRNA levels of some liver-specific genes such as albumin, or liver-enriched transcription factors such as C/EBPdelta, HNF-4 and HNF-1beta were slightly different in cultures supplemented with DMSO or TGF-beta1. However, no differences were found when DMSO or TGF-beta1 was added to the cultures supplemented with EGF. Western blotting analysis showed that TGF-beta1 decreased cytokeratin and increased vimentin levels, while DMSO decreased both cytokeratin and vimentin. When DMSO or TGF-beta1 was added in combination with EGF or HGF, both factors maintained the increase in albumin and cytokeratin induced by the growth factors although DMSO, but not TGF-beta1, inhibited vimentin expression. CONCLUSIONS Activation of vimentin expression produced in cultures supplemented with the mitogenic factors (EGF and HGF) is independent of the activation of cell growth, because DMSO but not TGF-beta1 can abolish vimentin synthesis, although both inhibited growth. Moreover, the vimentin expression in these cultures seems to be independent of the mRNA levels of transcription factors associated with the differentiated liver phenotype.

Fibronectin regulates morphology, cell organization and gene expression of rat fetal hepatocytes in primary culture

BACKGROUND/AIMS The extracellular matrix regulates hepatic development and regeneration, modulating the maintenance of liver architecture in the differentiated state. The aim of this work was to analyze how different extracellular matrix molecules modulate fetal hepatocyte morphology, growth and differentiation. METHODS We cultured fetal hepatocytes either on plastic or on different extracellular matrix proteins, i.e., collagen I, fibronectin or E-C-L (entactin-collagen IV-laminin) and we analyzed cell attachment, morphological organization, proliferative response and gene expression. RESULTS Cell attachment was increased by all the extracellular matrix proteins to a similar extent. However, only fibronectin facilitated the formation of elongated cord-like structures, reminiscent of liver plate organization. Immunocytochemical analysis of the cells in these structures revealed high levels of albumin and cytokeratin 18, phenotypical markers of parenchymal hepatocytes. Fibronectin did not block the mitogenic stimuli induced by epidermal growth factor in these cells and the elongated structures appeared either in the absence or in the presence of the mitogen. Cells cultured on fibronectin, regardless of whether epidermal growth factor was present or not, also presented the maximal levels of expression for liver specific genes, such as albumin or alpha-fetoprotein. This expression was coincident with an increased expression of hepatocyte nuclear factor (HNF)-4 and a higher HNF-1alpha/HNF-1beta ratio, when compared with those cells that were cultured on collagen or E-C-L extracellular matrix. CONCLUSIONS These results suggest that fibronectin might play a differential role, as compared to other extracellular matrix proteins, in fetal hepatocyte organization and gene expression.

Comparative study of tube assembly in three-dimensional collagen matrix and on Matrigel coats

This study compares phenotypic changes of human umbilical endothelial vein cells cultured in three-dimensional collagen matrixes in the presence of basic fibroblast growth factor or on Matrigel coats. Under both conditions, endothelial cells rapidly assembled into an irregular network of tubular structures with a high frequency of intercellular or lumen-like spaces. Tubular structures were characterized and compared by phase-contrast, confocal and electron microscopy. The dominant mechanism of lumen-like formation was highly model-dependent. Ultrastructural analyses of capillary-like structures and the mechanism of lumen-like formation indicated that the in vivo angiogenesis was better reproduced in the collagen model.

Alpha v integrin antagonists induce the disassembly of focal contacts in melanoma cells

In recent years, several antagonists of alpha(v)beta3 have been used to develop therapeutic approaches to the treatment of melanoma neoplasia. We studied the effects of anti-alpha(v)-integrin-blocking antibodies on attached M21 melanoma cells, the cellular distribution of alpha(v)-integrin and the molecular organization of focal structures. Anti-alpha(v)-integrin-blocking antibodies 17E6 and LM609, and an anti-alpha(v)beta3-integrin antagonist peptide cRGD 85189 induced detachment of M21 melanoma cells cultured for 24 hours on various substrates. cRGD was the most effective antagonist, reducing the number of adherent cells by 80%, while 17E6 reduced adhesion by only 30%. Light- and electron microscopy revealed attached cells with a flat shape and well-formed actin cytoskeleton. After treatment, cells became rounded and detached from the culture dish. alpha(v)-Integrins and focal-contact proteins were observed at adhesion sites in focal structures by immunocytochemistry. After treatment, however, cell rounding was accompanied by disorganization of the actin filaments and redistribution of alpha(v)-integrins and most of the focal proteins studied, except vinculin and tensin. Our results indicate that treatment of M21 melanoma cells with a(v)-integrin antagonists disrupts the actin cytoskeleton, redistributes a(v)-integrin and induces molecular disassembly of focal contacts.

Anti-migratory and anti-angiogenic effect of p16: A novel localization at membrane ruffles and lamellipodia in endothelial cells

Recent evidence has established different functions for the tumor suppressor protein, p16INK4A aside from controlling the cell cycle. Here we report that cdk4/6 inhibition blocked both human umbilical vein endothelial cells (HUVEC) spreading on a vitronectin matrix and HUVEC migration on vitronectin. p16 can also act as an anti-angiogenic molecule in vitro since HUVEC and HMEC cells transfected with Ad-p16 or treated with Antennapedia p16 peptides are unable to differentiate on a Matrigel matrix. Both, p16, cyclin D1, cdk4 and cdk6 were immuno-colocalized with Ezrin, Rac, Vinculin, αv-integrin, and FAK proteins in the ruffles and lamellipodia of migratory cells. Our results indicate that p16 is a key component of a new cytoplasmic pathway controlling angiogenesis of endothelial cells via the αvβ3-integrin-mediated migration.

Immunofluorescence and Immunoelectron Microscopy of Microinjected and Transfected Cultured Cells

Immunofluorescence microscopy is the most common method to analyze expression and localization of a given protein in cells that have been microinjected or transfected previously with the appropriate DNA constructs. It offers the advantage that it is quick, easy to perform, and allows examination of a large number of cells within a short time. However, illumination with UV light is often damaging for the cells, and the fluorescence tends to bleach as a result of the excitation of the fluorochrome by the UV light. Nowadays, these disadvantages have been overcome by sophisticated systems such as video intensification cameras and confocal microscopy. In addition, the information that can be obtained by immunofluorescence microscopy is also restricted by the limited resolution of the optical lens. Moreover, some fixation conditions can induce artifactual changes in the intracellular localization of a significant number of molecules (Melan and Sluder 1992; Griffiths et al. 1993). Therefore, if the precise localization of a protein is being studied, it will always be necessary to look at the fine structure of the cell, using immunoelectron microscopy techniques. Nevertheless, whenever possible, the utilization of immunofluorescence in combination with immunoelectron microscopy is preferable. In this chapter, we describe in detail protocols for immunofluorescence and immunoelectron microscopy analyses which are particularly suited for cells in culture.