Javier Turnay

Inhibition of Phosphatidylserine Recognition Heightens the Immunogenicity of Irradiated Lymphoma Cells In Vivo

Strategies to enhance the immunogenicity of tumors are urgently needed. Although vaccination with irradiated dying lymphoma cells recruits a tumor-specific immune response, its efficiency as immunogen is poor. Annexin V (AxV) binds with high affinity to phosphatidylserine on the surface of apoptotic and necrotic cells and thereby impairs their uptake by macrophages. Here, we report that AxV preferentially targets irradiated lymphoma cells to CD8+ dendritic cells for in vivo clearance, elicits the release of proinflammatory cytokines and dramatically enhances the protection elicited against the tumor. The response was endowed with both memory, because protected animals rejected living lymphoma cells after 72 d, and specificity, because vaccinated animals failed to reject unrelated neoplasms. Finally, AxV–coupled irradiated cells induced the regression of growing tumors. These data indicate that endogenous adjuvants that bind to dying tumor cells can be exploited to target tumors for immune rejection.

Bioactive sol-gel glasses with and without a hydroxycarbonate apatite layer as substrates for osteoblast cell adhesion and proliferation.

The biocompatibility of three sol-gel bioactive glasses with SiO(2) as the main constituent (75, 72.5 and 70 mol%), identical CaO content (25mol%), and without or with P(2)O(5) as third constituent (0, 2.5 and 5 mol%), have been analyzed (S75, S72.5P2.5, and S70P5 glasses). These studies were performed on both untreated glasses and on glasses coated with a hydroxycarbonate apatite (HCA) layer formed in vitro by soaking 7d in an acellular simulated body fluid. Cell attachment, spreading and proliferation were studied using neonatal rat calvaria osteoblasts. Cells attach to the three untreated glasses but show a higher efficiency on that with the higher phosphate content (S70P5). The formation of the HCA layer significantly enhances this process (1.7-fold). In all cases, attachment is followed by cell spreading on the surface of the materials, adopting the cells a flattened morphology and showing diverse anchoring cell projections. Mitotic activity has been detected on osteoblasts growing on the sol-gel glasses, being this process 2-4-fold higher when the apatite-like layer is already formed. Taking into account the results herein presented, these bioactive glasses can be considered biocompatible. In addition, their biocompatibility is greatly enhanced after induction of the formation of an HCA layer.

Influence of different chemical cross-linking treatments on the properties of bovine pericardium and collagen

The use of biological materials in the construction of bioprostheses requires the application of different chemical or physical procedures to improve the mechanical performance of the material without producing any undesirable effects. A number of cross-linking methods have been tested in biological tissues composed mainly of collagen. The basis for most of them is the use of glutaraldehyde (GA), which acts on the Lys or Hyl residues. We have studied the effects of alternative chemical treatments: diphenylphosphorylazide (DPPA) and ethyldimethylaminopropyl carbodiimide (EDAC). Their mechanism of action is based on the activation of the carboxyl groups, which then permits their cross-linking to amino groups. As a control, we employed conventional treatment with GA, applying it to bovine pericardium and collagen membranes removed from bovine pericardium. The analysis of the Lys and Hyl residues showed that DPPA and EDAC produced 50% of the chemical change provoked by GA. This value was even lower in the trials with collagen. In terms of the resistance to collagenase degradation, chemical cross-linking with GA provided much greater protection in both materials (3.81 +/- 3.47 nmol of amino acid/mg dry tissue for pericardium and 4.41 +/- 1.13 nmol of amino acid/mg dry tissue for collagen). Treatment with DPPA also protected pericardium (13.11 +/- 6.57 nmol amino acid/mg dry tissue) although the values for collagen was lower (50.0 +/- 32.4 nmol amino acid/mg dry tissue). Treatment with EDAC was much less protective than the other two chemical reagents (43.28 +/- 17.4 and 55.85 +/- 14.57 nmol amino acid/mg dry tissue for pericardium and collagen, respectively). The degree of tissue calcification after implantation of the chemically treated materials into young rats was considerably greater for GA and DPPA (32.9 +/- 18.8 and 36.3 +/- 13.3 mg g(-1) dry tissue, respectively) than with EDAC (18.0 +/- 7.2 mg g(-1) dry tissue; P < 0.001). After 60 days of implantation, the values for GA and EDAC were higher(124.1 +/- 31.3 and 124.6 +/- 21.0 mg g(-1) dry tissue, respectively) versus 34.6 +/- 19.2 mg g(-1) dry tissue for DPPA. There were no significant differences in collagen levels in samples treated with GA or EDAC after 30 days of implantation, although both groups showed significant differences when compared with DPPA-treated samples (P < 0.001). After 60 days of implantation, there were no significant differences among these three treatments in terms of the calcium accumulated on samples.

Annexin-Phospholipid Interactions. Functional Implications

Annexins constitute an evolutionary conserved multigene protein superfamily characterized by their ability to interact with biological membranes in a calcium dependent manner. They are expressed by all living organisms with the exception of certain unicellular organisms. The vertebrate annexin core is composed of four (eight in annexin A6) homologous domains of around 70 amino acids, with the overall shape of a slightly bent ring surrounding a central hydrophilic pore. Calcium- and phospholipid-binding sites are located on the convex side while the N-terminus links domains I and IV on the concave side. The N-terminus region shows great variability in length and amino acid sequence and it greatly influences protein stability and specific functions of annexins. These proteins interact mainly with acidic phospholipids, such as phosphatidylserine, but differences are found regarding their affinity for lipids and calcium requirements for the interaction. Annexins are involved in a wide range of intra- and extracellular biological processes in vitro, most of them directly related with the conserved ability to bind to phospholipid bilayers: membrane trafficking, membrane-cytoskeleton anchorage, ion channel activity and regulation, as well as antiinflammatory and anticoagulant activities. However, the in vivo physiological functions of annexins are just beginning to be established.

The tetraspanin CD9 inhibits the proliferation and tumorigenicity of human colon carcinoma cells

The implication of the tetraspanin CD9 in cancer has received much recent attention and an inverse correlation between CD9 expression and the metastatic potential and cancer survival rate has been established for different tumor types. In contrast to the well‐established role of CD9 in metastasis, very little is known about the involvement of this tetraspanin in the process of development of primary tumors. In the present study, we present evidence on the implication of CD9 in colon carcinoma tumorigenesis. We report here that ectopic expression of CD9 in colon carcinoma cells results in enhanced integrin‐dependent adhesion and inhibition of cell growth. Consistently with these effects, treatment of these cells with anti‐CD9‐specific antibodies resulted in (i) increased β1 integrin‐mediated cell adhesion through a mechanism involving clustering of integrin molecules rather than altered affinity; (ii) induction of morphological changes characterized by the acquisition of an elongated cell phenotype; (iii) inhibition of cell proliferation with no significant effect on cell survival; (iv) increased expression of membrane TNF‐α, and finally (v) inhibition of the in vivo tumorigenic capacity in nude mice. In addition, through the use of selective blockers of TNF‐α, we have demonstrated that this cytokine partly mediates the antiproliferative effects of CD9. These results clearly establish for the first time a role for CD9 in the tumorigenic process.

Bile acids in the colon, from healthy to cytotoxic molecules.

Bile acids are natural detergents mainly involved in facilitating the absorption of dietary fat in the intestine. In addition to this absorptive function, bile acids are also essential in the maintenance of the intestinal epithelium homeostasis. To accomplish this regulatory function, bile acids may induce programmed cell death fostering the renewal of the epithelium. Here we first discuss on the different molecular pathways of cell death focusing on apoptosis in colon epithelial cells. Bile acids may induce apoptosis in colonocytes through different mechanisms. In contrast to hepatocytes, the extrinsic apoptotic pathway seems to have a low relevance regarding bile acid cytotoxicity in the colon. On the contrary, these molecules mainly trigger apoptosis through direct or indirect mitochondrial perturbations, where oxidative stress plays a key role. In addition, bile acids may also act as regulatory molecules involved in different cell signaling pathways in colon cells. On the other hand, there is increasing evidence that the continuous exposure to certain hydrophobic bile acids, due to a fat-rich diet or pathological conditions, may induce oxidative DNA damage that, in turn, may lead to colorectal carcinogenesis as a consequence of the appearance of cell populations resistant to bile acid-induced apoptosis. Finally, some bile acids, such as UDCA, or low concentrations of hydrophobic bile acids, can protect colon cells against apoptosis induced by high concentrations of cytotoxic bile acids, suggesting a dual behavior of these agents as pro-death or pro-survival molecules.

The structure of human 4F2hc ectodomain provides a model for homodimerization and electrostatic interaction with plasma membrane.

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in amino acid transport and cell fusion, adhesion, and transformation. The structure of the ectodomain of human 4F2hc has been solved using monoclinic (Protein Data Bank code 2DH2) and orthorhombic (Protein Data Bank code 2DH3) crystal forms at 2.1 and 2.8 Å, respectively. It is composed of a (βα)8 barrel and an antiparallel β8 sandwich related to bacterial α-glycosidases, although lacking key catalytic residues and consequently catalytic activity. 2DH3 is a dimer with Zn2+ coordination at the interface. Human 4F2hc expressed in several cell types resulted in cell surface and Cys109 disulfide bridge-linked homodimers with major architectural features of the crystal dimer, as demonstrated by cross-linking experiments. 4F2hc has no significant hydrophobic patches at the surface. Monomer and homodimer have a polarized charged surface. The N terminus of the solved structure, including the position of Cys109 residue located four residues apart from the transmembrane domain, is adjacent to the positive face of the ectodomain. This location of the N terminus and the Cys109-intervening disulfide bridge imposes space restrictions sufficient to support a model for electrostatic interaction of the 4F2hc ectodomain with membrane phospholipids. These results provide the first crystal structure of heteromeric amino acid transporters and suggest a dynamic interaction of the 4F2hc ectodomain with the plasma membrane.

Differentiation of human colon adenocarcinoma cells alters the expression and intracellular localization of annexins A1, A2, and A5

Butyrate induces differentiation and alters cell proliferation in intestinal–epithelial cells by modulation of the expression of several genes. Annexins are a superfamily of ubiquitous proteins characterized by their calcium‐dependent ability to bind to biological membranes; their involvement in several physiological processes, such as membrane trafficking, calcium signaling, cell motility, proliferation, and differentiation has been proposed. Thus, we have analyzed changes in annexin A1 (AnxA1), annexin A2 (AnxA2), and annexin A5 (AnxA5) levels and localization in human colon adenocarcinoma cells differentiated by butyrate treatment or by culture in glucose‐free inosine‐containing medium. The acquired differentiated phenotype increased dipeptidyl peptidase‐IV (DPP‐IV) expression and alkaline phosphatase (ALP) activity, two well known brush border markers. Butyrate induces cell differentiation and growth arrest in BCS‐TC2, BCS‐TC2.2, HT‐29, and Caco‐2 cells, increasing the levels of AnxA1 and AnxA5, whereas AnxA2 decreases except in Caco‐2 cells. Inosine‐differentiated cells present increased amounts of the three studied annexins, as occurs in spontaneously differentiated Caco‐2 cells. AnxA2 down‐regulation is not due to proteasome activation and seems to be related to the butyrate‐induced cell proliferation arrest; AnxA1 and AnxA5 expression is growth‐state independent. AnxA1 and AnxA5 are mainly found in the cytoplasm while AnxA2 is localized underneath the plasma membrane in cell‐to‐cell contacts. Butyrate induces changes in subcellular localization towards a vesicle‐associated pattern. Human colon adenocarcinoma cell differentiation is associated with an up‐regulation of AnxA1, AnxA2, and AnxA5 and with a subcellular relocation of these proteins. No correlation between annexin levels and tumorigenicity was found. Up‐regulation of AnxA1 could contribute to the reported anti‐inflammatory effects of butyrate in colon inflammatory diseases.

Kinetic study of the cytotoxic effect of alpha-sarcin, a ribosome inactivating protein from Aspergillus giganteus, on tumour cell lines: protein biosynthesis inhibition and cell binding.

Abstractα-Sarcin is a ribosome inactivating protein produced by the mouldAspergillus giganteus. The effect of this protein on eight different tumour cell lines has been studied in the absence of any agent affecting membrane permeability. The protein is cytotoxic for all the tumour cell lines considered. α-Sarcin modifies the cell proliferation pattern by inhibiting the protein biosynthesis of the cultured cells. No membrane damage produced by α-sarcin has been observed by measuring lactic dehydrogenase leakage. Alteration on the cell mitochondrial activity has not been detected upon treatment with α-sarcin. Differences on the extent of the protein binding to the cells have been observed by flow cytometric measurements. The kinetic analysis of the protein biosynthesis inhibition produced by α-sarcin reveals an α-sarcin concentration-dependent lag phase followed by a first order decrease of the protein synthesis rate. This parameter is dependent on the external α-sarcin concentration. A saturable component for the action of α-sarcin is also deduced from these experiments. Results are discussed in terms of the protein passage across the cell membrane as the potential rate-limiting step for the action of α-sarcin.

Midregion Parathyroid Hormone‐Related Protein Inhibits Growth and Invasion In Vitro and Tumorigenesis In Vivo of Human Breast Cancer Cells

Parathyroid hormone‐related protein (PTHrP) is critical for normal mammary development and is overexpressed by breast cancers. PTHrP is a peptide hormone that undergoes extensive post‐translational processing, and PTHrP(38–94)‐amide is one of the mature secretory forms of the peptide. In this study, we explored the effect of PTHrP(38–94)‐amide in a panel of six breast cancer cell lines “in vitro” and in MDA‐MB231 cells “in vivo” specifically examining cell viability, proliferation, invasiveness, and growth in nude mice. PTHrP(38–94)‐amide markedly inhibited proliferation and also caused striking toxicity and accelerated cell death in breast cancer cells. In addition, direct injection of PTHrP(38–94)‐amide into MDA‐MB231 breast cancer cells passaged in immunodeficient mice produced a marked reduction in tumor growth. These studies (i) indicate breast cancer cells are one of the few tissues in which specific effects of midregion PTHrP have been established to date, (ii) support a role for midregion secretory forms of PTHrP in modulating not only normal but also pathological mammary growth and differentiation, (iii) add further evidence for the existence of a specific midregion PTHrP receptor, and (iv) provide a novel molecule for modeling of small molecule analogues that may have anti‐breast cancer effects.