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The enzyme PIMT methylates abnormal aspartyl residues in proteins. U-87 MG cells are commonly used to study the most frequent brain tumor, glioblastoma. Previously, we reported that PIMT isoform I possessed oncogenic features when overexpressed in U-87 MG and U-251 MG glioma cells. Higher levels of wild-type PIMT stimulated migration and invasion in both glioma cell lines. Conversely, PIMT silencing reduced these migratory abilities of both cell lines. These results indicate that PIMT could play a critical role in glioblastoma growth. Here, we investigated for the first time, molecular mechanisms involving PIMT in the regulation of epithelial to mesenchymal transition (EMT) upon TGF-β1 treatments. Gene array analyses indicated that EMT genes but not PIMT gene were regulated in U-87 MG cells treated with TGF-β1. Importantly, PIMT silencing by siRNA inhibited in vitro migration in U-87 MG cells induced by TGF-β1. In contrast, overexpressed wild-type PIMT and TGF-β1 had additive effects on cell migration. When PIMT was inhibited by siRNA, this prevented Slug induction by TGF-β1, while Snail stimulation by TGF-β1 was increased. Indeed, overexpression of wild-type PIMT led to the opposite effects on Slug and Snail expression dependent on TGF-β1. These data highlighted the importance of PIMT in the EMT response dependent on TGF-β1 in U-87 MG glioma cells by an antagonist regulation in the expression of transcription factors Slug and Snail, which are critical players in EMT.

The protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT) recognizes abnormal L-isoaspartyl and D-aspartyl residues in proteins. Among examined tissues, PIMT shows the highest level in the brain. The U-87 MG cell line is a commonly used cellular model to study the most frequent brain tumor, glioblastoma. Previously, we reported that PIMT amount increased when U-87 MG cells were detached from the extracellular matrix. Recently, we also showed that PIMT possessed pro-angiogenic properties. Together, these PIMT features led us to postulate that PIMT could play a critical role in glioblastoma growth. Here, we investigate PIMT role in U-87 MG cell viability, adhesion, migration, invasion, and colony formation and in the reorganization of the actin and tubulin cytoskeleton. PIMT inhibition by siRNA significantly reduced in vitro cell migration and invasion in various assays, including wound-healing assay, Boyden chambers coated with gelatin and Matrigel invasion assay. Conversely, in stably transfected U-87 MG cells overexpressing wild-type PIMT, cell migration, invasive capacity and colony formation significantly increased. However, in stably transfected cells with the gene encoding for mutated PIMT(D83V), despite of its overexpression, migration and invasion remained similar to those observed in control cells. In all these conditions, cell viability was unaffected. Importantly, overexpressed wild-type PIMT and mutated PIMT(D83V) have opposite effects on the organization of microtubules and actin cytoskeleton and thus on morphology of U-87 cells. These data highlighted the importance of PIMT level and its catalytic activity in migration and invasion of U-87 glioma cells and its possible contribution in cancer invasion during glioma growth.

Levels of soluble melanotransferrin in serum have been reported to be higher in patients with Alzheimer's disease than in control subjects. The present study investigated melanotransferrin in human body fluids in the light of these findings. To clarify the correlation between melanotransferrin and Alzheimer's disease, the melanotransferrin content was determined by non-reducing, denaturing SDS/PAGE and Western blotting. Under these conditions, serum melanotransferrin migrated at 79 and 82 kDa. Melanotransferrin antigenicity and the relative proportions of the two forms were very sensitive to factors that altered its conformation, including disulphide bridges, pH and bivalent cations. Serum melanotransferrin levels were not significantly different between control subjects and patients with Alzheimer's disease using whole serum, EDTA-supplemented serum or serum immunoglobulin-depleted by Protein G–Sepharose and enriched by affinity precipitation with the lectin from Asparagus pea. Glycosylated forms of serum melanotransferrin bound to Asparagus lectin manifested similar patterns on two-dimensional gel electrophoresis in samples from controls and Alzheimer's disease subjects. Melanotransferrin was also present in saliva and at a high level in urine, but contents were similar in controls and patients with Alzheimer's disease. Together, these results demonstrate that serum melanotransferrin exists in various conformations depending on the binding of bivalent cations or following post-translational modification. These data also indicate that human serum melanotransferrin levels are unchanged in subjects with Alzheimer's disease.

The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14-fold higher than that of holo-transferrin, with no apparent intra-endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration- and conformation-dependent, suggesting that the transport of P97 is due to receptor-mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low-density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.

Background: The increased synthesis of matrix metalloproteinases (MMPs) by aortic smooth muscle cells (SMCs) is thought to be involved in the etiopathogenesis of abdominal aortic aneurysms (AAAs), but the functional regulation and the activation states of these MMPs remain unclear. In this study, we assessed the expression levels and the functional regulation of several MMPs in the pathogenesis of AAAs. Methods: Human healthy aorta and AAA specimens were homogenized, and the proteolytic activities of MMP-2 and MMP-9 and of the macrophage metalloelastase (MMP-12) were assessed with zymography. Protein expression of MMP-1, MMP-12, membrane-type 1 MMP (MT1-MMP), tissue inhibitor of MMP 1 (TIMP-1), TIMP-2, TIMP-3, α-actin, and β-actin was analyzed with electrophoresis on sodium dodecyl sulfate gels and immunoblotting. Results: MMP-1, MMP-9, and MMP-12 zymogen levels and proteolytic activities were increased in AAAs when compared with healthy aorta. A severe reduction in α-actin–positive vascular SMCs was observed in all the AAA specimens and was correlated with an increase in TIMP-3 but not TIMP-1 or TIMP-2 potential activities. Although pro–MMP-2 activity was decreased, the extent of activated MMP-2 remained unaffected in the AAAs. In accordance with this result, a highly activated MT1-MMP form was also observed in AAAs. Conclusion: These data suggest that chronic aortic wall inflammation is mediated by macrophage infiltration, which may account for the destruction of medial elastin, as reflected by SMC down regulation, through increased levels of active MMP-1 and MMP-12. Moreover, altered MT1-MMP proteolytic turnover and differential regulation of TIMP expression in AAAs suggest that tight regulatory mechanisms are involved in the molecular regulation of MMP activation processes in the pathogenesis of AAAs. (J Vasc Surg 2002;35:539-46.)

The karyotypes of two bivalve molluscs, the giant scallop Placopecten magellanicus and the surf clam Spisula solidissima, were examined in unfertilized eggs and embryos. In both bivalves, a haploid number of 19 and a diploid number of 38 were determined in well-spread metaphase chromosomes of meiotic and mitotic cells respectively. Moreover, karyological analysis of induced triploid embryos in Placopecten magellanicus indicates a chromosome number of 57 and thus corroborates the haploid and diploid counts. The chromosomes of Spisula solidissima are shorter than those of Placopecten magellanicus. Their sizes vary from 1.5 to 4.4μm in surf clam while those in giant scallop range from 3.3 to 8.5μm. Both bivalve species have submetacentric, subtelocentric and telocentric chromosomes but only Spisula solidissima has metacentric chromosomes.

The ras oncogenes are among those most frequently found in human cancers. Blocking Ras farnesylation is a promising strategy for arresting cancer growth. Ras activates several signaling pathways with key roles in cellular proliferation, invasion, metastasis and angiogenesis. Furthermore, proteolytic activities of matrix proteinases such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs) are regulated by Ras isoforms. Thus, we investigated the effects of SCH-66336, a farnesyltransferase inhibitor, on secretion of components of the plasminogen activation system as well as on the gelatinases MMP-2 and MMP-9, which play pivotal roles in matrix remodeling. SCH-66336 up to 5 microM did not significantly alter the viability of prostate (PC-3) and renal (Caki-1) cancer cells incubated in serum-depleted medium. SCH-66336 partly inhibited the processing of H-Ras, while levels of mature N-Ras and K-Ras remained unaffected. Under these noncytotoxic conditions, uPA and tPA levels were lowered in culture medium but raised in cell lysates, suggesting inhibition of trafficking pathways. In contrast, SCH-66336 had no effect on uPAR expression or on secreted PAI-1 levels. As expected, the reduction of uPA and tPA activities by SCH-66336 inhibited the conversion of plasminogen to plasmin by about 25% in PC-3 cells. SCH-66336 also inhibited the levels of secreted pro-MMP-2 and pro-MMP-9 as well as the release of their inhibitors TIMP-1 and TIMP-2. SCH-66336 decreased both the adhesion and even more so the migration of PC-3 cells on gelatin. Thus, SCH-66336 inhibited farnesylation in both cancer cell types, and H-Ras functions should be reduced by the drug. In addition, the lower levels of secreted proteinases in the presence of SCH-66336 suggest that reduced matrix remodeling and cell migration should occur in treated tumors.

A key task for the multifunctional von Hippel-Lindau protein (pVHL) is regulation of the activity of hypoxia-inducible factor-1α (HIF-1α) by targeting it to the proteasome for degradation under normoxia. pVHL binding to HIF-1α is lost under low O2 tension, leading to transcription of several genes involved in the hypoxia response. However, regulation of pVHL by hypoxia remains to be investigated. We evaluated the effects of hypoxia on pVHL expression in carcinoma and endothelial cells. We showed that hypoxia stimulates pVHL levels (2.5-fold) in renal Caki-1 cells expressing wild-type VHL (VHL+/+). This upregulation was independent of VHL status, because hypoxia also increased pVHL expression in renal 786-O cells carrying mutated VHL (VHL-/-). Hypoxia did not affect pVHL expression in endothelial cells. Hypoxia-induced pVHL in Caki-1 cells was RhoA dependent, because inhibition by exotoxin C3 prevented pVHL stimulation. Furthermore, inhibition of Rho kinase by Y-27632 blocked pVHL induction by hypoxia. During normoxia, pVHL expression was also induced in cells transfected with dominant-active RhoA. Furthermore, disruption of actin organization by chemical agents or by hypoxia stimulated pVHL expression in kidney cells. On the other hand, inhibition of MAP kinases p38 and JNK, but not MAP kinase kinase (MEK1/2), reduced pVHL upregulation by 30 and 72%, respectively, during hypoxia, supporting a significant role for these signaling pathways. Expression and phosphorylation of c-Jun were stimulated in cells transfected with dominant-active RhoA. Together, these findings demonstrate that hypoxia induces pVHL expression in renal cancer cells, and this induction is mediated by RhoA-dependent pathways.

Recent evidence indicates that bone-marrow-derived stromal cells (MSCs) have a histology coherent with endothelial cells that may enable them to contribute to tumor angiogenesis through yet undefined mechanisms. In this work, we investigated the angiogenic properties of murine MSCs involved in extracellular matrix degradation and in neovascularization that could take place in a hypoxic environment such as that encountered in tumor masses. MSCs were cultured in normoxia (95% air and 5% CO(2)) or in hypoxia (1% oxygen, 5% CO(2), and 94% nitrogen). We found that hypoxic culture conditions rapidly induced MSC migration and three-dimensional capillary-like structure formation on Matrigel. In vitro, MSC migration was induced by growth-factor- and cytokine-enriched conditioned media isolated from U-87 glioma cells as well as from MSCs cultured in hypoxic conditions, suggesting both paracrine and autocrine regulatory mechanisms. Although greater vascular endothelial growth factor levels were secreted by MSCs in hypoxic conditions, this growth factor alone could not explain their greater migration. Interestingly, matrix metalloproteinase (MMP)-2 mRNA expression and protein secretion were downregulated, while those of membrane-type (MT)1-MMP were strongly induced by hypoxia. Functional inhibition of MT1-MMP by a blocking antibody strongly suppressed MSC ability to migrate and generate capillary-like structures. Collectively, these data suggest that MSCs may have the capacity to participate in tumor angiogenesis through regulation of their angiogenic properties under an atmosphere of low oxygen that closely approximates the tumor microenvironment.

The small G proteins of the Rho family are involved in reorganization of the actin cytoskeleton, cell migration and in the regulation of gene transcription. Hypoxia-induced ATP depletion results in the disruption of actin organization which could affect Rho functions. In solid tumors, regions with low oxygen tension stimulate angiogenesis in order to increase oxygen and nutrient supply. This process is mediated by stabilization of the transcriptional factor hypoxia inducible factor 1 (HIF-1), which increases vascular endothelial growth factor (VEGF) production. In this study, we investigated the activities of Rho proteins, which are key regulators of cytoskeleton organization during hypoxia in renal cell carcinoma. Caki-1 cells were exposed to hypoxia (1% O2) and exhibited increased Cdc42, Rac1 and RhoA protein expression. Immunoprecipitation of metabolically labelled RhoA showed that overexpression was at least due to neo-synthesis. The Rho GTPases overexpressed during hypoxia were mainly located at membranes and pull-down assays demonstrated that they were active since they bound GTP. RT-PCR analysis indicated that the increase in RhoA protein expression was also reflected at the mRNA level. Overexpression and activation of Rho proteins were downstream of, and dependent on, the production of reactive oxygen species (ROS) since, in the presence of an inhibitor, both the rise of ROS and upregulation of Rho proteins were abolished. Importantly,preincubation of cells with the toxin C3, which inhibits RhoA, reduced HIF-1α protein accumulation by 84% during hypoxia. Together, these results support a model where ROS upregulate Rho protein expression and where active RhoA is required for HIF-1α accumulation during hypoxia.

The Rho GDP dissociation inhibitor (RhoGDI) regulates the activation-inactivation cycle of Rho small GTPases, such as Cdc42 and RhoA, by extracting them from the membrane. To study the roles of Mg(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), ionic strength and phosphorylation on the interactions of RhoGDI with Cdc42 and RhoA, we developed a new, efficient and reliable method to produce prenylated Rho proteins using the yeast Saccharomyces cerevisiae. It has been previously reported that protein kinase A (PKA)-treatment of isolated membranes increased RhoA extraction from membranes by RhoGDI [Lang, Gesbert, Delespine-Carmagnat, Stancou, Pouchelet and Bertoglio (1996) EMBO J. 16, 510-519]. In the present study, we used an in vitro affinity chromatography system to show that phosphorylation of RhoA and Cdc42 significantly increased their interaction with RhoGDI under physiological conditions of ionic strength. This increase was independent of the nucleotide (GDP or guanosine 5'-[gamma-thio]triphosphate) loaded on to the Rho proteins, as well as of Mg(2+) and PIP(2). Moreover, dephosphorylation of rat brain membranes by alkaline phosphatase significantly decreased the extraction of RhoA and Cdc42 by RhoGDI. Subsequent re-phosphorylation by PKA restored the extraction levels, indicating the reversibility of this process. These results clearly demonstrate that the phosphorylation states of Cdc42 and RhoA regulate their interactions with RhoGDI and, consequently, their extraction from rat brain membranes. We therefore suggest that phosphorylation is a mechanism of regulation of Cdc42 and RhoA activity that is independent of GDP-GTP cycling.

The GTPases Rho regulate the assembly of polymerized actin structures. Their C-terminal sequences end with the CAAX motif that undergo a lipidation of the cysteine residue. Analogs to the C-terminal ends of Rho proteins, N-acetyl-S-all-trans, trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, wereused to analyze the role of prenylation in their membrane association. Silver-stained gels indicated that N-acetyl-S-all-trans-geranylgeranyl-L-cysteine treatment released only a few proteins of 20, 46, and 60 kDa. Western blot analysis showed that N-acetyl-S-all-trans-geranylgeranyl-L-cysteine released RhoB (10%), RhoA (28%), and Cdc42 (95%) from membranes, whereas N-acetyl-S-all-trans and trans-farnesyl-L-cysteine did not. Rab1, which possesses two geranylgeranyl groups, was also strongly extracted by N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, whereas Ras, which is farnesylated, was not. Furthermore, N-acetyl-S-all-trans-geranylgeranyl-L-cysteine was very efficient (95%) in dissociating actin and tubulin from membranes but not integral membrane protein P-glycoprotein and sodium/phosphate cotransporter NaP(i)-2. The extraction of Rho and cytoskeletal proteins occurred below the critical micellar concentration of N-acetyl-S-all-trans-geranylgeranyl-L-cysteine. Membrane treatments with 0.7 m KI totally extracted actin, whereas 70% of Cdc42 was released. Actin was, however, insoluble in Triton X-100-treated membranes, whereas this detergent extracted (80%) Cdc42. These data show that Rho proteins and actin are not physically bound together and suggest that their extraction from membranes by N-acetyl-S-all-trans-geranylgeranyl-L-cysteine likely occurs via different mechanisms.