Purification and in vitro analysis of exosomes secreted by malignantly transformed human cells

Exosomes are natural nanoparticles secreted by different cells and capable of carrying protein markers and genetic information, thus participating in cellular communication. There is good reason to think that quantitative and qualitative characterization of these microparticles produced by different tissues in normal and pathological states can give valuable diagnostic and prognostic information and be a biomarker of different diseases, including oncological ones. Elaboration of the purification of exosomes and their proteome analysis was the aim of the present work. An original approach to enhancing exosome production in cultured transformed human cells was developed. The data obtained allowed us to detect exosomes in cultural conditioned samples and control the quality of produced exosomes at all stages of their purification. Electrophoretic analysis of proteins obtained from exosomes of different origins shows differences in protein profiles. Proteins from exosomes of glioblastoma cell lines were separated by two-dimensional electrophoresis. Protein profiles were further analyzed by densitometry and mass spectrometry, which allowed more than 30 proteins, including specific tumor markers, to be identified.     

Enhanced albumin production by malignantly transformed hepatocytes during in vitro exposure to dimethylsulfoxide

The murine BW 1 and rat 32III 6/d tumor cell lines, derived from a spontaneous mouse hepatoma and a carcinogen-induced rat hepatocellular carcinoma, were used to investigate the effect of dimethylsulfoxide (DMSO) on liver cells in vitro. After a 4-day exposure to DMSO in final concentrations of 0.5 and 1.0%, BW 1 cell-associated albumin increased by 41.6 and 94.2%; extracellular albumin levels in these same cultures rose by 131.4 and 214.2%. Exposure of 32III 6/d cells to 2% DMSO produced increases in cell-associated and extra-cellular albumin concentrations of 67.8 and 188.7%, respectively. The lack of inducible gamma-glutamyl transpeptidase in BW 1 cells and its decrease in 32III 6/d cultures following DMSO treatment suggests that the DMSO-mediated enhancement of albumin production is not reflective of a random increase in the expression of cellular genes.     

Induction of human monocyte motility by lysyl oxidase

Lysyl oxidase highly purified from calf aorta was found to be a potent chemotactic agent for unstimulated human peripheral blood mononuclear cells, determined in in vitro assays in Boyden chambers. A typical chemotactic bell-shaped curve was observed, with a maximal migratory response of 237% of control occurring at 10⁻¹⁰ M lysyl oxidase. The chemotactic response was prevented by prior heat inactivation of the enzyme, by treatment of the enzyme with β-aminopropionitrile or ethylenediamine, which are active site-directed inhibitors of lysyl oxidase, and by a competing, lysine-containing peptide substrate of lysyl oxidase. The chemoattractant reponse to lysyl oxidases was characterized by both chemokinetic and chemotactic components. These results raise the possibility that extracellular lysyl oxidase may have important roles to play in biology in addition to its established function in the crosslinking of elastin and collagen.     

The selection of malignantly transformed rat liver cells using a hormonally defined medium

Summary A hormonally defined medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium and nutrient mixture F12 supplemented with 50 μg/ml each of insulin and transferrin was found to grow late passage malignantly transformed cells of the RL-PR-C rat liver cell culture. The same medium formulation would not grow the early passage, normal, diploid counterpart of the RL-PR-C cell culture. When mixtures of the early and late passage cells were made, only the late passage cells would grow, thus providing a selection system for the late passage cells. This work was supported by Grant PHS SO7 05429-20-3 from the National Institutes of Health, Bethesda, MD.     

The selection of malignantly transformed rat liver cells using a hormonally defined medium

A hormonally defined medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and nutrient mixture F12 supplemented with 50 micrograms/ml each of insulin and transferrin was found to grow late passage malignantly transformed cells of the RL-PR-C rat liver cell culture. The same medium formulation would not grow the early passage, normal, diploid counterpart of the RL-PR-C cell culture. When mixtures of the early and late passage cells were made, only the late passage cells would grow, thus providing a selection system for the late passage cells.     

Thiophene production in transformed roots cultures of Tagetes filifolia

Transformed root cultures of Tagetes filifolia were established by infection with Agrobacterium rhizogenes LBA 9402. Several clones were obtained with different growth index, content of thiophenes and pattern. The isolated clones showed considerable variations in total thiophene content (27 to 11764 g/g fresh weight) but all had similar patterns of the different thiophenes. Only one clone excreted thiophenes (about 80% of total amount). © Rapid Science Ltd. 1998     

Partial characterization of lysyl oxidase from several human tissues.

The extracellular release of mycobacillin from Bacillus subtilis first occurred in the medium at the onset of stationary phase and continued at a high rate even after 6 days. Mycobacillin synthetase activity appeared earlier than late-exponential phase in the cytosol of producer cells and was not sedimentable even at 105 000 g. The activity then quickly reached the maximum late in the stationary phase. With further increase in the age of the culture, the activity gradually disappeared from the cytosol, to reappear concomitantly in the membrane in an insoluble particulate form, even in absence of protein synthesis. The membrane-bound synthetase activity was sedimentable at 10 000 g and was fairly active even after 5 days.     

Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3^′ of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu²⁺. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by β-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.     

Alkaloid production by transformed root cultures of Catharanthus roseus

Transformed roots of Catharanthus roseus were obtained following infection of detached leaves with Agrobacterium rhizogenes. Roots would not grow in full strength Gamborg's B5 medium but would grow satisfactorily if the medium was diluted to one half strength. Little alkaloid appeared in the growth medium but root tissue contained a high level and wide variety of alkaloids. Ajmalicine, serpentine, vindolinine and catharanthine were prominent components. Vinblastine could also be detected by a combination of HPLC and radioimmunoassay, though at a level of only 0.05g/g dry weight.Abbreviations B5 Gamborg's B5 nutrient salts - LC/MS combined liquid chromatography/mass spectrometry - FW fresh weight - Kb kilobase     

beta-catenin signaling and regulation of cyclin D1 promoter in NRK-49F cells transformed by down-regulation of the tumor suppressor lysyl oxidase

Lysyl oxidase is the enzyme that is essential for collagen and elastin cross-linking. Previous investigations showed that lysyl oxidase is down-regulated in many human tumors and ras-transformed cells. Recently, we proved that antisense down-regulation of lysyl oxidase in NRK-49F cells induced phenotypic changes and oncogenic transformation, characterized by p21(ras) activation and beta-catenin/cyclin D1 up-regulation. In the present paper, we examined beta-catenin intracellular distribution and its association with E-cadherin. We observed an increased association between E-cadherin and beta-catenin in the lysyl-oxidase down-regulated cells during serum starvation. Moreover, we found that beta-catenin cytoplasmic and nuclear levels were increased, suggesting a failure of its down-regulation by the APC-GSK-3beta system, in particular the GSK-3beta phosphorylation of ser-33/37 and thr-41 of beta-catenin. Finally, we investigated the mechanisms leading to the observed cyclin D1 up-regulation. We showed that in the antisense lysyl oxidase cells the cyclin D1 promoter was activated through the LEF and the ATF/CRE sites in the proximal promoter. While the promoter activation through LEF is compatible with beta-catenin signaling, we investigated the possibility that the CRE-dependent activation might be linked to the down-regulation of lysyl oxidase. In fact, up-regulation of lysyl oxidase in a COS-7 cell model showed a significant diminution of the CREB protein binding to the cyclin D1 promoter, leading to a dramatic inhibition of its activity and a significant down-regulation of cyclin D1 protein level in vivo. Finally, our study describes some major anomalies occurring in lysyl oxidase down-regulated fibroblasts, related to beta-catenin signaling and cyclin D1 expression.     

Immunological characterization of bovine lysyl oxidase

Antibodies to homogeneously purified bovine aortic lysyl oxidase were prepared in chickens. The chicken anti-lysyl oxidase antiserum effectively inhibited bovine aortic lysyl oxidase activity. Non-immune antiserum from chickens, goats and humans was found to enhance bovine aortic lysyl oxidase activity, while non-immune rabbit serum inhibited enzyme activity. A competitive ELISA was developed to monitor immunoreactive lysyl oxidase during purification. Chromatography of bovine lysyl oxidase on Sephacryl S-200, the final step in purification, revealed two peaks of immunoreactive lysyl oxidase. The large molecular weight peak appears to contain inactive multimeric forms of the enzyme.     

Intracellular localization of the matrix enzyme lysyl oxidase in polarized epithelial cells.

Considerable evidence supports novel functions for lysyl oxidase (LOX) beyond its traditional role in initiating cross-linkages in collagen and elastin within the extracellular matrix. These novel roles are particularly relevant during the transition of malignant epithelial cells towards a migratory and invasive phenotype. However, knowledge on cellular and matrix functions of LOX has been generated almost exclusively in mesenchymal cell types. But it is becoming increasingly evident that these cell types are not adequate to address these novel and highly significant roles for LOX in epithelial tissues. In this initial report, we demonstrate that active LOX is expressed by polarized MDCK II kidney and MCF-10A breast epithelial cells. Furthermore, we show evidence for the presence of mature LOX in the cytoplasm and establish these cell lines as models for epithelial LOX studies.     

The propeptide domain of lysyl oxidase induces phenotypic reversion of ras-transformed cells

Lysyl oxidase is an extracellular enzyme critical for the normal biosynthesis of collagens and elastin. In addition, lysyl oxidase reverts ras-mediated transformation, and lysyl oxidase expression is down-regulated in human cancers. Since suramin inhibits growth factor signaling pathways and induces lysyl oxidase in ras-transformed NIH3T3 cells (RS485 cells), we sought to investigate the effects of suramin on the phenotype of transformed cells and the role of lysyl oxidase in mediating these effects. Suramin treatment resulted in a more normal phenotype as judged by growth rate, cell cycle parameters, and morphology. beta-aminopropionitrile, the selective inhibitor of lysyl oxidase enzyme activity, was remarkably unable to block suramin-induced reversion. By contrast, ectopic antisense lysyl oxidase demonstrated that lysyl oxidase gene expression mediated phenotypic reversion. Since lysyl oxidase is synthesized as a 50 kDa precursor and processed to a 30 kDa active enzyme and 18 kDa propeptide, the effects of these two products on the transformed phenotype of RS485 cells were then directly assessed in the absence of suramin. Here we report, for the first time, that the lysyl oxidase propeptide, and not the lysyl oxidase enzyme, inhibits ras-dependent transformation as determined by effects on cell proliferation assays, growth in soft agar, and Akt-dependent induction of NF-kappaB activity. Thus, the lysyl oxidase propeptide, which is released during extracellular proteolytic processing of pro-lysyl oxidase, functions to inhibit ras-dependent cell transformation.     

Stress-induced down-regulation of tumor-associated NADH oxidase during apoptosis in transformed cells

Tumor-associated NADH oxidase (tNOX) is a growth-related protein expressed in transformed cells. tNOX knockdown using RNA interference leads to a significant reduction in HeLa cell proliferation and migration, indicating an important role for tNOX in growth regulation and the cancer phenotype. Here, we show that tNOX is down-regulated during apoptosis in HCT116 cells. Treatment with diverse stresses induced a dose- and time-dependent decrease in tNOX expression that was concurrent with apoptosis. Moreover, shRNA-mediated tNOX knockdown rendered cells susceptible to apoptosis, whereas re-expression of tNOX partially recovered cell proliferation. Our results indicate that tNOX is suppressed during apoptosis and demonstrate that tNOX down-regulation sensitizes cells to stress-induced growth reduction, suggesting that tNOX is required for transformed cell growth.