Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors

Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13‐ to 16‐fold increased exosome yield and facilitated quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ proteomics. We identified several proteins linked to epithelial–mesenchymal transition, including increased abundance of vimentin and hepatoma‐derived growth factor in the membrane, and casein kinase II α and annexin A2 in the lumen of exosomes, respectively, from metastatic cells. The change in exosome protein abundance correlated little, although significant for FL3 versus T24, with changes in cellular mRNA expression. Our proteomic approach may help identification of proteins in the membrane and lumen of exosomes potentially involved in the metastatic process.     

The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca(2+) sensitization pathway in smooth muscle

Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. Here, we show that at least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. We crystallized the DH/PH fragment of PDZRhoGEF in complex with nonprenylated human RhoA and determined the structure at 2.5 A resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity.     

The molecular basis of RhoA specificity in the guanine nucleotide exchange factor PDZ-RhoGEF

The Dbl homology nucleotide exchange factors (GEFs) activate Rho family cytosolic GTPases in a variety of physiological and pathophysiological events. These signaling molecules typically act downstream of tyrosine kinase receptors and often facilitate nucleotide exchange on more than one member of the Rho GTPase superfamily. Three unique GEFs, i.e. p115, PDZ-RhoGEF, and LARG, are activated by the G-protein coupled receptors via the Galpha(12/13), and exhibit very selective activation of RhoA, although the mechanism by which this is accomplished is not fully understood. Based on the recently solved crystal structure of the DH-PH tandem of PDZ-RhoGEF in complex with RhoA (Derewenda, U., Oleksy, A., Stevenson, A. S., Korczynska, J., Dauter, Z., Somlyo, A. P., Otlewski, J., Somlyo, A. V., and Derewenda, Z. S. (2004) Structure (Lond.) 12, 1955-1965), we conducted extensive mutational and functional studies of the molecular basis of the RhoA selectivity in PDZ-RhoGEF. We show that while Trp(58) of RhoA is intimately involved in the interaction with the DH domain, it is not a selectivity determinant, and its interaction with PDZ-RhoGEF is unfavorable. The key selectivity determinants are dominated by polar contacts involving residues unique to RhoA. We find that selectivity for RhoA versus Cdc42 is defined by a small number of interactions.     

Novel peptide recognized by RhoA GTPase

A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA-R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+-and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein.     

Protective effects of melatonin and indole-3-propionic acid against lipid peroxidation, caused by potassium bromate in the rat kidney

Potassium bromate (KBrO(3)) is classified as a carcinogenic agent. KBrO(3) induces tumors and pro-oxidative effects in kidneys. Melatonin is a well known antioxidant and free radical scavenger. Indole-3-propionic acid (IPA), an indole substance, also reveals antioxidative properties. Recently, some antioxidative effects of propylthiouracil (PTU)-an antithyroid drug-have been found. The aim of the study was to compare protective effects of melatonin, IPA, and PTU against lipid peroxidation in the kidneys and blood serum and, additionally, in the livers and the lungs, collected from rats, pretreated with KBrO(3). Male Wistar rats were administered KBrO(3) (110 mg/kg b.w., i.p., on the 10th day of the experiment) and/or melatonin, or IPA (0.0645 mmol/kg b.w., i.p., twice daily, for 10 days), or PTU (0.025% solution in drinking water, for 10 days). The level of lipid peroxidation products-malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA)-was measured spectrophotometrically in thyroid homogenates. KBrO(3), when injected to rats, significantly increased lipid peroxidation in the kidney homogenates and blood serum, but not in the liver and the lung homogenates. Co-treatment with either melatonin or with IPA, but not with PTU, decreased KBrO(3)-induced oxidative damage to lipids in the rat kidneys and serum. In conclusion, melatonin and IPA, which prevent KBrO(3)-induced lipid peroxidation in rat kidneys, may be of great value as protective agents under conditions of exposure to KBrO(3).     

Sodium ascorbate and basic fibroblast growth factor protect muscle-derived cells from H2O2-induced oxidative stress

Engraftment of muscle-derived cells (MDCs) into the urethral sphincter may cure urinary incontinence. However, poor cell survival after injection prompted us to evaluate whether 24-h preincubation with sodium ascorbate (ASC) or basic fibroblast growth factor (bFGF) prior to cell transfer improves the survival of MDCs facing oxidative stress in vitro. We examined MDCs isolated from female rats for the presence of myogenic markers and for the ability to differentiate and respond to growth factors. Isolated cells were positive for desmin, M-cadherin, and myogenin. The fusion index exceeded 29%, and Akt kinase was phosphorylated at Ser473 residue upon exposure to insulin-like growth factor 1 (100 ng/ml). We then autologously transplanted MDCs transfected with lacZ marker gene into urethral wall of the rats; 2 wk later, the urethra and caudal wall of the urinary bladder were harvested from these animals. Serial cryosections revealed that transplanted cells formed multinuclear clusters at injection sites. In addition, we found that the viability of MDCs exposed to a cytotoxic concentration of H2O2 was higher after preincubation with 0.1 mM ASC (2.6-fold), 10 ng/ml bFGF (2.9-fold), or 20 ng/ml bFGF (3.5-fold) than that after exposure to H2O2 only. We conclude that preincubation with ASC or bFGF increases the resistance of MDCs to oxidative stress in vitro. Pretreatment with either agent might be used to enhance survival of MDCs after transplantation.     

Analysis of copy number variation in the normal human population within a region containing complex segmental duplications on 22q11 using high-resolution array-CGH

A previously detected copy number polymorphism (Ep CNP) in patients affected with neuroectodermal tumors led us to investigate its frequency and length in the normal population. For this purpose, a program called Sequence Allocator was developed and applied for the construction of an array that consisted of unique and duplicated fragments, allowing the assessment of copy number variation within regions of segmental duplications. The average resolution of this array was 11 kb and we determined the size of the Ep CNP to be 290 kb. Analysis of normal controls identified 7.7 and 7.1% gains in peripheral blood and lymphoblastoid cell line (LCL) DNA, respectively, while deletions were found only in the LCL group (7.1%). This array platform allows the detection of DNA copy number variation within regions of pronounced genomic complexity, which constitutes an improvement over available technologies.     

Age-related somatic structural changes in the nuclear genome of human blood cells

Structural variations are among the most frequent interindividual genetic differences in the human genome. The frequency and distribution of de novo somatic structural variants in normal cells is, however, poorly explored. Using age-stratified cohorts of 318 monozygotic (MZ) twins and 296 single-born subjects, we describe age-related accumulation of copy-number variation in the nuclear genomes in vivo and frequency changes for both megabase- and kilobase-range variants. Megabase-range aberrations were found in 3.4% (9 of 264) of subjects ≥60 years old; these subjects included 78 MZ twin pairs and 108 single-born individuals. No such findings were observed in 81 MZ pairs or 180 single-born subjects who were ≤55 years old. Recurrent region- and gene-specific mutations, mostly deletions, were observed. Longitudinal analyses of 43 subjects whose data were collected 7-19 years apart suggest considerable variation in the rate of accumulation of clones carrying structural changes. Furthermore, the longitudinal analysis of individuals with structural aberrations suggests that there is a natural self-removal of aberrant cell clones from peripheral blood. In three healthy subjects, we detected somatic aberrations characteristic of patients with myelodysplastic syndrome. The recurrent rearrangements uncovered here are candidates for common age-related defects in human blood cells. We anticipate that extension of these results will allow determination of the genetic age of different somatic-cell lineages and estimation of possible individual differences between genetic and chronological age. Our work might also help to explain the cause of an age-related reduction in the number of cell clones in the blood; such a reduction is one of the hallmarks of immunosenescence.     

Effect of 2-(4-fluorophenylamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole on the molecular organisation and structural properties of the DPPC lipid multibilayers

Interactions and complex formation between lipids and biologically active compounds are crucial for better understanding of molecular mechanisms occurring in living cells. In this paper a molecular organisation and complex formation of 2-(4-fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thiadiazole (FABT) in DPPC multibilayers are reported. The simplified pseudo binary phase diagram of this system was created based on the X-ray diffraction study and fourier transform infrared spectroscopic data. The detailed analysis of the refraction effect indicates a much higher concentration of FABT in the polar zones during phase transition. Both the lipid and the complex ripple after cooling. It was found that FABT occupied not only the hydrophilic zones of the lipid membranes but also partly occupied the central part of the non polar zone. The infrared spectroscopy study reveals that FABT strongly interact with hydrophilic (especially PO(2)(-)) and hydrophobic (especially "kink" vibrations of CH(2) group). The interactions of FABT molecules with these groups are responsible for changes of lipid multibilayers observed in X-ray diffraction study.