Proteomic characterization of the interstitial fluid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery

Clinical cancer proteomics aims at the identification of markers for early detection and predictive purposes, as well as to provide novel targets for drug discovery and therapeutic intervention. Proteomics-based analysis of traditional sources of biomarkers, such as serum, plasma, or tissue lyzates, has resulted in a wealth of information and the finding of several potential tumor biomarkers. However, many of these markers have shown limited usefulness in a clinical setting, underscoring the need for new clinically relevant sources. Here we present a novel and highly promising source of biomarkers, the tumor interstitial fluid (TIF) that perfuses the breast tumor microenvironment. We collected TIFs from small pieces of freshly dissected invasive breast carcinomas and analyzed them by two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Western immunoblotting, as well as by cytokine-specific antibody arrays. This approach provided for the first time a snapshot of the protein components of the TIF, which we show consists of more than one thousand proteins--either secreted, shed by membrane vesicles, or externalized due to cell death--produced by the complex network of cell types that make up the tumor microenvironment. So far, we have identified 267 primary translation products including, but not limited to, proteins involved in cell proliferation, invasion, angiogenesis, metastasis, inflammation, protein synthesis, energy metabolism, oxidative stress, the actin cytoskeleton assembly, protein folding, and transport. As expected, the TIF contained several classical serum proteins. Considering that the protein composition of the TIF reflects the physiological and pathological state of the tissue, it should provide a new and potentially rich resource for diagnostic biomarker discovery and for identifying more selective targets for therapeutic intervention.     

Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity

Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor-associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate-specific membrane antigen (PSMA), is up-regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N-glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N-glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N-glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG-hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question.     

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).     

Molecular architecture of Pipistrellus pipistrellus/Pipistrellus pygmaeus complex (Chiroptera: Vespertilionidae): further cryptic species and Mediterranean origin of the divergence

Previous genetic analyses have demonstrated that two phonic types of one of the most common European bats, the Common pipistrelle, belong to distinct species, although they are almost identical morphologically (45 kHz Pipistrellus pipistrellus and 55 kHz Pipistrellus pygmaeus). To reconstruct the history of the species complex and explain the codistribution of both forms in Europe and the Mediterranean, we performed phylogenetic analysis based on a 402-bp portion of the cytochrome b gene. Particular attention was paid to the eastern and southern parts of the range where no data were available. We found further distinctive allopatric haplotypes from Libya and Morocco. The difference of about 6-7% described in the Libyan population suggests the occurrence of a new species in the southern Mediterranean. The species status of Moroccan population is also discussed. The phylogeographic patterns obtained and analysis of fossil records support the hypothesis of expansion of both species into Europe from the Mediterranean region during the Holocene. The allopatric speciation model fits our data best. The paleobiographic scenario envisaged is corroborated also by molecular clock estimations and correlations with Late Neogene environmental changes in the Mediterranean region which ended with the Messinian salinity crisis.     

Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.     

Evidence for antibody-mediated enhancement of simian immunodeficiency virus (SIV) Gag antigen processing and cross presentation in SIV-infected rhesus macaques

By using the dominant simian immunodeficiency virus (SIV) Gag Mamu-A01 restricted major histocompatibility complex (MHC) class I epitope p11CM, we demonstrate antibody-mediated enhanced MHC class I cross presentation of SIV Gag. In vitro restimulation of peripheral blood mononuclear cells from SIV-infected rhesus macaques with recombinant full-length SIV Gag p55 plus p55 affinity-purified immunoglobulin G (p55 Gag/p55-IgG) led to the generation of markedly higher frequencies of p11CM specific precursor cytotoxic T lymphocytes (p-CTLs) compared with restimulation with (i) SIV Gag p55 alone or (ii) optimal concentrations of the p11CM peptide alone. These results, along with the finding that CD4 depletion abrogated the enhancement, suggest a prominent role for CD4(+) T cells. Testing for p-CTLs against other Mamu-A01-restricted SIV Gag epitopes suggested that this mechanism favored recognition of the dominant p11CM peptide, potentially further skewing of the CTL response. The p-CTL enhancing effect was also decreased or abrogated by pepsin digestion of the p55-specific IgG or by the addition of monoclonal antibodies to Fc receptor (FcR) II/III, suggesting that the effect was dependent on FcR-mediated uptake of the immune-complexed antigen. Finally, incubation of antigen-presenting cells with SIV Gag p55 immune complexes in the presence of lactacystin or of bafilomycin indicated that the mechanism of antibody-mediated enhancement of cross presentation required both the proteasomal and the endosomal pathways. These data demonstrate for the first time the cross presentation of antigens via immune complexes in lentiviral infection and indicate a heretofore-unrecognized role for antibodies in modulating the magnitude and potentially also the breadth of MHC class I-restricted antigen processing and presentation and CTL responses.     

Heat shock proteins hsp32 and hsp70 as biomarkers of an early response? In vitro induction of heat shock proteins after exposure of cell culture to carcinogenic compounds and their real mixtures

Heat shock proteins (hsp) are a highly conserved group of proteins that are synthesized as a response to different forms of stress (heat, toxic chemicals, diseases, non-physiological pH changes). Because of their high sensitivity to changes in the environment, these proteins were suggested as possible early biomarkers of exposure in ecotoxicological studies. The purpose of the present study was to check the suitability of hsp 32 and hsp70 as biomarkers of in vitro exposure to environmentally relevant carcinogens: polycyclic aromatic hydrocarbons (PAHs), their nitro-derivates, aromatic amines, acrylonitrile (ACN) and the mixture of organic compounds adsorbed onto ambient airborne particles (extractable organic matter, EOM).The expression of hsp 32 and hsp70 was studied in human diploid lung fibroblasts (HEL cells) and human monocytic leukaemia cells (THP-1 cells) incubated in vitro with different concentrations of dibenzo[a,l]pyrene (DB[a,l]P), 1-nitropyrene, (NP), 4-aminobiphenyl (ABP), ACN and EOM for different periods of time. The incubation of cells with DB[a,l]P, NP, ABP and EOM did not result in increased levels of hsp 32 or hsp70, either in dose- or time-dependent manner. ACN induced the expression of hsp 32 as well as hsp70 in HEL and THP-1 cells, which probably reflects its ability to induce oxidative stress. We conclude that hsp 32 and hsp70 are not suitable biomarkers of an early exposure to PAHs, their nitro-derivates, aromatic amines or EOM under the conditions used.     

Gallbladder carcinoma cells in cerebrospinal fluid as the first manifestation of a tumor. A case report

BACKGROUND: Meningeal carcinomatosis (MC) rarely occurs as the first evidence of a tumor. In such cases cytology of the cerebrospinal fluid is crucial to the diagnosis. The most frequent primary MCs are lung and breast cancers. MC from a gallbladder carcinoma is uncommon. CASE: A 58-year-old woman presented with paroxysmal headaches, seizures and coma. Analysis of the cerebrospinal fluid revealed carcinoma cells and a low protein concentration. Only postmortem examination discovered gallbladder adenocarcinoma to be the source of the tumor cells. CONCLUSION: A case with the onset of MC secondary to rare mucinous adenocarcinoma of the gallbladder is presented. Cytology of the cerebrospinal fluid was the only examination that uncovered malignancy. Nine similar cases were found in the literature. Low cerebrospinal fluid protein seems to be of diagnostic value.     

Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids

A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically measured using the Hoechst 33258 dye and the RNA concentration is calculated from these data. The procedure allows for accurate determination of DNA concentration in the range 10-1000 ng/ml in the presence of 200-fold excess of RNA and determination of RNA concentrations in the range 10-1000 ng/ml in the presence of large excess of DNA. An absence of the treatment of mixed samples with RNase-free DNase I provides rapid, reproducible, and accurate RNA quantification.     

Coenzyme Q releases the inhibitory effect of free fatty acids on mitochondrial glycerophosphate dehydrogenase

Data presented in this paper show that the size of the endogenous coenzyme Q (CoQ) pool is not a limiting factor in the activation of mitochondrial glycerophosphate-dependent respiration by exogenous CoQ(3), since successive additions of succinate and NADH to brown adipose tissue mitochondria further increase the rate of oxygen uptake. Because the inhibition of glycerophosphate-dependent respiration by oleate was eliminated by added CoQ(3), our data indicate that the activating effect of CoQ(3) is related to the release of the inhibitory effect of endogenous free fatty acids (FFA). Both the inhibitory effect of FFA and the activating effect of CoQ(3) could be demonstrated only for glycerophosphate-dependent respiration, while succinate- or NADH-dependent respiration was not affected. The presented data suggest differences between mitochondrial glycerophosphate dehydrogenase and succinate or NADH dehydrogenases in the transfer of reducing equivalents to the CoQ pool.