Protein alterations associated with temozolomide resistance in subclones of human glioblastoma cell lines

Temozolomide (TMZ) is the standard chemotherapeutic agent for human malignant glioma, but intrinsic or acquired chemoresistance represents a major obstacle to successful treatment of this highly lethal group of tumours. Obtaining better understanding of the molecular mechanisms underlying TMZ resistance in malignant glioma is important for the development of better treatment strategies. We have successfully established a passage control line (D54-C10) and resistant variants (D54-P5 and D54-P10) from the parental TMZ-sensitive malignant glioma cell line D54-C0. The resistant sub-cell lines showed alterations in cell morphology, enhanced cell adhesion, increased migration capacities, and cell cycle arrests. Proteomic analysis identified a set of proteins that showed gradual changes in expression according to their 50% inhibitory concentration (IC50). Successful validation was provided by transcript profiling in another malignant glioma cell line U87-MG and its resistant counterparts. Moreover, three of the identified proteins (vimentin, cathepsin D and prolyl 4-hydroxylase, beta polypeptide) were confirmed to be upregulated in high-grade glioma. Our data suggest that acquired TMZ resistance in human malignant glioma is associated with promotion of malignant phenotypes, and our reported molecular candidates may serve not only as markers of chemoresistance but also as potential therapeutic targets in the treatment of TMZ-resistant human malignant glioma, providing a platform for future investigations

Interstitial fluid: the overlooked component of the tumor microenvironment?

Background: The interstitium, situated between the blood and lymph vessels and the cells, consists of a solid or matrix phase and a fluid phase, together constituting the tissue microenvironment. Here we focus on the interstitial fluid phase of tumors, i.e., the fluid bathing the tumor and stromal cells. Novel knowledge on this compartment may provide important insight into how tumors develop and how they respond to therapy. Results: We discuss available techniques for interstitial fluid isolation and implications of recent findings with respect to transcapillary fluid balance and uptake of macromolecular therapeutic agents. By the development of new methods it is emerging that local gradients exist in signaling substances from neoplastic tissue to plasma. Such gradients may provide new insight into the biology of tumors and mechanistic aspects linked to therapy. The emergence of sensitive proteomic technologies has made the interstitial fluid compartment in general and that of tumors in particular a highly valuable source for tissue-specific proteins that may serve as biomarker candidates. Potential biomarkers will appear locally at high concentrations in the tissue of interest and will eventually appear in the plasma, where they are diluted. Conclusions: Access to fluid that reliably reflects the local microenvironment enables us to identify substances that can be used in early detection and monitoring of disease

Biomedical informatics and translational medicine

Biomedical informatics involves a core set of methodologies that can provide a foundation for crossing the "translational barriers" associated with translational medicine. To this end, the fundamental aspects of biomedical informatics (e.g., bioinformatics, imaging informatics, clinical informatics, and public health informatics) may be essential in helping improve the ability to bring basic research findings to the bedside, evaluate the efficacy of interventions across communities, and enable the assessment of the eventual impact of translational medicine innovations on health policies. Here, a brief description is provided for a selection of key biomedical informatics topics (Decision Support, Natural Language Processing, Standards, Information Retrieval, and Electronic Health Records) and their relevance to translational medicine. Based on contributions and advancements in each of these topic areas, the article proposes that biomedical informatics practitioners ("biomedical informaticians") can be essential members of translational medicine teams

Development and characterization of a novel mAb against bilitranslocase - a new biomarker of renal carcinoma.

BACKGROUND: Bilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues. MATERIALS AND METHODS: Mice were immunized with a multi-antigen peptide corresponding to segment 65-75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase. RESULTS: On the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use. CONCLUSIONS: In this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures

Transport flavonoidov kot indikator zrelosti grozdja sorte Refo\u161k

This research was performed with the aim of studying the influence of cluster thinning at v\ue9raison (CT) and pre-flowering leaf removal (PFLR) on some maturation and quality parameters of grape Refo\u161k. Polyphenol content was related to the pattern of a flavonoid transporter present in the skin. This protein cross-reacted with an antibody raised against a sequence of the mammalian bilitranslocase, an enzyme involved in dietary polyphenol assumption. This antibody was used in ELISA test to probe the presence of the transporter in ripening Refo\u161k grape berry. The bilitranslocase-like translocator expression was increased at the v\ue9raison and the onset of maturation by CT treatment, when compared to untreated grapevines, while PFLR showed the opposite trend. Nevertheless, at harvest, untreated samples showed an increment of protein concentration, which was significantly higher than in the other treatments. These results seem not strictly related with the total anthocyan content in grape berry, since total pigments increased more in CT and PFRL rather than in control samples, due to a favourable shift in photosynthetic area and reproductive organ balance induced by these agronomic practices. Similarly, also the phenolic potential, specifically evaluated at the harvest stage, showed a positive increase of all the parameters examined in the PFLR samples and of some of them in the CT ones. In conclusion, although the viticultural practices able to increase source/sink ratio in the cluster lead to a reduced expression of bilitranslocase-like translocator, its level and the one of other transporter proteins would be adequate enough to accomplish secondary metabolite translocation

Gene Set Based Integrated Data Analysis Reveals Phenotypic Differences in a Brain Cancer Model

A key challenge in the data analysis of biological high-throughput experiments is to handle the often low number of samples in the experiments compared to the number of biomolecules that are simultaneously measured. Combining experimental data using independent technologies to illuminate the same biological trends, as well as complementing each other in a larger perspective, is one natural way to overcome this challenge. In this work we investigated if integrating proteomics and transcriptomics data from a brain cancer animal model using gene set based analysis methodology, could enhance the biological interpretation of the data relative to more traditional analysis of the two datasets individually. The brain cancer model used is based on serial passaging of transplanted human brain tumor material (glioblastoma - GBM) through several generations in rats. These serial transplantations lead over time to genotypic and phenotypic changes in the tumors and represent a medically relevant model with a rare access to samples and where consequent analyses of individual datasets have revealed relatively few significant findings on their own. We found that the integrated analysis both performed better in terms of significance measure of its findings compared to individual analyses, as well as providing independent verification of the individual results. Thus a better context for overall biological interpretation of the data can be achieved