Analysis of Glioma Cell Platinum Response by Metacomparison of Two-dimensional Chromatographic Proteome Profiles

Abstract

Successful clinical development of cancer treatments is aided by the development of molecular markers that allow the identification of patients likely to respond. In the case of broadly cytotoxic drugs, such as the multinuclear series of platinum chemotherapeutic agents that we are evaluating for the treatment of glioma, one route to marker identification is proteomic profiling. We are using the two-dimensional chromatography system, the ProteomeLab PF2D, to compare proteomic profiles of glioma cells in culture before and after drug treatment. The existing software tools allowed the rapid identification of peaks increased by treatment of a given drug as compared with control untreated cells. To compare across these pairs, we developed new software, called the MetaComparison Tool (MCT). The MCT uses the chromatographic characteristics of peaks as identifiers, an approach that was validated by mass spectrometry of two independent isolations of a peak, from cells that were treated with two different platinum compounds. The MCT made it possible to rapidly query whether a given peak responded to more than one treatment and so allowed the identification of peaks that were specific to a given drug. As a result, this analysis greatly reduced the list of peaks whose isolation and downstream analysis by mass spectrometry is warranted, accelerating the search for protein markers of response. Molecular & Cellular Proteomics 5:35-42, 2006.The successful clinical deployment of anticancer drugs relies on being able to identify patients whose tumors are likely to respond. In the case of drugs developed against tumorspecific molecules, detection of the relevant target is the natural approach. For more broadly cytotoxic therapies, including platinum chemotherapeutic agents, such a direct approach is not possible, and so markers of likely response must be identified by molecular comparisons. In this work we present an approach to comparing the response of glioma cells to novel platinum drugs at the protein level as a first step toward this goal. We show that proteome displays generated by two-dimensional liquid chromatography can be compared using new computer software, the MetaComparison Tool (MCT), 1 and that this approach can rapidly lead to the identification of peaks worth further analysis as proteins whose expression is responsive to drug treatment.The need for new clinical agents for the treatment of glioma has led us to investigate the potential of a newer class of platinum compounds, which are multinuclear, and whose clinical profile and mechanism of action are quite distinct from the established platinum compounds. The first member of this class, the trinuclear platinum BBR3464, is effective against cells with an inherent or acquired resistance to cisplatin, including glioma cells (1, 2), suggesting that there are important differences in their mode of action. BBR compounds, exemplified by BBR3464, show increased rates of cellular uptake and more rapid formation of DNA adducts than cisplatin and a different pattern of ad...

Vandetanib alters the protein pattern in malignant glioma and normal brain in the BT4C rat glioma model

“…3, which further stresses the importance of designing experiments including control groups for each individual time-point of interest. In many previous proteomic studies of glioma in vitro the analyses of protein expression have been done at a single time-point after treatment (22,23,42,44). Based on our findings, together with the information from the earlier referred study (26), we believe that the issue of proteomic response in malignant glioma to treatment is complex and that potential markers may be expressed differently in relation to timing of treatment and sampling of specimens.…”

Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

“…For the tissues and blood samples used, written informed consent was obtained from patients according to the research proposals approved by the Institutional Review Board at the Medical Faculty of the University of Heidelberg. For PF2D analysis cells and tissues were lysed in 2 ml lysis buffer (12). Before lysis, tissues were mechanically dissected into small pieces by use of an Ultra-Turrax (IKA).…”

“…immunogenic epitope in more detail, we synthesized overlapping 20-meric peptides of the respective region ( Figure 6C) and identified S100A9 [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] as the immunogenic epitope ( Figure 6D). This epitope was recognized both by CD8 + and CD4 + T cells of patient NCH550 ( Figure 6E).…”

“…T cell activation by antigen-pulsed DCs is based on a complex and tightly regulated process involving multiple steps, including protein uptake, cleavage into small peptide fragments, their loading into the peptide-binding cleft of MHC I and II molecules, and their presentation at the cell surface to antigen-specific T cells. However, since PF2D technology has predominantly been used for identifying differentially expressed proteins (12)(13)(14)(15) and for validating protein arrays (16), so far, it is not known whether proteins subjected to PF2D fractionation under partially denaturing conditions maintain the features required for appropriate antigen processing by DCs. In addition, it is unclear whether concentrations of respective proteins in the eluates are sufficient for successful presentation to CD4 + and CD8 + T cells.…”

Development of barcode and proteome profiling of glioblastoma

“…Следует заметить, что за последние годы протеомный сравнительный анализ в области изучения глиобластомы стал очень популярен, поэтому есть возможность для сравнения наших и уже опубликованных результатов. Необходимо отметить, что по многим белкам наши и литературные данные совпадают, например, по пируваткиназе М1/М2 [24], триозофосфатизомеразе [25], кофилину, енолазе, виментину и его разным формам [26,27]. Появление мутантных форм р53 характерно для многих опухолей, есть публикации такого рода и по глиобластомам [28].…”