Browsing by Author "Honikel, Louise"

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    Protein expression profiles in hematopoietic stem/progenitor cells after exposure of mice to silicon (28Si) ions
    (Oxford University Press, 2014) Rithidech, Kanokporn Noy; Tungjai, Montree; Honikel, Louise; Gordon, Chris; Lai, Xianyin; Witzmann, Frank; Biochemistry and Molecular Biology, School of Medicine
    It has been well recognized that exposure to space radiation is a major challenge to space exploration. To protect astronauts in space environments, improvement in our knowledge of radiation-induced changes in specific target cells that may affect the health of astronauts is required. Cancer of blood cells, in particular myeloid leukemia (ML), is one of the major health concerns from exposure to space radiation. However, the predictions of risks for developing ML post-exposure to space radiation are unsatisfactory. To increase the reliability of predicting risk for ML, a much improved understanding of space radiation-induced changes in the target cells, i.e. hematopoietic stem/progenitor cells (HSPCs), is critically important. In vivo studies of humans are not possible. Thus, controlled and randomized animal experiments are critically important. Most proteomic applications mentioned above have used 2-DE or stable isotope-tagged mass spectrometry. Although the 2-DE has several advantages (e.g. the detection of potential post-translational modifications of proteins which can be readily visualized on the gel, although the exact type of modification requires determination by mass spectrometry), such technology is simply not as comprehensive and reliable as desired. To overcome these limitations, we recently developed a unique label-free quantitative mass spectrometry (LFQMS) platform [ 1]. This is an innovative, experimentally based method that accurately determines peptide peak retention-time and uses multiple filters for exclusion of unqualified peptides by peptide frequency, retention time, intensity coefficient of variation and intensity correlation to enhance protein quantification of qualified peptides and proteins. In this study, we used the LFQMS platform to examine protein expression-profiles in the colonies of HSPCs (the best population of cells for studying in vivo biological effects of radiation on hematopoietic stem cells) obtained at 6 months after exposure (at which radiation-induced genomic instability and chronic inflammation have been detected [ 2]) of CBA/CaJ mice whole-body to a total dose of 0, 0.1, 0.25 or 0.5 Gy of 300 MeV/nucleon 28Si ions, using a fractionated schedule (two exposures, 15 days apart that totaled each selected dose). These doses of 300 MeV/nucleon 28Si ions are comparable to what astronauts encounter in space. Mice exposed to 0 Gy of 28Si ions served as non-irradiated sham controls. The colonies of HSPCs were obtained from BM cells of five mice per treatment group, by means of an in vitro colony forming unit assay (CFU-A) using methylcellulose-based medium. Proteins were extracted from HSPC colonies and protein concentrations were determined by the Bradford Protein Assay. The trans-proteomic pipeline (TPP) was used to assign a probability of a particular protein being in the sample. A total of 1344 unique, non-redundant proteins were identified with ≥90% confidence from 3254 peptides, quantified and their abundances were compared statistically. Among the 1344 proteins, differential expression of 198 proteins was found to be statistically significant in HSPC colonies obtained from treated groups, in relation to those found in non-irradiated sham controls. A proof-of-concept-based Ingenuity Pathway Analysis (IPA, www.ingenuity.com) was used to characterize the functions and pathways of these 198 identified proteins. The majority of these proteins are cancer-related (P < 0.0001). Biochemical analyses of the molecular and cellular functions of these proteins reveal association with perturbation in cell survival, free radical scavenging, cell cycle, DNA repair, cellular assembly, hematological system development and inflammatory responses. These proteins are linked to two major molecular networks that are linked to cancer and inflammatory responses (i.e. nuclear factor-κ B and the protein phosphatase 2 A networks). Our results indicated 28Si ion-induced damage in HSPCs.
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    Proteomic Profiling of Hematopoietic Stem/Progenitor Cells after a Whole Body Exposure of CBA/CaJ Mice to Titanium (48Ti) Ions
    (MDPI, 2015-07-21) Rithidech, Kanokporn Noy; Tungjai, Montree; Jangiam, Witawat; Honikel, Louise; Gordon, Chris; Lai, Xianyin; Witzmann, Frank; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Myeloid leukemia (ML) is one of the major health concerns from exposure to radiation. However, the risk assessment for developing ML after exposure to space radiation remains uncertain. To reduce the uncertainty in risk prediction for ML, a much increased understanding of space radiation-induced changes in the target cells, i.e., hematopoietic stem/progenitor cells (HSPCs), is critically important. We used the label-free quantitative mass spectrometry (LFQMS) proteomic approach to determine the expression of protein in HSPC-derived myeloid colonies obtained at an early time-point (one week) and a late time-point (six months) after an acute whole body exposure of CBA/CaJ mice to a total dose of 0, 0.1, 0.25, or 0.5 Gy of heavy-ion titanium (48Ti ions), which are the important component of radiation found in the space environment. Mice exposed to 0 Gy of 48Ti ions served as non-irradiated sham controls. There were five mice per treatment groups at each harvest time. The Trans-Proteomic Pipeline (TPP) was used to assign a probability of a particular protein being in the sample. A proof-of-concept based Ingenuity Pathway Analysis (IPA) was used to characterize the functions, pathways, and networks of the identified proteins. Alterations of expression levels of proteins detected in samples collected at one week (wk) post-irradiation reflects acute effects of exposure to 48Ti ions, while those detected in samples collected at six months (mos) post-irradiation represent protein expression profiles involved in the induction of late-occurring damage (normally referred to as genomic instability). Our results obtained by using the IPA analyses indicate a wide array of signaling pathways involved in response to 1 GeV/n 48Ti ions at both harvest times. Our data also demonstrate that the patterns of protein expression profiles are dose and time dependent. The majority of proteins with altered expression levels are involved in cell cycle control, cellular growth and proliferation, cell death and survival, cell-to-cell signaling and interaction. The IPA analyses indicate several important processes involved in responses to exposure to 48Ti ions. These include the proteosme/ubiquination, protein synthesis, post-translation modification, and lipid metabolism. The IPA analyses also indicate that exposure to 1 GeV/n 48Ti ions affects the development and function of hematological system, immune cell trafficking, including the cytoskeleton. Further, the IPA analyses strongly demonstrate that the NF-κB and MAPKs (ERKs, JNKs, and p38MAPK) pathways play an essential role in signal transduction after exposure to 1 GeV/n 48Ti ions. At an early time-point (1 week), the top networks identified by the IPA analyses are related to metabolic disease, lipid metabolism, small molecule biochemistry, and development disorder. In contrast, the top networks identified in samples collected at a late time-point (6 mos post-irradiation) by the IPA analyses are related to cancer, hematological disorders, and immunological diseases. In summary, the proteomic findings from our study provide a foundation to uncover compounds potentially be highly effective in radiation countermeasures.