| 1. |
- Jitkaew, Siriporn, et al.
(författare)
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N(alpha)-tosyl-L-phenylalanine chloromethyl ketone induces caspase-dependent apoptosis in transformed human B cell lines with transcriptional down-regulation of anti-apoptotic HS1-associated protein X-1
- 2009
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:41, s. 27827-27837
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Tidskriftsartikel (refereegranskat)abstract
- N(alpha)-tosyl-L-phenylalanine chloromethylketone (TPCK) has been widely used to investigate signal transduction pathways that are involved in gene expression and cell survival/cell death. However, contradictory effects of TPCK on apoptosis have been reported, and the underlying signaling events leading to TPCK-induced promotion or prevention of apoptosis are not fully understood. Here, we show that TPCK induces caspase-dependent apoptosis in Epstein-Barr virus (EBV)-transformed human B cell lines with release of pro-apoptotic proteins from mitochondria. TPCK treatment also results in down-regulation of the anti-apoptotic proteins, cIAP1, cIAP2, and HAX-1, and caspase-dependent cleavage of the anti-apoptotic proteins, Bcl-2 and XIAP. Quantitative PCR analysis confirmed that the TPCK-induced down-regulation of HAX-1 occurred at the transcriptional level, and experiments using the specific pharmacological inhibitor, Bay 11-7082, suggested that HAX-1 expression is subject to regulation by the transcription factor, NF-kappaB. B cell lines derived from patients with homozygous HAX1 mutations were more sensitive to TPCK-induced apoptosis when compared with normal donor cell lines. Furthermore, N-acetylcysteine effectively blocked TPCK-induced apoptosis in EBV-transformed B cell lines and prevented the down-regulation or cleavage of anti-apoptotic proteins. Taken together, our studies demonstrate that TPCK induces apoptosis in human B cell lines and exerts multiple effects on pro- and anti-apoptotic factors.
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| 2. |
- Lewensohn, Rolf, et al.
(författare)
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S100a6 and/or s100a4 inhibitors for treating cancer
- 2008
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Patent (populärvet., debatt m.m.)abstract
- Aspects of this invention relate to the fields of molecular biology and medicine. More specifically, disclosed herein are several approaches to provide subjects suffering from cancer with an inhibitor of S100A6 and/or S 100A4 alone or in combination with other cancer therapies so as to improve the cancer therapy and/or more efficiently treat cancer, in particular forms of cancer that are resistant to other therapies. Also disclosed herein are approaches for using S 100A6 and/or S 100A4 as a biomarker for metastases. Further, disclosed herein are approaches for using S 100A6 and/or S 100A4 as a biomarker for cancer therapies, in particular, as a biomarker to determine individual responses to cancer therapies. In addition, disclosed herein are approaches to identifying S 100A6 and/or S 100A4 inhibitors, for example, that act synergistically with a cancer therapy.
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| 3. |
- Nordström, Anders, et al.
(författare)
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Multiple ionization mass spectrometry strategy used to reveal the complexity of metabolomics.
- 2008
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 80:2, s. 421-9
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Tidskriftsartikel (refereegranskat)abstract
- A multiple ionization mass spectrometry strategy is presented based on the analysis of human serum extracts. Chromatographic separation was interfaced inline with the atmospheric pressure ionization techniques electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive (+) and negative (-) ionization modes. Furthermore, surface-based matrix-assisted laser desorption/ionization (MALDI) and desorption ionization on silicon (DIOS) mass spectrometry were also integrated with the separation through fraction collection and offline mass spectrometry. Processing of raw data using the XCMS software resulted in time-aligned ion features, which are defined as a unique m/z at a unique retention time. The ion feature lists obtained through LC-MS with ESI and APCI interfaces in both +/- ionization modes were compared, and unique ion tables were generated. Nonredundant, unique ion features, were defined as mass numbers for which no mass numbers corresponding to [M + H](+), [M - H](-), or [M + Na](+) were observed in the other ionization methods at the same retention time. Analysis of the extracted serum using ESI for both (+) and (-) ions resulted in >90% additional unique ions being detected in the (-) ESI mode. Complementing the ESI analysis with APCI resulted in an additional approximately 20% increase in unique ions. Finally, ESI/APCI ionization was combined with fraction collection and offline-MALDI and DIOS mass spectrometry. The parts of the total ion current chromatograms in the LC-MS acquired data corresponding to collected fractions were summed, and m/z lists were compiled and compared to the m/z lists obtained from the DIOS/MALDI spectra. It was observed that, for each fraction, DIOS accounted for approximately 50% of the unique ions detected. These results suggest that true global metabolomics will require multiple ionization technologies to address the inherent metabolite diversity and therefore the complexity in and of metabolomics studies.
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| 4. |
- Orre, Lukas M., et al.
(författare)
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p53 is involved in clearance of ionizing radiation-induced RAD51 foci in a human colon cancer cell line
- 2006
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 342:4, s. 1211-7
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated p53-related differences in cellular response to DNA damaging agents, focusing on p53s effects on RAD51 protein level and sub-cellular localization post exposure to ionizing radiation. In a human colon cancer cell line, HCT116 and its isogenic p53-/- subcell line we show here p53-independent RAD51 foci formation but interestingly the resolution of RAD51 foci showed clear p53 dependence. In p53 wt cells, but not in p53-/- cells, RAD51 protein level decreased 48 h post irradiation and fluorescence immunostaining showed resolution of RAD51 foci and relocalization of RAD51 to nucleoli at time points corresponding to the decrease in RAD51 protein level. Both cell lines rejoined DNA double strand breaks efficiently with similar kinetics and p53 status did not influence sensitivity to DNA damaging agents. We suggest that p53 has a role in RAD51 clearance post DSB repair and that nucleoli might be sites of RAD51 protein degradation.
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| 5. |
- Pernemalm, Maria, et al.
(författare)
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Evaluation of three principally different intact protein prefractionation methods for plasma biomarker discovery.
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:7, s. 2712-2722
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to evaluate three principally different top-down protein prefractionation methods for plasma: high-abundance protein depletion, size fractionation and peptide ligand affinity beads, focusing in particular on compatibility with downstream analysis, reproducibility and analytical depth. Our data clearly demonstrates the benefit of high-abundance protein depletion. However, MS/MS analysis of the proteins eluted from the high-abundance protein depletion column show that more proteins than aimed for are removed and, in addition, that the depletion efficacy varies between the different high-abundance proteins. Although a smaller number of proteins were identified per fraction using the peptide ligand affinity beads, this technique showed to be both robust and versatile. Size fractionation, as performed in this study, focusing on the low molecular weight proteome using a combination of gel filtration chromatography and molecular weight cutoff filters, showed limitations in the molecular weight cutoff precision leading detection of high molecular weight proteins and, in the case of the cutoff filters, high variability. GeLC-MS/MS analysis of the fractionation methods in combination with pathway analysis demonstrates that increased fractionation primarily leads to high proteome coverage of pathways related to biological functions of plasma, such as acute phase reaction, complement cascade and coagulation. Further, the prefractionation methods in this study induces limited effect on the proportion of tissue proteins detected, thereby highlighting the importance of extensive or targeted downstream fractionation.
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| 6. |
- Ståhl, Sara, et al.
(författare)
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Proteomics and pathway analysis identifies JNK signaling as critical for high linear energy transfer radiation-induced apoptosis in non-small lung cancer cells
- 2009
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 8:5, s. 1117-1129
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Tidskriftsartikel (refereegranskat)abstract
- During the past decade, we have witnessed an explosive increase in generation of large proteomics data sets, not least in cancer research. There is a growing need to extract and correctly interpret information from such data sets to generate biologically relevant hypotheses. A pathway search engine (PSE) has recently been developed as a novel tool intended to meet these requirements. Ionizing radiation (IR) is an anticancer treatment modality that triggers multiple signal transduction networks. In this work, we show that high linear energy transfer (LET) IR induces apoptosis in a non-small cell lung cancer cell line, U-1810, whereas low LET IR does not. PSE was applied to study changes in pathway status between high and low LET IR to find pathway candidates of importance for high LET-induced apoptosis. Such pathways are potential clinical targets, and they were further validated in vitro. We used an unsupervised shotgun proteomics approach where high resolution mass spectrometry coupled to nanoflow liquid chromatography determined the identity and relative abundance of expressed proteins. Based on the proteomics data, PSE suggested the JNK pathway (p = 6.10(-6)) as a key event in response to high LET IR. In addition, the Fas pathway was found to be activated (p = 3.10(-5)) and the p38 pathway was found to be deactivated (p = 0.001) compared with untreated cells. Antibody-based analyses confirmed that high LET IR caused an increase in phosphorylation of JNK. Moreover pharmacological inhibition of JNK blocked high LET-induced apoptotic signaling. In contrast, neither an activation of p38 nor a role for p38 in high LET IR-induced apoptotic signaling was found. We conclude that, in contrast to conventional low LET IR, high LET IR can trigger activation of the JNK pathway, which in turn is critical for induction of apoptosis in these cells. Thus PSE predictions were largely confirmed, and PSE was proven to be a useful hypothesis-generating tool.
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| 7. |
- Wibom, Carl, 1977-
(författare)
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Multivariate analyses of proteomic and metabolomic patterns in brain tumors
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glioblastoma multiforme (GBM) is the most common primary brain tumor. Given the current standard of care, the prognosis for patients diagnosed with this disease is still poor. There consequently exists a need to improve current treatments, as well as to develop new ones. Many obstacles however need to be overcome to facilitate this effort and one of these involves the development of improved methods to monitor treatment effects. At present, the effects of treatment are typically assessed by radiological means several months after its initiation, which is unsatisfactory for a fast growing tumor like GBM. It is however likely that treatment effects can be detected on a molecular level long before radiological response, especially considering many of the targeted therapies that are currently being developed. Biomarkers for treatment efficacy may be of great importance in the future individualization of brain tumor treatment. The work presented herein was primarily focused on detecting early effects of GBM treatment. To this end, we designed experiments in the BT4C rat glioma model in which we studied effects of both conventional radiotherapy and an experimental angiogenesis inhibitor, vandetanib. Brain tissue samples were analyzed using a high throughput mass spectrometry (MS) based screening, known as Surface Enhanced Laser Desorption/Ionization - Time of Flight - Mass Spectrometry (SELDI-TOF-MS). The vast amounts of data generated were subsequently analyzed by established multivariate statistical methods, such as Principal Component Analysis (PCA), Partial Least Squares (PLS), and Orthogonal Partial Least Squares (OPLS), developed for analysis of large and complex datasets. In the radiotherapy study we detected a protein spectrum pattern clearly related to tumor progression. We notably observed how this progression pattern was hampered by radiotherapy. The vandetanib study also revealed significant alterations of protein expression following treatment of different durations, both in tumor tissue and in normal brain contralateral to the tumor. In an effort to further elucidate the pathophysiology of GBM, particularly in relation to treatment, we collected extracellular fluid (ECF) samples from 11 patients diagnosed with inoperable GBM. The samples were collected by means of stereotactic microdialysis, both from within the contrast enhancing tumor and the brain adjacent to tumor (BAT). Samples were collected longitudinally from each patient in a time span of up to two weeks, during which the patient received the first five fractions of radiotherapy. The ECF samples were then analyzed by Gas Chromatography Mass Spectrometry (GC-MS) to screen them with respect to concentrations of low molecular weight compounds (metabolites). Suitable multivariate analysis strategies enabled us to extract patterns of varying metabolite concentrations distinguishing between samples collected at different locations in the brain as well as between samples collected at different time points in relation to treatment. In a separate study, we also applied SELDI-TOF-MS and multivariate statistical methods to unravel possible differences in protein spectra between invasive and non-invasive WHO grade I meningiomas. This type of tumor can usually be cured by surgical resection however sometimes it grows invasively into the bone, ultimately causing clinical problems. This study revealed the possibility to differentiate between invasive and non-invasive benign meningioma based on the expression pattern of a few proteins. Our approach, which includes sample analysis and data handling, is applicable to a wide range of screening studies. In this work we demonstrated that the combination of MS screening and multivariate analyses is a powerful tool in the search for patterns related to treatment effects and diagnostics in brain tumors.
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