Current Pharmaceutical Biotechnology

ISSN: 1389-2010

Current Pharmaceutical Biotechnology
Volume 7, Number 3, June 2006

Contents

Proteomic Technologies in Translational Medicine
Guest Editor: Frode Selheim


Editorial Pp. 133


Proteomic and Computational Methods in Systems Modeling of Cellular Signaling Pp. 135-145
R. Kleppe, E. Kjaerland and F. Selheim
[Abstract]


Proteomic-Based Biomarker Discovery with Emphasis on Cerebrospinal Fluid and Multiple Sclerosis Pp. 147-158
F.S. Berven, K. Flikka, M. Berle, C. Vedeler and R.J. Ulvik
[Abstract]


Proteomic Strategies for Individualizing Therapy of Acute Myeloid Leukemia (AML) Pp. 159-170
G. Sjøholt, S.L. Bedringaas, A.P. Døskeland and B.T. Gjertsen
[Abstract]


Diagnostics, Prognostic and Therapeutic Exploitation of Telomeres and Telomerase in Leukemias Pp. 171-183
L. Deville, J. Hillion, M. Lanotte, P. Rousselot and E. Ségal Bendirdjian
[Abstract]


Proteomics Approaches to Elucidate Oncogenic Tyrosine Kinase Signaling in Myeloid Malignancies Pp. 185-198
E. Oveland, K.E. Fladmark, L. Wergeland, B.T. Gjertsen and R. Hovland
[Abstract]


Proteomics of p53 in Diagnostics and Therapy of Acute Myeloid Leukemia Pp. 199-207
N. Ånensen, I. Haaland, C. D'Santos, W. Van Belle and B.T. Gjertsen
[Abstract]

Algal Toxins as Guidance to Identify Phosphoproteins with Key Roles in Apoptotic Cell Death Pp. 209-215
T. Solstad and K.E. Fladmark
[Abstract]


Does Isoform Diversity Explain Functional Differences in the 14-3 3 Protein Family? Pp. 217-223
E. Kjarland, T.J. Keen and R. Kleppe
[Abstract]




Abstracts

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Editorial

Translational medicine concerns the elucidation of molecular mechanisms of disease processes, the mapping of pathological cellular network and identification of potential biochemical targets to develop new drug (“from the bench to bedside and bedside to bench”).

Proteomic based approaches are powerful tools with great promise in disease diagnosis and mechanistic profiling of therapeutic interventions. This special issue will focus on recent advances in the application of proteomic, including several aspects of clinical proteomic, biomarker discovery, systems biology using computational models, elucidation of cellular networks, and finally important functional and posttranslational modifications regulating cell signaling.

Cellular signaling is no longer considered as a linear pathway from the cells exterior to the nucleus. Stimulatory and inhibitory interconnected pathways are induced in parallel and often involve synergistic as well as antagonistic interplay between specific signaling pathway components. Thus, mapping of entire cellular signaling pathways seems to be required to elucidate how proteins are regulated in both healthy and pathological signaling.

To make sense of such complex network we must couple quantitative experimental data with computational modeling and bioinformatics. Dr. Kleppe et al. cover the use of proteomic and computational methods in systems modeling of cellular signaling.

Biomarkers discovery are the basis for early diagnosis and prognosis of disease. Dr. Berven and co-workers give a comprehensive overview of proteomic based biomarker discovery with emphasis on cerebrospinal fluid and multiple sclerosis.

The next four papers present diverse clinical proteomic-based methods for diagnosis and therapeutic interventions of acute myeloid leukemia.

Dr. Sjøholt et al. present proteomic strategies for individualizing therapy of myeloid leukemia.

Diagnostic, prognostic and therapeutic exploitation of telomeres and telomerase are covered by Dr. L. Deville and colleagues. Some of the anti-telomerase strategies show promising effects for the treatment of leukemia.

The tyrosine kinase inhibitor Imatinib mesylate is successfully used for targeting the fusion protein Bcr/Abl. Dr. Oveland et al. describe proteomic approaches to elucidate oncogenic tyrosine kinase signaling in myeloid malignancies.

An overview of proteomic of p53 in diagnostic and therapy of acute myeloid leukemia is presented by Dr. Ånesen and co-workers. The numerous posttranslational modifications of the p53 protein are given special attention.

Posttranslational phosphorylation has a crucial role in regulation of cellular signaling. Dr. Solstad and Dr. Fladmark make available a practical approach for identification of low abundance phosphoproteins by using algal toxins as guidance to identify phosphoproteins with key roles in apoptotic cell.

Proteomic studies on the 14-3-3 interactome are presented by Dr. Kjærland et al., and they pose the question "Does isoform diversity explain functional differences in the 14-3-3 protein family?".

Frode Selheim, Dr. Scient., PhD
Guest Editor
Department of Biomedicine, Proteomic Unit,
The University of Bergen,
Jonas Lies vei 91, N-5009 Bergen, Norway
E-mail: frode.selheim@biomed.uib.no


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Proteomic and Computational Methods in Systems Modeling of Cellular Signaling
R. Kleppe, E. Kjaerland and F. Selheim

Cellular signaling lies at the core of cellular behavior, and is central for the understanding of many pathologic conditions. To comprehend how signal transduction is orchestrated at the molecular level remains the ultimate challenge for cell biology. In the last years there has been a revolution in the development of high-throughput methodologies in proteomics and genomics, which have provided extensive knowledge about expression profiles and molecular interaction-networks. However, these methods have typically provided qualitative and static information. This is about to turn, and several high-throughput methods are now available that provide quantitative and temporal information. These data are well suited for analysis by computational methods and bioinformatics, which are becoming increasingly valuable tools to grasp the complexity of cellular networks. At present, several cellular pathways have been modeled in silico and the analysis provides new understanding of the underlying properties that contribute to their dynamic features. Here, we review methodologies that are used for in silico modeling as well as methods to obtain large-scale quantitative data, and discuss how they can be integrated to generate powerful and predictive models of cellular processes. We argue that the generation of such models provide powerful tools to understand how systems properties emerges in healthy and pathologic states, and to generate efficient strategies for pharmacological intervention.


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Proteomic-Based Biomarker Discovery with Emphasis on Cerebrospinal Fluid and Multiple Sclerosis
F.S. Berven, K. Flikka, M. Berle, C. Vedeler and R.J. Ulvik

Discovery of disease specific biomarkers in human body fluids has become an important challenge in clinical proteomics. Facing the increasing threat of degenerative and disabling diseases like cancer, cardiovascular, neurological and inflammatory diseases in large parts of the world’s population, there is an urgent need to improve early diagnostics. In this review we discuss possibilities and limitations connected to using mass spectrometry based proteomics in the search for novel biomarkers, with focus on multiple sclerosis as a typical representative for the large group of non-curable degenerative and disabling disease with the lack of specific tests for early diagnosis. Careful control of the pre-analytical phase including sampling, storage and fractionation of samples, in addition to a thoroughly considered patient selection, is important in order to avoid false biomarkers to appear in the resulting mass spectra. Furthermore, advanced computational tools are needed in order to discover potential biomarkers from the enormous data amounts generated by the mass spectrometers. The development of such computer tools is a research field currently in the start phase and could prove to be a bottle neck in the biomarker discovery the next years. Therefore, a rather detailed review of the most used computational and pre-analytical methods is given in this review. Mass spectrometry based biomarker discovery is undoubtedly still in its early infancy. However, in light of the potential of this technology to provide deep coverage of the body fluid proteomes, it will certainly consolidate its role in developing molecular medicine into clinical practice.


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Proteomic Strategies for Individualizing Therapy of Acute Myeloid Leukemia (AML)
G. Sjøholt, S.L. Bedringaas, A.P. Døskeland and B.T. Gjertsen

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by accumulating myeloid precursor cells in the bone marrow, with approximately 2-3 months 50% survival if left untreated. With current treatment modalities the five years overall survival hardly exceeds 50%. Cytogenetics and molecular diagnostics guide the clinician to select individualized therapy in certain subsets of AML, achieving long-term survival above 70% of these cases. However, approximately half of the AML patients have no risk stratifying features, and early reports indicate that proteomic approaches may be utilized for disease classification as well as development of novel biomarkers related to prognosis, diagnosis, and choice of therapeutic regimen. Proteomics, here defined as the analysis of all proteins in a cell, in a cell compartment or in a signaling pathway, has probably its greatest potential in investigating pathways that are easily targeted by small molecules or therapeutic antibodies. The major methodological challenges include detection sensitivity in a limited clinical material, a problem that in some cases can be solved through designated multiplexed protein assays based on single cells or cell extracts. In this review we will discuss pharmacoproteomic studies of drugs regulating leukemia specific targets like all-trans retinoic acid, histone deacetylase inhibitors, proteasome inhibitors and tyrosine kinase inhibitors, as well as studies on drug resistance and graft-versus-host studies during stem cell transplantations. These studies indicate new avenues in AML diagnostics, individualized therapy design and therapy response surveillance for the clinician.


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Diagnostics, Prognostic and Therapeutic Exploitation of Telomeres and Telomerase in Leukemias
L. Deville, J. Hillion, M. Lanotte, P. Rousselot and E. Ségal Bendirdjian

Telomeres are specialized structures at the end of human chromosomes. Telomere length decreases with each cell division, thus, reflecting the mitotic history of somatic cells. Telomerase, the ribonucleoprotein enzyme which maintains telomeres of eukaryotic chromosomes, is up-regulated in the vast majority of human neoplasia but not in normal somatic tissues. In contrast to other somatic cells, normal primitive human hematopoietic cells and some peripheral blood cells expressed low levels of telomerase activity. This activity is thought to play an important role in self-renewal of hematopoietic stem cells. In malignant disorders, telomere lengths are generally shortened and telomerase expression and activity enhanced with high differences in the levels. Although it is necessary to be cautious in interpreting these data, there are indications that telomere length and telomerase expression and activity can serve as a molecular marker of the clinical progression and prognosis of most leukemias. Approaches that directly target telomerase, telomeres or telomerase regulatory mechanisms have been developed. Some of these anti-telomerase strategies in combination with conventional drugs proved to be promising in some types of leukemias.


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Proteomics Approaches to Elucidate Oncogenic Tyrosine Kinase Signaling in Myeloid Malignancies
E. Oveland, K.E. Fladmark, L. Wergeland, B.T. Gjertsen and R. Hovland

Myeloid malignancies frequently harbor specific mutations in protein tyrosine kinases leading to oncogenic cell signaling. The most extensively investigated example is chronic myeloid leukemia, where the pathogenic tyrosine kinase fusion protein Bcr-Abl is a successful target for disease control by the specific inhibitor imatinib mesylate. In acute myeloid leukemia the receptor tyrosine kinase Flt3 is frequently mutated and inhibitors to impair the oncogenic signaling are in development. In this review we exemplify oncogenic signaling and how signal pathways can be unraveled with help from proteomics-based technologies. The distinction between cell extract and single cell approaches aiming at rigorous standardization and reliable quantitative aspects for future proteomics-based diagnostics is discussed.


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Proteomics of p53 in Diagnostics and Therapy of Acute Myeloid Leukemia
N. Ånensen, I. Haaland, C. D'Santos, W. Van Belle and B.T. Gjertsen

The anti-oncogene TP53 is frequently mutated in human cancer, but in hematological malignancies this is a rare feature. In acute myeloid leukemia (AML) more than 90% of the patients comprise wild type TP53 in their cancer cells, but if TP53 is mutated or deleted the disease is often found to be chemoresistant. In this review we define proteomics of the oncogene product p53 as the study of proteins in the p53 regulating signaling networks, as well as the protein study of members of the p53 family itself. Various messenger RNA splice forms as well as a multitude of post-translational modifications give a high number of protein isoforms in the p53 family. Some of the proteomic techniques allow detection of various isoforms, such as two-dimensional gel electrophoresis in combination with tandem mass spectrometry (MS/MS) and this methodology may therefore increasingly be used as a diagnostic tool in human disease. We introduce the p53 protein as an illustration of the complexity of post-translational modifications that may affect one highly connected protein and discuss the possible impact in AML diagnostics if the p53 profile is reflecting cell stress and status of signal transduction systems of the malignancy.


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Algal Toxins as Guidance to Identify Phosphoproteins with Key Roles in Apoptotic Cell Death
T. Solstad and K.E. Fladmark

The protein phosphatase inhibiting toxins microcystin and nodularin act rapidly to induce apoptotic cell death. Their inhibitory effect on protein phosphatases 1 and 2A can be utilized as tools to understand the phosphorylation-dependent regulatory mechanism underlying the early stage of apoptosis. The incubation of freshly isolated hepatocytes with these toxins results in a rapid hyperphosphorylation of cellular proteins before any morphological signs of apoptosis appears [Fladmark, K. E., Brustugun, O. T., Hovland, R., Boe, R., Gjertsen, B. T., Zhivotovsky, B. and Doskeland, S. O. (1999) Cell Death Differ. 6, 1099-108]. Proteins subjected to phosphorylation in this early phase of apoptosis may play key roles in this cellular process and become valuable targets for drug development. The ultra-rapid apoptosis-induction by microcystin and nodularin provides a unique amount of synchronized apoptotic cells with “large” amounts of mainly serine/threonine phosphorylated proteins. This ultra-rapid toxin-induced up-concentration of phosphorylated proteins reduces the material needed as well as simplifies our effort in order to obtain enough phosphoproteins for mass spectrometric identification and characterization. We will here give an overview of our strategy for identification of low-abundance phosphoproteins involved in algal toxin-induced apoptosis and most likely also in a general apoptotic pathway.


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Does Isoform Diversity Explain Functional Differences in the 14-3 3 Protein Family?
E. Kjarland, T.J. Keen and R. Kleppe

The 14-3-3 family of proteins was originally identified in 1967 as simply an abundant brain protein. However it took almost 25 years before the ubiquitous role of 14-3-3 in cell biology was recognized when it was found to interact with several signalling and proto-oncogene proteins. Subsequently 14-3-3 proteins were the first protein recognized to bind a discrete phosphoserine/threonine-binding motifs. In mammals the 14-3-3 protein family is comprised of seven homologous isoforms. The 14-3-3 family members are expressed in all eukaryotes and although no single conserved function of the 14-3-3s is apparent, their ability to bind other proteins seems a crucial characteristic. To date more than 300 binding partners have been identified, of which most are phosphoproteins. Consequently, it has become clear that 14-3-3 proteins are involved in the regulation of most cellular processes, including several metabolic pathways, redox-regulation, transcription, RNA processing, protein synthesis, protein folding and degradation, cell cycle, cytoskeletal organization and cellular trafficking. In this review we include recent reports on the regulation of 14-3-3 by phosphorylation, and discuss the possible functional significance of the existence of distinct 14-3-3 isoforms in light of recent proteomics studies. In addition we discuss 14-3-3 interaction as a possible drug target.