Current Pharmaceutical Biotechnology - Volume 7, Issue 3, 2006

Translational medicine concerns the elucidation of molecular mechanisms of disease processes, the mapping ofpathological cellular network and identification of potential biochemical targets to develop new drug ("from thebench to bedside and bedside to bench"). Proteomic based approaches are powerful tools with great promise in disease diagnosis and mechanistic profiling oftherapeutic interventions. This special issue will focus on recent advances in the application of proteomic, includingseveral aspects of clinical proteomic, biomarker discovery, systems biology using computational models,elucidation of cellular networks, and finally important functional and posttranslational modifications regulating cellsignaling. Cellular signaling is no longer considered as a linear pathway from the cells exterior to the nucleus. Stimulatory andinhibitory interconnected pathways are induced in parallel and often involve synergistic as well as antagonisticinterplay between specific signaling pathway components. Thus, mapping of entire cellular signaling pathwaysseems 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 computationalmodeling and bioinformatics. Dr. Kleppe et al. cover the use of proteomic and computational methods in systemsmodeling of cellular signaling. Biomarkers discovery are the basis for early diagnosis and prognosis of disease. Dr. Berven and co-workers give acomprehensive overview of proteomic based biomarker discovery with emphasis on cerebrospinal fluid andmultiple sclerosis. The next four papers present diverse clinical proteomic-based methods for diagnosis and therapeutic interventionsof acute myeloid leukemia. Dr. Sjoholt 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 andcolleagues. 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 myeloidmalignancies. An overview of proteomic of p53 in diagnostic and therapy of acute myeloid leukemia is presented by Dr. Ånesenand 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. Fladmarkmake available a practical approach for identification of low abundance phosphoproteins by using algal toxins asguidance 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 "Doesisoform diversity explain functional differences in the 14-3-3 protein family?".

Cellular signaling lies at the core of cellular behavior, and is central for the understanding of many pathologicconditions. To comprehend how signal transduction is orchestrated at the molecular level remains the ultimate challengefor cell biology. In the last years there has been a revolution in the development of high-throughput methodologies inproteomics 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, andseveral high-throughput methods are now available that provide quantitative and temporal information. These data arewell suited for analysis by computational methods and bioinformatics, which are becoming increasingly valuable tools tograsp the complexity of cellular networks. At present, several cellular pathways have been modeled in silico and theanalysis provides new understanding of the underlying properties that contribute to their dynamic features. Here, we re-view methodologies that are used for in silico modeling as well as methods to obtain large-scale quantitative data, and dis-cuss how they can be integrated to generate powerful and predictive models of cellular processes. We argue that the gen-eration of such models provide powerful tools to understand how systems properties emerges in healthy and pathologicstates, and to generate efficient strategies for pharmacological intervention.

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

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by accumulatingmyeloid precursor cells in the bone marrow, with approximately 2-3 months 50% survival if left untreated. With currenttreatment modalities the five years overall survival hardly exceeds 50%. Cytogenetics and molecular diagnostics guide theclinician to select individualized therapy in certain subsets of AML, achieving long-term survival above 70% of thesecases. However, approximately half of the AML patients have no risk stratifying features, and early reports indicate thatproteomic approaches may be utilized for disease classification as well as development of novel biomarkers related toprognosis, 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 eas-ily targeted by small molecules or therapeutic antibodies. The major methodological challenges include detection sensi-tivity in a limited clinical material, a problem that in some cases can be solved through designated multiplexed protein as-says based on single cells or cell extracts. In this review we will discuss pharmacoproteomic studies of drugs regulatingleukemia specific targets like all-trans retinoic acid, histone deacetylase inhibitors, proteasome inhibitors and tyrosinekinase 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 surveillancefor the clinician.

Telomeres are specialized structures at the end of human chromosomes. Telomere length decreases with eachcell division, thus, reflecting the mitotic history of somatic cells. Telomerase, the ribonucleoprotein enzyme which main-tains telomeres of eukaryotic chromosomes, is up-regulated in the vast majority of human neoplasia but not in normal so-matic tissues. In contrast to other somatic cells, normal primitive human hematopoietic cells and some peripheral bloodcells expressed low levels of telomerase activity. This activity is thought to play an important role in self-renewal of he-matopoietic stem cells. In malignant disorders, telomere lengths are generally shortened and telomerase expression andactivity 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 theclinical progression and prognosis of most leukemias. Approaches that directly target telomerase, telomeres or telomeraseregulatory mechanisms have been developed. Some of these anti-telomerase strategies in combination with conventionaldrugs proved to be promising in some types of leukemias.

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

The anti-oncogene TP53 is frequently mutated in human cancer, but in hematological malignancies this is arare feature. In acute myeloid leukemia (AML) more than 90% of the patients comprise wild type TP53 in their cancercells, but if TP53 is mutated or deleted the disease is often found to be chemoresistant. In this review we define pro-teomics of the oncogene product p53 as the study of proteins in the p53 regulating signaling networks, as well as the pro-tein 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 techniquesallow detection of various isoforms, such as two-dimensional gel electrophoresis in combination with tandem mass spec-trometry (MS/MS) and this methodology may therefore increasingly be used as a diagnostic tool in human disease. Weintroduce the p53 protein as an illustration of the complexity of post-translational modifications that may affect one highlyconnected protein and discuss the possible impact in AML diagnostics if the p53 profile is reflecting cell stress and statusof signal transduction systems of the malignancy.

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 hepatocyteswith these toxins results in a rapid hyperphosphorylation of cellular proteins before any morphological signs of apoptosisappears [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 playkey roles in this cellular process and become valuable targets for drug development. The ultra-rapid apoptosis-inductionby microcystin and nodularin provides a unique amount of synchronized apoptotic cells with "large" amounts of mainlyserine/threonine phosphorylated proteins. This ultra-rapid toxin-induced up-concentration of phosphorylated proteins re-duces the material needed as well as simplifies our effort in order to obtain enough phosphoproteins for mass spectromet-ric identification and characterization. We will here give an overview of our strategy for identification of low-abundancephosphoproteins involved in algal toxin-induced apoptosis and most likely also in a general apoptotic pathway.

The 14-3-3 family of proteins was originally identified in 1967 as simply an abundant brain protein. However ittook almost 25 years before the ubiquitous role of 14-3-3 in cell biology was recognized when it was found to interactwith several signalling and proto-oncogene proteins. Subsequently 14-3-3 proteins were the first protein recognized tobind a discrete phosphoserine/threonine-binding motifs. In mammals the 14-3-3 protein family is comprised of seven ho-mologous isoforms. The 14-3-3 family members are expressed in all eukaryotes and although no single conserved func-tion of the 14-3-3s is apparent, their ability to bind other proteins seems a crucial characteristic. To date more than 300binding partners have been identified, of which most are phosphoproteins. Consequently, it has become clear that 14-3-3proteins 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 andcellular trafficking. In this review we include recent reports on the regulation of 14-3-3 by phosphorylation, and discussthe possible functional significance of the existence of distinct 14-3-3 isoforms in light of recent proteomics studies. Inaddition we discuss 14-3-3 interaction as a possible drug target.