Blood cell production involves the commitment and differentiation of hematopoietic stem cells to committed progenitor cells which undergo a programmed development to form mature cells such as neutrophils, macrophages and lymphocytes. This complex process can be disrupted in diseases such as the leukemias and myeloproliferative disorders by oncogenes such as protein tyrosine kinases. The analysis of expression patterns for specific genes suggests that the regulation of protein expression can be achieved in a post-translational fashion. Post-translational protein modification, such as phosphorylation and acetylation govern events in blood cell production and yet cannot be measured using conventional molecular biology approaches. For this reason a suite of techniques in mass spectrometry need to be applied to define regulation and disregulation in normal and abnormal hematopoiesis. These approaches include discovery proteomics with relative quantification of thousands of proteins. Alternatively targeted examination of a single protein to identify its interaction partners or post-translational modifications using mass spectrometry reveals much mechanistic detail. The use of mass spectrometry and proteomics approaches in stem cell and leukemia studies has thus far revealed a good deal of information on hematopoiesis. Further application of the proteomics approach is a necessity to gain true insight into regulatory processes governing the production of billions of blood cells a day, and ways in which that process can be manipulated to therapeutic advantage.

A mass spectrometry-based plasma biomarker discovery workflow was developed to facilitate biomarker discovery. Plasma from either healthy volunteers or patients with pancreatic cancer was 8-plex iTRAQ labeled, fractionated by 2-dimensional reversed phase chromatography and subjected to MALDI ToF/ToF mass spectrometry. Data were processed using a q-value based statistical approach to maximize protein quantification and identification. Technical (between duplicate samples) and biological variance (between and within individuals) were calculated and power analysis was thereby enabled. An a priori power analysis was carried out using samples from healthy volunteers to define sample sizes required for robust biomarker identification. The result was subsequently validated with a post hoc power analysis using a real clinical setting involving pancreatic cancer patients. This demonstrated that six samples per group (e.g., pre- vs post-treatment) may provide sufficient statistical power for most proteins with changes>2 fold. A reference standard allowed direct comparison of protein expression changes between multiple experiments. Analysis of patient plasma prior to treatment identified 29 proteins with significant changes within individual patient. Changes in Peroxiredoxin II levels were confirmed by Western blot. This q-value based statistical approach in combination with reference standard samples can be applied with confidence in the design and execution of clinical studies for predictive, prognostic, and/or pharmacodynamic biomarker discovery. The power analysis provides information required prior to study initiation.

In response to DNA damage, transcription is blocked by inhibition of RNA polymerase II activity. The regulation of a preexisting pool of mRNAs, therefore, plays a key role in DNA repair, cell cycle arrest, or inhibition of differentiation. THOC5 is a member of the THO complex and plays a role in the export of a subset of mRNA, which plays an important role in hematopoiesis and maintaining primitive cells. Since three serine residues in the PEST domain of THOC5 have been shown to be directly phosphorylated by ataxia-telangiectasia-mutated (ATM) kinase, we examined the THOC5-dependent mRNA export under DNA damage. We show here that DNA damage drastically decreased the cytoplasmic pool of a set of THOC5-dependent mRNAs and impaired the THOC5/mRNA complex formation. The mRNP complex formed with nonphosphorylation mutant (S307/312/314A) THOC5, but not with a C-terminal deletion mutant after DNA damage, suggesting that the C-terminal domain of THOC5, but not its phosphorylation in the PEST domain, is necessary for the regulation of the mRNA-binding potency of THOC5. The cytoplasmic THOC5-dependent mRNAs were recovered by treatment with ATM kinase-specific or p53-specific siRNA, as well as by treatment with ATM kinase inhibitor, KU55933, under DNA damage conditions, suggesting that the ATM-kinase-p53 pathway is involved in this response to the DNA damage. Furthermore, the treatment with KU55933 blocked DNA damage-induced THOC5mRNP complex dissociation, indicating that activation of ATM kinase suppresses the ability of THOC5 to bind to its target mRNAs.

Stem cells are defined by their ability to self-renew and to differentiate, the processes whereby these events are achieved is the subject of much investigation. These studies include cancer stem cell populations, where eradication of this specific population is the ultimate goal of treatment. Whilst cellular signalling events and transcription factor complex-mediated changes in gene expression have been analysed in some detail within stem cells, full systematic understanding of the events promoting self-renewal or the commitment process leading to formation of a specific cell type require a systems biology approach. This in turn demands a need for proteomic analysis of post-translational regulation of protein levels, protein interactions, protein post-translational modification (e.g. ubiquitination, methylation, acetylation, phosphorylation) to identify networks for stem cell regulation. Furthermore, the phenomenon of induced pluripotency via cellular reprogramming also can be understood optimally using combined molecular biology and proteomics approaches; here we describe current research employing proteomics and mass spectrometry to dissect stem cell regulatory mechanisms.

In clinical analyses, the most appropriate biofluid should be analyzed for optimal assay performance. For biological fluids, the most readily accessible is blood, and metabolomic analyses can be performed either on plasma or serum. To determine the optimal agent for analysis, metabolic profiles of matched human serum and plasma were assessed by gas chromatography/time-of-flight mass spectrometry and ultrahigh-performance liquid chromatography mass spectrometry (in positive and negative electrospray ionization modes). Comparison of the two metabolomes, in terms of reproducibility, discriminative ability and coverage, indicated that they offered similar analytical opportunities. An analysis of the variation between 29 small-cell lung cancer (SCLC) patients revealed that the differences between individuals are markedly similar for the two biofluids. However, significant differences between the levels of some specific metabolites were identified, as were differences in the intersubject variability of some metabolite levels. Glycerophosphocholines, erythritol, creatinine, hexadecanoic acid, and glutamine in plasma, but not in serum, were shown to correlate with life expectancy for SCLC patients, indicating the utility of metabolomic analyses in clinical prognosis and the particular utility of plasma in relation to the clinical management of SCLC.

Acute leukaemias are commonly caused by mutations that corrupt the transcriptional circuitry of haematopoietic stem/progenitor cells. However, the mechanisms underlying large-scale transcriptional reprogramming remain largely unknown. Here we investigated transcriptional reprogramming at genome-scale in mouse retroviral transplant models of acute myeloid leukaemia (AML) using both gene-expression profiling and ChIP-sequencing. We identified several thousand candidate regulatory regions with altered levels of histone acetylation that were characterised by differential distribution of consensus motifs for key haematopoietic transcription factors including Gata2, Gfi1 and Sfpi1/Pu.1. In particular, downregulation of Gata2 expression was mirrored by abundant GATA motifs in regions of reduced histone acetylation suggesting an important role in leukaemogenic transcriptional reprogramming. Forced re-expression of Gata2 was not compatible with sustained growth of leukaemic cells thus suggesting a previously unrecognised role for Gata2 in downregulation during the development of AML. Additionally, large scale human AML datasets revealed significantly higher expression of GATA2 in CD34+ cells from healthy controls compared with AML blast cells. The integrated genome-scale analysis applied in this study represents a valuable and widely applicable approach to study the transcriptional control of both normal and aberrant haematopoiesis and to identify critical factors responsible for transcriptional reprogramming in human cancer.

Leukaemic transformation is frequently associated with the aberrant activity of a protein tyrosine kinase (PTK). As such it is of clinical relevance to be able to map the effects of these leukaemogenic PTKs on haemopoietic cells at the level of phosphorylation modulation. In this paradigm study we have employed a range of proteomic approaches to analyse the effects of one such PTK, BCR/ABL. We have employed phosphoproteome enrichment techniques allied to peptide and protein quantification to identify proteins and pathways involved in cellular transformation. Amongst the proteins shown to be regulated at the post-translational level were cofilin, an actin-severing protein thus linked to altered motility and Cbl an E3 ubiquitin ligase integrally linked to the control of tyrosine kinase signalling (regulated by 5 and 6 PTKs respectively). The major class of proteins identified however were molecular chaperones. We also showed that HSP90 phosphorylation is altered by BCR/ABL action and that HSP90 plays a crucial role in oncogene stability. Further investigation with another six leukaemogenic PTKs demonstrates that this HSP90 role in oncogene stability appears to be a common phenomenon in a range of leukaemias. This opens up the potential opportunity to treat different leukaemias with HSP90 inhibitors.

BACKGROUND: The transcription/export complex is evolutionarily conserved from yeast to man and is required for coupled transcription elongation and nuclear export of mRNAs. FMIP(Fms interacting protein) is a member of the THO (suppressors of the transcriptional defects of hpr1delta by overexpression) complex which is a subcomplex of the transcription/export complex. THO complex (THOC) components are not essential for bulk poly (A)+ RNA export in higher eukaryotes, but for the nuclear export of subset of mRNAs, however, their exact role is still unclear. RESULTS: To study the role of THOC5/Fms interacting protein in vivo, we generated THOC5/Fms interacting protein knockout mice. Since these mice are embryonic lethal, we then generated interferon inducible conditional THOC5/Fms interacting protein knockout mice. After three poly injections all of the mice died within 14 days. No pathological alterations, however, were observed in liver, kidney or heart. Thus we considered the hematopoietic system and found that seven days after poly injection, the number of blood cells in peripheral blood decreased drastically. Investigation of bone marrow cells showed that these became apoptotic within seven days after poly injection. Committed myeloid progenitor cells and cells with long term reconstituting potential were lost from bone marrow within four days after poly injection. Furthermore, infusion of normal bone marrow cells rescued mice from death induced by loss of THOC5/Fms interacting protein. CONCLUSION: THOC5/Fms interacting protein is an essential element in the maintenance of hematopoiesis. Furthermore, mechanistically depletion of THOC5/Fms interacting protein causes the down-regulation of its direct interacting partner, THOC1 which may contribute to altered THO complex function and cell death.

Two distinct groups of chronic lymphocytic leukaemia (CLL) are distinguished by the presence or absence of somatic hypermutation of the immunoglobulin heavy-chain gene. CLL without somatic hypermutation has an adverse outcome, but the precise biological differences that underlie this more aggressive clinical-course are unclear. Using a proteomic approach, we found that the two prognostic forms of CLL were consistently distinguished according to their protein expression pattern. The most important difference observed related to the different expression of nucleophosmin 1 between the two forms of CLL. This different expression was not related to apoptosis, proliferation or gene mutation. However, co-immunoprecipitation experiments identified an association between nucleophosmin 1 and ribosomal proteins. Using immunocytofluorescence, nucleophosmin 1 expression was identified in the nucleoli and nucleoplasm of all cells, but in a proportion of cells, nucleophosmin had been transferred from the nucleoplasm to the cytoplasm. Both the fluorescent intensity, and the frequency of cytoplasmic nucleophosmin 1 expression, was higher in CLL without somatic hypermutation. We propose therefore, that nucleophosmin 1, in association with ribosomal proteins, undergoes nucleo-cytoplasmic shuttling in CLL. This process is most prominent in un-mutated CLL and may signify altered protein biosynthesis.