Homing of bone marrow stromal cells (MSCs) to bone and bone marrow after transplantation, important for the correction of conditions such as metabolic storage disorders, can occur but with poor efficiency. Substantial improvements in engraftment will be required in order to derive a clinical benefit from MSC transplantation. Chemokines are the most important factors controlling cellular migration. Stromal-derived factor-1 (SDF-1) has been shown to be critical in promoting the migration of cells to the bone marrow, via its specific receptor CXCR4. The aim of our study was to investigate CXCR4 expression on MSCs and its role in mediating migration to bone marrow. We show that CXCR4, although present at the surface of a small subset of MSCs, is important for mediating specific migration of these cells to bone marrow. 4 Clinically, MSCs may be used to enhance HSC engraftment after transplantation, to correct inherited disorders of bone and cartilage, or as vehicles for gene therapy. 5,6 Crucial to the success of any of these strategies is efficient engraftment to bone and bone marrow. So far results have shown that although MSCs can engraft to those tissues, levels are at the limit of detection and clinically useful only in certain disorders such as osteogenesis imperfecta. 7 Higher levels will be required to achieve a therapeutic benefit in the majority of applications.The chemokine stromal-derived factor-1 (SDF-1) and its ligand CXCR4 play an important role in homing as shown by studies on engraftment of hematopoietic stem/progenitor cells 8 and on colonization of bone and bone marrow by metastatic breast and prostate cancer cells. 9 We have examined the expression of CXCR4 by MSCs and the migration of these cells to bone marrow stroma. Here we show for the first time that CXCR4 is important for MSC migration to bone marrow. However cell surface receptor levels are low, with large amounts found intracellularly. Study design Isolation of MSC cultures and CD34 ؉ cellsBone marrow aspirates were obtained, under local ethical approval, from the posterior iliac crest of donors aged 0 to 18 years after parental consent.MSCs were isolated by plastic adherence as previously described 10 and cultured in Dulbecco modified Eagle medium (DMEM; Invitrogen, Paisley, United Kingdom) ϩ 10% fetal calf serum (FCS; Stem Cell Technologies, Vancouver, BC). CD34 ϩ cells were isolated using the Minimacs system according to the manufacturer's instruction (Miltenyi Biotech, Bisley, United Kingdom) Reverse-transcription-polymerase chain reaction (RT-PCR)Total RNA from MSCs or peripheral blood mononuclear cells (PBMCs), the latter activated with 10 g/mL phytohemagglutinin (PHA; Abbott Murex, Dartford, United Kingdom), was extracted using RNAzol (AMS Biotechnology, Abington, United Kingdom) according to the manufacturer's instructions, then reverse transcribed using a First Strand cDNA Synthesis kit (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). cDNA was analyzed by PCR for CXCR4 using 2 sets of primers: 668-bp amplicon forwa...

There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL, TEL/PDGFR␤, FIP1/PDGFR␣, D816V KIT, NPM/ALK, and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFR␣. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin, although this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes, illustrating the utility of such a combined approach. Molecular & Cellular Proteomics 7: 853-863, 2008.

A calcium-binding soluble protein extracted from oyster shell suppresses calcium carbonate nucleation and decreases the rate of crystal growth in vitro. These findings suggest that soluble matrix may regulate shell growth.

The proteome is determined by rates of transcription, translation, and protein turnover. Definition of stem cell populations therefore requires a stem cell proteome signature. However, the limit to the number of primary cells available has restricted extensive proteomic analysis. We present a mass spectrometric method using an isobaric covalent modification of peptides for relative quantification (iTRAQ) , IntroductionDefinition of cell form and function is derived from the transcription of specific sets of genes, followed by their translation and post-translational regulation. Cellular development alters phenotype via such changes in transcription and translation, and in this respect post-translational control of protein levels is key in many cell regulatory pathways, such as cell cycling. 1 Stem cell commitment to differentiation is a critical step in development, where proteomic changes will be observed as a cell undergoes successive commitment and developmental steps. The precise nature of the elements of gene expression that contribute to stem cell characteristics have been assessed by transcriptional profiling and the elements of a stem cell signature refined via comparative expression analyses. [2][3][4] Alternatively, the critical stem cell characteristics can be defined by analysis of the function of specific genes (in the hematopoietic system, SCL, HOX-B4, and BMI-1 5-7 ). Many key regulators are transcriptional activators or suppressors that will inevitably affect the transcriptomic profile of a cell. However, this process is known to be subject to further regulation via post-translational mechanisms. 8,9 Lin Ϫ Sca ϩ Kit ϩ (LSK ϩ ) cells and Lin Ϫ Sca ϩ Kit Ϫ (LSK Ϫ ) cells display differential abilities to reconstitute hematopoiesis, and the latter is a more mature cell type than the former, only able to support hematopoiesis in the short term. 10,11 Comparison of these 2 cell populations can help us derive knowledge of the intracellular systems that regulate the ability to maintain pluripotency and long-term self-renewal. Transcriptomic analysis, previously used to define a stem cell "signature," will not detail the true differences between stem cells and their progeny at the proteome level because of the importance of post-translational regulation of protein levels. 9 We therefore sought to establish a method for defining the proteome of stem cell populations where only limited sample is available. Furthermore, the method should not rely on transcriptome data to infer changes at the protein level. Given the limited material (approximately 1 million LSK ϩ and LSK Ϫ cells, respectively, per experiment), we developed a procedure employing isobaric tags for relative quantification (iTRAQ). This allows 4 samples to be analyzed simultaneously, giving relative quantification 12,13 on hundreds of proteins at any one time. Samples are separately proteolytically digested, and 4 isotope-coded, isobaric reagents (using differential stable isotope distribution between reporter group and balance group moieties) ...

In addition to its role in bone turnover, osteoprotegerin (OPG) has been reported to bind to and inhibit Tumour necrosis factor-related apoptosis inducing ligand (TRAIL). TRAIL is produced in tumours by invading monocytes, inducing apoptosis in neoplastic cells sensitive to this cytokine. OPG production by tumour cells would therefore be a novel mechanism whereby cancer cells evade host defences and gain a growth advantage. In this study we show that OPG produced by breast cancer cells enhances tumour cell survival by inhibiting TRAIL-induced apoptosis. OPG expression by breast cancer cells (MDA-MB 436/231) grown in vitro was examined using PCR and ELISA, and the sensitivity of these cells to TRAIL was determined. The effects of OPG on TRAIL induced apoptosis was investigated by exposing MDA-MB 436 cells to TRAIL, in the presence or absence of OPG, followed by assessment of nuclear morphology. We found that the levels of OPG produced were sufficient to inhibit TRAIL-induced apoptosis, suggesting that OPG may play a role in tumour cell survival. We also examined the expression pattern of OPG in a selection of breast tumours (n=400) by immunohistochemistry, and related OPG expression to the clinico-pathological data for each tumour. OPG expression was found to be negatively correlated with increasing tumour grade. To our knowledge these results are the first to demonstrate that OPG can act as an endocrine survival factor for breast cancer cells, as well as reporting the expression patterns of OPG in a large cohort of human breast tumours.

Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression.

Lineage-marker depleted (Lin ؊ ) murine bone marrow cells expressing stem cell antigen 1 (Sca-1) were sorted on the basis of stem cell factor receptor (c-kit) expression to obtain Lin ؊ Sca ؉ Kit ؉ or Lin ؊ Sca ؉ Kit ؊ cells. Lin ؊ Sca ؉ Kit ؊ cells have a markedly greater chemotactic response to stromal derived factor-1 (SDF-1). Using a novel fluorescent stain, we show that both populations generate similar levels of a key messenger, phosphatidylinositol 3,4,5 trisphosphate (PIP 3 ), in response to SDF-1. Differences in motile behavior may therefore lie downstream of phosphatidylinositol 3-kinase (PI3-kinase) activation at the level of cytoskeleton regulation. The 2 cell populations were compared using 2-dimensional difference gel electrophoresis (2D-DIGE), with a maleimide CyDye fluorescent protein labeling technique that has enhanced sensitivity for low abundance samples. Comparative proteomic analysis of Cy3-and Cy5-labeled protein samples allows relative quantification of protein spots present in both cell populations; of these, 73% were common. Key protein differences were adseverin and gelsolin, actin microfilament splicing proteins, regulated by Rac, downstream of PI3-kinase activation. Adseverin was shown to be acetylated, a novel modification for this protein. Differences in major regulators of cell shape and motility between the 2 populations can explain the differential response to SDF-1. IntroductionHematopoietic stem cells have the capacity to self-renew and also to differentiate to form a number of mature cell types with distinct morphologies and functional activities. The distinctive features of the stem cell that give it longevity and the ability to balance its self-renewal and differentiation to the required degree in any circumstance remain unclear. These biologic properties are encoded in the transcriptional profile, or transcriptome, of stem cells. The properties of a stem cell can therefore be defined in terms of the gene expression profiles of stem cells. Retention of stem cell characteristics will rely in part on the cohort of genes specifically expressed in these primitive cells, but not in their maturing progeny. This axiom has led to transcriptional analysis of long-and short-term repopulating stem cells and other primitive populations (defined via expression of specific markers) to define the genes expressed for self-renewal and pluripotency. 1,2 The generation of these stem cell "blueprints" then offers the opportunity to investigate the functional activity of the proteins produced as a consequence of gene expression and to systematically study gene networks controlling stem cell biology.The relationship between messenger RNA production and protein synthesis is not, however, strictly correlative. 3 In a range of different organisms it has become clear that changes in the expression of a specific protein do not necessarily relate directly to altered transcription. Recent developments in protein separation and relative quantification allow populations of cells to be compared at the pr...

Butyrate is a critical cancer-preventive element in the colon milieu whose mechanism of action is unclear, but appears to be mediated through inhibition of histone deacetylases (HDACs) and consequent alterations in global protein acetylation. Cytokeratins (CKs) have roles in cytoskeletal function as components of the intermediate filaments (IFs) and this involves CKs in the regulation of tissue homeostasis of high-turnover epithelia such as the colon. We used a 2-D gel/MS analysis to characterise the proteome of IFs, and a novel monitoring-initiated detection and sequencing (MIDAS) approach to identify acetylation sites on principal proteins. We report that CKs are highly acetylated in a colon cancer cell line, with five acetylation sites characterised on CK8 and a further one on CK18. Acetylation of CK8 is responsive to butyrate. HDAC5 is the deacetylase associated with IFs. These data indicate a novel action of butyrate as a cancer preventive agent. Acetylation of CK8 may be associated with IFs stabilisation and thereby provide a candidate mechanism for the appropriate retention or loss of epithelial cells from the flat mucosa.