Total Proteome Analysis Identifies Migration Defects as a Major Pathogenetic Factor in Immunoglobulin Heavy Chain Variable Region (IGHV)-unmutated Chronic Lymphocytic Leukemia

The mutational status of the immunoglobulin heavy chain variable region defines two clinically distinct forms of chronic lymphocytic leukemia (CLL) known as mutated (M-CLL) and unmutated (UM-CLL). To elucidate the molecular mechanisms underlying the adverse clinical outcome associated with UM-CLL, total proteomes from nine UM-CLL and nine M-CLL samples were analyzed by isobaric tags for relative and absolute quantification (iTRAQ)-based mass spectrometry. Based on the expression of 3521 identified proteins, principal component analysis separated CLL samples into two groups corresponding to immunoglobulin heavy chain variable region mutational status. Computational analysis showed that 43 cell migration/adhesion pathways were significantly enriched by 39 differentially expressed proteins, 35 of which were expressed at significantly lower levels in UM-CLL samples. Furthermore, UM-CLL cells underexpressed proteins associated with cytoskeletal remodeling and overexpressed proteins associated with transcriptional and translational activity. Taken together, our findings indicate that UM-CLL cells are less migratory and more adhesive than M-CLL cells, resulting in their retention in lymph nodes, where they are exposed to proliferative stimuli. In keeping with this hypothesis, analysis of an extended cohort of 120 CLL patients revealed a strong and specific association between UM-CLL and lymphadenopathy. Our study illustrates the potential of total proteome analysis to elucidate pathogenetic mechanisms in cancer

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes

Background & Aims: Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Methods: Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. Results: HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. Conclusions: HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes

Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile

© 2016 The Author(s)The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems

Analysis of the rat primary hepatocyte nuclear proteome through sub-cellular fractionation

Characterising primary hepatocytes and their de-differentiation in culture is vital for the refinement of current culture techniques and for the development of new and improved in vitro hepatocyte models. We have performed multiplexed iTRAQ proteomics on whole cell preparations and further employed nuclear fractionation to expand the coverage of this important organelle. We identify many proteins that change in abundance during culture of rat hepatocytes for 48h and map their molecular functions. 431 proteins were identified and quantified in whole cell homogenates, mapping to 69 molecular functions using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system. In whole cell homogenates liver-associated functions, such as oxidoreductase activity, were enriched compared with the reference rat proteome dataset but some functions, such as transcriptional activity, were under-represented. Nuclear fractionation resulted in the identification of an additional 156 proteins which mapped to 31 molecular functions. These proteins included some associated with hepatic differentiation, such as HNF4alpha and CCAAT/enhancer-binding protein beta and others with less well-defined roles.   Hierarchical clustering of samples within each experiment showed segregation of fresh and cultured sample types and stringent statistical analysis demonstrated significant changes in 36% of proteins from the whole cell homogenates and 21% of proteins from the nuclear dataset (adjusted p valu