Insulin-producing cells derived from 'induced pluripotent stem cells' of patients with fulminant type 1 diabetes: Vulnerability to cytokine insults and increased expression of apoptosis-related genes.

AIMS/INTRODUCTION: The present study was carried out to generate induced pluripotent stem cells (iPSCs) from patients with fulminant type 1 diabetes, and evaluate the cytokine-induced apoptotic reactions of ß-like insulin-producing cells differentiated from the iPSCs. MATERIALS AND METHODS: iPSCs were generated from fibroblasts of patients with fulminant type 1 diabetes by inducing six reprogramming factors. Insulin-producing cells were differentiated from the iPSCs in vitro. The proportion of cleaved caspase-3-positive or terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling-positive cells among insulin (INS)-positive cells derived from fulminant type 1 diabetes iPSC and control human iPSC lines was evaluated under treatment with tumor necrosis factor-α, interleukin-1ß and interferon-γ. Ribonucleic acid sequencing was carried out to compare gene expressions in INS-positive cells derived from fulminant type 1 diabetes iPSC and control human iPSC lines. RESULTS: Two iPSC clones were established from each of three patients with fulminant type 1 diabetes. The differentiation of insulin-producing cells from fulminant type 1 diabetes iPSC was confirmed by immunofluorescence analysis and KCl-induced C-peptide secretion. After treatment with pro-inflammatory cytokines, these INS-positive cells showed higher expression of cleaved caspase-3 than those derived from control human iPSCs. Altered expression levels of several apoptosis-related genes were observed in INS-positive cells derived from the fulminant type 1 diabetes iPSCs by ribonucleic acid sequencing. CONCLUSIONS: We generated iPSCs from patients with fulminant type 1 diabetes and differentiated them into insulin-producing cells. This in vitro disease model can be used to elucidate the disease mechanisms of fulminant type 1 diabetes.

Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe.

Interactions between ATP and ATP-binding proteins (ATPome) are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes) that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014) [1]. As a result, we identified 539 proteins, including 178 novel ATP-binding protein candidates. We also established an ATPome selectivity profiling method for kinase inhibitors using our cataloged ATPome list. Normally only kinome selectivity is profiled in selectivity profiling of kinase inhibitors. In this data, we expand the profiled targets from the kinome to the ATPome through performance of ATPome selectivity profiling and obtained target profiles of staurosporine and (S)-crizotinib. The data accompanying the manuscript on this approach (Adachi et al., 2014) [1] have been deposited to the ProteomeXchange with identifier PXD001200.

Differential Proteome Analysis Identifies TGF-ß-Related Pro-Metastatic Proteins in a 4T1 Murine Breast Cancer Model.

Transforming growth factor-ß (TGF-ß) has a dual role in tumorigenesis, acting as either a tumor suppressor or as a pro-oncogenic factor in a context-dependent manner. Although TGF-ß antagonists have been proposed as anti-metastatic therapies for patients with advanced stage cancer, how TGF-ß mediates metastasis-promoting effects is poorly understood. Establishment of TGF-ß-related protein expression signatures at the metastatic site could provide new mechanistic information and potentially allow identification of novel biomarkers for clinical intervention to discriminate TGF-ß oncogenic effects from tumor suppressive effects. In the present study, we found that systemic administration of the TGF-ß receptor kinase inhibitor, SB-431542, significantly inhibited lung metastasis from transplanted 4T1 mammary tumors in Balb/c mice. The differentially expressed proteins in the comparison of lung metastases from SB-431542 treated and control vehicle-treated groups were analyzed by a quantitative LTQ Orbitrap Velos system coupled with stable isotope dimethyl labeling. A total of 36,239 peptides from 6,694 proteins were identified, out of which 4,531 proteins were characterized as differentially expressed. A subset of upregulated proteins in the control group was validated by western blotting and immunohistochemistry. The eukaryotic initiation factor (eIF) family members constituted the most enriched protein pathway in vehicle-treated compared with SB-43512-treated lung metastases, suggesting that increased protein expression of specific eIF family members, especially eIF4A1 and eEF2, is related to the metastatic phenotype of advanced breast cancer and can be down-regulated by TGF-ß pathway inhibitors. Thus our proteomic approach identified eIF pathway proteins as novel potential mediators of TGF-ß tumor-promoting activity.

Transcriptional Dynamics of Immortalized Human Mesenchymal Stem Cells during Transformation.

Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN); these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma) and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5), which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1), the cell surface receptor for hedgehog (Hh) signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells) through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional contribution of genes showed by expression profiling to the neoplastic transformation of human fibroblasts and human mesenchymal stem cells (hMSC).

Proteome-wide discovery of unknown ATP-binding proteins and kinase inhibitor target proteins using an ATP probe.

ATP-binding proteins, including protein kinases, play essential roles in many biological and pathological processes and thus these proteins are attractive as drug targets. Acyl-ATP probes have been developed as efficient probes for kinase enrichment, and these probes have also been used to enrich other ATP-binding proteins. However, a robust method to identify ATP-binding proteins with systematic elimination of nonspecific binding proteins has yet to be established. Here, we describe an ATP competition assay that permitted establishment of a rigorous ATP-binding protein list with virtual elimination of nonspecific proteins. A total of 539 ATP-binding protein candidates were identified, including 178 novel candidates. In informatics analysis, ribosomal proteins were overrepresented in the list of novel candidates. We also found multiple ATP-competitive sites for several kinases, including epidermal growth factor receptor, serine/threonine-protein kinase PRP4 homologue, cyclin-dependent kinase 12, eukaryotic elongation factor 2 kinase, ribosomal protein S6 kinase alpha-1, and SRSF protein kinase 1. Using our cataloged ATP-binding protein list, a selectivity profiling method that covers the kinome and ATPome was established to identify off-target binding sites of ATP-competitive kinase inhibitors, staurosporine and crizotinib.