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A major hurdle in stem cell therapy is the tumorigenic risk of residual undifferentiated stem cells. This report describes the design and evaluation of synthetic hybrid molecules that efficiently reduce the number of human induced pluripotent stem cells (hiPSCs) in cell mixtures. The design takes advantage of Kyoto probe 1 (KP-1), a fluorescent chemical probe for hiPSCs, and clinically used anticancer drugs. Among the KP-1-drug conjugates we synthesized, we found an exceptionally selective, chemically tractable molecule that induced the death of hiPSCs. Mechanistic analysis suggested that the high selectivity originates from the synergistic combination of transporter-mediated efflux and the cytotoxicity mode of action. The present study offers a chemical and mechanistic rationale for designing selective, safe, and simple reagents for the preparation of non-tumorigenic clinical samples.

SummaryA small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

Background and Aim: Transglutaminase 2 (TG2), catalyzing crosslinking between lysine and glutamine residues, is involved in many liver diseases. We previously reported that TG2, induced in the nucleus of ethanol- or free fatty acids (FFAs)-treated hepatic cells, crosslinks and inactivates a transcription factor Sp1, leading to reduced expression of c-Met and thereby caspase independent hepatic apoptosis in culture systems, animal models, and both alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH) patients. FFAs increase endoplasmic reticulum (ER) stress, NFkB activation and nuclear TG2 (nTG2) through pancreatic ER kinase (PERK)-dependent pathway, whereas ethanol induces nTG2 via retinoid signaling. However, the molecular mechanism by which ethanol/FFAs induce nuclear localization of TG2 has been unclear. Method: A similar nTG2-mediated cell death is induced in acyclic retinoid (ACR)-treated hepatocellular carcinoma. Using cultured cells, we investigated how to control this novel apoptotic pathway by regulating nuclear localization of TG2. Results: TG2 is composed of N-terminal b-sandwich, catalytic core, b-barrel 1, and C-terminal b-barrel 2 domains. In a previous work, we identified a 14 amino acid nuclear localization signal (NLS) within the b-barrel 1 domain and a putative leucine-rich nuclear export signal (NES) at position 657 to 664 (LHMGLHKL) near the C-terminus in the b-barrel 2 domain, and found that ACR downregulated exportin-1 levels, thereby accumulation of TG2 in the nucleus. Here, we found that both ethanol and FFAs provoked generation of truncated short form of TG2 (TG2-S) defects in the putative NES at least in part through alternative splicing, thereby causing accumulation of TG2-S in the nucleus. Conclusion: The generation of TG2-S in ethanol or FFAs-treated hepatic cells is a novel therapeutic target for prevention of hepatic cell death associated with ASH/NASH.

Non-alcoholic steatohepatitis (NASH), a progressive form of fatty liver, shares histological similarities with alcoholic steatohepatitis (ASH), including accumulated fat, hepatic apoptosis, and fibrous tissues in the liver, but the molecular mechanisms responsible for hepatic apoptosis remain unclear. We previously reported that transglutaminase 2 (TG2), induced in the nuclei of ethanol-treated hepatocytes, crosslinks and inactivates the transcription factor Sp1, leading to hepatic apoptosis. In this study, we investigated whether a similar change is involved in NASH, and if so, how TG2 and crosslinked Sp1 (CLSp1) are induced. Elevated nuclear TG2 and CLSp1 formation was demonstrated in NASH patients, as well as increased activation of apoptosis inducing factor (AIF) and release of cytochrome c. In Hc human normal hepatocytes treated with free fatty acids (FFAs), biochemical analyses revealed that ethanol and FFAs provoked fat accumulation, endoplasmic reticulum (ER) stress, increased nuclear factor kappa B (NFκB), and nuclear TG2. Salubrinal, a selective inhibitor of the ER stress-induced pancreatic ER kinase (PERK) signaling pathway, inhibited NFκB activation, nuclear TG2 expression, and apoptosis only if it was induced by FFAs, but not by ethanol. These results suggest that FFAs could increase ER stress and lead to nuclear NFκB activation and TG2 induction through PERK-dependent pathways, resulting in TG2-mediated apoptosis accompanying crosslinking and inactivation of Sp1, activation of AIF, and release of cytochrome c.

Transglutaminase 2 (TG2; EC 2.3.2.13) is the most abundantly expressed member of the transglutaminase family and exerts opposing effects on cell growth, differentiation and apoptosis via multiple activities, including transamidase, GTPase, cell adhesion, protein disulfide isomerase, kinase and scaffold activities. It is distributed in and around various parts of a cell, including the extracellular matrix, plasma membrane, cytosol, mitochondria and nucleus. Generally, nuclear TG2 represents only 5–7% of the total TG2 in a cell, and various stimuli will increase nuclear TG2 via cellular stress and/or an increased intracellular Ca2+ concentration. There is increasing evidence indicating the importance of nuclear TG2 in regulating gene expression via post-translational modification of (or interaction with) transcriptional factors and related proteins. These include E2F1, hypoxia inducible factor 1, Sp1 and histones. Through this mechanism, TG2 controls cell growth or survival, differentiation and apoptosis, and is involved in the pathogenesis and/or treatment of neurodegenerative diseases, liver diseases and cancers. The balance between import from the cytoplasm to the nucleus, and export from the nucleus to the cytoplasm, determines the level of TG2 in the nucleus. Selective regulation of the expression, activity or localization of nuclear TG2 will be important for basic research, as well as clinical applications, suggesting a new era for this long-studied enzyme.

Glucose-regulated protein 78 (Grp78) is an endoplasmic reticulum chaperone protein and is overexpressed in various cancers. However, it is unclear how significance of this molecule play an active role contributing to the oncogenic effect of head and neck cancer (HNC). To investigate the potential function of Grp78, six HNC cell lines were used. We found that Grp78 is highly expressed in all six cell lines and many of the proteins were localized in the periphery regions, implying other function of this molecule aside from endoplasmic reticulum stress response. Knockdown of Grp78 by small interfering RNA significantly reduced cell growth and colony formation to 53% to 12% compared with that of controls in all six HNC cell lines. Using in vitro wound healing and Matrigel invasion assays, we found that cell migration and invasive ability were also inhibited to 23% to 2% in all these cell lines tested. In vivo xenograft studies showed that administration of Grp78-small interfering RNA plasmid into HNC xenografts significantly inhibited both tumor growth in situ (>60% inhibition at day 34) and liver metastasis (>90% inhibition at day 20). Our study showed that Grp78 actively regulates multiple malignant phenotypes, including cell growth, migration, and invasion. Because knockdown Grp78 expression succeeds in the reduction of tumor growth and metastatic potential, this molecule may serve as a molecular target of therapeutic intervention for HNC. [Mol Cancer Ther 2008;7(9):2788–97]

One of the current obstacles to stem cell therapy is the tumorigenic potential of residual undifferentiated stem cells. The present study reports rediscovery of a synthetic derivative of okadaic acid, a marine polyether toxin, as a reagent that selectively induces the death of human pluripotent stem cells. Cell-based screening of 333 cytotoxic compounds identified methyl 27-deoxy-27-oxookadaate (molecule 1) as a substrate of two ATP-binding cassette (ABC) transporters, ABCB1 (MDR1) and ABCG2 (BCRP), whose expression is repressed in human embryonic stem cells and induced pluripotent stem cells. The results demonstrate that selective elimination of human pluripotent stem cells can be achieved by designing cytotoxic small molecules with appropriate ABC-transporter selectivity.

Transglutaminase 2 (TG2), catalyzing crosslinking between lysine and glutamine residues, is involved in many liver diseases. We previously reported that TG2, induced in the nucleus of ethanol- or free fatty acids (FFAs)-treated hepatic cells, crosslinks and inactivates a transcription factor Sp1, leading to reduced expression of c-Met and thereby caspase independent hepatic apoptosis in culture systems, animal models, and both alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH) patients. FFAs increase endoplasmic reticulum (ER) stress, NFkB activation and nuclear TG2 (nTG2) through pancreatic ER kinase (PERK)-dependent pathway, whereas ethanol induces nTG2 via retinoid signaling. However, the molecular mechanism by which ethanol/FFAs induce nuclear localization of TG2 has been unclear.A similar nTG2-mediated cell death is induced in acyclic retinoid (ACR)-treated hepatocellular carcinoma. Using cultured cells, we investigated how to control this novel apoptotic pathway by regulating nuclear localization of TG2.TG2 is composed of N-terminal b-sandwich, catalytic core, b-barrel 1, and C-terminal b-barrel 2 domains. In a previous work, we identified a 14 amino acid nuclear localization signal (NLS) within the b-barrel 1 domain and a putative leucine-rich nuclear export signal (NES) at position 657 to 664 (LHMGLHKL) near the C-terminus in the b-barrel 2 domain, and found that ACR downregulated exportin-1 levels, thereby accumulation of TG2 in the nucleus. Here, we found that both ethanol and FFAs provoked generation of truncated short form of TG2 (TG2-S) defects in the putative NES at least in part through alternative splicing, thereby causing accumulation of TG2-S in the nucleus.The generation of TG2-S in ethanol or FFAs-treated hepatic cells is a novel therapeutic target for prevention of hepatic cell death associated with ASH/NASH.

Transglutaminase 2 (TG2; EC 2.3.2.13) is the most abundantly expressed member of the transglutaminase family and exerts opposing effects on cell growth, differentiation and apoptosis via multiple activities, including transamidase, GTPase, cell adhesion, protein disulfide isomerase, kinase and scaffold activities. It is distributed in and around various parts of a cell, including the extracellular matrix, plasma membrane, cytosol, mitochondria and nucleus. Generally, nuclear TG2 represents only 5-7% of the total TG2 in a cell, and various stimuli will increase nuclear TG2 via cellular stress and/or an increased intracellular Ca(2+) concentration. There is increasing evidence indicating the importance of nuclear TG2 in regulating gene expression via post-translational modification of (or interaction with) transcriptional factors and related proteins. These include E2F1, hypoxia inducible factor 1, Sp1 and histones. Through this mechanism, TG2 controls cell growth or survival, differentiation and apoptosis, and is involved in the pathogenesis and/or treatment of neurodegenerative diseases, liver diseases and cancers. The balance between import from the cytoplasm to the nucleus, and export from the nucleus to the cytoplasm, determines the level of TG2 in the nucleus. Selective regulation of the expression, activity or localization of nuclear TG2 will be important for basic research, as well as clinical applications, suggesting a new era for this long-studied enzyme.

Alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH) share many histological similarities, but the molecular mechanisms responsible for hepatic apoptosis remain unclear. We previously reported that transglutaminase 2 (TG2), a protein cross-linking enzyme, is induced in the nucleus of ethanol-treated hepatocytes, and cross-links and inactivates a general transcription factor Sp1, which eventually leads to reduced expression of c-Met and caspase-independent hepatic apoptosis [Tatsukawa et al., Gastroenterology 2009;136:1783–1795]. In this study, we investigated if a similar change might be observed also in NASH and if yes how TG2 and cross-linked Sp1 (CLSp1) would be induced in NASH and ASH. We obtained elevated nuclear TG2 and CLSp1 formation in NASH patients, as well as in HepG2 cells treated with free fatty acids (FFAs). Biochemical analyses on this culture model revealed that both ethanol and FFAs provoked fat accumulation, endoplasmic reticulum (ER) stress, increased nuclear factor-ĸB (NFĸB) and nuclear TG2, but the synergistic effect was not obvious between FFA and ethanol. Salubrinal, a selective inhibitor against dephosphorylation of eukaryotic initiation factor-2α in ER stress-induced pancreatic ER kinase (PERK) signal pathway, inhibited NFĸB activation, nuclear TG2 expression and apoptosis only induced by FFAs, but not those induced by ethanol, while retinoid antagonist blocks ethanol induction of NFĸB and TG2. These results suggest that FFA and ethanol may increase ER stress and lead to nuclear NFĸB activation and TG2 induction through respectively distinctive pathways, leading to TG2-mediated apoptosis via cross-linking and inactivation of Sp1 and reduction in c-Met.

Background & Aims Despite high morbidity and mortality of alcoholic liver disease worldwide, the molecular mechanisms underlying alcohol-induced liver cell death are not fully understood. Transglutaminase 2 (TG2) is a cross-linking enzyme implicated in apoptosis. TG2 levels and activity are increased in association with various types of liver injury. However, how TG2 induces hepatic apoptosis is not known. Methods Human hepatic cells or primary hepatocytes from rats or TG2 +/+ and TG2 −/− mice were treated with ethanol. Mice were administered anti-Fas antibody or alcohol. Liver sections were prepared from patients with alcoholic steatohepatitis. Changes in TG2 levels, Sp1 cross-linking and its activities, expression of hepatocyte growth factor receptor, c-Met, and hepatic apoptosis were measured. Results Ethanol induced apoptosis in hepatic cells, enhanced activity and nuclear accumulation of TG2 as well as accumulation of cross-linked and inactivated Sp1, and reduced expression of the Sp1-responsive gene, c-Met . These effects were rescued by TG2 knockdown, restoration of functional Sp1, or addition of hepatocyte growth factor, whereas apoptosis was reproduced by Sp1 knockdown or TG2 overexpression. Compared with TG2 +/+ mice, TG2 −/− mice showed markedly reduced hepatocyte apoptosis and Sp1 cross-linking following ethanol or anti-Fas treatment. Treatment of TG2 +/+ mice with the TG2 inhibitors putrescine or cystamine blocked anti-Fas–induced hepatic apoptosis and Sp1 silencing. Moreover, enhanced expression of cross-linked Sp1 and TG2 was evident in hepatocyte nuclei of patients with alcoholic steatohepatitis. Conclusions TG2 induces hepatocyte apoptosis via Sp1 cross-linking and inactivation, with resultant inhibition of the expression of c-Met required for hepatic cell viability.