Transglutaminase (TGase) 2 is a ubiquitously expressed enzyme that modifies proteins by cross-linking or polyamination. An aberrant activity of TGase 2 has implicated its possible roles in a variety of diseases including age-related cataracts. However, the molecular mechanism by which TGase 2 is activated has not been elucidated. In this report, we showed that oxidative stress or UV irradiation elevates in situ TGase 2 activity. Neither the expression level nor the in vitro activity of TGase 2 appeared to correlate with the observed elevation of in situ TGase 2 activity. Screening a number of cell lines revealed that the level of TGase 2 activation depends on the cell type and also the environmental stress, suggesting that unrecognized cellular factor(s) may specifically regulate in situ TGase 2 activity. Concomitantly, we observed that human lens epithelial cells (HLE-B3) exhibited about 3-fold increase in in situ TGase 2 activity in response to the stresses. The activated TGase 2 catalyzed the formation of water-insoluble dimers or polymers of ␣B-crystallin, ␤B 2 -crystallin, and vimentin in HLE-B3 cells, providing evidence that TGase 2 may play a role in cataractogenesis. Thus, our findings indicate that in situ TGase 2 activity must be evaluated instead of in vitro activity to study the regulation mechanism and function of TGase 2 in biological and pathological processes.

SummaryThe core functions of stem cells (SCs) are critically regulated by their cellular redox status. Glutathione is the most abundant non-protein thiol functioning as an antioxidant and a redox regulator. However, an investigation into the relationship between glutathione-mediated redox capacity and SC activities is hindered by lack of probe. Here, we demonstrate that cyanoacrylamide-based coumarin derivatives are ratiometric probes suitable for the real-time monitoring of glutathione levels in living SCs. These probes revealed that glutathione levels are heterogeneous among subcellular organelles and among individual cells and show dynamic changes and heterogeneity in repopulating SCs depending on oxidative stress or culture conditions. Importantly, a subpopulation of SCs with high glutathione levels exhibited increased stemness and migration activities in vitro and showed improved therapeutic efficiency in treating asthma. Our results indicate that high glutathione levels are required for maintaining SC functions, and monitoring glutathione dynamics and heterogeneity can advance our understanding of the cellular responses to oxidative stress.

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Transglutaminase 2 (TGase 2) is one of a family of enzymes that catalyze protein modi®cation through the incorporation of polyamines into substrates or the formation of protein crosslinks. However, the physiological roles of TGase 2 are largely unknown. To elucidate the functions of TGase 2, we have searched for its interacting proteins. Here we show that TGase 2 interacts with E7 oncoprotein of human papillomavirus type 18 (HPV18) in vitro and in vivo. TGase 2 incorporates polyamines into a conserved glutamine residue in the zinc-binding domain of HPV18 E7 protein. This modi®cation mediates the inhibition of E7's Rb binding ability. In contrast, TGase 2 does not affect HPV16 E7, due to absence of a glutamine residue at this polyamination site. Using E7 mutants, we demonstrate that TGase 2-dependent inhibition of HPV E7 function correlates with the presence of the polyamination site. Our results indicate that TGase 2 is an important cellular interfering factor and de®ne a novel host±virus interaction, suggesting that the inability of TGase 2 to inactivate HPV16 E7 could explain the high prevalence of HPV16 in cervical cancer.

Cancer is a heterogeneous disease caused by diverse genomic alterations in oncogenes and tumor suppressor genes. Despite recent advances in high-throughput sequencing technologies and development of targeted therapies, novel cancer drug development is limited due to the high attrition rate from clinical studies. Patient-derived xenografts (PDX), which are established by the transfer of patient tumors into immunodeficient mice, serve as a platform for co-clinical trials by enabling the integration of clinical data, genomic profiles, and drug responsiveness data to determine precisely targeted therapies. PDX models retain many of the key characteristics of patients’ tumors including histology, genomic signature, cellular heterogeneity, and drug responsiveness. These models can also be applied to the development of biomarkers for drug responsiveness and personalized drug selection. This review summarizes our current knowledge of this field, including methodologic aspects, applications in drug development, challenges and limitations, and utilization for precision cancer medicine.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. Recent high-throughput analyses of genomic alterations revealed several driver genes and altered pathways in GC. However, therapeutic applications from genomic data are limited, largely as a result of the lack of druggable molecular targets and preclinical models for drug selection. To identify new therapeutic targets for GC, we performed array comparative genomic hybridization (aCGH) of DNA from 103 patients with GC for copy number alteration (CNA) analysis, and whole-exome sequencing from 55 GCs from the same patients for mutation profiling. Pathway analysis showed recurrent alterations in the Wnt signaling [APC, CTNNB1, and DLC1 (deleted in liver cancer 1)], ErbB signaling (ERBB2, PIK3CA, and KRAS), and p53 signaling/apoptosis [TP53 and BCL2L1 (BCL2-like 1)] pathways. In 18.4% of GC cases (19/103), amplification of the antiapoptotic gene BCL2L1 was observed, and subsequently a BCL2L1 inhibitor was shown to markedly decrease cell viability in BCL2L1-amplified cell lines and in similarly altered patient-derived GC xenografts, especially when combined with other chemotherapeutic agents. In 10.9% of cases (6/55), mutations in DLC1 were found and were also shown to confer a growth advantage for these cells via activation of Rho-ROCK signaling, rendering these cells more susceptible to a ROCK inhibitor. Taken together, our study implicates BCL2L1 and DLC1 as potential druggable targets for specific subsets of GC cases.gastric cancer | copy number alteration | whole-exome sequencing | patient-derived xenograft | druggable target G astric cancer (GC) is a highly prevalent malignancy and is the third leading cause of cancer-related deaths in the world (1). In unresectable and metastatic cases, the clinical outcome for this disease remains poor (median survival is 10-14 mo) (2), and other treatment options are often limited because of the lack of effective therapeutic approaches and molecular prognostic markers (2, 3). To date, with the exception of the application of trastuzumab [ERBB2 (ErbB2 receptor tyrosine kinase 2) antagonist] or ramucirumab [VEGFR2 (Vascular endothelial growth factor receptor 2) antagonist] for advanced GC cases (4, 5), drugs that target GC on a molecular level are limited.Recent genomic studies have demonstrated the heterogeneous genomic characteristics of GC (6-10). In addition, previous studies of patients with GC by whole-genome and whole-exome sequencing (WES) have identified frequent somatic mutations in tumor suppressors such as TP53, ARID1A, APC, and FAT4, and oncogenes including PI3KCA, KRAS, and RHOA (6-10). However, these findings, although academically meaningful, are far from ready for clinical applications, largely because of the lack of identified druggable molecular targets and the availability of reliable preclinical models for validation of potential target inhibitors.To identify novel therapeutic targets for GC, we explored genomic alterations in GC through an integrated genomic data set from WE...

Sprouty (Spry) is known to be a negative feedback inhibitor of growth factor receptor signaling through inhibition of the Ras/MAPK pathway. Several groups, however, have reported a positive role for Spry involving sequestration of the inhibitory protein c-Cbl. Thus, Spry may have various functions in the regulation of receptor-mediated signaling depending on the context. In the immune system, the function of Spry is unknown. In this study, we investigated the role of Spry1 in T cell activation. Spry1, among the four mammalian homologs, was specifically induced by TCR signaling of CD4+ murine T cells. In fully differentiated Th1 clones, overexpressed Spry1 inhibited TCR signaling and decreased IL-2 production while reducing expression with specific siRNA transfection had the opposite effect, increasing IL-2 production. In contrast, in naive T cells, Spry1 overexpression enhanced TCR signaling, and increased proliferation and IL-2 production, while siRNA transfection again had the opposite effect, reducing IL-2 production following activation. The enhancing effect in naive cells was abrogated by preactivation of the T cells with Ag and APC, indicating that the history of exposure to Ag is correlated with a hierarchy of T cell responsiveness to Spry1. Furthermore, both the NF-AT and MAPK pathways were influenced by Spry1, implying a different molecular mechanism from that for growth factor receptor signaling. Thus, Spry1 uses a novel mechanism to bring about differential effects on TCR signaling through the same receptor, depending on the differentiation state of the T cell.

Polyamine incorporation or cross-linking of proteins, post-translational modi¢cations mediated by transglutaminase 2 (TGase 2), have been implicated in a variety of physiological functions including cell adhesion, extracellular matrix formation and apoptosis. To better understand the intracellular regulation mechanism of TGase 2, the properties of biotinylated polyamines as substrates for determining in situ TGase activity were analyzed. We synthesized biotinylated spermine (BS), and compared the in vitro and in situ incorporation of BS with that of biotinylated pentylamine (BP), which is an arti¢cial polyamine derivative. When measured in vitro, BP showed a signi¢cantly higher incorporation rate than BS. In contrast, in situ incorporation of both BS and BP was not detected even in TGase 2-overexpressed 293 cells. Cells exposed to high calcium showed a marked increase of BP incorporation but not of BS. These data indicate that the in situ activity of TGase 2 gives di¡erent results with di¡erent substrates, and suggest the possibility of overrepresentation of in situ TGase 2 activity when assayed with BP. Therefore, careful interpretation or evaluation of in situ TGase 2 activity may be required. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.