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1
Lu, Peace Wun-Ang, Chia-Hsuan Chou, Jia-Sin Yang, Yi-Hsien Hsieh, Meng-Ying Tsai, Ko-Hsiu Lu, and Shun-Fa Yang. "HO-3867 Induces Apoptosis via the JNK Signaling Pathway in Human Osteosarcoma Cells." Pharmaceutics 14, no. 6 (June 13, 2022): 1257. http://dx.doi.org/10.3390/pharmaceutics14061257.
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Metastatic osteosarcoma often results in poor prognosis despite the application of surgical en bloc excision along with chemotherapy. HO-3867 is a curcumin analog that induces cell apoptosis in several cancers, but the apoptotic effect and its mechanisms on osteosarcoma cells are still unknown. After observing the decrease in cellular viability of three human osteosarcoma U2OS, HOS, and MG-63 cell lines, and the induction of cellular apoptosis and arrest in sub-G1 phase in U2OS and HOS cells by HO-3867, the human apoptosis array showed that heme oxygenase (HO)-1 and cleaved caspase-3 expressions had significant increases after HO-3867 treatment in U2OS cells and vice versa for cellular inhibitors of apoptosis (cIAP)1 and X-chromosome-linked IAP (XIAP). Western blot analysis verified the results and showed that HO-3867 activated the initiators of both extrinsic caspase 8 and intrinsic caspase 9, and significantly increased cleaved PARP expression in U2OS and HOS cells. Moreover, with the addition of HO-3867, ERK1/2, and JNK1/2 phosphorylation were increased in U2OS and HOS cells. Using the inhibitor of JNK (JNK in 8), HO-3867’s increases in cleaved caspases 3, 8, and 9 could be expectedly suppressed, indicating that JNK signaling is responsible for both apoptotic pathways, including extrinsic and intrinsic, in U2OS and HOS cells caused by HO-3867. Through JNK signaling, HO-3867 has proven to be effective in causing both extrinsic and intrinsic apoptotic pathways of human osteosarcoma cells.
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Yeh, Liang-Tsai, Chiao-Wen Lin, Ko-Hsiu Lu, Yi-Hsien Hsieh, Chao-Bin Yeh, Shun-Fa Yang, and Jia-Sin Yang. "Niclosamide Suppresses Migration and Invasion of Human Osteosarcoma Cells by Repressing TGFBI Expression via the ERK Signaling Pathway." International Journal of Molecular Sciences 23, no. 1 (January 1, 2022): 484. http://dx.doi.org/10.3390/ijms23010484.
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Osteosarcoma is a highly common malignant bone tumor. Its highly metastatic properties are the leading cause of mortality for cancer. Niclosamide, a salicylanilide derivative, is an oral antihelminthic drug of known anticancer potential. However, the effect of niclosamide on osteosarcoma cell migration, invasion and the mechanisms underlying have not been fully clarified. Therefore, this study investigated niclosamide’s underlying pathways and antimetastatic effects on osteosarcoma. In this study, U2OS and HOS osteosarcoma cell lines were treated with niclosamide and then subjected to assays for determining cell migration ability. The results indicated that niclosamide, at concentrations of up to 200 nM, inhibited the migration and invasion of human osteosarcoma U2OS and HOS cells and repressed the transforming growth factor beta-induced protein (TGFBI) expression of U2OS cells, without cytotoxicity. After TGFBI knockdown occurred, cellular migration and invasion behaviors of U2OS cells were significantly reduced. Moreover, niclosamide significantly decreased the phosphorylation of ERK1/2 in U2OS cells and the combination treatment of the MEK inhibitor (U0126) and niclosamide resulted in the intensive inhibition of the TGFBI expression and the migratory ability in U2OS cells. Therefore, TGFBI derived from osteosarcoma cells via the ERK pathway contributed to cellular migration and invasion and niclosamide inhibited these processes. These findings indicate that niclosamide may be a powerful preventive agent against the development and metastasis of osteosarcoma.
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Hsieh, Min-Hong, Jia-Sin Yang, Renn-Chia Lin, Yi-Hsien Hsieh, Shun-Fa Yang, Horng-Rong Chang, and Ko-Hsiu Lu. "Tomatidine Represses Invasion and Migration of Human Osteosarcoma U2OS and HOS Cells by Suppression of Presenilin 1 and c-Raf–MEK–ERK Pathway." Molecules 25, no. 2 (January 13, 2020): 326. http://dx.doi.org/10.3390/molecules25020326.
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Osteosarcoma, which is the most prevalent malignant bone tumor, is responsible for the great majority of bone cancer-associated deaths because of its highly metastatic potential. Although tomatidine is suggested to serve as a chemosensitizer in multidrug-resistant tumors, the anti-metastatic effect of tomatidine in osteosarcoma is still unknown. Here, we tested the hypothesis that tomatidine suppresses migration and invasion, features that are associated with metastatic process in human osteosarcoma cells and also investigate its underlying pathway. Tomatidine, up to 100 μM, without cytotoxicity, inhibited the invasion and migration capabilities of human osteosarcoma U2OS and HOS cells and repressed presenilin 1 (PS-1) expression of U2OS cells. After the knockdown of PS-1, U2OS and HOS cells’ biological behaviors of cellular invasion and migratory potential were significantly reduced. While tomatidine significantly decreased the phosphorylation of c-Raf, mitogen/extracellular signal-regulated kinase (MEK), and extracellular signal-regulated protein kinase (ERK)1/2 in U2OS cells, no obvious influences on p-Jun N-terminal kinase, p38, and Akt, including their phosphorylation, were observed. In ERK 1 silencing U2 OS cells, tomatidine further enhanced the decrease of their migratory potential and invasive activities. We conclude that both PS-1 derived from U2OS and HOS cells and the c-Raf–MEK–ERK pathway contribute to cellular invasion and migration and tomatidine could inhibit the phenomenons. These findings indicate that tomatidine might be a potential candidate for anti-metastasis treatment of human osteosarcoma.
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Dong, Feng, Tingting Liu, Hao Jin, and Wenbo Wang. "Chimaphilin inhibits human osteosarcoma cell invasion and metastasis through suppressing the TGF-β1-induced epithelial-to-mesenchymal transition markers via PI-3K/Akt, ERK1/2, and Smad signaling pathways." Canadian Journal of Physiology and Pharmacology 96, no. 1 (January 2018): 1–7. http://dx.doi.org/10.1139/cjpp-2016-0522.
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Epithelial-to-mesenchymal transition is a cellular process associated with cancer invasion and metastasis. However, the antimetastatic effects of chimaphilin remain elusive. In this study, we attempted to investigate the potential use of chimaphilin as an inhibitor of TGF-β1-induced epithelial-to-mesenchymal transition in U2OS cells. We found that TGF-β1 induced epithelial-to-mesenchymal transition to promote U2OS cell invasion and metastasis. Western blotting demonstrated that chimaphilin inhibited U2OS cell invasion and migration, increased the expression of the epithelial phenotype marker E-cadherin, repressed the expression of the mesenchymal phenotype marker vimentin, as well as decreased the level of epithelial-to-mesenchymal-inducing transcription factors Snail1 and Slug during the initiation of TGF-β1-induced epithelial-to-mesenchymal transition. In this study, we revealed that chimaphilin up-regulated the E-cadherin expression level and inhibited the production of vimentin, Snail1, and Slug in TGF-β1-induced U2OS cells by blocking PI-3K/Akt and ERK 1/2 signaling pathway. Additionally, the TGF-β1-mediated phosphorylated levels of Smad2/3 were inhibited by chimaphilin pretreatment. Above all, we conclude that chimaphilin represents an effective inhibitor of the metastatic potential of U2OS cells through suppression of TGF-β1-induced epithelial-to-mesenchymal transition.
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Chang, I.-Chang, Tsay-I. Chiang, Chun Lo, Yi-Hua Lai, Chia-Herng Yue, Jer-Yuh Liu, Li-Sung Hsu, and Chia-Jen Lee. "Anemone altaica Induces Apoptosis in Human Osteosarcoma Cells." American Journal of Chinese Medicine 43, no. 05 (January 2015): 1031–42. http://dx.doi.org/10.1142/s0192415x15500597.
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In the past decade, no significant improvement has been made in chemotherapy for osteosarcoma (OS). To develop improved agents against OS, we screened 70 species of medicinal plants and treated two human OS cell lines with different agent concentrations. We then examined cell viability using the MTT assay. Results showed that a candidate plant, particularly the rhizomes of Anemone altaica Fisch. ex C. A. Mey aqueous extract (AAE), suppressed the viability of HOS and U2OS cells in a concentration-dependent manner. Flow cytometry analysis revealed that AAE significantly increased the amount of cell shrinkage (Sub-G1 fragments) in HOS and U2OS cells. Moreover, AAE increased cytosolic cytochrome c and Bax, but decreased Bcl-2. The amount of cleaved caspase-3 and poly-(ADP-ribose) polymerase-1 (PARP-1) were significantly increased. AAE suppressed the growth of HOS and U2OS through the intrinsic apoptotic pathway. Data suggest that AAE is cytotoxic to HOS and U2OS cells and has no significant influence on human osteoblast hFOB cells. The high mRNA levels of apoptosis-related factors (PPP1R15A, SQSTM1, HSPA1B, and DDIT4) and cellular proliferation markers (SKA2 and BUB1B) were significantly altered by the AAE treatment of HOS and U2OS cells. Results show that the anticancer activity of AAE could up-regulate the expression of a cluster of genes, especially those in the apoptosis-related factor family and caspase family. Thus, AAE has great potential as a useful therapeutic drug for human OS.
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Hancock, Meaghan H., Anna R. Cliffe, David M. Knipe, and James R. Smiley. "Herpes Simplex Virus VP16, but Not ICP0, Is Required To Reduce Histone Occupancy and Enhance Histone Acetylation on Viral Genomes in U2OS Osteosarcoma Cells." Journal of Virology 84, no. 3 (November 25, 2009): 1366–75. http://dx.doi.org/10.1128/jvi.01727-09.
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ABSTRACT The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.
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Jamin, Augusta, Nouhou Ibrahim, April Wicklund, Kaitlin Weskamp, and Matthew S. Wiebe. "Vaccinia Virus B1 Kinase Is Required for Postreplicative Stages of the Viral Life Cycle in a BAF-Independent Manner in U2OS Cells." Journal of Virology 89, no. 20 (July 29, 2015): 10247–59. http://dx.doi.org/10.1128/jvi.01252-15.
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ABSTRACTThe vaccinia virus B1R gene encodes a highly conserved protein kinase that is essential for the poxviral life cycle. As demonstrated in many cell types, B1 plays a critical role during viral DNA replication when it inactivates the cellular host defense effector barrier to autointegration factor (BAF or BANF1). To better understand the role of B1 during infection, we have characterized the growth of a B1-deficient temperature-sensitive mutant virus (Cts2 virus) in U2OS osteosarcoma cells. In contrast to all other cell lines tested to date, we found that in U2OS cells, Cts2 viral DNA replication is unimpaired at the nonpermissive temperature. However, the Cts2 viral yield in these cells was reduced more than 10-fold, thus indicating that B1 is required at another stage of the vaccinia virus life cycle. Our results further suggest that the host defense function of endogenous BAF may be absent in U2OS cells but can be recovered through either overexpression of BAF or fusion of U2OS cells with mouse cells in which the antiviral function of BAF is active. Interestingly, examination of late viral proteins during Cts2 virus infection demonstrated that B1 is required for optimal processing of the L4 protein. Finally, execution point analyses as well as electron microscopy studies uncovered a role for B1 during maturation of poxviral virions. Overall, this work demonstrates that U2OS cells are a novel model system for studying the cell type-specific regulation of BAF and reveals a role for B1 beyond DNA replication during the late stages of the viral life cycle.IMPORTANCEThe most well characterized role for the vaccinia virus B1 kinase is to facilitate viral DNA replication by phosphorylating and inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral life cycle have been postulated for decades but are difficult to examine directly due to the importance of B1 during DNA replication. Here, we demonstrate that in U2OS cells, a B1 mutant virus escapes the block in DNA replication observed in other cell types and, instead, this mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral life cycle in a manner that is both dependent on and independent of BAF.
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Lu, Peace Wun-Ang, Renn-Chia Lin, Jia-Sin Yang, Eric Wun-Hao Lu, Yi-Hsien Hsieh, Meng-Ying Tsai, Ko-Hsiu Lu, and Shun-Fa Yang. "GO-Y078, a Curcumin Analog, Induces Both Apoptotic Pathways in Human Osteosarcoma Cells via Activation of JNK and p38 Signaling." Pharmaceuticals 14, no. 6 (May 24, 2021): 497. http://dx.doi.org/10.3390/ph14060497.
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Osteosarcoma is the most common primary bone malignancy in teenagers and continues to confer a generally poor prognosis due to its highly metastatic potential. Poor solubility in water and instability of curcumin limits its bioavailability for use in the adjuvant situation to improve the prognosis and the long-term survival of patients with osteosarcoma. To further obtain information regarding the apoptosis induced by a new curcumin analog, GO-Y078, in human osteosarcoma cells, flow cytometric analysis, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. GO-Y078 dose-dependently decreased viabilities of human osteosarcoma U2OS, MG-63, 143B, and Saos-2 cells and induced sub-G1 fraction arrest and apoptosis in U2OS and 143B cells. In addition to the effector caspase 3 and poly adenosine diphosphate-ribose polymerase, GO-Y078 significantly activated both initiators of extrinsic caspase 8 and intrinsic caspase 9, whereas cellular inhibitors of apoptosis 1 (cIAP-1) and X-chromosome-linked IAP (XIAP) in U2OS and 143B cells were significantly repressed. Moreover, GO-Y078 increased phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2, and p38 in U2OS and 143B cells. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), GO-Y078′s increases in cleaved caspases 8, 9, and 3 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Altogether, GO-Y078 simultaneously induces both apoptotic pathways and cell arrest in U2OS and 143B cells through activating JNK and p38 signaling and repressing IAPs. These findings contribute to a better understanding of the mechanisms responsible for GO-Y078′s apoptotic effects on human osteosarcoma cells.
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Lu, Ko-Hsiu, Jia-Sin Yang, Yi-Hsien Hsieh, Hsiao-Ju Chu, Chia-Hsuan Chou, Eric Wun-Hao Lu, Chiao-Wen Lin, and Shun-Fa Yang. "Lipocalin-2 Inhibits Osteosarcoma Cell Metastasis by Suppressing MET Expression via the MEK–ERK Pathway." Cancers 13, no. 13 (June 25, 2021): 3181. http://dx.doi.org/10.3390/cancers13133181.
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Higher neutrophil-derived cytokine lipocalin-2 (LCN2) expression possesses a versatile role in a myriad of cancers, but little is known about the role of LCN2 on osteosarcoma metastasis. In this study, we demonstrated that higher LCN2 inhibited cellular motility, migration, and invasion of osteosarcoma cells. Moreover, using RNA sequencing technology, we found that LCN2 repressed MET gene expression in U2OS cells. Manipulation of LCN2 levels influenced the migratory potential of osteosarcoma cells as cellular migration was enhanced by transfecting with vectors containing a constitutively active LCN2 cDNA and recombinant human LCN2. Moreover, the phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2 and ERK 1/2 was decreased by LCN2 knockdown. Furthermore, the use of ERK inhibitor (U0126) and activator (tBHQ) confirmed that the pharmaceutic inhibition of MEK–ERK augmented the LCN2-mediated MET suppression and migration of U2OS and HOS cells. Conclusively, LCN2 inhibits osteosarcoma cell metastasis by suppressing MET via the MEK–ERK pathway.
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Wang, Xiaohui, Ying Yu, Lingna Zang, Peng Zhang, Jinfeng Ma, and Dong Chen. "Targeting Clusterin Induces Apoptosis, Reduces Growth Ability and Invasion and Mediates Sensitivity to Chemotherapy in Human Osteosarcoma Cells." Current Pharmaceutical Biotechnology 21, no. 2 (February 12, 2020): 131–39. http://dx.doi.org/10.2174/1389201020666190821151120.
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Objective: The aim of the study was to investigate the expression of sCLU in relation to the clinicopathological features and prognosis of patients with untreated High-Grade Osteosarcoma (HGOS) and to evaluate sCLU as a target for osteosarcoma (OS) therapies. Methods: The expression of sCLU in 98 patients of HGOS enrolled from April 2005 to March 2015 at the affiliated hospital of Qingdao University was evaluated by immunohistochemistry. The sCLU expression, clinical data and survival were compared. siRNA-mediated sCLU gene silencing on cell apoptosis, viability, invasion and chemosensitivity to doxorubicin in U2OS cells in vitro was evaluated. Results: sCLU expression was found in 59 (60%) of the 98 patients. A positive correlation was observed between sCLU expression and metastatic disease (P = 0.036) and a negative correlation between sCLU expression and response to chemotherapy (P = 0.002). Targeting sCLU expression in U2OS cells induced significant reduction in cellular growth and higher rates of spontaneous endogenous apoptosis. In addition, targeting sCLU expression inhibited the invasion of U2OS cells. Furthermore, targeting sCLU expression significantly sensitized to chemotherapeutic drug, doxorubicin. Conclusions: The overexpression of sCLU was significantly correlated with metastasis and chemosensitivity in patients with HGOS. sCLU may be a promising therapeutic or chemopreventive target for human OS treatment.
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Rogatsky, I., J. M. Trowbridge, and M. J. Garabedian. "Glucocorticoid receptor-mediated cell cycle arrest is achieved through distinct cell-specific transcriptional regulatory mechanisms." Molecular and Cellular Biology 17, no. 6 (June 1997): 3181–93. http://dx.doi.org/10.1128/mcb.17.6.3181.
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Glucocorticoids inhibit proliferation of many cell types, but the events leading from the activated glucocorticoid receptor (GR) to growth arrest are not understood. Ectopic expression and activation of GR in human osteosarcoma cell lines U2OS and SAOS2, which lack endogenous receptors, result in a G1 cell cycle arrest. GR activation in U2OS cells represses expression of the cyclin-dependent kinases (CDKs) CDK4 and CDK6 as well as their regulatory partner, cyclin D3, leading to hypophosphorylation of the retinoblastoma protein (Rb). We also demonstrate a ligand-dependent reduction in the expression of E2F-1 and c-Myc, transcription factors involved in the G1-to-S-phase transition. Mitogen-activated protein kinase, CDK2, cyclin E, and the CDK inhibitors (CDIs) p27 and p21 are unaffected by receptor activation in U2OS cells. The receptor's N-terminal transcriptional activation domain is not required for growth arrest in U2OS cells. In Rb-deficient SAOS2 cells, however, the expression of p27 and p21 is induced upon receptor activation. Remarkably, in SAOS2 cells that express a GR deletion derivative lacking the N-terminal transcriptional activation domain, induction of CDI expression is abolished and the cells fail to undergo ligand-dependent cell cycle arrest. Similarly, murine S49 lymphoma cells, which, like SAOS2 cells, lack Rb, require the N-terminal activation domain for growth arrest and induce CDI expression upon GR activation. These cell-type-specific differences in receptor domains and cellular targets linking GR activation to cell cycle machinery suggest two distinct regulatory mechanisms of GR-mediated cell cycle arrest: one involving transcriptional repression of G1 cyclins and CDKs and the other involving enhanced transcription of CDIs by the activated receptor.
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Peng, Yu-Cai, Felix Kuo, David E. Breiding, Yu-Fang Wang, Claire P. Mansur, and Elliot J. Androphy. "AMF1 (GPS2) Modulates p53 Transactivation." Molecular and Cellular Biology 21, no. 17 (September 1, 2001): 5913–24. http://dx.doi.org/10.1128/mcb.21.17.5913-5924.2001.
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ABSTRACT We have reported that the papillomavirus E2 protein binds the nuclear factor AMF1 (also called G-protein pathway suppressor 2 or GPS2) and that their interaction is necessary for transcriptional activation by E2. It has also been shown that AMF1 can influence the activity of cellular transcription factors. These observations led us to test whether AMF1 regulates the functions of p53, a critical transcriptional activator that integrates stress signals and regulates cell cycle and programmed cell death. We report that AMF1 associates with p53 in vivo and in vitro and facilitates the p53 response by augmenting p53-dependent transcription. Overexpression of AMF1 in U2OS cells increases basal level p21WAF1/CIP1 expression and causes a G1 arrest. U2OS cells stably overexpressing AMF1 show increased apoptosis upon exposure to UV irradiation. These data demonstrate that AMF1 modulates p53 activities.
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Lo Cigno, Irene, Marco De Andrea, Cinzia Borgogna, Silvia Albertini, Manuela M. Landini, Alberto Peretti, Karen E. Johnson, Bala Chandran, Santo Landolfo, and Marisa Gariglio. "The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters." Journal of Virology 89, no. 15 (May 13, 2015): 7506–20. http://dx.doi.org/10.1128/jvi.00013-15.
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ABSTRACTThe human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) infection by inhibiting both viral-DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict human papillomavirus 18 (HPV18) replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used, in which the IFI16 protein was knocked down through the use of small interfering RNA (siRNA) technology and overexpressed following transduction with the adenovirus IFI16 (AdVIFI16) vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral-load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral-gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinized HPV DNA through epigenetic modifications and plays a broad surveillance role against viral DNA in the nucleus that is not restricted to herpesviruses.IMPORTANCEIntrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as a sensor of foreign DNA and an antiviral restriction factor, as recently demonstrated by our group for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1). Here, we provide the first evidence that IFI16 inhibits HPV18 replication by repressing viral-gene expression and replication. This antiviral restriction activity was observed in immortalized keratinocytes transfected with the religated genomes and in U2OS cells transfected with HPV18 minicircles, suggesting that it is not cell type specific. We also show that IFI16 promotes the assembly of heterochromatin on HPV DNA. These changes in viral chromatin structure lead to the generation of a repressive state at both early and late HPV18 promoters, thus implicating the protein in the epigenetic regulation of HPV gene expression and replication.
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von Fallois, Moritz, Friederike Katharina Kosyna, Markus Mandl, Yosef Landesman, Jürgen Dunst, and Reinhard Depping. "Selinexor decreases HIF-1α via inhibition of CRM1 in human osteosarcoma and hepatoma cells associated with an increased radiosensitivity." Journal of Cancer Research and Clinical Oncology 147, no. 7 (April 15, 2021): 2025–33. http://dx.doi.org/10.1007/s00432-021-03626-2.
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Abstract Background The nuclear pore complexes (NPCs) are built of about 30 different nucleoporins and act as key regulators of molecular traffic between the cytoplasm and the nucleus for sizeable proteins (> 40 kDa) which must enter the nucleus. Various nuclear transport receptors are involved in import and export processes of proteins through the nuclear pores. The most prominent nuclear export receptor is chromosome region maintenance 1 (CRM1), also known as exportin 1 (XPO1). One of its cargo proteins is the prolyl hydroxylase 2 (PHD2) which is involved in the initiation of the degradation of hypoxia-inducible factors (HIFs) under normoxia. HIFs are proteins that regulate the cellular adaptation under hypoxic conditions. They are involved in many aspects of cell viability and play an important role in the hypoxic microenvironment of cancer. In cancer, CRM1 is often overexpressed thus being a putative target for the development of new cancer therapies. The newly FDA-approved pharmaceutical Selinexor (KPT-330) selectively inhibits nuclear export via CRM1 and is currently tested in additional Phase-III clinical trials. In this study, we investigated the effect of CRM1 inhibition on the subcellular localization of HIF-1α and radiosensitivity. Methods Human hepatoma cells Hep3B and human osteosarcoma cells U2OS were treated with Selinexor. Intranuclear concentration of HIF-1α protein was measured using immunoblot analysis. Furthermore, cells were irradiated with 2–8 Gy after treatment with Selinexor compared to untreated controls. Results Selinexor significantly reduced the intranuclear level of HIF-1α protein in human hepatoma cells Hep3B and human osteosarcoma cells U2OS. Moreover, we demonstrated by clonogenic survival assays that Selinexor leads to dose-dependent radiosensitization in Hep3B-hepatoma and U2OS-osteosarcoma cells. Conclusion Targeting the HIF pathway by Selinexor might be an attractive tool to overcome hypoxia-induced radioresistance.
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Ferreira de Oliveira, José Miguel P., Joana Filipa D. Almeida, Maria Martins, Carina Proença, Helena Oliveira, Eduarda Fernandes, and Conceição Santos. "3′,4′-Dihydroxyflavonol Modulates the Cell Cycle in Cancer Cells: Implication as a Potential Combination Drug in Osteosarcoma." Pharmaceuticals 14, no. 7 (July 3, 2021): 640. http://dx.doi.org/10.3390/ph14070640.
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New agents are demanded to increase the therapeutic options for osteosarcoma (OS). Although OS is the most common bone cancer in children and adolescents, it is considered a rare disorder. Therefore, finding adjuvant drugs has potential to advance therapy for this disease. In this study, 3′,4′-dihydroxyflavonol (DiOHF) was investigated to assess the effects in OS cellular models in combination with doxorubicin (Dox). MG-63 and U2OS human OS cells were exposed to DiOHF and Dox and tested for cell viability and growth. To elucidate the inhibitory effects of DiOHF, additional studies were conducted to assess apoptosis and cell cycle distribution, gene expression quantification of cell cycle regulators, and cytokinesis-block cytome assay to determine nuclear division rate. DiOHF decreased OS cell growth and viability in a concentration-dependent manner. Its combination with Dox enabled Dox dose reduction in both cell lines, with synergistic interactions in U2OS cells. Although no significant apoptotic effects were detected at low concentrations, cytostatic effects were demonstrated in both cell lines. Incubation with DiOHF altered cell cycle dynamics and resulted in differential cyclin and cyclin-dependent kinase expression. Overall, this study presents an antiproliferative action of DiOHF in OS combination therapy via modulation of the cell cycle and nuclear division.
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Ko, Aram, Eun-Woo Lee, Jung-Yong Yeh, Mi-Ran Yang, Wonkyung Oh, Jin-San Moon, and Jaewhan Song. "MKRN1 Induces Degradation of West Nile Virus Capsid Protein by Functioning as an E3 Ligase." Journal of Virology 84, no. 1 (October 21, 2009): 426–36. http://dx.doi.org/10.1128/jvi.00725-09.
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ABSTRACT West Nile virus capsid protein (WNVCp) displays pathogenic toxicity via the apoptotic pathway. However, a cellular mechanism protective against this toxic effect has not been observed so far. Here, we identified Makorin ring finger protein 1 (MKRN1) as a novel E3 ubiquitin ligase for WNVCp. The cytotoxic effects of WNVCp as well as its expression levels were inhibited in U2OS cells that stably expressed MKRN1. Immunoprecipitation analyses revealed an interaction between MKRN1 and WNVCp. Domain analysis indicated that the C terminus of MKRN1 and the N terminus of WNVCp were required for the interaction. MKRN1 could induce WNVCp ubiquitination and degradation in a proteasome-dependent manner. Interestingly, the WNVCp mutant with amino acids 1 to 105 deleted WNVCp was degraded by MKRN1, whereas the mutant with amino acids 1 to 90 deleted was not. When three lysine sites at positions 101, 103, and 104 of WNVCp were replaced with alanine, MKRN1-mediated ubiquitination and degradation of the mutant were significantly inhibited, suggesting that these sites are required for the ubiquitination. Finally, U2OS cell lines stably expressing MKRN1 were resistant to cytotoxic effects of WNV. In contrast, cells depleted of MKRN1 were more susceptible to WNVCp cytotoxicity. Confirming this, overexpression of MKRN1 significantly reduced, but depletion of MKRN1 increased, WNV proliferation in 293T cells. Taken together, our results suggest that MKRN1 can protect cells from WNV by inducing WNVCp degradation.
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Caccianini, Laura, Davide Normanno, Ignacio Izeddin, and Maxime Dahan. "Single molecule study of non-specific binding kinetics of LacI in mammalian cells." Faraday Discussions 184 (2015): 393–400. http://dx.doi.org/10.1039/c5fd00112a.
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Many key cellular processes are controlled by the association of DNA-binding proteins (DBPs) to specific sites. The kinetics of the search process leading to the binding of DBPs to their target locus are largely determined by transient interactions with non-cognate DNA. Using single-molecule microscopy, we studied the dynamics and non-specific binding to DNA of the Lac repressor (LacI) in the environment of mammalian nuclei. We measured the distribution of the LacI–DNA binding times at non-cognate sites and determined the mean residence time to be τ1D = 182 ms. This non-specific interaction time, measured in the context of an exogenous system such as that of human U2OS cells, is remarkably different compared to that reported for the LacI in its native environment in E. coli (<5 ms). Such a striking difference (more than 30 fold) suggests that the genome, its organization, and the nuclear environment of mammalian cells play important roles on the dynamics of DBPs and their non-specific DNA interactions. Furthermore, we found that the distribution of off-target binding times follows a power law, similar to what was reported for TetR in U2OS cells. We argue that a possible molecular origin of such a power law distribution of residence times is the large variability of non-cognate sequences found in the mammalian nucleus by the diffusing DBPs.
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Petrovic, Voin, Camilla Olaisen, Animesh Sharma, Anala Nepal, Steffen Bugge, Eirik Sundby, Bård Helge Hoff, Geir Slupphaug, and Marit Otterlei. "Changes in cellular signaling proteins in extracts from A549, H460, and U2OS cells treated with cisplatin or docetaxel." Data in Brief 12 (June 2017): 18–21. http://dx.doi.org/10.1016/j.dib.2017.03.023.
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Wang, Yi-Xiang J., Ken Cham-Fai Leung, Wing-Hoi Cheung, Hao-Hao Wang, Lin Shi, De-Feng Wang, Ling Qin, and Anil T. Ahuja. "Low-intensity pulsed ultrasound increases cellular uptake of superparamagnetic iron oxide nanomaterial: Results from human osteosarcoma cell line U2OS." Journal of Magnetic Resonance Imaging 31, no. 6 (May 24, 2010): 1508–13. http://dx.doi.org/10.1002/jmri.22173.
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Gao, Xinwei, Yanfeng Liu, Jia Zhang, Luwei Wang, Yong Guo, Yinru Zhu, Zhigang Yang, Wei Yan, and Junle Qu. "Nanodrug Transmembrane Transport Research Based on Fluorescence Correlation Spectroscopy." Membranes 11, no. 11 (November 19, 2021): 891. http://dx.doi.org/10.3390/membranes11110891.
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Although conventional fluorescence intensity imaging can be used to qualitatively study the drug toxicity of nanodrug carrier systems at the single-cell level, it has limitations for studying nanodrug transport across membranes. Fluorescence correlation spectroscopy (FCS) can provide quantitative information on nanodrug concentration and diffusion in a small area of the cell membrane; thus, it is an ideal tool for studying drug transport across the membrane. In this paper, the FCS method was used to measure the diffusion coefficients and concentrations of carbon dots (CDs), doxorubicin (DOX) and CDs-DOX composites in living cells (COS7 and U2OS) for the first time. The drug concentration and diffusion coefficient in living cells determined by FCS measurements indicated that the CDs-DOX composite distinctively improved the transmembrane efficiency and rate of drug molecules, in accordance with the conclusions drawn from the fluorescence imaging results. Furthermore, the effects of pH values and ATP concentrations on drug transport across the membrane were also studied. Compared with free DOX under acidic conditions, the CDs-DOX complex has higher cellular uptake and better transmembrane efficacy in U2OS cells. Additionally, high concentrations of ATP will cause negative changes in cell membrane permeability, which will hinder the transmembrane transport of CDs and DOX and delay the rapid diffusion of CDs-DOX. The results of this study show that the FCS method can be utilized as a powerful tool for studying the expansion and transport of nanodrugs in living cells, and might provide a new drug exploitation strategy for cancer treatment in vivo.
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Gerin, Isabelle, Marina Bury, Francesca Baldin, Julie Graff, Emile Van Schaftingen, and Guido T. Bommer. "Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines." Biochemical Journal 476, no. 4 (February 19, 2019): 629–43. http://dx.doi.org/10.1042/bcj20180435.
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Abstract Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.
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Pan, Yu, Richard L. Bennett, and W. Stratford May. "RAX Activates Tumor Suppressor p53." Blood 108, no. 11 (November 16, 2006): 1449. http://dx.doi.org/10.1182/blood.v108.11.1449.1449.
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Abstract Recently, our laboratory identified RAX as a unique cellular activator for the interferon-induced double-stranded RNA-dependent protein kinase, PKR. Cellular stresses such as IL-3 withdrawal from factor dependent hematopoietic cells, inflammatory cytokine and chemotherapy treatment or viral infection promote RAX phosphorylation with activation of PKR leading to inhibition of new protein synthesis. In addition, PKR can also regulate the transcription factors p53, STAT1 and NF-kB. Now we report a novel activity for RAX in regulating the expression and activity of the tumor suppressor, p53. Results indicate that increased RAX expression in p53 expressing cells, such as HEK293, mouse embryo fibroblast (MEF) or human osteosarcoma U2OS promotes an increase in the steady-state level of endogenous p53 and its activation of the p53 target gene, p21. Furthermore, when RAX is expressed in U2OS cells or coexpressed with p53 in p53 null H1299 cells, enhanced, dose-dependent p53 transcriptional activity is observed as assessed using a p53 luciferase reporter. Consistent with these findings, flow cytometry demonstrates that either RAX or PKR can stimulate p53-dependent G1 arrest that precedes apoptosis. Since p53 is a major ‘sensor’ for DNA damage leading to cell cycle arrest and apoptosis, we also examined whether RAX enhances p53’s response to DNA damage. Following gamma irradiation or Cisplatin treatment of p53 expressing cells, expression of exogenous RAX augments while ‘knock down’ of endogenous RAX by siRNA or expression of the dominant-negative, RAX (S18A) mutant, potently blocks any increase in p53 and p21 expression compared to vector-only control cells. While we find no evidence that RAX directly interacts with p53, results indicate that RAX promotes p53 SUMOylation but does not affect p53 ubiquitination or interaction with MDM2. We now propose that RAX augments the p53 stress-dependent cellular response potentially by a PKR-dependent mechanism involving SUMOylation. Since p53 is only mutated in apparently 15% of hematologic malignancies versus more than 50% in solid tumors, it is envisioned that a more detailed understanding of the mechanism by which RAX activates p53 may serve as a basis for novel antileukemic strategies.
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Aratyn-Schaus, Yvonne, Patrick W. Oakes, and Margaret L. Gardel. "Dynamic and structural signatures of lamellar actomyosin force generation." Molecular Biology of the Cell 22, no. 8 (April 15, 2011): 1330–39. http://dx.doi.org/10.1091/mbc.e10-11-0891.
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The regulation of cellular traction forces on the extracellular matrix is critical to cell adhesion, migration, proliferation, and differentiation. Diverse lamellar actin organizations ranging from contractile lamellar networks to stress fibers are observed in adherent cells. Although lamellar organization is thought to reflect the extent of cellular force generation, understanding of the physical behaviors of the lamellar actin cytoskeleton is lacking. To elucidate these properties, we visualized the actomyosin dynamics and organization in U2OS cells over a broad range of forces. At low forces, contractile lamellar networks predominate and force generation is strongly correlated to actomyosin retrograde flow dynamics with nominal change in organization. Lamellar networks build ∼60% of cellular tension over rapid time scales. At high forces, reorganization of the lamellar network into stress fibers results in moderate changes in cellular tension over slower time scales. As stress fibers build and tension increases, myosin band spacing decreases and α-actinin bands form. On soft matrices, force generation by lamellar networks is unaffected, whereas tension-dependent stress fiber assembly is abrogated. These data elucidate the dynamic and structural signatures of the actomyosin cytoskeleton at different levels of tension and set a foundation for quantitative models of cell and tissue mechanics.
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Ramabhadran, Vinay, Farida Korobova, Gilbert J. Rahme, and Henry N. Higgs. "Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture." Molecular Biology of the Cell 22, no. 24 (December 15, 2011): 4822–33. http://dx.doi.org/10.1091/mbc.e11-05-0457.
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INF2 is a unique formin that can both polymerize and depolymerize actin filaments. Mutations in INF2 cause the kidney disease focal and segmental glomerulosclerosis. INF2 can be expressed as two C-terminal splice variants: CAAX and non-CAAX. The CAAX isoform contains a C-terminal prenyl group and is tightly bound to endoplasmic reticulum (ER). The localization pattern and cellular function of the non-CAAX isoform have not been studied. Here we find that the two isoforms are expressed in a cell type–dependent manner, with CAAX predominant in 3T3 fibroblasts and non-CAAX predominant in U2OS, HeLa, and Jurkat cells. Although INF2-CAAX is ER localized in an actin-independent manner, INF2–non-CAAX localizes in an actin-dependent meshwork pattern distinct from ER. INF2–non-CAAX is loosely attached to this meshwork, being extracted by brief digitonin treatment. Suppression of INF2–non-CAAX causes fragmentation of the Golgi apparatus. This effect is counteracted by treatment with the actin monomer–sequestering drug latrunculin B. We also find discrete patches of actin filaments in the peri-Golgi region, and these patches are reduced upon INF2 suppression. Our results suggest that the non-CAAX isoform of INF2 serves a distinct cellular function from that of the CAAX isoform.
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Watanabe, Reiko, Shin-ichiro Kanno, Amaneh Mohammadi Roushandeh, Ayako Ui, and Akira Yasui. "Nucleosome remodelling, DNA repair and transcriptional regulation build negative feedback loops in cancer and cellular ageing." Philosophical Transactions of the Royal Society B: Biological Sciences 372, no. 1731 (August 28, 2017): 20160473. http://dx.doi.org/10.1098/rstb.2016.0473.
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Nucleosome remodelling (NR) regulates transcription in an ATP-dependent manner, and influences gene expression required for development and cellular functions, including those involved in anti-cancer and anti-ageing processes. ATP-utilizing chromatin assembly and remodelling factor (ACF) and Brahma-associated factor (BAF) complexes, belonging to the ISWI and SWI/SNF families, respectively, are involved in various types of DNA repair. Suppression of several BAF factors makes U2OS cells significantly sensitive to X-rays, UV and especially to cisplatin, and these BAF factors contribute to the accumulation of repair proteins at various types of DNA damage and to DNA repair. Recent cancer genome sequencing and expression analysis has shown that BAF factors are frequently mutated or, more frequently, silenced in various types of cancer cells. Thus, those cancer cells are potentially X-ray- and especially cisplatin-sensitive, suggesting a way of optimizing current cancer therapy. Recent single–stem cell analysis suggests that mutations and epigenetic changes influence stem cell functionality leading to cellular ageing. Genetic and epigenetic changes in the BAF factors diminish DNA repair as well as transcriptional regulation activities, and DNA repair defects in turn negatively influence NR and transcriptional regulation. Thus, they build negative feedback loops, which accelerate both cellular senescence and transformation as common and rare cellular events, respectively, causing cellular ageing. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.
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Weglarz-Tomczak, Ewelina, Demi J. Rijlaarsdam, Jakub M. Tomczak, and Stanley Brul. "GEM-Based Metabolic Profiling for Human Bone Osteosarcoma under Different Glucose and Glutamine Availability." International Journal of Molecular Sciences 22, no. 3 (February 2, 2021): 1470. http://dx.doi.org/10.3390/ijms22031470.
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Cancer cell metabolism is dependent on cell-intrinsic factors, such as genetics, and cell-extrinsic factors, such nutrient availability. In this context, understanding how these two aspects interact and how diet influences cellular metabolism is important for developing personalized treatment. In order to achieve this goal, genome-scale metabolic models (GEMs) are used; however, genetics and nutrient availability are rarely considered together. Here, we propose integrated metabolic profiling, a framework that allows enriching GEMs with metabolic gene expression data and information about nutrients. First, the RNA-seq is converted into Reaction Activity Score (RAS) to further scale reaction bounds. Second, nutrient availability is converted to Maximal Uptake Rate (MUR) to modify exchange reactions in a GEM. We applied our framework to the human osteosarcoma cell line (U2OS). Osteosarcoma is a common and primary malignant form of bone cancer with poor prognosis, and, as indicated in our study, a glutamine-dependent type of cancer.
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Nonga, Olivier E., Darja Lavogina, Erki Enkvist, Katrin Kestav, Apirat Chaikuad, Sarah E. Dixon-Clarke, Alex N. Bullock, et al. "Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family." Molecules 26, no. 14 (July 19, 2021): 4353. http://dx.doi.org/10.3390/molecules26144353.
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We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.
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Zhang, Yichong, Ji-Hoon Lee, Tanya T. Paull, Sarah Gehrke, Angelo D’Alessandro, Qianhui Dou, Vadim N. Gladyshev, et al. "Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity." Science Signaling 11, no. 538 (July 10, 2018): eaaq0702. http://dx.doi.org/10.1126/scisignal.aaq0702.
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Mitochondria are integral to cellular energy metabolism and ATP production and are involved in regulating many cellular processes. Mitochondria produce reactive oxygen species (ROS), which not only can damage cellular components but also participate in signal transduction. The kinase ATM, which is mutated in the neurodegenerative, autosomal recessive disease ataxia-telangiectasia (A-T), is a key player in the nuclear DNA damage response. However, ATM also performs a redox-sensing function mediated through formation of ROS-dependent disulfide-linked dimers. We found that mitochondria-derived hydrogen peroxide promoted ATM dimerization. In HeLa cells, ATM dimers were localized to the nucleus and inhibited by the redox regulatory protein thioredoxin 1 (TRX1), suggesting the existence of a ROS-mediated, stress-signaling relay from mitochondria to the nucleus. ATM dimer formation did not affect its association with chromatin in the absence or presence of nuclear DNA damage, consistent with the separation of its redox and DNA damage signaling functions. Comparative analysis of U2OS cells expressing either wild-type ATM or the redox sensing–deficient C2991L mutant revealed that one function of ATM redox sensing is to promote glucose flux through the pentose phosphate pathway (PPP) by increasing the abundance and activity of glucose-6-phosphate dehydrogenase (G6PD), thereby increasing cellular antioxidant capacity. The PPP produces the coenzyme NADPH needed for a robust antioxidant response, including the regeneration of TRX1, indicating the existence of a regulatory feedback loop involving ATM and TRX1. We propose that loss of the mitochondrial ROS-sensing function of ATM may cause cellular ROS accumulation and oxidative stress in A-T.
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Lin, Hui-Hsien, Kelly L. Robertson, Heather A. Bisbee, and Michelle E. Farkas. "Oncogenic and Circadian Effects of Small Molecules Directly and Indirectly Targeting the Core Circadian Clock." Integrative Cancer Therapies 19 (January 2020): 153473542092409. http://dx.doi.org/10.1177/1534735420924094.
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Circadian rhythms are essential for controlling the cell cycle, cellular proliferation, and apoptosis, and hence are tightly linked to cell fate. Several recent studies have used small molecules to affect circadian oscillations; however, their concomitant cellular effects were not assessed, and they have not been compared under similar experimental conditions. In this work, we use five molecules, grouped into direct versus indirect effectors of the circadian clock, to modulate periods in a human osteosarcoma cell line (U2OS) and determine their influences on cellular behaviors, including motility and colony formation. Luciferase reporters, whose expression was driven via Bmal1- or Per2-promoters, were used to facilitate the visualization and quantitative analysis of circadian oscillations. We show that all molecules increase or decrease the circadian periods of Bmal1 and Per2 in a dose-dependent manner, but period length does not correlate with the extent of cell migration or proliferation. Nonetheless, molecules that affected circadian oscillations to a greater degree resulted in substantial influence on cellular behaviors (ie, motility and colony formation), which may also be attributable to noncircadian targets. Furthermore, we find that the ability and extent to which the molecules are able to affect oscillations is independent of whether they are direct or indirect modulators. Because of the numerous connections and feedback between the circadian clock and other pathways, it is important to consider the effects of both in assessing these and other compounds.
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Boutell, Chris, and Roger D. Everett. "Herpes Simplex Virus Type 1 Infection Induces the Stabilization of p53 in a USP7- and ATM-Independent Manner." Journal of Virology 78, no. 15 (August 1, 2004): 8068–77. http://dx.doi.org/10.1128/jvi.78.15.8068-8077.2004.
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ABSTRACT The major oncoprotein p53 regulates several cellular antiproliferation pathways that can be triggered in response to a variety of cellular stresses, including viral infection. The stabilization of p53 is a key factor in the ability of cells to initiate an efficient transcriptional response after cellular stress. Here we present data demonstrating that herpes simplex virus type 1 (HSV-1) infection of HFFF-2 cells, a low-passage-number nontransformed human primary cell line, results in the stabilization of p53. This process required viral immediate-early gene expression but occurred independently of the viral regulatory protein ICP0 and viral DNA replication. No specific viral protein could be identified as being solely responsible for the effect, which appears to be a cellular response to developing HSV-1 infections. HSV-1 infection also induced the phosphorylation of p53 at residues Ser15 and Ser20, which have previously been implicated in its stabilization in response to DNA damage. However, an HSV-1 infection of ATM−/− cells, which lack a kinase implicated in these phosphorylation events, did not lead to the phosphorylation of p53 at these residues, but nonetheless p53 was stabilized. We also show that the wild-type p53 expressed by osteosarcoma U2OS cells can be stabilized in response to DNA damage induced by UV irradiation, but not in response to HSV-1 infection. These data suggest that multiple cellular mechanisms are initiated to stabilize p53 during an HSV-1 infection. These mechanisms occur independently of ICP0 and its ability to sequester USP7 and may differ from those initiated in response to DNA damage.
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Doege, Kathrin, Sandra Heine, Inga Jensen, Wolfgang Jelkmann, and Eric Metzen. "Inhibition of mitochondrial respiration elevates oxygen concentration but leaves regulation of hypoxia-inducible factor (HIF) intact." Blood 106, no. 7 (October 1, 2005): 2311–17. http://dx.doi.org/10.1182/blood-2005-03-1138.
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Abstract The transcription factor hypoxia-inducible factor-1 (HIF-1) is critical for erythropoietin and other factors involved in the adaptation of the organism to hypoxic stress. Conflicting results have been published regarding the role of the mitochondrial electron transport chain (ETC) in the regulation of HIF-1α. We assessed cellular hypoxia by pimonidazole staining and blotting of the O2-labile HIF-1 α-subunit in human osteosarcoma cell cultures (U2OS and 143B). In conventional, gas-impermeable cell culture dishes, ETC inhibitors had no effect on pimonidazole staining or HIF-1α abundance in a 20% O2 atmosphere; both parameters were undetectable. Pimonidazole staining and HIF activity were substantial in 0.1% O2 irrespective of ETC inhibition. At an intermediate oxygen concentration (3% O2) pimonidazole staining and HIF-α expression were detectable but strongly reduced after ETC inhibition in conventional cell cultures. All effects of ETC inhibition on HIF-1α regulation were eliminated in gas-permeable dishes. As shown in a 143B subclone deficient in mitochondrial DNA (206ρ0), genetic inactivation of the ETC led to similar responses with respect to HIF-1α regulation as ETC inhibitors. Our data demonstrate that reduction of oxygen consumption reduces the O2 gradient in conventional cell cultures, causing elevation of the cellular O2 concentration, which leads to degradation of HIF-α.
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Pietras, Paulina, Marta Leśniczak-Staszak, Aldona Kasprzak, Małgorzata Andrzejewska, Karol Jopek, Mateusz Sowiński, Marcin Rucinski, Shawn M. Lyons, Pavel Ivanov, and Witold Szaflarski. "MVP Expression Facilitates Tumor Cell Proliferation and Migration Supporting the Metastasis of Colorectal Cancer Cells." International Journal of Molecular Sciences 22, no. 22 (November 9, 2021): 12121. http://dx.doi.org/10.3390/ijms222212121.
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Cancer cells show significant dysregulation of genes expression, which may favor their survival in the tumor environment. In this study, the cellular vault’s components MVP (major vault protein), TEP1 (telomerase-associated protein 1) and vPARP (vault poly(ADP-ribose) polymerase) were transiently or completely inhibited in U2OS cells (human bone osteosarcoma epithelial cells) to evaluate their impact on the cell proliferative and migratory capacity as well as on the development of their resistance to the drug vinorelbine. Comparative analysis of MVP protein expression level in normal colon tissue, primary colorectal tumor, and metastasis showed that the expression of this protein does not increase significantly in the primary tumor, but its expression increases in metastatic cells. Further comparative molecular analysis using the whole transcriptome microarrays for MVP-positive and MVP-negative cells showed that MVP is involved in regulating proliferation and migration of cancer cells. MVP may facilitate metastasis of colon cancer due to its impact on cell migration. Moreover, two vault proteins, MVP and TEP1, contribute the resistance to vinorelbine, while vPARP does not.
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Campbell, K. J., N. R. Chapman, and N. D. Perkins. "UV stimulation induces nuclear factor κB (NF-κB) DNA-binding activity but not transcriptional activation." Biochemical Society Transactions 29, no. 6 (November 1, 2001): 688–91. http://dx.doi.org/10.1042/bst0290688.
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The cellular response to DNA-damaging agents is partly mediated by DNA-binding transcription factors such as p53 and nuclear factor κB (NF-κB). Typically NF-κB activation is associated with resistance to apoptosis. Following stimulation with UV light however, NF-κB activation has been shown to be required for programmed cell death. To study this effect further and to determine the relationship between NF-κB and p53 function, we have examined the effect of UV light on U2OS cells. UV stimulation resulted in the activation of NF-κB DNA-binding and the induction of p53. Surprisingly, and in contrast with tumour necrosis factor α stimulation, this UV-induced NF-κB was transcriptionally inert. These observations suggest a model in which the NF-κB switch from an anti-apoptotic to a pro-apoptotic role within the cell results from modulation of its ability to stimulate gene expression, possibly as a result of the ability of p53 to sequester transcriptional co-activator proteins such as p300/CREB (cAMP-response-element-binding protein)-binding protein.
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Iaea, David B., Shu Mao, Frederik W. Lund, and Frederick R. Maxfield. "Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane." Molecular Biology of the Cell 28, no. 8 (April 15, 2017): 1111–22. http://dx.doi.org/10.1091/mbc.e16-07-0499.
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Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and nonvesicular sterol transport processes. Using the fluorescent cholesterol analogue dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a t1/2 =12–15 min. Approximately 70% of sterol transport is ATP independent and therefore is nonvesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. A soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of nonvesicular sterol transport between the plasma membrane and ERC. This study shows that nonvesicular sterol transport mechanisms and STARD4 in particular account for a large fraction of sterol transport between the plasma membrane and the ERC.
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Fulcher, Luke J., Thomas Macartney, Polyxeni Bozatzi, Annika Hornberger, Alejandro Rojas-Fernandez, and Gopal P. Sapkota. "An affinity-directed protein missile system for targeted proteolysis." Open Biology 6, no. 10 (October 2016): 160255. http://dx.doi.org/10.1098/rsob.160255.
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The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate.
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Schultz, Linda B., Nabil H. Chehab, Asra Malikzay, and Thanos D. Halazonetis. "P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks." Journal of Cell Biology 151, no. 7 (December 25, 2000): 1381–90. http://dx.doi.org/10.1083/jcb.151.7.1381.
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p53 binding protein 1 (53BP1), a protein proposed to function as a transcriptional coactivator of the p53 tumor suppressor, has BRCT domains with high homology to the Saccharomyces cerevisiae Rad9p DNA damage checkpoint protein. To examine whether 53BP1 has a role in the cellular response to DNA damage, we probed its intracellular localization by immunofluorescence. In untreated primary cells and U2OS osteosarcoma cells, 53BP1 exhibited diffuse nuclear staining; whereas, within 5–15 min after exposure to ionizing radiation (IR), 53BP1 localized at discreet nuclear foci. We propose that these foci represent sites of processing of DNA double-strand breaks (DSBs), because they were induced by IR and chemicals that cause DSBs, but not by ultraviolet light; their peak number approximated the number of DSBs induced by IR and decreased over time with kinetics that parallel the rate of DNA repair; and they colocalized with IR-induced Mre11/NBS and γ-H2AX foci, which have been previously shown to localize at sites of DSBs. Formation of 53BP1 foci after irradiation was not dependent on ataxia-telangiectasia mutated (ATM), Nijmegen breakage syndrome (NBS1), or wild-type p53. Thus, the fast kinetics of 53BP1 focus formation after irradiation and the lack of dependency on ATM and NBS1 suggest that 53BP1 functions early in the cellular response to DNA DSBs.
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Piazzi, Manuela, Snezana Kojic, Cristina Capanni, Nemanja Stamenkovic, Alberto Bavelloni, Oriano Marin, Giovanna Lattanzi, William Blalock, and Vittoria Cenni. "Ectopic Expression of Ankrd2 Affects Proliferation, Motility and Clonogenic Potential of Human Osteosarcoma Cells." Cancers 13, no. 2 (January 6, 2021): 174. http://dx.doi.org/10.3390/cancers13020174.
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Ankrd2 is a protein known for being mainly expressed in muscle fibers, where it participates in the mechanical stress response. Since both myocytes and osteoblasts are mesenchymal-derived cells, we were interested in examining the role of Ankrd2 in the progression of osteosarcoma which features a mechano-stress component. Although having been identified in many tumor-derived cell lines and -tissues, no study has yet described nor hypothesized any involvement for this protein in osteosarcoma tumorigenesis. In this paper, we report that Ankrd2 is expressed in cell lines obtained from human osteosarcoma and demonstrate a contribution by this protein in the pathogenesis of this insidious disease. Ankrd2 involvement in osteosarcoma development was evaluated in clones of Saos2, U2OS, HOS and MG63 cells stably expressing Ankrd2, through the investigation of hallmark processes of cancer cells. Interestingly, we found that exogenous expression of Ankrd2 influenced cellular growth, migration and clonogenicity in a cell line-dependent manner, whereas it was able to improve the formation of 3D spheroids in three out of four cellular models and enhanced matrix metalloproteinase (MMP) activity in all tested cell lines. Conversely, downregulation of Ankrd2 expression remarkably reduced proliferation and clonogenic potential of parental cells. As a whole, our data present Ankrd2 as a novel player in osteosarcoma development, opening up new therapeutic perspectives.
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Campbell, Amy E., Catarina Ferraz Franco, Ling-I. Su, Emma K. Corbin, Simon Perkins, Anton Kalyuzhnyy, Andrew R. Jones, Philip J. Brownridge, Neil D. Perkins, and Claire E. Eyers. "Temporal modulation of the NF-κB RelA network in response to different types of DNA damage." Biochemical Journal 478, no. 3 (February 10, 2021): 533–51. http://dx.doi.org/10.1042/bcj20200627.
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Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3ε binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA.
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Byrne, Dominic P., Christopher J. Clarke, Philip J. Brownridge, Anton Kalyuzhnyy, Simon Perkins, Amy Campbell, David Mason, Andrew R. Jones, Patrick A. Eyers, and Claire E. Eyers. "Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics." Biochemical Journal 477, no. 13 (July 2, 2020): 2451–75. http://dx.doi.org/10.1042/bcj20200309.
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Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely ‘non-canonical’ substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.
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Egorova, Olga, Heather HC Lau, Kate McGraphery, and Yi Sheng. "Mdm2 and MdmX RING Domains Play Distinct Roles in the Regulation of p53 Responses: A Comparative Study of Mdm2 and MdmX RING Domains in U2OS Cells." International Journal of Molecular Sciences 21, no. 4 (February 15, 2020): 1309. http://dx.doi.org/10.3390/ijms21041309.
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Dysfunction of the tumor suppressor p53 occurs in most human cancers. Mdm2 and MdmX are homologous proteins from the Mdm (Murine Double Minute) protein family, which play a critical role in p53 inactivation and degradation. The two proteins interact with one another via the intrinsic RING (Really Interesting New Gene) domains to achieve the negative regulation of p53. The downregulation of p53 is accomplished by Mdm2-mediated p53 ubiquitination and proteasomal degradation through the ubiquitin proteolytic system and by Mdm2 and MdmX mediated inhibition of p53 transactivation. To investigate the role of the RING domain of Mdm2 and MdmX, an analysis of the distinct functionalities of individual RING domains of the Mdm proteins on p53 regulation was conducted in human osteosarcoma (U2OS) cell line. Mdm2 RING domain was observed mainly localized in the cell nucleus, contrasting the localization of MdmX RING domain in the cytoplasm. Mdm2 RING was found to possess an endogenous E3 ligase activity, whereas MdmX RING did not. Both Mdm2 and MdmX RING domains were able to dimerize with endogenous full-length Mdm2 and MdmX protein and affect their cellular function. The results showed that overexpression of the Mdm2 or MdmX RING domains interfered with the endogenous full-length Mdm2 and MdmX activity and resulted in p53 stabilization and p53 target gene activation. However, both Mdm RING domains showed oncogenic activity in a colony formation assay, suggesting that the Mdm RING domains possess p53-independent oncogenic properties. This study highlights the distinct structural and functional traits of the RING domain of Mdm2 and MdmX and characterized their role in cellular responses through interfering with p53 dependent signaling pathway.
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Micheletti, Gabriele, Natalia Calonghi, Giovanna Farruggia, Elena Strocchi, Vincenzo Palmacci, Dario Telese, Silvia Bordoni, Giulia Frisco, and Carla Boga. "Synthesis of Novel Structural Hybrids between Aza-Heterocycles and Azelaic Acid Moiety with a Specific Activity on Osteosarcoma Cells." Molecules 25, no. 2 (January 18, 2020): 404. http://dx.doi.org/10.3390/molecules25020404.
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Nine compounds bearing pyridinyl (or piperidinyl, benzimidazolyl, benzotriazolyl) groups bound to an azelayl moiety through an amide bond were synthesized. The structural analogy with some histone deacetylase inhibitors inspired their syntheses, seeking new selective histone deacetylase inhibitors (HDACi). The azelayl moiety recalls part of 9-hydroxystearic acid, a cellular lipid showing antiproliferative activity toward cancer cells with HDAC as a molecular target. Azelayl derivatives bound to a benzothiazolyl moiety further proved to be active as HDACi. The novel compounds were tested on a panel of both normal and tumor cell lines. Non-specific induction of cytotoxicity was observed in the normal cell line, while three of them induced a biological effect only on the osteosarcoma (U2OS) cell line. One of them induced a change in nuclear shape and size. Cell-cycle alterations are associated with post-transcriptional modification of both H2/H3 and H4 histones. In line with recent studies, revealing unexpected HDAC7 function in osteoclasts, molecular docking studies on the active molecules predicted their proneness to interact with HDAC7. By reducing side effects associated with the action of the first-generation inhibitors, the herein reported compounds, thus, sound promising as selective HDACi.
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Wu, Xiang, Shao-ming Zhou, Yi-Ling Ding, Ying-ping Gong, Weiqi Zeng, and Yi Cui. "Nexrutine Inhibits Cancer Cell Growth as a Consequence of Mitochondrial Damage and Mitophagy." Cellular Physiology and Biochemistry 36, no. 2 (2015): 763–72. http://dx.doi.org/10.1159/000430136.
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Background/Aims: Nexrutine is an herbal extract of Phellodendron amurense and has been used as nutrient supplement in China as well as America. Potential protection effect of Nexrutine has been reported. Methods: To investigate the mechanism of Nexrutine, we used the HeLa, U2OS and HCT116 as a model. Based on the acidification of cell culture media, we examined the lactate, mitochondria damage as well as mitophagy status by corresponding assay. Results: Our data suggest that Nexrutine alters the cellular glucose metabolism to promote lactate production. This effect is caused by mitochondrial damage, not an alteration to lactate dehydrogenase activity. As a result of the mitochondrial damage, cell proliferation was inhibited and was associated with an elevation in p21/p27 proteins, which are both important cell cycle inhibitors. As another consequence of the mitochondrial damage, mitophagy was highly activated in Nexrutine-treated cells in a dose-dependent manner. When the autophagy pathway was blocked by siRNAs against BECN1 or ATG7, the growth inhibition caused by Nexrutine was reversed. Conclusion: Our study revealed that autophagy plays an important role in the inhibition of cancer cell proliferation by Nexrutine.
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Kong, Derek K., Serban P. Georgescu, Carla Cano, Mark J. Aronovitz, Juan Lucio Iovanna, Richard D. Patten, John M. Kyriakis, and Sandro Goruppi. "Deficiency of the Transcriptional Regulator p8 Results in Increased Autophagy and Apoptosis, and Causes Impaired Heart Function." Molecular Biology of the Cell 21, no. 8 (April 15, 2010): 1335–49. http://dx.doi.org/10.1091/mbc.e09-09-0818.
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Autophagy is a cytoprotective pathway used to degrade and recycle cytoplasmic content. Dysfunctional autophagy has been linked to both cancer and cardiomyopathies. Here, we show a role for the transcriptional regulator p8 in autophagy. p8 RNA interference (RNAi) increases basal autophagy markers in primary cardiomyocytes, in H9C2 and U2OS cells, and decreases cellular viability after autophagy induction. This autophagy is associated with caspase activation and is blocked by atg5 silencing and by pharmacological inhibitors. FoxO3 transcription factor was reported to activate autophagy by enhancing the expression of autophagy-related genes. P8 expression represses FoxO3 transcriptional activity, and p8 knockdown affects FoxO3 nuclear localization. Thus, p8 RNAi increases FoxO3 association with bnip3 promoter, a known proautophagic FoxO3 target, resulting in higher bnip3 RNA and protein levels. Accordingly, bnip3 knockdown restores cell viability and blocks apoptosis of p8-deficient cells. In vivo, p8 −/− mice have higher autophagy and express higher cardiac bnip3 levels. These mice develop left ventricular wall thinning and chamber dilation, with consequent impaired cardiac function. Our studies provide evidence of a p8-dependent mechanism regulating autophagy by acting as FoxO3 corepressor, which may be relevant for diseases associated with dysregulated autophagy, as cardiovascular pathologies and cancer.
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Wang, Kun, Ruilan Yan, Katrina F. Cooper, and Randy Strich. "Cyclin C mediates stress-induced mitochondrial fission and apoptosis." Molecular Biology of the Cell 26, no. 6 (March 15, 2015): 1030–43. http://dx.doi.org/10.1091/mbc.e14-08-1315.
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Mitochondria are dynamic organelles that undergo constant fission and fusion cycles. In response to cellular damage, this balance is shifted dramatically toward fission. Cyclin C–Cdk8 kinase regulates transcription of diverse gene sets. Using knockout mouse embryonic fibroblasts (MEFs), we demonstrate that cyclin C directs the extensive mitochondrial scission induced by the anticancer drug cisplatin or oxidative stress. This activity is independent of transcriptional regulation, as Cdk8 is not required for this activity. Furthermore, adding purified cyclin C to unstressed permeabilized MEF cultures induced complete mitochondrial fragmentation that was dependent on the fission factors Drp1 and Mff. To regulate fission, a portion of cyclin C translocates from the nucleus to the cytoplasm, where it associates with Drp1 and is required for its enhanced mitochondrial activity in oxidatively stressed cells. In addition, although HeLa cells regulate cyclin C in a manner similar to MEF cells, U2OS osteosarcoma cultures display constitutively cytoplasmic cyclin C and semifragmented mitochondria. Finally, cyclin C, but not Cdk8, is required for loss of mitochondrial outer membrane permeability and apoptosis in cells treated with cisplatin. In conclusion, this study suggests that cyclin C connects stress-induced mitochondrial hyperfission and programmed cell death in mammalian cells.
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Zhao, S., W. Xiong, and K. Xu. "MiR-663a, regulated by lncRNA GAS5, contributes to osteosarcoma development through targeting MYL9." Human & Experimental Toxicology 39, no. 12 (July 7, 2020): 1607–18. http://dx.doi.org/10.1177/0960327120937330.
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Osteosarcoma is characterized by high malignancy and high metastasis rate, resulting in high mortality and disability. MiR-663a has been reported in a variety of tumors to promote tumorigenesis. However, miR-663a has not been reported in the pathogenesis of osteosarcoma. Bioinformatics analysis and experiments including real-time quantitative polymerase chain reaction (RT-qPCR), luciferase reporter, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Western blot, RNA immunoprecipitation, and flow cytometry assay were applied to explore the function and mechanism of miR-663a in MG63, U2OS, Saos-2, SF-86, and hFOB1.19 cells. In this study, we found that miR-663a is highly expressed in osteosarcoma. At the same time, we discovered that miR-663a facilitates cell proliferation and migration, whereas suppresses cell apoptosis in osteosarcoma. Through a series of biological experiments, it was found that miR-663a regulates the cellular process in osteosarcoma by modulating the expression of MYL9. In addition, we also found that long noncoding RNA (lncRNA) GAS5 serves as a molecular sponge for miR-663a and regulates the progression of osteosarcoma via the ceRNA mechanism. We uncover that miR-663a promotes osteosarcoma development through targeting MYL9, which was regulated by lncRNA GAS5.
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Kong, Raymond, Sherree Friend, Brian Hall, David Basiji, and Haley Pugsley. "An innovative method for assessing autophagy using the FlowSight imaging flow cytometer (P3253)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 192.12. http://dx.doi.org/10.4049/jimmunol.190.supp.192.12.
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Abstract Autophagy is a process in which normal cellular components that accumulate during growth and differentiation are degraded via the lysosome; it is a survival mechanism that reallocates nutrients from unnecessary processes to more vital processes in the cell. Basal levels of autophagy are usually low but can be up-regulated by numerous stimuli including starvation, physiological stress, pharmacological agents and infections. In addition, suppression of autophagy has been associated with cancer, neurodegenerative disorders, infectious diseases and inflammation. During autophagy, cytoplasmic LC3 is processed and recruited to the autophagosomal membranes; therefore, cells undergoing autophagy can be identified by visualizing LC3 puncta using immunofluorescence microscopy. While manual microscopy allows visual identification of autophagy on a per-cell basis, an objective and statistically rigorous assessment is difficult to obtain. To overcome these problems, we used the FlowSight imaging cytometry platform collect imagery of large numbers of cells to assess autophagy in an objective, quantitative, and statistically robust manner. In this study, we demonstrate a method for determining the best image-based parameter for assess the LC3 puncta in starved and non-starved U2OS RFP-LC3 human osteosarcoma reporter cells.
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Yu, Yongjun, and James C. Alwine. "Interaction between Simian Virus 40 Large T Antigen and Insulin Receptor Substrate 1 Is Disrupted by the K1 Mutation, Resulting in the Loss of Large T Antigen-Mediated Phosphorylation of Akt." Journal of Virology 82, no. 9 (February 27, 2008): 4521–26. http://dx.doi.org/10.1128/jvi.02365-07.
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ABSTRACT The cellular kinase Akt is a key controller of cellular metabolism, growth, and proliferation. Many viruses activate Akt due to its beneficial effects on viral replication. We previously showed that wild-type (WT) simian virus 40 (SV40) large T antigen (TAg) inhibits apoptosis via the activation of PI3K/Akt signaling. Here we show that WT TAg expressed from recombinant adenoviruses in U2OS cells induced the phosphorylation of Akt at both T308 and S473. In contrast, Akt phosphorylation was eliminated by the K1 mutation (E107K) within the retinoblastoma protein (Rb) binding motif of TAg. This suggested that Akt phosphorylation may depend on TAg binding to Rb or one of its family members. However, in Rb-negative SAOS2 cells depleted of p107 and p130 by using small hairpin RNAs (shRNAs), WT TAg still mediated Akt phosphorylation. These results suggested that the K1 mutation affects another TAg function. WT-TAg-mediated phosphorylation of Akt was inhibited by a PI3K inhibitor, suggesting that the effects of TAg originated upstream of PI3K; thus, we examined the requirement for insulin receptor substrate 1 (IRS1), which binds and activates PI3K. Depletion of IRS1 by shRNAs abolished the WT-TAg-mediated phosphorylation of Akt. Immunoprecipitation studies showed that the known interaction between TAg and IRS1 is significantly weakened by the K1 mutation. These data indicate that the K1 mutation disrupts not only Rb binding but also IRS1 binding, contributing to the loss of activation of PI3K/Akt signaling.
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Jardin, Fabrice, Anais Pujals, Laura Pelletier, Elodie Bohers, Vincent Camus, Sylvain Mareschal, Sydney Dubois, et al. "Recurrent Mutations of the Exportin 1 Gene (XPO1) in Primary Mediastinal B-Cell Lymphoma: A Lysa Study." Blood 126, no. 23 (December 3, 2015): 129. http://dx.doi.org/10.1182/blood.v126.23.129.129.
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Abstract Background and aim of the study Primary mediastinal B-cell lymphoma (PMBL) is an entity of aggressive B-cell lymphoma that is clinically and biologically distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We recently detected by Whole exome sequencing a recurrent point mutation in the XPO1 (exportin 1) gene (also referred to as chromosome region maintenance 1; CRM1), which resulted in the Glu571Lys (p.E571K) missense substitution in 2 refractory/relapsed PMBL (Dubois et al., ICML 2015; Mareschal et al. AACR 2015). XPO1 is a member of the Karyopherin-b superfamily of nuclear transport proteins. XPO1 mediates the nuclear export of numerous RNAs and cellular regulatory proteins, including tumor suppressor proteins. This mutation is in the hydrophobic groove of XPO1 that binds to the leucine-rich nuclear export signal (NES) of cargo proteins. In this study, we investigated the prevalence, specificity, and biological / clinical relevance of XPO1 mutations in PMBL. Patients and methods High-throughput targeted or Sanger sequencing of 117 PMBL patients and 3 PMBL cell lines were performed. PMBL cases were defined either molecularly by gene expression profile (mPMBL cohort) or by standard histological method (hPMBL cohort) and enrolled in various LYSA (LYmphoma Study Association) clinical trials. To assess the frequency and specificity of XPO1 mutations, cases of classical Hodgkin lymphoma (cHL) and primary mediastinal grey zone lymphoma (MGZL) were analysed. Cell experiments were performed to assess the impact of the E571 mutation on the activity of selective inhibitor of nuclear export (SINE) molecules. Results XPO1 mutations were present in 28/117 (24%) PMBL cases but were rare in cHL cases (1/19, 5%) and absent from MGZL cases (0/20). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in PMBL cases defined by gene expression profiling (n = 32), as compared to hPMBL cases (n = 85, 13%). No difference in age, International Prognostic Index (IPI) or bulky mass was observed between the PMBL patients harboring mutant and wild-type XPO1 in the overall cohort whereas a female predominance was noticed in the mPMBL cohort. Based on a median follow-up duration of 42 months, XPO1 mutant patients exhibited significantly decreased PFS (3y PFS = 74% [CI95% 55-100]) compared to wild-type patients (3y PFS = 94% [CI95% 83-100], p=0.049) in the mPMBL cohort. In 4/4 tested cases, the E571K variant was also detected in cell-free circulating plasmatic DNA, suggesting that the mutation can be used as a biomarker at the time of diagnosis and during follow-up. Importantly, the E571K variant was detected as a heterozygous mutation in MedB-1, a PMBL-derived cell line, whereas the two other PMBL cell lines tested, Karpas1106 and U-2940, did not display any variants in XPO1 exon 15. KPT-185, the SINE compound that blocks XPO1-dependent nuclear export, induced a dose-dependent decrease in cell proliferation and increased cell death in the PMBL cell lines harbouring wild type or mutated alleles. To test directly if XPO1 mutation from E571 to E571K alters XPO1 inhibition by SINE compounds, the mutated protein was tested in vitro. The E571XPO1 mutated allele was transiently transfected into osteosarcoma U2OS cells which stably express the fluorescently labelled XPO1 cargo REV. Cells were treated with the clinical SINE compound selinexor, which is currently in phase I/II clinical trials and nuclear localization of REV-GFP was analysed in red transfected cells. The results showed that the nuclear export of the mutated XPO1 protein was inhibited by selinexor similarly to the wild-type XPO1 protein (Figure 1). Conclusion Although the oncogenic properties of XPO1 mutations remain to be determined, their recurrent selection in PMBL strongly supports their involvement in the pathogenesis of this curable aggressive B-cell lymphoma. XPO1 mutations were primarily observed in young female patients who displayed a typical PMBL molecular signature. The E571K XPO1 mutation represents a novel hallmark of PMBL but does not seem to interfere with SINE activity. Rev-GFP (green fluorescent) expressing U2OS cells were transfected with wild type XPO1-RFP (red fluorescent protein), XPO1-C528S-RFP, XPO1-E571K-mCherry, and XPO1-E571G-mCherry. The cells were then treated with 1µM KPT-330 for 8 hours. Figure 1. Rev-GFP expressing U2OS cells transfected with XPO1 variants. Figure 1. Rev-GFP expressing U2OS cells transfected with XPO1 variants. Disclosures Landesman: Karyopharm Therapeutics: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Argueta:Karyopharm Therapeutics: Employment. Milpied:Celgene: Honoraria, Research Funding.
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Ledinski, Maja, Ivan Marić, Petra Peharec Štefanić, Iva Ladan, Katarina Caput Mihalić, Tanja Jurkin, Marijan Gotić, and Inga Urlić. "Synthesis and In Vitro Characterization of Ascorbyl Palmitate-Loaded Solid Lipid Nanoparticles." Polymers 14, no. 9 (April 26, 2022): 1751. http://dx.doi.org/10.3390/polym14091751.
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Antitumor applications of ascorbic acid (AA) and its oxidized form dehydroascorbic acid (DHA) can be quite challenging due to their instability and sensitivity to degradation in aqueous media. To overcome this obstacle, we have synthesized solid lipid nanoparticles loaded with ascorbyl palmitate (SLN-AP) with variations in proportions of the polymer Pluronic F-68. SLNs were synthesized using the hot homogenization method, characterized by measuring the particle size, polydispersity, zeta potential and visualized by TEM. To investigate the cellular uptake of the SLN, we have incorporated coumarin-6 into the same SLN formulation and followed their successful uptake for 48 h. We have tested the cytotoxicity of the SLN formulations and free ascorbate forms, AA and DHA, on HEK 293 and U2OS cell lines by MTT assay. The SLN-AP in both formulations have a cytotoxic effect at lower concentrations when compared to ascorbate applied the form of AA or DHA. Better selectivity for targeting tumor cell line was observed with 3% Pluronic F-68. The antioxidative effect of the SLN-AP was observed as early as 1 h after the treatment with a small dose of ascorbate applied (5 µM). SLN-AP formulation with 3% Pluronic F-68 needs to be further optimized as an ascorbate carrier due to its intrinsic cytotoxicity.
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Huang, Wenjing, Tsubasa S. Matsui, Takumi Saito, Masahiro Kuragano, Masayuki Takahashi, Tomohiro Kawahara, Masaaki Sato, and Shinji Deguchi. "Mechanosensitive myosin II but not cofilin primarily contributes to cyclic cell stretch-induced selective disassembly of actin stress fibers." American Journal of Physiology-Cell Physiology 320, no. 6 (June 1, 2021): C1153—C1163. http://dx.doi.org/10.1152/ajpcell.00225.2020.
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Cells adapt to applied cyclic stretch (CS) to circumvent chronic activation of proinflammatory signaling. Currently, the molecular mechanism of the selective disassembly of actin stress fibers (SFs) in the stretch direction, which occurs at the early stage of the cellular response to CS, remains controversial. Here, we suggest that the mechanosensitive behavior of myosin II, a major cross-linker of SFs, primarily contributes to the directional disassembly of the actomyosin complex SFs in bovine vascular smooth muscle cells and human U2OS osteosarcoma cells. First, we identified that CS with a shortening phase that exceeds in speed the inherent contractile rate of individual SFs leads to the disassembly. To understand the biological basis, we investigated the effect of expressing myosin regulatory light-chain mutants and found that SFs with less actomyosin activities disassemble more promptly upon CS. We consequently created a minimal mathematical model that recapitulates the salient features of the direction-selective and threshold-triggered disassembly of SFs to show that disassembly or, more specifically, unbundling of the actomyosin bundle SFs is enhanced with sufficiently fast cell shortening. We further demonstrated that similar disassembly of SFs is inducible in the presence of an active LIM-kinase-1 mutant that deactivates cofilin, suggesting that cofilin is dispensable as opposed to a previously proposed mechanism.