Temporal Splicing Switches in Elements of the TNF-Pathway Identified by Computational Analysis of Transcriptome Data for Human Cell Lines

Alternative splicing plays an important role in numerous cellular processes and aberrant splice decisions are associated with cancer. Although some studies point to a regulation of alternative splicing and its effector mechanisms in a time-dependent manner, the extent and consequences of such a regulation remains poorly understood. In the present work, we investigated the time-dependent production of isoforms in two Hodgkin lymphoma cell lines of different progression stages (HD-MY-Z, stage IIIb and L-1236, stage IV) compared to a B lymphoblastoid cell line (LCL-HO) with a focus on tumour necrosis factor (TNF) pathway-related elements. For this, we used newly generated time-course RNA-sequencing data from the mentioned cell lines and applied a computational pipeline to identify genes with isoform-switching behaviour in time. We analysed the temporal profiles of the identified events and evaluated in detail the potential functional implications of alterations in isoform expression for the selected top-switching genes. Our data indicate that elements within the TNF pathway undergo a time-dependent variation in isoform production with a putative impact on cell migration, proliferation and apoptosis. These include the genes TRAF1, TNFRSF12A and NFKB2. Our results point to a role of temporal alternative splicing in isoform production, which may alter the outcome of the TNF pathway and impact on tumorigenesis.

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Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress

Toxins ◽

10.3390/toxins13110787 ◽

2021 ◽

Vol 13 (11) ◽

pp. 787

Author(s):

Enrique García-Pérez ◽

Dojin Ryu ◽

Hwa-Young Kim ◽

Hae Dun Kim ◽

Hyun Jung Lee

Keyword(s):

Oxidative Stress ◽

Ochratoxin A ◽

Cell Lines ◽

Time Dependent ◽

Mrna Levels ◽

Dependent Manner ◽

In Vitro System ◽

Glutathione Peroxidase 1 ◽

Glucose 6 Phosphate Dehydrogenase

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

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Binding of Delta1, Jagged1, and Jagged2 to Notch2 Rapidly Induces Cleavage, Nuclear Translocation, and Hyperphosphorylation of Notch2

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6913-6922.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6913-6922 ◽

Cited By ~ 122

Author(s):

Kiyoshi Shimizu ◽

Shigeru Chiba ◽

Noriko Hosoya ◽

Keiki Kumano ◽

Toshiki Saito ◽

...

Keyword(s):

Ligand Binding ◽

Notch Signaling ◽

Time Course ◽

Nuclear Translocation ◽

Reporter Genes ◽

Time Dependent ◽

Dependent Manner ◽

Notch Receptors ◽

Notch Protein ◽

Membrane Spanning

ABSTRACT Delta1, Jagged1, and Jagged2, commonly designated Delta/Serrate/LAG-2 (DSL) proteins, are known to be ligands for Notch1. However, it has been less understood whether they are ligands for Notch receptors other than Notch1. Meanwhile, ligand-induced cleavage and nuclear translocation of the Notch protein are considered to be fundamental for Notch signaling, yet direct observation of the behavior of the Notch molecule after ligand binding, including cleavage and nuclear translocation, has been lacking. In this report, we investigated these issues for Notch2. All of the three DSL proteins bound to endogenous Notch2 on the surface of BaF3 cells, although characteristics of Jagged2 for binding to Notch2 apparently differed from that of Delta1 and Jagged1. After binding, the three DSL proteins induced cleavage of the membrane-spanning subunit of Notch2 (Notch2TM), which occurred within 15 min. In a simultaneous time course, the cleaved fragment of Notch2TMwas translocated into the nucleus. Interestingly, the cleaved Notch2 fragment was hyperphosphorylated also in a time-dependent manner. Finally, binding of DSL proteins to Notch2 also activated the transcription of reporter genes driven by the RBP-Jκ-responsive promoter. Together, these data indicate that all of these DSL proteins function as ligands for Notch2. Moreover, the findings of rapid cleavage, nuclear translocation, and phosphorylation of Notch2 after ligand binding facilitate the understanding of the Notch signaling.

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Inhibition of Wnt/β-Catenin Signaling in Neuroendocrine Tumors In Vitro: Antitumoral Effects

Cancers ◽

10.3390/cancers12020345 ◽

2020 ◽

Vol 12 (2) ◽

pp. 345

Author(s):

Xi-Feng Jin ◽

Gerald Spöttl ◽

Julian Maurer ◽

Svenja Nölting ◽

Christoph Josef Auernhammer

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Cell Lines ◽

Neuroendocrine Tumors ◽

Density Lipoprotein ◽

Time Dependent ◽

Dependent Manner ◽

Antitumor Properties

Background and aims: Inhibition of Wnt/β-catenin signaling by specific inhibitors is currently being investigated as an antitumoral strategy for various cancers. The role of Wnt/β-catenin signaling in neuroendocrine tumors still needs to be further investigated. Methods: This study investigated the antitumor activity of the porcupine (PORCN) inhibitor WNT974 and the β-catenin inhibitor PRI-724 in human neuroendocrine tumor (NET) cell lines BON1, QGP-1, and NCI-H727 in vitro. NET cells were treated with WNT974, PRI-724, or small interfering ribonucleic acids against β-catenin, and subsequent analyses included cell viability assays, flow cytometric cell cycle analysis, caspase3/7 assays and Western blot analysis. Results: Treatment of NET cells with WNT974 significantly reduced NET cell viability in a dose- and time-dependent manner by inducing NET cell cycle arrest at the G1 and G2/M phases without inducing apoptosis. WNT974 primarily blocked Wnt/β-catenin signaling by the dose- and time-dependent downregulation of low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation and non-phosphorylated β-catenin and total β-catenin, as well as the genes targeting the latter (c-Myc and cyclinD1). Furthermore, the WNT974-induced reduction of NET cell viability occurred through the inhibition of GSK-3-dependent or independent signaling (including pAKT/mTOR, pEGFR and pIGFR signaling). Similarly, treatment of NET cells with the β-catenin inhibitor PRI-724 caused significant growth inhibition, while the knockdown of β-catenin expression by siRNA reduced NET tumor cell viability of BON1 cells but not of NCI-H727 cells. Conclusions: The PORCN inhibitor WNT974 possesses antitumor properties in NET cell lines by inhibiting Wnt and related signaling. In addition, the β-catenin inhibitor PRI-724 possesses antitumor properties in NET cell lines. Future studies are needed to determine the role of Wnt/β-catenin signaling in NET as a potential therapeutic target.

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Post-translational alterations in newly synthesized cartilage proteoglycans induced by the glutamine analogue 6-diazo-5-oxo-l-norleucine. Time course of inhibition and recovery

Biochemical Journal ◽

10.1042/bj2730283 ◽

1991 ◽

Vol 273 (2) ◽

pp. 283-288 ◽

Cited By ~ 5

Author(s):

C C Clark ◽

C F Richards ◽

R V Iozzo

Keyword(s):

Time Course ◽

Chondroitin Sulphate ◽

Time Dependent ◽

Dependent Manner ◽

Cartilage Proteoglycan ◽

Chondroitin Sulphate Proteoglycan ◽

Cartilage Proteoglycans ◽

Matrix Free ◽

Subsequent Addition

Incorporation of [35S]sulphate by cultures of matrix-free cells from chick embryo sterna in the presence of the glutamine analogue 6-diazo-5-oxo-L-norleucine (0.58 mM) was inhibited in a time-dependent manner to less than 15% of that in control cultures after 2 h. Characterization of the major cartilage proteoglycan synthesized under these conditions showed that it contained few, if any, normal-sized chondroitin sulphate chains and only about half of the normal complement of substituted serine residues. Subsequent addition of D-glucosamine hydrochloride (final concn. 2 mM) resulted in a time-dependent recovery of [35S]sulphate incorporation to 90% of control cultures after 2 h, but restored the chondroitin sulphate chains to normal size within 15 min. On the basis of these results, it is concluded that a 2 h preincubation is necessary to deplete the chondrocytes of the endogenous supply of UDP-N-acetyl-D-glucosamine required for optimal glycoconjugate synthesis, and that this situation results in the synthesis of a chondroitin sulphate proteoglycan with significantly altered properties, owing to the paucity of glycosaminoglycan chains; however, this condition is completely reversible if the D-glucosamine pool is repleted.

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The Potential Role of STAT's in Anti-Leukemic Therapy with Different Drugs

Blood ◽

10.1182/blood.v120.21.5120.5120 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5120-5120

Author(s):

Hatice Demet Kiper ◽

Burcin Tezcanli Kaymaz ◽

Ozlem Purclutepe ◽

Ceyda Tunakan Dalgic ◽

Nur Selvi ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Drug Exposure ◽

Conflicts Of Interest ◽

Time Dependent ◽

Blue Dye ◽

Dependent Manner ◽

Down Regulation ◽

Expression Levels ◽

Induced Apoptosis

Abstract Abstract 5120 STAT pathways play a pivotal role in oncogenesis and leukemogenesis, thus targeting STAT signalling appears to be an effective anticancer treatment strategy. It has been described that constitutive activation of STAT3 and STAT5 plays a pro-oncogenic role both in acute and chronic myeloid neoplasms. In this study, we aimed to clarify the potential relationship between drug-induced apoptosis with different agents and STAT pathway. A third-generation bisphosphonate; zoledronate, an angiotensin-converting enzyme inhibitor (ACE-I); enalapril, a proteasome inhibitor which is used for treatment of multiple myeloma; bortezomib and a second-generation tyrosine kinase inhibitor; dasatinib were examined in this goal. Cell viability and cytotoxicity tests were conducted by using Trypan blue dye exclusion and XTT assays, respectively. Apoptotic analyses were performed by AnnexinV-EGFP staining method and fluorescence microscopy. Expression levels of STAT3, −5A and −5B genes were analysed in myeloid cell lines by qRT-PCR. The results showed that zoledronate; bortezomib and dasatinib decreased viability and proliferation and induced apoptosis in CML cell line K562 in a dose- and time-dependent manner which is associated by prominent decrease of STAT3, STAT5A and STAT5B mRNA expressions. Enalapril was also found to be cytotoxic and induced apoptosis in APL cell line HL60 in a dose- and time-dependent manner and the expression levels of STAT5A gene have significantly reduced in enalapril-treated HL60 cells as compared to untreated controls. Treatments of cell lines with other drugs were also associated with significant apoptosis in certain time points. The results and changes in expression of STAT's in mRNA level at 72nd hours are summarized in table. Taken together all these data showed that targeting STAT pathways by different drugs may be an appropriate approach in anti-leukemic therapy. This finding is important to propose that discovery or identification of novel agents targeted STATs may open new windows to the other hematological and solid malignancies which are associated with aberrant STAT expression. Table: The changes in STAT expressions after drug exposure in time-dependent manner with the dose of IC50. DRUGS CELL LINE IC50 APOPTOSIS (%) STAT3 mRNA Down Regulation (%) STAT5A mRNA Down Regulation (%) STAT5B mRNA Down Regulation (%) ENALAPRIL HL-60 7 μM 20 20* 76 5* ZOLEDRONATE K562 60 μM 34 63 31 57 BORTEZOMIB K562 177 μM 37 98 100 99 DASATINIB K562 3,314 nM 75 NA 33 78 * : Not significant NA: not applied Disclosures: No relevant conflicts of interest to declare.

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Biologic Sequelae of CDC2 Inhibition in MM.

Blood ◽

10.1182/blood.v110.11.4797.4797 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4797-4797

Author(s):

Mariateresa Fulciniti ◽

Pierfrancesco Tassone ◽

Teru Hideshima ◽

Kenneth C. Anderson ◽

Nikhil C. Munshi

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Cellular Proliferation ◽

Cell Cycle Checkpoint ◽

Time Dependent ◽

Thymidine Uptake ◽

Maximal Effect ◽

Dependent Manner ◽

Apoptotic Pathway ◽

M Phase

Abstract Multiple Myeloma (MM) is a malignant proliferation of plasma cells characterized by disruption of cell cycle checkpoint controls which maintain G2M transition and/or mitosis. CDC2 is the cyclin-dependent kinase that normally drives cells into mitosis and is universally expressed in MM. To examine the biologic role of CDC2 in MM, we evaluated cellular and molecular effects of Terameprocol (M4N, tetra-O-methyl nordihydroguaiaretic acid) that has been shown to inhibit cell cycle progression at the G2/M phase by inhibiting the transcription of sp-1 dependent expression of CDC2. We observed that Terameprocol downregulated the expression of cdc2 in a time-dependent manner, with a maximal effect at 24h. This was associated with induction of G2/M growth arrest in a panel of MM cell lines (INA6, OPM1, OPM2, MM1S, RPMI-8226, U266), as determined by PI staining. Interestingly, Terameprocol treatment led to increase in p21waf1 protein levels. Importantly, we observed inhibition of DNA synthesis by Terameprocol in a dose- and time-dependent manner, with IC50 range from 1–20 uM for a 24 hours period of treatment, as assessed by 3H-thymidine uptake. Longer exposure of MM cells to Terameprocol resulted in cytoxicity, as assessed by MTT assay, via induction of apoptosis, evidenced by Annexin V+ /PI staining, in all the MM cell lines tested. Terameprocol -induced apoptosis is predominantly associated with caspase-9 and caspase-3, but not caspase-8 activation, suggesting that Terameprocol triggers intrinsic apoptotic pathway in MM cells. Our results show that genes that control entry and progression of G2/M phase, especially cdc2, may be an attractive target for MM therapy and Terameprocol represents a prototypic agent for the control of unregulated cellular proliferation in MM.

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Inhibition of Phosphodiesterase 5 and Increasing the Level of Cyclic Guanosine 3′,5′ Monophosphate by Hydroalcoholic Achillea wilhelmsii C. Koch Extract in Human Breast Cancer Cell Lines MCF-7 and MDA-Mb-468

Breast Cancer Basic and Clinical Research ◽

10.1177/1178223417690178 ◽

2017 ◽

Vol 11 ◽

pp. 117822341769017 ◽

Cited By ~ 2

Author(s):

Ramin Saravani ◽

Hamid Reza Galavi ◽

Ali Shahraki

Keyword(s):

Cell Lines ◽

Messenger Rna ◽

Colorimetric Assay ◽

Time Dependent ◽

Breast Cancer Cell Lines ◽

Dependent Manner ◽

Cgmp Signaling ◽

Achillea Wilhelmsii ◽

Phosphodiesterase 5 ◽

Mcf 7

This study aimed to investigate the effect of hydroalcoholic Achillea wilhelmsii C. Koch extract (HAWE) on phosphodiesterase 5 (PDE5) gene expression and cyclic guanosine 3′,5′ monophosphate (cGMP) signaling in the MCF-7 and MDA-Mb-468 cell lines. The effective dose (ED50) of HAWE was examined in both cell lines using a 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide viability test, and the type of cell death was detected by flow cytometry. The expression of PDE5 and the concentration of cGMP were measured in a time-dependent manner in the ED50 by real-time polymerase chain reaction and a colorimetric assay, respectively. Treatment with HAWE showed 25 µg/mL to be the ED50 for both cell lines, and HAWE led to a reduction in the PDE5 messenger RNA expression. The intracellular cGMP increased in a time-dependent manner. The results showed that HAWE has an antiproliferative property in MCF-7 and MDA-Mb-468 cell lines through the cGMP pathway. Therefore, HAWE is a potential source to effectively isolate inhibitory PDE5.

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Alternative Splicing in the Voltage-Sensing Region of N-Type CaV2.2 Channels Modulates Channel Kinetics

Journal of Neurophysiology ◽

10.1152/jn.00048.2004 ◽

2004 ◽

Vol 92 (5) ◽

pp. 2820-2830 ◽

Cited By ~ 44

Author(s):

Yingxin Lin ◽

Stefan I. McDonough ◽

Diane Lipscombe

Keyword(s):

Alternative Splicing ◽

Cell Lines ◽

Time Course ◽

Voltage Dependence ◽

Sympathetic Neurons ◽

Charge Movement ◽

Gating Currents ◽

Splice Isoforms ◽

Tsa201 Cell ◽

Kinetics Of

The CaV2.2 gene encodes the functional core of the N-type calcium channel. This gene has the potential to generate thousands of CaV2.2 splice isoforms with different properties. However, the functional significance of most sites of alternative splicing is not established. The IVS3-IVS4 region contains an alternative splice site that is conserved evolutionarily among CaVα1 genes from Drosophila to human. In CaV2.2, inclusion of exon 31a in the IVS3-IVS4 region is restricted to the peripheral nervous system, and its inclusion slows the speed of channel activation. To investigate the effects of exon 31a in more detail, we generated four tsA201 cell lines stably expressing CaV2.2 splice isoforms. Coexpression of auxiliary CaVβ and CaVα2δ subunits was required to reconstitute currents with the kinetics of N-type channels from neurons. Channels including exon 31a activated and deactivated more slowly at all voltages. Current densities were high enough in the stable cell lines co-expressing CaVα2δ to resolve gating currents. The steady-state voltage dependence of charge movement was not consistently different between splice isoforms, but on gating currents from the exon 31a-containing CaV2.2 isoform decayed with a slower time course, corresponding to slower movement of the charge sensor. Exon 31a-containing CaV2.2 is restricted to peripheral ganglia; and the slower gating kinetics of CaV2.2 splice isoforms containing exon 31a correlated reasonably well with the properties of native N-type currents in sympathetic neurons. Our results suggest that alternative splicing in the S3-S4 linker influences the kinetics but not the voltage dependence of N-type channel gating.

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Effect of external Ca2+ concentration on stretch-augmented natriuretic peptide secretion by rat atria

AJP Cell Physiology ◽

10.1152/ajpcell.1991.260.4.c756 ◽

1991 ◽

Vol 260 (4) ◽

pp. C756-C762 ◽

Cited By ~ 50

Author(s):

E. Page ◽

J. Upshaw-Earley ◽

G. E. Goings ◽

D. A. Hanck

Keyword(s):

Sarcoplasmic Reticulum ◽

Natriuretic Peptide ◽

Time Course ◽

Rate Coefficient ◽

Time Dependent ◽

Dependent Manner ◽

Dependent Inactivation ◽

Long Time ◽

Peptide Secretion

An in vitro noncontracting rat atrial preparation stretched at 37 degrees C by a distending pressure of 5.1 mmHg was used to examine effects of external Ca2+ concentration ([Ca2+]out, 0.05-3.0 mM) on secretion of immunoreactive atrial natriuretic peptide (ANP) in presence of saxitoxin (STX) and in presence or absence of ryanodine. Under these conditions, the time course of the amount (y) of ANP secreted per milligram dry atrium during 44 min could be approximated by a rate coefficient (k) according to the relation y = s[1 - e(-kt)], where s is the maximal amount secreted after a long time (t). Although k, the rate coefficient for stretch-augmented secretion, increased significantly as [Ca2+]out was raised, secretion inactivated progressively in a time- and [Ca2+]out-dependent manner. This time-dependent decrease was not prevented by ryanodine. We conclude that a component of ANP secreted by quiescent atria in vitro is positively modulated by [Ca2+]out and does not require ryanodine-sensitive Ca2+ release from sarcoplasmic reticulum. The [Ca2+]out-sensitive processes underlying time-dependent inactivation of secretion remain undetermined.

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Abnormal 45Ca Fluxes in Dispersed Submandibular Acini of Rats Treated with Reserpine

Journal of Dental Research ◽

10.1177/00220345870660080101 ◽

1987 ◽

Vol 66 (8) ◽

pp. 1294-1299 ◽

Cited By ~ 7

Author(s):

R.M. Müller ◽

J.R. Martinez

Keyword(s):

Time Course ◽

Chronic Treatment ◽

Tracer Uptake ◽

Time Dependent ◽

Coupling Mechanism ◽

Dependent Manner ◽

Isotopic Tracer ◽

Stimulus Response ◽

Ca Uptake ◽

Submandibular Acini

The uptake and efflux of the isotopic tracer 45Ca were compared in dispersed submandibular acini of both control rats and rats treated with seven daily doses of reserpine (0.5 mglkg, i.p.). Tracer uptake occurred in a time-dependent manner in both types of acini and reached 8.4 ± 0.2 and 8.0 ± 0.2 pmol/mg protein, respectively, in acini from control and treated animals after 60 min of incubation. Uptake of tracer was 2.35 nmol/mg DNA in control cells and 4 nmol/mg DNA in cells from treated rats at 60 min. 45Ca uptake (per mg protein) was enhanced in control acini 48% by 20 μmol/L epinephrine: 38% by 50 μmol/L carbachol; and 23% by 10 μmol/L isoproterenol. A similar order of potency was observed when uptake was expressed per mg DNA. In acini from reserpine-treated rats, 45Ca uptake (per mg protein) was increased 53% by epinephrine, 39% by isoproterenol, and only 8% by carbachol. The same enhanced effect of isoproterenol and lack of effect of carbachol were observed when uptake was calculated per mg DNA. In the absence of secretagogue, efflux of 45Ca from tracer-pre-loaded acini was larger in acini from reserpine-treated rats (53%) than in control acini (36%). Whether expressed in terms of mg protein or mg DNA, this efflux was increased in control acini 35% by epinephrine, from 25 to 28% by isoproterenol, and 17% by carbachol. In acini of reserpine-treated rats, epinephrine increased 45Ca efflux 20%, isoproterenol from 25 to 28%, and carbachol from 14 to 15%. The time course of epinephrine- and isoproterenol-induced efflux was also different from that in control cells. Thus, chronic treatment with reserpine altered uptake and efflux of 45Ca in rat submandibular acini. This suggests alterations in secretagogue-sensitive Ca++ pools or gating mechanisms and is likely to underlie disturbances in the Ca++-mediated events of the stimulus-response coupling mechanism, such as fluid, electrolyte, and protein secretion.

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