Gene Regulatory Network Characterization of Gastric Cancer's Histological Subtypes: Distinctive Biological and Clinically Relevant Master Regulators.

Gastric cancer (GC) molecular heterogeneity represents a major determinant for clinical outcomes, and although new molecular classifications have been introduced, they are not easy to translate from bench to bedside. We explored the data from GC public databases by performing differential gene expression analysis (DEGs) and gene network reconstruction to identify master regulators (MRs), as well as a gene set analysis (GSA) to reveal their biological features. Moreover, we evaluated the association of MRs with clinicopathological parameters. According to the GSA, the Diffuse group was characterized by an epithelial-mesenchymal transition (EMT) and inflammatory response, while the Intestinal group was associated with a cell cycle and drug resistance pathways. In particular, the regulons of Diffuse MRs, such as Vgll3 and Ciita, overlapped with the EMT and interferon-gamma response, while the regulons Top2a and Foxm1 were shared with the cell cycle pathways in the Intestinal group. We also found a strict association between MR activity and several clinicopathological features, such as survival. Our approach led to the identification of genes and pathways differentially regulated in the Intestinal and Diffuse GC histotypes, highlighting biologically interesting MRs and subnetworks associated with clinical features and prognosis, suggesting putative actionable candidates.

A novel member of Prame family, Gm12794c, counteracts retinoic acid differentiation through the methyltransferase activity of PRC2.

Embryonic stem cells (ESCs) fluctuate among different levels of pluripotency defined as metastates. Sporadically, metastable cellular populations convert to a highly pluripotent metastate that resembles the preimplantation two-cell embryos stage (defined as 2C stage) in terms of transcriptome, DNA methylation, and chromatin structure. Recently, we found that the retinoic acid (RA) signaling leads to a robust increase of cells specifically expressing 2C genes, such as members of the Prame family. Here, we show that Gm12794c, one of the most highly upregulated Prame members, and previously identified as a key player for the maintenance of pluripotency, has a functional role in conferring ESCs resistance to RA signaling. In particular, RA-dependent expression of Gm12794c induces a ground state-like metastate, as evaluated by activation of 2C-specific genes, global DNA hypomethylation and rearrangement of chromatin similar to that observed in naive totipotent preimplantation epiblast cells and 2C-like cells. Mechanistically, we demonstrated that Gm12794c inhibits Cdkn1A gene expression through the polycomb repressive complex 2 (PRC2) histone methyltransferase activity. Collectively, our data highlight a molecular mechanism employed by ESCs to counteract retinoic acid differentiation stimuli and contribute to shed light on the molecular mechanisms at grounds of ESCs naive pluripotency-state maintenance.

TRPV2 Calcium Channel Gene Expression and Outcomes in Gastric Cancer Patients: A Clinically Relevant Association.

Gastric cancer (GC) is characterized by poor efficacy and the modest clinical impact of current therapies. Apoptosis evasion represents a causative factor for treatment failure in GC as in other cancers. Since intracellular calcium homeostasis regulation has been found to be associated with apoptosis resistance, the aberrant expression of intracellular calcium regulator genes (CaRGs) could have a prognostic value in GC patients. We analyzed the association of the expression levels of 98 CaRGs with prognosis by the log-rank test in a collection of 1524 GC samples from four gene expression profiling datasets. We also evaluated differential gene expression in comparison with normal stomach tissue, and then we crossed results with tissue microarrays from the Human Protein Atlas. Among the investigated CaRGs, patients with high levels of TRPV2 expression were characterized by a shorter overall survival. TRPV2 expression was found to increase according to tumor stage. Both mRNA and protein levels were significantly higher in tumor than normal stomach samples. TRPV2 was also associated with poor prognosis in the Lauren's intestinal type GC and in patients treated with adjuvant therapy. Overall, we highlighted the relevance of TRPV2 not only as a prognostic biomarker but also as a potential therapeutic target to improve GC treatment efficacy.

Synthesis, characterization and antibacterial activity of colloidal NiO nanoparticles.

The Colloidal solutions of nickel oxide (NiO) nanoparticles synthesized via Nd-Yag pulse ablation of nickel immersed in H2O were studied. The created nanoparticles were characterized by UV-VIS absorption, Fourier transform infrared spectroscopy (FTIR) and transmission electron microscope (TEM). FTIR characterization confirms the formation of nickel oxide nanoparticles. The optical band gap values, determined by UV-VIS absorption measurements, are found to be (4.5 ev). TEM shows that nanoparticles size ranged from 2-21 nm. The antimicrobial activity was carried out against pseudomonas aurogenisa, Escherichia coli (gram negative bacteria), Staphylococcus aureus and Streptococcus pneumonia (gram positive bacteria). The NiO nanoparticles showed inhibitory activity in both strains of bacteria with best selectivity against gram-positive bacteria. The findings of present study indicate that NiO nanoparticles could potentiate the permeability of bacterial cell wall, and remarkably increase the accumulation of amoxicillin in bacteria, suggesting that NiO nanoparticles together with amoxicillin would facilitate the synergistic impact on growth inhibition of bacterial strains.

Cell cycle-dependent resolution of DNA double-strand breaks.

DNA double strand breaks (DSBs) elicit prompt activation of DNA damage response (DDR), which arrests cell-cycle either in G1/S or G2/M in order to avoid entering S and M phase with damaged DNAs. Since mammalian tissues contain both proliferating and quiescent cells, there might be fundamental difference in DDR between proliferating and quiescent cells (or G0-arrested). To investigate these differences, we studied recruitment of DSB repair factors and resolution of DNA lesions induced at site-specific DSBs in asynchronously proliferating, G0-, or G1-arrested cells. Strikingly, DSBs occurring in G0 quiescent cells are not repaired and maintain a sustained activation of the p53-pathway. Conversely, re-entry into cell cycle of damaged G0-arrested cells, occurs with a delayed clearance of DNA repair factors initially recruited to DSBs, indicating an inefficient repair when compared to DSBs induced in asynchronously proliferating or G1-synchronized cells. Moreover, we found that initial recognition of DSBs and assembly of DSB factors is largely similar in asynchronously proliferating, G0-, or G1-synchronized cells. Our study thereby demonstrates that repair and resolution of DSBs is strongly dependent on the cell-cycle state.

Chemical characterization, antioxidant and cytotoxic activities of the methanolic extract of Hymenocrater longiflorus grown in Iraq.

Hymenocrater longiflorus was collected from northern Iraq, and the chemical composition and antioxidant and cytotoxic activities of this plant were investigated. Ten compounds detected by HPLC-ESI/MS were identified as flavonoids and phenolic acids. The free radical scavenging activity of the 70% methanol extract was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The antioxidant activities of the extract may be attributed to its polyphenolic composition. The cytotoxicity of the plant extract against the osteosarcoma (U2OS) cell line was assessed with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The extract significantly reduced the viability of cells in a concentration- and time-dependent manner. Cells were arrested during the S-phase of the cell cycle, and DNA damage was revealed by antibodies against histone H2AX. The apoptotic features of cell shrinkage and decrease in cell size were also observed. Western blot analysis revealed cleavage of poly (ADP-ribose)-polymerase 1 (PARP-1), in addition to increases in the proteins p53, p21, and γ-H2AX. Collectively, our findings demonstrate that the H. longiflorus extract is highly cytotoxic to U2OS cells, most likely due to its polyphenolic composition.

MYC impairs resolution of site-specific DNA double-strand breaks repair.

Although it is established that when overexpressed, the MYC family proteins can cause DNA double-stand breaks (DSBs) and genome instability, the mechanisms involved remain unclear. MYC induced genetic instability may result from increased DNA damage and/or reduced DNA repair. Here we show that when overexpressed, MYC proteins induce a sustained DNA damage response (DDR) and reduce the wave of DSBs repair. We used a cell-based DSBs system whereby, upon induction of an inducible restriction enzyme AsiSI, hundreds of site-specific DSBs are generated across the genome to investigate the role of MYC proteins on DSB. We found that high levels of MYC do not block accumulation of γH2AX at AsiSI sites, but delay its clearance, indicating an inefficient repair, while the initial recognition of DNA damage is largely unaffected. Repair of both homologous and nonhomologous repair-prone segments, characterized by high or low levels of recruited RAD51, respectively, was delayed. Collectively, these data indicate that high levels of MYC proteins delay the resolution of DNA lesions engineered to occur in cell cultures.

Biocompatibility, uptake and endocytosis pathways of polystyrene nanoparticles in primary human renal epithelial cells.

Recent years have witnessed an unprecedented growth in the number of applications­such as drug delivery, nutraceuticals and production of improved biocompatible materials­in the areas of nanoscience and nanotechnology. Engineered nanoparticles (NPs) are an important tool for the development of quite a few of these applications. Despite intense research activity, mechanisms regulating the uptake of NPs into cells are not completely defined, being the phenomenon dramatically influenced by physico-chemical properties of NPs and cell-specific differences. Since the cellular uptake of NPs is a prerequisite for their use in nanomedicine, the definition of their internalization pathway is crucial. For this reason, we used 44 nm polystyrene NPs as a model to analyze the uptake and endocytosis pathways in primary human renal cortical epithelial (HRCE) cells, which play a key role in the clearance of drugs. NPs were found not to affect the viability and cell cycle progression of HRCE cells. Distinct internalization pathways were analyzed by the use of drugs known to inhibit specific endocytosis routes. Analyses, performed by confocal microscopy in combination with quantitative spectrofluorimetric assays, indicated that NPs enter HRCE cells through multiple mechanisms, either energy-dependent (endocytosis) or energy-independent.

Role of noncoding RNAs in the regulation of P-TEFb availability and enzymatic activity.

P-TEFb is a transcriptional factor that specifically regulates the elongation step of RNA polymerase II-dependent transcription and its activity strictly required for Human Immunodeficiency Virus (HIV) infection and during cardiac differentiation. P-TEFb role has emerged as a crucial regulator of transcription elongation and its activity found finely tuned in vivo at transcriptional level as well as posttranscriptionally by dynamic association with different multisubunit molecular particles. Both physiological and pathological cellular signals rapidly converge on P-TEFb regulation by modifying expression and activity of the complex to allow cells to properly respond to different stimuli. In this review we will give a panoramic view on P-TEFb regulation by noncoding RNAs in both physiological and pathological conditions.

RNA polymerase II CTD modifications: how many tales from a single tail.

Eukaryote's RNA polymerases II (RNAPII) have the feature to contain, at the carbossi-terminal region of their largest subunit Rpb1, a unique CTD domain. Rpb1-CTD is composed of an increasing number of repetitions of the Y1 S2 P3 T4 S5 P6 S7 heptad that goes in parallel with the developmental level of organisms. Because of its composition, the CTD domain has a huge structural plasticity; virtually all the residues can be subjected to post-translational modifications and the two prolines can either be in cis or trans conformations. In light of these features, it is reasonable to think that different specific nuances of CTD modification and interacting factors take place not only on different gene promoters but also during different stages of the transcription cycle and reasonably might have a role even if the polymerase is on or off the DNA template. Rpb1-CTD domain is involved not only in regulating transcriptional rates, but also in all co-transcriptional processes, such as pre-mRNA processing, splicing, cleavage, and export. Moreover, recent studies highlight a role of CTD in DNA replication and in maintenance of genomic stability and specific CTD-modifications have been related to different CTD functions. In this paper, we examine results from the most recent CTD-related literature and give an overview of the general function of Rpb1-CTD in transcription, transcription-related and non transcription-related processes in which it has been recently shown to be involved in.

Sequence-specific double strand breaks trigger P-TEFb-dependent Rpb1-CTD hyperphosphorylation.

Double strand DNA breaks (DSBs) are one of the most challenging forms of DNA damage which, if left unrepaired, can trigger cellular death and can contribute to cancer. A number of studies have been focused on DNA-damage response (DDR) mechanisms, and most of them rely on the induction of DSBs triggered by chemical compounds or radiations. However, genotoxic drugs and radiation treatments of cultured cell lines induce random DSBs throughout the genome, thus heterogeneously across the cell population, leading to variability of the cellular response. To overcome this aspect, we used here a recently described cell-based DSBs system whereby, upon induction of an inducible restriction enzyme, hundreds of site-specific DSBs are generated across the genome. We show here that sequence-specific DSBs are sufficient to activate the positive transcription elongation factor b (P-TEFb), to trigger hyperphosphorylation of the largest RNA polymerase II carboxyl-terminal-domain (Rpb1-CTD) and to induce activation of p53-transcriptional axis resulting in cell cycle arrest.

Caffeine prevents transcription inhibition and P-TEFb/7SK dissociation following UV-induced DNA damage.

BACKGROUND: The mechanisms by which DNA damage triggers suppression of transcription of a large number of genes are poorly understood. DNA damage rapidly induces a release of the positive transcription elongation factor b (P-TEFb) from the large inactive multisubunit 7SK snRNP complex. P-TEFb is required for transcription of most class II genes through stimulation of RNA polymerase II elongation and cotranscriptional pre-mRNA processing. METHODOLOGY/PRINCIPAL FINDINGS: We show here that caffeine prevents UV-induced dissociation of P-TEFb as well as transcription inhibition. The caffeine-effect does not involve PI3-kinase-related protein kinases, because inhibition of phosphatidylinositol 3-kinase family members (ATM, ATR and DNA-PK) neither prevents P-TEFb dissociation nor transcription inhibition. Finally, caffeine prevention of transcription inhibition is independent from DNA damage. CONCLUSION/SIGNIFICANCE: Pharmacological prevention of P-TEFb/7SK snRNP dissociation and transcription inhibition following UV-induced DNA damage is correlated.

Camptothecin releases P-TEFb from the inactive 7SK snRNP complex.

An immediate effect of DNA Topoisomerase I inhibitors camptothecin (CPT) and its derivates is the inhibition of transcription. These fast-acting drugs are believed to inhibit transcription by blocking topoisomerase-mediated relief of DNA supercoiling that occurs during transcription elongation. The CPT effects are commonly considered to be due to a collision between the drug-trapped enzyme on the DNA template and the elongating RNAPII. Here we present evidences that CPT treatment induces an early affect on the positive elongation factor b (P-TEFb). The P-TEFb activity is tightly and dynamically regulated, and a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. We found that, shortly after treatment, CPT disrupts the large inactive P-TEFB complex, and such effect is reversible and independent from DNA replication. Thus, CPT modulates P-TEFb equilibrium in a manner similar to Flavopiridol (FP), a pan-Cdk inhibitor proposed as chemotherapeutic agents against cancers. We determined that while FP inhibits Cdk9 leading to hypo-phosphorylation of RNA polymerase II, CPT-mediated release of free P-TEFb correlates with a concomitant hyper-phosphorylation of RNAPII, which in turn alters the levels and distribution of the RNAPII along transcribed genes. The findings that CPT affects P-TEFb activity provide a direct evidence of the mechanism of this drug to inhibit transcription.

Increased HEXIM1 expression during erythroleukemia and neuroblastoma cell differentiation.

The HEXIM1 protein, in association with 7SK snRNA, binds and inhibits the kinase activity of P-TEFb (CDK9/cyclin T). P-TEFb activity is crucial for efficient transcription elongation of viral and cellular genes. HEXIM1 was originally isolated as a protein up-regulated by hexamethylene bisacetamide (HMBA), a prototypical inducer of differentiation. To determine the causative role of HEXIM1 during cell differentiation we analyzed the biochemical and functional consequences of HEXIM1 protein levels in several in vitro differentiation systems. We found that HEXIM1 mRNA and protein levels are up-regulated during differentiation of murine erythroleukemia cells upon treatment with HMBA or DMSO. Stimulation of HEXIM1 is not restricted to hematopoietic cells, as induction of phenotypic differentiation of neuroblastoma cells by retinoic acid results in up-regulation of HEXIM1. Moreover, ectopic expression of HEXIM1 causes growth inhibition and promotes neuronal differentiation. These findings highlight a crucial role of HEXIM1 protein during cell differentiation.

Transcription-dependent association of multiple positive transcription elongation factor units to a HEXIM multimer.

The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb.HEXIM1.7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb.HEXIM1.7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.

Inhibition of Tat activity by the HEXIM1 protein.

BACKGROUND: The positive transcription elongation factor b (P-TEFb) composed by CDK9/CyclinT1 subunits is a dedicated co-factor of HIV transcriptional transactivator Tat protein. Transcription driven by the long terminal repeat (LTR) of HIV involves formation of a quaternary complex between P-TEFb, Tat and the TAR element. This recruitment is necessary to enhance the processivity of RNA Pol II from the HIV-1 5' LTR promoter. The activity of P-TEFb is regulated in vivo and in vitro by the HEXIM1/7SK snRNA ribonucleic-protein complex. RESULTS: Here we report that Tat transactivation is effectively inhibited by co-expression of HEXIM1 or its paralog HEXIM2. HEXIM1 expression specifically represses transcription mediated by the direct activation of P-TEFb through artificial recruitment of GAL4-CycT1. Using appropriate HEXIM1 mutants we determined that effective Tat-inhibition entails the 7SK snRNA basic recognition motif as well as the C-terminus region required for interaction with cyclin T1. Enhanced expression of HEXIM1 protein modestly affects P-TEFb activity, suggesting that HEXIM1-mediated repression of Tat activity is not due to a global inhibition of cellular transcription. CONCLUSION: These results point to a pivotal role of P-TEFb for Tat's optimal transcription activity and suggest that cellular proteins that regulate P-TEFb activity might exert profound effects on Tat function in vivo.

Identification of proteins interacting with the RNAPII FCP1 phosphatase: FCP1 forms a complex with arginine methyltransferase PRMT5 and it is a substrate for PRMT5-mediated methylation.

FCP1, a phosphatase specific of the carboxyl-terminal-domain of the large subunit of the RNA polymerase II (RNAPII), stimulates transcription elongation and it is required for general transcription and cell viability. To identify novel interacting proteins of FCP1, we used a human cell line expressing an epitope flagged FCP1 and proteins, which formed complexes with FCP1, were identified by mass spectrometry. We identified four proteins: RPB2 subunit of the RNAPII, the nuclear kinase, NDR1, the methyltransferase PRMT5 and the enhancer of rudimentary homologue (ERH) proteins. Intriguingly, both the PRMT5 and ERH proteins are interacting partners of the SPT5 elongation factor. Interactions of RPB2, ERH, NDR1 and PRMT5 with FCP1 were confirmed by co-immunoprecipitation or in vitro pull-down assays. Interaction between PRMT5 and FCP1 was further confirmed by co-immunoprecipitation of endogenous proteins. We found that FCP1 is a genuine substrate of PRMT5-methylation both in vivo and in vitro, and FCP1-associated PRMT5 can methylate histones H4 in vitro.

Functional inactivation of Cdk9 through oligomerization chain reaction.

The oligomerization chain reaction (OCR) strategy is a recently described technique for inactivation of target proteins that function as homoassociate complexes. This novel strategy is based on the fusion of self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins. Here, we present the successful application of the OCR strategy for inactivation of the heterodimeric Cdk9/cyclin T1 complex. Cyclin T1/Cdk9 (P-TEFb) complex is a positive regulator of gene transcription, whose function is underlined by the ability to phosphorylate the carboxyl-terminal domain (CTD) of the RNA polymerase II conferring productive transcript elongation. Fusion of the CC domain to Cdk9 leads to the formation of high molecular complexes to which the endogenous cyclin T1 is recruited. The CC-Cdk9 chimera effectively inhibits HIV-1 Tat activation, whose transcription activity is exquisitely dependent upon cyclin T1/Cdk9 function. Furthermore, expression of CC-Cdk9 protein inhibits cell proliferation, as shown by colony-formation assay. Collectively, our findings add further support to the OCR strategy for functional inactivation of hetero-associated factors such as the Cdk9/cyclin T1 complex, and highlight a putative function of Cdk9 in cell growth control.

Catalytic activity of Cdk9 is required for nuclear co-localization of the Cdk9/cyclin T1 (P-TEFb) complex.

Cdk9 and its binding partner cyclin T1 comprise the positive elongation factor b (P-TEFb). P-TEFb phosphorylates the RNA polymerase II carboxyl-terminal-domain (CTD) allowing efficient transcription elongation. Recent studies showed that Cdk9 is a predominant nuclear protein, and here we investigated the functional requirement for nuclear localization of Cdk9. We found that the catalytic inactive kinase mutant (Cdk9dn) fails to accumulate in the nucleus showing a diffuse sub-cellular localization. In addition to the catalityc activity, nuclear localization of Cdk9 protein requires the presence of the phospho-acceptor sites at the C-terminus tail. Finally, enforced expression of wild-type cyclinT1, which enhances nuclear localization of Cdk9wt, fails to direct the Cdk9 mutants to the nucleus. Collectively, these findings implicate that nuclear localization of Cdk9 requires auto-phosphorylation of the kinase, and highlight the presence of a regulatory mechanism underlying the nuclear localization of the P-TEFb complex.