A Monoclonal Antibody against p53 Cross-Reacts with Processing Bodies

The p53 tumor suppressor protein is an important regulator of cell proliferation and apoptosis. p53 can be found in the nucleus and in the cytosol, and the subcellular location is key to control p53 function. In this work, we found that a widely used monoclonal antibody against p53, termed Pab 1801 (Pan antibody 1801) yields a remarkable punctate signal in the cytoplasm of several cell lines of human origin. Surprisingly, these puncta were also observed in two independent p53-null cell lines. Moreover, the foci stained with the Pab 1801 were present in rat cells, although Pab 1801 recognizes an epitope that is not conserved in rodent p53. In contrast, the Pab 1801 nuclear staining corresponded to genuine p53, as it was upregulated by p53-stimulating drugs and absent in p53-null cells. We identified the Pab 1801 cytoplasmic puncta as P Bodies (PBs), which are involved in mRNA regulation. We found that, in several cell lines, including U2OS, WI38, SK-N-SH and HCT116, the Pab 1801 puncta strictly colocalize with PBs identified with specific antibodies against the PB components Hedls, Dcp1a, Xrn1 or Rck/p54. PBs are highly dynamic and accordingly, the Pab 1801 puncta vanished when PBs dissolved upon treatment with cycloheximide, a drug that causes polysome stabilization and PB disruption. In addition, the knockdown of specific PB components that affect PB integrity simultaneously caused PB dissolution and the disappearance of the Pab 1801 puncta. Our results reveal a strong cross-reactivity of the Pab 1801 with unknown PB component(s). This was observed upon distinct immunostaining protocols, thus meaning a major limitation on the use of this antibody for p53 imaging in the cytoplasm of most cell types of human or rodent origin

Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells.

The spontaneously immortalised DF-1 cell line is rapidly replacing its progenitor primary chicken embryo fibroblasts (CEFs) for studies on avian viruses such as avian influenza but no comprehensive study has as yet been reported comparing their innate immunity phenotypes. We conducted microarray analyses of DF-1 and CEFs, under both normal and stimulated conditions using chicken interferon-α (chIFN-α) and the attenuated infectious bursal disease virus vaccine strain PBG98. We found that DF-1 have an attenuated innate response compared to CEFs. Basal expression levels of Suppressor of Cytokine Signalling 1 (chSOCS1), a negative regulator of cytokine signalling in mammals, are 16-fold higher in DF-1 than in CEFs. The chSOCS1 “SOCS box” domain (which in mammals, interacts with an E3 ubiquitin ligase complex) is not essential for the inhibition of cytokine-induced JAK/STAT signalling activation in DF-1. Overexpression of SOCS1 in chIFN-α-stimulated DF-1 led to a relative decrease in expression of interferon-stimulated genes (ISGs; MX1 and IFIT5) and increased viral yield in response to PBG98 infection. Conversely, knockdown of SOCS1 enhanced induction of ISGs and reduced viral yield in chIFN-α-stimulated DF-1. Consequently, SOCS1 reduces induction of the IFN signalling pathway in chicken cells and can potentiate virus replication

Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks

During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair. © 2014 Nakajima et al

Retinoblastoma Loss Modulates DNA Damage Response Favoring Tumor Progression

Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS), crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRasV12. Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies

Cyclin-Dependent Kinase Activity Controls the Onset of the HCMV Lytic Cycle

The onset of human cytomegalovirus (HCMV) lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE) gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK) inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2) cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state

ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence

Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22phox that produces H2O2. Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H2O2, we detected an increase in H2O2 in the nucleus correlated with NOX4-p22phox perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1β foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence

Cellular processes of v-Src transformation revealed by gene profiling of primary cells - Implications for human cancer

<p>Abstract</p> <p>Background</p> <p>Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. In this study, we took advantage of several strains of the Rous sarcoma virus (RSV) to characterize the patterns of v-Src-dependent gene expression in two different primary cell types, namely chicken embryo fibroblasts (CEF) and chicken neuroretinal (CNR) cells. We identified a common set of v-Src regulated genes and assessed if their expression is associated with disease-free survival using several independent human tumor data sets.</p> <p>Methods</p> <p>CEF and CNR cells were infected with transforming, non-transforming, and temperature sensitive mutants of RSV to identify the patterns of gene expression in response to v-Src-transformation. Microarray analysis was used to measure changes in gene expression and to define a common set of v-Src regulated genes (CSR genes) in CEF and CNR cells. A clustering enrichment regime using the CSR genes and two independent breast tumor data-sets was used to identify a 42-gene aggressive tumor gene signature. The aggressive gene signature was tested for its prognostic value by conducting survival analyses on six additional tumor data sets.</p> <p>Results</p> <p>The analysis of CEF and CNR cells revealed that cell transformation by v-Src alters the expression of 6% of the protein coding genes of the genome. A common set of 175 v-Src regulated genes (CSR genes) was regulated in both CEF and CNR cells. Within the CSR gene set, a group of 42 v-Src inducible genes was associated with reduced disease- and metastasis-free survival in several independent patient cohorts with breast or lung cancer. Gene classes represented within this group include DNA replication, cell cycle, the DNA damage and stress responses, and blood vessel morphogenesis.</p> <p>Conclusion</p> <p>By studying the v-Src-dependent changes in gene expression in two types of primary cells, we identified a set of 42 inducible genes associated with poor prognosis in breast and lung cancer. The identification of these genes provides a set of biomarkers of aggressive tumor behavior and a framework for the study of cancer cells characterized by elevated Src kinase activity.</p

Iscador Qu inhibits doxorubicin-induced senescence of MCF7 cells

Chemotherapy in patients with inoperable or advanced breast cancer inevitably results in low-dose exposure of tumor-cell subset and senescence. Metabolically active senescent cells secrete multiple tumor promoting factors making their elimination a therapeutic priority. Viscum album is one of the most widely used alternative anti-cancer medicines facilitating chemotherapy tolerance of breast cancer patients. The aim of this study was to model and investigate how Viscum album extracts execute additive anti-tumor activity with low-dose Dox using ER + MCF7 breast cancer cells. We report that cotreatment of MCF7 with Viscum album and Dox abrogates G2/M cycle arrest replacing senescence with intrinsic apoptotic program. Mechanistically, this switch was associated with down-regulation of p21, p53/p73 as well as Erk1/2 and p38 activation. Our findings, therefore, identify a novel mechanistic axis of additive antitumor activity of Viscum album and low dose-Dox. In conclusion, ER + breast cancer patients may benefit from addition of Viscum album to low-dose Dox chemotherapy due to suppression of cancer cell senescence and induction of apoptosis

Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence

Additional file 3: Figure S3. Regulation of genes of Arrhythmogenic right ventricular cardiomyopathy pathway during senescence induction in HFF strains Genes of the “Arrhythmogenic right ventricular cardiomyopathy” pathway which are significantly up- (green) and down- (red) regulated (log2 fold change >1) during irradiation induced senescence (120 h after 20 Gy irradiation) in HFF strains. Orange color signifies genes which are commonly up-regulated during both, irradiation induced and replicative senescence