Thomas R. Jackson

Boronate affinity saccharide electrophoresis: A novel carbohydrate analysis tool

The incorporation of specialised carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for fluorophore‐assisted carbohydrate electrophoresis greatly improved the effective separation of saccharides that show similar mobilities in standard electrophoresis. Polyacrylamide gel electrophoresis using methacrylamido phenylboronic acid in low loading (typically 0.5–1% dry weight) was unequivocally shown to alter retention of labelled saccharides depending on their boronate affinity. While conventional fluorophore‐assisted carbohydrate electrophoresis of 2‐aminoacridone labelled glucose oligomers showed an inverted parabolic migration, an undesired trait of small oligosaccharides labelled with this neutral fluorophore, boron affinity saccharide electrophoresis separation of these carbohydrates completely restored their predicted running order, based on their charge/mass ratio, and resulted in improved separation of the analyte saccharides. These results exemplify boron affinity saccharide electrophoresis as an important new technique for analysing carbohydrates and sugar‐containing molecules.

DNA damage causes TP53-dependent coupling of self-renewal and senescence pathways in embryonal carcinoma cells

Recent studies have highlighted an apparently paradoxical link between self-renewal and senescence triggered by DNA damage in certain cell types. In addition, the finding that TP53 can suppress senescence has caused a re-evaluation of its functional role in regulating these outcomes. To investigate these phenomena and their relationship to pluripotency and senescence, we examined the response of the TP53-competent embryonal carcinoma (EC) cell line PA-1 to etoposide-induced DNA damage. Nuclear POU5F1/OCT4A and P21CIP1 were upregulated in the same cells following etoposide-induced G2M arrest. However, while accumulating in the karyosol, the amount of OCT4A was reduced in the chromatin fraction. Phosphorylated CHK2 and RAD51/γH2AX-positive nuclear foci, overexpression of AURORA B kinase and moderate macroautophagy were evident. Upon release from G2M arrest, cells with repaired DNA entered mitoses, while the cells with persisting DNA damage remained at this checkpoint or underwent mitotic slippage and gradually senesced. Reduction of TP53 using sh- or si-RNA prevented the upregulation of OCT4A and P21CIP1 and increased DNA damage. Subsequently, mitoses, micronucleation and senescence were all enhanced after TP53 reduction with senescence confirmed by upregulation of CDKN2A/P16INK4A and increased sa-β-galactosidase positivity. Those mitoses enhanced by TP53 silencing were shown to be multicentrosomal and multi-polar, containing fragmented and highly deranged chromosomes, indicating a loss of genome integrity. Together, these data suggest that TP53-dependent coupling of self-renewal and senescence pathways through the DNA damage checkpoint provides a mechanism for how embryonal stem cell-like EC cells safeguard DNA integrity, genome stability and ultimately the fidelity of self-renewal.

Macroautophagy-aided elimination of chromatin

How tumor cells process damaged or unwanted DNA is a matter of much interest. Recently, Rello-Varona et al. (Cell Cycle 2012; 11:170–76) reported the involvement of macroautophagy (hereon autophagy) in the elimination of micronuclei (MN) from osteosarcoma cells. Prior to that, diminution of whole nuclei from multinucleated TP53-mutant tumor cells was described. Here, we discuss these two kinds of chromatin autophagy evoked after genotoxic stress in the context of the various biological processes involved: (1) endopolyploidy and the ploidy cycle; (2) the timing of DNA synthesis; (3) DNA repair; (4) chromatin:nuclear envelope interactions; and (5) cytoplasmic autophagy. We suggest that whereas some MN can be reunited with the main nucleus (through interactions with envelope-limited chromatin sheets) and participate in DNA repair, failure of repair serves as a signal for the chromatin autophagy of MN. In turn, autophagy of whole sub-nuclei in multi-nucleated cells appears to favor de-polyploidization, mitigation of aneuploidy with its adverse effects, thereby promoting the survival fitness of descendents and treatment resistance. Thus, both kinds of chromatin autophagy provide tumor cells with the opportunity to repair DNA, sort and resort chromatin, reduce DNA content, and enhance survival.

Detection of candidate biomarkers of prostate cancer progression in serum: a depletion-free 3D LC/MS quantitative proteomics pilot study

Background:Prostate cancer (PCa) is the most common male cancer in the United Kingdom and we aimed to identify clinically relevant biomarkers corresponding to stage progression of the disease.Methods:We used enhanced proteomic profiling of PCa progression using iTRAQ 3D LC mass spectrometry on high-quality serum samples to identify biomarkers of PCa.Results:We identified >1000 proteins. Following specific inclusion/exclusion criteria we targeted seven proteins of which two were validated by ELISA and six potentially interacted forming an ‘interactome’ with only a single protein linking each marker. This network also includes accepted cancer markers, such as TNF, STAT3, NF-κB and IL6.Conclusions:Our linked and interrelated biomarker network highlights the potential utility of six of our seven markers as a panel for diagnosing PCa and, critically, in determining the stage of the disease. Our validation analysis of the MS-identified proteins found that SAA alongside KLK3 may improve categorisation of PCa than by KLK3 alone, and that TSR1, although not significant in this model, might also be a clinically relevant biomarker.

Role of stress-activated OCT4A in the cell fate decisions of embryonal carcinoma cells treated with etoposide

Tumor cellular senescence induced by genotoxic treatments has recently been found to be paradoxically linked to the induction of “stemness.” This observation is critical as it directly impinges upon the response of tumors to current chemo-radio-therapy treatment regimens. Previously, we showed that following etoposide (ETO) treatment embryonal carcinoma PA-1 cells undergo a p53-dependent upregulation of OCT4A and p21Cip1 (governing self-renewal and regulating cell cycle inhibition and senescence, respectively). Here we report further detail on the relationship between these and other critical cell-fate regulators. PA-1 cells treated with ETO display highly heterogeneous increases in OCT4A and p21Cip1 indicative of dis-adaptation catastrophe. Silencing OCT4A suppresses p21Cip1, changes cell cycle regulation and subsequently suppresses terminal senescence; p21Cip1-silencing did not affect OCT4A expression or cellular phenotype. SOX2 and NANOG expression did not change following ETO treatment suggesting a dissociation of OCT4A from its pluripotency function. Instead, ETO-induced OCT4A was concomitant with activation of AMPK, a key component of metabolic stress and autophagy regulation. p16ink4a, the inducer of terminal senescence, underwent autophagic sequestration in the cytoplasm of ETO-treated cells, allowing alternative cell fates. Accordingly, failure of autophagy was accompanied by an accumulation of p16ink4a, nuclear disintegration, and loss of cell recovery. Together, these findings imply that OCT4A induction following DNA damage in PA-1 cells, performs a cell stress, rather than self-renewal, function by moderating the expression of p21Cip1, which alongside AMPK helps to then regulate autophagy. Moreover, this data indicates that exhaustion of autophagy, through persistent DNA damage, is the cause of terminal cellular senescence.

Chondroprotection by urocortin involves blockade of the mechanosensitive ion channel Piezo1.

Osteoarthritis (OA) is characterised by progressive destruction of articular cartilage and chondrocyte cell death. Here, we show the expression of the endogenous peptide urocortin1 (Ucn1) and two receptor subtypes, CRF-R1 and CRF-R2, in primary human articular chondrocytes (AC) and demonstrate its role as an autocrine/paracrine pro-survival factor. This effect could only be removed using the CRF-R1 selective antagonist CP-154526, suggesting Ucn1 acts through CRF-R1 when promoting chondrocyte survival. This cell death was characterised by an increase in p53 expression, and cleavage of caspase 9 and 3. Antagonism of CRF-R1 with CP-154526 caused an accumulation of intracellular calcium (Ca2+) over time and cell death. These effects could be prevented with the non-selective cation channel blocker Gadolinium (Gd3+). Therefore, opening of a non-selective cation channel causes cell death and Ucn1 maintains this channel in a closed conformation. This channel was identified to be the mechanosensitive channel Piezo1. We go on to determine that this channel inhibition by Ucn1 is mediated initially by an increase in cyclic adenosine monophosphate (cAMP) and a subsequent inactivation of phospholipase A2 (PLA2), whose metabolites are known to modulate ion channels. Knowledge of these novel pathways may present opportunities for interventions that could abrogate the progression of OA.

MultiElec: A MATLAB Based Application for MEA Data Analysis.

We present MultiElec, an open source MATLAB based application for data analysis of microelectrode array (MEA) recordings. MultiElec displays an extremely user-friendly graphic user interface (GUI) that allows the simultaneous display and analysis of voltage traces for 60 electrodes and includes functions for activation-time determination, the production of activation-time heat maps with activation time and isoline display. Furthermore, local conduction velocities are semi-automatically calculated along with their corresponding vector plots. MultiElec allows ad hoc signal suppression, enabling the user to easily and efficiently handle signal artefacts and for incomplete data sets to be analysed. Voltage traces and heat maps can be simply exported for figure production and presentation. In addition, our platform is able to produce 3D videos of signal progression over all 60 electrodes. Functions are controlled entirely by a single GUI with no need for command line input or any understanding of MATLAB code. MultiElec is open source under the terms of the GNU General Public License as published by the Free Software Foundation, version 3. Both the program and source code are available to download from http://www.cancer.manchester.ac.uk/MultiElec/.

DNA methylation of the Oct4A enhancers in embryonal carcinoma cells after etoposide treatment is associated with alternative splicing and altered pluripotency in reversibly senescent cells

ABSTRACT The epigenetic mechanisms underlying chemoresistance in cancer cells resulting from drug-induced reversible senescence are poorly understood. Chemoresistant ESC-like embryonal carcinoma PA1 cells treated with etoposide (ETO) were previously found to undergo prolonged G2 arrest with transient p53-dependent upregulation of opposing fate regulators, p21CIP1 (senescence) and OCT4A (self-renewal). Here we report on the analysis of the DNA methylation state of the distal enhancer (DE) and proximal enhancer (PE) of the Oct4A gene during this dual response. When compared to non–treated controls the methylation level increased from 1.3% to 12.5% and from 3% to 19.4%, in the DE and PE respectively. It included CpG and non-CpG methylation, which was not chaotic but presented two patterns in each enhancer. Discorrelating with methylation of enhancers, the transcription of Oct4A increased, however, a strong expression of the splicing form Oct4B was also induced, along with down-regulation of the Oct4A partners of in the pluripotency/self-renewal network Sox2 and Lin28. WB demonstrated disjoining of the OCT4A protein from the chromatin-bound fraction. In survival clones, methylation of the DE was considerably erased, while some remnant of methylation of the PE was still observed. The alternative splicing for Oct4B was reduced, Oct4A level insignificantly decreased, while the expression of Sox2 and Lin28 recovered, all three became proportionally above the control. These findings indicate the involvement of the transient patterned methylation of the Oct4A enhancers and alternative splicing in the adaptive regulation of cell fate choice during the p53-dependant dual state of reversible senescence in ESC-like cancer stem cells.

Urocortin suppresses endometrial cancer cell migration via CRFR2 and its system components are differentially modulated by estrogen

Urocortin (UCN1) peptide shares structural and functional homology with corticotropin‐releasing factor (CRF). UCN1 is significantly reduced in endometrial adenocarcinoma compared to healthy controls. However, there are no data which evaluate the effects of UCN1 in the endometrium, or how it is modulated. We used proliferation and transwell assays to determine the effect of UCN1 on the proliferation and migration of Ishikawa and HEC1A cells. We also determined the expression levels of UCN1 and its receptors produced by estrogen receptor agonists, and the effect of UCN1 on estrogen receptor expression, using quantitative polymerase chain reaction. UCN1 suppressed migration of endometrial cancer cells in vitro. This effect appears to be specific to CRF receptor 2 (CRFR2), as selective antagonism of CRFR2 but not CRFR1 completely eliminated suppression of migration. Activation of ERA reduced UCN1 expression, but only had a small effect on the expression of CRFR1. However, expression of CRFR2 was more notably reduced at both the mRNA and protein levels by activation of ERB. UCN1 in turn reduced both ERA and ERB expression, as assessed by real‐time quantitative PCR. We demonstrate that UCN1 significantly suppresses the migration of endometrial cancer cells but has no effect on their proliferation. Thus, loss of UCN1 in endometrial cancer may promote invasion and metastatic spread. There is a complex relationship between the UCN1 system and estrogen receptors, which may provide insights into endometrial carcinogenesis, a disease known to be driven by estrogen excess.

Proteomics in Prostate Cancer Research

Early detection of disease possible through the detection of biological markers (biomarkers) is critical for the healthcare journey of all patients within the twenty-first century. Biomarkers are defined by the National Institutes of Health as ‘characteristics that are objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention’. Therefore, to reduce mortality rates, it is imperative new disease-linked biomarkers are discovered in order to develop future diagnostics and the therapeutics. Once identified, it may for instance enable diagnoses, staging and treatment of haematological cancers (such as leukaemia) and solid tumours (such as prostate). Identification of these disease-linked biomarkers can be a slow and arduous process, although the market will be worth an estimate