Kristina Warton

The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle

1 NCC27 is a nuclear chloride ion channel, identified in the PMA‐activated U937 human monocyte cell line. NCC27 mRNA is expressed in virtually all cells and tissues and the gene encoding NCC27 is also highly conserved. Because of these factors, we have examined the hypothesis that NCC27 is involved in cell cycle regulation. 2 Electrophysiological studies in Chinese hamster ovary (CHO‐K1) cells indicated that NCC27 chloride conductance varied according to the stage of the cell cycle, being expressed only on the plasma membrane of cells in G2/M phase. 3 We also demonstrate that Cl− ion channel blockers known to block NCC27 led to arrest of CHO‐K1 cells in the G2/M stage of the cell cycle, the same stage at which this ion channel is selectively expressed on the plasma membrane. 4 These data strongly support the hypothesis that NCC27 is involved, in some as yet undetermined manner, in regulation of the cell cycle.

Functional characterization of the NCC27 nuclear protein in stable transfected CHO-K1 cells

NCC27 belongs to a family of small, highly conserved, organellar ion channel proteins. It is constitutively expressed by native CHO‐K1 and dominantly localized to the nucleus and nuclear membrane. When CHO‐K1 cells are transfected with NCC27‐expressing constructs, synthesized proteins spill over into the cytoplasm and ion channel activity can then be detected on the plasma as well as nuclear membrane. This provided a unique opportunity to directly compare electrophysiological characteristics of the one cloned channel, both on the nuclear and cytoplasmic membranes. At the same time, as NCC27 is unusually small for an ion channel protein, we wished to directly determine whether it is a membrane‐resident channel in its own right. In CHO‐K1 cells transfected with epitope‐tagged NCC27 constructs, we have demonstrated that the NCC27 conductance is chloride dependent and that the electrophysiological characteristics of the channels are essentially identical whether expressed on plasma or nuclear membranes. In addition, we show that a monoclonal antibody directed at an epitope tag added to NCC27 rapidly inhibits the ability of the expressed protein to conduct chloride, but only when the antibody has access to the tag epitope. By selectively tagging either the amino or carboxyl terminus of NCC27 and varying the side of the membrane from which we record channel activity, we have demonstrated conclusively that NCC27 is a transmembrane protein that directly forms part of the ion channel and, further, that the amino terminus projects outward and the carboxyl terminus inward. We conclude that despite its relatively small size, NCC27 must form an integral part of an ion channel complex.—Tonini, R., Ferroni, A., Valenzuela, S. M., Warton, K., Campbell, T. J., Breit, S. N., Mazzanti, M. Functional characterization of the NCC27 nuclear protein in stable transfected CHO‐K1 cells. FASEB J. 14, 1171–1178 (2000)

Methylation of cell-free circulating DNA in the diagnosis of cancer.

A range of molecular alterations found in tumor cells, such as DNA mutations and DNA methylation, is reflected in cell-free circulating DNA (circDNA) released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. Unlike mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. Since DNA methylation is reflected within circDNA, detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA) for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker.

An in vitro study of the effects of exposure to a GSM signal in two human cell lines: Monocytic U937 and neuroblastoma SK-N-SH

The use of mobile phones is increasing, which also increases the populations exposure to global system of mobile communications (GSM) signals. Questions of safety and possible biological effects are of concern and to date, remain largely unanswered. In order to examine possible biological effects of a GSM‐like signal at a cellular level, we exposed two human cell lines (one of neuronal (SK‐N‐SH) and the other of monocytoid (U937) origin) to a 900 MHz RF signal, pulsed at 217 Hz, producing a specific absorption rate (SAR) of 0.2 W/kg. Putative effects were assessed by comparing radiofrequency‐exposed cells to sham‐exposed cells using a variety of assay techniques. For the cell line SK‐N‐SH, effects were specifically assessed by gene microarray, followed by real‐time PCR of the genes of interest, Western blot analysis was used to measure heat shock protein levels, and flow cytometry to measure cell cycle distributions and apoptosis. Effects of radiofrequency on the cell line U937 were assessed by cell viability and cell cycle analysis. From our study of these two cell lines, we found no significant difference between sham‐exposed versus radiofrequency‐exposed cells in any of the assays or conditions examined.

Prognostic and diagnostic significance of DNA methylation patterns in high grade serous ovarian cancer

OBJECTIVE Altered DNA methylation patterns hold promise as cancer biomarkers. In this study we selected a panel of genes which are commonly methylated in a variety of cancers to evaluate their potential application as biomarkers for prognosis and diagnosis in high grade serous ovarian carcinoma (HGSOC); the most common and lethal subtype of ovarian cancer. METHODS The methylation patterns of 10 genes (BRCA1, EN1, DLEC1, HOXA9, RASSF1A, GATA4, GATA5, HSULF1, CDH1, SFN) were examined and compared in a cohort of 80 primary HGSOC and 12 benign ovarian surface epithelium (OSE) samples using methylation-specific headloop suppression PCR. RESULTS The genes were variably methylated in primary HGSOC, with HOXA9 methylation observed in 95% of cases. Most genes were rarely methylated in benign OSE, with the exception of SFN which was methylated in all HGSOC and benign OSE samples examined. Methylation of DLEC1 was associated with disease recurrence, independent of tumor stage and suboptimal surgical debulking (HR 3.5 (95% CI:1.10-11.07), p=0.033). A combination of the methylation status of HOXA9 and EN1 could discriminate HGSOC from benign OSE with a sensitivity of 98.8% and a specificity of 91.7%, which increased to 100% sensitivity with no loss of specificity when pre-operative CA125 levels were also incorporated. CONCLUSIONS This study provides further evidence to support the feasibility of detecting altered DNA methylation patterns as a potential diagnostic and prognostic approach for HGSOC.

Methylated circulating tumor DNA in blood: power in cancer prognosis and response

Circulating tumor DNA (ctDNA) in the plasma or serum of cancer patients provides an opportunity for non-invasive sampling of tumor DNA. This ‘liquid biopsy’ allows for interrogations of DNA such as quantity, chromosomal alterations, sequence mutations and epigenetic changes, and can be used to guide and improve treatment throughout the course of the disease. This tremendous potential for real-time ‘tracking’ in a cancer patient has led to substantial research efforts in the ctDNA field. ctDNA can be distinguished from non-tumor DNA by the presence of tumor-specific mutations and copy number variations, and also by aberrant DNA methylation, with both DNA sequence and methylation changes corresponding to those found in the tumor. Aberrant methylation of specific promoter regions can be a very consistent feature of cancer, in contrast to mutations, which typically occur at a wide range of sites. This consistency makes ctDNA methylation amenable to the design of widely applicable clinical assays. In this review, we examine ctDNA methylation in the context of monitoring disease status, treatment response and determining the prognosis of cancer patients.

Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma

BackgroundFree circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality.ResultsUse of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37×106-86×106 unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing.ConclusionsOur optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

Comparison of 4 commercial kits for the extraction of circulating DNA from plasma

The utility of circulating DNA as a source of clinical biomarkers in blood is limited by its low concentration and small fragment size. Effective purification methods can maximize circulating DNA yield and contribute to the success of downstream protocols. We describe the evaluation of 4 commercial DNA purification kits-QIAamp Circulating Nucleic Acids kit, QIAamp DNA Blood Mini kit, QIAamp Ultrasens Virus kit and the QIASymphony DSP Virus kit-for the extraction of high and low molecular weight DNA from blood plasma. Using qPCR to quantitate endogenous Alu sequences, as well as spiked exogenous high and low molecular weight zebrafish DNA, we found that the Circulating Nucleic Acids kit and the DSP kit were both efficient at purifying DNA from plasma regardless of fragment size, whereas the DNA Blood Mini kit was only able to effectively extract high molecular weight DNA. The Ultrasens Virus Kit produced the lowest yields for both large and small fragments. The use of carrier RNA with the Circulating Nucleic Acids and the DSP kits improved yields. Appropriate choice of kit can be an important factor in determining experiment outcome.

Multiplexed tandem polymerase chain reaction identifies strong expression of oestrogen receptor and Her-2 from single, formalin-fixed, paraffin-embedded breast cancer sections

Aim: To establish the suitability of multiplex tandem polymerase chain reaction (MT‐PCR) for rapid identification of oestrogen receptor (ER) and Her‐2 status using a single, formalin‐fixed, paraffin‐embedded (FFPE) breast tumour section. Methods: Tissue sections from 29 breast tumours were analysed by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). RNA extracted from 10 μm FFPE breast tumour sections from 24 of 29 tumours (14 ER positive and 5 Her‐2 positive) was analysed by MT‐PCR. After establishing a correlation between IHC and/or FISH and MT‐PCR results, the ER/Her‐2 status of a further 32 randomly selected, archival breast tumour specimens was established by MT‐PCR in a blinded fashion, and compared to IHC/FISH results. Results: MT‐PCR levels of ER and Her‐2 showed good concordance with IHC and FISH results. Furthermore, among the ER positive tumours, MT‐PCR provided a quantitative score with a high dynamic range. Threshold values obtained from this data set applied to 32 archival tumour specimens showed that tumours strongly positive for ER and/or Her‐2 expression were easily identified by MT‐PCR. Conclusion: MT‐PCR can provide rapid, sensitive and cost‐effective analysis of FFPE material and may prove useful as triage to identify patients suited to endocrine or trastuzumab (Herceptin) treatment.

Abstract A4: Whole genome sequencing of free circulating DNA from plasma of HGSOC patients

Genomic analysis of free circulating DNA (fcDNA) has a number of potential clinical applications, including cancer studies. Tumor-derived fcDNA harbors the same molecular aberrations, including mutations and methylation, as the derivative tumor. As it can be collected by non-invasive means, fcDNA is particularly promising as a cancer detection tool. In addition, molecular characterization of fcDNA in cancer patients is holds promise for determining tumor sub-types and monitoring response to treatment. However, because fcDNA concentrations are relatively low in the circulation, whole-genome analysis for molecular characterization is quite challenging. Standardization of fcDNA collection and processing for downstream analysis is imperative to help address these challenges. Our studies focus on the development of an early detection test for high-grade serous ovarian cancer (HGSOC), using whole-genome methylation analysis in fcDNA from HGSOC patients. DNA methylation plays a key role in the development of many cancer types, and thus carries great potential as a cancer diagnostic biomarker. We propose that DNA methylation changes in HGSOC tumors can be detected in the patient9s fcDNA and can be used as a blood-based test to detect HGSOC. We have carried out Next Generation Sequencing (NGS) of methylation-enriched fcDNA from 3 HGSOC patients and 5 healthy controls with the aim of identifying a list of differentially methylated loci in fcDNA that can distinguish between HGSOC and control plasma. We also sought to generate standard protocols for collection and processing of fcDNA for whole-genome studies. First we analyzed effects of blood storage time on fcDNA recovery and quality and determined that storage for ≥8 hrs prior to plasma separation leads to increased DNA concentrations, as well as an appearance of a band that co-migrates with high-molecular weight genomic DNA. This increased DNA concentration likely represents genomic DNA contamination as a result of leukocytes lysis during storage. We next analyzed the effects of processing and scaling on enriched fcDNA recovery and quality. We used a methyl-binding protein-based (MBD2) protocol to enrich for methylated sequences in fcDNA isolated from plasma samples. Following application of a modified protocol due to low concentrations of fcDNA in plasma, we obtained a 10.2-14.9% enrichment of methylated fragments. In preparing the sample libraries for NGS, the predominant fcDNA size of ~180 base pairs allows minimal sample loss during the size restriction step. NGS analysis provided 37-86x10^6 unique mappable reads per fcDNA sample, representing >50% of the total mappable reads. These read counts indicate a satisfactory level of library complexity was achieved from minimal fcDNA input. Our standardization protocols provide the ability to comprehensively sequence patient fcDNA using half of the recommended sample input. This optimized approach allows in-depth whole genome characterization and comparison of fcDNA isolated from HGSOC and healthy controls to develop diagnostic tools and investigate biological mechanisms driving tumor progression. Citation Format: Kristina Warton, Lin Vita, Nicola J. Armstrong, Warren Kaplan, Kevin Ying, Helena Mangs, Neville F. Hacker, Robert L. Sutherland, Susan J. Clark, Goli Samimi. Whole genome sequencing of free circulating DNA from plasma of HGSOC patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A4.