a b s t r a c tReal-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples such as biopsies. TDP1 removes covalent 3′DNA adducts in DNA single-strand break repair. This enzymatic activity forms the basis of the design of the TDP1-biosensor, which consists of a short hairpin-forming oligonucleotide having a 5′fluorophore and a 3′quencher brought in close proximity by the secondary structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only "signal amplification" the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1 activity in pure enzyme fractions as well as in crude cell extracts. In the present study we demonstrate the specificity of the biosensor, its ability to quantitatively detect up-or down-regulated TDP1 activity, and that it may be used for measuring and for analyzing the mechanism of TDP1 inhibition.
Minimal residual disease (MRD) is defi ned as the persistence of residual tumor cells in patients during or after treatment, when the patient is in remission. Th e monitoring of MRD has been shown to be an excellent tool for optimization of patient care and therapy response. Formal proof of its benefi cial eff ects has been published for patients with diff erent types of leukemia [1], usually employing quantitative polymerase chain reaction (qPCR) methodology, which currently is the most sensitive approach for disease monitoring. In patients with non-Hodgkin lymphoma (NHL), however, the impact of MRD monitoring is less well defi ned.Mantle cell lymphoma (MCL) is a heterogeneous disease representing about 5 -10% of all cases of NHL [2]. Approximately 90% of patients with MCL are diagnosed by pathology, with overexpression of the cyclin D1 gene ( CCND1 ) caused by fusion of the BCL1 gene on chromosome 11 to the immunoglobulin heavy chain ( IgH ) locus on chromosome 14 [2,3]. In addition to t(11;14), clonal rearrangements of the IgH locus can be identifi ed in most MCLs [4]. Recently, overexpression of the intron-less SOX11 gene was reported as a diagnostic marker highly associated with MCL, with the added advantage of identifying CCND1-negative patients with MCL [5,6].Th e overexpression of SOX11 and CCND1 and the presence of t(11;14) and/or clonal rearrangements are all previously described as potent MRD markers for MCL [7 -10]. However, the literature is lacking direct comparisons of these four key biomarkers.We previously reported assays for sensitive and mRNA specifi c quantifi cation of the overexpression of SOX11 and CCND1 as well as the genomic fusion of BCL1/IgH [7,11]. Expanding the latter assay with patient-specifi c forward primers targeting IgH-VDJ we were, in addition, able to cover the clonal rearrangements of IgH@ .Given these prerequisites, we aimed to compare the four MRD markers for MCL in order to determine their relative values as MRD vehicles in MCL. A total of 213 samples from 78 patients were analyzed with a median number of 2 samples per patient (range 1 -12): 44 patients each contributed a diagnostic peripheral blood (PB) sample, 21 patients with relapse each contributed a PB sample, whereof seven of these patients also included a diagnostic sample in the diagnostic cohort of 44 patients, and 148 follow-up PB samples from 47 patients (20 of these patients had no diagnostic or relapse sample available) were examined for both SOX11 and CCND1 expression; a subgroup of these samples was additionally analyzed for BCL1/IgH and clonal IgH-VDJ allelic burden. Part of the patient material has been used in other studies [7,8,11]. Figure 1(A) shows a strong correlation ( r 2 value: 0.7867) between SOX11 and CCND1 expression in samples ranging from diagnosis to complete remission (CR) and relapse. This was in agreement with a previous study, performed by Meggendorfer et al. , targeting mRNA expression levels at diagnosis [12]. Also apparent in the present study is the low background expression of CCND1...
BackgroundThe current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized that such a correlation assay would provide an approach to address the latter issues, utilizing the leukemic cell line OCI-AML3 with a known set of genetic aberrations.ResultsBy analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution we stringently assessed the signal-to-noise ratio (SNR) from single as well as a discrete number of cells based on a multiple displacement amplification method, with whole exome sequencing as signal readout. In this setting, known OCI-AML3 mutations as well as large copy number alterations could be identified, adding to the current knowledge of cytogenetic status. The presence of DNMT3A R882C, NPM1 W288 fs and NRAS Q61L was consistent, in spite of uneven allelic read depths. In contrast, at the level of single cells, we observed that one-third to half of all variants were not reproduced in the replicate sample, and this allelic mismatch displayed an exponential function of cell input. Large signature duplications were discernible from 5 cells, whereas deletions were visible down to the single cell. Thus, even under highly optimized conditions, single cell whole genome amplification and interpretation must be taken with considerable caution, given that allelic change is frequent and displays low SNR. Allelic noise is rapidly alleviated with increased cell input, and the SNR is doubled from 2 to 50 cells.ConclusionsIn conclusion, we demonstrate noisy allele distributions, when analyzing genetic aberrations within single cells relative to multiple cells. Based on the presented data we recommend that single cell analyses should include replicate cell dilution assays for a given setup for relative assessment of procedure-specific SNR to ensure that the resolution supports the specific hypotheses.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5063-5) contains supplementary material, which is available to authorized users.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.