scholarly journals Resolution of DNA damage at the single-cell level shows largely different actions of X-rays and bleomycin.

1995 ◽  
Vol 43 (2) ◽  
pp. 229-235 ◽  
Author(s):  
M I Affentranger ◽  
W Burkart
Keyword(s):  
Single Cell ◽  
Cell Population ◽  
Large Cell ◽  
Dna Strand Breaks ◽  
Single Cell Level ◽  
X Rays ◽  
Cell Level ◽  
Strand Breaks ◽  
Two Agents ◽  
Dna Strand

Both X-rays and the radiomimetic agent bleomycin (BLM) induce DNA strand breaks, predominantly via reactive radicals. To compare the induction of breaks with the two agents in Chinese hamster (CHO-K1) cells, two different alkaline unwinding methods, a 3H tracer-based analysis of large cell populations and an optical adaption allowing measurement of single cells, were applied. Radiation and BLM show qualitatively similar dose responses when the average number of DNA strand breaks is measured in a large cell population. However, the breakage pattern at the single-cell level indicates large discrepancies between the actions of the two agents. Irradiated cells show a uniform distribution of DNA strand breaks over the cell population. Effects of treatment with 30 micrograms x ml-1 BLM for 2 hr vary from practically zero in some cells to high levels of DNA strand breakage in others. Unlike the repair of radiation-induced DNA breaks, the repair efficiency of BLM-induced DNA strand breaks, as measured at the single-cell level, varies strongly among cells of the same population. Such heterogeneity at the cellular level potentially reduces BLM's usefulness for tumor therapy because the appearance of BLM-resistant subpopulations may critically impair treatment outcome.

scholarly journals Single-cell transcriptome provides novel insights into antler stem cells, a cell type capable of mammalian organ regeneration

2018 ◽  
Author(s):  
Hengxing Ba ◽  
Datao Wang ◽  
Weiyao Wu ◽  
Hongmei Sun ◽  
Chunyi Li
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Large Cell ◽  
Stem Cell Markers ◽  
Single Cell Level ◽  
Cell Type ◽  
Cell Level ◽  
Homogeneous Population ◽  
Organ Regeneration

AbstractAntler regeneration, a stem cell-based epimorphic process, has potential as a valuable model for regenerative medicine. A pool of antler stem cells (ASCs) for antler development is located in the antlerogenic periosteum (AP). However, whether this ASC pool is homogenous or heterogeneous has not been fully evaluated. In this study, we produced a comprehensive transcriptome dataset at the single-cell level for the ASCs based on the 10x Genomics platform (scRNA-seq). A total of 4,565 ASCs were sequenced and classified into a large cell cluster, indicating that the ASCs resident in the AP are likely to be a homogeneous population. The scRNA-seq data revealed that tumor-related genes were highly expressed in these homogeneous ASCs: i.e. TIMP1, TMSB10, LGALS1, FTH1, VIM, LOC110126017 and S100A4. Results of screening for stem cell markers suggest that the ASCs may be considered as a special type of stem cell between embryonic (CD9) and adult (CD29, CD90, NPM1 and VIM) stem cells. Our results provide the first comprehensive transcriptome analysis at the single-cell level for the ASCs, and identified only one major cell type resident in the AP and some key stem cell genes, which may hold the key to why antlers, the unique mammalian organ, can fully regenerate once lost.

scholarly journals Assessment of Heterogeneity of Cytochrome P450 Activity in Cancer-Cell Population by Cytometry of Reaction Rate Constant is Robust to Variation in Substrate Concentration

2020 ◽  
Author(s):  
Mariana Bleker de Oliveira ◽  
Vasilij Koshkin ◽  
Christopher G. R. Perry ◽  
Sergey N. Krylov
Keyword(s):  
Cytochrome P450 ◽  
Single Cell ◽  
Cell Population ◽  
Rate Constant ◽  
Substrate Concentration ◽  
Reaction Rate ◽  
Reaction Rate Constant ◽  
Single Cell Level ◽  
Cell Level ◽  
Key Players

ABSTRACTEnzymes of the cytochrome P450 (CYP) family catalyze the metabolism of chemotherapeutic agents and are among the key players in primary and acquired chemoresistance of cancer. The activity of CYP is heterogeneous in tumor tissues, and the quantitative characteristics of this heterogeneity can be used to predict chemoresistance. Cytometry of reaction rate constant (CRRC) is a kinetic approach to assess cell population heterogeneity by measuring rates of processes at the single-cell level via time-lapse imaging. CRRC was shown to be an accurate and robust method for assessing the heterogeneity of drug-extrusion activity catalyzed by ABC transporters, which are also key players in cancer chemoresistance. We hypothesized that CRRC is also a reliable method for assessing the heterogeneity of CYP activity. Here, we evaluated the robustness of assessing the heterogeneity of CYP activity by CRRC with respect to controlled variation in the concentration of a CYP substrate by comparing CRRC with non-kinetic approaches. We found that changing the substrate concentration by 20% resulted only in minimal changes in the position, width, and asymmetry of the peak in the CRRC histogram, while these parameters varied greatly in the non-kinetic histograms. Moreover, the Kolmogorov-Smirnov statistical test showed that the distribution of the cell population in CRRC histograms was not significantly different; the result was opposite for non-kinetic histograms. In conclusion, we were able to demonstrate the robustness of CRRC with respect to changes in substrate concentration when evaluating CYP activity at the single-cell level.

Determining DNA strand breaks using the laser scanning microscope

1993 ◽  
Vol 51 ◽  
pp. 272-273
Author(s):  
M.K. Winters ◽  
D.W. Fairbairn ◽  
M.D. Standing ◽  
K.L. O’Neill
Keyword(s):  
Dna Damage ◽  
Single Cell ◽  
Ethidium Bromide ◽  
Laser Scanning ◽  
Accurate Method ◽  
Dna Strand Breaks ◽  
Scanning Microscope ◽  
Strand Breaks ◽  
Dna Breakage ◽  
Dna Strand

The single cell gel assay has been proven to be an effective, fast and accurate method of determining the amount of DNA single or double stranded breaks. The possible uses of this assay have already reached into some aspects of DNA damage caused by chemicals or other destructive agents. It has yet to reach even deeper into mutagenesis and carcinogenesis, both of which may show single or double stranded breaks to the DNA. Here we explore the effects that different concentrations of H2O2 have on the DNA of cells. The assay is performed by suspending cells in a low melt point agarose, spreading the agarose out on a frosted slide and letting it gel over ice. The cells are then treated with H2O2 to induce single strand DNA breakage. The cells are lysed in an alkaline environment, which separates the DNA into single strands and then electrophoresed. The gel is then stained with ethidium bromide.

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