Abstract
Cultured cell populations are composed of heterogeneous cells, and previous single-cell lineage tracking analysis of individual HeLa cells provided empirical evidence for significant heterogeneity of the rate of cell proliferation and induction of cell death. Nevertheless, such cell lines have been used for investigations of cellular responses to various substances, resulting in incomplete characterizations. This problem caused by heterogeneity within cell lines could be overcome by investigating the spatiotemporal responses of individual cells to a substance. However, no approach to investigate the responses by analyzing spatiotemporal data is currently available. Thus, this study aimed to analyze the spatiotemporal responses of individual HeLa cells to cytotoxic, sub-cytotoxic, and non-cytotoxic doses of the well-characterized carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although cytotoxic doses of MNNG are known to induce cell death, the single-cell tracking approach revealed that cell death occurred following at least four different cellular events, suggesting that cell death is induced via multiple processes. We also found that HeLa cells exposed to a sub-cytotoxic dose of MNNG were in a state of equilibrium between cell proliferation and cell death, with cell death again induced through different processes. However, exposure of cells to a non-cytotoxic dose of MNNG promoted growth by reducing the cell doubling time, thus promoting the growth of a sub-population of cells. A single-cell lineage tracking approach could dissect processes leading to cell death in a spatiotemporal manner and the results suggest that spatiotemporal data obtained by tracking individual cells can be used as a new type of bioinformatics data resource that enables the examination of cellular responses to various external substances.
Highlights
Cellular responses to genotoxic insults have been investigated using various end-point analyses that measure alterations induced in cells at a specific moment in time, and deduce the likely cellular responses by evaluating the data obtained at different time points
We referred to cells identified at time point 1 as progenitor cells, and these cells and their progeny were tracked over time and cellular events were identified by morphological observation, which allowed visual detection of all cellular events recorded on the videos, regardless of the molecular events occurring in the cells, to create cell lineage maps (S1–S5 Figs) and a cell lineage database
The cell doubling times of Imaging 2 of Control and MNNG-1μM cells were normalized by a factor of 1.124, and the cell doubling time of Imaging 1 and that of normalized Imaging 2 were subjected to statistical analysis
Summary
Cellular responses to genotoxic insults have been investigated using various end-point analyses that measure alterations induced in cells at a specific moment in time, and deduce the likely cellular responses by evaluating the data obtained at different time points. We used computer-controlled microscopes to create live cell imaging videos, and extracted critical information on individual cells by single-cell tracking to create a cell lineage database [1] Using this database, we empirically showed significant cellto-cell heterogeneity in cultured HeLa cells and demonstrated that the fates of individual cells were diverse, indicating that the HeLa cell line comprises a highly heterogeneous population of cells [1]. The A549 lung carcinoma cell line and mouse embryonic fibroblasts have been shown to comprise heterogeneous cells [3, 4] These observations suggest that individual cells within a cell line may not share similar characteristics, and an accurate determination of cellular responses to genotoxic insults requires investigation of the spatiotemporal responses of individual cells to the insults.
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