Kinetics of the cell biological changes occurring in the progression of DNA damage-induced senescence

Abstract

Cellular senescence is characterized by cell-cycle arrest accompanied by various cell biological changes. Although these changes have been heavily relied on as senescence markers in numerous studies on senescence and its intervention, their underlying mechanisms and relationship to each other are poorly understood. Furthermore, the depth and the reversibility of those changes have not been addressed previously. Using flow cytometry coupled with confocal microscopy and Western blotting, we quantified various senescence-associated cellular changes and determined their time course profiles in MCF-7 cells undergoing DNA damage-induced senescence. The examined properties changed with several different kinetics patterns. Autofluorescence, side scattering, and the mitochondria content increased progressively and linearly. Cell volume, lysosome content, and reactive oxygen species (ROS) level increased abruptly at an early stage. Meanwhile, senescence associated β-galactosidase activity increased after a lag of a few days. In addition, during the senescence progression, lysosomes exhibited a loss of integrity, which may have been associated with the accumulation of ROS. The finding that various senescence phenotypes matured at different rates with different lag times suggests multiple independent mechanisms controlling the expression of senescence phenotypes. This type of kinetics study would promote the understanding of how cells become fully senescent and facilitate the screening of methods that intervene in cellular senescence.

Fig. 1.. Cell cycle arrest in the adriamycin-pulsed MCF-7 cells. MCF- 7 cells were treated with 0.25 μM adriamycin for 4 h and further incubated in fresh medium. At indicated time points, cells were collected and stored in 70% ethanol before being stained with propidium iodide and applied to flow cytometry. (A) The change during the 5-day chase period in the percentage of G1 (—•—), G2/M (---▲---), S (-·-△-·-), and subG1 (···◇···) phases, respectiGely, was plotted. (B) The portions of the G2GM, G, G1, and subG1 phases at 0, 6, 12, 24 h after the adriamycin pulse were plotted in bars. An increase in the number of cells at G2GM phases as well as a dramatic decrease in the G phase were apparent at 12 h point. (C) Cells were lysed in RIPA buffer and the extracts were applied to Western blotting for p53, p21WAF1, E2F1, and Erk (a loading control). Both the time course analyses on the cell cycle distribution and Western blottings were carried out for three biological samples, and representatiGe figures were presented.

Fig. 2.. Expression of GA β-Gal actiGity. (A) The adriamycin-treated MCF-7 cells were stained for GA β-Gal actiGity in situ at day 8. A group of cells stained well in a bright field were taken photo. From this photo, it is clear that a majority of the chased cells underwent remarkable change in size and shape as well as the positiGity for GA β-Gal actiGity clearly demonstrating their being at senescence. (B) GA β-Gal assay was carried out at the indicated time points, and the number of cells positiGe for the actiGity was counted, and the percentage was plotted. The decision on the positiGity is a subjectiGe matter, and therefore, the percentage may get higher, but, the shape of the curGe does not change in a more generous decision on the positiGity. (C) During the chase, 1.8 × 10⁵ cells were collected at indicated time points and lysed, and the extracts were applied to β-galactosidase assay in solution (pH 6.0) using CPRG. The obser- Gance of light at 570 nm was plotted. For both (B, C), more than three independent biological samples were analyzed, and the most representatiGe figures were presented.

Fig. 3.. Increase in cell volume (FSC) and cellular granule content (SSC). (A) MCF-7 cells were pulsed either with 0.25 μM (upper boxes) or 2 μM (lower boxes) adriamycin and chased in its absence for indicated time period and applied to flow cytometry. (A) Dot scatter plots show an increase in both FSC (X-axis) and SSC (Y-axis) in the population pulsed with 0.25 μM, but not in the population pulsed with 2 μM adriamycin. (B, D) Relative mean values of the FSC (B) or SSC (D) of the cells collected at the indicated points were plotted. The graphs were plotted with the numbers averaged from three independent biological repeats. (C) Cells were collected at the indicated time points and their extracts were applied to Western blotting analysis for phosphorylated- or total proteins of S6K and 4EB-BP1. Western analyses were repeated for three independent biological samples, and a typical blot was presented.

Fig. 4.. Increase in autofluorescence and lysosome content. (A, C) The adriamycin-pulsed cells were collected at indicated time points during the chase and applied to flow cytometry either directly or after staining with LysoTracker Red for 30 min. In (C), typical fields were photographed. The graphs were plotted with the numbers averaged from three independent biological repeats. (B) Untreated cells or the cells adriamycin-pulsed and chased for 6 days (day 6) were stained with LysoTracker Red, and applied to confocal microscopy.

Fig. 5.. Loss of lysosomal integrity. (A) MCF-7 cells treated with NH₄Cl or pulse-chased with 0.25 μM adriamycin for 3 or 5 days were stained with acridine orange for 15 min, and processed for confocal microscopy after excitation at 488 nm. Fluorescence emission was observed either through the channels for FITC (for green fluorescence) or Texas Red (for red fluorescence). (B) Human fibroblasts at either an early passage (13) or a late passage (38.5) were processed in the same way.

Fig. 6.. Increase in mitochondrial content and superoxide level, and a decrease in MMP. (A) The adriamycin-pulsed MCF-7 cells were chased for indicated time period, and stained with either MTG (—•—) or NAO (—*—) and applied to flow cytometry to monitor cellular content of mitochondria. MTG and NAO gave rise to the kinetics curves similar to each other except for the abruptly high value for the NAO measurement at day 2 point. This value might have originated from an experimental error since no such high value was obtained in repetition. (B) mRNA isolated from the cells chased for the indicated time points were converted to cDNA. And, the same volume of the cDNA reaction was applied to PCR using primers for PGC-1α, TFAM, NRF-1. The levels of the bands for Lamp1 and β-actin, nonmitochondrial genes, did not change serving as control. (C) MCF-7 cells were chased for indicated time period, stained with JC-1 or MitoSox, and applied to flow cytometry. In the case of the JC-1-stained cells, the ratio of FL2/FL1 was obtained, and mean values were plotted to monitor the change in MMP (C), and for the cells stained with MitoSox, the mean values were directly applied to plot a kinetics curve to monitor the change of the mitochondrial superoxide level (D). All the graphs were plotted with the numbers averaged from three independent biological repeats.

Fig. 7.. Confocal images of the increased mitochondria content and their size of the adriamycinpulsed MCF-7 cells. The adriamycin-pulsed cells were stained with MTG (and counter-stained with DAPI) at indicated time points during the chase period and processed for confocal microscopy.