High concentrations of NaCl induce cell swelling leading to senescence in human cells

Abstract

Cell swelling and retardation in DNA replication are always observed in senescent cells. When DNA replication is slowed down with RNA and protein syntheses unchanged in proliferating cells, it causes a phenomenon known as unbalanced growth. The purpose of this study is to assess the role of cell swelling in unbalanced growth in terms of senescence and investigate the mechanism underlying this phenomenon. We tried to induce cell swelling with minimum damage to cells in this study. We perturbed the osmoregulatory functions to induce cell swelling under hypotonic and hypertonic conditions in normal human fibroblasts. Addition of excess NaCl was found to induce significant cell and nuclear swelling in dose- and time-dependent manners. Excess NaCl immediately retarded DNA replication, accumulated cells at G1 phase of the cell cycle, and eventually deprived division potential of the cells. Such cells showed typical senescent cell shape followed by expression of the typical senescence-associated genes. Excess NaCl also activated ERK1/2, p38, and JNK of the mitogen activated protein kinase family. Addition of U0126, an inhibitor of ERK1/2, prevented appearance of senescent features induced by excess NaCl. These results suggest that hypertonic conditions induce cell swelling due to unbalanced growth, thereby leading to cellular senescence.

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Correspondence to Dai Ayusawa.

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Yoshimi Yamakami and Ryuzo Yonekura have contributed equally to this work.

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a

Supplementary material 1 (PPT 378 kb) Growth properties in SUSM-1 and HeLa cells cultured under hypertonic conditions. Cells were cultured for the time indicated, and growth (), viability (b), and the activity of DNA synthesis (c) were determined as described in “Materials and methods” section. Open circles, no addition; closed circles, addition of 0.1 M NaCl. Averages ± SD for three separate measurements are shown relative to control

a

Supplementary material 2 (PPT 430 kb) Measurements of size markers in SUSM-1 and HeLa cells cultured under hypertonic conditions. Cells were cultured for 2 weeks, and processed to determine cell size (), protein content per cell (b), and nuclear size (c) as described in “Materials and methods” section. None, control; NaCl, 0.1 M NaCl. The values are the means of 100 cells and the error bars represent standard deviations

Supplementary material 3 (PPT 604 kb) Visualization of actin filaments in HeLa cells cultured under hypertonic conditions. Cells stably expressing GFP-actin were cultured for 2 weeks with addition of 0.1 M NaCl, and subjected to fluorescence microphotography. Bars, 100 μm. (n = 2)

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Supplementary material 4 (PPT 308 kb) Analysis of the senescence-associated genes in SUSM-1 and HeLa cells cultured under hypertonic conditions. Cells were cultured for the time indicated, and Northern blot analysis was performed with the probes indicated as described in “Materials and methods” section (). Relative mRNA levels were normalized with those of GAPDH and expressed relative to day 0 (b). Two independent experiments gave similar results

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Yamakami, Y., Yonekura, R., Matsumoto, Y. et al. High concentrations of NaCl induce cell swelling leading to senescence in human cells. Mol Cell Biochem 411, 117–125 (2016). https://doi.org/10.1007/s11010-015-2573-1

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Keywords

  • Cell swelling
  • Cellular senescence
  • Hypertonic stress
  • Unbalanced growth