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High-dose copper activates p53-independent apoptosis through the induction of nucleolar stress in human cell lines

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Abstract

Copper is an essential micronutrient involved in many redox reactions in human cells. However, a high concentration of copper, intake from the environment or abnormal accumulation within cells because of genetic mutation, leads to cell toxicity. This is attributable to oxidative damage, altered gene expression, and functional impairment of the mitochondria. Copper stress also alters the morphology of the nucleolus, but the process has not been fully elucidated. In this study, cells were treated with copper sulfate at 3–9 ppm and examined if a high dose of copper would block ribosome biogenesis. With the incorrect distribution of nucleolar proteins nucleophosmin and fibrillarin to the nucleoplasm, ribosomal RNA (rRNA) processing was impaired; 34S rRNA from an abnormal A2 cut increased, and downstream pre-rRNAs decreased. The under-accumulation of 60S subunits was detected using sucrose gradients. From transcriptome analysis, ribosome synthesis–related genes were misregulated. Blockage in ribosome synthesis under copper-treatment induced nucleolar stress and triggered p53-independent apoptosis pathways. Thus, nucleolar stress is one cause of cell death under copper exposure.

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All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Acknowledgments

We thank the Join Center for Instruments and Researches, and the College of Bioresources and Agriculture of National Taiwan University, as well as the staff of the Technology commons, College of Life Science, National Taiwan University for technical support. We thank Dr. Louis Grillet for editing this article. This work was financially supported by the Ministry of Science and Technology of Taiwan under contracts No. MOST 106-2313-B-002-031-MY3, MOST 109-2313-B-002-023-MY3 and National Taiwan University Career Development Project (108L7862; 109L7862; 110L7862).

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Authors and Affiliations

  1. Department of Agricultural Chemistry, National Taiwan University, 1 Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan

    Chieh-Hsin Chen, Yi-Ting Chou & Kai-Yin Lo

  2. Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan

    Ya-Wen Yang

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KYL conceived and coordinated the study and wrote the paper. CHC, YTC, and YWY designed, performed, and analyzed the experiments. All the authors reviewed the results and approved the final version of the manuscript.

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Correspondence to Kai-Yin Lo.

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10495_2021_1692_MOESM1_ESM.tif

Supplementary file1 Fig S1. The cell cycle of HEK293T cells is unaffected by Cu(II) exposure Cells treated with different concentrations of Cu(II) were stained with propidium iodide (PI) and analyzed by flow cytometry (n=3) (TIF 5104 kb)

10495_2021_1692_MOESM2_ESM.tif

Supplementary file2 Fig S2 Cell viability of MDA-MB-231 revealed by MTT assay. MDA-MB-231 cells were treated with different concentrations of Cu(II) for 24 h and cell viability was examined by MTT assay (n=6) (TIF 3823 kb)

10495_2021_1692_MOESM3_ESM.tif

Supplementary file3 Fig S3 The protein levels of p53, p21, p27, and E2F1 in HEK293T treated with Cu(II). The protein levels of p53, phospho-p53 (Ser46), phopsho-p53(Ser15), p21, p27, and E2F1 detected by Western blot from three independent experiments were shown (TIF 7584 kb)

10495_2021_1692_MOESM4_ESM.tif

Supplementary file4 Fig S4. KEGG pathway enrichment of 3 ppm Cu(II) DEGs. A graphical representation of the 20 most represented KEGG pathways in DEGs of cells treated with 3 ppm Cu(II). Gene ratio is the ratio of DEGs counts to this pathway in the annotated genes counts. Q-value is the adjusted p-value after multiple hypothesis testing, and its range is [0, 1]. When the Q-value is close to zero, this means the enrichment is more significant (TIF 6951 kb)

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Chen, CH., Chou, YT., Yang, YW. et al. High-dose copper activates p53-independent apoptosis through the induction of nucleolar stress in human cell lines. Apoptosis 26, 612–627 (2021). https://doi.org/10.1007/s10495-021-01692-y

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