Abstract
After cells have completed a sufficient number of cell divisions, they exit the cell cycle and enter replicative senescence. Here, we report that beryllium causes proliferation arrest with premature expression of the principal markers of senescence. After young presenescent human fibroblasts were treated with 3 μM BeSO4 for 24 h, p21 cyclin-dependent kinase inhibitor mRNA increased by >200%. Longer periods of exposure caused mRNA and protein levels to increase for both p21 and p16(Ink4a), a senescence regulator that prevents pRb-mediated cell cycle progression. BeSO4 also caused dose-dependent induction of senescence-associated β-galactosidase activity (SA-β-gal). Untreated cells had 48 relative fluorescence units (RFU)/μg/h of SA-β-gal, whereas 3 μM BeSO4 caused activity to increase to 84 RFU/μg/h. In chromatin immunoprecipitation experiments, BeSO4 caused p53 protein to associate with its DNA binding site in the promoter region of the p21 gene, indicating that p53 transcriptional activity is responsible for the large increase in p21 mRNA elicited by beryllium. Forced expression of human telomerase reverse transcriptase (hTERT) rendered HFL-1 cells incapable of normal replicative senescence. However, there was no difference in the responsiveness of normal HFL-1 fibroblasts (IC50 = 1.9 μM) and hTERT-immortalized cells (IC50 = 1.7 μM) to BeSO4 in a 9-day proliferation assay. The effects of beryllium resemble those of histone deacetylase-inhibiting drugs, which also cause large increases in p21. However, beryllium produced no changes in histone acetylation, suggesting that Be2+ acts as a novel and potent pharmacological inducer of premature senescence.
Footnotes
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This study was supported by National Institutes of Health Grant P20 RR-016464 from the Idea Networks of Biomedical Research Excellence (INBRE) Program of the National Center for Research Resources.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.118018.
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ABBREVIATIONS: CDK, cyclin-dependent kinase; SA-β-gal, senescence-associated β-galactosidase; HDAC, histone deacetylase; PD, population doubling; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; MUG, 4-methylumbelliferyl-β-d-galactopyranoside; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; TRAP, telomere repeat amplification protocol; PCR, polymerase chain reaction; ChIP, chromatin immunoprecipitation; gapdh, glyceraldehyde-3-phosphate dehydrogenase; Ct, threshold cycle; RFU, relative fluorescence unit(s); RNAP, RNA polymerase II; hTERT, human telomerase reverse transcriptase.
- Received November 30, 2006.
- Accepted March 28, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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