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Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
Published on: September 28, 2012
DNA damage induces p21 protein expression by inhibiting ubiquitination in ML-1 cells
K Fukuchi1, S Tomoyasu, T Nakamaki
1Department of Clinical Pathology, Showa University, School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan. kfukuchi@med.showa-u.ac.jp
Abstract:
We previously reported that deferoxamine, an iron chelating agent, induced p53 and cell accumulation in the G1 phase of ML-1 cells in the same way as the DNA damaging agent, etoposide. Etoposide treatment increased expression of the p21 gene, a cyclin kinase inhibitor, at both the mRNA and protein levels. However, deferoxamine treatment only increased the p21 mRNA level without the appearance of a detectable protein product. A substrate for cyclin kinase, pRB, was unphosphorylated by etoposide treatment, but remained unaffected by deferoxamine, indicating that p21 was functional after etoposide, but not after deferoxamine treatment. Therefore, in the present study, we investigated the involvement of the ubiquitin proteasome pathway in post-transcriptional regulation of p21. By the addition of lactacystin, a proteasome inhibitor, to deferoxamine treatment, the level of unubiquitinated p21 protein product was similar to that induced by etoposide treatment, and the ubiquitinated p21 bands became apparent. After etoposide treatment, the level of ubiquitinated p21 was diminished and a high level of unubiquitinated p21 expression was observed. We concluded that (1) efficient expression of p21 protein requires inhibition of the ubiquitin-proteasome pathway, and (2) DNA damage inhibits the ubiquitination of p21.
Insights
Deferoxamine increases p21 gene expression but not protein. Inhibiting the ubiquitin-proteasome pathway with lactacystin restores p21 protein, revealing its role in post-transcriptional regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Deferoxamine, an iron chelator, induces cell cycle arrest similar to DNA damaging agents like etoposide.
- Etoposide increases p21 gene and protein expression, while deferoxamine only increases p21 mRNA, suggesting post-transcriptional regulation differences.
Purpose of the Study:
- Investigate the role of the ubiquitin-proteasome pathway in the post-transcriptional regulation of p21 protein.
- Clarify why deferoxamine treatment leads to p21 mRNA increase but not detectable protein.
Main Methods:
- Utilized ML-1 cells treated with deferoxamine, etoposide, and lactacystin (a proteasome inhibitor).
- Analyzed p21 mRNA and protein levels, ubiquitination status, and pRB phosphorylation.
- Compared the effects of etoposide and deferoxamine on p21 expression and degradation pathways.
Main Results:
- Lactacystin treatment with deferoxamine restored p21 protein levels and showed ubiquitinated p21 bands.
- Etoposide treatment diminished ubiquitinated p21 and resulted in high levels of unubiquitinated p21.
- Deferoxamine alone did not lead to functional p21 protein, unlike etoposide.
Conclusions:
- Efficient p21 protein expression is dependent on the inhibition of the ubiquitin-proteasome pathway.
- DNA damage, as induced by etoposide, inhibits the ubiquitination of p21, allowing for protein accumulation.
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