DNA damage induces p21 protein expression by inhibiting ubiquitination in ML-1 cells.
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.
Fukuchi
K
K
Department of Clinical Pathology, Showa University, School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan. kfukuchi@med.showa-u.ac.jp
Tomoyasu
S
S
Nakamaki
T
T
Tsuruoka
N
N
Gomi
K
K
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis* / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003561:Cytarabine / Q000494:pharmacology; D004249:DNA Damage; D003676:Deferoxamine / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006918:Hydroxyurea / Q000494:pharmacology; D011132:Polyribosomes / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000037:antagonists & inhibitors* / Q000378:metabolism
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D015853:Cysteine Proteinase Inhibitors; D012333:RNA, Messenger; D014452:Ubiquitins; D003561:Cytarabine; C067713:lactacystin; D005047:Etoposide; D003676:Deferoxamine; D000111:Acetylcysteine; D006918:Hydroxyurea