ABSTRACT
The promyelocytic leukemia (PML) can selectively and dynamically recruit a number of proteins including p53 to form a sub-nuclear multiprotein chamber named PML-NBs. In DNA damage response, p53 is recruited into PML-NBs and modified by phosphorylations and acetylations, which in turn potentiate its transcriptional and pro-apoptotic activities. In contrast, in carcinoma cells, the role of PML in the irradiation induced p53-mediated apoptosis is not precisely understood. In this study, we have used the breast carcinoma cell line, MCF-7, and stably suppressed the expression of PML. Inhibition of PML expression had no detectable effect on the expression of endogenous p53 at the mRNA level; however, a significant decrease of p53 protein was observed. There was also an increase in the p53-MDM2 complexes, which may facilitate p53 protein degradation by the ubiquitin-proteasome pathway, also in irradiation treated cells. The p53 transcriptional activity was attenuated both in unstressed and 10 Gy irradiation treated cells. Moreover, inhibition of PML expression in MCF-7 cells significantly reduced p53 downstream genes, cell cycle arrest gene p21(WAF/cip-1) and pro-apoptotic gene Bax expression, then irradiation-induced apoptosis. These results suggest that PML is a key regulator in the irradiation activated p53 apoptotic pathway in breast carcinoma cells.
Subject(s)
Apoptosis/radiation effects , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Neoplasm Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Signal Transduction , Transcription Factors/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/antagonists & inhibitors , Cobalt Radioisotopes , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Down-Regulation , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic , Humans , Luciferases/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Promyelocytic Leukemia Protein , Proteasome Endopeptidase Complex , Proto-Oncogene Proteins c-mdm2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Ubiquitin/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.
