Increased mdm-2 expression in a p53-independent manner blocks UV-induced cell cycle arrest and apoptosis in human osteosarcoma cells.
DNA damage results in an increase in P53 levels, which is required to initiate a P53-mediated cell cycle arrest and/or apoptosis. P53 and MDM-2 form a feedback control loop: while P53 can transactivate the MDM-2 gene, high levels of MDM-2 inhibit P53 transactivation as well as promote rapid degradation of P53. In the present study, we investigated the interaction between endogenous MDM-2 and P53 following UV-induced DNA damage in an MDM-2 overexpression cell line. A human osteosarcoma cell line (OsACL, which contains wild-type P53 and overexpresses MDM-2 protein) was used in this study. Here we show that following UV treatment, P53 levels increased in the OsACL cells despite the presence of high-level endogenous MDM-2; however, CAT assays using a P53 reporter system revealed that this P53 was transcriptionally inactive. Although P53 transactivation was inhibited, MDM-2 levels rose markedly following UV irradiation. Northern blot analysis revealed that the increase in MDM-2 protein levels was a result of increased levels of MDM-2 mRNA, possibly due to increased transcription. Cell cycle analysis revealed that OsACL cells were markedly resistant to UV-induced apoptosis. Transfection of OsACL cells with an anti-sense MDM-2 plasmid dowregulated MDM-2 expression and increased UV-induced apoptosis. In conclusion, MDM-2 overexpression can block UV-induced cell cycle arrest and apoptosis by inhibiting P53 transcriptional activity. Furthermore, increased expression of MDM-2 in OsACL cells following UV irradiation appears to be related to P53-independent mechanisms.
Dong
Yan Bin
YB
Department of Surgery, University of Louisville, James Graham Brown Cancer Center, Louisville, KY 40202, USA.
Yang
Hai Liang
HL
Elliott
Mary Jane
MJ
McMasters
Kelly M
KM
D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology; D012516:Osteosarcoma / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*; D014466:Ultraviolet Rays; D015854:Up-Regulation
D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D015500:Chloramphenicol O-Acetyltransferase; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2