The mdm2 proto-oncogene sensitizes human medullary thyroid carcinoma cells to ionizing radiation.
We have analysed the radiation response of a human medullary thyroid carcinoma cell line (MTT), characterized by the absence of a functional p53 protein, and the consequences of MDM2 overexpression in this process. We show that the product of the mdm2 proto-oncogene is able to sensitize MTT cells to ionizing radiation. After radiation treatment, MTT cells display histograms consistent with a G2M arrest. MTT cells expressing MDM2 (MTT-mdm2) are unable to respond to DNA damage with G2M arrest, and display a high percentage of apoptosis. MTT-mdm2 cells show high levels of E2F-1 protein, suggesting that the induction of apoptosis observed upon MDM2 overexpression could be dependent on E2F-1. This observation is further supported with assays showing that E2F-1 binding to specific DNA sequences is enhanced in MTT-mdm2 cells. Likewise, transactivation of reporter constructs exclusively dependent on E2F-1 is also elevated after transfection with MDM2. This effect can be reverted by transient transfection with p19ARF. To link the expression of E2F-1 with the induction of apoptosis, we generated clonal cell lines overexpressing E2F-1. Transfection with E2F-1 results in a low number of outgrowing colonies with reduced proliferation rates, indicating that E2F-1 is deleterious for cell growth. This negative regulation correlates with an increase in the percentage of the cell population with DNA content below 2N, suggesting that E2F-1 promotes apoptosis. Finally, overexpression of E2F-1 sensitizes MTT cells to radiation exposure. We conclude that the effects observed by MDM2 overexpression could be mediated by E2F-1.
Dilla
Tatiana
T
Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Romero
Jesús
J
Sanstisteban
Pilar
P
Velasco
Juan A
JA
D017209:Apoptosis; D018276:Carcinoma, Medullary / Q000235:genetics / Q000378:metabolism / Q000532:radiotherapy*; D002453:Cell Cycle / Q000528:radiation effects; D018797:Cell Cycle Proteins; D002455:Cell Division; D002470:Cell Survival / Q000528:radiation effects; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D004307:Dose-Response Relationship, Radiation; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D006801:Humans; D009687:Nuclear Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D051736:Proto-Oncogene Proteins c-mdm2; D011839:Radiation, Ionizing; D011838:Radiation-Sensitizing Agents / Q000378:metabolism*; D013964:Thyroid Neoplasms / Q000235:genetics / Q000378:metabolism / Q000532:radiotherapy*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D025803:Tumor Suppressor Protein p14ARF / Q000502:physiology; D015854:Up-Regulation
C417743:Cdkn2a protein, mouse; D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C000715470:MAS1 protein, human; D009687:Nuclear Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011838:Radiation-Sensitizing Agents; D014157:Transcription Factors; D025803:Tumor Suppressor Protein p14ARF; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2