p53-dependent activation of microRNA-34a in response to etoposide-induced DNA damage in osteosarcoma cell lines not impaired by dominant negative p53 expression

• Published in: PloS one Vol. 9; no. 12; p. e114757
• Main Authors: Novello, Chiara, Pazzaglia, Laura, Conti, Amalia, Quattrini, Irene, Pollino, Serena, Perego, Paola, Picci, Piero, Benassi, Maria Serena
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 09.12.2014; Public Library of Science (PLoS)
• Subjects: Antineoplastic Agents - pharmacology; Apoptosis; Base Sequence; Biocompatibility; Biology and Life Sciences; Biotechnology; Blotting, Western; Bone cancer; Bone Neoplasms - drug therapy; Bone Neoplasms - genetics; Bone Neoplasms - pathology; Bone tumors; Breakage; Cell Cycle; Cell Proliferation; Chemotherapy; Chromatin Immunoprecipitation; Conformation; Cyclin-dependent kinase 4; Damage assessment; Deoxyribonucleic acid; DNA; DNA damage; DNA Damage - drug effects; DNA Damage - genetics; DNA Methylation; DNA topoisomerase inhibitors; Etoposide; Etoposide - pharmacology; G1 phase; Gene expression; Gene Expression Regulation, Neoplastic - drug effects; Genes, Dominant; Humans; Medicine and Health Sciences; Methylation; MicroRNA; MicroRNAs - genetics; miRNA; Molecular Sequence Data; Mutation - genetics; Osteosarcoma; Osteosarcoma - drug therapy; Osteosarcoma - genetics; Osteosarcoma - pathology; p53 Protein; Promoter Regions, Genetic - genetics; Real-Time Polymerase Chain Reaction; Research and Analysis Methods; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA, Messenger - genetics; Sarcoma; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured; Tumor proteins; Tumor Suppressor Protein p53 - antagonists & inhibitors; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism; Tumors; United Kingdom > UK; Italy
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0114757

Osteosarcoma (OS) is the most common primary malignant bone tumor and prevalently occurs in the second decade of life. Etoposide, a chemotherapeutic agent used in combined treatments of recurrent human OS, belongs to the topoisomerase inhibitor family and causes DNA breakage. In this study we evaluated the cascade of events determined by etoposide-induced DNA damage in OS cell lines with different p53 status focusing on methylation status and expression of miR-34a that modulate tumor cell growth and cell cycle progression. Wild-type p53 U2-OS cells and U2-OS cells expressing dominant-negative form of p53 (U2- OS175) were more sensitive to etoposide than p53-deficient MG63 and Saos-2 cells, showing increased levels of unmethylated miR-34a, reduced expression of CDK4 and cell cycle arrest in G1 phase. In contrast, MG63 and Saos-2 cell lines presented aberrant methylation of miR-34a promoter gene with no miR-34a induction after etoposide treatment, underlining the close connection between p53 expression and miR-34a methylation status. Consistently, in p53siRNA transfected U2-OS cells we observed loss of miR-34a induction after etoposide exposure associated with a partial gain of gene methylation and cell cycle progress towards G2/M phase. Our results suggest that the open and unmethylated conformation of the miR-34a gene may be regulated by p53 able to bind the gene promoter. In conclusion, cell response to etoposide-induced DNA damage was not compromised in cells with dominant-negative p53 expression.