title: p53-dependent activation of microRNA-34a in response to etoposide-induced DNA damage in osteosarcoma cell lines not impaired by dominant negative p53 expression.
abstract: 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.
authors.affiliation: Laboratory of Experimental Oncology, Rizzoli Orthopaedic Institute, Bologna, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Novello
authors.affiliation: Laboratory of Experimental Oncology, Rizzoli Orthopaedic Institute, Bologna, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Pazzaglia
authors.affiliation: Laboratory of Experimental Oncology, Rizzoli Orthopaedic Institute, Bologna, Italy.
authors.forename: Amalia
authors.initials: A
authors.lastname: Conti
authors.affiliation: Laboratory of Experimental Oncology, Rizzoli Orthopaedic Institute, Bologna, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Quattrini
authors.affiliation: Laboratory of Experimental Oncology, Rizzoli Orthopaedic Institute, Bologna, Italy; Centre for Molecular and Translational Oncology & Department of Life Sciences- University of Parma, Parma, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Pollino
authors.affiliation: Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Perego
authors.affiliation: Laboratory of Experimental Oncology, Rizzoli Orthopaedic Institute, Bologna, Italy.
authors.forename: Piero
authors.initials: P
authors.lastname: Picci
authors.affiliation: Laboratory of Experimental Oncology, Rizzoli Orthopaedic Institute, Bologna, Italy.
authors.forename: Maria Serena
authors.initials: MS
authors.lastname: Benassi
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D002453:Cell Cycle; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D004249:DNA Damage / Q000187:drug effects* / Q000235:genetics; D019175:DNA Methylation; D005047:Etoposide / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005799:Genes, Dominant; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D012516:Osteosarcoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D005047:Etoposide