title: Mechanisms of gene amplification and evidence of coamplification in drug-resistant human osteosarcoma cell lines.
abstract: Gene amplification and copy number changes play a pivotal role in malignant transformation and progression of human tumor cells by mediating the activation of genes and oncogenes, which are involved in many different cellular processes including development of drug resistance. Since doxorubicin (DX) and methotrexate (MTX) are the two most important drugs for high-grade osteosarcoma (OS) treatment, the aim of this study was to identify genes gained or amplified in six DX- and eight MTX-resistant variants of the human OS cell lines U-2OS and Saos-2, and to get insights into the mechanisms underlying the amplification processes. Comparative genomic hybridization techniques identified amplification of MDR1 in all six DX-resistant and of DHFR in three MTX-resistant U-2OS variants. In addition, progressive gain of MLL was detected in the four U-2OS variants with higher resistance levels either to DX or MTX, whereas gain of MYC was found in all Saos-2 MTX-resistant variants and the U-2OS variant with the highest resistance level to DX. Fluorescent in situ hybridization revealed that MDR1 was amplified in U-2OS and Saos-2/DX-resistant variants manifested as homogeneously staining regions and double minutes, respectively. In U-2OS/MTX-resistant variants, DHFR was amplified in homogeneously staining regions, and was coamplified with MLL in relation to the increase of resistance to MTX. Gene amplification was associated with gene overexpression, whereas gene gain resulted in up-regulated gene expression. These results indicate that resistance to DX and MTX in human OS cell lines is a multigenic process involving gene copy number and expression changes.
authors.affiliation: Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna, Italy.
authors.forename: Claudia M
authors.initials: CM
authors.lastname: Hattinger
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Stoico
authors.forename: Francesca
authors.initials: F
authors.lastname: Michelacci
authors.forename: Michela
authors.initials: M
authors.lastname: Pasello
authors.forename: Isabella
authors.initials: I
authors.lastname: Scionti
authors.forename: Daniel
authors.initials: D
authors.lastname: Remondini
authors.forename: Gastone C
authors.initials: GC
authors.lastname: Castellani
authors.forename: Marilù
authors.initials: M
authors.lastname: Fanelli
authors.forename: Katia
authors.initials: K
authors.lastname: Scotlandi
authors.forename: Piero
authors.initials: P
authors.lastname: Picci
authors.forename: Massimo
authors.initials: M
authors.lastname: Serra
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D055028:Comparative Genomic Hybridization; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005784:Gene Amplification; D018628:Gene Dosage; D015972:Gene Expression Regulation, Neoplastic; D052138:Genes, Neoplasm; D016259:Genes, myc; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008727:Methotrexate / Q000494:pharmacology; D051788:Myeloid-Lymphoid Leukemia Protein / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D012516:Osteosarcoma / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; C497422:KMT2A protein, human; D051788:Myeloid-Lymphoid Leukemia Protein; D004317:Doxorubicin; D013762:Tetrahydrofolate Dehydrogenase; D011495:Histone-Lysine N-Methyltransferase; D008727:Methotrexate