Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques.
Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.
Hattinger
Claudia M
CM
Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna, Italy. genetica.onc@ior.it
Reverter-Branchat
Gemma
G
Remondini
Daniel
D
Castellani
Gastone C
GC
Benini
Stefania
S
Pasello
Michela
M
Manara
Maria C
MC
Scotlandi
Katia
K
Picci
Piero
P
Serra
Massimo
M
D000970:Antineoplastic Agents / Q000494:pharmacology; D001859:Bone Neoplasms / Q000188:drug therapy / Q000235:genetics*; D002869:Chromosome Aberrations; D002877:Chromosomes, Human / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D006801:Humans; D007621:Karyotyping; D026901:Membrane Transport Proteins / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D012516:Osteosarcoma / Q000188:drug therapy / Q000235:genetics*; D011003:Ploidies; D058978:Reduced Folate Carrier Protein; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate