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Updated: Aug 30, 2026
Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts
Published on: July 25, 2020
Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative
Claudia M Hattinger1, Gemma Reverter-Branchat, Daniel Remondini
1Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna, Italy. genetica.onc@ior.it
Abstract:
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.
Insights
Genetic aberrations, including gene amplifications, are linked to methotrexate resistance in osteosarcoma (OS). Identifying these genetic changes may help predict treatment response in OS patients.
Area of Science:
- Oncology
- Genetics
- Pharmacology
Background:
- Methotrexate (MTX) is a key drug for osteosarcoma (OS) treatment.
- Understanding genetic factors driving MTX resistance is crucial for improving patient outcomes.
Purpose of the Study:
- To identify genetic aberrations associated with MTX resistance in OS cells.
- To explore the role of specific genes (DHFR, RFC, MLL, MYC) in MTX response.
Main Methods:
- Comparative Genomic Hybridization (CGH) on chromosomes and microarrays.
- Fluorescence In Situ Hybridization (FISH) for gene confirmation.
- Analysis of gene status (DHFR, MLL, MYC, RFC) in OS cell lines.
Main Results:
- MTX resistance in U-2OS cells correlated with gains in 5q12-q15, 11q14-qter, and the MLL gene (11q23).
- MTX resistance in Saos-2 cells associated with gain in 8q22-qter, MYC (8q24.12-q24.13), and decreased RFC expression.
- Amplification/overexpression of DHFR and gain of MLL were linked to inherent lower MTX sensitivity.
Conclusions:
- Genetic analyses can identify candidate genes for predicting MTX resistance in OS.
- Early identification of MTX-unresponsive tumors may guide treatment strategies.
