Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques
Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques
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DOI：
10.1078/0171-9335-00336
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发表时间：
2003-09-01
影响因子：
6.6
通讯作者：
Serra, M
中科院分区：
文献类型：
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作者：
Hattinger, CM;Reverter-Branchat, G;Serra, M
甲氨蝶呤（MTX）是治疗骨肉瘤的重要药物之一。为了鉴定与OS细胞中MTX耐药性发展相关的遗传畸变，除了先前报道的二氢叶酸还原酶（DHFR）和还原叶酸载体（RFC）基因的表达变化外，还使用了基于比较基因组杂交（CGH）的技术。通过染色体上的CGH直接比较U-20 S或Saos-2人OS细胞系的MTX耐药变异体与其各自的亲本细胞系，揭示MTX耐药的发展与U-20 S变异体中染色体区域5 q12-q15和11 q14-qter的获得相关，并且与Saos-2变异体中8 q22-qter的获得相关。通过微阵列上的CGH进一步分析表明，除了FGR（1 p36）的获得、DHFR的扩增/过表达和RFC表达的轻微降低之外，在U-20 S MTX耐药变体中混合谱系白血病（MLL）基因（11 q23）的获得也逐渐增加，这也通过荧光原位杂交（FISH）证实。在Saos-2 MTX耐药变异体中，检测到MYC（8q24.12-q24.13）的增加，以及RFC表达的显著降低。在另外四种人OS细胞系中进一步分析DHFR、MLL、MYC和RFC基因状态，发现只有DHFR和MLL的获得与固有的对MTX的较低敏感性相关。这些数据表明，互补技术的遗传分析有助于识别新的候选基因，这可能被认为是早期识别MTX无反应的肿瘤。
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-20S 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-20S 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-20S 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.