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.

Influence of Reduced Folate Carrier and Dihydrofolate Reductase Genes on Methotrexate-Induced Cytotoxicity

“…Several resistance mechanisms of MTX have been proposed; impaired cellular uptake and efflux, decreased formation of MTXpolyglutamates (MTX-PGs) and reduced binding affinity to its target, DHFR etc (13,14). These mechanisms could eventually be classified into two major phenomenons: 1) the increased DHFR enzyme level due to DHFR gene over-expression and 2) the reduced intracellular transport of MTX at the cell membrane (3).…”

Targeting polo-like kinase 1 by NMS-P937 in osteosarcoma cell lines inhibits tumor cell growth and partially overcomes drug resistance

“…Since the most relevant mechanism of drug resistance in these cell lines is overexpression of ABCB1 [19,16], we verified whether NMS-P937 can interact with this membrane transporter. Inhibition of ABCB1 expression and activity obtained, respectively, through gene silencing and treatment with ABCB1 inhibitor CBT-1 [20,21], produced an increased sensitivity to NMS-P937, clearly showing that ABCB1 negatively interferes with activity of this drug.…”

“…Groups of high expressors (HIGH) included patients showing PLK1 expression levels equal or higher than the cutoff value DX and NMS-P937 were used in association in DX-resistant variants. Since the most relevant mechanism of drug resistance developed by these cell lines is overexpression of ABCB1 [19,16], additional experiments were performed in order to verify whether NMS-P937 may interact with this membrane transporter.…”

Osteosarcoma: From Molecular Biology to Mesenchymal Stem Cells

“…Overexpression of Myc in bone marrow stromal cells leads to osteosarcoma development and loss of adipogenesis [32]. This factor is ampliied in U2OS osteosarcoma cell line variants with the highest resistance to doxorubicin and gain of Myc was found in SaOS-2 methotrexate-resistant variants [33].…”