Dihydrofolate reductase amplification and sensitization to methotrexate of methotrexate-resistant colon cancer cells

Abstract

Gene amplification is one of the most frequent manifestations of genomic instability in human tumors and plays an important role in tumor progression and acquisition of drug resistance. To better understand the factors involved in acquired resistance to cytotoxic drugs via gene amplification, we have analyzed the structure and dynamics of dihydrofolate reductase (DHFR) gene amplification in HT29 cells treated with methotrexate (MTX). Analysis of the DHFR gene amplification process shows that the amplicon exhibits a complex structure that is consistently reproduced in independent treatments. The cytogenetic manifestation of the amplification in advanced stages of the treatment may be in the form of double minutes or as a homogeneously stained region. To get insights into the mechanisms of resistance, we have also investigated the sensitization to MTX of MTX-resistant cells after drug withdrawal and reexposure to MTX. Passive loss of the DHFR amplicon by withdrawal of the drug results in MTXsensitive cells exhibiting a substantial reduction of their capacity or even an incapacity to generate resistance when submitted to a second cycle of MTX treatment. On a second round of drug administration, the resistant cells generate a different amplicon structure, suggesting that the formation of the amplicon as in the first cycle of treatment is not feasible. These results indicate that DHFR gene amplification is a ''wear and tear'' process in HT29 cells and that MTX-resistant cells may become responsive to a second round of treatment if left untreated during a sufficient period of time. [Mol Cancer Ther 2009; 8(2):424 -32]

Frequency of double minute chromosomes and combined cytogenetic abnormalities and their characteristics

“…The hypodiploid group included the most DMspositive cases (27.2%). Morales et al reported that passive loss of the DHFR amplicon by the withdrawal of MTX results in MTX-sensitive cells exhibiting a substantial reduction of their capacity or even an incapacity to generate resistance when submitted to a second cycle of MTX treatment (Morales et al 2009). This phenomenon may be due to chromosome loss.…”

Drug rechallenge and treatment beyond progression—implications for drug resistance

“…Indeed, loss of drug resistance is occasionally reported following drug withdrawal in vitro , 127,129–133 although this mechanism is difficult to demonstrate in vivo . In xenograft models of RCC and hepatocellular carcinoma, tumours that had acquired resistance to sorafenib or sunitinib were shown to become resensitized to treatment after being transplanted into new hosts.…”

Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells

“…Methotrexate (MTX) is an essential drug in curative chemotherapy regimens and is used to treat patients with certain types of cancer including acute lymphoblastic leukemia, osteosarcoma, and choriocarcinoma. MTX works by disrupting DNA synthesis (Barnhart et al 2001); specifically, MTX inactivates the dihydrofolate reductase (DHFR) enzyme, which leads to the depletion of tetrahydrofolate cofactors that are required for DNA and RNA syntheses (Morales et al 2009). …”