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 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. 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.
Highlights
Tumor cells arise from normal cells through the accumulation of multiple genetic and epigenetic alterations that positively and negatively regulate aspects of cell proliferation, apoptosis, genome stability, angiogenesis, invasion, and metastasis
homogeneously staining regions (HSR) were the main form of dihydrofolate reductase (DHFR) amplification in response to doses of MTX ranging from 3 Â 10À8 mol/L to 10À6 mol/L as illustrated in fluorescence in situ hybridization (FISH) experiments (Fig. 2C and D)
Most chemotherapeutic failures are due to the development of drug resistance by the tumor cells [3], which usually leads to giving up the chemotherapy regimen
Summary
Tumor cells arise from normal cells through the accumulation of multiple genetic and epigenetic alterations that positively and negatively regulate aspects of cell proliferation, apoptosis, genome stability, angiogenesis, invasion, and metastasis (reviewed in ref. 1). Genetic alterations include point mutations, deletions, inversions, translocations, and DNA sequence amplifications and result in the activation or inactivation of proto-oncogenes or tumor suppressor genes, respectively [1]. Amplified chromosomal regions (amplicons) contain multiple copies of one or more genes and result in their overexpression [2, 3]. Cytogenetic manifestations of gene amplification are mainly of two types: double minutes (DM) and homogeneously staining regions HSRs may be located at the same locus of the original gene or translocated to another chromosome. The relationship between these two types of amplification has been widely discussed but remains uncertain and controversial [2]. All of them have been associated with chromosome double-strand breaks [6] or activation of fragile sites [7]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
