Published OnlineFirst February 3, 2009; DOI: 10.1158/1535-7163.MCT-08-0759 424 Dihydrofolate reductase amplification and sensitization to methotrexate of methotrexate-resistant colon cancer cells Cristina Morales,1 Maria J. Garcı´a,4 Maria Ribas,1 withdrawal and reexposure to MTX. Passive loss of the Rosa Miro´,2 Mar Mun˜oz,3 Carlos Caldas,4 DHFR amplicon by withdrawal of the drug results in MTX- and Miguel A. Peinado1,3 sensitive cells exhibiting a substantial reduction of their capacity or even an incapacity to generate resistance when 1Institut d’Investigacio´Biome`dica de Bellvitge, L’Hospitalet; submitted to a second cycle of MTX treatment. On a 2 Institut de Biotecnologia i Biomedicina, Departament de Biologia second round of drug administration, the resistant cells CelÁlular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, Bellaterra; 3Institut de Medicina Predictiva i generate a different amplicon structure, suggesting that the Personalitzada del Ca`ncer, Badalona, Barcelona, Spain and formation of the amplicon as in the first cycle of treatment 4Breast Cancer Functional Genomics Laboratory, Cancer is not feasible. These results indicate that DHFR gene Research UK Cambridge Research Institute and Department of amplification is a ‘‘wear and tear’’ process in HT29 cells Oncology, University of Cambridge, Li Ka Shing Centre, and that MTX-resistant cells may become responsive to a Cambridge, United Kingdom second round of treatment if left untreated during a sufficient period of time. [Mol Cancer Ther 2009; Abstract 8(2):424–32] Gene amplification is one of the most frequent manifes- tations of genomic instability in human tumors and plays an important role in tumor progression and acquisition of Introduction drug resistance. To better understand the factors involved Tumor cells arise from normal cells through the accumu- in acquired resistance to cytotoxic drugs via gene ampli- lation of multiple genetic and epigenetic alterations that fication, we have analyzed the structure and dynamics of positively and negatively regulate aspects of cell prolifer- dihydrofolate reductase (DHFR) gene amplification in HT29 ation, apoptosis, genome stability, angiogenesis, invasion, cells treated with methotrexate (MTX). Analysis of the and metastasis (reviewed in ref. 1). Genetic alterations DHFR gene amplification process shows that the amplicon include point mutations, deletions, inversions, transloca- exhibits a complex structure that is consistently repro- tions, and DNAsequence amplifications and result in the duced in independent treatments. The cytogenetic mani- activation or inactivation of proto-oncogenes or tumor festation of the amplification in advanced stages of the suppressor genes, respectively (1). Gene amplification is treatment may be in the form of double minutes or as a one of the most frequent manifestations of genomic homogeneously stained region. To get insights into the instability in human tumors and plays an important role mechanisms of resistance, we have also investigated the in tumor progression and in the acquisition of drug sensitization to MTX of MTX-resistant cells after drug resistance. 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 (HSR; Received 8/5/09; revised 10/27/09; accepted 11/20/09; ref. 2, 4). DMs are small, spherical, acentric, atelomeric, published OnlineFirst 02/03/2009. usually paired, and autonomously replicating extrachro- Grant support: Spanish Ministryof Science and Innovation (SAF2008/ mosomal structures. HSRs appear as light disruption 1409, SAF2006/0351, and Consolider-Ingenio 2010 CSD2006/49). regions in a normal pattern of alternating dark and light C. Morales was a fellow of Institut d’Investigacio´Biome`dica de Bellvitge. bands in Giemsa staining of metaphase chromosomes. The costs of publication of this article were defrayed in part by the HSRs may be located at the same locus of the original gene payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solelyto or translocated to another chromosome. The relationship indicate this fact. between these two types of amplification has been widely Note: Current address for C. Morales: Biotech Research and Innovation discussed but remains uncertain and controversial (2). Centre, Universityof Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark. Current address for M.J. Garcı´a: Centro Nacional Different mechanisms have been proposed to explain the de Investigaciones Oncolo´gicas, Human Cancer Genetics Programme, dynamics of gene amplification, including breakage- C/Melchor Ferna´ndez Almagro, 3, E-28029 Madrid, Spain. fusion-bridge cycles, unequal sister chromatid exchange, Requests for reprints: Miguel A. Peinado, Institut de Medicina Predictiva i unscheduled rounds of DNAreplication during cell Personalitzada del Ca`ncer, Ctra Can Ruti, Camı´de les Escoles Badalona, 08916 Barcelona, Spain. Phone: 34-935543050; Fax: 34-934978697. division, or replication fork arrest followed by recombina- E-mail: [email protected] tion (3, 5). All of them have been associated with Copyright C 2009 American Association for Cancer Research. chromosome double-strand breaks (6) or activation of doi:10.1158/1535-7163.MCT-08-0759 fragile sites (7). Mol Cancer Ther 2009;8(2). February 2009 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst February 3, 2009; DOI: 10.1158/1535-7163.MCT-08-0759 Molecular Cancer Therapeutics 425 Methotrexate (MTX) and its polyglutamate forms are Our objective was to identify the appropriate chemother- potent competitive inhibitors of the dihydrofolate reduc- apeutic strategy that may improve the efficacy of the tase (DHFR) enzyme, which plays a key role in intracel- treatment for each case. We have focused in MTX resistance lular folate metabolism and is essential for DNAsynthesis through DHFR gene amplification because it is the most and cell growth (8). Combination therapies including MTX common mechanism of resistance (10) and can be easily are currently used to treat several tumor types, including detected at molecular level. In a first setting, we have breast, bladder, and head and neck cancers, osteogenic analyzed the structure and dynamics of DHFR amplifica- sarcoma, and leukemias. MTX is also notable for its use in tion in HT29 MTX-treated cells. Afterwards, we have inflammatory disease, rheumatoid arthritis, and dermato- investigated the sensitization to MTX of MTX-resistant cells logic disorders (9). However, the usefulness of treatment after drug withdrawal and reexposure to MTX (Fig. 1). with MTX is limited by the development of drug These studies shed more light on the mechanisms involved resistance as occurs in most colorectal cancers. Resistance in the generation of drug resistance by gene amplification to MTX may be acquired through different ways, and indicate that chemoresistance may be spontaneously including increased expression of the target gene DHFR reversed and partially impaired, opening new perspectives via gene amplification (10–13), down-regulation of the in pharmacologic regimens. reduced folate carrier (SLC19A1; refs. 14, 15), inefficient polyglutamylation of MTX because of decreased activity of folylpolyglutamate synthase (16), up-regulation of Materials and Methods glutamate hydrolase (17), mutation of DHFR gene result- Cell Lines and MTX Treatment ing in a decreased affinity for MTX (18, 19), or by HT29 colon cancer cell line was obtained from the À epigenetic modulation of genes involved in drug resis- American Type Culture Collection. Three HT29 10 6 tance (20). mol/L MTX-resistant clones (clones 2, 3, and 6) were In colon cancer cells, multiple mechanisms operate in generated in a previous study (22). Cells were propagated MTX resistance. The capacity to develop resistance and the and maintained in T25 flasks (25 cm2) with DMEM-F12 mechanism(s) involved are associated with the genetic (parental cells) and DMEM (clones) culture medium (Life features of the tumor, namely, the karyotype and the Technologies Ltd.) supplemented with 10% fetal bovine presence of a microsatellite mutator phenotype (21–23). serum (Life Technologies) at 37jC in an atmosphere of 5% Figure 1. Schematic diagram of the MTX treatment, generation of resistance, and resensitization to MTX in HT29 colon cancer cells. Left, HT29 cells were submitted to a first round of treatment with stepwise increasing doses of MTX. Five clones generated in two independent treatments (onlyone experiment is represented for simplicity) were characterized at karyotypic and genetic level. Parental HT29 cells contained three intact copies of chromosome, which exhibited instabilityin intermediate states of the treatment ending up in one or two intact chromosome 5 and the appearance of HSR and/or DMs. Middle, after passaging the MTX-resistant cells in the absence of the drug, DHFR copynumber decreased to normal levels and the HSRs and DMs were lost. A single intact chromosome 5 remained. Right, cell clones reexposed to MTX exhibited a decreased capacityto develop resistance as illustrated bythe number of resistant foci. New amplicon structures, including HSR and DMs, were formed in resistant cells. Mol Cancer Ther 2009;8(2). February 2009 Downloaded from mct.aacrjournals.org