Dihydrofolate Reductase Amplification and Sensitization to Methotrexate of Methotrexate-Resistant Colon Cancer Cells

Published OnlineFirst February 3, 2009; DOI: 10.1158/1535-7163.MCT-08-0759 Published Online First on February 3, 2009 as 10.1158/1535-7163.MCT-08-0759

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

Cristina Morales,1 Maria J. Garcıá,4 Maria Ribas,1 withdrawal and reexposure to MTX. Passive loss of the Rosa Miro´,2 Mar Munõz,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 manifestations 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. Garcıá: 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 Fernańdez 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).

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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.

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CO2. MTX treatment was done in triplicate experiments by control genes. cDNAs were prepared by reverse transcrip- exposure to stepwise increasing concentrations of MTX tion with Moloney murine leukemia virus (Invitrogen) (Supplementary Table S1).5 MTX-resistant clones were using random hexamer primer (Amersham) according to À maintained with 10 6 mol/L MTX. standard procedures. Primer sequences (Supplementary For sensitization studies, MTX-resistant cells (clone 2) Table S4)5 were designed using PrimerSelect 3.1 program. were passaged 35 times in the absence of MTX. Afterwards, Duplicate PCR was done in a LightCycler as described clones were obtained by limiting dilution (clones E, M, N, above and using 62 ng cDNA(except for 18S and PPIAthat and P), grown independently, and passaged six more times were 0.2 and 7.8 ng, respectively). in the absence of the cytotoxic drug. For the second cycle of Fine Characterization of the Breakage Site treatment, 105 cells of each clone and the parental HT29 PCR amplification was done using 20 ng DNAin a cells (as a control) were seeded in 25 cm2 T-flasks and a reaction volume of 25 AL containing 1 Amol/L of each second cycle of MTX treatment was applied. This treatment primer, 1 unit Taq DNApolymerase (Roche Diagnostics), was done in triplicate as a stepwise treatment with 1 mmol/L of deoxynucleotide triphosphate mix, and the À increasing concentrations of MTX from 10 8 mol/L to PCR buffer recommended by the Taq manufacturer. Primer À 10 7 mol/L in identical conditions to that of the first round sequence and PCR conditions are detailed in Supplemen- of treatment. tary Table S5.5 PCR products were resolved on a 6% Fluorescence In situ Hybridization polyacrylamide denaturing gel and silver stained, purified Chromosomes 8q22qter, 5p12, and 5q13-14 probes used using a Concert Rapid PCR Purification System (Life for fluorescence in situ hybridization (FISH; Supplementary Technologies), and sequenced using a BigDye version Table S2)5 were obtained from RPCI-11 human male 3.1 sequence kit (Applied Biosystems) according to the bacterial artificial chromosome (BAC) libraries [Wellcome manufacturer’s instructions. Sequence homologies were Trust Sanger Institute (Hinxton, United Kingdom) and the searched using the Blat engine.6 Children’s Hospital (Oakland, CA)] and BAC clone collections of Invitrogen Life Technologies. FISH was done as described (24). Abrief description is provided as Results Supplementary Data. Characterization of a Preexisting Amplicon DNA and RNA Extraction Previous studies have shown the presence of an DNAwas obtained using proteinase K/RNase treatment amplification at 8q24 involving MYC oncogene in HT29 and phenol-chloroform extraction according to standard cells (28). We have confirmed the karyotypic features of this procedures (25). Total RNAwas obtained by using the cell line by using conventional comparative genomic guanidinium-isothiocyanate method followed by phenol- hybridization (CGH) and FISH (22) and CGH array (data chloroform extraction and isopropanol precipitation (26). not shown). Ahigh-level gain affecting a 25-Mb region at RNAwas reeluted in water, quantified spectrophotomet- À j 8q23qter was observed (Supplementary Fig. S1; Supple- rically, and stored at 80 C. mentary Table S6).5 FISH analysis revealed MYC inter- Real-time PCR Quantification of Gene Copy Number spersed signals in both arms of a metacentric marker Gene copy number at the amplicon boundaries was chromosome 8 with four to five copies per arm (Fig. 2A determined by quantitative PCR using a LightCycler and B). Amplicon repeats seem to have head-tail orienta- (Roche Diagnostics). Primer sequences (Supplementary MYC 5 tion (Fig. 2Aand B). overexpression (as compared Table S3) were designed using PrimerSelect 3.1 program with normal colonic mucosa) was detected by real-time (DNASTAR, Inc.). PCR was done in duplicate and using PCR (data not shown). Interestingly, the amplicon also A 4 ng DNA, 2.5 mmol/L MgCl2, 0.25 mol/L forward and included other genes that may be involved in proliferation reverse primer, and the LightCycler Mix (FastStart DNA and tumorigenesis (EIF3S3, RAD21, TAF2, MTBP, MLZE, Master SYBR Green I; Roche Diagnostics) as recommended and WISP1). by the manufacturer. Standard PCR conditions were used Dynamics of Amplicon Formation and Resistance 5 B2M (Supplementary Table S3). was used as control gene to MTX and the copy number was calculated using a standard The DHFR genomic region was characterized at cytoge- curve derived from serial dilutions of normal human À2 À3 À4 À5 À6 netic and molecular level in HT29 cells treated with control DNA(10 ,10 ,10 ,10 ,and10 )and increasing doses of MTX. To determine the dynamics of normalized as described (27). the process, the intermediate stages were investigated Real-time PCR Quantification of Gene Expression (Fig. 1). HSRs were the main form of DHFR amplification DHFR, hMSH3 RASGRF2 À Gene expression of genes , and in response to doses of MTX ranging from 3 Â 10 8 mol/L À included in the amplicon was determined by quantitative to 10 6 mol/L as illustrated in FISH experiments (Fig. 2C 18S cyclophilin 1 PPIA real-time PCR. and ( ) were used as and D). HSRs appeared as different combinations of asymmetric chromosome 5, some of them with more copies

5 Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). 6 http://genome.ucsc.edu/

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of DHFR in one chromatid than in the other (Fig. 2C and sudden increase of the DHFR copy number that reached up D). DMs were only prevalent after cells were maintained in to 80-fold (clone 1). DHFR gene expression levels paralleled the higher dose of drug for 3 months (Supplementary the degree of amplification reaching up to 100-fold in one Table S1).5 On the other hand, one of the clones (clone 1) of the clones (Supplementary Table S1).5 exhibited DMs containing the DHFR amplicon at relatively Characterization of 5q14 Amplification and Its À low doses of MTX (10 7 mol/L), which extended to near Boundaries À 100% of the cells when 10 6 mol/L MTX doses were Analysis of HT29 MTX-resistant clone 2 with Spectral reached (Supplementary Table S1).5 Genomics BAC array (1-Mb resolution) showed significant À At the highest MTX dose (10 6 mol/L), the DHFR gene gains in two BACs (Supplementary Fig. S2),5 indicating that copy number amplification was 4-fold (clone 4) in the form the size of the amplicon was below 2 Mb. The low- of HSR, whereas the appearance of DMs resulted in a resolution CGH array platform (472-clone genomic array

Figure 2. FISH analysis of 5q14.1 and 8q23qter amplicons in MTX- resistant HT29 cell clones. FISH analysis of 8q23qter amplicon using RP11-28I2 (8q24.21) and RP11- 65A5 (8q24.3) labeled in green and red, respectively( A), and RP11- 2K18 (8q23.3) and RP11-440N18 (8q24.21) labeled in green and red, respectively( B). Two normal chromosome 8 and a metacentric marker chromosome8withfourtofive amplicon copies per arm are observed in both images. C, asymmetric distribution of DHFR label (green) could be observed in chromosome 5 in earlystages of the MTX treatment. MYC probe (red) hybridization shows multiple signals in a metacentric marker chromosome 8. D, additional examples of asymmetric copy number distribution of DHFR labels in chromosome 5 are illustrated, including a telomeric break point of amplicon 5q14.1 down of the DHFR signal (red arrowhead). E, FISH analysis of 5q14.1 in advanced stages of the treatment. RP11- 241J12 (green) and RP11-79I10 (red) probes cohybridize over all DMs, mapping inside the amplified region. F, BAC CTC-512J14 (green) maps outside the amplified region and delimits the centromeric break point of 5q14.1 amplicon, giving a single signal per chromosome.

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Figure 3. Strategyfor the characterization of 5q14.1 amplicon boundaries and breakage sites. PCRs with different set of primers were used to delimit the amplicon boundaries and to identifypossible fusion points. A, PCRs P5, P6, P7, P8, P9, P10, and P11 result in specific products from the linear conformation of the genomic DNA included in the amplicon. B, PCRs P1, P2, P3, and P4 will onlyproduce a specific PCR band if two breaking points near the boundaries of the amplicon are fused or form circular structures. The telomeric breakage point was fused with a duplicated sequence ATTTTAC at the joint point. PCR P4 confirmed the absence of this fusion point in parental HT29 cells and its reiterative formation in all the HT29 clones treated once with MTX. Noteworthy, this fusion point was not detected in clones M and P obtained after a second treatment with MTX, indicating the loss of the preexisting reorganization and the formation of a new amplicon with a different structure.

from Hutchinson/MRC Research Center) was not infor- that are amplified and CTC-512J14 and CTD-2113H21 that mative at this level of resolution (1 Mb). FISH analysis with show no coamplification with the DHFR locus; Supple- a collection of 11 BACs (Supplementary Table S2)5 was mentary Fig. S2).5 Astepwise targeting strategy was used done to get a more accurate definition of the involved to set the limits of the amplicon. These corresponded to two region. The amplified region was common to all three regions of 1,130 and 353 bp representing the putative MTX-resistant clones (2, 3, and 6), spanned f1.078 Mb centromeric and telomeric break points, respectively (Supplementary Fig. S2),5 and included BACs CTD-2327L5, (Supplementary Table S8).5 Analysis of the features of RP11-241J12, and RP11-79I10 (Fig. 2E and F). The amplicon these two genomic regions revealed the presence of included the genes ZFYVE16, DP58, DHFR, MSH3, different repetitive elements in both. The centromeric side RASGRF2, CKMT2, CACH-1, ZCCHC9, UNQ9217,and contained an AluSx element flanked by two LINE SSBP2 (Supplementary Fig. S2).5 Real-time reverse tran- sequences. In the telomeric side, two inverted Alu repeats scription-PCR showed the concomitant overexpression of of the Jb and Sx families were separated by 75 bp (Fig. 3). DHFR, MSH3, and RASGRF2 genes in all the three clones In an attempt to characterize the fusion points of the (Supplementary Table S7).5 amplicon, different PCRs were done using primers Amore precise characterization of the amplicon bound- designed to amplify putative circularized or inverted aries was done by quantitative real-time PCR of sequences structures (this is by using one primer on each side of the at the amplicon limits (BACs CTD-2327L5 and RP11-79I10 amplicon but oriented outwards; Supplementary Table S5;5

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À Fig. 3). Only in one case (P0 PCR; Supplementary Table (3 Â 10 7 mol/L) was administered and all cells died after S5),5 a specific PCR product was obtained allowing us to 2 weeks, indicating a loss of the MTX resistance capacity. identify an inverted reorganization at the telomeric Four different clones were submitted to a new MTX breakage site of the amplicon, near the two inverted Alu treatment that was applied in triplicate experiments and repeats (Fig. 3). The presence of this reorganized sequence in identical conditions than the first treatment. Two of the was confirmed with a second set of primers (PCR P4; clones (E and N) were unable to develop resistance and all Supplementary Table S5).5 The resulting fusion point cells died after 48 and 28 days, respectively. An average of between the two reorganized regions is a duplication of seven to eight resistant colonies was observed in the other the sequence ATTTTAC present in both regions. two MTX-treated clones (M and P). Parental HT29 cells Sensitization to MTX of HT29 MTX-Resistant Clones submitted to the same treatment presented a higher Because gene amplification involves reorganization of survival rate with an average of 42 resistant clones per the affected chromosome, we wondered if this de novo flask (Table 1). structure will be stable in cells after withdrawal of the drug, As expected, quantitative PCR analysis of resistant clones or rather, the copy number of the DHFR gene would be confirmed an increased DHFR copy number in response to restored to normal levels. And if this were the case, would MTX (Table 1). G-banding and FISH analysis of resistant resulting cells be more or less resistant to a second cycle of M and P clones showed a reorganized chromosome 5 treatment? As explained above, the different MTX-resistant [del(5)(q11.2)], one normal chromosome 5 with its clones exhibited a high level of DHFR amplification in the corresponding DHFR signal, and DHFR amplification as form of DM structures (Table 1). Moreover, two cell DMs (Fig. 4D and E) or as a HSR in 14.3% and 21.4% of populations could be observed with regard to chromosome clones M and P metaphases, respectively (Fig. 4F). HSR 5: type A, containing one normal and two reorganized mapped in chromosome 5. Moreover, DHFR signals at chromosome 5 (Fig. 4A), and type B, displaying two normal chromosomes other than chromosome 5 were observed and one reorganized chromosome 5 (Fig. 4B). Because all along with chromosome 5 without a DHFR signal, MTX-resistant clones exhibited the same amplicon struc- suggesting the reorganization of chromosome 5 (data not ture, similar levels of amplification, and MTX resistance, shown). the sensitization studies were done in clone 2 cells. Next, we wondered if the amplicon generated during the Single-cell clones were obtained from MTX-resistant cells second MTX treatment had the same structure that the one by limiting dilution, and those exhibiting the type A produced in the first treatment. Initially, we investigated if karyotype were cultured for 41 passages in the absence of clones M and P (second MTX treatment) contained the MTX. FISH analysis using RP11-241J12 BAC showed that fusion point present in the amplicon of all clones analyzed all clones contained a single complete chromosome 5 and in the first MTX treatment. PCR P4 that specifically had lost all DM chromosomes (Fig. 4C). Quantitative PCR amplifies the fused sequence (see above) failed to produce also confirmed that DHFR copy number was restored to a product from clones E and M (Fig. 3), indicating the levels similar to untreated parental cells (Table 1). To de novo nature of the amplicon resulting from the second ascertain the sensitivity of these cells to MTX, a single dose cycle of MTX treatment. These results suggest the involve- ment of a different mechanism or different sequences in the reorganization process. By quantitative real-time PCR of Table 1. Copy number of DHFR in parental and MTX-resistant HT29 cells receiving a second round of MTX treatment multiple sequences distributed along 6 Mb around the DHFR gene, we showed that MTX-resistant clones M and P DHFR copy DHFR copy Resistant had a larger amplicon than clones generated in the first number at the number after focic treatment (Fig. 5). Moreover, unlike resistant clones initiation of the second obtained in the first treatment that exhibited identical the second treatment* amplicon extension, clones M and P displayed different treatment amplicon size (Fig. 5).

Parental 0.51/0.80 ND 42 b Clone 2 23.92 19.16 Full Discussion Clone E 0.62 Not resistant 0 Clone M 0.87 10.16 7 Gene amplification as a mechanism of drug resistance is a Clone N 1.18 Not resistant 0 stepwise selection process in which cells become resistant Clone P 1.53 6.89 8 through repeated cycles of cell death and proliferation accompanied by genomic instabilization and successive NOTE: Relative values respect peripheral blood DNA(1 corresponds to a cycles of gene copy number gains (29). Most chemothera- diploid DHFR copy number). No correction for the ploidy of the cell has been applied. peutic failures are due to the development of drug Abbreviation: ND, not determined. resistance by the tumor cells (3), which usually leads to À *Increasing concentrations of MTX up to 10 7 mol/L as described in giving up the chemotherapy regimen. To design alternative Materials and Methods. strategies, it is necessary to investigate the mechanisms and cNumber of colonies in a T25 flask seeded with 105 cells after the treatment À with 10 7 mol/L MTX. dynamics of gene amplification, together with its repeat- bMTX-resistant clone maintained in MTX. ability and reversibility.

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Figure 4. Analysis of 5q14.1 amplicon in MTX-resistant HT29 cell clones after one or two rounds of treatment. A, FISH analysis using RP11-241J12 BAC probe revealed the presence of two cell subpopulations in HT29 MTX-resistant clone 2. One of the subpopulations presents a single chromosome 5 (A), whereas the other shows two normal chromosome 5 (B). Additional copies of the locus appear in the form of DMs in both cases. Clone M cells derived from MTX-resistant cells were maintained in the absence of the drug. C, DMs were lost and a single chromosome 5 remained (arrow). After a new round of MTX treatment, new DMs were generated as shown after Leishman staining (D) or byFISH ( E). F, alternatively, some cells exhibited HSR carrying the DHFR amplicon.

DHFR gene amplification occurs through a preferential DMs probably occurs following HSR breakdown, but the process in HT29 cells as shown by the common structure of presence of DMs at intermediate doses of MTX and a low the amplicon generated in independent treatments done frequency of previous HSRs suggests that DMs do not with parental or derived clones. Interestingly, besides this necessarily represent a later stage of the amplification common structure at genomic level, the cytogenetic process. manifestation may be different and HSR and DM may HSRs formed at the intermediate steps of MTX treatment appear in different phases of the treatment. In any case, often seemed associated with different chromosome 5 with DMs are associated with maintained resistance to high asymmetric signals for DHFR at the two chromatids doses of MTX. Although the concurrence of HSR and DMs (Fig. 2D). This fact points to an unequal sister chromatid was observed in some cells, the alternative prevalence of homologue recombination as the initial amplification one or the other form of gene amplification was the most mechanism (30, 31). It is possible that the inverted Alus common situation. Our data indicate that the formation of found near the telomeric breakage point confer instability

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Figure 5. Accurate analysis of 5q14.1 amplicon boundaries by real-time PCR. Light squares denote low copy number similar to that observed in parental HT29 cells. Dark squares denote high copynumber and indicate the extent of the amplicon after a first cycleof MTX treatment (clones 2, 3, and 6) and after two cycles of MTX treatment (clones M and P). to this region, promoting complementary interactions in reorganized, whereas the other will remain stable (39). the same strand and facilitating a hairpin or a secondary Analysis of HT29 MTX-resistant clones 2, 3, 6, 1, and 4 structure formation leading to a unequal recombination showed that the generation of the amplicon involved the between chromatids (Fig. 2C and D; refs. 32–34). The small reorganization as marker chromosomes of one or (more asymmetric intrachromosomal repeats would ultimately often) two of the intact chromosome 5. As expected, MTX- split out by recombination mechanisms (homologous resistant clones deprived of MTX reverted to a low copy recombination or nonhomologous end joining) to smaller number of the DHFR gene and maintained a unique intact extrachromosomal DMs, which suggests the participation chromosome 5. Interestingly, these cells showed a of different mechanisms during the amplification process decreased ability to generate resistance in a second cycle (35, 36). The telomeric boundary of the amplicon involved a of treatment: two clones died and the other two generated direct repeat sequence, which suggests an inverted recom- a fifth of resistant colonies compared with HT29 parental bination at the breakage point (36, 37). The instability cells undergoing a first cycle of treatment (Fig. 1). Beyond observed in the 5q14.1 region was independent of the the decreased efficiency, the structure of the amplicon was DHFR copy number or the MTX dose, as the proportion also different (Figs. 3 and 5), suggesting that retreated of chromosome 5 with asymmetric signal of DHFR with cells cannot generate the amplicon structure borne during respect to the number of chromosome 5 with normal copies the first round of MTX. Alternatively, some of the cells of DHFR was maintained at all stages of the process (data develop resistance through the generation of a different not shown). The complexity of the amplification process structure but at a lower rate than previously untreated is also revealed by the structure of the amplicon at 8q23.3- cells. q24.3, which formation involves secondary rearrangements As a whole, our data show that, besides the complexity (this study and ref. 38). of the mechanisms involved in the generation of The number of intact chromosome 5 bearing the amplicons, HT29 cells develop resistance to the cytotoxic amplification target gene (DHFR) may play an important drug MTX through a consistent process resulting in a role in early stages of drug resistance. As reported in a common amplicon structure (Fig. 5). Moreover, after previous study, SW480 and SK-CO-1 colon cancer cell lines passive loss of the amplicon by withdrawal of the drug, that show amplification capability are unable to survive cells become MTX sensitive and exhibit a substantial MTX treatment even at low doses of the cytotoxic drug (22). reduction of their capacity to generate de novo resistance It is of note that they only contained a single intact in a second cycle of MTX treatment. When they reacquire chromosome 5, whereas the untreated HT29 cells had three resistance is through the generation of an amplicon with intact copies. It has been hypothesized that at least two a different structure, suggesting that the formation of the homologue chromosomes are needed in the amplification first amplicon is a ‘‘wear and tear’’ process. Obviously, event (4, 7, 22, 35). One chromosome 5 would be we cannot make a direct extrapolation of the conclusions

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of this study to clinical settings. Nevertheless, our results 18. BlakleyRL, Sorrentino BP. In vitro mutations in dihydrofolate reductase that confer resistance to methotrexate: potential for clinical prompt us to postulate that a second round of treatment application. Hum Mutat 1998;11:259 – 63. may be considered even in patients that have developed 19. Dicker AP, Waltham MC, Volkenandt M, et al. Methotrexate chemoresistance by gene amplification, provided a recess resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate in the therapeutic regimen is feasible to allow the resensi- reductase mutation. Proc Natl Acad Sci U S A 1993;90:11797 – 801. In vivo 20. Wielinga P, Hooijberg JH, Gunnarsdottir S, et al. The human multidrug tization of the cells. investigations are needed to resistance protein MRP5 transports folates and can mediate cellular validate such strategies. resistance against antifolates. Cancer Res 2005;65:4425 – 30. 21. Snijders AM, Fridlyand J, Mans DA, et al. Shaping of tumor and drug-resistant genomes byinstabilityand selection. Oncogene 2003;22: Disclosure of Potential Conflicts of Interest 4370 – 9. No potential conflicts of interest were disclosed. 22. Morales C, Ribas M, Aiza G, Peinado MA. Genetic determinants of methotrexate responsiveness and resistance in colon cancer cells. Oncogene 2005;24:6842 – 7. Acknowledgments 23. Snijders AM, Hermsen MA, Baughman J, et al. Acquired genomic We thank Gemma Aiza for technical support. aberrations associated with methotrexate resistance varywith background genomic instability. Genes Chromosomes Cancer 2008;47:71 – 83. References 24. Masramon L, Ribas M, Cifuentes P, et al. Cytogenetic characterization of two colon cell lines byusing conventional G-banding, comparative 1. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. genomic hybridization, and whole chromosome painting. Cancer Genet Nat Med 2004;10:789 – 99. Cytogenet 2000;121:17 – 21. 2. Schwab M. Oncogene amplification in solid tumors. Semin Cancer Biol 25. Nakano H, Yamamoto F, Neville C, Evans D, Mizuno T, Perucho M. 1999;9:319 – 25. Isolation of transforming sequences of two human lung carcinomas: 3. Albertson DG. Gene amplification in cancer. Trends Genet 2006;22: structural and functional analysis of the activated c-K-ras oncogenes. Proc 447 – 55. Natl Acad Sci U S A 1984;81:71 – 5. 4. Singer MJ, Mesner LD, Friedman CL, Trask BJ, Hamlin JL. Amplifica- 26. Chomczynski P, Sacchi N. Single-step method of RNA isolation by tion of the human dihydrofolate reductase gene via double minutes is acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem initiated bychromosome breaks. Proc Natl Acad Sci U S A 2000;97: 1987;162:156 – 9. 7921 – 6. 27. Bustin SA. Absolute quantification of mRNA using real-time reverse 5. Myllykangas S, Knuutila S. Manifestation, mechanisms and mysteries transcription polymerase chain reaction assays. J Mol Endocrinol 2000; of gene amplifications. Cancer Lett 2006;232:79 – 89. 25:169 – 93. 6. Pipiras E, Coquelle A, Bieth A, Debatisse M. Interstitial deletions and 28. Corzo C, Petzold M, Mayol X, et al. RxFISH karyotype and MYC intrachromosomal amplification initiated from a double-strand break amplification in the HT-29 colon adenocarcinoma cell line. Genes targeted to a mammalian chromosome. EMBO J 1998;17:325 – 33. Chromosomes Cancer 2003;36:425 – 6. 7. Coquelle A, Pipiras E, Toledo F, Buttin G, Debatisse M. Expression of 29. de Anta JM, Mayo C, Sole F, et al. Methotrexate resistance in vitro is fragile sites triggers intrachromosomal mammalian gene amplification and achieved bya dynamicselection process of tumor cell variants emerging sets boundaries to earlyamplicons. Cell 1997;89:215 – 25. during treatment. Int J Cancer 2006;119:1607 – 15. 8. Chen MJ, Shimada T, Moulton AD, et al. The functional human 30. Omasa T. Gene amplification and its application in cell and tissue dihydrofolate reductase gene. J Biol Chem 1984;259:3933 – 43. engineering. J Biosci Bioeng 2002;94:600 – 5. 9. Krajinovic M, Moghrabi A. Pharmacogenetics of methotrexate. Phar- 31. Herrick J, Conti C, Teissier S, et al. Genomic organization of amplified macogenomics 2004;5:819 – 34. MYC genes suggests distinct mechanisms of amplification in tumorigen- 10. Banerjee D, Mayer-Kuckuk P, Capiaux G, Budak-Alpdogan T, Gorlick esis. Cancer Res 2005;65:1174 – 9. R, Bertino JR. Novel aspects of resistance to drugs targeted to 32. Johnson RD, Jasin M. Sister chromatid gene conversion is a dihydrofolate reductase and thymidylate synthase. Biochim Biophys Acta prominent double-strand break repair pathwayin mammalian cells. EMBO 2002;1587:164 – 73. J 2000;19:3398 – 407. 11. Nunberg JH, Kaufman RJ, Schimke RT, Urlaub G, Chasin LA. 33. Nag DK, Suri M, Stenson EK. Both CAG repeats and inverted DNA Amplified dihydrofolate reductase genes are localized to a homogeneously repeats stimulate spontaneous unequal sister-chromatid exchange in staining region of a single chromosome in a methotrexate-resistant Saccharomyces cerevisiae. Nucleic Acids Res 2004;32:5677 – 84. Chinese hamster ovarycell line. Proc Natl Acad Sci U S A 1978;75: 34. Mirkin EV, Mirkin SM. Replication fork stalling at natural impediments. 5553 – 6. Microbiol Mol Biol Rev 2007;71:13 – 35. 12. Rots MG, Pieters R, Kaspers GJ, Veerman AJ, Peters GJ, Jansen G. 35. Carroll SM, DeRose ML, GaudrayP, et al. Double minute chromo- Classification of ex vivo methotrexate resistance in acute lymphoblastic somes can be produced from precursors derived from a chromosomal and myeloid leukaemia. Br J Haematol 2000;110:791 – 800. deletion. Mol Cell Biol 1988;8:1525 – 33. 13. Schimke RT. Methotrexate resistance and gene amplification. 36. Morris T, Thacker J. Formation of large deletions byillegitimate Mechanisms and implications. Cancer 1986;57:1912 – 7. recombination in the HPRT gene of primaryhuman fibroblasts. Proc Natl 14. Bosson G. Reduced folate carrier: biochemistryand molecular biology Acad Sci U S A 1993;90:1392 – 6. of the normal and methotrexate-resistant cell. Br J Biomed Sci 2003;60: 37. Mangano R, Piddini E, Carramusa L, Duhig T, Feo S, Fried M. Chimeric 117 – 29. amplicons containing the c-myc gene in HL60 cells. Oncogene 1998;17: 15. MatherlyLH. Molecular and cellular biologyof the human reduced 2771 – 7. folate carrier. Prog Nucleic Acid Res Mol Biol 2001;67:131 – 62. 38. Pole JC, Courtay-Cahen C, Garcia MJ, et al. High-resolution analysis 16. Rots MG, Pieters R, Peters GJ, et al. Role of folylpolyglutamate of chromosome rearrangements on 8p in breast, colon and pancreatic synthetase and folylpolyglutamate hydrolase in methotrexate accumulation cancer reveals a complex pattern of loss, gain and translocation. and polyglutamylation in childhood leukemia. Blood 1999;93:1677 – 83. Oncogene 2006;25:5693 – 706. 17. Cole PD, Kamen BA, Gorlick R, et al. Effects of overexpression of 39. Hastings PJ, Bull HJ, Klump JR, Rosenberg SM. Adaptive amplifica- g-glutamyl hydrolase on methotrexate metabolism and resistance. Cancer tion: an inducible chromosomal instabilitymechanism. Cell 2000;103: Res 2001;61:4599 – 604. 723 – 31.

Mol Cancer Ther 2009;8(2). February 2009

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

Mol Cancer Ther Published OnlineFirst February 3, 2009.

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