Hydroxyurea Accelerates Loss of Extrachromosomally Amplified Genes from Tumor Cells1

(CANCER RESEARCH 51, 6273-6279, December I, 1991) Hydroxyurea Accelerates Loss of Extrachromosomally Amplified Genes from Tumor Cells1

Daniel D. Von Hoff,2 Tracey Waddelow, Barbara Forseth, Karen Davidson, Jeff Scott, and Geoffrey Wahl

Division of Oncology, Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 {D. D. V. H., T. W., B. F., K. D., J. S.], and The Salk Institute for Biological Studies, La lolla, California 92138 Â¡G.W.Â¡

ABSTRACT recently been shown to multimerize over time (15,16) providing one mechanism for the generation of the heterogeneously sized Gene amplification is one mechanism mediating the resistance of tumor DMs in tumor cells. A second type of structure containing cells to antineoplastic agents and the overexpression of a variety of oncogenes in diverse tumor types. There is mounting evidence that amplified genes consists of chromosomal regions which typi cally fail to exhibit banding by using the trypsin-Giesma acentric extrachromosomal elements such as double minute chromosomes are common intermediates in the amplification process. These acentric method. Such structures have been referred to as HSRs. In structures partition unequally at mitosis and can be lost in the absence many cases, HSRs result from the integration of extrachromo of selection. In the present study, we used human and hamster cell lines somal elements (15-17). Since episomes and DMs lack cen- documented to contain extrachromosomally amplified drug resistance tromers, they are typically lost in the absence of selection. By genes to investigate the feasibility of enhancing the loss rate of the contrast, amplified genes in HSRs are typically stable, although extrachromosomally amplified genes to make the cells more sensitive to a few studies have reported very gradual loss of genes from drug treatment. The results show that treatment of each of these lines such structures (18). with hydroxyurea accelerates the loss of the extrachromosomally ampli Previous work has documented that when the selective pres fied drug resistance genes. Loss of these extrachromosomal genes was associated with a corresponding increase in the drug sensitivity in the sure of a drug is removed from mammalian cells that carry cases examined. The mechanism of accelerated loss does not appear to unstably amplified genes on extrachromosomal sites, the cells involve a differential effect on the replication of extrachromosomal DNA gradually lose those amplified genes (lose their DMs or epi sequences. The results suggest that hydroxyurea treatment may provide somes) (11-13, 15, 19-21). In order to assess whether general a valuable tool for the general accelerated elimination of extrachromo procedures can be developed to enhance the loss of extrachro somally amplified genes. mosomally amplified genes, we have examined the elimination rates of MDR1 genes from vinblastine resistant human squa- INTRODUCTION mous tumor cells, CAD genes from PALA resistant Chinese hamster ovary cells, and dihydrofolate reducÃase(DHFR) genes The donai evolution which occurs during the development from methotrexate resistant human squamous tumor cells. of malignancy is often mediated by both subtle gene mutations Snapka and Varshavsky (22) previously showed that hydrox and large scale genetic changes. Among the latter, increases in yurea could increase rate of loss of unstably amplified DHFR the number of copies of genomic regions ranging from 100 genes from mouse cells. In this report, we confirm and extend kilobases to more than IO6 base pairs are commonplace (1-4). the observations of Snapka and Varshavsky by showing that The end results of such "gene amplifications" are the overpro hydroxyurea dramatically increases the loss rate of episomes duction of the protein products encoded by the genes within and DMs from each cell line analyzed. Our results indicate, the target region. Amplification of drug resistant genes has been however, that the effects of hydroxyurea are not likely to be due associated with both in vitro and in vivo resistance of patients' to inhibition of DNA synthesis. Alternative possibilities for the tumors to antineoplastic agents (3-9). mechanism of action of hydroxyurea are discussed. Amplified genes have been localized to two types of cytoge- netically distinguishable structures. In biopsies of human tu MATERIALS AND METHODS mors, the predominant structure consists of paired chromatin bodies referred to as double minute chromosomes (1-6, 10). Cell Lines. The human squamous cell KB cell line resistant to Typically DMs3 are observed as having heterogeneous sizes (11, vinblastine (KBV1) was a gift from Dr. Igor Roninson, University of 12). Recently, it has been found that in many cases, amplified Illinois (23). The line was grown in minimal essential medium supple drug resistance genes can be localized to submicroscopic, cir mented with 10% FBS plus 1.0 Mg/ml of vinblastine sulfate (Lilly Research Laboratories, Indianapolis, IN) in T25 flasks in a 5% CO2, cular autonomously replicating DNA molecules (referred to as 37Â°Cincubator. The cells were harvested with 0.25% trypsin and 1 mM "episomes" or "amplisomes") (13, 14, 47). Episomes have EDTA and passaged at confluency by diluting them 1:10 about every 4 Received 8/3/90; accepted 9/23/91. days. The line has approximately 50 copies of the multidrug resistance The costs of publication of this article were defrayed in part by the payment gene (MDR1) which has previously been shown to reside in autono of page charges. This article must therefore be hereby marked advertisement in mously replicating submicroscopic circular extrachromosomal DNA accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by research grants from the Bristol Myers Drug Resistance Pro molecules (episomes) approximately 600 and 750 kilobase pairs in size gram (100-RI46) (D. V. H.) and the Department of Health and Human Services (24). This line contains an average of 3 double minutes/cell. (Grant U01CA48405) (G. W., D. V. H.), as well as by Medical Oncology Training The human squamous cell HEp2 cell line resistant to methotrexate Grant CA09434 from Department of Health and Human Services (J. S.). Pre (Hep2 R19) was derived in our laboratory (17). The line was grown in sented in abstract form at the American Association for Cancer Research, May enriched minimal essential medium supplemented with 10% dialyzed 1990(46). 2To whom request for reprints should be addressed. FBS plus 120 ng/ml of methotrexate (Lederle Laboratories, Pearl 3The abbreviations used are: DMs, double minute chromosomes; HSR, ho River, NY). The cells were carried in T25 flasks in a 5% CO2, 37Â°C mogeneously staining region; DHFR, dihydrofolate reducÃase;PALA, A"-(phos- incubator. The cells were harvested with 0.25% trypsin and 1 mM phonacetyl)-L-aspartic acid); FBS, fetal bovine serum; CAD, carbamylphosphate EDTA and passaged at confluency by diluting them 1:10 about every 3 synthetase, aspartate transcarbamylase, dihydroorotase; PBS, phosphate buffered saline; CHEF, contour clamped homogeneous electrical field electrophoresis; days. The line has approximately 30 copies of the DHFR gene which MOKI, multidrug resistance gene 1; kbp, kilobase pairs; Bull nl. bromodeoxy- have been shown to reside in autonomously replicating circular mole uridine;TE, 1 mM EDTA-10 mM Tris-HCl, pH 8.0. cules ranging in size from approximately 300 kbp (submicroscopic) to 6273

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1991 American Association for Cancer Research. HYDROXYUREA AND AMPLIFIED GENE LOSS over 4000 kbp in size (visible with light microscope as double minutes). capillary transfer according to the manufacturer's protocol. Membranes This line contains an average of 5-9 double minutes/cell. were baked 30 min at 80Â°Cin a vacuum. Membranes were hybridized The cell lines C5R500 and T5S1-3 are Chinese hamster ovary cell with specific DNA probes labeled with 32P by nick translation (Boeh- lines containing about 50 copies/cell of a transfected, amplified CAD ringer Mannheim) to a specific activity of greater than 10s dpm/>g as gene (13, 15). In the T5S1 -3 cells the CAD genes are amplified intrach- described previously (29). Hybridized membranes were matted with X- romosomally, while in C5R500 cells the amplified CAD genes are ray film (Kodak X-omat) between two intensifying screens (DuPont localized to episomes approximately 250 kbp in size (13, 15). Both cell Lightning Plus) and placed at â€”¿70Â°Ctogenerate autoradiograms. lines were grown in Dulbecco's modified Eagle's medium supplemented Isolation and Identification of Episomal DNA. Alkaline lysis was used with 10% dialyzed FBS, Ix nonessential amino acids (GIBCO), and to isolate episomal DNA in each of the cell lines (13). Cells were 200-500 pM PALA (a specific inhibitor of CAD). The cells were carried trypsinized, washed with PBS, counted, and 2.0 x IO7cells were lysed in T25 flasks in a 5% CO2 37Â°Cincubator and were passaged at under alkaline conditions. Cells were disrupted by vortexing at a pH of confluence by diluting them 1:10 about every 4 days. 12.45 for 2 min in 2 ml of 1% sodium dodecyl sulfate-50 mM NaCl-25 Exposure of Cells to Hydroxyurea. All cells were continuously ex mM EDTA (13, 16). Samples were incubated for 30 min at 30Â°Cand posed to concentrations of hydroxyurea (Squibb, Princeton, NJ) of 50, then neutralized with 0.4 ml 1.0 M Tris-HCl, pH 7.O. Lysates were 100, and 200 pM. treated with 0.3 ml of 5 M NaCl and 14 pi of 20 mg/ml proteinase K Determination of Gene Copy Number. The cells were washed with (Sigma) and incubated for 30 min at 37Â°C.Phenol (saturated with 0.2 PBS, trypsinized, and DNA was isolated as previously described (13). M NaCl-0.2 M Tris-HCI at pH 8) (0.8 ml) was added, samples were DNA was resuspended in 1.0 ml of TE buffer, pH 8.0, and sheared by gently mixed, and lysates were centrifuged at 10,000 x g for 10 min at 10 passages through a 27-gauge needle. DNA content was assayed in 4Â°C.Aqueous phases were transferred to new tubes, 2.5 volumes of some of the samples by use of a fluorimeter (Hoeffer). Gene copy 100% ethanol were added, and samples were stored at -20Â°Covernight. number was estimated by immobilizing, on nitrocellulose filters, ap DNA was collected by centrifugation at 10,000 x g for 10 min at 4"C, proximately 900 ng of DNA in 110 /il of TE buffer. The DNA, in 1.5- rinsed with 1 ml 70% ethanol, spin dried, and resuspended in TE buffer. ml microfuge tubes, was denatured by adding 220 /jl of 0.5 N NaOH, DNA Replication Studies. Exponentially growing cells were labeled and heating to 65"C for 1 h. The tubes were cooled in ice, 330 n\ of with 30 pM BrdUrd for 1 generation as determined by cell counting. cold 2 M ammonium acetate were added, and 150-^1 aliquots of the Replication of the gene of interest was then determined either from mixture were spotted on nitrocellulose (Schleicher & Schuell), prewet- total high molecular weight DNA prepared by standard procedures (13) ted with l M ammonium acetate, using a dot-blot manifold (Schleicher or from episome enriched DNA prepared by the alkaline lysate tech & Schuell) attached to wall suction. The nitrocellulose membrane was nique outlined above. DNA from 1-5 x IO7 cells prepared by either removed from the apparatus and baked 30 min at 80Â°Cin a vacuum method was sheared by 10 passages through a 27-gauge needle and oven. The membrane was hybridized to the various DNA probes as sedimented to equilibrium in 5-ml gradients (45,000 rpm in a Beckman described below. Following hybridization the membranes were matted Vti 80 rotor at 15Â°Cfor 16 h) of CsCl (initial density, 1.75 g/ml). Each with X-ray film (Kodak X-omat) between two intensifying sheets gradient was fractionated into 24- to 200-^1 aliquots and the refractive (DuPont Lighting Plus) and placed in a -70Â°C freezer to provide index of every other fraction was determined. A 100-pl portion of each autoradiograms. fraction was denatured by adding NaOH to 0.3 M and heating at 65Â°C Analysis of Dot-Blot Data. The intensity of the dots on the X-ray for 15 min and neutralized by adding an equal volume of 2 M ammo film was assessed using a densitometer image analysis system (Macin nium acetate. Samples were applied to nitrocellulose in the dot-blot tosh II with video camera and the Image 1.4 software system). Each apparatus as outlined above. After baking at 80Â°Cfor 30 min to membrane was first hybridized with a probe specific for the drug immobilize the DNA, the membranes were hybridized with the drug resistant gene of interest. After densitometry was performed on the resistance gene probe or to an internal control gene (lla-2 ribosomal film, the membranes were stripped of the drug resistance gene probe sequence). The dots were evaluated on the densitometry apparatus (by washing in a shaking bath of 95Â°C0.1 x SSPE buffer for 5 min 3 described above. times). The membranes were then rehybridized with a control gene DNA Probes. Membranes (nylon or nitrocellulose) were probed with sequence (lla-2, a chromosomally located ribosomal gene). The film 32P-labeled probes prepared by nick translation. MDR1 specific se provided with that rehybridization was Â«scanned on the densitometer. quences were detected using the pmdrl probe from the American Type This scanning provided a recheck on how much DNA was loaded on Culture Collection. DHFR-specific sequences were detected by hybrid each dot. The drug resistance gene copy number determinations were ization to the 900-base pair insert of the plasmid 5'-hdhfr (a gift from expressed per amount of DNA loaded on each dot. Anna Hill, University of Arizona). That probe does not recognize Preparation and Irradiation of DNA in Agarose Blocks. Cells were pseudogenes (32). The CAD gene was detected by the 102f probe derived harvested by trypsinization, rinsed with PBS, and suspended in PBS at from the Syrian hamster C4Z)gene4 (30). The 1la-2 ribosomal sequence a density of 2.5 x IO7 cells/ml. Cell suspensions were mixed with an probe4 was used to detect the amount of chromosomal DNA loaded on equal volume of molten (55Â°C)1% low melting point agarose (FMC SeaPlaque in PBS) and pipeted in 40-^1 aliquots (5 x 10* cells) into a the dot blot (30). rubber-based block-forming mold (25). Blocks were allowed to harden and were placed in 15-ml culture tubes containing 2 ml of 1% Sarkosyl- RESULTS 100 HIMEDTA. Proteinase K (2 mg/ml) was added, the blocks were incubated for 24 h at 55Â°C,rinsed twice with 10 ml of TE, and stored Hydroxyurea Causes Accelerated Loss of Extrachromosomal in TE at 4Â°C.Blocks were placed in 100 n\ TE in 1.5-ml microfuge Drug Resistance Genes. Hydroxyurea was utilized because it tubes and irradiated by a '"Cs source (Gamma Cell 40) at rates of 1.2 was previously documented to accelerate the loss of amplified Gy/min for variable exposure times prior to electrophoresis. DHFR genes located on double minutes in methotrexate resist DNA Electrophoresis. Episomal DNA molecules embedded in agarose blocks and linearized by '"Cs irradiation were fractionated by ant 3T6 mouse cells (22). For the present experiments each one of the cell lines had the selective agent removed from the media contour clamped homogeneous electrical field electrophoretic strategy (24, 26, 27), using a Bio-Rad CHEF-II DR apparatus with 0.6% agarose (Vinblastine for the KBVI cell line; methotrexate for the gels at 14Â°Cwith 60-min pulses for 158 h at 50 V. Molecular weight HEp2R19 cell line, and PALA for the C5R500 and T5S1-3 standards between 200 and 1100 kbp were provided by Saccharomyces cell lines). Each of the cell lines was then carried in media cerevisiae chromosomes (Beckman) (28). Gels were stained for 30 min without hydroxyurea or with hydroxyurea at concentrations of in 1.0 Mg/ml ethidium bromide and photographed with a red filter by 50, 100, or 200 piw (continuous exposure). At various time transillumination with a 300 nm UV light source. points (usually days 7, 14, and 21), cells were removed from DNA Transfer and Hybridization. DNA was transferred from agarose gels to nylon-66 membranes (Schleicher & Schuell, Nytran) by alkaline â€¢¿G.Wahl,Salk Institute. 6274

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1991 American Association for Cancer Research. HYDROXYIJREA AND AMPLIFIED GENE LOSS the flasks and the copy number of the drug resistance gene of yurea. The cells were then washed and exposed to various interest was determined by using the dot-blot technique de vinblastine concentrations for 1 h. The cells were then washed scribed above. The amount of DNA loaded on each dot was and plated at a concentration of 2500 cells/35-mm Petri dish internally controlled by probing with the internal standard by using a soft agar cloning technique (31). The fraction of ribosomal DNA probe. treated and untreated cells that survived the vinblastine treat As expected for acentric, extrachromosomal elements, with ment and clonal selection are shown in Fig. 3. The cells treated drawal of the selective pressure resulted in a gradual loss in the with 100 fiM hydroxyurea, which were shown to have a decrease copy number of these unstably amplified genes from all three in MDR1 gene copy number and episome content (Fig. 2), did cell lines as a function of the cumulative cell doublings. The have an increased sensitivity to low concentrations of vinblas loss rate appears to be different for each cell line under the tine. However, treatment with 1 Mg/ml vinblastine did not reveal nonselective growth conditions used. Continuous exposure of a significant difference in the sensitivity of cells that were each cell line to hydroxyurea dramatically accelerated the rate treated with hydroxyurea or those that were not treated with of loss of their extrachromosomally amplified genes (Fig. 1; hydroxyurea. Since the KBVI cells used for this experiment Table 1). The loss rate was dependent on the concentration of had not been subcloned recently, it is conceivable that the hydroxyurea used as shown in Fig. \A for the C5R500 line population contained a subset of cells containing chromo- which contains CAD gene episomes and DMs. Similar concen somally integrated genes which gave resistance to all concentra tration effects were noted for the loss of MDR1 and DHFR tions of vinblastine including 1 pg/ml. Thus, the magnitude of genes (see below). the hydroxyurea effects in these experiments are underesti To determine whether the effect of hydroxyurea was specific mated, given the possibility of an intrachromosomally amplified for extrachromosomally amplified genes, we utilized a cell line region containing MDR1 genes. called T5S1-3 which is isogenic with C5R500 except that the Effects of Hydroxyurea on Cell Growth Kinetics. Previous CAD genes are amplified at a single chromosomal site (13, 15). studies with hydroxyurea (used at the concentrations used in Fig. l B shows that there is no loss of CAD gene sequences from the present study) indicated that it can increase the apparent the T5S1-3 cells upon the withdrawal of the selective pressure in the presence or absence of hydroxyurea. C5R 500 T5SI-3 R The effects of hydroxyurea on the MDR1 episomes from (Extrachromosomal CAD Gene) (Chromosomal CAD Gene) Â° KBV1 cells were also determined. KBVI cells embedded in agarose blocks were treated with X-rays and then fractionated by using CHEF electrophoresis (see "Materials and Methods"). The technique generates linear molecules from circular epi somes and easily resolves DNA molecules <1000 kbp in size. Fig. 2 shows such a gel run on cells exposed to 100 /Â¿M hydroxyurea for 11 and 21 days. Fig. 2A reveals that the abundance of MDR1 episomes decreases with time in passage

in the absence of vinblastine, and that 100 UM hydroxyurea 200(1Â«

accelerates the loss of MDR1 genes and reduces the number of O 2 4 6 8 IO o 2 4 e e 10 MDR1 episomes. Fig. 2B shows the results of probing the same Number of Cell Doublings Number of Cell Doublings blot as shown in Fig. 1A with a chromosomal sequence. The Fig. 1. A. loss of amplified CAD genes from an extrachromosomal site as a diffuse chromosomal smears in each lane contrast with the function of cumulative cell doublings. The selective agent (PALA) was withdrawn from the C5R500 cell culture and cells were exposed to various concentrations discrete bands produced by the MDR1 probe in Fig. 2A and of hydroxyurea. Copy numbers of the CAD gene were measured at subsequent confirm that the discrete bands in Fig. 2A represent linearized time intervals as described in "Materials and Methods." The results are expressed MDR1 episomes as described previously (24). The intensity of as the percentage of original (at time of removal of the selective agent) gene copy number. Conditions included: no added hydroxyurea (â€¢);hydroxyurea added at hybridization to each lane in Fig. 2B reveals that approximately 50 jiM (O); hydroxyurea at 100 Â¡Â¡M(â€¢);and hydroxyurea at 200 UM (O) as equal numbers of cells were analyzed for each sample with the continuous exposures. As shown in B, hydroxyurea does not affect the stability exception ofthat used for the 11-day hydroxyurea treated lane. of chromosomally amplified CAD genes. T5S1-3 cells were grown in the absence of PALA and either were not treated with hydroxyurea (â€¢)or were passaged in The time necessary for the loss of the drug resistance genes 50 JIMhydroxyurea (O), 100 Â¡Â¡Mhydroxyurea(â€¢).or200 fiMhydroxyurea (O) for can be expressed as the number of cell doublings required to the number of cell divisions indicated. have a 50% reduction in gene copy number. We refer to this parameter as the 50% elimination time. Table 1 summarizes Table I Elimination times for drug resistance genes 50% elimination time data for three concentrations of hydrox Concentration of yurea for each of the cell lines. It is clear from Table 1 that ETÂ»*KBVBIC5R500HEp2R,905010020005010020005010020010.89.17.04.5NotCell line hydroxyurea (>IM) hydroxyurea can significantly affect the rate of decrease in the copy number of all three drug resistance genes over and above the spontaneous loss rate observed in the absence of selection. Increased Drug Sensitivity following Hydroxyurea-stimulated doublingsNotreached at 10.3 Loss of MDR1 Genes. The data shown in Fig. 2 reveal that doublings5.23.6Notreached at 9.2 hydroxyurea produced a substantial decrease in the number of MDR 1episomes in the human squamous tumor cell line KBV1. We then determined if this loss correlated with an increased doublingsNotreached at 9.6 sensitivity of the cells to vinblastine. KBV1 cells which were reached at 8.2doublings4.52.5 grown continuously in culture in 1 Â¿Â¿g/rnlvinblastine were grown for an additional 12 cell doubling days in medium â€¢¿ET50,elimination time 50. the number of cell doublings needed to have without vinblastine and either with or without 100 Â¿Â¿Mhydrox 50% reduction in gene copy number (from base line). 6275

â€¢¿oO (O â€¢¿oo B â€”¿ OJ M>>â€¢oâ€”0CWco.OCwCOâ€¢oevi0C'â€¢RCO.0CCOâ€¢or=D+0C^CO_QCCM o D â€¢¿OO â€¢¿o co iÂ» O) W 0 0 C C W CO CO IO M ce Fig. 2. Hydroxyurea (A/I/) eliminates (O _o o o o JD MDR1 episomes from KBVI cells (A). KBVI -Q -Q -Q cells were grown under various conditions and C C C the cells were embedded in agarose blocks and -y-irradiated (40 Gy) followed by electropho- > III resis used to fractionate the linearized epi I somes. After electrophoresis and Southern transfer, the blot was hybridized with an MORI probe. Lane 1. KBVI cell line carried in 1.0 /jg/ml vinblastine; Lanes 2 and 3. the same line carried in the absence of vinblastine for 11 and 21 days, respectively. Lanes 4 and 5, KBVI cells carried in the absence of vin blastine but with 100 fiM hydroxyurea for 11 and 21 days, respectively. Bands I and 2, epi somes approximately 750 and 600 kbp in size, respectively (24). B. rehybridization of the blot shown in A with a chromosomal sequence. The blot shown in A was boiled in 0.1 x SSPE-0.1 % sodium dodecyl sulfate to remove hybridized MDRI probe and then rehybridized with a Band probe containing ribosomal DNA sequences (I la-2). The diffuse hybridization in each lane is produced by random fragmentation of chro mosomal DNA by -y-rays. The intensity of the smear indicates that the amount of DNA pres ent in each lane was approximately the same with the exception of the sample containing hydroxyurea treated cells for 11 days.

population doubling time (22). The doubling time of each cell line was, therefore, determined at 50 Â¿Â¿Mhydroxyurea.All cell lines continued to grow during continuous hydroxyurea expo sure, but increases in doubling times were noted which were 100 different for each cell line (Fig. 4). For example, 100 ^M hydroxyurea prolonged the population doubling time of KBVI cells 3.8-fold, HEp2R19 cells 2.8-fold, and C5R500 cells 1.9- fold. We also noted that cell size increased with hydroxyurea treatment. Such increases in cell size following hydroxyurea treatment were noted previously (22). As suggested previously (22), it is conceivable that the larger cell size could cause contact inhibition of cell proliferation (22, 23, 25-35) to occur when fewer cells are present and results in an apparent increase in the estimated population doubling time. As a reference point, 0.001 0.01 0.1 1 in patients receiving p.o. daily doses of hydroxyurea, serum Vinblastine Concentration (meg/ml) concentrations range from 300 to 2000 ÃŸM(36). Fig. 3. Increased vinblastine sensitivity of KBVI cells following hydroxyurea Effects of Hydroxyurea on I pisolimi DNA Replication. Hy treatment. KBVI cells grown in the absence (O) or in the presence of 100 Â¿IM droxyurea accelerated loss of extrachromosomal elements could hydroxyurea (â€¢)werewashed and exposed to various concentrations of vinblastine sulfate for 1 hr. The cells were then plated in an agar colony forming system to be mediated by its ability to interfere with either some aspect determine their percentage of survival with 5 log concentrations of vinblastine. of the segregation of extrachromosomal elements at mitosis, to The cloning efficiencies in soft agar for the hydroxyurea pretreated versus the disrupt their molecular integrity, or to preferentially inhibit nonpretreated cells were 35.9 and 33.3%, respectively, without exposure to their replication. We have begun to investigate these possibili vinblastine. Shaded area, clinically achievable concentration of vinblastine. ties by determining whether hydroxyurea inhibits the replica tion of extrachromosomal DNA sequences to a greater extent 6276

DISCUSSION The results of these studies indicate that hydroxyurea can accelerate the loss of extrachromosomally amplified drug re sistance genes from vinblastine and methotrexate resistant hu man tumor cells, and from resistant hamster cells. The accel erated loss is specific for extrachromosomally amplified genes since no effect was observed on the stability of chromosomally amplified regions. Elimination of extrachromosomally ampli fied resistance genes was correlated with an increase in drug sensitivity. The loss rate of extrachromosomal DNA noted in the three cell lines analyzed in these studies was not as great as that reported previously (22). We also found that higher concentra tions of hydroxyurea were required to accelerate the loss of extrachromosomal DNA than reported previously (22). We have repeated the studies initially performed by Snapka and Varshavsky (22) with the mouse cell lines they used and we obtained quantitatively similar results to the ones they reported (data not shown). Christen et al. (38) have also recently reported that 150 ÃŸMhydroxyurea accelerated the loss of unstably am plified MDRl genes from KBVI cells and reduced the 50% inhibitory concentration of vinblastine from 68 Â±4 to 26 Â±3 nM. Thus, the results reported here are quantitatively similar to those reported in independent studies and document the reproducibility of the elimination protocol. We infer that the differences observed in the effectiveness of hydroxyurea to eliminate extrachromosomally amplified genes depends on the cell lines used and/or the amplified sequences studied. While we and others (22, 38) have found that hydroxyurea concentrations less than 200 /Â¿Mcanstimulate the elimination of extrachromosomally amplified DNA, other reports indicate Days in Culture that higher concentrations of hydroxyurea can actually increase

Fig. 4. Effect of hydroxyurea on growth rate kinetics. The number of cell doublings was determined for each cell line in the absence or presence of hydroxyurea. Growth of KBVI (A), C5R500 (B), and HEP2R9 (C) cells in absence of hydroxyurea (â€¢)and in the presence of 50 UMhydroxyurea (O) and 100 liM hydroxyurea (D). f0 '^ Â§ 01 Â«j than chromosomal sequences. We have previously analyzed the â€¢¿oa. replication characteristics of extrachromosomal DNA by meas C Â« uring the incorporation of BrdUrd into DNA according to the strategy first elaborated by Meselson and Stahl (37). Cell line C5R500 was used for the current experiments since the 250- D TJ 'S 3 kbp CAD episome that it contains can be isolated with a higher efficiency than the episomes in the other cell lines analyzed, d> and because hydroxyurea produces a more rapid loss of the o extrachromosomal elements from this line than the others o> O. tested. Thus, if hydroxyurea accelerates episome loss by inter fering with DNA replication, such effects should be detected most sensitively in the C5R500 cell line. Ribo, on Chromosome CAD on episome C5R500 cells were either not treated with hydroxyurea, or with 100 pM hydroxyurea for 4 days. They were then labeled Gene Type and Location with BrdUrd as described in "Materials and Methods," and Fig. 5. Effect of hydroxyurea on synthesis of episomal and chromosomal DNA. C5R500 cells were grown without (â€¢)orwith (^) 100 Â¿IMhydroxyureafor 4 days. total and episomal DNA was isolated and fractionated accord BrdUrd was added at a concentration of 30 JAI to each of the cultures. Both total ing to density on CsCl gradients. Fig. 5 reveals that 100 /Â¿M DNA and episomal DNA were isolated from each of the populations at 1 cell doubling time (sufficient rime to generate heavy light DNA) and the DNA was hydroxyurea did not substantially reduce the incorporation of then fractionated according to density on CsCIs gradients. The amount of the BrdUrd into either chromosomal DNA or extrachromosomal CAD gene sequence present in the heavy light portion of the total cell and alkaline lysate (episomal) DNA was determined by probing dot blots made with each DNA molecules. This experiment reveals that, at the concen gradient fraction with the 102f (CAD gene) probe. The amount of ribosomal gene trations tested, hydroxyurea did not profoundly affect cellular present in the heavy light fraction of total cell DNA was determined by probing duplicate blots with the lla-2 (ribosomal) gene probe. No substantial effect of DNA synthesis, and that there was no preferential inhibition hydroxyurea on either a chromosomally located ribosomal gene or on the episo- of extrachromosomal DNA replication. mally located CAD gene was noted. 6277

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1991 American Association for Cancer Research. HYDROXYUREA AND AMPLIFIED GENE LOSS the frequency of cells which undergo gene amplification (39, Stark, G. R. DNA amplification in drug resistant cells and in tumors. Cancer Surv., 5:1-23, 1986. 40). While such data were initially interpreted in favor of a Trent, J., Buick, R. N., Olson, S., Horns, R. C., and Schimke, R. T. Cytologie paradoxical effect of hydroxyurea to stimulate DNA replication evidence for gene ampliation in methotrexate resistant cells obtained from (39,40), more recent studies provide compelling evidence which patients with ovarian adenocarcinomas. J. Clin. Oncol., 2:8-15, 1984. does not support such a hypothesis (17, 41, 42). Rather, it is Curt, G. A., Carney, D. N., Cowan, K. H., Jolivet, J., Bailey, B. D., Drake, J. C., Chiensong, K. S., Minna, J. D., and Chabner, B. A. Unstable metho more likely that high concentrations of hydroxyurea inhibit trexate resistance in human small cell carcinomas associated with double DNA synthesis and lead to the accumulation of fragile struc minute chromosomes. N. Engl. J. Med., 308: 199-202, 1983. tures which may be prone to chromosome breakage. Studies in Carman, M. D., Schornagel, J. H., Rivert, R. S., Srimatkandada, S., Portlock, C. S., Duffy, T., and Berlino, J. R. Resistance to methotrexate due to gene the past several years indicate that conditions that cause chro amplication in a patient with acute leukemia. J. Clin Oncol., 2: 16-26, 1984. mosome breakage can significantly increase the frequency of 8. Horns, R. C., Dower, W. J., and Schimke, R. T. Gene amplification in a cells with amplified sequences, presumably by generating acen leukemia patient treated with methotrexate. J. Clin Oncol., 2: 2-7, 1984. 9. Clark, J. L.. Reizer, S. H., Mittleman, A., and Berger, F. G. Thymidylate tric chromosomal intermediates (41, 42). Thus, depending on synthase gene amplification in a colon tumor resistant to fluoropyrimidine the concentration of hydroxyurea used, two different conse chemotherapy. Cancer Treat. Rep., 71: 261-265, 1987. quences can result; low concentrations which are insufficient to 10. Brenner, J. E., Wahl., G. M., and VonHoff, D. D. Double minute chromo somes and homogeneously staining regions in tumors taken directly from inhibit DNA replication can cause elimination of extrachro patients versus in human tumor cell lines. Anticancer Drugs, 2:11-25, 1991. mosomal elements, while higher concentrations can precipitate 11. Barker, P. E. Double minutes in human tumor cells. Cancer Genet. Cytoge- the formation of amplification intermediates. net., 5:81-94, 1982. The precise mechanism for hydroxyurea induced accelerated 12. Cowell, J. K. Double minutes and homogeneously staining regions: gene amplification in mammalian cells. Annu. Rev. Genet., 16: 21-59, 1982. loss of extrachromosonal amplified genes is still unclear. While 13. Carroll, S. M., Gaudray, P., DeRose, M. L., Emery, J. F., Meinkoth, J. L., hydroxyurea inhibits ribonucleotide reducÃase(35), an enzyme Nakkin, F., Subler, M., Von Hoff, D. D., and Wahl, G. M. Characterization involved in the de novo synthesis of deoxyribonucleotide tri- of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication phosphates required for DNA synthesis (34), we found no origin. Mol. Cell. Biol., 7: 1740-1750, 1987. evidence that inhibition of DNA synthesis occurred at the 14. Pauletti, G., Lai, E., and Attardi, G. Early appearance and long-term per hydroxyurea concentrations tested. This is consistent with ob sistence of the submicroscopic extrachromosomal element amplisomes con taining the amplified DHFR genes in human cell lines. Proc. Nati. Acad. servations of McClarty et al. (43, 44) that hydroxyurea concen Sci. USA, 87: 2955-2959, 1990. trations similar to those used here actually increased ribonucle 15. Carroll, S. M., DeRose, M. L., Gaudray, P., Moore, C. M., Needham- otide reducÃase levels by a postlranscriptional mechanism. VanDevanter, D. R., Von Hoff, D. D., and Wahl, G. M. Double minute Studies to be presented elsewhere indicale lhal one componenl chromosomes can be produced from precursors derived from a chromosomal deletion. Mol. Cell. Biol., 8:1525-1533, 1988. of Ihe mechanism by which hydroxyurea enhances the loss of 16. Von Hoff, D. D., Forseth, B. J., Clare, N., Hansen, K. L., and VanDevanter, extrachromosomal elemenis involves Ihe formalion of micro- D. R. Double minutes arise from extrachromosomal DNA intermediates nuclei.5 which integrate into chromosomal sites in HL60 leukemia cells. J. Clin. The abilily of hydroxyurea lo accelerale Ihe loss of exlra- Invest., Â«5:1887-1895, 1990. 17. Wahl, G. M. The importance of circular DNA in mammalian gene amplii! chromosomal elemenis comprising differenl genomic regions cation. Cancer Res., 49: 1333-1340, 1989. in human cell lines suggesls new clinical applicalions for this 18. Biedler, J. L. Evidence for a transient prolonged extrachromosomal existence drug. While Ihe available evidence indÃcaleslhal drug resislance <>tamplified DNA sequences in antifolate resistant, vincristine resistant, and human neuroblastoma cells. In: R. T. Schimke, (ed.), Gene Amplification, due lo gene amplificaron is probably rarer in vivo lhan il is in pp. 39-45. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1980. cell cullure, reversal of amplificalion medialed drug resislance 19. Schimke, R. T., Brown, P. C., Kaufman. R. J., McGregor, M., and Slate, D. in vivo might be achievable by hydroxyurea irealmenl and L. Chromosomal and extrachromosomal localization of amplified dihydrofolate reducÃasegenes in cultured mammalian cells. Cold Spring Harbor deserves furlher invesligalion. In Ihis regard, it is imporlanl lo Symp. Quant. Biol., 45: 785-798, 1980. noie lhal while only modesl reduclions in MDR1 gene copy 20. Kaufman, R. J., Brown. P. C., and Schimke, R. T. Loss and stabilization of number were achieved wilh Ihe KBVI cell line, Ihe increased amplified dihydrofolate reducÃasegenes in mouse sarcoma S 180 cell lines. sensitivity of the cells following hydroxyurea treatmenl would Mol. Cell. Biol., /: 1084-1093, 1981. 21. Masters, J., Keeley, B., Gay, H., and Attardi, G. Variable content of double have broughl ihem wilhin Ihe range of clinically achievable minute chromosomes is not correlated with degree of phenotype instability plasma concenlralions (see Fig. 3; Ref. 45). On Ihe olher hand, in methotrexate-resistant human cell lines. Mol. Cell Biol. 2:498-507,1982. Ihe broad diversity of tumors with oncogene amplification, and 22. Snapka, R. M., and Varshavsky, A. Loss of unstably amplified dihydrofolate reducÃasegenes from mouse cells is greatly accelerated by hydroxyurea. Proc. the prevalence of extrachromosomal amplicons in such cells, Nati. Acad. Sci. USA, 80: 7533-7537, 1983. also make ihem prime targets for attempting hydroxyurea 23. Fogo, A. T., Wang-Peng, J., Gottesman, M. M., and Pastan, 1. Amplification therapy or olher slralegies designed lo accelerale Ihe loss of of DNA sequences in human multidrug resistant KB carcinoma cells. Proc. exlrachromosomally amplified DNA. Nati. Acad. Sci. USA, 82: 7661-7665, 1985. 24. Ruiz, J. C., Choi, K., Von Hoff, D. D., Roninson, I. B., and Wahl, G. M. Autonomously replicating episomes containing imiti genes in a multidrug resistant human cell line. Mol. Cell. Biol., 9: 109-115, 1989. ACKNOWLEDGMENTS 25. VanDevanter, D. R., Trammell, H. M., and Von Hoff, D. D. 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Daniel D. Von Hoff, Tracey Waddelow, Barbara Forseth, et al.

Cancer Res 1991;51:6273-6279.

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