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[CANCER RESEARCH 50. 2803-2807. May I. 1990) Determination of the DNA Content of a Six-Gene Amplicon in the Multidrug- resistant Chinese Hamster Cell Line CHRB3 by Flow Karyotyping A. P. M. Jongsma,1 A. Riethorst, and J. A. Aten Department of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan ¡21,1066 C.V Amsterdam [A. P. M. J., A. R.J, and Laboratory for Radiobiology, University of Amsterdam, Academic Medical Centre, FO-212, Meibergdreef9, 1105 AZ Amsterdam, [J. A. A.J, The Netherlands ABSTRACT cover the full length of the amplicon (16, 23). In the present study we estimated the DNA content of the Acquired multidrug resistance in cultured cells is often due to ampli amplified 6-gene region by flow fluorometric chromosome anal fication of pgp genes, which gives rise to overproduction of P-glycopro- yses of a precursor MDR cell line of CHRC5, CHRB3, and some teins that confer resistance by reducing the intracellular drug accumula of its revenants. The amplified DNA was cytogenetically visible tion. The size of these amplicons varies between multidrug resistant cell lines and is often much larger than the gene selected for. Amplicons of as a large HSR (24, 25) on chromosome 7q+. The size of such the multidrug resistant Chinese hamster ovary cell line CHRC5 and its a HSR reflects the degree of amplification (2, 5, 26, 27) and is progenitor CHRB3, for example, span at least five different genes besides proportional to the relative resistance of the cells. the pgp genes. Linkage of these gene classes with pgp had been shown We used Southern blots to analyze the number of amplicons, by In situ hybridization and by long distance mapping using pulsed field including the pgp genes, that were present in the cell lines. We gradient gel electrophoresis. Because the boundaries of the larger ampli showed by cytogenetic and flow karyotypic analyses that in one cons could not be determined, the size of such amplicons is not yet known, of the revenants only DNA of the 7q+ HSR was lost, whereas even though the six genes span at least 1500 kilobases. the remaining genome was unchanged. The amplicon size, In the present study we have determined the amplicon size in B.V. a eventually, was calculated from the missing HSR-DNA and the subclone of CHRB3 with a homogeneously staining region on chromosome lost amplicon copies in this revenant cell line. This combination 7q+ that harbors the amplified genes. We estimated the amplicon size in revenant clones by correlating the decreased DNA content of the 7q+ of experimental methods provides a size estimate for amplicons homogeneously staining region with the number of lost amplicons. The that are too large to measure by physical mapping of restriction reduction of the homogeneously staining region DNA that accompanied enzyme fragments using pulsed field gradient gel electropho reversion was determined by flow cytometry of propidium iodide stained resis. chromosome suspensions of the various cell lines. We found that about 107 megabase pairs were lost together with 11-24 P-glycoprotein gene copies, suggesting that the mean amplicon size is in the range of 4.5- MATERIALS AND METHODS 10.1 megabase pairs. Cell Lines and Culture. The multidrug resistant cell lines CHRB3 and CHRC5 were both developed by Ling and Thompson (17) from the AuxBl Chinese hamster ovary cells, by stepwise selection on increasing INTRODUCTION concentrations of colchicine. Since CHRB3 had become heterogeneous with respect to its colchicine resistance after 3 months of drug free Amplified genes have been observed in certain tumor tissues culture in our laboratory, three subclones B3+, B3-4, and B3-5 were (1-4), and in cells with acquired drug resistance (2, 5-7). The isolated according to the dilution method (28). The Chinese hamster finding that these genes are often flanked by large expanses of cells V79 and CHNF22 (29) were used as diploid controls. All cell lines coamplified DNA sequences (8, 9) suggests that other genes were cultured routinely in (. minimal essential medium (Cat No. 072- may be coamplified with the selected gene because they happen 1900; Giòco Europe Limited, Paisley, Scotland) supplemented with to be nearby (10). Examples are sequences coamplified with 10% fetal bovine serum and antibiotics in a humidified atmosphere carbamylphosphate synthetase, aspartate transcarbamylase, supplied with 5% CO2 at 37°C.During the experimental period the cell and dehydroorotase containing multifunctional protein genes lines were grown with permissive doses of colchicine to maintain (11), dehydrofolate reducíasegenes (12), and MDR2 genes (5, resistance. 13-15). Assay of Drug Resistance. Colchicine resistance was determined in triplicate from the colony formation dose-response curves (17), after Previous investigations in this laboratory have demonstrated plating 200 cells in 6-well dishes (tissue culture cluster 6; Costar, that at least six different genes (classes 1-6) are coamplified in Cambridge, MA) in the presence of different concentrations of colchi the MDR Chinese hamster ovary cell line CHRC5 (14-16) cine. After an incubation period of 8-10 days at 37°C,clones were isolated by Ling and Thompson (17). The amplified sequences fixed, stained with 0.2% crystal violet (Merck 820603) in 3.7% gluta- include the pgp genes encoding the M, 170,000 P-glycoproteins raldehyde, and counted. Cell survival was calculated from the percent (15) which are responsible for the MDR phenotype (18-20) by age of cells that were able to produce a clone of at least 100 cells. As a reducing the intracellular drug accumulation (17,21). The other measure of the resistance the D,0 was used. The relative resistance was amplified genes coding for sorcin and unknown products were calculated from the ratio of the D,0 of the resistant cells to the D,0 of linked to the pgp genes as demonstrated by pulsed field gradient the sensitive, parental cell line. gel electrophoresis and in situ hybridization (14, 15, 22). A Determination of the Amplicon Copy Number. DNA from the differ reliable estimate of the size of this large amplicon in the CHRC5 ent cell lines was digested with EcoRI, serially diluted, and size- fractionated on 0.6% agarose gels. The separated products were blotted cells by summation of restriction fragments failed because the onto nitrocellulose (30) and sequentially hybridized io pgp\ (class 2c) fragments (spanning at least 1500 kilobases) detected did not and sorcin (class 4) genes with the probes cp28 and cp6 (see Refs. 14 and 22), respectively, that were 32P-labeled according to the method of Received 9/18/89; revised 12/27/89. The costs of publication of this article were defrayed in part by the payment Feinberg and Vogelstein (31). The gene copy number was estimated of page charges. This article must therefore be hereby marked advertisement in from the DNA concentrations, giving similar hybridization signals. The accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed. amount of DNA loaded on the gels was checked by the ethidium 2The abbreviations used are: MDR, multidrug resistance; HSR, homogene bromide-staining of the gel and by rehybridization to an actin probe. ously staining region; PI, propidium iodide; Mb, megabase pairs; I'm, drug Karyotyping by Microscopy. Cells for karyotype studies were pre concentration that reduces survival to 10%. pared and stained with trypsin-Giemsa by standard procedures accord- 2803 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1990 American Association for Cancer Research. DNA CONTENT OF A SIX-GENE AMPLICON ing to the method of Seabright (32). Karyotypes were based on at least 15 photographs of well spread metaphases. For identification of the chromosomes, the nomenclature of Deaven and Peterson (33) was used. Chromosome Isolation. Cell cultures were treated with vindesin for 4 hours. The concentrations used to arrest the cells in mitosis were 0.14, 0.14, 0.07, and 0.035 mg/ml with B3% B3-4, B3-5, and AuxBl cells, respectively. The chromosomes were isolated by the procedure de scribed by Aten eíal. (34). The mitotic cells were selectively detached by shake-off, centrifuged at 200 x g for 5 min, resuspended in 0.4 ml of a hypotonie solution containing 75 mM KCI and 45 UM PI, and incubated for 20 min at room temperature. Subsequently, 0.2 ml of a solution containing 57 m\i KCI, 45 /JM PI, and 2% Triton X-100 was added. The cells were incubated for 5 min to partially dissolve the cell t t t membrane and to allow the PI to enter the cells. Finally, the cell suspension was syringed twice through a 22-gauge needle to release the 20 10 10 20 20 10 7,5 10 7,5 5 1 3.5 20 10 7,5 10 7,5 i i 3,5 chromosomes from the metaphase cells. Chromosome suspensions were stored at -18°C after the addition of ethanol to a final concentration C* B of 30% until measurements. Flow Karyotyping. The chromosomes were analyzed at a rate of 200/ Fig. 2. Amplification of P-glycoprotein and sorcin genes in the MDR cell s using a System 30 cytofluorograph (Beckton Dickinson). The PI lines. The slots of a single 0.7% agarose gel, ABC, were loaded with EcoRI digested genomic DNA of the cell lines as indicated. The DNA was size fraction stained chromosomes were irradiated with 500 mW of 488 nm light ated, transferred to nitrocellulose paper, and hybridized with 3¡P-Iabeledprobes: from a 2000 Spectra Physics argon ion laser. The fluorescence signal A, B, and C for pgp\ genes; and rehybridized for sorcin genes (C*).

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