Determination of the DNA Content of a Six-Gene Amplicon in the Multidrug- Resistant Chinese Hamster Cell Line CHRB3 by Flow Karyotyping

Home , Amplicon

[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 Â¡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; GiÃ²co 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 37Â°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 reducÃasegenes (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 37Â°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

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 eÃal. (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 -18Â°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 3Â¡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*). Equal signals was detected at >630 nm. The electrical pulses from the photomultiplier for 20 /ig AuxBl, 10 /jg B3-4, and 2 /jg B3* (large arrows. A and B) indicate that tubes were individually integrated to obtain a frequency distribution of the pgp\ copy numbers are in the proportion of 1/20 to 1/10 to 1/2, suggesting chromosome fluorescence. 2, 4, and 20 copies, respectively. The refined dilution series for the revenants suggests a ratio of 3/4 (small arrows, C) indicating 3 and 4 copies of pgp\ in B3- Analysis of the Karyotypes. Using the X-chromosome as internal 5 and B3-4, respectively. The same ratio of sorcin genes (small arrows, C*) standard, the relative chromosome lengths were determined for each of suggests 3 and 4 copies of the sorcin gene as well. the cell lines from at least 15 photographed metaphase spreads to identify the peaks in the flow karyotype produced from the chromosome suspension. By grouping types of equal length, we obtained 12 classes 'Ã¨ corresponding to the 12 different peaks in the flow karyotypes. The a areas of the peaks were calculated by integrating the contents of the 1 100 channels corresponding to each peak. Mean and frequency of each chromosome group that produced a peak in the flow karyotypes were checked by the data of the cytogenetic analyses. The calibration of the DNA content of the chromosomes was based on the flow cytometric analysis of the nuclear DNA content of AuxBl cells in G, phase which was found to be 74% ofthat of normal human diploid cells, i.e., about 4.4 x 10" kilobases. 10

RESULTS B3-4 Relative Resistance to Colchicine and Gene Amplification. Fig. 1 presents the colony formation dose-response curves of the B3-5

AuxBl 00. 234 10 20 60 100 gene copy number Fig. 3. Correlation of the relative resistance to colchicine and the pgpl copy number. Relative resistance: D10MDR cells/D,0 AuxBl.

io- sensitive parental cell line AuxBl, the three subclones of CHRB3, e.g., B3+, B3-4, and B3-5, and the control diploid Chinese hamster cell lines V79 and CHNF22. The values for the relative resistance to colchicine (1, 40, 5, 3, 1, and 1, respectively) suggest that B3-4 and B3-5 were revenants with decreased levels of P-glycoprotein, whereas subclone B3+ was 1-2 comparable with the original CHRB3 (35). The sensitivity of AuxBl to colchicine was similar to that of the control Chinese hamster cells. The amplification of the class 2c (P-glycoprotein 1 = pgpl) and class 4 (sorcin) genes was estimated from Southern blots. Fig. 2 shows the final blot loaded with various DNA concentra 0.01 0.1 10 tions that were selected to produce at least a number of lanes colchicine(ug/ml) with bands of equal density between the cell lines (arrows). Fig. 1. Survival of B3*, B3-4, B3-5, AuxBl, and the control cells V79 and From these lanes the ratios of the number of gene copies were CHNF22 in increasing concentrations of colchicine. Resistance to colchicine as determined. Presuming that AuxBl is diploid for these genes, expressed by the DIOdetermined by colony formation assay was 1.8, 0.23, 0.12, B3+, B3-4, and B3-5 contain 20, 4, and 3 pgpl gene copies, and 0.045 Â¿igcolchicine per ml resulting in a relative resistance compared to AuxBl of 41, 5.2, and 2.8 for B3*, B3-4, and B3-5, respectively. respectively (Fig. 2, arrows). The same copy numbers were 2804

u * t Fig. 4. Representative karyograms of B3*, II K B3-4, B3-5, and AuxBl. The decrease in length of the 7q+ (frame) HSR is in agreement with the decrease in the relative resistance to colchicine. Other differences between the cell lines are an altered banding pattern in one of the homologues of chromosome 1 in the re 11 â€¢ 4 sistant cell lines and a deleted chromosome X OÃr in AuxBl (arrows).

I* Â¡

Z22 Z4Z3 Z5Z7 5 Z9Z8 7 8 9 10 X ZioZi2Zi3 found after rehybridization for the sorcin gene (shown for B3- lost amplified (HSR) DNA content by the subtracting proce 4 and B3-5). The quantitative similarity of the pgpl and sorcin dure. In contrast, B3-4 lost only DNA of the HSR which was gene copy numbers in the various cell lines suggests that the approximately 107 x 10' kilobases. As in the other chromo partial loss of the HSR involves multiples of the full length somes no DNA changes could be detected, the missing 107-Mb amplicon. Thus the amplicon copy numbers were approxi DNA from the HSR contained the 16 lost amplicons. If the mately 2, 3, 4, and 20 in AuxBl, B3-5, B3-4, and B3+, respec HSR harbors amplicons of one size, the size of the 16 amplicon tively. The correlation between the relative resistance (D,0 copies lost should be approximately 6.7 Mb. resistant cells/Dio AuxBl) and the gene copy number (Fig. 3) suggests that these numbers were rather accurately estimated. Karyotypes. Representative karyograms of B3+, B3-4, B3-5, DISCUSSION and AuxBl are shown in Fig. 4. We found numerically and We have shown by flow and cytogenetic karyotyping that structurally very stable chromosome complements which were 107-Mb HSR DNA harbor 16 pgpl copies and 16 sorcin gene nearly identical for all cell lines. The most remarkable cytoge- copies. We propose that the DNA fragment contains 16 copies netic difference between the MDR cell lines was observed in of the complete amplicon suggesting that the mean amplicon the lengths of the HSR in chromosome 7q+ (Fig. 4, bars). size comprises approximately 6.7 Mb. An exact estimate of the Close examination of the various HSR bearing chromosomes copy numbers is critical for accurate calculation of the amplicon revealed, however, that one or both of the bands adjacent to the size. Our estimation of a confidence interval of Â±50%is based HSR were affected in the revenant B3-5 and in the AuxBl on the small titration factors of 1.5 used in the dilution series subclone indicating that also nonamplified DNA was missing. to detect the gene copy numbers and the nearly linear relation Consistent chromosome abberations in the resistant cell lines, ship between gene copy number and the relative resistance. as compared to the sensitive parental AuxBl, were (a) the loss Thus the amplicon size is in the range of 4.5 to 10.1 Mb. of a dark band in the terminal part of the long arm of one of Amplicon sizes estimated from the relative length of HSRs the homologues of chromosome 1 and (b) an intact X-chro- which are known to be the cytogenetic representations of am mosome suggesting that the chromosome X deletion in the plified DNA (24, 25) indicate that the amplicons might indeed subclone of AuxBl originated after the development of colchi- be as large as 10 Mb (4, 36), which is much larger than the cine resistance. gene selected for. Such estimates, however, are based on two Table 1 presents the chromosomes classified into 12 groups assumptions: (a) the DNA content per HSR length is equal to (A-L) according to their relative lengths to identify the peaks the DNA content per length of the native chromosomes; and in the flow karyotypes (Fig. 5; see corresponding characters). (b) all amplified DNA sequences reside in the visible HSR. By The flow karyotypes of the various cell lines were quite similar, studying revenant clones we could avoid the need of these as was expected from the karyograms. Only the HSR bearing assumptions as we determined the DNA content (instead of the chromosome 7q+, moves from peak J in B3+, via G in B3-4 relative length) of HSR material that was actually lost from the and F in B3-5, to E in AuxBl (Fig. 5, arrows) suggesting that cells, whereas the remaining genome was unchanged. Thus the this chromosome loses its DNA during the process of reversion 16 amplicon copies lost must have been located in the lost HSR to colchicine sensitivity. material, suggesting that the mean length of an amplicon is 107 Table 2 presents the flow karyotypic estimate of the DNA Mb. That the HSR DNA was lost rather than translocated to content of chromosome 7q+ together with the number of am- other chromosomes in B3-4 and that this material was the only plicons present in the various cell lines. As the chromosome DNA that was lost from the genome at all were strongly 7q+ in B3-5 and AuxBl lacks also material from the adjacent suggested by the identical banding patterns and similar DNA bands outside the HSR, they are not suitable to determine the contents for all chromosomes but No. 7q+ in B3+ and B3-4 2805

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1990 American Association for Cancer Research. DNA CONTENT OF A SIX-GENE AMPLICON Table I Analysis of the flow karyotypes of AuxBl, B3-5, B3-4, and B3+ A shift in the position of chromosome 7q+ is observed from peak J in B3* to the peaks G. F, and E in B3-4. B3-5, and AuxBl, respectively (Column 4). The B3 peak characters in Column 1 refer to the characters in the flow karyotypes (Fig. 5). Columns 2 and 3 show the number of the chromosomes in each peak and the corresponding chromosome types. Column 4 gives the changing position of the changing chromosome 7q+ for the different cell lines. For other karyotypic differences see footnotes. of chromosomes PeakABC"DEFG*HIJKL"No.(7q+ excluded)111405023012ChromosomeidentificationZ131098,7q+incloneAuxBlB3-5B3-4B3*

8, ZIO,Z127.1,

5, Z8.Z9Z4,

Z5Z2, Z3,Xe21.

1Position " A deleted chromosome 9. resulting in the shift of peak C under peak B in all resistant cell lines. * A background peak of unknown origin at position G in all cell lines. c The deleted chromosome X in AuxBl, resulting in a shift in its position from I toH. Ã A slight difference in the DNA content between the chromosome 1 homo logues in the resistant cell lines.

Table 2 DNA content of chromosome 7q+ and the copy number of the amplicon sequence in the cell lines The revertan! B3-4 lost 16 amplicon copies accompanied with 107-Mb HSR 0) DNA, resulting in a size of 6.7 Mb/amplicon. .Ã› DNA E CelllineB3*B3-4 (x 103/kilobases)328 no.20

221 4 B3-5Â° 180 32 AuxBl"7q+ 0) 119Amplicon " B3-5 and AuxBl were excluded from the calculation of the amplicon size since the banding pattern of chromosome 7q+ suggests differences in the chro mosome regions adjacent to the HSR.

should be considered as a maximum value for the amplicon size. During successive selection rounds under drug pressure, new AuxB1 and shortened amplicons may be formed (23, 36, 37), as had been shown also in the more resistant cell line CHRC5 in which the gene classes 1, 2a, and 2b failed to coamplify in the last amplification round (14, 22). During the amplicogenesis in this series, initially 4 amplicons were generated besides the native sequences and in a second step the total number of copies increased to 20 in CHRB3. Whether the latter 14 amplicon copies were of the same size as the 4 that were generated in an earlier stage (16) is not known. If not, our size estimate would refer to the majority of the 16 amplicons lost. Previous attempts to estimate the size of the 6-gene amplicon fluorescence by constructing a long-range physical map of the amplicons in Fig. 5. Flow karyotypes of B3*, B3-4. B3-5, and AuxBl. The shift of chro the more resistant CHRC5 cells (16, 23) have yielded 1.5 Mb mosome 7q+ from position J via G and F to E between the cell lines spans 107 x IO3, 41 x IO3, and 61 x IO3 kilobase DNA, respectively. Arrows, position of as a minimal estimate, but the boundaries of the amplicon were 7q+. For explanation of the peaks see Table 1. not reached. More recent experiments, however, have raised this minimal estimate to 3 Mb,3 which is close to our maximal (Figs. 4 and 5, respectively). However, if nonamplified sequence estimate of approximately 6.7 Mb. had been inserted in the HSR and therefore counted as ampli As revenants may originate spontaneously, the cell lines were fied DNA, the mean size would be less. Such small insertions subcloned prior to the experiments to obtain optimal flow could not be excluded. With in situ hybridization we found a karyotypes and reliable estimates for the copy numbers. Sub- region in the much larger HSR in CHRC5 that did not hybridize clone B3* was comparable with the parental CHRB3 line, with the tested probes for four of the six amplicon genes (22). However, the HSR in B3* that is much smaller than that in inasmuch as the 10-fold amplification (16) was confirmed and the relative resistance factor of 40 was in good agreement with CHRC5 can hardly harbor 16 silver grains, suggesting that the previously estimated 30 in CHRB3 (35). The isolated AuxBl possible insertions of unknown origin are far below the detec tion level of such a technique. Therefore the estimated 6.7 Mb 3 P. Borst, personal communication. 2806

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1990 American Association for Cancer Research. DNA CONTENT OF A SIX-GENE AMPLICON was obviously a subpopulation of the previously described cell 14. de Bruijn, M. H. L., van der Bliek, A. M., Biedler. J. L., and Borst, P. Differential amplification and disproportionate expression of five genes in line (see for karyotypes 29 and 33), showing some chromosomal three multidrug-resistant Chinese hamster lung cell lines. Mol. Cell. Biol.. 6: alterations such as, e.g., the deleted X-chromosome which was 4717-4722, 1986. 15. van der Bliek. A. M., van der Velde-Koerts, T.. Ling. V.. and Borst, P. intact in the AuxBl derived resistant cell lines. The AuxBl Overexpression and amplification of five genes in a multidrug-resistant cells were supposed to be diploid for the pgp genes because of Chinese hamster ovary cell line. Mol. Cell. Biol., 6: 1671-1678. 1986. the presence of two identical, normal chromosome 1 homo 16. Borst, P., and van der Bliek. A. M. Amplification of several different genes in multidrug-resistant Chinese hamster cell lines. In: 1. B. Roninson (ed.). logues, the native carriers of the amplified genes (29, 38, 39). Molecular and Cellular Biology of Multidrug-Resistance in Tumor Cells, in This was confirmed by the sensitivity to colchicine that was press. 17. Ling, V., and Thompson. L. H. Reduced permeability in CHO cells as a similar to that of the control cells V79 and CHNF22 and by mechanism of resistance to colchicine. J. Cell. Physiol., 83: 103-116. 1974. the nearly linear relationship between the pgp gene copy number 18. Juliano. R. L., and Ling. V. A surface glycoprotein modulating drug perme and the resistance in the AuxBl-B3 series (Fig. 3). ability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta, 455: 152-162, 1976. Our data demonstrate that the two karyotyping techniques, 19. Riordan. J. R.. Deuchars. K., Kartner. N.. AlÃ³n,N., Trent. J., and Ling. V. cytogenetics and flow cytometry, are complementary. Cytoge- Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell netics is required to identify the peaks of the flow karyotype lines. Nature (Lond.). 316: 817-819. 1985. 20. Shen. D-W.. Cardarelli. C.. Hwang. J., Cornwell, M.. Richer!. N.. Ishii, S., and to determine the chromosome regions that are translocated Pastan, I., and Gottesman, M. M. Multiple drug-resistant human KB carci or deleted as indicated by shifts of peaks. Flow cytometry is a noma cells independently selected for high-level resistance to colchicine, Adriamycin, or vinblastine show changes in expression of specific proteins. more sensitive technique that can be used for detecting very J. Biol. Chem., 261: 7762-7770, 1986. small deletions or insertions (40). The accuracy obtained by the 21. Roninson. I. B. Molecular mechanism of multidrug resistance in tumor cells. combination of these methods makes them useful for estimating Clin. Physiol. Biochem., S: 140-151. 1987. 22. Jongsma. A. P. M., Spengler, B. A., van der Bliek. A. M.. Borst. P., and amplicons that are too large to be measured by physical map Biedler. J. L. Chromosomal localization of three genes coamplified in the ping using pulsed field gradient electrophoresis. The evidence multidrug-resistant CHRC5 Chinese hamster ovary cell line. Cancer Res., 47: that the HSR region was the only DNA that was lost from the 2875-2878, 1987. 23. Borst, P., van der Bliek, A. M., van der Velde-Koerts, T., and Hes, E. cells justifies the conclusion that this region contains the 16 Structure of amplified DNA. analyzed by pulsed field gradient gel electro lost amplicons, resulting in the maximal estimation of 4.5-10.1 phoresis. J. Cell. Biochem., 34: 247-258. 1987. 24. Biedler, J. L.. Melera. P. W., and Spengler. B. A. Chromosome abnormalities Mb/amplicon. and gene amplification: comparison of antifolate-resistant and human neuro- blastoma cell systems. In: J. D. Rowley and J. E. Ultmann (eds.). Chromo somes and Cancer: From Molecules to Man. Bristol-Myers Cancer Symposia, ACKNOWLEDGMENTS Vol. 5, pp. 117-138. New York: Academic Press. 1983. 25. Meyers, M. B., Spengler, B. A.. Chang, T-D., Melera, P. W., and Biedler, J. We thank Dr. P. Borst for advice and critical comments, J. Stap for L. Gene amplification-associated cytogenetic aberrations and protein changes technical assistance, and our colleagues in the laboratory for reviewing in vincristine-resistant Chinese hamster, mouse, and human cells. J. Cell the manuscript. The cell lines CHRB3 and CHNF22 were kindly Biol.. 700:588-597. 1985. 26. Biedler, J. L.. and Peterson. R. H. F. Altered plasma membrane glycocon- provided by Dr. V. Ling, Toronto, and Dr. J. L. Biedler, New York, jugates of Chinese hamster cells with acquired resistance to actinomycin D. respectively. daunorubicin. and vincristine. In: A. C. Sartorelli, J. S. Lazo, and J. R. Berlino (eds.). Molecular Actions and Targets for Cancer Chemotherapeutic Agents, pp. 453-482. New York: Academic Press. 1981. REFERENCES 27. Teeter, L. D.. Sanford. J. A., Sen, S-, Stallings, R. L.. Siciliano, M. J., and Kuo, M. T. Multidrug-resistant phenotype cosegregales with an amplified gene in somatic cell hybrids of drug-resistant Chinese hamster ovary cells 1. Cox, D.. Yuncken, C., and Spriggs, A. I. Minute chromatin bodies in and drug-sensitive murine cells. Mol. Cell. Biol.. 6: 4268-4273, 1986. malignant tumours of childhood. Lancet, 2: 55-58. 1965. 2g Brouwer, M., Smets, L. A., and Jongsma, A. P. M. Isolation and character 2. Biedler. J. L., and Spengler. B. A. Metaphase chromosome anomaly: asso ization of subclones of LI 210 murine leukemia with different sensitivities to ciation with drug resistance and cell-specific products. Science (Wash. DC), various cytotoxic agents. Cancer Res.. 43: 2884-2888. 1983. 191: 185-187, 1976. 29 Biedler. J. L.. Chang. T.-D.. Scotto. K. W., Melera. P. W., and Spengler. B. 3. Trent. J.. Meltzer. P.. Rosenblum. M., Harsh. G.. Kinzler, K., Mashal. R., A. Chromosomal organization of amplified genes in multidrug-resistant Feinberg, A., and Vogelstein. B. Evidence for rearrangement, amplification, Chinese hamster cells. Cancer Res., 48: 3179-3187. 1988. and expression of c-myc in a human glioblastoma. Proc. Nati. Acad. Sci. -,Q Maniatis, T., Fritsch, E. F., and Sambrook. J. Molecular Cloning: A Labo USA, 83: 470-473, 1986. ratory Manual. Cold Spring Harbor. NY: Cold Spring Harbor Laboratory, 4. Brodeur, G. M., and Seeger. R. C. Gene amplification in human neuroblas- 1982. tomas: basic mechanisms and clinical implications. Cancer Genet. Cytoge- j| Feinberg, A. P., and Vogelstein. B. A technique for radiolabeling DNA net., 19: 101-111. 1986. restriction endonuclease fragments to high specific activity. Anal. Biochem., 5. Roninson, I. B.. Abelson. H. T.. Housman. D. E., Howell, N., and Varshav- I32:6-\ÃŒ, 1983. sky, A. Amplification of specific DNA sequences correlates with multi-drug 32 Seabright, M. A rapid banding technique for human chromosomes. Lancet. resistance in Chinese hamster cells. Nature (Lond.). 309: 626-628. 1984. 2:971-972, 1971. 6. Harker, W. G., and Sikic, B. I. Multidrug (pleiotropic) resistance in doxo- 33 Deaven, L. L., and Petersen. D. F. The chromosomes of CHO. an aneuploid rubicin-selected variants of the human sarcoma cell line MES-SA. Cancer Chinese hamster cell line: G-band, C-band. and autoradiographic analyses. Res., Â«.-4091-4096, 1985. Chromosoma (Bed.). 41: 129-144. 1973. 7. Meltzer, P. S., Cheng. Y.-C., and Trent, J. M. Analysis of dihydrofolate 34 Aten. J. A., Buys. C. H. C. M., van der Veen, A. Y., Mesa, J. R.. Yu, L. C., reducÃasegene amplification in a methothrexate-resistant human tumor cell Gray. J. W., Osinga. J.. and Stap. J. Stabilization of chromosomes by DNA line. Cancer Genet. Cytogenet., 17: 289-300. 1985. Â¡ntercalatorsfor flow karyotyping and identification by banding of isolated 8. Federspiel, N. A., Beverley, S. M.. Schilling. J. W.. and Schimke, R. T. Novel chromosomes. Histochemistry, 87: 359-366, 1987. DNA rearrangements are associated with dihydrofolate reducÃasegene am- 35. Kartner. N., Evernden-Porelle, D.. Bradley. G.. and Ling, V. Detection of P- plification. J. Biol. Chem.. 259: 9127-9140, 1984. glycoprotein in multidrug-resistant cell lines by monoclonal antibodies. Na 9. Krawczun, M. S.. Camargo, M., and Cenenka. J. Patterns of BrdU incor ture (Lond.). 316: 820-823. 1985. poration in homogeneously staining regions and double minutes. Cancer 36. Stark. G. R. DNA amplification in drug resistant cells and in tumours. Genet. Cytogenet., 21: 257-265. 1986. Cancer Surv.. S: 1-23, 1986. 10. Fox, M. Gene amplification and drug resistance. Nature (Lond.), 307: 212- 37. Guilotto. E., Saito. I., and Stark. G. R. Structure of DNA formed in the first 213. 1984. step of CAD gene amplification. EMBO J., J: 2II5-2121. 1986. 11. Wahl, G. M., de Saint Vincent, B. R., and DeRose, M. L. Effect of chro- 38. Sen. S.. Teeter. L. D., and Kuo. T. Specific gene amplification associated mosomal position on amplification of transfected genes in animal cells. with consistent chromosomal abnormality in independently established mul Nature (Lond.), 307: 516-520, 1984. tidrug-resistant Chinese hamster ovary cells. Chromosoma (Beri.). 95: 117- 12. Lewis, J. A., Biedler. J. L.. and MÃªlera. P. W. GÃ¨neamplification accom 125. 1987. panies low level increases in the activity of dihydrofolate reducÃasein ami- 39. Teeter. L. D., Atsumi. S.-I., Sen, S., and Kuo. T. DNA amplification in folate-resistant Chinese hamster lung cells containing abnormally banding multidrug. cross-resistant Chinese hamster ovary cells: molecular character chromosomes. J. Cell Biol.. 94:418-424. 1982. ization and cytogenetic localization of the amplified DNA. J. Cell Biol., 103: 13. Ling, V., Kartner, N., Sudo. T., Siminovitch. L.. and Riordan, J. R. Multi- 1159-1166.1986. drug-resistance phenotype in Chinese hamster ovary cells. Cancer Treat. 40. Groen. F. C. A., and van der Ploeg. M. DNA cytophotometry of human Rep.. 67: 869-874. 1983. chromosomes. J. Histochem. Cytochem.. 27: 436-440. 1979. 2807

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1990 American Association for Cancer Research. Determination of the DNA Content of a Six-Gene Amplicon in the Multidrug-resistant Chinese Hamster Cell Line CH RB3 by Flow Karyotyping

A. P. M. Jongsma, A. Riethorst and J. A. Aten

Cancer Res 1990;50:2803-2807.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/50/9/2803

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/50/9/2803. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.