Hereditas 106: 97-105 (1987)

Isolation of selectively amplified DNA sequences from multidrug-resistant SEWA cells FREDRIK STAHL, PETER SANDBERG, TOMMY MARTINSSON, JAN SKOOG, BJC)RN DAHLLOF, YVONNE WETTERGREN, GUNNAR BJURSELL' and GORAN LEVAN Departments of Genetics and Medical Chemistry', Goteborg University, Sweden

STAHL F., SANDBERG, P., MARTINSSON,T., SKOOG, J., DAHLLOF,B., WETTERGREN, Y..BJURSELL. G. and LEVAN.G. 1987. Isolation of selectively amplified DNA sequences from multidrug-resistant SEWA cells - Hereditas 106.9-105. Lund, Sweden. ISSN 0018-0661. Received May 1,1986 A double minute (DM)-fraction was enriched by centrifugation from actinomycin D (AMD)-resistant SEWATC13 murine cells. Among DNA sequences cloned from the DM-fraction and exhibiting specific amplification, 3 were chosen to probe the amplicons of 8 multidrug-resistant (MDR) SEWA lines, 6 related AMD-resistant sublines and 2 independently selected sublines resistant to vincristine and colcemid, respectively. It was determined that some common DNA sequences had been amplified in all the lines, but that the total DNA region amplified varied among lines. We compared the DM-containing AMD-resistant line to a line with homogeneously staining regions (HSR),which had been derived from it by prolonged maintenance under constant selection in AMD. With 1 probe it was found that some previously unamplified DNA sequences were amplified in the HSR-carrying line, suggesting a complex mechanism behind the transition from DM to HSR. In contrast, identical Southern blot patterns were found in the AMD-resistant line with DM and 4 subclones with different HSR, indicating that in this case the HSR arose by integration of an amplicon present in the DM (without extensive rearrangement) rather than by a second amplification event. The tentative conclusion from our experiments is that in SEWA cells MDR is accomplished by the amplification of DNA sequences. The primary amplified DNA resides in DM, which under constant selection tend to be replaced by HSR. This second step may or may not be accompanied by amplification of previously unamplified DNA sequences.

GBran Levan, Department of Genetics, Box 33031, S-400 33 Goteborg, Sweden

Multidrug resistance (MDR, also called pleiotropic 1981; POLOTSKAYAet al. 1983; DAHLLoFet al. 1984). drug resistance) is a common response to selection The function of the overexpressed proteins is still in vitro to a group of structurally unrelated com- unknown and it is not clear whether all of them pounds such as actinomycin D (AMD), vincristine contribute to the resistance phenotype or if some (VCR) and colcemid (COL). Resistant cells are are overexpressed just as a result of co-amplificacharacterized by a wide cross-resistance to drugs tion. other than the selective agent. In many cases the Most lines exhibiting MDR display the cytogeresistance has been correlated to a reduced net up- netic signs of gene amplification; double minutes take of drug (BIEDLER et al. 1975; BECK1983; MAR- (DM) and homogeneously staining regions (HSR). We have established MDR lines from the murine TINS SON^^ al. 1985). Selective gene amplification (for rewiew, see SEWA ascites tumor exhibiting both DM and HSR et al. 1984; MARTINSSON et a1 1985). All STARK and WAHL1984; HAMLIN et al. 1984) is an (DAHLL~F important step in the MDR development and it was lines but 2 overexpress the low M W cytoplasmic recently shown that, in one case, at least 5 different protein. In this paper, we report on the molecular genes were amplified (VANDERBLIEKet al. 1986). cloning of amplified DNA sequences from DM of One of these is the gene for a 170 kDa glycoprotein, an AMD-resistant SEWA cell line. Some of the found to be overexpressed in many instances of amplified DNA-sequences of the AMD-resistant MDR (JULIANO and LING1976; BIEDLER et al. 1983; line were also amplified in independently derived BECK1983), whereas another may be the gene for a VCR- and COL-resistant SEWA lines. low MW cytoplasmic protein (MEYERS and BIEDLER

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Material and methods Cell material and culturing conditions. -All cell lines were carried as stationary suspension cultures in McCoy 5A medium with 16 'YO fetal calf serum, supplemented with drug as needed. The cell lines used were all derivatives of the murine SEWATC13K line, which is sensitive to the drugs employed. Resistant derivative lines were produced by stepwise selection in the drug-containing media (Table 1).

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Cloning of DM-DNA in pATl53. - The DM-DNA was digested with BamHI and Hind111 and was mixed with 0.4 p g of DNA from the plasmid pAT153, which had been digested with the same restriction enzymes. The DNA was precipitated with ethanol, dissolved in ligation buffer (50 mM MgC12,5 mM 1,4-dithiothreitol, 1mM ATP, 66 mM Tris-HC1, pH 7.6) and ligated in 10 pl for 2 hours at 10°C with T4-ligase. The regenerated plasmids were used to transform E. coli K12 FLOl (MM294/recA, F. Lindberg, unpublished).

Two-dimensional PAGE. - High-resolution two- Differential screening of transformed bacteria. - All dimensional protein electrophoresis was performed tetracycline-sensitive colonies were replicated onto according to the method of O'FARRELL (1975) with nitrocellulose filters by blotting (BENTONand minor modifications as described earlier (DAHLLOF DAVIS1977). DNA from a DM-enriched fraction of et al. 1984). Gels were silver stained by the method TC13AMD0.1~14and total DNA from the sensideveloped by Ansorge as modified by MOSELARSEN tive TC13K were nick translated with 02P)dCTP (RIGBY et al. 1977) and hybridized to the replica et al. (1983). filters. Colonies showing marked hybridization to DNA from the resistant line but not the sensitive Enrichment and preparation of DM-DNA. - line were selected for further study. DM-enrichment was made through centrifugations. Similar methods have been described earlier Dot-blot analysis. - The Biorad dot blot apparatus (BARKER 1982; GEORGEand POWERS 1981). Cells was used and essentially the method of KAFATOS growing under optimal conditions were treated et al. (1979) was followed. The nitrocellulose filters with 2 pg colcemid/ml for 12 hours. A mitotic were hybridized to the DM-DNA clone Te7 as deindex of about 30 YO was reached. Approximately scribed below. los TC13AMD'O. lc14 cells were pelleted by centrifugation in a swing-out rotor at 1000 x g for 5 min. Isolation of DNA, Southern blotting and hybridizaAfter centrifugations, as described above, the cells tion. -Total DNA from different SEWA lines were were resuspended in medium, polyamin buffer extracted, essentially as described by BLINand (Youmet al. 1981) and in hypotonic solution (10 ml STAFFORD (1976). After digestion with EcoRl the of 75 mM KC1) and left on ice for 10 min. There- fragments were separated on 1 YO agarose gels and after the cells were washed with polyamin buffer transferred to nitrocellulose filters (SOUTHERN and finally lysed by dropwise addition of 4 ml of 1975). Plasmids from the colonies selected by diffepolyamin buffer with 0.3 'YO digitonin during rential screening were isolated by the method of vigorous mixing. The mixture was pushed 6 times HOLMES and QUIGLEY (1981), nick translated (RIGBY through an 18 gauge needle before use. The chro- et al. 1977) with ('2P)dCTP and hybridized to filters mosome suspension was layered on a 4 ml cushion in a first screening. For the final analysis the DMof 20 YOsucrose in polyamin buffer and centrifuged DNA clones Ie7, If8 and IIa5, were restricted with for 30 min at 3000 X g. The supernatant was diluted Bam H1 and Hind I11 and separated on an agarose with an equal volume of polyamin buffer and centri- gel. Inserts were cut out from the gel and labeled fuged at 3000 x g for 1 hour. The pellet was resu- with (j2P)dCTP according to FEINBERG and VOGELspended in 0.5 M EDTA and 5 YO sarcosyl, pH 8.0 STEIN (1984). Hybridization conditions were as and treated with proteinase K (100 pg/ml) at 50°C follows: Probes were boiled for 10 min before added over night. After several phenol and chloroform to 10 ml hybridization buffer: 6XSSC (Standard extractions the DNA was precipitated with ethanol Saline Citrate, 1XSSC: 0.15 M NaCl and 15 mM and dissolved in TE-buffer (1 mM EDTA and Nacitrate pH 7.0), 0.2 Yo NaDodS04, 1XDenhart's 10 mM Tris, pH 8.0) with 100 pg/ml of pancreatic solution (0.02% bovine serum albumin, 0.02 % RNase A. After 2 hours the DNA was reextracted, polyvinylpyrrolidon 0.01 70 Ficoll 400) and 0.25 precipitated and redissolved in TE-buffer. The mg/ml herring sperm DNA). Hybridization to procedure yielded about 1 p g of DM-enriched nitrocellulose filters was over night at 65°C. Before DNA. hybridization filters were incubated in hybridization

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buffer at 65°C for at least 3 hours. Filters were washed in 3 changes of 0.6XSSC and 0.2 % NaDodSO, at 65°C. In situ hybridization. - Chromosome preparations were made according to a standard method (MARTINSSON et al. 1982) and hybridization in situ was performed as described by HARPER et al. (1981) with some modifications. The slides were treated with pancreatic RNase A at 50 pg/ml in 1XSSC at 37°C for 1 hour, denatured in a mixture of 70 YO formamide in 2XSSC at 70°C for 2 min and dehydrated in ethanol. The probe (Ie7) was radioactively labeled by nick translation (RIGBY et al. 1977) with ('H)dCTP (62 Ci/mmol), eH)dTTP (96 Ci/mmol) and 'H)dATP (73 Ci/mmol) to a specific activity of 2-4x lo7cpmlpg DNA. The hybridization mixture, containing 0.1 pg labeled probe, 50 % formamide, 2XSCP (2XSSC,0.04 M NaHP04, pH 6.0), 1XDenhart's solution, 100 pg of herring sperm DNA and 10 % dextran sulphate per ml was denatured at 70°C for 5 min and then cooled on ice. Ten ~l of hybridization mixture was added to each slide, a coverslip was applied and mounted, and the slides were incubated at 37°C for 15 hours. The coverslip was taken off and unhybridized probe was removed by sequential washings, 3 times 10 rnin in 50 YO formamide/2XSSC at 39°C and 3 times 10 min in 2XSSC at 39"C, followed by dehydration in ethanol. The slides were dipped in Ilford Nuclear Research K2 emulsion diluted 1:l with water and exposed for

5-11 days. After developing, the slides were stained with Wright's stain (SANCHEZ et al. 1973) and analyzed.

Results Cytogenetics and protein analysis of the cell lines used The cell lines were all derivates of the SEWATC13K line. This line is sensitive to AMD, VCR and COL, and exhibits 2 HSRs, one of which was shown to contain amplification of the c-myc oncogene (SCHWAB et al. 1985; unpublished data). Independent selection experiments yielded lines resistant to AMD, VCR och COL. Each of these lines displayed numerous DM in additon to the HSRs already present in the parental line and, except for the COL-resistant line, they all overexpressed a 21 kDa protein. On prolonged maintenance in constant AMD selection, the DM of the AMD-resistant line were replaced by 2 new HSR, and concomitantly ceased to overexpress the 21 kDa protein (TC13AMD'O.l). At an early stage, the DMcontaining AMD-resistant line was subcloned in AMD-free medium. The subclones had very few DM, low level expression of the 21 kDa protein, and greatly reduced AMD resistance. One of the subclones was subjected to AMD selection, and a high level of DM content and of 21 kDa protein

Table 1. Culturing conditions, cytogenetics and protein data on SEWA cell lines. Cell line

Selective agent

Maintenance drug conc.

&ml TC13K TC13AMD'O. lh TC13AMDO. lc14 TC13AMD'O. l ~ 1 4 : 6 ~ TC13AMDr0.1c14 13" TC13AMDQ. 1 ~ 1 4 1 7 ~ TC13AMDO.l~14:19~ TC13COL'O. lh TC13VCR'l

AMD AMD

AMD AMD AMD AMD

COL VCR

99

0.1 0.1 0.01 0.01 0.01 0.01 0.1 1.0

"In addition all lines exhibited HSR representing amplification of the c-myc oncogene bIndependently selected from TC13K 'Subclone of TC13AMDQ.1, used for DM enrichment dSubclone of TC13AMD0.1~14 'Approximately 15 % of the cells also displayed a newly developed HSR

Expression of 21 kDa protein

Cytogenetic" feature related to resistance

HSR DM

HSR HSR HSR

HSR DM DM

100

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Fig. 2 A and B. Dot blot analysis. Hybridization of ('3')labeled DNA from the DM-DNA clone Ie7, to equal amounts of total DNA from (A) TC13AMD0.1~14and (B) a DM-enriched fraction of TC13AMD'O.lcl4. The values in the upper row are amount of DNA in nanogram.

in propidium iodide and visually inspected in the fluorescence microscope. The efficiency of the DMenrichment was evaluated by comparisons to a parallel procedure performed on TC13K cells (containing no DM). In Fig. 1A a mitotic cell of TC13AMDr0.1c14 is shown. The cell contains 46 chromosomes and about 70 DM. Fig. 1B shows a sample after the DM-enrichment procedure. It contains only DM, although other samples would contain an occasional chromosome. A dot blot was made of equal amounts of DNA from TC13AMDr0.1c14 and a DM-preparation on the same material (Fig. 2) When using one of our DMFig. 1 A and B. Efficiency of DM-enrichment. Propidium DNA clones (Ie7) as a probe an &fold enrichment iodide stained slides before and after DM-enrichment. was found in the DM-preparation. Fig. A. Metaphase plate of TC13AMD9.1~14.The DM A DNA library was prepared in pAT1.53 plasmids are seen as small dots between the chromosomes. Fig. B. Sample from an DM-enriched fraction of TC13AMDO.lc14 from the DM-enriched fraction, and the plasmids were used to transform bacteria. Out of a total of with a high frequency of DM-like structures. 300 colonies, 162 tetracycline-sensitive transformants were isolated and tested in the differential screening. Forty-two colonies showing an increased overexpression was restored (TC13AMD0.1~14). hybridization to DM-DNA from TC13AMD0.1~14 On maintenance of this line in medium with ADM, as compared to DNA from the sensitive TC13K cells appeared where the DM had been replaced by were selected (Fig. 3). HSR. At this stage 20 single cell clones were isolatTo finally determine which of the isolated DMed, all of which proved to exhibit new HSR and no DNA clones contained selectively amplified seDM. Four subclones displaying a unique set quences, 19 of them were radioactively labelled of HSRs were studied in detail. In contrast to and hybridized to Southern blots of EcoRl digested TC13AMDB. 1, these subclones continued to over- DNA from TC13K and TC13AMD0.1~14.Fourexpress the 21 kDaprotein at a high level (Table 1). teen of them gave rise to at least 1 band of greatly increased intensity in the latter line as compared to the former. In Fig. 4, 6 examples are shown. As Enrichment of DM seen, the pattern is not identical among the DMCells of TC13AMD10.1c14 carrying about 50 DNA clones. Some of them recognized only 1fragDM/cell were used in the DM-enrichment proce- ment, highly amplified compared to a corresponddure. The different steps during enrichment were ing band in the control line (lane 1). Others recogmonitored by taking samples which were stained nized several fragments, all of which were amplified

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101

Fig. 4. Test of DNA clones selected in differential screening. Total DNA from (a) TC13K and (b) TC13AMD'O. lc14, restricted with EcoRl and hybridized to 6 DM-DNA clones (1. IIall, 2. I e l l , 3. IIc4, 4. TIcl, 5. IIdl, and 6. IIb4) in Southern blots.

Fig. 3. A and B. Differential screening. After cloning DNA from a DM-enriched fraction into a pAT153 plasmid, 162 recombinants were picked and replicas hybridized to ("P)-labeled DNA from (A) TC13K and (B) a DM-enriched fraction of TC13AMDO. lc14, respectively. Colonies (Ie7, If8 and If9) showing excess hybridization to TCl3AMDcO.lcl4 compared to TC13K (circles) were candidates for containing DM specific sequences and after further testing were found to be specifically amplified in TC13AMD'O. lc14.

compared to the control (lanes 5,6). Others still recognized 1or several fragments which seemed to be repetitive both in control cells and in the resistant line (lanes 2, 3, 4). In these cases the bands were more intense in the resistant line generally, or there were specific bands, which were much more intense. Generally, the specifically amplified bands

represented true amplification of existing DNA sequences since they all appeared to be present in a lower copy number in the sensitive line. Out of the 14 DM-DNA clones 4 appeared to hybridize to the same EcoRl fragments, whereas the other 10 exhibited unique hybridization patterns. When adding together specifically amplified bands detected with the 11 different DM-DNA clones hybridized to EcoRl digested TC13AMD'O. lc14, approximately 130 kb was found to be covered.

Similarities and differencesin DNA amplification in different MDR SEWA cell lines Three of the DM-DNA clones were used individually to probe 8 SEWA lines exhibiting MDR and 2 controls, mouse liver and TC13K (Fig. 5, 6 and 7). The first DM-DNA clone (Ie7) recognized a common amplified fragment in all 8 PDR lines (Fig. 5) This quite clearly shows that MDR cell lines independently derived from TC13K amplify some common DNA sequences. The second DMDNA clone (If8) gave rise to the same 3-band pattern in TC13AMDr0.1c14 and its sublines. In

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Fig. 5. Hybridization with ("'P)-labeled DM-DNA clone Ie7 to EcoRl restricted total DNA from (a) mouse liver, (b) TC13K, (c) TC13AMDr0.1c14, (d) TC13AMDr0.1, (e) TC13AMDr0.1c14:6, ( f ) TC13AMDr0.1c14:13, (g) TC13AMDr0.1c14:17, (h) TC13AMDO.lc14:19, (i) TC13COL'O.l and (j) TC13VCR1. Size marker is Hind 111digested lambda DNA (C1857 Sam7).

Fig. 6. Hybridization of ('2P)-labeled DM-DNA clone from If8 to EcoRl restricted total DNA. Lanes as in Fig. 5.

Discussion From the present experiments it can be estimated that about 19 YO of the isolated DM-DNA clones represented sequences specifically amplified in the TC13AMDO.l only 2 of these bands were ampli- resistant line. This figure shows that our DMfied, whereas If8 did not detect any amplified se- enrichment procedure has led to an enrichment of quences in TC13VCRl or TC13COL'O.l (Fig. 6). amplified sequences. Estimates of the amount of The third DM-DNA clone tested (IIa5) again gave DM chromatin in TC13AMD'O. lc14 cells indicate rise to indistinguishable banding patterns for that the relative amount of amplified chromosome TC13AMD0.1~14and its subclones (Fig. 7). As material is not over 2 YO,so the enrichment in the with the If8 DM-DNA clone TC13AMD'O.l differ- DM fraction is 10-fold, which also fits the data ed from the other lines; this time showing amlifica- obtained in the dot blot experiment. Most data from cell lines where drug resistance tion of a number of bands that were not amplified or amplified to a lesser degree in TC13AMD0.1~14 has developed after stepwise selection indicate that and its subclones. Some of the TC13AMDB.l amp- there is a reasonably good correlation between such lified bands were also amplified in TC13COLO. 1 parameters as average number of DM per cell, degree of amplification, degree of gene expression, and in TC13VCR'l (Fig. 7). The Ie7 DM-DNA clone, showing amplification and level of resistance. Several workers have in all the MDR lines, was used for an in situ hybri- attempted to estimate other parameters, e.g., dization experiment. TC13AMDO.lc14:13 is one number of gene copies per DM or the size of the of the subclones of TC13AMD0.1~14and exhibits amplicon. At best, these estimates have been very an HSR located terminally in chromosome 11(Fig. rough. The figures obtained have also been very 8B), which arose concomitant with loss of the DM variable. Thus, the number of gene copies per single present in the parental line. As seen in Fig. 8A and minute might be 1-5, and the size of the amplicon C there is a very clear localization of silver grains somewhere between 150 and 3000 kb (for review, et al. 1984; and STARK and WAHL1984). to the distal part of the HSR chromosome. De- see HAMLIN tailed analysis of 38 metaphases showed that out of For instance, in SEWA cells carrying myc-amplifia total of 901 silver grains 449 (49.8 YO)were local- cation in C-bandless chromosomes (CM, chromosomes consisting entirely of HSR; LEVAN and LEVAN ized to the HSR.

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103

Fig. 7. Hybridization of pP)-labeled DM-DNA clone from IIa5 to EcoRl restricted total DNA. Lanes as in

Fig. 5 .

1982), we have estimated that 1 CM chromosome contains about 40 copies of the c-myc oncogene (unpublished data). Assuming that the CM chromosomes contain nothing but amplified sequences, a simple calculation based on the mouse total genome size indicates that the amplicon size in these cells is in the neighborhood of 3000 kb. In summary, estimates of number of gene copies per DM and of amplicon size vary considerably. This could be because of inaccuracies in the estimations, but, on the other hand, there is no obvious reason to believe that either of these parameters is constant in different inductions. In our present investigation we have found that, after EcoRl digestion of total DNA from TC13AMD0.1~14, our DM-DNA clones cover about 130 kb of DNA, which therefore is the present minimum estimate of the amplicon size in this material. At least 1of the DM-DNA clones (Ie7) detected a similar EcoRl fragment, amplified in all 8 lines exhibiting MDR. Three of these lines were selected independently using different selective agents. This clearly indicates that amplification of identical or similar genes occurs during the development of MDR. Equally clear is that the amplified regions in the independently selected cell lines are not identical, since the banding pattern differs between these lines when probed with 2 of the 3 DM-DNA clones tested. Similar observations have been made in other materials by other groups (RONINSON et a1

Fig. 8. A-C. In situ hybridization with the (“H)-labeled DM-DNA clone Ie7 to metaphase chromosomes of TC13AMDO. lc14: 13. Fig. A. Metaphase plate showing selective hybridization to the HSR chromosome, which is depicted in G-staining in (B)(the HSR is indicated by the bar to the right. Fig. C. Cutout HSR chromosomes from other metaphases displaying selective hybridization to the distal part of the HSR chromosome.

1984; FOJO et al. 1985; GuDKovet al. 1985;RIORDAN et d.1985; VANDERBLIEK~~ al. 1986). The TC13AMD0.1~14derivatives were cloned at a time when the parental line was starting to lose DM, and each subclone exhibited a unique set of HSR. In these lines every DM-DNA clone tested gave rise to identical banding patterns, indicating that DM-DNA sequences had been integrated into HSR without extensive rearrangement. In contrast, when HSR developed in TC13AMD’O.1there were changes in the banding patterns obtained with 2 of the 3 DM-DNA clones tested, suggesting that this HSR emerged by a different mechanism. Our results emphasize that there is an intimate relationship between the DM and the HSR in this material. This is corroborated by the fact that a probe derived from the DM-fraction hybridizes in situ to an HSR.

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16-24 HAMLIN, J.L., M I ~ R A N D T ,J.D., HEINTZ. N, H, and AZIZKHAN, J.C. 1984. DNA sequence amplification in mammalian cells. - h t . Rev. Cyto/ 90: 31-82 HARPER, M.E., ULLRICH, A. and SAUNDERS. G.F. 1981. Localization of the human insulin gene to the distal end of the short arm of chromosome 11. - Proc. Nutl. Acad. Sci. U,S.A,78:445m HOLMES, D.S. and QUICLEY, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. - Anal. Biochem. 114; 193-197 JULIANO, R.L.and LING, V.A. 1976. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. - Biochint. Biophys. A c a . 455: 152-162 KAFATOS.F.C., JONES, C.W. and EFSTRATIADIS. A. 1979. Determination of nucleic acid sequence homologies and relative concentration by a dot hybridization procedure. Nucleic Acids Res. 7: 1541-1552 LEVAN,G. and LEVAN. A. 1982. Transition of double minutes into homogeneously staining regions and C-bandless chromosomes in the SEWA tumor. - In Gene Amplification ( E d . R. T. Acknowledgements. - Devoted technical assistance by Ms. Britt SCHIMKE), Cold Spring Harbor Laboratory, p. 91-97 EmegArd is gratefully acknowledged. This work was supported by MARTINSSON,T . , TENNING,P., LUNDH,L. and LEVAN. G. grants from the Swedish Cancer Society, the Erik Philip-Sorensen 1982. Methotrexate resistance and double minutes in a cell Foundation and the Nilsson-Ehle Foundation. TM is a recipient line from the SEWA mouse ascites tumor. - Hereditas 97: of a fellowship from the Swedish Cancer Society. 123-137 MARTINSSON. T., DAHLLOF,B..WETTERGREN. Y., LEFFLER. H. and LEVAN. G. 198.5. Pleiotropic drug resistance and gene amplification in a SEWA mouse tumor cell line. - E.rp. CellRes. Literature cited 158: 382-394 BARKER, P. 1982. Isolation of double minutes from a human MEYERS, M.B. and BIEDLER,J.L. 1981. Increased synthesis of a low molecular weight protein in vincristine resistant cells. tumor cell line. - I n Gene Amplification ( E d . R.T. SCHIMKE), Cold Spring Harbor Laboratory, p. 205-212 Biochem. Biophys. Res. Conimun. 99: 228-235 BECK. T. B. 1983. Vinca alkaloid-resistant phenotype in cultured MOSE LARSEN, P . , FEY, S.J.. BRAVO, R. and CELIS, J.E. 1983. human leukemic lymhoblasts. - Cancer Treat. Rep. 67: 875-882 Mouse mitochondria1 protein IEF 24: Idenfication and BENTON, W. D. and DAVIS, R. W. 1977. Screening )r gt recombiimmunohistochemical localization of mitochondria in various tissues. - Electrophoresis 4: 211-256 nant clones by hybridization to single plaques in situ. - Science 196: 18&182 O'FARRELL,P.H. 1975. High resolution two-dimensionalelectroBIEDLER, J. L., RIEHM. H., PETERSON, R.H.F. and SPENGLER. phoresis of proteins. - 1. Biol. Chem. 250: 4007-4021 POLOTSKAYA, A.V.. GUDKOV. A . V . and KOPNIN, B.P. 1983. B.A. 1975. 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Resistgenes in multidrug-resistant cell lines. -Nature 316: 817-XI0 ance to actinomycin D and to vincristine induced in a SEWA RONINSON,I.B., ABELSON, H.T., HOUSMAN, D.E.. HOWELL. mouse tumor cell line with concomitant appearance of N. and VARSHAVSKY.A. 1984. Amplification of specific DNA double minutes and a low molecular weight protein. - Exp. sequences correlates with multidrug resistance in Chinese Cell Res. 152: 415426 hamster cells. - Nature 309: 626-628 FEINBERG, A. P. and VOGELSTEIN, B. 1984. Addendum. A SANCHEZ, ESCOBAR,J . I . and YUNIS, J.J. 1973. A simple Gtechnique for radiolabeling DNA restriction endonucleases banding technique. -Lancet (2):269 fragments to high specific activity. - Anal. Biochem. 137: SCHWAB, M., RAMSAY, G., ALITALO, K., VARMUS, H.E.. 266-267 BISHOP, J.M., MARTINSSON, T., LEVAN, G. and LEVAN. A. FOJO, A. T., WHANG-PENG, J., GO-ITESMAN, M. M. and 1985. Amplification and enhanced expression of the c-myc PASTAN, I. 1985. 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ed with-ihe disappearance Of the 21 kDa protein overexpression found in Other multidrugresistant lines. Simultaneously there was a change in the Southern blot patterns, indicating loss of part of the ampiicon and amplification af additional DNA sequences. The MDR phenotype was retained. The simplest explanation of these data is that the gene encoding the 21 kDa protein usually is coamplified together with other genes responsible for the MDR. However, the repeated observations of overexpression of a low M W acidic protein in many independent MDR lines seems to contradict this notion (MEYERSand BIEDLER1981; BECK1983; POLOTSKAYA et al. 1983; DAHLLOF et al. 1984; TWENTY MAN^^ al. 1986).

-

o.,

Hereditas 106 (1987) V A N D E R BLIEK, A., V A N D E R VELDE-KOERTS, T ., LING, V. and BORST, P. 1986. Overexpression and amplification of

five genes in a multidrug-resistant Chinese hamster ovary cell line. - Mol. Cell. Biol. 6: 1611-1618

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