GENES, CHROMOSOMES 8 CANCER 4:314-320 (1992)

Detection of Amplified DNA Sequences in Human Tumor Cell Lines by Fluorescence In Situ Hybridization lrit Bar-Am, Orna Mor, Herman Yeger, Yosef Shiloh, and Lydia Avivi Department of Human Genetics, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel (1.B.-A,,O.M.. Y.S., L.A.);Department of Pathology, The Hospital for Sick Children, Toronto, Ontario, Canada (H.Y.)

An unambiguous and rapid characterization of amplified D N A sequences in tumor cells is important for the understandingof neoplastic progression. This study was conducted t o evaluate the potential of fluorescence in situ hybridization (FISH) t o identify such amplified DNA sequences in human tumor cell lines. Applying this technique, we followed the metaphase location and interphase position of amplified DNA sequences corresponding t o the SAMK, MYC, and MYCN genes in four cell lines derived from human tumors: two gastric carcinoma lines (KATO 111 and SNU- I6), a neuroblastoma (NUBJ), and a neuroepithelioma (NUB-20) line. In metaphase cells of KATO 111, NUB-7, and NUB-20 lines, the amplified regions were clearly visible and easily identified at an intrachrornosornal location: in KATO 111 and NUB-7 at a terminal position and in NUB-20 at an interstitial position. In SNU- 16, on the other hand, the amplified SAMK and MYC sequences were identified in extrachromosoma1 double minute chromosomes (DMs). In this line, the SAMK and MYC sequences were coamplified in the same cells and were colocated on the same DMs. FISH also allowed the identification of amplified D N A sequences in nondividingcells, enabling us t o distinguish, at interphase, whether the amplification gave rise t o intrachromosomal amplified regions (IARs) o r to extrachromosomal DMs. The FISH technique also allowed us t o determine at metaphase as well as at interphase the extent of amplification and the size of the IARs. Genes Chrorn Cancer 4:314-320 (1992). 1992 WiIey-Lirs, Inc. @

INTRODUCTION

Unscheduled DNA amplification, a common genomic alteration in cancer cells, has been shown to be associated with in vivo neoplastic development. Thus far, molecular studies have revealed amplification of at least 15cellular protooncogenes in different tumors. Usually only a single protooncogene is amplified in any given type of tumor (Alitalo and Schwab, 1986; Taya et al., 1987; Zhou et al., 1988;Slamon et al., 1989 Schimke, 1990; Schwab and Amler, 1990). DNA amplification is usually manifested either by generation of additional intrachromosomal regions, abnormal banding regions (ABRs) or homogeneously staining regions (HSRs) or by the formation of extrachromosomal (centromere-free) double minute chromosomes (DMs) (see Hamlin et al., 1984, 1991; Schimke, 1988; for reviews). These cytogenetic manifestations of DNA amplification are common markers of many malignant tumors (Brieux de Salum et al., 1984; Gebhart et al., 1986; Uehara et al., 1987; Limon et al., 1989; Bruderlein et al., 1990). Identification of amplified DNA sequences in tumor cells contributes to the understanding of the neoplastic process and provides a useful tool for tumor classification. Moreover, the “amplified status” of an oncogene in a given tumor specimen may serve as a clinical marker to estimate neoplastic progression and disease prognosis (Alitalo and Schwab, 1986; Martinsson et al., 1988; Slamon et al., 1989; Schwab, 1990; Heerdt et al., 1991). 0 1992 WILEY-LISS, INC.

To date most of the information on the clear-cut identification of amplified DNA sequences in tumor cells has been gathered from molecular analysis, which is time consuming and laborious and provides no information on the cellular level. Conventional cytogenetic technique, too, has severe limitations: Although it allows a rapid assessment of amplified sequences, it fails to provide for their direct identification. Moreover, the information obtained with traditional cytogenetic tools is ambiguous due to the low mitotic indices and poor quality (low banding efficiency)of the few metaphases that often characterize primary tumors and some tumor cell lines. Therefore, the need for a rapid assessment and unambiguous identification of the amplified DNA sequences in tumor cells is obvious. Fluorescence in situ hybridization (FISH) is rapidly emerging as a useful technique with which structural and numerical chromosomal aberrations can be detected. The technique is applicable to interphase nuclei as well as metaphase cells in different in vivo and in vitro systems (see Lichter and Ward, 1990; Ferguson-Smith, 1991; Lichter et al., 1991; Trask, 1991, for reviews). In the present paper we report the use of FISH for the rapid and unambiguous assessment of the amplified DNA sequences in ~

Received October 22, 1991; accepted November 22, 1991. Address reprint requests to Lydia Avivi, Department of Human Genetics, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel.

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DETECTION OF AMPLIFIED DNA SEQUENCES BY FISH

dividing and nondividing cells derived from different human tumors. MATERIALS AND METHODS Cell Cultures

Four different cell lines derived from human tumors were used in this study: two gastric carcinoma lines, KATO I11 (Sekiguchi et al., 1978), obtained from the American Type Culture Collection, and SNU-16(Park et al., 1990),a gift from Dr. A. F. Gazdar; a neuroblastoma cell line, NUB-7 (Yeger et al., 1988);and a neuroepithelioma cell line, NUB-20 (Yeger et al., 1990). Each of these cell lines is known to contain amplified DNA sequences (Yeger et al., 1988,1990;Hattori et al., 1991; Mor et al., 1991).All cell lines were maintained in RPMI 1640medium supplemented with 15%fetal calf serum. Sample Preparation

Confluent cell cultures were split at a 1:4 ratio, and 48 hr later the cells were blocked with 10 - 6 M colchicine for 1 h. Slides were prepared according to stan-

dard techniques. Probes

A 6.1 kb EcoRI-XbaI fragment of the SAMK gene (Hattori et al., 1991) was isolated in our laboratory from a genomic region at chromosome band 10q26, which is amplified in the cell lines KATO I11 and SNU-16(Mor et al., 1991).The MYCN probe NB-19-21, a 2.0 kb fragment containing the second exon of the gene, was obtained from Dr. F. Alt; the MYC probe, a 1.1kb EcoRI-ClaI fragment containing the third exon of the gene, was obtained from Dr.M. Schwab. The probes were labeled by nick translation using biotin1CdATP according to the instructions provided by the supplier (Bethesda Research Laboratories). In Situ Hybridization

The hybridization protocol followed that of Pinkel et al. (1986), with slight modifications. Following RNase treatment, slides were dehydrated in an ethanol series and denatured in 70% formamide/2 x S C at 70°C for 2 min (except for slides containing metaphase cells and interphase nuclei with DMs from cell line SNU-16, which were denatured for 4 min in the same buffer). The hybridization mix consisted of 50% formamide, 2 x S C , 10% dextran sulfate, 500 pg/ml of carrier DNA, and biotin-labeledprobes ($10 ng/pl). A sample of 30 p1 of this mixture was applied to each slide under a coverslip. Following overnight incubation at 37"C, the slides were washed at 43°C in 50% formamide/2 x SSC for 20 min, in 2 x SSC at

37°C for 10 min, and in PN buffer (0.1 M Na2HP04,0.1 M NaH,PO, + "-40, pH 8.0) for 5 min. Detection

All probes were detected using fluorescein isothiocyanate (F1TC)-conjugatedavidin DCS. The signals were enhanced with biotinylated goat antiavidin, followed by another layer of FITC avidin. The slides were mounted with antifade medium containing propidium iodide. Slides containing metaphases and interphases were examined with either an Olympus BH2 or a Zeiss fluorescence microscope with appropriate filter combination. RESULTS

FISH was used for the detection of amplified DNA sequences in four tumor lines using three biotinylated DNA probes corresponding to the SAMK, MYC, and MYCN genes. The results obtained in the two gastric carcinoma cell lines following hybridization with the SAMK and MYC probes are shown in Figure 1. Clearly, in KATO I11 cells, the SAMK probe hybridized intensely to a large terminal intrachromosomal segment (Fig. la, b). A bright fluorescent signal at a similar chromosomal location appeared in all metaphase cells of this cell line. Moreover, the fluorescent signal from the amplified chromosomal region in KATO I11 cells was also easily recognized at interphase. Almost all the interphase nuclei derived from KATO I11 exhibited a clear, compact, and well-defined signal following hybridization with the S A M probe (Fig. lc). On the other hand, following hybridization of SNU16 cells with the SAMK probe, signals were scattered all over the interphase nuclei as well as the metaphase cells (Fig. le, 6. Each cell revealed a large number of minute fluorescent signals, which in metaphase cells were located exclusively on DMs. The finding of fluorescent signals in each individual DM (compare Fig. Id, e) indicated that all DMs in SNU-16 cells retained amplified sequences of the SAMK probe. Unexpectedly, following hybridization with the MYC probe, SNU-16 cells exhibited the same pattern of hybridization as observed with SAMK (Fig. lg-i), indicating that, apparently, in SNU-16cells the two DNA regions that hybridized to SAMK and MYC are coamplified and tightly linked in the DMs. No intense hybridization signals were obtained in KATO I11 cells following hybridization with biotinylated MYC probe nor in KATO III and SNU-16 cells hybridized with MYCN (not shown). The biotinylated MYCN and MYC probes were also hybridized to the neuroblastoma NUB-7 and the neuroepithelioma NITB-20 cell lines, respectively (Fig.

Figure I. Fluorescent signals of amplified D N A sequences in cells of two gastric carcinoma lines: KATO 111, hybridized with SAMK probe (a-c), and SNU- 16, hybridized with SAMK (d-9 and with MYC (6). a. b Metaphase cells showing terminal IARs. c: lnterphase nuclei showing well defined amplified regions. d, g: Metaphase cells stained with propidium iodide exhibiting DMs. e, h: Metaphase cells (same as in d and g, respectively) showing signals in each of the DMs. f, i: lnterphase nuclei showing scattered signals.

DETECTION OF AMPLIFIED DNA SEQUENCES BY FISH

2). A large fluorescent signal at a terminal position of a metaphase chromosomewas observed in each of the NUB-7 cells (Fig. 2a, b). Well-defined signals were easily recognized at interphase,appearing one per cell in almost the entire interphase cell population (Fig. 2c). Similarly, one fluorescentsignal per cell appeared in cells of NUB-20, being as easily detectable at interphase as at metaphase (Fig. 2d-f). At metaphase, each signal was confined to a narrow interstitial chromosomal region (Fig. 2d, e). Cells of NUB-20, however, although subjected to the same hybridization and detection protocol as KATO 111 and w-?, yielded weaker signals compared with those obtained from the latter two cell lines. Thus the interphase as well as metaphase analyses

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allowed the identification of an amplified domain and also established unequivocally the location of the amplified sequences in discrete intrachromosomal positions or in the extrachromosomalDMs. Furthermore, these analyses provide information on the level of amplification,the size of the amplified region, and the degree of cell variation. DISCUSSION

The use of FISH for the detection of amplified DNA sequences has been shown in this study to be a fast, reliable technique, leading to the unambiguous characterization of amplified DNA sequences in human tumor cell lines. FISH confirmed that the HSRs in KATO I11 (Mor et al., 1991)and in NUB-20cells (Yeger

Figure 2. Fluorescent signals of amplified D N A sequences in cells of two lines: NUB-7, hybridized with MYCN probe (a-c), and NUB-20, hybridized with MYC (69. a, b Metaphase cells showing terminal IARs. d. e: Metaphase cells showing interstitial IARs. c, f: lnterphase nuclei showing well-defined amplified regions. Note the difference between NUB-7 and NUB-20 in the size of the IARs and intensity of signals.

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et al., 1990) and the DMs in SNU-16 cells (Park et al., 1990) indeed carried amplified DNA sequences. Hybridization with a biotinylated SAMK probe revealed intrachromosomal amplified regions (IARs) at terminal positions in metaphase cells derived from the cell line KATO 111. This finding supports previous information obtained by classical cytogenetic analysis and radioactive in situ hybridization (ISH) showing that the amplified regions in KATO I11 cells were located on the distal part of chromosome 11(Mor et al., 1991). FISH, however, provided this information more quickly, more clearly, and more directly. The fluorescent signals were exclusively within the IARs, with 95% of metaphase cells exhibiting one IAR per cell and almost no background in the field. In contrast, following radioactive ISH (Mor et al., 1991), silver grains were scattered within as well as near IARs, less than 60% of the metaphases exhibited hybridization signals on the IARs, and the background was usually high. Information concerning the chromosomal location of amplified DNA regions in many types of cancer cells indicated that the IARs, at advanced stages of the amplification process, were found on chromosomes other than those carrying the original single copy sequences (Hamlin et al., 1984,1991;Schwab and Amler, 1990;Smith et al., 1990).KATO In is a typical example, showing amplification of 10q26sequences in an HSR on chromosome 11 (Mor et al., 1991). Previous information on the cytogenetic manifestation of MYCN amplification in NUB-7 cells was scarce, due mostly to the low mitotic index of this cell line. Using FISH, we could easily and unambiguously identify one terminal IAR in each of the few metaphases found in the field. In NUB20 cells, classical cytogenetic analysis detected the presence of an HSR on chromosome 14 but failed to provide information on between-cell variation or on the exact boundaries of the amplified regions (Yeger et al., 1990). FISH, on the other hand, confirmed the presence of one IAR per cell in NLTB-20, with minimal cell-to-cellvariation, and allowed us to determine its interstitial chromosomal position. It is noteworthy that very few IARs had been found to date in an interstitial position, most of them being at the termini of chromosomes (Trask and Hamlin, 1989). The intensity and size of the fluorescent signals enabled us to verify in metaphase cells the low amplification level (< 20-fold) of the MYC gene in NUB-20 cells (Yeger et al., 1990) as well as the high levels (5&75-fold) of SAMK amplification in KATO 111 (Mor et al., 1991) and of MYCN (30-50-fold) in NUB-7 (0.Mor, unpublished). Because of the difficulty in applying ISH to DMs, only little direct evidence exists for the presence of

amplified sequences in these extrachromasomal structures (Dolf et al., 1991).Conventionalcytogenetic analysis in SNU-16 cells revealed the presence of four to over 20 DMs per cell (Park et al., 1990),but radioactive ISH failed to detect unequivocally the amplified sequences in these DMs (Mor et al., 1991).Using FISH, it was possible to identify the hybridization signals of the amplified sequences on the DMs clearly and simultaneously to determine the variation in the number of DMs on SNU-16metaphases. SNU-16 is one of the few cases known to carry amplification of more than a single genomic domain. Molecular analysis showed that both MYC and SAMK sequences were amplified in this cell line (Mor et al., 1991; Shiloh et al., 1992).Whether the two genes were coamplified in the same cells or in two different subpopulations of cells had been unclear. This study showed that MYC, originally located on chromosome band 8q24 (Nee1 et al., 1982; Taub et al., 1982), and SAMK, normally found on chromosome band lOq26 (Mor et al., 1991; Shiloh et al., 1992),were coamplified in the same cells and in the same DMs. The clear visibility of the fluorescent hybridization signals throughout the cell cycle was exploited for the analysis of interphase nuclei. Much of the information obtained in this study from metaphase cells could also be obtained from analysis of interphase nuclei: KATO 111, NUB-7, and NU3-20 cells that contained one IAR per metaphase cell also exhibited one intense hybridization signal per interphase nucleus. The interphase signal occupied a discrete and well-defined domain in the nucleus, and its size and fluorescence intensity were related to the molecular level of DNA amplification known from the studies of Yeger et al. (1990),Mor et al. (1991), and 0. Mor (unpublished).On the other hand, SNU-16 cells, which revealed at metaphase a large number of DMs, showed at interphase a large number of minute fluorescent signals scattered throughout the nucleus. The use of interphase nuclei for chromosomal and DNA studies, an approach termed interphase cytogenetics (Cremer et al., 1986), allows the examination of a large number of cells from different cell populations. This is an obvious advantage to cancer researchers, since tumors, in addition to their low mitotic indices and poor quality of metaphase spreads, are known to contain several subpopulations of cells differing in cytogenetic and molecular behavior. Indeed, interphase cytogenetics has been widely used recently for the detection of structural and numerical chromosomal aberrations in cancer cells (Cremer et al., 1988a,b; Devilee et al., 1988; Nederlof et al., 1989; Anastasi et al., 1990; Tkachuk et al., 1990;van Dekken et al., 1990; Arnoldus et al., 1991; Poddighe et al.,

DETECTION OF AMPLIFIED DNA SEQUENCES BY FISH

1991).Interphase cytogenetics might also provide useful clinical tools for prenatal diagnosis and for the study of noncancerous cell systems prone to chromosomal aberrations (Cremer et al., 1986 Pinkel et al., 1988; Lichter et al., 1990). The FISH technique offers a convenient and useful tool to follow chromosomes and chromosomal segments at different stages of the cell cycle. It is easily applicable to the study of DNA amplification in different cell systems. In tumor cells it can be applied to metaphase cells and interphase nuclei in both shortand long-term cultures as well as to direct preparations. These analyses are expected to gain importance in the classification and characterizationof malignant diseases. ACKNOWLEDGMENTS

We thank Dr. Adi F. Gazdar of the US. National Cancer Institute and Dr. J.-G. Park of Seoul National University, Seoul, Korea, for the cell line SNU16. This study was supported by a joint research grant from the Deutsches Krebsforschungszentrum (DKFZ) and the Israel national Council for Research and Development. This paper is based on a portion of a dissertation to be submitted by I. B.-A.in partial fulfillment of the requirements for the PhD degree from the Sackler School of Medicine at Tel Aviv University. REFERENCES Alitalo D, Schwab M (1986) Oncogenic amplification in tumor cells. Adv Cancer Res 47B5281. Anastasi J, Le Beau MM, Vardiman JW, Westbrook CA (1990)Detection of numerical chromosomal abnormalities in neoplastic hematopietic cells by in situ hybridization with a chromosome-specificprobe. Am J Pathol 136:131-139. Arnoldus EPJ, Noordmeer IA, Peters ACB, Voormolen JHC, Bots GTAM, Raap AK, van der Ploeg M (1991) Interphase cytogenetics of brain tumors. Genes Chrom Cancer 3:lOl-107. Brieux de Salum S, Slavutsky I, Besuchio S, Pavlovsky A (1984)Homogeneously staining regions (HSR) in a human malignant melanoma. Cancer Genet Cytogenet 11:5340. Bruderlein S, Van der Bosch K, Schlag P, Schwab M (1990) Cytogenetics and DNA amplification in colorectal cancers. Genes Chrom Cancer 2:6370. Cremer T, Landegent J, Bruckner A, Scholl HP, Schardin M, Hager HD, Devilee P, Pearson P, van der Ploeg M (1986) Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: Diagnosis of trisomy 18with probe L1.84. Hum Genet 7434&352. Cremer T, Lichter P, Borden J, Ward DC, Manuelidis L (1988a) Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Hum Genet 80235246. Cremer T, Tesin D, Hopman AHN, Manuelidis L (1988b) Rapid interphase and metaphase assessment of specific chromosome changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes. Exp Cell Res 176199-220. Devilee P, Thierry RF, Kievits T, Kolluri R, Hopman AHN, Willard HF, Pearson PL, Comelisse CJ (1988) Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumors using chromosome-specific repetitive DNA probes. Cancer Res 48: 58255830.

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Detection of amplified DNA sequences in human tumor cell lines by fluorescence in situ hybridization.

An unambiguous and rapid characterization of amplified DNA sequences in tumor cells is important for the understanding of neoplastic progression. This...
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