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Chromosomal localization of amplified c-myc in a human colon adenocarcinoma cell line with a biotinylated probe
C h r o m o s o m a l Localization of Amplified cmyc in a H u m a n Colon A d e n o c a r c i n o m a Cell Line with a Biotinylated Probe D. Cherif, M. Le Coniat, H. G. Suarez, A. Bernheim, and R. Berger
ABSTRACT: A c-myc amplified sequence has been localized on a chromosome marker 19q+ with a biotin-labeled probe in the human colon adenocarcinoma SW480 cell line. The advantages of the technique for the localization of amplified DNA sequences are discussed.
INTRODUCTION Amplification of c-oncogenes is one of the mechanisms of protooncogene activation related to cancerogenesis. N u m e r o u s examples of oncogene amplification have been reported in h u m a n and animal malignancies in either in vivo or in vitro established cell lines (for a review see [1]). Double m i n u t e ch r o m o so m es (drain), homogeneously staining regions (HSRs), and abnormal banded regions (ABRs) are cytogenetic expressions of gene amplification [2]. We report the use of a biotinylated probe to localize the amplification of a c-myc oncogene on metaphase c h r o m o s o m e s of a cell line established from a h u m a n colon adenocarcinoma.
MATERIAL AND METHODES Cell Line The SW480 cell line was established from a h u m a n adenocarcinoma [3]. It was cultured in RPMI-1640 m e d i u m , s u p p l e m e n t e d with 10% fetal calf serum, glutamine, and antibiotics.
Cytogenetics C h r o m o s o m e preparations were performed in the exponential growth phase of the cell culture after 6 hours incubation with 0.5 ~g/ml colchicine. Mechanical suspensions were obtained from the cultures, and hypotonic solution (KC1 0.07 M) was used for 15 minutes before fixation with 3:1 methanol: acetic acid fixative. Cell From the UnitOINSERM301 and L. O. I. (CNRS), H6pital Saint-Louis, Paris, France (D. C., M. L., A. B., R. B.) and the Institut de Recherches Scientifiquessur le Cancer (CNRS).Villejuif,France (H. G. S.). Address requests for reprints to: D. Cherif, Laboratoire de CytogOnOtique, Centre G. Hayem, HOpital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Paris Cede× 10, France. Received November 20, 1987; accepted March 8, 1988,
245 © •988 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genet Cytogenet 33:245-249 (1988) 0165-4608/88/$03.50
246
D. Cherif et al.
suspensions were spread, and the slides were kept at room temperature for 1 week before hybridization. A G band with Wright staining [4] and R-band techniques I5] were performed.
DNA Probe and Nonradioactive Labeling The probe was a 1.4-kb ClaI-EcoRI fragment (third exon) of h u m a n c-myc gene excised from pKH47. As a result of the T 4 DNA ligase (Appligene-Strasbourg, France) action, a mixture of DNA fragments was obtained, most of them 2.8-7 kb long. This technique was a p p l i e d to encourage the formation of a network. Biotin-11-dUTP was incorporated according to the r a n d o m - p r i m i n g m e t h o d [6, 7] to reach a final concentration of nucleotides and biotin 11dUTP of 0.02 mM. Traces of 3H-dATP were also incorporated to allow the identification of the fractions containing biotinylated segments after purification of the probe on G50 Sephadex column. It was estimated that about 25% of the 3H-dATP was incorporated.
In Situ Hybridization Procedure In situ h y b r i d i z a t i o n was performed according to Pinkel et al. [8]. Briefly, cells and chromosomes were treated with RNase (100 ~g/ml; 37°C; 1 hour) and denatured (70% formamide/2 x SSC; 70°C; 2 minutes). The h y b r i d i z a t i o n mix (50% formamide, 2 x SSC, 10% dextran sulfate, 500 ~g/ml salmon sperm DNA, and 100 ng/ml probe DNA) was applied, and the slides were incubated overnight at 37°C. The slides were washed at 45°C in 50% formamide/2 x SSC (pH 7), then in 2 x SSC and finally in I x BN buffer (Sodium bicarbonate 0.1 M; Nonidet P40, Sigma, 0.05%, pH 8).
Probe Detection Detection of the biotin-labeled probe was performed with a layer of fluoresceinavidin DCS (5 p~g/ml). The fluorescence intensity was amplified with biotinylated goat antiavidin followed by an additional layer of a v i d i n DCS (avidin and antiavidin were obtained from Vector Laboratories, Burlingame, CA). Finally, a thin layer of antifade solution [9] containing the DNA counterstains [ 4 , 6 - d i a m i d i n o - 2 - p h e n y l indole or DAPI (0.8 ~g/ml, and p r o p i d i u m i o d i d e (0.4 p~g/ml)] was applied. The slides were screened using a Leitz fluorescence microscope. The fluorescein and p r o p i d i u m i o d i d e were excited at 450-490 nm (Leitz filter combination I2/3). The h y b r i d i z e d probe a p p e a r e d as yellow-green spot, while the rest of chromosomes were red. The DAPI-stained chromosomes were observed with the Leitz filter combination A.
RESULTS Cytogenetics The m o d a l n u m b e r of chromosomes of the SW480 cells was 56-57 (53 metaphases of 193 metaphases), with a wide dispersion from 53 to 96, and a second peak in the h y p e r t e t r a p l o i d range (35 metaphases). The karyotype also varied, but at least ten markers were almost constant, i n c l u d i n g a del(8)(q22), a der(8)t(8;?)(p13;?), and a rearranged chromosome 19. This 19q + marker had two a d d i t i o n a l dark bands separated by a light band with G-banding technique (Fig. 1). One normal chromosome 8 was present in the majority of the metaphases.
Chromosomal Localization of Amplified c-myc
247
Figure 1 G-banded metaphase of SW480 cell line. The 19q+ marker chromosome is arrowed; the normal chromosome 19 is shown by a black triangle.
In Situ Hybridization A positive signal was observed as a yellow-green spot on both chromatids of the 19q+ marker (Fig. 2) in 65 of 70 metaphases (93%). The localization on the 19q+ corresponded to the additional light G band. No consistent signal was observed on other chromosomes and, more specifically, on normal or rearranged chromosomes 8.
DISCUSSION The SW480 cell line was established from a h u m a n adenocarcinoma more than lO years ago [3]. Previous studies have s h o w n that this cell line contains an actived cKi-ras gene [10] accompanied by a 5 - 1 0 x amplification of c-myc [11]. Based on restriction m a p p i n g experiments with an exon 3 probe it was shown that in these cells c-myc was highly rearranged and amplified. This amplification was confirmed by Northern and dot blot experiments showing that the SW480 cells had a 10-20 x greater amount of c-myc messages than normal controls [11]. Our cytogenetic studies on this cell line showed various numerical as well as structural rearrangements resulting in several markers. Among them, two rearranged chromosomes 8 and an
248
D. C h e r i f et al.
F i g u r e 2 A. In situ hybridization of SW480 cell line with a c-myc probe. The 19q+ marker chromosome exhibits a bright signal revealing the localization of c-myc amplification (arrow). B. The same metaphase stained with DAPI.
Chromosomal Localization of Amplified c-myc
249
abnormal c h r o m o s o m e 19 (19q+) were found in the majority of metaphases. One normal c h r o m o s o m e 8 was also present in most of the cells. In situ hybridization experiments using a biotinylated c-myc probe showed that the amplified c-myc sequences were localized on the 19q+ chromosome. No significant signals were observed on normal and rearranged c h r o m o s o m e s 8. The use of a biotinylated probe was chosen because of its advantages compared to the radioactive in situ hybridization technique. The positive signals that are strongly fluorescent twin spots on sister chromatids are easily recognized from background, w h i c h appears as single spots. We found that this technique is very efficient because a positive signal was observed in 93% of the metaphases studied. From these results and those of others, it appears that the use of biotinylated probes is highly r e c o m m e n d e d to localize amplified sequences on metaphase chromosomes. We sincerely acknowledge Miss C. Czapek for secretarial assistance, Mrs D. Mathieu-Mahul for providing the exon 3 c-myc probe and Mrs J. Stevenet for their expert technical assistance. D. C. was a recipient of a grant from the "Fondation Fran~aise contre la Leuc~mie" and from INSERM.
REFERENCES 1. Taya Y, Terada M, Sugimura T (1987): Role of oncogene amplification in tumor progression. Adv Viral Oncol 7:141-153. 2. Schimke RT (ed.) (1982): Gene Amplification. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 3. Leibovitz A, Stinson JC, Mc Combs WB, Mc Coy CE, Mazur KC, Mabry ND (1976): Classification of human colorectal adenocarcinoma cell lines. Cancer Res 36:4562-4569. 4. Yunis JJ, Sawyer JR, Ball DW (1978): Characterization of banding patterns of metaphaseprophase G-banded chromosomes and their use in gene mapping. Cytogenet Cell Genet 22:679-683. 5. Bernheim A, Berger R (1981): A simple method for improving the reproducibility of the R banding technique. Human Genet 57:432-433. 6. Feinberg AP, Vogelstein B (1983): A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal Biochem 132:6-13. 7. Feinberg AP, Vogelstein B (1984): A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal Biochem 137:266-267. 8. Pinkel D, Straume T, Gray JW (1986): Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934-2938. 9. Johnson GD, de C. Nogueira Aranjo GM (1981): A simple method for reducing the fading of immunofluorescence during microscopy. J Immunol Meth 43:349-350. 10. Der Ch Y, Cooper GM (1983): Altered gene products are associated with activation of cellular ras k genes in human lung and colon carcinoma. Cell 32:201-208. 11. Suarez HG, Nardeux PC, Andeol Y, Sarasin A (1987): Multiple actived oncogenes in human tumors. Oncogene Res 1:201-207.
ABSTRACT: A c-myc amplified sequence has been localized on a chromosome marker 19q+ with a biotin-labeled probe in the human colon adenocarcinoma SW480 cell line. The advantages of the technique for the localization of amplified DNA sequences are discussed.
INTRODUCTION Amplification of c-oncogenes is one of the mechanisms of protooncogene activation related to cancerogenesis. N u m e r o u s examples of oncogene amplification have been reported in h u m a n and animal malignancies in either in vivo or in vitro established cell lines (for a review see [1]). Double m i n u t e ch r o m o so m es (drain), homogeneously staining regions (HSRs), and abnormal banded regions (ABRs) are cytogenetic expressions of gene amplification [2]. We report the use of a biotinylated probe to localize the amplification of a c-myc oncogene on metaphase c h r o m o s o m e s of a cell line established from a h u m a n colon adenocarcinoma.
MATERIAL AND METHODES Cell Line The SW480 cell line was established from a h u m a n adenocarcinoma [3]. It was cultured in RPMI-1640 m e d i u m , s u p p l e m e n t e d with 10% fetal calf serum, glutamine, and antibiotics.
Cytogenetics C h r o m o s o m e preparations were performed in the exponential growth phase of the cell culture after 6 hours incubation with 0.5 ~g/ml colchicine. Mechanical suspensions were obtained from the cultures, and hypotonic solution (KC1 0.07 M) was used for 15 minutes before fixation with 3:1 methanol: acetic acid fixative. Cell From the UnitOINSERM301 and L. O. I. (CNRS), H6pital Saint-Louis, Paris, France (D. C., M. L., A. B., R. B.) and the Institut de Recherches Scientifiquessur le Cancer (CNRS).Villejuif,France (H. G. S.). Address requests for reprints to: D. Cherif, Laboratoire de CytogOnOtique, Centre G. Hayem, HOpital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Paris Cede× 10, France. Received November 20, 1987; accepted March 8, 1988,
245 © •988 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genet Cytogenet 33:245-249 (1988) 0165-4608/88/$03.50
246
D. Cherif et al.
suspensions were spread, and the slides were kept at room temperature for 1 week before hybridization. A G band with Wright staining [4] and R-band techniques I5] were performed.
DNA Probe and Nonradioactive Labeling The probe was a 1.4-kb ClaI-EcoRI fragment (third exon) of h u m a n c-myc gene excised from pKH47. As a result of the T 4 DNA ligase (Appligene-Strasbourg, France) action, a mixture of DNA fragments was obtained, most of them 2.8-7 kb long. This technique was a p p l i e d to encourage the formation of a network. Biotin-11-dUTP was incorporated according to the r a n d o m - p r i m i n g m e t h o d [6, 7] to reach a final concentration of nucleotides and biotin 11dUTP of 0.02 mM. Traces of 3H-dATP were also incorporated to allow the identification of the fractions containing biotinylated segments after purification of the probe on G50 Sephadex column. It was estimated that about 25% of the 3H-dATP was incorporated.
In Situ Hybridization Procedure In situ h y b r i d i z a t i o n was performed according to Pinkel et al. [8]. Briefly, cells and chromosomes were treated with RNase (100 ~g/ml; 37°C; 1 hour) and denatured (70% formamide/2 x SSC; 70°C; 2 minutes). The h y b r i d i z a t i o n mix (50% formamide, 2 x SSC, 10% dextran sulfate, 500 ~g/ml salmon sperm DNA, and 100 ng/ml probe DNA) was applied, and the slides were incubated overnight at 37°C. The slides were washed at 45°C in 50% formamide/2 x SSC (pH 7), then in 2 x SSC and finally in I x BN buffer (Sodium bicarbonate 0.1 M; Nonidet P40, Sigma, 0.05%, pH 8).
Probe Detection Detection of the biotin-labeled probe was performed with a layer of fluoresceinavidin DCS (5 p~g/ml). The fluorescence intensity was amplified with biotinylated goat antiavidin followed by an additional layer of a v i d i n DCS (avidin and antiavidin were obtained from Vector Laboratories, Burlingame, CA). Finally, a thin layer of antifade solution [9] containing the DNA counterstains [ 4 , 6 - d i a m i d i n o - 2 - p h e n y l indole or DAPI (0.8 ~g/ml, and p r o p i d i u m i o d i d e (0.4 p~g/ml)] was applied. The slides were screened using a Leitz fluorescence microscope. The fluorescein and p r o p i d i u m i o d i d e were excited at 450-490 nm (Leitz filter combination I2/3). The h y b r i d i z e d probe a p p e a r e d as yellow-green spot, while the rest of chromosomes were red. The DAPI-stained chromosomes were observed with the Leitz filter combination A.
RESULTS Cytogenetics The m o d a l n u m b e r of chromosomes of the SW480 cells was 56-57 (53 metaphases of 193 metaphases), with a wide dispersion from 53 to 96, and a second peak in the h y p e r t e t r a p l o i d range (35 metaphases). The karyotype also varied, but at least ten markers were almost constant, i n c l u d i n g a del(8)(q22), a der(8)t(8;?)(p13;?), and a rearranged chromosome 19. This 19q + marker had two a d d i t i o n a l dark bands separated by a light band with G-banding technique (Fig. 1). One normal chromosome 8 was present in the majority of the metaphases.
Chromosomal Localization of Amplified c-myc
247
Figure 1 G-banded metaphase of SW480 cell line. The 19q+ marker chromosome is arrowed; the normal chromosome 19 is shown by a black triangle.
In Situ Hybridization A positive signal was observed as a yellow-green spot on both chromatids of the 19q+ marker (Fig. 2) in 65 of 70 metaphases (93%). The localization on the 19q+ corresponded to the additional light G band. No consistent signal was observed on other chromosomes and, more specifically, on normal or rearranged chromosomes 8.
DISCUSSION The SW480 cell line was established from a h u m a n adenocarcinoma more than lO years ago [3]. Previous studies have s h o w n that this cell line contains an actived cKi-ras gene [10] accompanied by a 5 - 1 0 x amplification of c-myc [11]. Based on restriction m a p p i n g experiments with an exon 3 probe it was shown that in these cells c-myc was highly rearranged and amplified. This amplification was confirmed by Northern and dot blot experiments showing that the SW480 cells had a 10-20 x greater amount of c-myc messages than normal controls [11]. Our cytogenetic studies on this cell line showed various numerical as well as structural rearrangements resulting in several markers. Among them, two rearranged chromosomes 8 and an
248
D. C h e r i f et al.
F i g u r e 2 A. In situ hybridization of SW480 cell line with a c-myc probe. The 19q+ marker chromosome exhibits a bright signal revealing the localization of c-myc amplification (arrow). B. The same metaphase stained with DAPI.
Chromosomal Localization of Amplified c-myc
249
abnormal c h r o m o s o m e 19 (19q+) were found in the majority of metaphases. One normal c h r o m o s o m e 8 was also present in most of the cells. In situ hybridization experiments using a biotinylated c-myc probe showed that the amplified c-myc sequences were localized on the 19q+ chromosome. No significant signals were observed on normal and rearranged c h r o m o s o m e s 8. The use of a biotinylated probe was chosen because of its advantages compared to the radioactive in situ hybridization technique. The positive signals that are strongly fluorescent twin spots on sister chromatids are easily recognized from background, w h i c h appears as single spots. We found that this technique is very efficient because a positive signal was observed in 93% of the metaphases studied. From these results and those of others, it appears that the use of biotinylated probes is highly r e c o m m e n d e d to localize amplified sequences on metaphase chromosomes. We sincerely acknowledge Miss C. Czapek for secretarial assistance, Mrs D. Mathieu-Mahul for providing the exon 3 c-myc probe and Mrs J. Stevenet for their expert technical assistance. D. C. was a recipient of a grant from the "Fondation Fran~aise contre la Leuc~mie" and from INSERM.
REFERENCES 1. Taya Y, Terada M, Sugimura T (1987): Role of oncogene amplification in tumor progression. Adv Viral Oncol 7:141-153. 2. Schimke RT (ed.) (1982): Gene Amplification. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 3. Leibovitz A, Stinson JC, Mc Combs WB, Mc Coy CE, Mazur KC, Mabry ND (1976): Classification of human colorectal adenocarcinoma cell lines. Cancer Res 36:4562-4569. 4. Yunis JJ, Sawyer JR, Ball DW (1978): Characterization of banding patterns of metaphaseprophase G-banded chromosomes and their use in gene mapping. Cytogenet Cell Genet 22:679-683. 5. Bernheim A, Berger R (1981): A simple method for improving the reproducibility of the R banding technique. Human Genet 57:432-433. 6. Feinberg AP, Vogelstein B (1983): A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal Biochem 132:6-13. 7. Feinberg AP, Vogelstein B (1984): A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal Biochem 137:266-267. 8. Pinkel D, Straume T, Gray JW (1986): Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934-2938. 9. Johnson GD, de C. Nogueira Aranjo GM (1981): A simple method for reducing the fading of immunofluorescence during microscopy. J Immunol Meth 43:349-350. 10. Der Ch Y, Cooper GM (1983): Altered gene products are associated with activation of cellular ras k genes in human lung and colon carcinoma. Cell 32:201-208. 11. Suarez HG, Nardeux PC, Andeol Y, Sarasin A (1987): Multiple actived oncogenes in human tumors. Oncogene Res 1:201-207.