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Address reprint requests to: Dr. Nancy Wang, Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Box 608, 601 Elmwood Avenue, Rochester, NY 14642
Affiliations
Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine, Rochester, NY, USA
Homogeneous staining region (hsr), a cytogenetic indicator of gene amplification, has been frequently found in ovarian carcinoma (ovc). To identify the origin of the hsr, chromosome microdissection combined with polymerase chain reaction and fluorescence in situ hybridization (FISH) was applied to two human ovarian cancer cell lines, GR and MLS/P. The hsr probes were labeled with biotin or digoxigenin and hybridized to normal metaphase spreads to elucidate the chromosomal origin and regional localization of the amplified genes. FISH to normal metaphase spreads with the probe generated from the whole hsr-bearing chromosome from GR hybridized to 8q24, 2p13→2q11.2, 10pter→10p15, 10p12→10q11.2, 5q23→5q31, and 5q33→5qter. For MLS/P, the hsr-bearing marker chromosome hybridized to 8q and 15q. In both cases, detailed FISH analysis revealed enhanced signal intensity at the 8q24 locus, which coincides with the chromosomal location of the C-MYC oncogene. To verify the involvement of C-MYC in hsr formation, in situ hybridization with a probe specific for the C-MYC oncogene was conducted and confirmed the amplification of C-MYC as the origin of the hsr. The whole hsr-bearing chromosome for GR is designated as rev ish der(10) (10pter→10p15::8q24hsr:: 10p12→10q11.2::8q24::2q11.2→2p13::2p13→2q11.2::8q24::10q11→10p11.2::5q23→5q31::5q33→5qter (wcp10+,D10Z1++,wcp2+,D2Z++,wcp5+,wcp8+,C-MYC++/hsr). The hsr-bearing marker for MLS/P is designated as rev ish der(8)(qter→8q24::8q24::8q24→8q10::8q10→8q24::8q24::8q24::8q24→8qter:: 15q11→15qter)(wcp8+,D8Z1+,wcp15+,C-MYC+++++++). FISH with the probe generated from the hsr of GR also painted the hsr in MLS/P, indicating that the two hsrs have shared homology, which indicates that the amplification of 8q24/C-MYC as the origin of hsr may be a nonrandom genomic alteration in ovc.
Introduction
Homogeneous staining regions (hsrs), cytogenetic indicators of gene amplification, have been frequently identified in a variety of fresh tumors and tumor cell lines [
Simultaneous existence of double minute chromosomes and homogeneously staining region in a retinoblastoma cell line (Y79) and amplification of N-myc at HSR.
]. To understand the genetic mechanisms of the genesis and progression of ovarian carcinomas, it is crucial to identify the origin of these hsrs.
However, routine chromosome banding analysis and fluorescence in situ hybridization (FISH) cannot identify these complex structural aberrations unequivocally. The recent advent of chromosome microdissection in conjunction with FISH has greatly facilitated the identification of chromosomal structural aberrations that cannot be identified by conventional banding techniques [
]. This combined method of microdissection and FISH (termed micro-FISH) can be applied to the dissection of any cytologically visible manifestation to unequivocally define the chromosomal constituents and origin of the aberration. Using micro-FISH, Guan et al. [
] identified the origin and composition of the hsr regions from seven ovarian cancer cases. They found DNA sequence amplification at 19q13.1∼q13.2, which is the candidate site for AKT2, a serine threonine kinase gene, in three of seven ovarian cancers. In the present study, we employed the micro-FISH approach to demonstrate the amplification of C-MYC as the origin of an hsr in two ovarian carcinoma cell lines. Amplification of the C-MYC gene has been detected in ovarian cancer by Southern hybridization and PCR [
Gene structure and expression analysis of the epidermal growth factor receptor, transforming growth factor-alpha, myc, jun, and metallothioneine in human ovarian carcinomas classification in malignant phenotypes.
]. Our study demonstrates that C-MYC amplification is involved in hsr formation.
Materials and methods
Ovarian Cancer Cell Culture
Microdissection of chromosomal DNA was performed on ovarian carcinoma cell lines GR, derived from a papillary adenocarcinoma, and MLS/P, derived from a stage III serous adenocarcinoma. Cells were grown in RPMI medium (GIBCO) supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 humidified incubator. The cells were arrested by treatment with Colcemid (GIBCO) for 2–3 hours, harvested for metaphase chromosomes, and G banded as described previously [
Identification of chromosomal structural alterations in human ovarian carcinoma cell lines using combined GTG-banding and repetitive fluorescence in situ hybridization (FISH).
Chromosome Microdissection and Amplification of Dissected DNA
The procedure for chromosome microdissection and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) was performed by following the method of Guan et al. [
]. Briefly, microdissection was performed with glass microneedles controlled by Narashige micromanipulators attached to an inverted microscope. Five to ten copies of the entire hsr-bearing marker chromosome were dissected from the metaphase spreads of GR and MLS/P cell lines, respectively. The dissected material was transferred to a 0.2-mL microcentrifuge tube containing 5 μL of collection buffer [40 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 50 mM NaCl, 200 μM of each dNTP, 1 unit TopoI, and 5 pmol of a universal primer (6MW) (CCGACTCGAGNNNNNNATGTGG)]. The collection buffer was covered with 45 μL of mineral oil and incubated at 37°C for 30 minutes followed by a heat soak at 94°C for 30 minutes. An initial 8 cycles of PCR (94°C, 1 min; 30°C, 2 min 37°C, 2 min) was performed by adding 0.3 unit T7 DNA polymerase at each cycle. Then, 50 μL of PCR reaction mixture (10 mM Tris-HCl, pH 8.4, 2 mM MgCl2, 50 mM KCl, 0.1 mg/mL gelatin, 200 μM of each dNTP, 50 pmol primer 6MW, and 2 units Taq DNA polymerase) was added to the same tube. The reaction was heated to 95°C for 3 minutes, followed by 35 cycles at 94°C for 1 minute, 56°C for 1 minute, and 72°C for 2 minutes, with a final extension at 72°C for 5 minutes.
Reverse Chromosome Painting
A 2-μL aliquot of the first PCR product was then labeled for 15 cycles in a secondary PCR as heretofore described, except for the addition of 20 μM digoxigenin/biotin-11-dUTP. Labeled PCR products were purified by using a Centricon-100 filter, and 400 ng of this probe was added to 10 μL hybridization mixture containing 55% formamide, 10% Dextran, 1 × SSC, and 5–10 μg of human Cot-1 DNA. The micro-FISH probes generated from GR and MLS/P cell lines were painted back to the respective metaphase spreads to verify the specificity of the probe to the marker and to spreads from normal persons to identify the origin of the marker. The micro-FISH probe generated from the hsr marker in GR was also hybridized to MLS/P metaphase spreads to determine the sequence homology between the hsrs of the two cell lines.
Forward Chromosome Painting
To identify the structure of the hsr-bearing marker chromosome, whole-chromosome painting (wcp) probes (Oncor) for chromosomes 5 (P5209-DIG), 8 (P5201-DIG), 2 (P5206-DIG), and 10 (P5212-DIG) were used to paint metaphase spreads from GR, whereas wcp probes for chromosomes 15 (P5216-DIG) and 8 (P5201-DIG) were used to paint spreads from MLS/P. In addition, centromeric probes (Oncor) specific for chromosomes 2 (D2Z) and 10 (D10Z1) were hybridized to GR. A probe specific for the C-MYC oncogene (P5117-DIG, Oncor) was used for FISH to verify the involvement of C-MYC amplification in the hsr formation in both GR and MLS/P cells. The FISH procedure employed was essentially the same as that described by the manufacturer.
Metaphase cells were viewed with a Zeiss Axioplan microscope equipped with an epifluorescence filter. Photomicrographs were taken on Kodak 400 color film.
Results
As shown in Figure 1b and d (arrowhead), the presence of a large hsr-bearing marker chromosome was detected in every metaphase spread of GR and MLS/P, respectively. To elucidate the chromosomal composition of the hsr marker chromosome, from five to ten copies of the marker chromosome from GR and MLS/P were dissected and amplified by PCR. The amplified DNA was then labeled with either biotin or digoxigenin-11-dUTP and used as a FISH probe to hybridize to metaphase spreads corresponding to the respective tumor cell lines. The micro-FISH probe generated from the hsr marker painted homogeneously along the whole target chromosome in GR (Fig. 1a) and MLS/P (Fig. 1c) metaphase spreads, confirming that the microdissected DNA was derived from the hsr-bearing marker chromosome.
Figure 1FISH with the probe generated from the hsr-bearing chromosomes from GR and MLS/P were hybridized to G-banded metaphase spreads from GR (a) and MLS/P(c), respectively, to confirm the specificity of the microdissected chromosome. (a and b) The probe generated from GR hybridized to the marker of origin (arrowhead) as well as i(5q) chromosomes (arrows). (c and d) The hsr probe from MLS/P hybridized completely to the entire native marker (arrowhead) as well as chromosome 15
The micro-FISH probes were then hybridized to normal metaphase chromosomes to elucidate the chromosomal composition and the regional localization of the hsr-bearing markers. As shown in Figure 2a, the hsr marker from GR hybridized to the 8q24, 5q23→5q31, 5q33→5qter, 2q11.2→2p13, and 10p12→10q11.2 in normal metaphase. To identify the detailed structural rearrangement of the hsr-bearing marker from GR, commercially available wcp probes specific to chromosomes 8, 2, 10, and 5 were hybridized to GR metaphase spreads. As shown in Fig. 3, FISH with wcp 8 (Fig. 3a) and wcp 5 (Fig. 3c) to GR cells revealed that the hsr region of the marker as well as two narrow bands at the short arm are derived from chromosome 8 with a segment of chromosome 5 translocated to the distal end of the short arm of the marker. FISH with wcp 2 showed a segment of chromosome 2 inserted into the short arm of the marker between the two narrow bands painted by wcp 8 (Fig. 4a). FISH with wcp 10 painted a subterminal band in the p arm, the centromere, and the distal end of the long arm on the marker (Fig. 4c). In addition, centromeric probes specific for chromosome 2 (D2Z) and chromosome 10 (D10Z1) were hybridized to GR. As shown in Figure 5, these paints revealed the presence of two chromosome 2 centromeres (Fig. 5a) and two chromosome 10 centromeres (Fig. 5c) on the hsr marker.
Figure 2FISH to normal metaphase spreads with hsr probes generated from GR (a) and MLS/P (c) to elucidate the chromosomal composition of the putative hsrs. (a and b) The GR marker hybridized to 5q (large arrowheads), centromeric 2 (small arrowheads), 10p (short arrow), and 8q24 (long arrows) regions. (c and d) The hsr marker from MLS/P hybridized to chromosomes 8q (arrowheads) and 15 (arrows). Increased signal intensity is observed at the 8q24 region
Figure 3The wcp probes specific to chromosomes 8 (P5201-DIG) and 5 (P5209-DIG) were hybridized to GR metaphase spreads to elucidate the regional localization of rearranged and amplified genes along the hsr-bearing marker. (a and b) wcp 8 probe painted two narrow bands at the short arm as well as the entire long arm of the GR marker (arrowhead). Arrows indicate hybridization to two additional chromosomes with chromosome 8 involved in rearrangement. (c and d) wcp 5 probe painted the marker at the terminal region of the short arm (arrowhead) and four other chromosomes (arrows) with chromosome 5 involved in translocation
Figure 4wcp probes 2 (P5206-DIG) and 10 (P5212-DIG) were hybridized to GR metaphase spreads. (a and b) Chromosome 2 painting in the midsection of the short arm of the marker. Arrows indicate other chromosomes painted either entirely or partly by wcp 2. (c and d) Chromosome 10 painting profiles, showing bands along the marker at the centromeric region, the distal end of the long arm, and the subterminal end of the short arm (arrowhead). Arrow shows hybridization to a partial chromosome 10
Figure 5FISH with centromeric probes, D2Z and D10Z1, specific for chromosomes 2 and 10, respectively, to metaphase spreads of GR revealed the presence of two chromosome 2 centromeres (a and b, arrowhead) and two chromosome 10 centromeres (c and d, arrowhead) on the hsr marker
The chromosomal composition and structural rearrangements of the MLS/P marker were determined by similar FISH approaches. As shown in Figure 2c, when the probe generated from the hsr-bearing marker from MLS/P was hybridized to normal metaphase spreads, it painted the entire chromosome segments of 15q (arrow) and 8q (arrowhead) with intense signal at the 8q24 region. The chromosomal structural arrangement of the MLS/P hsr marker was determined by painting metaphase spreads from MLS/P with wcp probes 8 and 15. Most of the hsr marker was painted by wcp 8 (Fig. 6a, arrowhead), whereas the distal end of the long arm was painted by wcp 15 (Fig. 6c, arrowhead), indicating that the marker is a derived chromosome 8 with chromosome 15 translocated to the distal end of the long arm.
Figure 6The wcp probes for chromosomes 8 and 15 (P5216-DIG) were hybridized to MLS/P metaphase spreads. (a and b) wcp 8 probe paints most of the hsr-bearing marker (arrowhead) except for the terminal region of the long arm and the normal chromosome 8 (arrow). (c and d) FISH with wcp 15 paints the terminal end of the long arm of the hsr marker (arrowhead) and the normal chromosome 15 (arrow)
Microdissection and FISH results for both GR and MLS/P revealed the hsr-bearing marker regions to be amplifications of chromosome 8 with enhanced signal intensity at the 8q24 region, which coincides with the site of the C-MYC oncogene. To verify the involvement of C-MYC amplification in the hsr formation, a digoxigenin-labeled probe specific for the C-MYC oncogene was hybridized to both GR and MLS/P metaphase spreads. The c-myc probe hybridized homogeneously to the entire hsr as well as to two narrow bands at the short arm of the marker chromosome in GR (Fig. 7a), which is identical with that obtained from FISH with wcp 8 (Fig. 3a and 3b, arrowhead). For MLS/P, however, hybridization was observed in tandem repeat patterns on the short and long arms of the hsr-bearing marker (Fig. 7c). These results confirm the involvement of C-MYC amplification in the hsr formation of both GR and MLS/P marker chromosomes. The micro-FISH probe generated from the GR hsr marker also hybridized to the hsr marker in MLS/P metaphase (Fig. 8), indicating that the two hsrs from GR and MLS/P shared homology.
Figure 7FISH with a probe specific for C-MYC (P5117-DIG) to metaphase spreads of GR (a) and MLS/P(c) was done to determine the involvement of C-MYC amplification in hsr formation. (a and b) The C-MYC probe hybridized to the entire long arm of the GR marker and two narrow bands in the short arm (arrowhead). (c and d) C-MYC hybridized in tandem repeat patterns along both the long and short arms of the MLS/P marker (arrowheads) and q24 region of chromosome 8 (arrow)
Figure 8The micro-FISH probe generated from the hsr of the GR cell line hybridized to MLS/P metaphase. The GR probe hybridized to chromosome 8 (arrow) and to a significantly large part of the MLS/P hsr marker (arrowhead), indicating that the hsrs from GR and MLS/P share homology
Homogeneous staining regions are extended chromosome regions that are cytologically visible by conventional banding techniques. Hsrs were first found by Biedler and Spengler [
] in chromosomes of antifolate-resistant Chinese hamster cells and neuroblastoma cell lines with the use of trypsin-Giemsa staining methods. They are considered a product of gene amplification [
Simultaneous existence of double minute chromosomes and homogeneously staining region in a retinoblastoma cell line (Y79) and amplification of N-myc at HSR.
]. However, studies on hsrs in tumors have been limited owing to the lack of a direct approach to isolate the hsr DNA and to identify the chromosomal location of the hsr. The combined method of microdissection and FISH, which encompasses both cytogenetic and molecular genetic techniques, has enabled a direct and detailed structural analysis on aberrant chromosomes in neoplastic cells. With the use of this method, the hsr region(s) can be dissected and used as FISH probes to elucidate the native chromosomal origin of the amplified sequences. Applying this strategy, Trent and co-workers [
] identified the amplification of the AKT2 (19q13.1∼q13.2), IGF1R (15q26), and ERBB2 (17q12) genes in ovarian, melanoma, and breast tumor samples, respectively. Using the same strategy, we identified the origin of a ring chromosome and marker chromosome in a hematological malignancy [
In the present study, we applied the combined strategy of chromosome microdissection and FISH to identify the chromosomal origin and compostion of an hsr-bearing marker chromosome in two human ovarian carcinoma cell lines, MLS/P and GR. The FISH probe generated from MLS/P hybridized to the entire hsr marker chromosome, which confirms the specificity of the probe to the hsr marker. Parallel G banding and FISH studies on normal metaphase spreads revealed the involvement of 8q and 15q in the hsr marker formation. FISH with commercially available wcp probes specific to chromosome 8 (wcp 8) and 15 (wcp 15) to MLS/P metaphase spreads demonstrated that the marker is mainly composed of chromosome 8 with chromosome 15q translocated to the distal end of the short arm. We also noted a greater amplification intensity of the 8q24 region, which coincides with the site for the C-MYC oncogene. These observations and the fact that C-MYC amplification has been found to be associated with a number of malignant tumors, including ovarian carcinoma, led us to further FISH analysis with the use of a probe specific to C-MYC. Our results showed C-MYC to be amplified in a tandem repeat pattern on the short and long arms of the hsr marker, confirming the involvement of C-MYC in hsr formation. When the results obtained from the G banding, FISH with wcp 8, 15, and C-MYC, and micro-FISH analysis are combined, the whole hsr-bearing marker chromosome in MLS/P can be designated as rev ish der(8)(8qter→8q24::8q24:8q24→8q10::8q10→8q24:: 8q24:8q24→8qter::15q11→15qter)(wcp8+,D8E1+,c-myc +++++++, wcp 15+).
In contrast with the marker from MLS/P, the hsr-bearing marker in GR revealed a different structural rearrangement, even though C-MYC amplification was observed in both cell lines. Reverse FISH to G-banded normal metaphase spread with the use of the probe generated from the hsr marker in GR indicates that the components of the marker are q23→q31 and q33→qter of chromosome 5, pter→p15 and p12→q11.2 of chromosome 10, q11.2→2p13 of chromosome 2, and the q24 region of chromosome 8. FISH with wcp probes 5, 2, 10, and 8, centromeric probes D2Z and D10Z1, and C-MYC was applied to GR metaphase spreads to determine the structural rearrangement of the different components within the marker. As shown in Figure 3, Figure 7, an identical painting pattern was observed by FISH with wcp 8 and c-myc probes, indicating that C-MYC is either the sole or major component of the chromosome 8 part of the marker. Regarding the involvement of chromosome 2, the region on the marker painted by wcp 2 has a size twice that of the region on the normal chromosome 2 painted by the micro-FISH-generated marker-specific probe (Figure 2, Figure 4). This finding indicates the duplication of 2p13→2q11.2 on the hsr marker. Additionally, FISH with the chromosome 2 centromere-specific probe to the marker revealed the presence of two centromeres located at both the distal and the proximal ends of the duplicated chromosome 2 segments. This finding indicates that the two segments of 2p13→2q11.2 are in reverse order. The GR marker is identified as: rev ish der(10)(10pter→ 10p15::8q24hsr::10p12→10q11.2::8q24::2q11.2→2p13:: 2p13→2q11.2::8q24::10q11.2→10p11.2::5q23→5q31::5q33→5qter(wcp 10+,D10Z1++,wcp 2+,D2Z++,wcp 5+,wcp 8+,C-MYC++/hsr). The identification of 5q23→ 5q31::5q33→5qter on the marker is based on the fact that FISH with the hsr marker-specific probe to the normal chromosome 5 painted both the region of 5q23→5q31 and the region of 5q33→5qter, whereas FISH with wcp 5 to the marker painted a single region at the distal end of the short arm of the marker, suggesting fusions of the two segments 5q23→5q31 and 5q33→5qter on the marker.
In this study, chromosome microdissection and FISH have enabled us to identify and characterize hsr, an important genetic manifestation in ovarian cancer that would have been unidentifiable by routine banding analysis or FISH analysis or both. Our results corroborate previous studies on C-MYC amplification and overexpression in ovarian cancer with the use of Southern hybridization and PCR [
Gene structure and expression analysis of the epidermal growth factor receptor, transforming growth factor-alpha, myc, jun, and metallothioneine in human ovarian carcinomas classification in malignant phenotypes.
]. Our study demonstrates for the first time that C-MYC amplification is directly linked to hsr formation in ovarian cancer. The micro-FISH probe generated from the GR hsr also hybridized to the hsr in MLS/P, indicating that the two hsrs share some degree of homology. However, even though the hsr markers in both GR and MLS/P cell lines show C-MYC amplification, it is evident that the intensity and the distribution of the oncogene on each of the markers vary significantly. Investigators have reported that hsrs frequently integrate into new chromosomal locations rather than being formed at the resident locus [
]. This seems to be the likely case for the hsr markers in both GR and MLS/P cell lines, which show the translocation of extraneous chromosomal segments to the region of amplification. The molecular genetic significance of these translocations is unknown; however, it is possible that they may play an active role in transcription of the hsr. To evaluate the clinical significance of the presence or absence of hsr, the origin and gene(s) amplified, and the structural rearrangement in hsr formation, this molecular cytogenetic study should be extended to a larger number of fresh tumor specimens and correlated with their pathological classifications.
Acknowledgements
This work was supported in part by NIH grant no. CA 52761.
Simultaneous existence of double minute chromosomes and homogeneously staining region in a retinoblastoma cell line (Y79) and amplification of N-myc at HSR.
Gene structure and expression analysis of the epidermal growth factor receptor, transforming growth factor-alpha, myc, jun, and metallothioneine in human ovarian carcinomas.
Identification of chromosomal structural alterations in human ovarian carcinoma cell lines using combined GTG-banding and repetitive fluorescence in situ hybridization (FISH).
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