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Frequent Alterations of Evolutionarily Conserved Regions of Chromosome 1 in Human Malignant Melanoma
Frequent Alterations of Evolutionarily Conserved Regions of Chromosome 1 in Human Malignant Melanoma Ji Zhang, Arthur A. Glatfelter, Raymond Taetle, and Jeffrey M. Trent
ABSTRACT: Recurring alterations of chromosome 1 represent the most frequent site of structural chromosome abnormalities across all human solid tumors, including human cutaneous malignant melanoma. In melanoma, breakpoints involving chromosome 1 appear to accumulate most frequently at the paracentromeric regions, and secondly, to cluster within 1p36. Of interest, these three band regions (1p11–12, 1q21, and 1p36) were simultaneously recognized by a single YAC clone which was isolated from sequences mapping to 1q21. This observation indicates the common and highly conserved nature of sequences residing within these three bands. Because of this finding, we have examined the possible association of these recurring sites of rearrangements of chromosome 1 in malignant melanoma. To elucidate genomic alterations in these regions, we have analyzed melanoma samples simultaneously by fluorescence in situ hybridization (FISH) using both the YAC clone encoding 1p11, 1q21, and 1p36 homologous sequences, and an a-satellite probe for the chromosome 1 centromere. Twelve of 20 (60%) randomly selected melanoma cell lines showed detectable rearrangements in one or more of the chromosome 1 band regions. These results provide support for the notion that the homology between these regions is associated with chromosomal instability, and possibly, is of biologic relevance in malignant melanoma. © Elsevier Science Inc., 1999. All rights reserved.
INTRODUCTION The mechanisms underlying the formation of recurring chromosome abnormalities in most human malignancies (especially human solid tumors) is largely indeterminate. Nevertheless, efforts using molecular approaches to investigate these cytological abnormalities have led to the identification of a number of cellular genes which play an important role in either positively (oncogenes) or negatively (tumor suppressor genes) regulating cell growth [1–3]. In melanoma, chromosome abnormalities are most frequently characterized by rearrangements of chromosomes 1, 6, 7, and 9, accounting for 82%, 64%, 61%, and 46%, respectively, of tumor samples reported [4, 5]. Specific breakpoints identified from these alterations tend to be clustered at 1p36, 1p22–q21, 6p11–q21, 7p, and 9p [4]. Also, consistent gain of 7p is frequently observed in mela-
From the Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health (J. Z., A. A. G., J. M. T.), Bethesda, Maryland, USA; and the Department of Internal Medicine, Section of Hematology/Oncology, Arizona Comprehensive Cancer Center (R. T.), Tucson, Arizona, USA. Address reprint requests to: Jeffrey M. Trent, Cancer Genetics Branch, NHGRI, 49 Convent Drive MSC 4470, National Institutes of Health, Bethesda, MD 20892-4470. Received May 11, 1998; accepted August 7, 1998. Cancer Genet Cytogenet 111:119–123 (1999) Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
nomas and may relate to the observation of consistent overexpression of epidermal growth factor receptor (EGFR, which maps to 7p12) in this disease [6]. Consistent loss of 1p, 6q, and 9p has been observed, suggesting the locations of tumor suppressor genes important in melanoma, and the biological relevance of these chromosome changes has been corroborated by both loss of heterozygosity (LOH) [7– 9] and microcell transfer experiments [10, 11]. For 9p, LOH as high as 86% has been detected within the interval IFNA [D9S171], and a putative tumor suppressor gene (p16) has been identified in this interval [8, 12]. In the case of chromosomes 1 and 6, a single specific region of deletion or rearrangement has not yet been identified, although numerous research studies have pinpointed common regions of loss or structural alteration to 1p36, 1p11–13, 1q21, and 6q12–21, suggesting these regions are likely to harbor genes contributing to the development or progression of malignant melanoma [9, 10]. Our laboratory has recently reported the physical mapping of a non-reciprocal translocation t(1;6)(q21;q14) resulting in net loss of 6q in a melanoma cell line [13, 14]. In the course of these experiments, we have isolated a YAC clone (954E4) assigned by STS contig mapping to band 1q21. Fluorescence in situ hybridization analysis of this YAC hybridized to normal human metaphase chromosomes revealed hybridization to three distinct chromo-
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Figure 1 FISH of YAC 954E4 to normal and tumor metaphases. (A) Hybridization of biotinylated YAC 954E4 to a normal human metaphase illustrating the concurrent hybridization to bands 1q21, 1p11–12, and 1p36, as evaluated by DAPI staining. The arrow at 1q21 indicates the band where the STS used to select the YAC originated, while arrowheads at 1p11–12 and 1p36 denote chromosomal loci of homologous DNA sequences. (B–H) Two-color FISH of rearrangement involving 1p11–12, 1q21, and 1p36 in malignant melanoma using SpectrumOrange-labeled YAC 954E4 (red) combined with a biotinylated chromosome 1 a-satellite (green) as probes. (B–C) Demonstration of chromosome rearrangements involving 1p11. The arrow in B denoted a 1q involved isochromosome in which two green signals are well separated, indicating the presence of two copies of a-satellite sequences. The arrow in C illustrated another 1p11 involved translocation in which the a-satellite is retained but the adjacent p arm material is missing. (D–F) Detection of chromosome rearrangements involving 1q21. Arrows indicate chromosome translocations where neither the a-satellite nor the heterochromatin sequences are present. (A greenish stain near the constriction of the arrowed chromosome in 1F represents a light cross hybridization between different a-satellite sequences.) These translocations were also evaluated by chromosome banding analysis. (G–H) Illustration of rearrangements involving both 1q21 and 1p11–12 in single abnormalities. The arrow in G denotes a translocation chromosome involving both 1p11–12 and 1q21. The covered sequences (minor red signal) within 1p11–12 are clearly visualized between two blocks of a-satellite DNA. The arrow in H indicates a ring chromosome which retains part of 1q21 and 1p11–12 material with a main component of heterochromatin. The insert demonstrates the same ring chromosome with reverse DAPI-banding.
some regions on chromosome 1. Further analysis of 20 additional tumor cases has provided evidence for instability of these chromosomal segments in malignant melanoma.
MATERIALS AND METHODS Tumor Cell Lines Tumor cell lines for FISH analysis were obtained from The Tissue Culture Cell Service of the Arizona Cancer Center. YAC Isolation and Labeling YAC 954E4 was isolated from the CEPH YAC library based on a 1q21 STS [14]. The YAC DNA was first purified from a low-melting point agarose gel, using pulsed-field gel
electrophoresis, and labeled for FISH by a PCR-labeling strategy described previously [15]. Briefly, 1 ng purified YAC DNA was added to make up a 5 ml reaction mix (containing 40 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 50 mM NaCl, 200 mM of each deoxynucleotide triphosphate, and 5 pmol of a degenerate universal primer termed UN-1 [CCGACTCGAGNNNNNNATGTGG] [16]. The reaction mix was covered with a drop of mineral oil and incubated at 948C for 5 minutes. Then, an initial six cycles of PCR (denaturation at 948C for 1 minute, annealing at 308C for 1 minute, and extension at 378C for 3 minutes) was conducted by adding 0.3 U of T7 DNA polymerase (Sequenase version 2.0, USB) at each cycle. Following this preamplification step, a conventional PCR catalyzed by Taq DNA polymerase was performed in the same tube with a vol-
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Recurring Alteration of Chromosome 1 ume of 50 ml. The reaction solution consisted of 50 pmol UN-1, 14 mM Tris-HCl, pH 8.3, 3.5 mM MgCl2, 50 mM KCl, and 200 mM of each dNTP (either 20 mM biotin-11dUTP or 50 mM SpectrumOrange-dUTP (Vysis, Naperville, IL, USA) was used to replace 10% or 25% of dTTP in the reaction mixture). Following 5 minutes denaturation at 948C, the reaction was cycled 30 times for 1 minute at 948C, 1 minute at 568C, 3 minutes at 728C, and completed by a final extension step at 728C for 5 minutes. FISH The detailed procedure for two-color FISH has been described previously [15]. In brief, metaphase spreads were prepared by conventional cytogenetic methods either from phytohemagglutinin-stimulated lymphocyte culture from a normal individual, or from tumor cell lines with chromosomal DNA denatured by immersion into 70% formamide/2 3 SSC at 708C for 2 minutes. The slides were then rinsed in ice-cold 2 3 SSC and dehydrated in a graded ethanol series. The non-isotopically labeled PCR product was then added to a hybridization solution containing 50% formamide, 1 3 SSPE, 1 3 SSC, 10% dextran sulfate, 1% Tween 20, 100 mg/mL sonicated total human DNA, and 0.9 mg/mL sonicated salmon sperm DNA at a concentration of 5–10 mg/mL. The probe DNA was denatured by heating the hybridization mix at 758C for 5 minutes, and then preannealed with repetitive sequences at 378C for 10 minutes prior to hybridization. For the a-satellite probe following overnight hybridization, detection of the hybridization was performed with two layers of FITC-conjugated avidin and one layer of anti-avidin antibody. Chromosomes were counterstained with DAPI (0.5 mg/mL) and fluorescent signals were visualized under a Zeiss Axiophot microscope equipped with a dual bandpass filter.
Table 1 Chromosome 1 alterations in 20 malignant melanoma cell lines Cell line UACC91 UACC375 UACC383 UACC612 UACC639 UACC647 UACC827 UACC930 UACC972 UACC1022 UACC1097 UACC1227 UACC1237 UACC1529 UACC2387 UACC2427 UACC2565 UACC2796 UACC2993 UACC3093
1p11–12
1q21
1p36
1 0 0 0 1 0 1 0 0 1 1 0 0 1 0 0 0 1 1 0
0 1 0 0 0 0 1 0 0 0 1 0 1 1 0 1 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0
onstrate rearrangements involving both 1q21 and 1p11–12 in abnormalities from two different cases. In Figure 1G, the hybridizing sequences within 1p11–12 are clearly visualized between blocks of a-satellite DNA. In Figure 1H, the breakages occurred between 1p11–12 and 1q21, leading to the generation of a ring chromosome (insert). Table 1 summarizes the bands involved in alterations of all 20 cell lines. DISCUSSION
RESULTS DNA from YAC 954E4 was isolated from a CEPH library based upon STS-content mapping [14]. As shown in Figure 1A, when the biotinylated YAC probe was hybridized to normal human metaphases and detected with avidinconjugated FITC, three distinct chromosome regions [1q21, 1p11–p12, and 1p36] were simultaneously highlighted. The strongest signal was localized to 1q21, representing the chromosomal origin of the STS used to isolate the YAC, with cross-hybridizing signal at 1p11–12 and 1p36. This YAC was then hybridized to a series of 20 melanoma cell lines to evaluate simultaneously genomic rearrangements at all three chromosomal sites. As shown in Figures 1B–1H, the labeled YAC probe (red), when combined with a biotinylated a-satellite probe (green), identified a series of rearrangements involving 1q21, 1p11–p12, or 1p36. Figures 1B and 1C demonstrate the 1p11 involvement in chromosome abnormalities by either production of an isochromosome of 1q (Fig. 1B) or translocation involving 1p11 (Fig. 1C). Figures 1D, 1E, and 1F demonstrate the involvement of 1q21 in rearrangements where neither the a-satellite sequences nor the 1q paracentromeric heterochromatin sequences (as evaluated by DAPIbanding analysis) were observed. Figures 1G and 1H dem-
A single YAC, selected by a single STS mapping to 1q21, has been shown, following FISH, to hybridize to three distinct regions (1q21, 1p11–12, and 1p36) of human chromosome 1. This observation unequivocally indicates the presence on this single YAC of homologous sequences found between these three chromosome regions. This observation gains significance following an examination of the comparative chromosome map between human and mouse. Specifically, the characterization of linkage groups with extensive homology between mouse chromosome 3 (42.6–54.6 cM), and the pericentric regions (1p11–p22 and 1q21–q31) of human chromosome 1 have previously been recognized [17]. Based upon this observation, a model of human chromosome 1 based on insertion of centromeric and heterochromatin DNA interrupting the ancestral mouse linkage group has been proposed [18]. Interestingly, 1p36 has recently been confirmed to contain sequences homologous to those present in the pericentromeric region of human 1, and a human gene (RUN1) sharing homology with a member (RNU1) of the same linkage group on mouse chromosome 3 has been identified [19]. Finally, comparative mapping of human and mouse genomes have indicated the preservation of gene order within the 45.1-cM segment of DNA encompassing
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Figure 2 Ideograms illustrating the relationship between the conserved loci identified by YAC 954E4 and “hot spots” for chromosome rearrangement in malignant melanoma. A detailed genetic map of the central part (42.6 cM to 54.6 cM) of mouse chromosome 3 is shown on the left, demonstrating a linear relationship among 12 genetic loci (22 genes) in mouse genome. Shadow boxes indicate physical locations of the human counterparts. Highly conserved genomic regions detected by YAC 954E4 are shown by vertical bars. The blocks on the right of the chromosome 1 ideogram are the breakpoints on chromosome 1 from 136 cases of malignant melanoma noted in the Catalog of Chromosome Aberrations in Cancer [5].
bands p22 to q32 on human chromosome 1, corresponding in the mouse to two conserved linkage groups located on chromosomes 1 and 3 [17–19]. The relationship between the mouse genetic chromosome map and the human cytogenetic map predicted by this model is depicted in Figure 2 [20]. The concurrence of these conserved sequences and this frequent association in melanoma may suggest an etiologic role for these evolutionarily conserved and homologous segments in the generation of chromosome 1 rearrangements. In support of
this notion, it is interesting to note the fusion of sequences encoded within 1q21 and 1p11–12, resulting in the formation of a ring chromosome (Fig. 1H). These results provide support for a mechanism of homologous recombination participating directly in the generation of this chromosome rearrangement. Finally, although this study has focused upon malignant melanoma, chromosome 1 alterations (again most frequently involving 1q21, 1p11–12, and 1p36) are found in a large number of additional tumor types [21–23]. We be-
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lieve that detailed physical and transcriptional mapping of the YAC 954E4 may provide insights into the possible role of these chromosome-specific homologous sequences, as well as possibly identifying a gene(s) which may be biologically relevant to tumor progression.
12. Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stokert E, Day RS III, Johnson BE, Skolnick MH (1994): A cell cycle regulator potentially involved in genesis of many tumor types. Science 264:436–440.
REFERENCES
14. Zhang J, Cui P, Glatfelter AA, Cummings LM, Meltzer PS, Trent JM (1995): Microdissection based cloning of a translocation breakpoint in a human malignant melanoma. Cancer Res 55:4640–4656.
1. Rabbitts TH (1994): Chromosomal translocations in human cancer. Nature 372:143–149. 2. Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RSR, Johnson DE, Skolnick MA (1994): A cell cycle regulator potentially involved in genesis of many tumor types. Science 264:436–440. 3. Cooper GM (1995): Oncogene and Chromosome Translocation. Oncogene, 2nd Ed. Jones and Bartlett Publishers, Boston. pp. 99–112. 4. Thompsom FH, Emerson J, Olson S, Weinstein R, Laevitt SA, Leong SP, Emerson S, Trent JM, Nelson MA, Salmon SE, Taetle R (1995): Cytogenetics of 158 patients with regional or disseminated melanoma. Subset analysis of near-diploid and simple karyotypes. Cancer Genet Cytogenet 83:93–104. 5. Mitelman F, Johansson B, Mertens F (1994): Catalog of Chromosome Aberrations in Cancer, 5th Ed. Wiley-Liss, New York. 6. Rodeck U (1995): Growth factor independence and growth regulatory pathways in human melanoma development. Cancer Metastasis Rev 12:219–226. 7. Healy E, Rehman I, Angus B, Rees JL (1995): Loss of heterozygosity in sporadic primary cutaneous melanoma. Genes Chromosom Cancer 12:152–156. 8. Weaver-Feldhaus J, Gruis NA, Neuhausen S, LePaslier D, Stockert E, Skilnick MH, Kamb A (1994): Localization of a putative tumor suppressor gene by using homozygous deletions in melanomas. Proc Natl Acad Sci USA 91:7563–7567. 9. Walker GJ, Palmer JM, Walters MK, Hayward NK (1991): A genetic model of melanoma tumorigenesis based on allelic losses. Genes Chromosom Cancer 12:134–141. 10. Trent JM, Stanbridge EJ, McBride HL, Meese EU, Casey G, Araujo DE, Witkowski CM, Nagle RB (1990): Tumorigenicity in human melanoma cell lines controlled by introduction of human chromosome 6. Science 247:568–571. 11. Welch DR, Chen P, Miele ME, McGary CT, Bower JM, Stanbridge EJ, Weissman BE (1994): Microcell-mediated transfer of chromosome 6 into metastatic human C8161 melanoma cells suppresses metastasis but does not inhibit tumorigenicity. Oncogene 9:255–262.
13. Trent JM, Thompson FH, Meyskens FL Jr (1989): Identification of a recurring translocation site involving chromosome 6 in human malignant melanoma. Cancer Res 49:420–423.
15. Zhang J, Meltzer P, Jenkins R, Guan XY, Trent J (1993): Application of chromosome microdissection probes for elucidation of BCR-ABL fusion and variant Philadelphia chromosome translocations in chronic myelogenous leukemia. Blood 81:3365–3371. 16. Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BA, Tunnacliffe A (1992): Degenerate oligonucleotideprimed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13:718–725. 17. Moseley WS, Seldin MF (1989): Definition of mouse chromosome 1 and 3 gene linkage groups that are conserved on human chromosome 1: evidence that a conserved linkage group spans the centromere of human chromosome 1. Genomics 5:899–905. 18. Hardas BD, Zhang J, Trent JM, Elder JT (1994): Direct evidence for homologous sequences on the paracentric regions of human chromosome 1. Genomics 21:359–363. 19. Romani M, Baldini A, Volpi EV, Casciano I, Nobile C, Muresu R, Siniscalco M (1994): Concurrent mapping of an adenovirus 5/SV40 integration site and the U1 snRNA cluster (RNU1) within 400 kb of the chromosome region 1P36.1. Cytogenet Cell Genet 67:37–40. 20. Seldin MF (1993): Mouse Chromosome 3, Mammalian Genome 4. Springer International. p. S47–S57. 21. Nagai H, Negrini M, Carter SL, Gillum DR, Rosenberg AL, Schwartz GF, Croce CM (1995): Detection and cloning of a common region of loss of heterozygosity at chromosome 1p in breast cancer. Cancer Res 55:1752–1757. 22. Cryns VL, Yi SM, Tahara H, Gaz RD, Arnold A (1995): Frequent loss of chromosome arm 1p DNA in parathyroid adenomas. Genes Chromosom Cancer 13:9–17. 23. Mathew H, Murty VVVS, Bosl GJ, Chaganti RSK (1994): Loss of heterozygosity identifies multiple sites of allelic deletions on chromosome 1 in human male germ cell tumors. Cancer Res 54:6265–6269.
ABSTRACT: Recurring alterations of chromosome 1 represent the most frequent site of structural chromosome abnormalities across all human solid tumors, including human cutaneous malignant melanoma. In melanoma, breakpoints involving chromosome 1 appear to accumulate most frequently at the paracentromeric regions, and secondly, to cluster within 1p36. Of interest, these three band regions (1p11–12, 1q21, and 1p36) were simultaneously recognized by a single YAC clone which was isolated from sequences mapping to 1q21. This observation indicates the common and highly conserved nature of sequences residing within these three bands. Because of this finding, we have examined the possible association of these recurring sites of rearrangements of chromosome 1 in malignant melanoma. To elucidate genomic alterations in these regions, we have analyzed melanoma samples simultaneously by fluorescence in situ hybridization (FISH) using both the YAC clone encoding 1p11, 1q21, and 1p36 homologous sequences, and an a-satellite probe for the chromosome 1 centromere. Twelve of 20 (60%) randomly selected melanoma cell lines showed detectable rearrangements in one or more of the chromosome 1 band regions. These results provide support for the notion that the homology between these regions is associated with chromosomal instability, and possibly, is of biologic relevance in malignant melanoma. © Elsevier Science Inc., 1999. All rights reserved.
INTRODUCTION The mechanisms underlying the formation of recurring chromosome abnormalities in most human malignancies (especially human solid tumors) is largely indeterminate. Nevertheless, efforts using molecular approaches to investigate these cytological abnormalities have led to the identification of a number of cellular genes which play an important role in either positively (oncogenes) or negatively (tumor suppressor genes) regulating cell growth [1–3]. In melanoma, chromosome abnormalities are most frequently characterized by rearrangements of chromosomes 1, 6, 7, and 9, accounting for 82%, 64%, 61%, and 46%, respectively, of tumor samples reported [4, 5]. Specific breakpoints identified from these alterations tend to be clustered at 1p36, 1p22–q21, 6p11–q21, 7p, and 9p [4]. Also, consistent gain of 7p is frequently observed in mela-
From the Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health (J. Z., A. A. G., J. M. T.), Bethesda, Maryland, USA; and the Department of Internal Medicine, Section of Hematology/Oncology, Arizona Comprehensive Cancer Center (R. T.), Tucson, Arizona, USA. Address reprint requests to: Jeffrey M. Trent, Cancer Genetics Branch, NHGRI, 49 Convent Drive MSC 4470, National Institutes of Health, Bethesda, MD 20892-4470. Received May 11, 1998; accepted August 7, 1998. Cancer Genet Cytogenet 111:119–123 (1999) Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
nomas and may relate to the observation of consistent overexpression of epidermal growth factor receptor (EGFR, which maps to 7p12) in this disease [6]. Consistent loss of 1p, 6q, and 9p has been observed, suggesting the locations of tumor suppressor genes important in melanoma, and the biological relevance of these chromosome changes has been corroborated by both loss of heterozygosity (LOH) [7– 9] and microcell transfer experiments [10, 11]. For 9p, LOH as high as 86% has been detected within the interval IFNA [D9S171], and a putative tumor suppressor gene (p16) has been identified in this interval [8, 12]. In the case of chromosomes 1 and 6, a single specific region of deletion or rearrangement has not yet been identified, although numerous research studies have pinpointed common regions of loss or structural alteration to 1p36, 1p11–13, 1q21, and 6q12–21, suggesting these regions are likely to harbor genes contributing to the development or progression of malignant melanoma [9, 10]. Our laboratory has recently reported the physical mapping of a non-reciprocal translocation t(1;6)(q21;q14) resulting in net loss of 6q in a melanoma cell line [13, 14]. In the course of these experiments, we have isolated a YAC clone (954E4) assigned by STS contig mapping to band 1q21. Fluorescence in situ hybridization analysis of this YAC hybridized to normal human metaphase chromosomes revealed hybridization to three distinct chromo-
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Figure 1 FISH of YAC 954E4 to normal and tumor metaphases. (A) Hybridization of biotinylated YAC 954E4 to a normal human metaphase illustrating the concurrent hybridization to bands 1q21, 1p11–12, and 1p36, as evaluated by DAPI staining. The arrow at 1q21 indicates the band where the STS used to select the YAC originated, while arrowheads at 1p11–12 and 1p36 denote chromosomal loci of homologous DNA sequences. (B–H) Two-color FISH of rearrangement involving 1p11–12, 1q21, and 1p36 in malignant melanoma using SpectrumOrange-labeled YAC 954E4 (red) combined with a biotinylated chromosome 1 a-satellite (green) as probes. (B–C) Demonstration of chromosome rearrangements involving 1p11. The arrow in B denoted a 1q involved isochromosome in which two green signals are well separated, indicating the presence of two copies of a-satellite sequences. The arrow in C illustrated another 1p11 involved translocation in which the a-satellite is retained but the adjacent p arm material is missing. (D–F) Detection of chromosome rearrangements involving 1q21. Arrows indicate chromosome translocations where neither the a-satellite nor the heterochromatin sequences are present. (A greenish stain near the constriction of the arrowed chromosome in 1F represents a light cross hybridization between different a-satellite sequences.) These translocations were also evaluated by chromosome banding analysis. (G–H) Illustration of rearrangements involving both 1q21 and 1p11–12 in single abnormalities. The arrow in G denotes a translocation chromosome involving both 1p11–12 and 1q21. The covered sequences (minor red signal) within 1p11–12 are clearly visualized between two blocks of a-satellite DNA. The arrow in H indicates a ring chromosome which retains part of 1q21 and 1p11–12 material with a main component of heterochromatin. The insert demonstrates the same ring chromosome with reverse DAPI-banding.
some regions on chromosome 1. Further analysis of 20 additional tumor cases has provided evidence for instability of these chromosomal segments in malignant melanoma.
MATERIALS AND METHODS Tumor Cell Lines Tumor cell lines for FISH analysis were obtained from The Tissue Culture Cell Service of the Arizona Cancer Center. YAC Isolation and Labeling YAC 954E4 was isolated from the CEPH YAC library based on a 1q21 STS [14]. The YAC DNA was first purified from a low-melting point agarose gel, using pulsed-field gel
electrophoresis, and labeled for FISH by a PCR-labeling strategy described previously [15]. Briefly, 1 ng purified YAC DNA was added to make up a 5 ml reaction mix (containing 40 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 50 mM NaCl, 200 mM of each deoxynucleotide triphosphate, and 5 pmol of a degenerate universal primer termed UN-1 [CCGACTCGAGNNNNNNATGTGG] [16]. The reaction mix was covered with a drop of mineral oil and incubated at 948C for 5 minutes. Then, an initial six cycles of PCR (denaturation at 948C for 1 minute, annealing at 308C for 1 minute, and extension at 378C for 3 minutes) was conducted by adding 0.3 U of T7 DNA polymerase (Sequenase version 2.0, USB) at each cycle. Following this preamplification step, a conventional PCR catalyzed by Taq DNA polymerase was performed in the same tube with a vol-
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Recurring Alteration of Chromosome 1 ume of 50 ml. The reaction solution consisted of 50 pmol UN-1, 14 mM Tris-HCl, pH 8.3, 3.5 mM MgCl2, 50 mM KCl, and 200 mM of each dNTP (either 20 mM biotin-11dUTP or 50 mM SpectrumOrange-dUTP (Vysis, Naperville, IL, USA) was used to replace 10% or 25% of dTTP in the reaction mixture). Following 5 minutes denaturation at 948C, the reaction was cycled 30 times for 1 minute at 948C, 1 minute at 568C, 3 minutes at 728C, and completed by a final extension step at 728C for 5 minutes. FISH The detailed procedure for two-color FISH has been described previously [15]. In brief, metaphase spreads were prepared by conventional cytogenetic methods either from phytohemagglutinin-stimulated lymphocyte culture from a normal individual, or from tumor cell lines with chromosomal DNA denatured by immersion into 70% formamide/2 3 SSC at 708C for 2 minutes. The slides were then rinsed in ice-cold 2 3 SSC and dehydrated in a graded ethanol series. The non-isotopically labeled PCR product was then added to a hybridization solution containing 50% formamide, 1 3 SSPE, 1 3 SSC, 10% dextran sulfate, 1% Tween 20, 100 mg/mL sonicated total human DNA, and 0.9 mg/mL sonicated salmon sperm DNA at a concentration of 5–10 mg/mL. The probe DNA was denatured by heating the hybridization mix at 758C for 5 minutes, and then preannealed with repetitive sequences at 378C for 10 minutes prior to hybridization. For the a-satellite probe following overnight hybridization, detection of the hybridization was performed with two layers of FITC-conjugated avidin and one layer of anti-avidin antibody. Chromosomes were counterstained with DAPI (0.5 mg/mL) and fluorescent signals were visualized under a Zeiss Axiophot microscope equipped with a dual bandpass filter.
Table 1 Chromosome 1 alterations in 20 malignant melanoma cell lines Cell line UACC91 UACC375 UACC383 UACC612 UACC639 UACC647 UACC827 UACC930 UACC972 UACC1022 UACC1097 UACC1227 UACC1237 UACC1529 UACC2387 UACC2427 UACC2565 UACC2796 UACC2993 UACC3093
1p11–12
1q21
1p36
1 0 0 0 1 0 1 0 0 1 1 0 0 1 0 0 0 1 1 0
0 1 0 0 0 0 1 0 0 0 1 0 1 1 0 1 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0
onstrate rearrangements involving both 1q21 and 1p11–12 in abnormalities from two different cases. In Figure 1G, the hybridizing sequences within 1p11–12 are clearly visualized between blocks of a-satellite DNA. In Figure 1H, the breakages occurred between 1p11–12 and 1q21, leading to the generation of a ring chromosome (insert). Table 1 summarizes the bands involved in alterations of all 20 cell lines. DISCUSSION
RESULTS DNA from YAC 954E4 was isolated from a CEPH library based upon STS-content mapping [14]. As shown in Figure 1A, when the biotinylated YAC probe was hybridized to normal human metaphases and detected with avidinconjugated FITC, three distinct chromosome regions [1q21, 1p11–p12, and 1p36] were simultaneously highlighted. The strongest signal was localized to 1q21, representing the chromosomal origin of the STS used to isolate the YAC, with cross-hybridizing signal at 1p11–12 and 1p36. This YAC was then hybridized to a series of 20 melanoma cell lines to evaluate simultaneously genomic rearrangements at all three chromosomal sites. As shown in Figures 1B–1H, the labeled YAC probe (red), when combined with a biotinylated a-satellite probe (green), identified a series of rearrangements involving 1q21, 1p11–p12, or 1p36. Figures 1B and 1C demonstrate the 1p11 involvement in chromosome abnormalities by either production of an isochromosome of 1q (Fig. 1B) or translocation involving 1p11 (Fig. 1C). Figures 1D, 1E, and 1F demonstrate the involvement of 1q21 in rearrangements where neither the a-satellite sequences nor the 1q paracentromeric heterochromatin sequences (as evaluated by DAPIbanding analysis) were observed. Figures 1G and 1H dem-
A single YAC, selected by a single STS mapping to 1q21, has been shown, following FISH, to hybridize to three distinct regions (1q21, 1p11–12, and 1p36) of human chromosome 1. This observation unequivocally indicates the presence on this single YAC of homologous sequences found between these three chromosome regions. This observation gains significance following an examination of the comparative chromosome map between human and mouse. Specifically, the characterization of linkage groups with extensive homology between mouse chromosome 3 (42.6–54.6 cM), and the pericentric regions (1p11–p22 and 1q21–q31) of human chromosome 1 have previously been recognized [17]. Based upon this observation, a model of human chromosome 1 based on insertion of centromeric and heterochromatin DNA interrupting the ancestral mouse linkage group has been proposed [18]. Interestingly, 1p36 has recently been confirmed to contain sequences homologous to those present in the pericentromeric region of human 1, and a human gene (RUN1) sharing homology with a member (RNU1) of the same linkage group on mouse chromosome 3 has been identified [19]. Finally, comparative mapping of human and mouse genomes have indicated the preservation of gene order within the 45.1-cM segment of DNA encompassing
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Figure 2 Ideograms illustrating the relationship between the conserved loci identified by YAC 954E4 and “hot spots” for chromosome rearrangement in malignant melanoma. A detailed genetic map of the central part (42.6 cM to 54.6 cM) of mouse chromosome 3 is shown on the left, demonstrating a linear relationship among 12 genetic loci (22 genes) in mouse genome. Shadow boxes indicate physical locations of the human counterparts. Highly conserved genomic regions detected by YAC 954E4 are shown by vertical bars. The blocks on the right of the chromosome 1 ideogram are the breakpoints on chromosome 1 from 136 cases of malignant melanoma noted in the Catalog of Chromosome Aberrations in Cancer [5].
bands p22 to q32 on human chromosome 1, corresponding in the mouse to two conserved linkage groups located on chromosomes 1 and 3 [17–19]. The relationship between the mouse genetic chromosome map and the human cytogenetic map predicted by this model is depicted in Figure 2 [20]. The concurrence of these conserved sequences and this frequent association in melanoma may suggest an etiologic role for these evolutionarily conserved and homologous segments in the generation of chromosome 1 rearrangements. In support of
this notion, it is interesting to note the fusion of sequences encoded within 1q21 and 1p11–12, resulting in the formation of a ring chromosome (Fig. 1H). These results provide support for a mechanism of homologous recombination participating directly in the generation of this chromosome rearrangement. Finally, although this study has focused upon malignant melanoma, chromosome 1 alterations (again most frequently involving 1q21, 1p11–12, and 1p36) are found in a large number of additional tumor types [21–23]. We be-
Recurring Alteration of Chromosome 1
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lieve that detailed physical and transcriptional mapping of the YAC 954E4 may provide insights into the possible role of these chromosome-specific homologous sequences, as well as possibly identifying a gene(s) which may be biologically relevant to tumor progression.
12. Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stokert E, Day RS III, Johnson BE, Skolnick MH (1994): A cell cycle regulator potentially involved in genesis of many tumor types. Science 264:436–440.
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