Distinct Deletions of Chromosome 9P Associated with Melanoma Versus Glioma, Lung Cancer, and Leukemia 1

[CANCER RESEARCH54, 344--348, January 15,1994] Advances in Brief

Distinct Deletions of Chromosome 9p Associated with Melanoma versus Glioma, Lung Cancer, and Leukemia 1

Aaron Coleman, Jane W. Fountain, Tsutomu Nobori, Olufunmilayo I. Olopade, Gavin Robertson, David E. Housman, and Tracy G. Lugo 2 Division of Biomedical Sciences, University of California, Riverside, California 92521 [A. C., G. R., T. G. L.]; Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 [J. W. F, D. E. H.]; Department of Medicine, University of California, San Diego, California 92093 [I. N.]; and Section of Hematology/Oncology, Department of Medicine, University of Chicago, Chicago, Illinois 60637 [0. L 0.]

Abstract lines using probes that detect the IFN-a and D9S126 loci confirms the location of the telomeric 9p breakpoint in line AH between IFN-a and Deletions of DNA on chromosome 9p21-22 are frequently observed in MTAP and suggests that the analogous breakpoint in line FF resides cells derived from melanomas, gliomas, non-small cell lung cancers, and in a location centromeric to MTAP. acute lymphoblastic leukemia. The minimal deletion shared by the latter three cancers extends from the interferon-a locus towards the centromere; Materials and Methods its centromeric end is flanked by the gene encoding methylthioadenosine phosphorylase. We have determined that the telomeric end of the minimal Cell Lines and Culture Conditions. The origin of the human malignant homozygous deletion shared by two melanoma cell lines does not include melanoma cell lines FF (SK-Mel-133) and AH (SK-Mel-13) has been de- the methylthioadenosine phosphorylase locus. Thus, a distinct region of scribed (8). Ceils were routinely maintained in Dulbecco's modified Eagle's DNA is lost in melanoma. The physical size of this region remains to be medium plus 5% fetal calf serum without antibiotics. In some experiments, the defined precisely, but it may extend over several million base pairs. culture medium was supplemented with 10/xM AZ and 20 p~M MTA (both from Introduction Sigma Chemical Co.). Immunoblot Analyses. The presence of protein reactive with antiserum Frequent, nonrandom chromosomal breaks and deletions that are directed against human MTAP was determined by an immunoblot method as characteristic of particular cancers may indicate the sites of tumor described previously (9). Crude cell extracts (10-150/xg/lane) were separated suppressor genes. Such a pattern has been observed on chromosome by electrophoresis in a 12.5% polyacrylamide gel containing 0.1% sodium 9 in human malignant melanoma. Fountain et aI. (1) have detected dodecyl sulfate. Proteins were transferred by electroblotting to a nitrocellulose filter; nonspecific binding sites were blocked with 3% powdered milk in BBS loss of heterozygosity for DNA markers within chromosome band (0.2 Msodium borate-0.15 MNaC1, pH 8.5), and the filter was incubated for 16 9p21 in more than 80% of melanoma tumor and cell line DNAs tested. h at room temperature with anti-MTAP antiserum diluted 1:500 in BBS con- The critical region is defined by the overlap between homozygous taining 3% powdered milk. After the filter was washed with BBS, antibody was deletions in two melanoma cell lines, FF and AH, and is flanked by the detected by binding for 1 h to 125I-labeled protein A (1 mCi/ml; ICN Radio- IFN-ot3 locus on the telomeric end and the D9S3 marker on the chemicals, Irvine, CA), and the filter was exposed to Kodak XAR-5 film at centromeric end (Fig. 1). Rearrangements on chromosome 9 are also -70~ This procedure detects the Mr 32,000 homotrimeric subunit of the characteristic of other cancers, including gliomas (2), acute lympho- MTAP protein. blastic leukemia (3), and certain forms of lung cancer (4). The region Chromosome Studies. Metaphase chromosome spreads were prepared and of minimal overlap between deletions in a series of these malignancies subjected to Giemsa-trypsin banding according to procedures described pre- has been defined by Olopade et al. (5-7) and by James et al. (2). The viously (10). Photomicroscopy was performed on a Nikon Optiphot micro- scope equipped with a 100X planapochromatic objective. Cytogenetic abnor- critical region here extends from the IFN-ot locus towards the centro- malities are described according to the International System for Cytogenetic mere with its centromeric end flanked by the gene encoding MTAP. Nomenclature (11). In situ hybridization was carried out according to methods The homozygous deletion in the melanoma cell line AH extends into described in Ref. 12 using the chromosome 9-specific probe pBL-9 (kindly the centromeric portion of the interferon gene cluster, raising the provided by Dr. Daniel Pinkel, University of California at San Francisco, San possibility that a single locus in the vicinity of MTAP might be Francisco, CA). Following in situ hybridization, the biotin-labeled probe was implicated in all of these diverse cancers. However, we have deter- visualized by binding to streptavidin conjugated with fluorescein isothiocya- mined that the telomeric end of the deletion found in the melanoma nate (Vector Laboratories). The chromosomes were counterstained with prop- cell line FF does not include the MTAP locus. Thus, the region of idium iodide (Sigma) and observed under ultraviolet illumination through a overlap between the two melanoma deletions lies centromeric to Nikon B-2A filter (450-490 nm). MTAP and is distinct from that involved in glioma, lung cancer, and PFGE and DNA Hybridizations. PFGE and Southern blotting were carried out as described (13). High molecular weight DNA (5/xg) was prepared acute lymphoblastic leukemia. We have examined the chromosomes in low melting temperature agarose plugs, digested with an excess (>30 units) of these two melanoma cell lines and confirmed that a microscopically of NotI or MluI restriction enzyme, and loaded into a contour-clamp homoge- visible loss of chromosome 9p material has occurred in both of them. neous field electrophoresis gel consisting of 1.0% agarose. The gel was elec- Pulsed-field gel analysis of DNA restriction fragments from these cell trophoresed in TBE buffer (44.5 mM Tris-borate, 1 mM EDTA) at 10~ using a Pulsaphor unit (LKB) for 5 days at 90 V with a constant pulse time of 400 Received 10/7/93; accepted 11/24/93. s. Afterward, the gel was exposed briefly to UV radiation (330 nm transmitted The costs of publication of this article were defrayed in part by the payment of page and 254 nm reflected for 2.5 min each), denatured in 0.5 ra NaOH and 1.5 M charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. NaC1, and neutralized in 1 M Tris-HC1 and 1.5 M NaCI, pH 7.5. Transfer to a 1 This work was supported in part by Grants CA56910 (T. G. L.), HG00297 (D. E. H.), Zetabind membrane (AMF Cuno) was performed overnight in 10X SSC (1X = CA17575 (D. E. H.), and CA08985 (J. W. E) from the Cancer Research Coordinating 150 mM NaCI-15 mM sodium citrate). Prehybridizations and hybridizations Committee of the University of California and from the NIH. were carried out in a solution consisting of 50% formamide, 1 si NaCI, 25 mM 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: IFN-ot, a-interferon; MTA, methylthioadenosine;MTAP, Tris (pH 7.5), IX Denhardt's solution, 6.7% dextran sulfate, 0.1% sodium methylthioadenosinephosphorylase; PFGE, pulsed-field gel electrophoresis;BBS, borate- dodecyl sulfate, and 500/xg/ml sheared DNA from salmon testes. Probes were buffered saline; SSC, standard saline citrate; AZ, azaserine. labeled by random primer extension, purified through a Sephadex G50-150 344

hybridization (1). Since the D9S126 locus, centromeric to MTAP, is also homozygously deleted in cell line AH, we predicted that the MTAP gene would be lost as well. If so, this deletion would overlap the region identified in lung cancer, glioma, and acute lymphoblastic leukemia (Refs. 6 and 7; see Fig. 1). However, the telomeric extent of the homozygous deletion in cell line FF, which includes the D9S126 and D9S3 loci, has yet to be determined. No DNA probes corresponding to the MTAP gene were available for hybridization studies. We therefore examined the two melanoma Fig. 1. Overlap of homozygous9p deletions in human melanomacell lines. Genotypes cell lines for evidence of the MTAP gene product. A preliminary of lymphoblastoid(B) and melanoma (Mel) cell lines from patients FF and AH are shown experiment tested the ability of the cells to survive in culture medium as describedpreviously (1). Gene loci are depicted in order from 9pter to centromeric9p; allele symbols are the same as those in Ref. 1 except for MTAP. Both melanomacell lines containing MTA and AZ. AZ is an inhibitor of de novo purine bio- have suffered loss of heterozygosity in these regions: AH at D9S33, IFNB1, IFNA, and synthesis. The MTAP enzyme permits a cell to use MTA as a source D9S3, and FF at D9S33 and IFNB1. Dashes, homozygous deletions; *, presence of of purine nucleotides for DNA synthesis via nucleotide salvage path- rearranged DNA fragments. The deleted or rearranged region shared by FF and AH melanoma cells is boxed. The shaded areas show the regions of overlap between homo- ways, and thus it confers the ability to survive under these selective zygous deletions in these melanoma cells and a separate region defined by overlapping conditions (15). The two cell lines exhibited markedly different be- deletions in glioma, lung cancer, and leukemiacells as describedby Olopade et aL (5-7). haviors in this selective medium. AH cells failed to grow in MTA+AZ MTA~, position of the MTAP locus separating these two regions. The presence (+) or absence (-) of MTAP protein in the two melanomacell lines is indicated (see "Results"). at any cell density tested but grew well under nonselective conditions. FF cells grew poorly at low cell densities whether or not the inhibitor was present, but at higher cell densities they grew vigorously even in column, and added directly to the hybridization solution following 3 or more h of prehybridization at 42~ Hybridizations were incubated overnight at the presence of MTA+AZ. This indicated that FF cells might retain 42~ and washed the following day under high stringency conditions (0.1X the MTAP gene. SSC, 0.1% sodium dodecyl sulfate at 65~ Blots were exposed to Kodak To confirm the presence of the MTAP gene product, we performed XAR-5 film for 1-5 days at -70~ For subsequent hybridizations, blots were immunoblots of melanoma cell extracts with an antibody directed stripped in hybridization solution at 65~ for 1 h, followed by a 30-min wash against human MTAP (Fig. 2). No immunoreactive protein was found in 0.1X SSC and 0.1% sodium dodecyl sulfate at 65~ in extracts of AH cells, consistent with their inability to survive in The probe p72-0.9 was used to detect the D9S126 locus (13), and a 320- MTA+AZ. Protein reactive with the anti-MTAP antibody was, how- base pair polymerase chain reaction product was used to detect the IFN-o~ gene ever, clearly evident in the extract of FF cells. The ability of FF cells cluster. The primers used to amplify this product were (14):IFNA 203: 5'-GGC to survive in MTA+AZ indicates that this immunoreactive material is AAC CAG TIC CAG AAG GC-3'IFNA 523: 5'-GGA TCT CAT GAT Trc functional MTAP. TGC TC-3' These primers were chosen from highly homologous regions found between Chromosome 9 Abnormalities in Melanoma Cell Lines AH and the IFN-o~ genes in an effort to maximize the number of IFN-o~ genes detected FF. Both of these cell lines have sustained loss of heterozygosity and by the probe. On conventional Southern blots, this probe recognizes essentially homozygous deletions of polymorphic DNA markers within chromo- the same pattern as that depicted in Fountain et al. (1) where, alternatively, a some band 9p21-22 (1). We examined the chromosomes of these two cloned IFN-a fragment was employed as a probe (data not shown). cell lines using G-banding and confirmed the identity of marker chromosomes derived from chromosome 9 by in situ hybridization with a Results probe specific for human chromosome 9 (Fig. 3). As suspected, nei- Fig. 1 shows the order of marker loci in chromosome band 9p21- ther cell line contains an intact copy of chromosome 9. The melanoma 22. The order of the markers D9S33, IFNB, IFNA, D9S126, D9S3, and cell line AH contains two marker chromosomes derived from chro- D9S19 has been established by a combination of multipoint linkage mosome 9; one resembles a copy of chromosome 9 with a terminal analysis, fluorescence in situ hybridization, and PFGE (13). The IFNB deletion of the short arm (Fig. 3A, marker chromosome M8), and the gene and the IFNA gene cluster, which includes about 20 genes and other is an apparent iso9q chromosome in which a portion of one arm pseudogenes, reside on a single, 1400-kilobase Nod restriction frag- has been duplicated (Fig. 3A, marker chromosome M1). The homo- ment (6). The physical distances between the other markers remain zygous deletion in this cell line includes the D9S126 locus, and there undetermined. The position of the MTAP locus in relation to the IFN genes has been established through an analysis of deletions in a series of leukemia, glioma, and lung cancer cell lines and tumors (6, 7). Cytogenetic analysis of del(9p) chromosomes or unbalanced translo- cations involving 9p in cell lines with homozygous partial deletions of IFN DNA sequences localized the IFNB gene telomeric to the IFNA gene cluster, and cell lines with deletion breakpoints within the IFNA gene cluster established the order of some of the genes within the cluster. Many of the cell lines with deletions of the centromeric portion of the IFNA gene cluster also lost MTAP enzyme activity as determined by a radiochemical assay (6, 7). The cell lines with the smallest deletions indicated that the minimal deleted region shared by all these tumors lies between the centromeric end of the IFNA gene cluster and the MTAP gene. Location of the MTAP Locus Relative to Homozygous Deletions Fig. 2. Immunoblotanalysis of MTAP protein expression in melanomacell lines. Crude in Melanoma Cell Lines. The homozygous deletion in melanoma cell extracts of AH and FF melanomacells were separated by denaturingpolyacrylamide cell line AH extends as far as the centromeric portion of the interferon gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with anti- MTAP antiserum (9). Bands of protein reactive with the antibody were bound to lzsI- gene complex. This cell line retains only one allele of the IFNA locus, labeled protein A, and the filters were autoradiographed.Extracts from the cell lines CEM and this allele has suffered a rearrangement detectable by Southern and MDBK were included as negative and positive controls, respectively. 345

Fig. 3. Deletions of chromosome 9 in melanoma cell lines AH and FF. (A) Giemsa banded karyotype of AH. Arrows, two marker chromosomes derived from chromosome 9. One representative metaphase spread is depicted. In twenty-five metaphase spreads examined, the total number of chromosomes varied from 76 to 102. The chromosome abnormalities we observed in AH melanoma cells can be summarized: XX[1]; XXX[7]; XXXX[14]; XXXXX[3]; +117]; +1x2[17]; +Ix3[1]; +2118]; +2x213]; +3[9]; +3x2[15]; +3x6[1];-4[22]; -4x212]; +5[8]; +5x2[17];-6[3]; +7[6]; +7x2[13]; +7x4[1]; +7x5[1]; +8[20]; +8x212];-9x21251;-1011]; +1013]; +11113]; +11x218]; +llx3[1];-1214]; +1212]; +12x2[1]; -1312]; -13x2123]; -1411]; -14x2124]; + 15111]; + 15x2[11]; + 15x3[1]; + 15x4[1]; + 1618]; + 16x2116]; + 16x3[1]; -1711]; + 1716]; + 17x2[17]; + 17x3[1]; + 18119]; + 18x212]; -1911]; +19115]; +19x2[1]; +19x3[1]; +20x4[11]; +20x5[10]; +20x614]; +2113]; +21x214]; +21x318]; +21x414]; +21x513];-22[2]; +22115]; +22x2[1]; +22x3[1]; +M1124]; +Mlx2[1]; +M2120]; +M3124]; +M3x2[1]; +M4[1]; +M4x2[ll]; +M4x3[ll]; +M4x412]; +M5122]; +M5x2[1]; +M6125]; +M7124]; +M7x2[l]; +M8123]; +M8x211]; +M916]; +M9x2[19]; +M1013]; +M10x2121]; +M10x3[1]; +Mll[19]; +Mllx2[1]; +M12121]; +M12x2[1]. (Aplus sign denotes the presence of extra copies of a chromosome; for example, + 19x2 signifies two copies of chromosome 19 in addition to the normal diploid complement. A minus sign denotes the loss of a chromosome. The numbers in square brackets indicate the number of spreads in which the abnormality was observed. M1, M2, etc. refer to the marker chromosomes depicted in the photograph. The abnormalities involving chromosome 9 and the marker chromosomes derived from it have been underlined.) (B) Metaphase chromosomes of AH melanoma cells hybridized in situ to chromosome 9-specific probe pBL-9. The biotin-labeled probe was visualized with avidin conjugated to fluorescein isothiocyanate (bright fluorescence), and the chromosomes were counterstained with propidium iodide. Material derived from chromosome 9 is evident on marker chromosomes M1 and M8. (C) Giemsa banded karyotype of FE Arrows, copies of chromosome 9 with deletions of a portion of the short arm. This karyotype is a composite; it includes chromosomes from one representative metaphase spread plus marker chromosomes Mll, M17, M18, and M19 from additional spreads. The number of chromosomes per cell in twenty-five metaphase spreads varied from 71 to 83. The summary of karyotypic abnormalities we observed in FF melanoma cells is as follows: XY[20]; XXY[1]; XYY[1]; X[1]; Y[2]; +1117];-2[4];-2x2[1]; +3[23]; +4[22]; +5115]; +6[22]; +7[20]; +7x214]; +8121]; +8x2[1];-9x2125]; +10[20]; +10x2[1]; +11t231; +llx2[l]; +1214]; +12x2120]; +1319]; +13x2[12]; +14122]; +15121]; +15x212]; +1616];-1612]; +1713];-1716]; +18119];-1811]; +1918]; +19x2[11]; +19x315]; +20114]; +20x212]; +21111]; +21x219]; +21x312]; +21x4[1];-2111]; +22110]; +22x2[1];-22[7];-22x2[1]; +M1122]; +Mix2[1]; +M2120]; +M2x2[1]; +M314]; +M4[19]; +M4x212]; M5121]; +M6124]; +M7124]; +M8124]; +M8x21U; +M9[16]; +M10[18]; +M10x212]; +Mll[5]; +M12[18]; +M13122]; +M14[19]; +M15[16]; +M15x2[1]; +M16123]; +M17[14]; +M1815]; +M1912]. (D) Metaphase chromosomes of FF melanoma cells hybridized in situ to pBL-9. Material derived from chromosome 9 is evident on at least two chromosomes (arrows) in addition to marker chromosomes M7 and M8.

is loss of heterozygosity at loci both centromeric (D9S3) and telo- cell line also retains alleles at loci both centromeric and telomeric to meric (IFNA, IFNB1, and D9S33) to D9S126. However, since at least the homozygous deletion, but in contrast to AH, in situ hybridization one allele at both of these flanking loci is retained, the appearance of revealed a complex arrangement of material derived from chromo- the chromosomes cannot be accounted for by a simple deletion of both some 9 that was translocated onto other chromosomes (Fig. 3D). 9p termini. Cell line FF has a homozygous deletion that encompasses Twenty-five metaphase chromosome spreads from each cell line two loci, D9S126 and D9S3. It contains two nearly identical marker were karyotyped. Both of these cell lines are near-tetraploid with a chromosomes that resemble terminal deletions of 9p (Fig. 3C). This highly variable number (70--100) of chromosomes. No intact copies 346

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1994 American Association for Cancer Research. CHROMOSOME 9p DELETIONS IN MELANOMA of chromosome 9 were observed, and one or two copies of each Discussion chromosome 9-derived marker chromosome were present in all metaphases. There are now several lines of evidence for the involvement of a PFGE Analysis of IFNA and D9S126 Restriction Fragments. chromosome 9 tumor suppressor in human malignant melanoma. Cy- To provide an additional estimate of the physical location of 9p togenetic studies have revealed rearrangements of chromosome 9 in breakpoints in AH and FF, these melanoma DNAs were digested with both melanomas and precursor lesions, suggesting that damage to this NotI and MluI restriction endonucleases, separated on a PFGE, and chromosome may be an early event in melanocytic tumor progression hybridized to probes for IFN-o~ and D9S126 (Fig. 4). All of the genes (16). This suggestion was also supported by early molecular studies corresponding to the IFN-c~ locus reside on a single 1.4-megabase performed on multiple metastatic cell lines derived from a melanoma NotI restriction fragment (5). A fragment of 1.4 megabases is seen in patient (17). In recent studies, the targeted region has been more FF (Mel), but in AH (Mel) a faster-migrating band of 1.2-1.3 mega- precisely defined on 9p21-22. Attention has focused on the DNA bases is evident. The IFN-o~ probe hybridized to this blot detects marker D9S126 since loss of heterozygosity has most frequently been IFN-a genes located up to and within approximately 100 kilobases detected by this marker in the vast majority of melanoma cell lines and from the centromeric end of the 1.4-megabase NotI fragment. Thus the tumors examined (1). In addition, a melanoma patient has been de- breakpoint within the IFN-c~ locus in AH (Mel) probably occurred scribed who harbors a constitutional deletion of a portion of chromo- 100-200 kilobases telomeric to this site. In contrast, the breakpoint in some 9p21; she has developed eight primary melanomas, atypical FF (Mel) must lie centromeric to this NotI site. The MluI restriction moles, and a plexiform neurofibroma (18). This patient is hemizygous fragment detected by the IFNA probe in FF (Mel) is larger than 1.6 for chromosome 9p21 DNA loci between D9S33 and D9S19, markers megabases and could not be resolved by PFGE. This DNA is probably which lie just telomeric and just centromeric to the region of homo- hypermethylated at least one MluI site since control lymphoblastoid zygous deletion detected in the tumor cell lines AH and FE Finally, a DNA derived from the same individual [Fig. 4, FF(B)] yielded two hereditary predisposition to melanoma has been linked to chromo- smaller MluI fragments. Thus no indication of a deletion in FF (Mel) some 9p21 markers in several melanoma pedigrees (19), although is clearly indicated by this probe. In AH (Mel), all of the hybridizing there is also evidence for genetic heterogeneity in this disease (20). fragments are considerably smaller, which could be due either to a The discovery of deletions at the interferon locus in a high propor- break within the primary MluI fragment detected by our IFN-c~ probe tion of acute lymphoblastic leukemias (3), and later in gliomas (2) and or to DNA hypomethylation or MluI restriction site polymorphism(s) non-small cell lung cancers (4), led to the proposal that loss of inter- within this region. feron gene function might play a role in the development of these When the same PFGE blot was hybridized to a probe corresponding cancers. However, careful mapping of the deletions shared by a large to the D9S126 locus, only one very faint MluI fragment was observed number of tumor samples has placed the critical region in an area within the zone of resolution. Since D9S126 is homozygously deleted centromeric to the interferon gene cluster (5-7). In a substantial num- in both FF (Mel) and AH (Mel), no signal was expected in either one ber of tumors with smaller deletions, the MTAP locus is also retained, of these two lanes. However, D9S126 fragments are present in DNA and at present this locus marks the centromeric boundary of the region from the lymphocytes of patient FF (1), and a faint MluI band is seen of overlap. It now appears that the region frequently deleted in these in the FF(B) lane in Fig. 4. In general, however, the NotI and MluI tumors is distinct from that in melanoma. This may indicate that two restriction fragments corresponding to D9S126 are larger than 1.6 different tumor suppressor genes are targeted in these cancers, al- megabases and, therefore, were not resolved on this blot. though the possibility that a single very large locus extends through the MTAP gene has not been excluded. Current estimates of the size of the homozygous deletion shared by the FF and AH melanomas range from 2-5 megabases. The present PFGE study demonstrates only that the IFNA and D9S126 loci reside on separate, large restriction fragments. The karyotypes of the two cell lines raise the possibility that the deletions could be quite large since both have large-scale deletions and clearly visible cytogenetic rearrangements of telomeric chromosome 9p. However, these rearrangements are certainly more complex than simple terminal deletions since some 9p21-22 DNA loci are retained in both cell lines. Further physical mapping surrounding the IFNA, MTAP, and D9S126 loci will be required to determine the precise extent of the critical region for each cancer.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1994 American Association for Cancer Research. Distinct Deletions of Chromosome 9p Associated with Melanoma versus Glioma, Lung Cancer, and Leukemia

Aaron Coleman, Jane W. Fountain, Tsutomu Nobori, et al.

Cancer Res 1994;54:344-348.

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