Distinct Deletions of Chromosome 9P Associated with Melanoma Versus Glioma, Lung Cancer, and Leukemia 1
Total Page：16
File Type：pdf, Size：1020Kb
[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 car- ried 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 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1994 American Association for Cancer Research. CHROMOSOME 9p DELETIONS IN MELANOMA 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 melanoma cell 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).