llpl3 have been detected in retinoblastoma (5) and Wilms tumor (6-9). ... tion and strip-washed a maximum of six times without de- tectable decrease in signal or ...
Proc. Natl. Acad. Sci. USA
Vol. 82, pp. 1470-1474, March 1985 Genetics
Loss of polymorphic restriction fragments in malignant melanoma: Implications for tumor heterogeneity (cancer/somatic mutations/DNA polymorphisms)
NICHOLAS C. DRACOPOLI, ALAN N. HOUGHTON, AND LLOYD J. OLD Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021
Contributed by Lloyd J. Old, October 26, 1984
broblasts and EBV-transformed B cells were grown in RPMI 1640 medium supplemented with 15% fetal bovine serum.
Loss of genetic material at certain chromoABSTRACT somal sites is implicated in the etiology of retinoblastoma and Wilms tumor. Whether specific chromosomal deletions are associated with other types of human cancer needs to be explored. We have examined 24 melanoma cell lines, derived from 21 patients with nonfamilial malignant melanoma, for evidence of somatically induced hemizygosity or homozygosity. Twelve DNA probes, recognizing single-copy restriction fragment length polymorphisms (RFLP) determined by loci on 11 different chromosomes, were used to screen autologous combinations of melanoma cells and either B cells or fibroblasts. Loss of heterozygosity in melanoma cells was identified at 27 of 100 informative loci. These losses occurred at loci on 8 different chromosomes, and the frequency of loss at individual loci varied between 8% and 67%. We conclude that somatic mutations resulting in homozygosity or hemizygosity are common in melanoma and evidently not restricted to specific chromosomes.
DNA Isolation and Southern Blot Analysis. High molecular weight DNA was prepared from B cells, fibroblasts, and melanomas (passages 13-80) (12). In addition, DNA was prepared from early-passage melanoma cells derived from patients CZ (passage 6), EL (passage 6), CL (passage 6), and DX-2 (passage 5). Fifteen micrograms of DNA was digested with 75 units of Msp I, Taq I, HindIII, or EcoPI (Bethesda Research Laboratories and New England Biolabs) for 4 hr according to the supplier's specifications. After being tested for complete digestion on minigels, the DNA fragments were separated by electrophoresis in 0.8% agarose gels for 16 hr and transferred to Zeta-probe membrane (Bio-Rad) (13). For hybridization, the membranes were treated with nick-translated 32P-labeled plasmid DNA (14) (1-2 x 106 cpm/ml) and incubated at 42°C in 20 mM sodium phosphate, pH 6.5/50% (vol/vol) formamide/5x NaCV/Cit (lx = 0.15 M NaCl/15 mM sodium citrate, pH 7)/0.04% Ficoll/0.04% polyvinylpyrrolidone/0.04% bovine serum albumin/10% (wt/vol) dextran sulfate/salmon sperm DNA (100 ,ug/ml). Membranes were washed for a maximum of 120 min in two changes of 2x NaClICit/0.1% NaDodSO4 and then for 90 min in three changes of O.1x NaClI/Cit/0.1% NaDodSO4. The last two washes were done at 65C. The membranes were dried and exposed to Kodak XAR-5 film with Dupont Cronex intensifying screens at -70°C. After exposure, the membranes were "strip-washed" for 30 min at 42°C in 0.4 M NaOH and then for 30 min at 42°C in 0.1 x NaCl/Cit/0.5% NaDodSO4 (13). Membranes have been used for hybridization and strip-washed a maximum of six times without detectable decrease in signal or increase in background. Plasmid Clones. Twelve cloned DNA probes that recognize different RFLPs determined by loci on chromosomes 1, 3, 5, 7, 10, 13, 15, 17, 18, 20, and 22 are described in Table 1.
Malignant melanoma occurs in both sporadic and hereditary forms, and at least 5% of cases of melanoma are familial (1). Genetic analysis suggests the presence of a melanoma susceptibility gene that is linked to the Rh blood group locus on chromosome 1 and inherited as an autosomal dominant trait with incomplete penetrance (2). In addition, karyotypic analysis of melanoma cells has revealed nonrandom chromosomal deletions in regions lp and 6q (3, 4). Nonrandom deletions of chromosomal regions 13q14 and llpl3 have been detected in retinoblastoma (5) and Wilms tumor (6-9). It has been proposed that these rare childhood cancers result from the deletion of dominant-acting genes, permitting the expression of tumorigenic recessive alleles. Whether this model can be extended to include other types of malignancies remains to be investigated. In this study, we have used 12 DNA probes that identify restriction fragment length polymorphisms (RFLP) determined by loci on 11 human chromosomes to search for evidence of somatically induced homozygosity or hemizygosity in cells derived from 21 patients with malignant melanoma.
RESULTS The probes used in this study (Table 1) detect RFLPs assigned to 11 different chromosomes. Eleven of these probes detected polymorphic sites in cellular DNA digested with a single restriction enzyme. Another probe, p7F12, was used to detect polymorphisms at both Msp I and Taq I restriction sites. The cell panel in Table 2 consists of sets of autologous melanoma and B cells (or fibroblasts) derived from 21 individuals. RFLP analysis of cultured EBV-transformed B cells or, in the case of patient AU, cultured skin fibroblasts established the constitutional genotype at these loci. The possibil-
MATERIALS AND METHODS Cell Lines. The cell panel consists of autologous pairs of cultured melanoma and Epstein-Barr virus (EBV)-transformed B cells derived from 20 individuals. Cultured skin fibroblasts were derived from patient AU. In the case of patient DX, melanoma cell lines DX-2, DX-3, DX-4, DX-6 were established from distinct metastases occurring at four separate sites (10). The derivation and culture conditions of the melanoma cell lines have been described (11). Melanoma cells were grown in Eagle's minimum essential medium supplemented with 7.5% fetal bovine serum. Cultured skin fi-
ity that incorporation of viral DNA into the B-cell genome has affected the genotypes at these loci is largely eliminated because the restriction fragment sizes are identical to those of the commonly occurring human polymorphisms (Table 1). The genotype and gene frequencies of the polymorphic loci
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Abbreviations: RFLP, restriction fragment length polymorphism; EBV, Epstein-Barr virus.
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Table 1. Description of 12 probes used to detect humail RFLP: Chromosomal assignment and polymorphic fragment sizes Polymorphic fragment Restriction Chromosomal Ref. Allele location Locus* Probe size, kb enzyme 15 Al 2.3 HindIll DISI lp36 H3 2.0 A2 15 3.8 Al Msp I NRAS lp31-p22 pNP1 2.7 A2 16 12.0 Al EcoRI SST 3q27-q28 pgHS7-2.7 6.4 A2 15 0.72 Al EcoRI 5 D5S4 L1.4 0.62 A2 17 1.6 Al HindIII 7 D7SJ pA2H3 A2 7.4 2.8 A3 15 6.3 I Al 10 Taq 5-1 DIOS] 3.6 A2 18 Al 4.3 Msp I D13SI 13ql2-ql4 p7F12 3.4, 0.9 A2 Bi 6.9 Taq I B2 5.9, 1.4 Al 12.0 I Msp 15, 19 15 DISSI pMS1-14 4.3 A2 Al 2.9 Msp I 19, 20 D17SJ 17pter-pl3 p12-2 2.1 A2 Al 6.0 19, 20 18 Taq I D18SJ p12-62 A2 2.6 Al 1.5 Msp I 15, 19 20 D20S4 pMS1-27 A2 6.5 Al 2.6 19, 20 Taq I 22pter-ql3 D22S1 pMS3-18 1.5, 1.0 A2 *The locus and allele designations have been defined in the "Report of the Committee on Human Gene Mapping by Recombinant DNA Techniques" (15).
restriction sites, respectively, in the 21 melanoma patients. Allele frequencies at each of the 11 polymorphic restriction sites are not significantly different from those expected at Hardy-Weinburg equilibrium. A total of 100 heterozygous genotypes (designated A1/A2
in the constitutional cells in these 21 melanoma patients are presented in Table 3. The gene frequencies at these loci are similar to those described in other surveys of North American populations (15). Two loci, NRAS and D22SI, were monomorphic for the common alleles at Msp I and Taq I A
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