Amplification ofc-myc oncogene and absence ofc-Ha-ras point mutation in human bone sarcoma

The genomic organization of four oncogenes, c-myc, c-myb, c-Ha-ras, and v-fms, was analyzed in 21 patients with malignant bone tumors. Amplification of the c-myc proto-oncogene without rearrangement was the sole abnormality detected in four tumors:
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  Journal of Orthopaedic Research zyxwvusr k556 563 zyxwvutsrqponm he Journal of Bone and Joint Surgery, Inc. zyxwvutsrq   1993 Orthopaedic Research zyxwvutsrqp ociety Amplification of c-myc Oncogene and Absence of c-Ha-rus Point Mutation in Human Bone Sarcoma Carlos Barrios, 'fJavier S. Castresana, *Juan Ruiz, and Andris Kreicbergs Departments of Orthopedics and *Tumor Pathology, Karolinska Hospital, Stockholm, Sweden, and f Molecular Neuro-Oncology Laboratory, Massachusetts General Hospital, Boston, Massachusetts, zy : . zy . z Summary: The genomic organization of four oncogenes, c-myc, c-myb, c-Ha-ras, and v-fms, was analyzed in 21 patients with malignant bone tumors. Amplifi- cation of the c-myc proto-oncogene without rearrangement was the sole abnor- mality detected in four tumors: two chondrosarcomas, one osteosarcoma, and one lymphoma of bone. DNA hybridizations with c-myb, c-Ha-ras, and v-fms probes disclosed no structural gene abnormalities. Point mutations at the 12th codon of the c-Ha-ras gene were investigated with the polymerase chain reaction technique; no alterations were detected. The observed amplification of the c-myc there was not related to histologic type, grade, surgical stage, or ploidy level of the tumors. The results indicated that c-myc amplification, pre- sumed to be involved in the development of malignancy in a variety of solid tumors, is encountered sporadically in malignant bone tumors; however, this occurs without relation to common histopathologic features. The clinical sig- nificance of oncogene amplification in bone sarcoma remains to be established. Acting through protein products at different cel- lular levels, oncogenes have been implicated in the promotion and control of the eukaryotic cell cycle as growth regulatory genes (3,4). In tumor research, analysis of oncogenes has focused on the detection of either structural or functional changes (19). The former comprise a group of anomalies at the DNA level, such as gene amplifications, point mutations, delections, and rearrangements of genes. The latter refer to the level of expression of the gene, ribo- nucleic acid (RNA) formation, which is related to the final production of proteins. In fact, one of the mechanisms for overexpression of oncogene pro- teins is the amplification of its coding gene (23,36). All of these genetic changes are presumed to be Received May 6,1991; accepted December 9,1992. Address correspondence and reprint requests to Dr. C. Barrios at Orthopedics and Trauma Institute, Clinica Quiron, Blasco IbaAez 14,46010 Valencia, Spain. involved in the initiation and progression of human cancer, through transformation of the physiological function of oncogenes during cell differentiation z 4). Although our understanding of how structural and functional alterations of genes play a role in ma- lignant transformation is far from clear, there is a growing body of evidence suggesting that charac- terization of oncogenes may offer pertinent clinical information. Thus, N-myc oncogene amplification in human neuroblastomas has been found to be asso- ciated with a poor prognosis and rapid progression of the tumor. Stage 1 and 2 neuroblastomas usually do not present amplification of the N-myc oncogene (9,40). Rapid progression of tumors as well as short survival times have been observed in small cell lung carcinoma cell lines showing amplification of one or more members of the myc gene family (c-myc, N-myc, and L-myc) (25). Amplification of neu/erB- 2, myc, and int-2 oncogenes has been associated with poor prognosis in patients with breast cancer 556  AMPLIFICATION OF zyxwvuts -myc ONCOGENE AND ABSENCE OF c-Ha-ras POINT MUTATION 557 z TABLE zyxwvuts   Clinicopathologic eatures of bone zyxwv arcomas screened for oncogene amplifcation Case Sex 4%(yrs) Histology Type Site Grade 1 2 3 4 5 6 7 S 9 10 11 12 13 14 15 16 17 18 19 20 21 M 76 M 60 M 77 M 40 M 52 M 47 M 70 F 82 M 73 F 74 F 25 M 45 F 13 F 16 F 36 F 18 M 44 M 13 M 68 M 10 F 20 Chondrosarcoma Chondrosarcoma Chondrosarcoma Chondrosarcoma Chondrosarcoma Chondrosarcoma Chondrosarcoma Chondrosarcoma Chondrosarcoma Chordoma Lymphoma of bone Osteosarcoma Osteosarcoma Osteosarcoma Osteosarcoma Osteosarcoma Osteosarcoma Osteosarcoma Osteosarcoma Ewing’s sarcoma Ewing’s sarcoma P P P LR LR P P P LR LR P P P P P P P P P P LR Proximal femur Distal femur Proximal femur Iliac bone Abdominal wall zyx   Pelvis Ischium Calcaneus Sacrum Distal femur Proximal femur Distal femur Pubis 11 I1 I I I1 I I I1 I1 111 IV I11 IV 111 I11 IV I11 IV I11 111 I11 P = primary and LR = local recurrence. (48,52). Amplification, rearrangement, and point mu- tations of genes of the ras family have been detected in human bladder carcinoma (21), colorectal cancer (6), epidermoid lung cancer (32), and embryonal carcinoma (49). Oncogene anomalies have been identified in a small number of studies of bone sarcomas. The find- ings include amplification of c-myc in a human os- teosarcoma (51) and an osteosarcoma cell line (41); overexpression of the fos gene in transgenic mice, leading to the generation of bone lesions, half of which progressed to bone tumors, mainly chondro- sarcomas (38); specific association of one allele of the L-myc oncogene in a male patient with develop- ment of musculoskeletal sarcomas (27); involvement of the retinoblastoma tumor suppressor gene in the genesis of sarcomas (35); inactivation of the p53 gene by delections or mutations, or both, in human sarco- mas (33); and a consistent t(11;22)(q24:q12) translo- cation in Ewing’s sarcoma (47). So far, however, no consistent pattern of oncogene involvement has been demonstrated in bone tumors. This work analyzes the genomic organization of four oncogenes: c-myc, c-myb, c-Ha-ras, and v-fms, in fresh tumor samples taken from patients with ma- lignant bone tumors. Furthermore, a screening for point mutations at codon 12 of the c-Ha-ras onco- gene performed in bone sarcomas by restriction frag- ment length polymorphism RFLP) analysis with the polymerase chain reaction (PCR) technique (14) is described. MATERIAL AND METHODS Patients and Clinicopathologic Features Tumor specimens were obtained from 25 patients who had malignant bone tumors treated by surgery at the Karolinska Hospital from 1986-1989. Eighteen tumors were primary lesions and six were local re- currences. There were 11 chondrosarcomas, 10 oste- osarcomas, two Ewing’s sarcomas, one chordoma, and one lymphoma of bone. After extraction, DNA was available for oncogene screening in 21 tumors. Their clinicopathological characteristics are given in Table 1. The tumors were classified histologically accord- ing to the principles of the World Health Organi- zation (39). Histologic evidence of malignancy was graded as proposed by Broders et al. 8), but grading J Orthop Res Vol. 11, No. 4 993  558 C. BARRIOS ET AL. of chondrosarcomas (1-111) was performed according to the schema of O'Neal and Ackerman (34). Surgi- cal stage (IA/B-IIA/B-111) was defined according to Enneking (16). The nuclear DNA content of each tumor was de- termined routinely byflow DNA cytometry. The method used was described in detail previously (28,46). zyxwvutsr Purification of DNA and Blotting Analyses Immediately after surgical removal, the tumor samples were frozen in liquid nitrogen, and they were stored at -70°C until extraction of the DNA. High molecular weight DNA was isolated by lysis of small pieces of tumor tissue in a buffer containing 0.5 M NaC1, 50 mM Tris-C1 (pH 7.6), zyxwvu   mM EDTA, 0.5% sodium dodecyl sulphate (SDS), and 2 x 250 pg/ml of proteinase K at +56 C overnight. The DNA was purified by repeated extractions with phe- nolkhloroformhsoamyl alcohol (25243 and precip- itation in 95% ethanol after RNase treatment. Equal amounts of DNA were digested with the restriction enzymes EcoRI, HindIII, and RsaI, subjected to electrophoresis in 1% horizontal agarose gels with Tris-C1, boric acid, and EDTA (TBE) buffer, stained with 0.5 yg/ml ethidium bromide in TBE buffer, and photographed. The gels then were soaked in 500 ml of 0.5 M NaOH and 1.5 M NaCl for half an hour to denature the DNA and were transferred to ny- lon membrane (Zeta-probe; Bio-Rad Laboratories, Richmond, CA, U.S.A.) by the Southern method (44), with use of a solution of 0.4 M NaOH as a blotting buffer. The three restriction enzymes selected are the ones most commonly used for assessment of struc- tural anomalies of the oncogenes involved in this study. The combination of these enzymes displays different sets of autoradiographic bands which give substantial information about the existence of gross rearrangements shown by the appearance of anom- alous bands. For all samples, dot blot DNA analysis was performed in addition to confirm gene amplifi- cation and estimation of the gene copy number as described by Kafatos et al. (26). Hybridization Protocol Filters were hybridized to c-DNA probes labeled with al~ha-~~P y random priming (18) to a specific activity of >4 x lo6 cpm/yg of DNA. Hybridizations TABLE zyx . Proto-oncogene analysis in relation to histologic type C-myc am- Histologic type No. plification Intensity Chondrosarcoma 9 2 4-8 Osteosarcoma 8 1 16 Ewing's sarcoma 2 Lymphoma of bone 1 1 4 Chordoma 1 Total 21 4 4-16 were carried out in 50 formamide, 4x sodium chlo- ride, sodium phosphate, and EDTA (SSPE), and 7% SDS at +42 C overnight. Subsequently, the nylon membranes were rinsed briefly three times in 7% SDS at room temperature and then were washed at 50°C in 7% SDS 4x SSPE for 15 min. The filters were washed once at 50°C in 50 formamide, 4x SSPE, and 7% SDS for 30 min and twice at 50 in 0.1~ SSC and 1 SDS for 30 min. Finally, the filters were rinsed twice in Ix NaCl and sodium citrate and were exposed to X-mat film (Kodak, Rochester, NY, U.S.A.) at -70°C for at least 48 h. For both the dot blot and the Southern procedures, a hybridization signal was considered to be amplified if it showed at least a threefold increase in band intensity relative to an equal amount of control DNA, following common criteria (22,51). The con- trols were normal tissue from the same individ- ual (when available) or DNA from cultured human diploid fibroblasts. The intensity of the bands was assessed by scanning densitometry (model 1650; Bio- Rad Laboratories). PCR The oligonucleotide primers for PCR were syn- thesized with the use of a DNA synthesizer (Perkin- Elmer Cetus, Norwalk, CT, U.S.A.). This was done according to sequences that correspond to nucle- otides 31-50 upstream and downstream of codon 12 of c-Hums (SAGGCCCCTGAGGAGCGATGA 3' and SCAAAATGGTTCTGGATCAGC 3'). DNA was amplified in vitro to produce a fragment of 100 bp containing the 12th codon of the c-Ha-rus onco- gene. Briefly, 1 yg of DNA from the tumor was ad- ded to 50 y1 of a buffer containing 50 mM Tris (pH 7.4), 50 mM KC1,2 pgiml of bovine serum albumin, 1.5 mM MgClZ, 00 yM dATPs, 200 yM dGTPs, 200 yM dTTPs, 200 yM dCTPs, 0.4 yg of each primer, and J Orthop Res Vol. zyxwvutsrqp 1 No. 4 I993  AMPLIFICATION OF c-myc ONCOGENE AND ABSENCE OF c-Ha-ras POINT MUTATION 559 FIG. 1 zyxwvutsrqp outhern blots of seven of the 21 bone sarcomas analyzed for zyxwv -rnyc amplification. Ten micrograms of DNA from fresh surgical specimens was digested with Hindlll restriction endonuclease. The c rnyc gene was detected in a 11.5 kb band. Phage lambda DNA Hindlll fragments were used as weight markers. Amplification was observed in one osteosarcoma Osl) 16-fold) and in the only case of primary lymphoma of bone LyB) fourfold). DNAfrom cultured human diploid fibroblast HDF) was used as a control. 0.5 unit of Taq DNA polymerase (Perkin-Elmer Cetus), with addition of mineral oil on top of the solution. The mixture was incubated at 94°C for 2 min in an automated heat block to denaturate the DNA; this was followed by 30 cycles of denaturation (94°C for 1 min) and annealing (55°C for 2 min). Primer extension was performed at 72°C for 2 min. The mineral oil was removed by the addition of chlo- roform, and the DNA of the aqueous phase was pre- cipitated with ethanol and 0.3 M sodium acetate. The DNA was dried and dissolved in zy 4 zy 1 of Tris-EDTA buffer. Restriction enzyme digestion was performed by the addition of Msp I to half of the amplified DNA, 7 p1, in a total volume of 12 pl. After overnight incu- bation at 37 C, the restriction fragments were re- solved in a 5% agarose gel. Msp I recognizes the sequence 5' ... C/CGG ... 3' placed in the middle of the amplified fragment. Any mutation in the first or sec- ond base of codon 12 will make it impossible for FIG. 2. Msp I cleavage pattern of 100 bp amplified DNA fragment containing the 12th codon of the zyx -Ha-ras oncogene. Lines 1, 3 5, 7, 9 and 11 are DNA fragments from different tumors not treated by the restriction enzyme. Lines 2, 4 6, 8, 10, and 12 show the fragments from the same tumors now digested with Msp I, appearing as a 50 bp band. This indicates that no mutation is present at the 12th codon. zyxwvutsrq J Orthop Res Vol. 11 No. 4 1993  560 C. BARRIOS ET AL. TABLE 3. zyxwvutsr -myc amplification and histopathologic features C-myc amplification No. zyxwvutsrq   - value DNA content Diploid 10 1 9 0.33 Aneuploid 11 3 8 High 12 2 10 Malignancy grade Low 9 2 7 0.58 Surgical stage Intracompartmental 12 1 11 0.18 Extracompartmental 9 3 6 P value according to Fisher's test. Msp I to recognize its sequence; it would appear then as a single 100 bp fragment after the electro- phoresis, instead of as two 50 bp bands. The 12th codon, GGC, has its two first bases in common with the two last bases of the Msp I recognition site. c-DNA Probes The following specific probes were used: (a) a 1.2 kb PstI fragment excised from a pBR322 clone (Ryc 7.4) and used to cover the third exon of the human c-myc (50), kindly provided by C. M. Croce; (b) a 2.0 kb EcoRI fragment from a pBR322 clone (F8) of the c-myb DNA sequence (29), kindly provided by R. D. Gallo; (c) a 1.4 kb PstI fragment of a pBR322 clone containing the v-fms gene (12), kindly provided by I. M. Verma; and (d) a 3 kb Sac1 fragment excised from a pBR322 recombinant of the human c-Ha-ras gene (lo), kindly provided by M. Perucho. RESULTS In four of 25 tumor specimens, DNA isolation failed because of either complete degradation (one chondrosarcoma) or insufficient yield (two osteosar- comas and one chondrosarcoma); 21 specimens were left for further analysis. The degradation of DNA appeared to be related to the degree of tumor necro- sis. Insufficient yield occurred predominantly in tu- mors with lower cellularity. Results of DNA molecular analysis in relation to histologic type of tumor are given in Table 2. With use of a cDNA probe spanning the sequence of the third exon of the c-myc oncogene, a single 11.5 kb long restriction band was observed in HindIII-digested DNA isolated from the tumor samples. Amplification of the c-myc proto-oncogene, ranging from four to 16-fold, was the sole abnormality detected in four of the 21 tumors analyzed: two chondrosarcomas (Cases 1 and 7), one osteosarcoma (Case 16), and one lym- phoma of bone (Case 11) (Fig. 1). Histologically, the osteosarcoma with c-myc amplification disclosed a chondroblastic type pattern, according to Dahlin's criteria (13). The results of dot blot analysis were in accordance with the results obtained by the Southern technique. To analyze whether the c-myc oncogene also was rearranged in the tumors, new hybridizations were performed following digestion of DNA with addi- tional restriction enzymes. In the EcoRI-cleaved DNA, a typical single band of 12.5 kb was observed in all samples. Since no anomalous or additional bands were found with the different restriction en- zymes, it appears that no gene rearrangements were present in the screened tumors. Hybridization with c-myb, c-Ha-ras, and v-fms re- vealed no increases in the number of gene copies in the tumors analyzed. These hybridizations also were used as a test to confirm that equivalent amounts of DNA were loaded in each gel line. Point mutations at codon 12 of the c-Hu-rus onco- gene were investigated by the PCR-RFLP method in 11 of the 20 tumors; no alterations were detected (Fig. 2). In all cases studied, the restriction enzyme Msp I could cut the amplified 100 bp DNA fragments at the recognition site. This indicates that the 12th codon sequence GGC of the c-Hu-ras gene was intact. Closer analysis of the four c-myc amplified tumors showed no relationship to various clinicopathologic features such as histologic type, grade of malignancy, or ploidy level (Table 3). According to flow DNA cytometry, one tumor was diploid and three were aneuploid. The diploid tumor corresponded to a chondrosarcoma. All four tumors with gene amplifi- cation were primary lesions. DISCUSSION Although it must be emphasized that the present study was confined to four oncogenes in 21 lesions, our findings indicate that structural oncogene ab- normalities are rare in malignant bone tumors. The amplification of the c-myc oncogene was the sole abnormality to be encountered even occasionally. Notably, this seems to be the first report of c-myc amplification in lymphoma of bone. We were not J Orthop Res Vol. zyxwvutsrq 1 No. 4 1993
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