Document 08JpJDNpZJe6vK3LEwQ1rO7x

Alteration and Abnormal in Human Expression Multiple of the c-myc Myeloma Oncogene By Peter Selvanayagam. Raymond Mark Blick, Franca Narni, Peter van Tuinen, David Alexanian. Grady F. Saunders, and Bart Barlogie H. Ledbetter, Structural alterations of the c-myc oncogene in human Burkitt's lymphoma and mouse plasmacytoma suggest that this oncogene is involved in several B cell neoplasms. The possibility of c-myc alterations in human myeloma has not been explored, probably because the low proliferative activity characteristic of this tumor impairs the propaga- tion of representative cell lines for the performance of adequate cytogenetic studies. This report tions in the c-myc locus with concomitant describes elevated alteraexpres- sion of mRNA in the tumor cells of two of 37 patients with multiple myeloma. In one case. somatic cell hybrid studies M ULTIPLE MYELOMA is a neoplasm of differen- tiated B (plasma) cells that is characterized by monoclonal immunogbobulin (Ig) production.' There is grow- ing evidence that cellular oncogenes are altered or overex- pressed in several human malignancies, although their role in neoplastic transformation remains to be determined.2 Onco- genes are activated by several mechanisms, most notably by chromosomal translocation, as has been observed frequently in human lymphoma and leukemia.3'5 Similar data are not available for myeloma, probably because no consistent chro- mosomal anomalies have been found.' Myeboma plasma cells typically have a low proliferative activity that renders karyotypic studies difficult, and only a few representative cell lines have been established?9 Yet, an unequivocal increase in the nuclear DNA content ofplasma cells has been observed in 80% of patients.'#{176}We analyzed the structure and expression of the c-myc oncogene in myeboma plasma cells because c-myc is involved frequently in other human and animal B cell neoplasms."2 We report here alterations in the c-myc gene locus with a concomitant elevated expression of mRNA in the tumor cells of two of 37 patients. Seven other myeloma samples also showed an abnormally high expression of c-myc mRNA without alteration in gene structure. These data indicate that the c-myc oncogene is involved in human myeloma. From the Departments ofHematology, Biochemistry and Molec- u/ar Biology. and Clinical Immunology and Biological Therapy. The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute; and the Institute for Molecular Genetics, Baylor College ofMedicine. Houston. Submitted December 1. /986; accepted August 15, 1987. Supported in part by Grants No. CA37161. CA28771, CA16672 from the National Institute of Health and the Cullen Trust Foundationfor Health Care. Address reprint requests to Bart Barlogie. MD. Department of Hematology, University of Texas System Cancer Center. M.D. Anderson Hospital and Tumor Institute, /515 Holcombe Blvd. Houston, TX 77030. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance indicate this fact. with 18 U.S.C. 1 734 solely to 1 988 by Grune & Stratton, Inc. 0006-4971/88/7101-0002$3.00/0 revealed that the cloned rearranged DNA was entirely derived from chromosome 8. thus indicating a novel mech- anism of c-myc activation different from that in Burkitt's lymphoma. Seven other patients exhibited five- to 12-fold overexpression of c-myc RNA when compared with normal marrow cells. Elevated mRNA expression in about one fourth of our patients suggests that the c-myc oncogene has a pathogenetic role in the evolution of multiple myelo- ma. e 1988 by Grune & Stratton. Inc. MATERIALS AND METHODS Bone marrow aspirates were studied in 37 patients, in eight at the time of diagnosis and in 29 during later advanced stages of disease. Informed consent was obtained before the bone marrow aspiration procedures. The degree of marrow plasmacytosis was usually marked (median, 55%). Myeloma protein was phenotyped by immu- noelectrophoresis, and cytogenetic studies were conducted on several samples (Table I ). Bone marrow cells and placental tissue from normal healthy individuals served as controls. HL-60 cells with abundant c-myc mRNA were used as a positive control for RNA expression studies. Informed consent was obtained before bone marrow aspiration procedures. Nucleic acid analysis. High-molecular weight DNA and total RNA were coextracted from bone marrow cells by the method of Erisman et al.'3 DNA was digested with restriction enzymes as recommended by the manufacturer. Size fractionation in agarose gel and transfer to nitrocellubose filter were carried out by the method of Southern.'4 For Northern blot analysis, 20 zg of total RNA was denatured in 1 mob/L glyoxal, 50% dimethylsulfoxide, and 10 mmol/L phosphate buffer, pH 7.0, at 50#{176}fCor one hour; size- fractionated in I .2% agarose gel; and transferred to nitrocellulose filter by the method of Thomas." Hybridization of the DNA and RNA filters with 32P-labeled gene probes and subsequent washing were carried out according to the method of Maniatis et al." Autoradiography on x-ray films was performed at - 70#{176}fCor various durations. Gene expression was quantitated by densitometric scanning of autoradiograms. Isolation of the rearranged gene. Tumor DNA digested with Hind III enzyme was fractionated in a sucrose density gradient, and the fraction enriched for the rearranged myc fragment was cloned into X2001 phage vector.'7 Screening of the library and purification of the recombinant phage were performed by published protocols.'8 Somatic cell hybrid analysis. Construction and analysis of Chinese hamster-human and mouse-human somatic cell hybrids have been reported previously.'9'#{176} G-banding analysis was per- formed on at least 20 cells in each hybrid and was followed by sequential G- I 1 staining of selected G-banded cells. Chromosomes were scored as absent if they were found in less than 20% of the cells scored. Gene probes. The c-myc probe used in our experiments is a I .6 kibobase (kb) CIa I-Eco RI fragment (MC 41'3rC) consisting of the third exon and the 3' flanking sequences of the human c-myc gene.2' To detect structural abnormalities at the 5' portion of myc, we used a 0.8-kb Pvu II fragment consisting of the first exon and the 5' flank of the c-myc gene.22 Immunogbobulin gene rearrangements were exam- med with human JH,2' 24 C5,24 C,,,,2' C,,,2' Ck,27 and Ch28 probes. For determining the proliferative activity, an S-phase-specific gene encoding histone H3 was used.29 30 Blood. Vol 7 1 . No 1 (January), 1 988: pp 30-35 C-MYC ALTERATION IN MULTIPLE MVELOMA 31 Table 1 . Clinical. Cytogenetic. and Molecular Features in 37 Patients With Multiple Myeloma c-myc RNA DNA No. % PC Expression ( 1 5%I Rearrangement 1 76 2 10 3 65 4 90 5 21 6 26 7 21 8 86 9 43 10 48 11 73 12 48 13 80 14 77 10 10 12 10 8 8 8 6 5 1 1 1 1 1 + - 15 16-25 26-37 44 >10 >10 1 1 1 - .1,, Rearrangement + + + + + + + + + + + + - + + +. 8;-, + 2 Ig L Rearrangement ic K x K A K K K K K K nd K X k.6; Xl; nd, 3 k.8; nd, 2; ND, 2 M Protein A, G, Ak A, A, A, A, G, G, A, G, A, 4A A, A A,,1;A,2;G,5;BJP,2 A,,4; G,.4; G5.4 Chromosome8 Anomalies +8 ND t(8;14)(q24;q32) -8 Normal Normal Normal ND Normal t(8;14)(q24;q32) +8;+8 der(8);t(8;?)(q24;?) der(8);(8;?)(q22;?l der(8); -8; t(1;8)(pl 1;p23) -8 Normal ND Abbre viations : X. -fold increase over normal marrow; nd, not detected; ND, not done; BJP. Bence Jones protein; % PC, plas ma cells. RESULTS Analysis of Ig (and more recently of T cell receptor) gene rearrangement has helped determine the clonality and differ- entiation stage in lymphoproliferative disorders.23'#{176} When applied to bone marrow samples from 37 myeboma patients, discrete rearrangements of Ig heavy- and light-chain genes were observed in most cases and corresponded to immuno- electrophoretic results (Table I and Fig 1). The autoradio- graphic signal intensity of rearranged Ig gene bands corre- sponded with the degree of bone marrow plasmacytosis. For c-myc gene analysis, DNA was digested with Barn HI, Eco RI and Hind III restriction enzymes and hybridized to the probe MC 4l'3rC. One of the 37 samples (myeboma I), digested with Hind III, showed a rearranged 14-kb c-rnyc fragment as well as the germline 1 1 .6-kb band. This finding was corroborated by additional analysis with Kpn I and Bgl II, which revealed rearranged myc fragments of I 5 and 9 kb in size, respectively (Fig 2A). Further analysis of the gene locus by using additional restriction enzymes localized the aberration to a site between the Xba I and BgI II at the 3' flanking region of the gene (Fig 2B). In comparison with their germline counterparts, the rearranged myc fragments always exhibited a reduced signal intensity (Fig 2A). The germline bands had the same size as those from normal human placental DNA analyzed in parallel (data not shown). To examine whether the rearranged myc DNA in myeboma was associated with Ig genes frequently observed in other B cell malignancies," filters were rehybridized to several Ig heavy- and light-chain gene probes. As seen in Fig 3, the rearranged myc fragment did not react with C5 and JH as well as S, Cai, C.y4, Ck, and C probes (data not shown), thereby implying a novel rearrangement of c-rnyc in myeboma I . To confirm this, the novel DNA situated at the 3' flank ofthe altered rnyc was isolated by means ofcboning into a phage vector and probed with a panel of hamster-human Normal Marrow L. I Myeloma 1 p\J\ Myeloma 1 C') Normal Marrow Fig 1 . Detection marrow of a patient of monoclonal plasma cells in the bone with IgAx meyloma. BgI II- and Barn HI- digested DNA from myeloma 1 was hybridized to P-labeled H and C, probes which detected 5.5-kb and 1 0.5-kb rearranged Ig heavy- and K light-chain gene bands. respectively (arrows). The probes also revealed 4.2-kb heavy-chain and 1 2-kb light-chain germline gene bands. probably from the unrearranged alleles of the 75% plasma cells and normal hemopoietic cells. Hybridization to the C5 probe revealed a germline configuration (not shown). DNA from bone marrow cells of a healthy donor was used as control. 32 SELVANAVAGAM ET AL I E0 CD 0 Lu =- - :,::C CD > a) .Q_ I C I + 0 w I.," Is.') - 23.7 - 9.5 - 67 ` - 4.,; 2.25 e LLjx A --t =- :e .S Ix -`it -f - :,-, _Q OOwl E 0! .. . _a e )coII `1' .. . _ ea E X 1---- If ca i BI ii -I - I ai ` `lii XSHR PROBES B - .-i Pv-Pv `-4 MC4 l3Rc I `-4 humm 1 Fig 2. (Top) Southern blot analysis of myeloma 1 DNA with c-myc probe MC 41 `3rC. Kpn I. BgI II, and Hind Ill restriction enzyme digestions revealed a rearranged c-myc fragment (arrows) with reduced signal intensity when compared with germline bands. No rearranged c-myc band was detectable after Barn HI digestion. probably reflecting a similar or large DNA fragment size. The germline fragments resulting from digestion with different enzymes matched the sizes of control human placental DNA analyzed in parallel (not shown). (Bottom) Partial restriction map of the human germline c-myc gene (A) and the rearranged myc DNA from myeloma 1 showing the alteration in the 3' flanking region of the gene (B; open bar). The location of the probes used is indicated. XSHR is the cloned Hind Ill fragment. and mouse-human somatic hybrid cell DNA. As seen in Table 2, the probe hurnrn I isolated from the novel sequences in myeloma I (Fig 2B) was localized to chromosome 8 with 100% concordance. The probe hybridized to an 18-kb human DNA fragment generated by Barn HI. The probe also hybridized to a 23-kb mouse band, whereas hamster DNA failed to react with this probe (Fig 4). In a second more recently studied case (myeloma 2), the tumor DNA revealed additional myc fragments 21, 5, and 10 kb in size after digestion with Barn HI, Eco RI and Kpn I, respectively (Fig 5). Due to a shortage of material, detailed characterization of this rearrangement by using primary tumor DNA was not possible. When DNA was digested with Pvu II enzyme and hybrid- ized to the first exon-specific rnyc probe,22 all 37 samples exhibited a germline configuration indicating the absence of structural changes characteristic of endemic Burkitt's lym- phoma. There was no gene amplification evident in any of these other samples investigated. The two cases with rnyc DNA rearrangement showed increased transcriptional activity on Northern analysis (Fig 6A). Seven other patients, or a total of 24%, also exhibited higher myc RNA expression in comparison with that of normal bone marrow (Fig 6B). RNA overexpression ranged from five- to I 2-fold using slot blot analysis (Table 1). Bone marrow cells from a healthy individual were chosen as reference tissue in the absence of significant quantities of normal plasma cells. Elevated c-rnyc expression in our myeloma samples was not proliferation related because the expression of the S-phase-specific histone H3 gene was consistently lower in myeloma samples compared with nor- mal bone marrow (Fig 6C). Although (8;14) translocations have recently been observed in human myeloma,3' our two cases (see Table 1 ), high myc expression including was also noted with other chromosome 8 abnormalities; conversely, only one of the two cases with t(8;14) translocations was associated with elevated myc transcriptional activity. Although there was no apparent clinical association with increased c-rnyc expression, the IgA myeboma protein type was more frequent among such patients in comparison with the remaining 28 individuals with low myc gene activity (67% V 39%, P = .29). DISCUSSION There is growing evidence that cellular oncogenes are altered or amplified in several human malignancies. The c-myc oncogene, which is closely associated with Burkitt's lymphoma,3 is also altered in other human B cell tumors.32 In human plasma cell myeboma, however, gross alterations of the myc gene were infrequent, with only two of 37 patients showing rearrangement within the 30-kb region analyzed. Sumegi et al33 found no alteration of the c-rnyc locus among 21 patients with myeloma.33 Recently, Gazdar et al observed c-rnyc gene rearrangement in a myeloma cell line that they considered responsible for maintaining continuous growth in culture.9 Interestingly, this cell line as well as six of our nine patients with elevated rnyc expression secreted IgA myeloma protein (Table 1). Similarly, recent cytogenetic studies from this laboratory revealed that all of four t(8;14) anomalies were associated with an IgA phenotype.3' The c-myc gene alteration in myeloma 1 with 75% marrow plasmacytosis appeared to be tumor specific because the hybridization signal intensities of the rearranged rnyc frag- ments (and of the rearranged Ig gene bands) were consis- tently less than those of their germline counterparts (Figs I and 2). This observation ruled out the possibility of a constitutive c-rnyc aberration and indicated involvement of only one allele. The 5' portion of the rnyc gene, which is frequently altered in endemic Burkitt's lymphoma, was intact in myeloma. In contrast to similar alterations in a few variant Burkitt's lymphomas" and mouse plasmacytomas34 with chromosomal translocations involving Ig light-chain genes, alteration at the 3' flank of the rnyc gene from C-MYC ALTERATION IN MULTIPLE MYELOMA 33 kb . #{216}i: ---- - 11 .. , - 1! :::= Cp I c-myc .1 Fig 3. Cohybridization analysis demonstrating the lack of Ig gene elements within the rearranged c-myc fragment in myeloma 1 . The filter containing BgI Il-digested DNA was hybridized to the c-myc probe MC41 `3rC and revealed a 9-kb altered myc fragment (center). After the c-myc probe was stripped from the filter. the filter was rehybridized to C8 (left) and J, (right) probes. Similar negative results were obtained after hybridization to the 5,,. C,.1. and C. subunits of the Ig heavy-chain gene as well as the Cic and CX light-chain gene probes (not shown). The less intense germline C,, signal is probably due to deletion of the C,, region during B cell maturation of myeloma plasma cells. Equal amounts of placental DNA from a normal individual were used as control. Ta ble 2. Som atic Cell H ybrid Map ping of the hum m 1 Probe Hybrid Probe 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X V C,1A 1.4 8.2 16.1 MR2.2 MR7.11 MR1.21 SA-5 MH-18 - - - + + + + - - - - + + - ++ ++ + - - + + + - - - - ++ +- - +- +- +- + - + + + - - + +- ++ +- ++ +- - - +- - +- - - + + + + + - -- - + - - -- +- ++- +- ++ - - - + - - - + -- ++ - + +- + +- ++-- + - + -- - - +- + -- +- - - +++- + -- - - + - - + -+ - - + - - - - + - - + - + + + + + - + + + ++ - + - - + + - - - This hybrid contains tmis hybrid contains the translocation the translocation chromosome chromosome 1 Sqter - 1 Sql 5: : 1 7p13 -` 1 7qter. 9qter -` 9q 1 2: : 1 7p 1 1 -` 1 7qter. C CD Cl) EE CD Ill 234 5 678 CD Cl) 0 9 kb 23 - 18 Fig 4. High-molecular weight DNA from hamster-human and mouse-human somatic cell hybrids was digested with Barn HI and transferred to nitrocellulose filter. Hybridization conditions with the humm 1 probe was as described in Materials and Methods. The size of the human and mouse fragments hybridizing to the probe was determined by comparison with standard size markers resolved in parallel. Lanes 1 to 9 contain DNA from somatic cell hybrids described in Table 2. Discrete bands reacting with the humm 1 probe can be detected in lanes 3 and 9. 34 SELVANAYAGAM ET AL TN i, TN TN kb - 23.1 9.4 ,.`. .t.11Q_, `p-, riM 6.6 4.3 __a_!p HI #{231}#R{2I 31}p cP!i I Fig 5. Detection of c-myc gene alteration in myeloma 2. DNA from the bone marrow cells of the patient (T) and of a healthy donor (N) was analyzed with the probe MC41'3rC by methods described earlier. Rearranged myc fragments in the patient's DNA are indicated by arrows. myeboma 1 did not show cohybridization with either light- or heavy-chain gene probes. Instead, somatic cell hybridization experiments revealed that the cloned rearranged DNA origi- nated entirely from chromosome 8, thus suggesting inversion or deletion as a possible novel mechanism of rnyc gene deregulation. Similar alterations at the 3' portion of the myc gene, associated with enhanced transcriptional activity, have been reported in T cell leukemia lines.35'3' Enhancer elements are capable of controlling gene transcription in an orienta- lion-independent manner over long distances,37 and the possi- bility that the novel DNA isolated from the 3' part of the rearranged myc gene in myeboma 1 has enhancer properties is being investigated. A second case of rnyc gene alteration with associated RNA overexpression likewise revealed a lack of Ig gene involvement. The high myc RNA expression in the absence of DNA rearrangement could be due to alterations at large distances from the analyzed area.36 The occurrence of elevated c-myc expression in about one fourth of our patients with or without demonstrable gene alterations mdi- cates a possible pathogenetic role of the c-rnyc oncogene in multiple myeloma. ACKNOWLEDGMENT We thank Trenton Lewin for his technical Thomas for her secretarial assistance. assistance and Mattie -. c'J CD CD EE .2 co .2 CD I CD >, __l > A EI E kb - 2.3 - 1.1 0 CD B- E L- 34 0- Cl) C 0 2---- 0 :tz<8- 16- myeloma 56 78 9 0 (0 I I PC% 76 0 C Cl) 2. - 2A 0` <0 z I `J E 0 z 10 CD E 0 CD ;. a \. .,c...; 0 (0 I #{149} ., . -. ,. ; #{182}.-... Fig 6. (A) Abnormal expression of the c-myc gene in bone marrow cells of myeloma 1 and 2. Equal amounts of total RNA (20 i.tg) were size-fractionated. transferred to nitrocellulose filter. and hybridized to the probes MC41 `3rC and C,. The myc probe revealed 2.3-kb transcripts. and the C, gene probe detected 1 .1 -kb transcripts in myeloma samples. The human promyelocytic cell line H160 showed abundant myc expression but lacked the C, gene expression. PC%. percentage of myeloma plasma cells. (B) RNA overexpression in myeloma cases was determined by slot blot analysis in comparison with normal marrow control. (C) Slot blot analysis of histone H3 gene expression in myeloma 1 . Normal marrow and H160 cells revealed higher proliferative activity than myeloma 1. C-MVC ALTERATION IN MULTIPLE MYELOMA 35 REFERENCES I . Barlogie B, Alexanian R: Cellular aspects of myeloma: Bio- logic and clinical implications, in Delamore 1W (ed): Multiple Myeloma and Other Paraproteinemias. Edinburgh, Churchill Liv- ingstone, 1986, p 154 2. Duesberg PH: Activated proto-onc genes: Sufficient or neces- sary for cancer. Science 228:669, 1985 3. Dalla-Favera R, Bregni M, Erikson J, Patterson D, Gallo RC, Croce CM: Human c-myc oncogene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci USA 79:7824, 1982 4. Tsujimoto Y, Jaffe E, Cossman J, Gorham J, Nowell PC, Croce CM: Clustering of breakpoints on chromosome I 1 in human B-cell neoplasms with the t( 1 I : 14) chromosome translocation. Nature 315:340, 1985 5. Bashkshi A, Jensen TP, Goldman P. 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