Vol. 4, 1065-1070, December 1993 Cell Growth & Differentiation 1065

Mapping of Amplification Units in the qi 3-1 4 Regionof Chromosome 1 2 in Human Sarcomas: Some

. 1Amplica Do Not Include MDM2

Anne Forus, Vivi A. Fl#{248}renes,Gunhild M. Maelandsmo,Paul S. Meltzer, Oystein Fodstad, and Ola Myklebost2

Department of Tumor Biology, Institute for Cancer Research, TheNorwegian Radium Hospital, Montebello, N-0310 Oslo, Norway [A. F.,V. A. F., G. M. M., 0. F., 0. M.J, and University of Michigan MedicalCenter, Ann Arbor, Michigan 48109 [P. 5. M.J

Abstract

Amplification of cellular oncogenes may be importantfor the development and progression of malignanttumors. In human sarcomas, amplification of severalgenes located to the qi 3-1 4 region of chromosome 12has been reported. Because the mdm2 protein seems toinactivate the tumor suppressor protein p53, a selectivegrowth advantage of 1 2q1 3-1 4 amplification haspreviously been assigned to increased copy number andexpression of the MDM2 gene. We have analyzed apanel of 98 human sarcomas of different subtypes tocharacterize the 1 2q1 3-1 4 amplica and determine whichof the genes GLI, A2MR, SAS, MDM2, and GADD153(CHOP) in this region was most consistently amplified.MDM2 was amplified in 9 of the tumors, SAS in 10,GADD153 in 4, GLI in 2, and A2MR in 2. Amplificationwas, in most cases, associated with increased expressionof the corresponding gene. SAS and MDM2 werecoamplified in 8 of the tumors, whereas GADD153, GLI,and A2MR were only amplified together with SAS. Oneliposarcoma showed amplification of MDM2 alone,whereas two osteosarcomas and a rhabdomyosarcomacell line showed amplification of SAS and GADD153(CHOP) but not MDM2. It is suggested that the selectivetarget for these amplica may be an as yet unidentifiedgene localized between SAS and MDM2.

Introduction

The ql 3-1 4 segment ofchromosome 12 is frequently alteredin human sarcomas. Both rearrangement and amplificationof genes localized to this chnomosomal region have beenobserved (1-6). Amplification ofoncogenes has been shownto give tumor cells a selective growth advantage (7, 8), andthe observed aberrations involving possible oncogenes inthe chromosome l2ql3-l4 region may indicate a role forthese in tumor development or progression.

Two genes in the 1 2q1 3 region, GLI, a putative oncogene,and A2MR3 (LRP), codingfonthe a2-macroglobulmn receptor

Received 9/10/93; accepted 10/6/93.1 This work was supported by the Norwegian Cancer Society, the Norwegian

Research Council for Science and the Humanities, and the University of Oslo.2 To whom requests for reprints should be addressed.

3 The abbreviations used are: A2MR, a2-macroglobulin receptor; GADD,growth arrest and DNA damage; MDM2, mouse double minute 2; SAS, sar-coma amplified sequence; APOB, apolipoprotein B; C/EBP, CCAAT/enhancer-binding protein; CHOP, C/EBP homologous protein; MFH, malig-nantfibrous histiocytoma; LS, liposarcoma; MS, malignant schwannoma; OS,

(low-density lipoprotein receptor-related protein) (9), areamplified in some sarcomas (1, 2). However, the low am-plification frequency speaks against these being importantfor tumor development. We therefore suggested that someunknown gene(s) close to CLI and A2MR might be moreconsistently amplified (10).

The MDM2 gene, also localized to this region, was re-cently reported to be frequently amplified in human sarco-mas (3). This gene encodes a protein that binds to and prob-ably inactivates the tumor suppressor protein p53 (3, 1 1 , 12).Thus, a priori, it seemed most likely that MDM2 would bethe driving force for selection of the amplica. However, theSAS gene, located in the same segment, was amplified in ahigh proportion of human MFHs (6, 1 3), and the frequencyofSASamplification was similarto that reported for MDM2.Moreover, GADD1S3 (CHOP), a DNA damage-induciblegene ofthe C/EBP family (14, 1 5), is consistently rearrangedin liposarcomas with the (1 2;1 6)(ql 3;pl 1) translocation (4,1 6) and is localized close to MDM2 and SAS (1 7, 18).

We have examined a panel of human sarcomas of varioussubtypes for amplifications of MDM2, SAS, GADDJ53, GLI,and A2MR, and we have also studied the expression of thegenes because this is likely to be affected by amplification.

Results

The amplification pattern of A2MR, GLI, GADD153, SAS,and MDM2 was analyzed in 98 human sarcomas. In the 20cases for which a matched blood sample was available, Ieu-kocyte DNA was included as a control for a normal genecopy number. Southern blots were sequentially hybridizedto probes for each of the loci and then to three differentcontrol probes (from the short and long arms of chromosome1 2 and from chromosome 2). After correcting for unequalsample loading, a signal at least 3-fold more intense thansignals from samples with a normal copy number ofthe genewas scored as an amplification. In all cases, there was agree-ment between the results obtained with the three controlprobes. The results are shown in Tables 1 and 2 and Figs. 1and 3, together with data for the cell lines RMS1 3/Rh30 andOSA, with previous reported amplifications in this region(1-3). We also determined whether gene amplification wasassociated with increased expression in the 88 samples fromwhich RNA was available. These results are shown in Table2 and Fig. 2, also showing data for the RMS1 3/Rh30 andOSA cell lines.

Amplification of GLI or A2MR was found in liposarcomaLS21 (both genes), LS1 1 (3’ end of A2MR), and osteosar-coma OS1 1 (GLI). In addition, amplifications were previ-ously found in RMS1 3/Rh30 (both genes; Fig. 1 ) and OSA(GLI; Fig. 1) (1 , 2). Although the samples with amplification

osteosarcoma; NCS, nonclassified sarcoma; HP, hemangiopericytoma; RMS,rhabdomyosarcoma; FS, fibrosarcoma; cDNA, complementary DNA; kb,kilobase(s).

1066 Amplification of l2q13-l4 in Sarcoma

4 V. A. Fl#{248}renes,unpublished observations.

Table 1 Human sarcoma samples with amplification of MDM2, SAS,

GADD!53, GL 1, or A2MR

Ninety-eighthuman sarcomaswereanalyzed foramplification of the genesMDM2, SAS, GADD153, CL !, and A2MR. For each subgroup, the number

of sarcomas examined and the samples with amplifications of any gene areshown. The OSA and RMS1 3/Rh30 cell lines are not included among the 98samples, as they were selected because of previously reported amplificationsof 12q1 3-1 4. Numbers in parentheses indicate the number of xenografts ineach subgroup, x indicates that sample is a xenograft. In three cases, bothpatient material and xenograft were analyzed, showing identical results(e.g., MS8/8x).

No. of Samples withTumor samples . .

samples amplification

Carcinosarcoma 2 0Chondrosarcoma 3 0Fibrosarcoma 2 FSl

FS2

Hemangiopericytoma 1 (1) HP1x

Leiomyosarcoma 14 (4) 0Liposarcoma 1 9 (2) LS6

LSl 1

LS21

Malignant fibrous histiocytoma 20 (5) 0Malignant schwannoma 6 (1 ) M54

MS8/8xOsteosarcoma 1 7 (1 0) OS6x

0�11Rhabdomyosarcoma 5 (1) 0Synovial sarcoma 3 (2) 0Non classified sarcomas 3 (2) NCS2x

Other soft tissue sarcomas 3 0

Total 98 11

Other samplesOsteosarcoma 1 OSARhabdomyosarcoma 1 a RMS1 3/Rh30

a The RMS1 3 and Rh30 cell lines were established independently from the

same tumor as a cell line (RMS13) and a xenograft line (Rh30) (1, 23).

ofA2MR had high levels ofA2MR mRNA (e.g., RMS1 3; Fig.2), many sarcomas with a normal gene copy number, benignlipomas, and also normal fat expressed similar amounts ofthis transcript [e.g., LS19 (Fig. 2) and results not shown].High levels ofGL/mRNA were detected only in samples withGLI amplification, but one sample with GLI amplification(OS1 1) did not express the gene at a detectable level (Table2; Fig. 2).

GADD1S3 was amplified in 4 of the 98 sarcomas (HP1 x,LS21 , OS6x, and OSl 1 ) and also in the RMS1 3/Rh30 andOSA cell lines (Fig. 1 ). As can be seen in Table 2, GADD1S3was only amplified in samples with SAS amplification(HP1 x, LS21 , OS6x, OS1 1, RMS1 3, and OSA), and in threeof these (HP1 x, LS21 , and OSA), MDM2 was also includedin the amplica (Table 2). All of these samples expressed de-tectable amounts of GADD153 mRNA (Table 2; Fig. 2), andin 4 (including RMS13 and OSA), the levels were highlyincreased. However, some clinical samples without ampli-fication (4 of67) also expressed large amounts of GADD 153mRNA (data not shown). Since GADD153 is induced byDNA damage and growth arrest, this increase in expressioncould have been caused by preoperative radiation or che-motherapy. However, this was not the case for any of thesepatients. It is possible, however, that inflammation, hypoxia,on nutrient deprivation (1 9, 20) within the tumor could alsoinduce expression of the GADDJ53 gene.

In our sarcoma panel, SAS and MDM2 were the mostfrequently amplified genes (1 0 and 9%, respectively). In ad-

dition, both genes were amplified in the OSA cell line,whereas SAS but not MDM2 was amplified in RMS1 3/Rh30.The two genes were coamplified in most but not all cases (8of 1 1 samples; Table 2; Fig. 1). In the liposancoma LS6, onlyMDM2 amplification was detected, whereas the osteosar-comas OS6x and OS1 1 had amplifications of SAS (andGADD!53) but not MDM2. The SAS mRNA was not de-tectable in cases without amplification (Table 2). However,all but one sample (HP1x; Table 2) with amplification ex-pressed the gene at detectable levels, and in most cases,multiple bands could be seen (0.8, 1 .7, and 4 kb; Fig. 2).Amplification of the MDM2 gene was also associated withincreased expression (Fig. 2), but some tumors without am-plifications also expressed detectable amounts of MDM2mRNA, although generally at a low level (data not shown).

As shown in Fig. 3, the sizes of the different amplica andthe number of genes included were highly variable. In oneliposarcoma (Fig. 3a), only MDM2 was found to be ampli-fied, whereas another liposarcoma (Fig. 3d) showed ampli-fication of all five genes studied. It is possible that the areabetween SAS and MDM2 could be amplified in all cases.

Discussion

The many aberrations ofthe 1 2q1 3-1 4 region in human can-cers suggest that it harbors genes important for tumor de-velopment or progression. Several genes localized to thischromosomal segment have been reported to be amplifiedin a variety of sarcomas (1-3, 5, 6, 12, 13). Such amplifi-cations could be nonspecific products of the structural in-stability of this region, but the absence of MDM2 amplifi-cation, e.g., in Ewing’s sarcomas (21 ) and melanomas,4suggests that these aberrations are more specific. Moreover,when putative oncogenes are involved, selection toward in-creased gene dosage may be of relevance for the develop-ment of a transformed phenotype, as reported for other tu-mon types (7, 8). The aim of the present study was todetermine the amplification frequency of A2MR, GLI,GADD1S3, SAS, and MDM2 in human sarcomas, to findwhich gene might be the most likely selective target for theamplifications observed in these tumors.

Amplification of CL! on A2MR was found in 2% of thesamples (Table 1), and also in the cell lines RMS1 3/Rh30 andOSA, as reported previously (1 , 2). GLI is a putative onco-gene that can transform cells in vitro in cooperation with theadenovinus E1A gene (22). Nevertheless, the low amplifi-cation frequency of GLI and A2MR suggested that bothgenes are incidentally included in the amplica, and thereforenot important for the development or progression of sarco-mas.

It is known that aberrations ofthe GADD153 gene may beinvolved in the tumonigenesis of at least one sarcoma sub-type. As a result of the very specific t(l 2;l 6)(ql 3;pl 1) trans-location in myxoid liposarcomas (23), the entire coding ne-gion of GADD1S3 is fused to the promoter and 5’ end of agene encoding a nuclear RNA-binding protein (4, 16). Al-though the overall biological consequences of the translo-cation are still unclean, it is likely that other alterations of thisoncogene, such as amplification and overexpression, couldcontribute to tumor progression. Furthermore, the responseof tumors to DNA-damaging cancer therapy might be af-fected by aberrations of this DNA damage response gene.

Cell Growth & Differentiation 1067

5 P. 5. Meltzer, unpublished results.

Table 2 Amplification and expression of MDM2, SAS, GADD1S3, GLI, and A2MR in the samples with amplified genes

Gene amplification and mRNA levels were scored from autoradiograms of DNA and RNA blots hybridized to probes from each locus. A, amplified gene; -,

normal gene dosage (DNA) or no expression detected (RNA); +, + +, etc., expression levels of the gene (+, low/normal level; + +, etc., increased levels); ND,RNA was not available for testing.

MDM2 SAS GADD153 GLI A2MRSample

DNA RNA DNA RNA DNA RNA DNA RNA DNA RNA

FS1 A +++ A ++ - - - - - +

FS2 A +++ A + - + - (+) - +

HPlx A ++ A - A +(+) - - - -

LS6 A +(+) - - - + - ND - ND

LS11 A +++ A ++ - + - ND 3’end ND

LS21 A ++ A + A + A (+) A ++

MS4 A ND A ND - ND - ND - ND

MS8/8x A + A (+) - - - - - (+)

NCS2x A +++ A (+) - (+) - - - +

OS6x - - A (+) A +++ - - - +

OS11 - (+) A (+) A ++ A - - ++

No.ofsampleswith 9 10 4 2 2

amplification

Other samplesOSA A ++ A + A +++ A ++ - -

RMS13/Rh30 - - A +(+) A ++++ A ++ A

GADD1S3 was amplified in 4% ofthe sarcomas studied here(Table 2), and also in the cell lines RMS1 3/Rh30 and OSA(Fig. 1).

The two genes with the highest amplification frequency inour panel, SAS and MDM2 (1 0 and 9%, respectively; Table2), have been reported to be even more frequently (14-30%)amplified in other sarcoma materials (3, 6, 12). Most notably,SAS (6) and MDM2 (3) were amplified in about 30% of theMFH samples analyzed previously, whereas no amplifica-tions were detected in our 20 MFHs. SAS amplification hasbeen found only in large, intraabdominal MFHs (6), perhapsas a result of a long period of growth prior to diagnosis. Inthis context, it is noteworthy that among the MFHs studiedhere, only one was a deeply situated intraabdominal tumor.The other 1 9 were located more superficially, or to the ex-tremities. Interestingly, unlike Ladanyi et al. (1 2), we de-tected MDM2 amplifications in primary tumors as well as inrecurrent and metastatic lesions, independent of site ofgrowth (data not shown).

Other investigators have shown that amplifications arisingin different tumors may have the same “core,” containing theselective target gene, but the sizes of the amplica may behighly variable (24, 25). Except for A2MR, all of the genesanalyzed in this study generally showed elevated expressionwhen amplified (Table 2), a prerequisite for being a candi-date selective gene. A summary ofour results is given in Fig.3, showing a map of the relevant chromosomal region andthe extensions of the various amplica. As can be seen, thesizesofthe amplica and the numberofgenes included vary.lnonecase(LS11; Fig. 3c), amplificationofMDM2, SAS, andthe 3’ end of A2MR was observed, whereas the GLI andGADD1S3 genes were excluded, showing thatthe amplifiedsequences are not necessarily continuous. Similar observa-tions have previously been reported, e.g., forthe 1 1ql 3.5-14region in breast cancer (26).

SASand MDM2were the mostfrequently amplified genes,and because overexpression of MDM2 probably representsan alternative way of inactivating p53, MDM2 seems a priorito be the most likely selective target ofthe amplica. SAS was,

however, amplified more frequently than MDM2, and sev-eral samples (OS6x, OS1 1 , and RMS1 3/Rh30) had amplifi-cation of SAS but not MDM2. Although little is known aboutthe SASgene and the function of its product, it is possible thatSAS might be important for tumor development.

It is conceivable, but not very likely, that MDM2 was mi-tially included in all ofthe amplica and has been lost at laterstages of tumor progression. An alternative explanation isthat amplica containing MDM2could have been selected by

that gene, whereas the other amplica were selected by theactivityofanothergene, probably localized between MDM2and GLI. There are two known candidates for such a role:SAS, a type II membrane protein without known function,5and the GADD153 gene, which has shown oncogenic po-tential in myxoid liposarcomas. However, other unde-scnibed genes are probably also localized to this region.Moreover, it is possible that the area between SAS andMDM2 may be amplified in all ofthe samples (Fig. 3, shadedarea). Hence, the simplest explanation would be that a corn-mon selective target for 1 2q1 3-1 4 amplification in sarcomacould be an as yet unknown gene located there. It will there-fore be of interest to clone new sequences from this regionand to study the amplification patterns in even larger panelsof human sarcomas.

Materials and Methods

Specimens. Tumor tissue was obtained from 67 patientswith sarcomas of various subtypes and from 28 different san-comas grown s.c. as xenognafts in nude mice (Table 1 ). Thetissues were cut into small pieces, frozen in liquid nitrogenimmediately after surgery, and stored at -70 or -1 35#{176}C.Three cell lines established from an osteosarcoma (U2OS)and two rhabdomyosarcomas (Rh28 and R’�/A) were alsoincluded in the material. A matched blood sample was avail-

4�/ 4/ � � 4�%\

AA- ri�: A

$4�\ �

A A A A

A2MR

GLI

. GADD153

a� 4e4GADDJ53

MDM2

A

SAS

MDM2

18SrRNA

Fig. 2. Expression ofMDM2, SAS, GADD1S3, CLI, and A2MRin individual

sarcoma samples. A blot containing total RNA was sequentially hybridizedto probes for A2MR, GLI, GADD1S3, SAS, MDM2, and the 1 85 rRNA cali-bration probe. Samples were the malignant schwannoma MS8, fibrosarcoma

FS1 ,osteosarcoma cell line OSA, osteosarcoma OS6x and 051 1,rhabdo-myosarcoma cell line Rh30, liposarcoma LS19 and nonclassified sarcomaNCS2x. Samples with amplifications of a gene are indicated by an A below

the corresponding panel. None of the genes were amplified in LS19.

Fig. 1. Amplification of MDM2, SAS, GADD153, CLI, and A2MR in sar-

coma samples. Left and right panels are from autoradiograms of a blot se-quentially hybridized to probes for A2MR, CLI, GADD1S3, SAS, and MDM2.

Samples were DNA from the rhabdomyosarcoma cell line RMS1 3, xenograftRh30, osteosarcoma cell line OSA, liposarcoma LS1 9, and fibrosarcoma FS1,cut with HinDIll. None of the loci were amplified in LS1 9. Amplification isscored by comparing relative signal intensities across the lanes, and sampleswith amplification of a gene are indicated by an A below the corresponding

panel.

I

1068 Amplification of 12q13-14 in Sarcoma

A A A - - A

able from 20 of the patients. DNA from these samples wasincluded in the analysis as a control showing a normal gene

copy.In addition, the rhabdomyosancoma cell lines RMS1 3 and

Rh30, established independently from the same patient, and

the osteosarcoma cell line OSA were studied, as they pre-viously have been reported to harbor amplifications of GLItogether with A2MR (RMS1 3/Rh30) on MDM2 (OSA) (1-3).

The osteosancoma cell line OSA and the rhabdomyosan-

coma cell line RMS13 were kindly provided by Dr. E. C.Douglass (1), and the rhabdomyosancoma cell lines Rh28,Rh30, and RD/A were provided by Dr. P. J. Houghton (27,28).

Preparation and Analysis of DNA. Solid tumor sampleswere pulverized in liquid nitrogen and incubated for severalhours at 65#{176}Cin 0.25 M EDTA, 1 % sodium sancosyl, and 500pg/mI proteinase K (Boehninger Mannheim). DNA was ex-tracted by an Applied Biosystems 340A automatic DNA ex-

tractor as described by the manufacturer. DNA from leuko-cytes and cell suspensions was prepared by standardmethods (29).

Seven pg of each sample were electrophoresed through0.8% agarose gels. Blotting of the gels and hybridization offilters were performed as described previously (30). Southernblots were sequentially hybridized to DNA probes for eachlocus and then to three different control probes to correct forunequal sample loading. Probes were labeled with 32P bythe random priming technique (31), and hybridization con-ditions were as described previously (32). After autoradiog-naphy, the probes were removed by incubation for 1 5 mmin 1 00 mM NaOH-1 m� EDTA at room temperature.

A signal at least 3-fold more intense than signals fromsamples with a normal copy number ofthe gene (leukocytes)

l2qter � I

MDM2

0 250 500 750 1000I I I I

SAS GADD153 GLI

a) (1)

b) (5)

c) (1)

d) (1)

l2cenA2MR

=

� � E

e) (!) �

f) (!) �

g)(1)

h)(1)

I) (1) .u

Cell Growth & Differentiation 1069

Fig. 3. The variable size andgene content of the 1 2q1 3-1 4 am-

plica in sarcomas. Upper part or-der of the genes along chromo-some 1 2 and the approximate

distances between the genes (14,

15). Black boxes, genes; dottedlines, regions that may be ampli-fied (no probes available); shadedarea, the possible “common re-

gion of overlap” that may be in-

cluded in all ofthe amplica. As wedo not have probes flankingMDM2 and A2MR outside themapped region, we cannot deter-

mine whether the amplica in casesa, b, c, d, 1, and i may extend in thedirection of the arrows. The differ-

ent amplica represent the follow-ing samples: a, L56; b, FS1, FS2,

M54, MS8/MS8x, and NCS2x; c,LS11; d, LS21; e, OSA; 1,HP1x;g,

OS6x; h, OS1 1 ; i, RMS1 3/Rh30.

Approximate distances (kb)

was scored as amplification. Densitometnic analysis of theautonadiognams was used to decide on cases that were notobvious.

Densitometric Analysis of DNA. Autoradiognams fromhybridized blots were scanned two dimensionally on a Mo-lecular Dynamics laser densitometer. The net signals from

specific bands were connected for unequal loading of thesamples by calibration relative to the signal obtained fromcontrol probes. Signals from Di254 on 1 2cen-ql 4, A2M on12pl 2-1 3, and APOB on chromosome 2 were used as stan-dards for the analysis of DNA amplification.

RNA Expression Analysis. Total cellular RNA was pun-fied from fresh on frozen tissues by extraction into 5 M gua-nidine thiocyanate and sedimentation through a 5.7 M ce-sium chloride cushion (29). Precipitation from lithiumchloride-urea was used to purify RNA from tissue culturecells (33). Five pg total RNA from each sample were elec-trophonesed through 1 % agarose gels containing 6.6% form-aldehyde (0.02 M NaH2PO4, pH 6.6) and transferred by cap-illary action onto nylon membranes in 1 Ox standard salinecitrate (29). The RNA was fixed to the membranes by bakingat 80#{176}Cfor 2 h and UV irradiation for 5 mm under a gen-micidal lamp. Hybridization conditions were as describedfor DNA.

Probes. The genomic probe for GLI (pKK36P1 ) waskindly provided by Drs. K. W. Kinzlen and B. Vogelstein (34).As probes for A2MR were used either partial cDNA clonesor a complete 1 5-kb cDNA, joined together from the originalpartial clones (35). cDNA probes for MDM2 were kindlyprovided by Dns. D. L. George and B. Vogelstemn (3).

A/u repeats were removed from the full length cDNA bypurification ofa 3.1 -kb fragment after excision by ScaI. Vec-ton sequences were then removed by XhoI scission followedby a second purification. The human GADDJ53 cDNAclone was kindly provided by Dr. N. J. Holbrook (14). Theprobe for SASwas the pSJP2 clone described previously (1 3).

To calibrate for unequal loading of DNA, we used theA2M clone pHLA2M.1 from the American Type Culture Col-lection, the anonymous segment D12S4, American TypeCulture Collection clone p9Fl 1 , and the APOB clone pB8

kindly provided by Dr. J. L. Breslow (36). The probe for ni-bosomal RNA was complementary to nucleotides 287 to305 in human 18S rRNA. When hybridized as described forDNA blots, but at 55#{176}C,the probe does not detect mouseRNA.

Acknowledgments

We are grateful to Oystein Eskeland and Martina Skrede for excellent tech-nical assistance, Drs. E. C. Douglass and P. 1. Houghton for provision of cell

lines, Dr. I. Hoie and Professor K-E. Giercksky for the provision of clinical

samples, and Dr. A. E. Stenwig for the histological classification ofthe tumors.

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