us dI

Proc.Nat!.Acad.Sci. USA Vol. 89, pp.1616-1620, March 1992 Medical Sciences

Characterization of an inversion on the long arm of chromosome 10 juxtaposing DlOS170 and RET and creating the oncogenic sequence RET/PTC

(locus identifikedby probe H4)

MARCO A. PIEROTrIt,

MASSIMO

SANTOROt,

ROBERT B.

JENKINS§,

GABRIELLA

SOZZIt,

ITALIABONGARZONEt,

MICHELE GRIECOt, NICOLETTA MONZINIt,

MONICA

MIOZZOt,

MARIE A.

HERRMANN§,

ALFREDO Fusco¶, IAN D.

HAY§, GIUSEPPE DELLA PORTAt,

AND

GIANCARLO VECCHIOt

tCentrodiEndocrinologia ed Oncologia SperimentaledelConsiglio Nazionaledelle Ricerche,c/oDipartimento di Biologia e PatologiaCellulare eMolecolare, Universiti diNapoli, Naples, Italy;tDivisionediOncologiaSpermntale A, Istituto Nazionale Tumori, Milan, Italy;tDivisionsofLaboratory Medicine, Endocrinology and Internal Medicine, Mayo Clinic and Foundation, Rochester, MN 55905; and1DipartimentodiMedicina Sperimentale eClinica, Facoltadi Medicina eChirurgia di Catanzaro, Universiti di Reggio Calabria, Catanzaro, Italy

CommunicatedbyJ. EdwardRall, October 4, 1991

ABSTRACT RET/PTCisatransformingseque cated by thefusion of the tyrosine inase domain of the RET protoon- cogene with the5' end of thelocus DIOS170 dI by probe H4andis frequently

found

activatedin human

papillary

thyroid carcinomas.RETand

DIOS)

70 have been

mappe

to us

region

ofthe long arm of chromosome 10: qll.2 and q21,

respectively.

To

identify

the mechanis leadin tothegeneration ofthe oncogenicseqe RET/PTC,acombined

cytogenetic

and molecular

analysis

ofseveralcas ofpapillary thyroid cadno- mas wasdone. In four cases the results tedthatthesetumors hadRET/PTCactivatlon andaparacentric inversion of the long arm of chromosome 10,

inv(lO)(qll.2q21),

with

breakpoints

coinddentwith the

regions

where RET andDIOSi70 arelocated.

Therefore, a chromosome lOq inversion provides thestural basisfor the DIOS170-RET fusion that forms thehybridtrans- form sequence RET/PTC.

Insomeforms ofcancer,chromosomerearrangementshave providedthe structuralbasis oftheirmolecularpathogenetic mechanisms. In particular, in hematologic malignancies, chromosomal translocations result in deregulation of the expression ofthecell-growth-relatedgenesc-MYC and BCL2 in Burkittandin follicularlymphomas, respectively (1-3).In other instances, the translocation may generate chimeric transforming transcripts as in the case of the BCR-ABL fusionprotein found in chronicmyelocytic leukemia (4).

In solid tumors,however, only rarelyhave chromosome aberrations beenanalyzedatthemolecular level. Themost relevantexamplesinclude the chromosomal deletions found toinactivateone orbothcopiesof tumor-suppressor genes- suchas RBinretinoblastoma,WTIin Wilms tumor, and DCC incolon carcinoma (5).

Theonly example ofgenerationof chimerictransforming sequences in solid tumors is

tie

activation of c-MET on chromosome 7bytheTPRgene that mapstochromosome 1 (6). However,this event, mostlikelyachromosomaltrans- location, has been generated in vitro by treatment of the original human osteogenic sarcoma cell line (HOS) with a chemicalcarcinogen (7). In othercasesthe rearrangements giving rise to the transforming fusion products have been generatedduringthetransfectionprocedures,asin thecase of RET(8, 9). Whenasimilargeneticeventoccurredsomat- icallyintheoriginaltumorDNA, like the fusion betweenthe nonmuscle tropomyosin sequence and the tyrosine kinase domainof proto-TRK gene (10),nochromosomal aberrations

were identified to provide amodel for therelative mecha- nism.

Wehavereported (11) the occurrence in papillary thyroid carcinomasofspecificrearrangementsof the tyrosine kinase domain of the proto-RET gene with an unknown "gene"

DJOS170, identified by probe H4 and originally designated thechimerictransformingsequence as

PTC11,

butheredes- ignateit asRET/PTC. Intwo distinct setsofexperiments, RET/PTC activationwasfoundin=25%ofpapillarythyroid carcinomas (12, 13) andrepresented atumor-specificsomatic event, beingdetected in theoriginal DNA of both primary tumorsand metastasesbutnotinnormal cellsfrom thesame patients(12, 13). WehavemappedDIOSi70tochromosome 10q21 (14, 15) andconfirmedthe RETlocus at10qll.2(15, 16).

From oneofour laboratories (R.B.J.,M.A.H., and I.D.H.) the occurrenceofasimplecytogeneticaberration inpapillary thyroid carcinomas has also previously been reported: an inv(10)(qll.2q21) (17) with breakpoints where RET and DIOS170 are located. Among 18 cases of this tumor histo- type, we have, todate, identified five cases that have this identical abnormality. Here we report thecytogenetic and molecularcharacterizationof fourof thesetumorsand dem- onstrate that the cytogenetic inversion has provided the structuralbasis fortheDIOS]70-RETfusion leadingtothe generationof the chimerictransformingsequenceRET/PTC.

MATERIALS AND

METHODS

ChromosomePreparation. Specimens fromprimarytumor ormetastaticlymphnodeswereobtainedatsurgeryandwere histologically diagnosed as papillary thyroid carcinomas.

Cases 1, 3, and 4 corresponded to pure papillary thyroid carcinomas;case 2mightbeconsideredadiffusesclerosing variantofpapillarycarcinoma. The samplesweremechani- callydisaggregatedandincubatedin0.8%collagenasetype II (Sigma)at370Cin5%CO2 overnight.Thecellsuspensionwas seeded andcultured inRPMI1640medium(WhittakerBio- products) supplemented with 5% fetal calf serum (Flow Laboratories), penicillin(200 units perml),streptomycin (200

Ag/ml),

transferrin(10

jug/ml),

hydrocortisone (0.5

,ug/ml),

epidermal growth factor (5

ng/ml)

(Sigma), and insulin (5

,tg/ml)

(Sigma).

IIPTC,

thesymbolusedoriginallytodesignatethispapillarythyroid carcinomatransforming sequence, has anothermeaning (phenyl- thiocarbamidetasting)for the HumanGeneMappingNomenclature Committee;thepapillary thyroidcarcinomatransformingsequence isdesignatedRET/NPC.

1616 Thepublicationcostsofthis articleweredefrayedin partbypagecharge payment. This articlemust therefore beherebymarked "advertisement"

inaccordance with 18 U.S.C.§1734 solelytoindicatethis fact.

Chromosome preparations were performed on primary culture of 10 days, according to standardtechniques.G-band- ingwas obtained by using Wright's stain.

Southern and Northern (RNA) Blot. High-Mr DNA was digested with the appropriate restriction enzymes, electro- phoresed, blotted on nitrocellulose or nylon filters, and hybridizedtonick-translatedmolecular probes, as described (11).

Northern blot analysisused 10

Ag

of total mRNA prepared from the cell lines indicatedin Fig. 3 and was done following described procedures (11) by using as a probe a cDNA fragment correspondingto thetyrosine kinase domain of the RET oncogene (probe C3 of ref. 11).

Transfection Assay. The transfection assay was done on NIH 3T3 cells with high-Mr DNA obtained from the meta- static lymph nodeof patient 1, byfollowing described tech- niques (13).

PCR. RNA amplification was obtained by following the method described by E. S.Kawasaki (18) starting from 1.5

ttg

of total mRNA. The map in Fig. 4 indicates the nucleotide position oftheprimers relatedtoDIOS170 (196-213) and RET (485-502) sequences, respectively. The breakpoint ofthe RET/PTCsequence between nucleotides 351 and 352 and the EcoRI site (371) are also indicated.

RESULTS

The cytogenetic analysesofthe tumor cellsrevealed as the specific acquiredabnormality a paracentric inversion ofthe longarmof chromosome10withbreakpointsatqll.2 andq21 in 15 outof 15 metaphases of case 1, 8 out of 10 metaphases ofcase2, 3 out of30metaphases ofcase 3,and 29 outof30 metaphasesofcase 4. Fig. 1Ashows arepresentativeexam- ple of the inversion from patients 1 and 2. An identical inversion was seen in patients 3 and 4. The karyotype of patient 4 has been reported in detail elsewhere (17). The peripheral lymphocytes of patients 1, 2, and 4 showed a normal 46,XX karyotype. The peripheral lymphocytes of patient 3 were notstudied.

The rearrangement of the RET gene, leading to the gener- ationofRET/PTChas beenshowntooccurina6.3-kilobase (kb) EcoRI fragment of the proto-RET gene located imme-

A B

0

v

- '4

CASE 1

-s-i a

CASE 2

3,

5 RET.PTC

(RET-D1OS170)

10 inv(10)(q11.2 q21) FIG. 1. Chromosome 10q inversion in papillary thyroid carci-

noma.(A)Tworepresentativechromosome 10homologs fromtumor cells ofpatients 1 and 2 showing inv(10)(qll.2q21) (arrows). (B) Schematic view ofthe paracentric inversion of chromosome lOq

generatingthetransformingsequenceRET/PTC. Thegeneorderon

chromosomelOqhasbeen previouslydetermined(16).The indicated directionofgenetranscriptionisoneof thetwopossibilities (diver- gentvs. convergent).

diately upstream of its tyrosine kinase-encoding domain, whereasDIOS]70truncation was found at different pointsin differenttumors(11). The DNAfrom the fourtumors carry- ing thelOq inversionwas analyzed bySouthern blot witha 1-kilobase pair (kbp) BglII-BamHI probe(Fig. 2, probe2), specific forthe 6.3-kbp EcoRIfragment ofproto-RET,where allthe characterizedRET rearrangements have been dem- onstrated. The probe detected an extra band in the four tumorsandnotin the related normal DNA (Figs. 2and 3A), indicating the tumor-specificRETactivation (11). To dem- onstratetheinvolvement of DJOS170 sequencesin theRET rearrangementof the fourtumors, weanalyzedtheirDNAs withtheavailableDIOS]70-specificAlu-freegenomic probes.

Inpatients2and 3using the 0.6-kbpBamHI-Ava IDIOS170 probe (Fig. 2, probe 3)arearrangedbandwasclearlydetected (Fig. 2). Inpatient3 the6-kbp BamHI-rearranged DIOS170 bandwasof the same sizeas oneof theBamHI-rearranged RET bands. This finding indicates that the two genes are fused together. Incases 1 and 4 we were unable to showa DIOSJ70rearrangement(Fig. 3B and datanotshown).How- ever,becausecase 1provided enoughtumortissue,a trans- fectionassay onNIH 3T3 cellswasdone.After14days1.05 fociper

Ag

ofDNA wasobserved. TheNIH3T3 transform- antspossessed both humanRET(Fig. 3A, lane NIH-T1) and DIOSJ70(Fig.3B,laneNIH-T1) sequences. Asexpected,the humanDIOSJ7O andRET sequences detected in the trans- formants were of the same size as the DJOSJ70-related fragment and the RET-rearranged bandpresentintheoriginal tumor DNA(Fig.3A,laneT). Asanticipated,thesizeofthe DIOS]70fragmentswas notdistinguishable from that of the DIOSI70fragmentseeninnormalDNA(Fig. 3B).Moreover, Fig. 3D showsaNorthern blotobtained withaRET-specific cDNAprobe; inthetransformants obtained withDNAfrom thistumor,theREToncogene wastranscribed intruncated mRNAshaving sizes identicaltothosepreviously detectedin aRET/PTC-positivepapillary thyroid carcinoma (11). Tran- scripts of thesame sizewere detected inthe same cell lines with a

DJOSJ70-specific

probe (data not shown). A PCR analysiswasperformed with primers derived from boththe RETand the

DJOSJ70

portion ofRET/PTC to amplify a cDNAfragmentthatcontainedthe RET-DIOSI 70breakpoint (11)(Fig. 4). This experiment showed that theRET-DIS0170 rearrangement, although occurring in different intronic re-

gions,produced identicalmRNAspecies. Infact,theRET/

PTCcDNAsderivedfromNIH3T3transfectants ofthe case reported (11) andfrom thetumorofcase 1contained thesame predicted fragment of 306 base pairs. ComplementaryDNA directly prepared from thetumorofpatient1 contained the same PCR fragment (Fig. 4), thus confirming RET/PTC activation. Thespecificity of theamplifiedbandswas ascer- tainedbyEcoRIdigestion ofthePCRproductsthatresulted inthepredicted fragments (Fig. 4, bandsAandB).Thesame resultwas also obtained in patient 3, confirmingthe above conclusions (data notshown); moreover, the specificity of the PCR was ascertained by hybridizing its products with PTC-specific probes and by sequencing the PCR products (data notshown).

Thecytogeneticandmoleculargenetic data indicate thatan inversion on chromosome lOq has activated RET/PTC by fusing together

DIOSJ70

and RETsequences. Suchamech- anism wouldpredictthat intumorDNA the sequenceof the RETgene locatedupstreamfromthebreakpointisrearranged and fused to the 3' end sequence of

DIOSJ70.

The tumor DNAs from the four patients were analyzed with a probe locatedupstreamfrom thebreakpointin theproto-RETgene (Fig. 2, probe 1), and an extraband was detected demon- stratingthat the 5' endofproto-RET wasrearranged in all thesecases(Figs. 2and 3C).

Moreover,in

patients

2and3,wedetecteda

rearranged

band ofthesamesize, 10.5kbpand7.2kbp,respectively,with both

1618 Medical Sciences: Pierotti et al.

N T

Probe 2

N T

Probe 3

N T

Probe 1

N T

_ - -10.5

- --6 3

- _ -3.7

EcoRI BarnHI

Probe 2

N T N T

mm _ --6.3

Patient 3

Eco RI

vw -a-6 lp 4.8

'mm 3.7

Bam HI

- - -6.3

Eco RI

Probe 3

N T

-- -9.5

t -6 Bam HI

Probe 4

w _7.2

c -R3

Eco RI

Probe 2

N T N T

EcoRI

Probe 1

N T

EcoRI

Probe1

N T

_w -9_ 3

I

_ -6.3

_ _-4.5

. _ -3.7

Hirid= Eco RI

BlB 2

El E E E

Proto RET

3

4

E

EE E EE EEE EE

E E IE E

I,tI, 7 1- I

Proto Dl OS107 4Kb

FIG. 2. Southern blotanalysis of normal (peripheral blood cells) (lanes N) and tumor (lanes T) DNA ofpatients 2, 3, and 4 with papillary thyroid carcinomasdisplayinginv(10Xq1l.2q21).Theanalysiswasdone afterdigestion with the indicated restrictionenzymesandby usingasprobes: (i)a1-kb BglII(Bg)-BamHI (B) probe specific for the 6.3-kbp EcoRl (E) fragment of proto-RET(probe 2); (ii)a0.6-kbBamHl-AvaI(A)DIOS170fragment (probe 3) for patients 2 and 3, for patient 3 also aO.6-kbBamHl-EcoRIDJOS17O-specificsequence(probe 4); and (iii)a1.8-kbpBamHI-BamHIfragmentrelated to the 5' end ofproto-RET (probe 1). Size of normal and of rearranged (arrows) bands is givenaskbp.

3'-end DIOS]70 and 5'-end proto-RET-related probes (Fig. 2).

This bandrepresentsthe fusioneventreciprocaltothatgener-

ating the RET/PTC oncogenicsequence(see Fig. 1B).

DISCUSSION

Oncogene activation by chromosome rearrangements has been described insomehematopoietic malignancies (1, 2, 4).

Thecytogenetic and molecular analyses of the fourcasesof papillary thyroid carcinomas reported here provideanexample

of oncogene activation in a solid tumor due to an acquired chromosomalabnormality. This abnormality leadstoachimeric transforming transcript,RET/PTC,inanalogytotheproduction of the chimeric protein that follows the 9;22translocation in chronic granulocytic leukemia (4). Infact, RET/PTC isorigi- natedby the fusion oftwo unrelated sequences, aportion of proto-RET includingitstyrosine kinase domain andDJOS170, whichprovides the 5' endpromoterregion (11).

ThetransformingsequenceRET/PTC has beenidentified in -25% ofpatients withpapillary thyroid carcinomas (12, Patient 2

Patient4

BarniFHI

Proc. Natl. Acad. Sci. USA 89

(1992)

_m.0 _.-9. 8

-_a .4

ft. -12

--10-5

Proc.Natl. Acad. Sci. USA 89 (1992) 1619

H I I T N

z-

6.3- _

I

T N Z Z

12- - -- _

B

1.7-_ _

A

8.5- -

6.3- __

C

28S- ^{- f}

6ON4

^{" "'I}

_-28S

18S-

-18S

D

FIG. 3. Southern blot analysis of normal (lanesN)and tumor(lanes T) DNA of patient 1 and of NIH3T3-untransfected (lanes NIH) or transformed (lanesNIH-Ti)by the tumor DNA ofpatient 1. AfterEcoRIdigestion, the DNAs were hybridized to the molecularprobes described inFig. 2forpatient2: RETprobe (A),DIOS1 70 probe (B), and 5' end of proto-RET probe (C). The latter probe does not recognizespecific sequence either in control or in RET/PTC-transfected NIH 3T3 (NIH-Ti) because this region of proto-RET was truncated in the RET/PTC-transforming sequence formation. (D) Northern blot analysis of transcripts from a neuroblastoma cell line (lane SK-N-SH), representative of proto-RET mRNAs (10),NIH 3T3transformed by the DNA from patient 1 metastatic tissue (laneNIH-Ti)andfrom NIH-3T3 transformedby clonedand sequenced

RETIPTC

(lane NIH-T2) (11).

13). Recently, Ishizaka et al. (19) have cloned the same

hybrid sequence, generated by DJOS170 and RETfusion, Eco RI

N

_ I

C T z z

I- I-N

I I

C T z z

RET EcoRI

(371)

351 352

19

^-174 12-

213 ^A

^bp-

³⁰⁶345^{! bp 132Bbp=485 502}

FIG.4. PCRamplification of the fusioncDNAfragment containing theDOSJ170-RETbreakpoint region,asdeduced from thepartial map (Lower)derived from RET/PTC sequence(11). (Upper) The starting RNAs were from a control human melanoma cell line (lanes C), a

lymph-node metastatic specimen from patient 1 (lanes T), NIH 3T3 transformedby thetumorDNAofpatient1(lanesNIH-Ti),andNIH3T3 transformedby thesequencedRET/PTC (lanesNIH-T2).Bacteriophage 0X-174RFDNA-HaeIIIdigestswereusedasmolecular size markers.

froma papillary thyroidcarcinomacell line. Thesefindings make RET/PTC the most frequently detected oncogene in humansolidtumors, immediatelyfollowing the detection of oncogenes belongingtotheRASgenefamily.

A pathogenetic role of RET/PTC in papillary thyroid carcinomas issupportedby the observation of its activation in both primary andmetastatic tumorlesions and notin the normal DNA of the same patient (11-13). In addition, the normal thyroid epithelium does not express RET-related transcripts (11), andaninappropriateRETexpressiondriven by a mousemammary tumorvirus promoter/enhancer has been recently reported (20)to form mammary and salivary glandadenocarcinomas in transgenic mice.

The presentdata, reporting aparacentric inversion ofthe longarm ofchromosome 10,allow us topropose the model depicted in Fig. 1B for the generation ofthe RET/PTC- transformingsequence.Themolecularanalysisalsosupports themodel byidentifying therearrangementbands duetothe fusioneventreciprocaltothatgeneratingRET/PTC. Because we have determined the gene order on lOq as cen-RET- DJOSJ70-qter(15)and because the inversion resulted inthe generationof functional chimerictranscripts,we candeduce thatRETandDIOS]70arenormally transcribedonopposite DNA strands. In the model we have considered only the possibility of RET-DJOSJ70 divergent transcription. How- ever, our data cannot exclude the opposite possibility of convergent transcripts. The most relevant consequence of this model isthat every caseof RET/PTCformation intumor cells should involve a chromosomal inversion. It has been recently reported (21) that thegenes of theimmunoglobulin Klightchainsareassembledbysomaticrecombination that in someinstances canincludeamegabase-sized chromosomal inversion, demonstrating that in physiological conditions such an event can leadto afunctionalproduct.

We areindebtedtoDr.Silvana Pilotti foradvice and forsupplying thepathologic specimens. The assistance ofMr.MarioAzzini,Mrs.

CarlaCalderone, Mrs. AnnaGrassi,Ms.PieraMondellini,andMr.

Chris R. Shad is alsoacknowledged.This workwassupported bythe AssociazioneItalianaper la Ricerca sul Cancro andbytheProgetto FinalizzatoBiotecnologieeBiostrumentazione,ConsiglioNazionale delleRicerche.

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Proc.Natl. Acad. Sci. USA 89

(1992)