Proc. Nati. Acad. Sci. USAVol. 82, pp. 4448-4452, July 1985Genetics

Human urokinase gene is located on the long arm ofchromosome 10

(plasminogen activator/somatic cell hybrids/in situ hybridization)

PASQUALE TRIPPUTI*, FRANCESCO BLASIt, PASQUALE VERDEt, LINDA A. CANNIZZAROt§,BEVERLY S. EMANUEL0, AND CARLO M. CROCE**The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, PA 19104; tliternational Institute of Genftics and Biophysics, ConsiglioNazionale delle Ricerche, via Marconi 10, 80123 Naples, Italy; and tDepartment of Pathology and Laboratory Medicine and Department of Pediatrics,University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Communicated by DeWitt Stetten, Jr., January 17, 1985

ABSTRACT Urokinase is one of the two plasminogenactivators that catalyze the conversion of inactive plasminogento plasmin. By combining somatic cell genetics, in situ hybridi-zation, and Southern hybridization, we localized the humanurokinase gene on the distal third of the long arm (q24-qter) ofchromosome 10.

Plasminogen activators catalyze plasminogen-dependent fi-brinolysis, a key reaction controlling extracellularproteolysis (1). Two forms of plasminogen activator havebeen recognized: urokinase, isolated from human urine, andtissue plasminogen activator. Urokinase is a Mr 54,000two-chain protein (2) derived from a single-chain precursor(3-5). Tissue plasminogen activator is a single-chain proteinof Mr 65,000 (6). Both tissue plasminogen activator (7) andurokinase (8) have been cloned. Increased production ofplasminogen activator is associated with spontaneous humantumors (reviewed in ref. 9) and experimental tumors (10).Plasminogen activator production in malignant tumors isbelieved to be part of the biosynthetic program induced byhormones that stimulate growth in vivo (10). Increases inplasminogen activatormRNA are an early and direct effect ofhormones (11) and of the potent tumor promoter phorbolester (refs. 12 and 13; Stoppelli et al., unpublished results).These data are consistent with and may explain early findingsthat plasminogen activator production is an early oncogene-dependent effect of transformation with tumor viruses(14-17). Finally, recent data indicate that urokinase is re-quired for the metastatic activity of human tumors (18).Because of the possible role of the genes encoding humanplasminogen activator in tumor biology, it is important todetermine their chromosomal location. We have combinedsomatic genetics, in situ hybridization, and Southern blothybridization to localize the human urokinase gene on thedistal third of the long arm (q24-qter) of chromosome 10.

MATERIALS AND METHODS

Somatic Cell Hybrids. The cell lines and hybrid clones usedfor the mapping of urokinase sequences have been exten-sively described in previous studies (19, 20). The chromo-somal content of hybrids was determined by enzymaticanalysis using starch gel electrophoresis according to stan-dard methods (21) and by karyotypic analysis using a com-bination of trypsin Giemsa and G11 banding techniques asdescribed (22).DNA Extraction, Southern Blot Analysis, and Hybridiza-

tion. DNA from human, mouse, and hybrid cell lines wasprepared by cell lysis, proteinase K digestion, extraction with

phenol, and precipitation with ethanol as described (23).Fifteen micrograms of DNA were digested with 30 units ofthe appropriate restriction endonuclease in standard condi-tions recommended by the supplier (New England Biolabs).Fragments were separated by electrophoresis on a 1%agarose gel. DNA was denatured and transferred to nitrocel-lulose as described by Southern (24). DNA on nitrocellulosesheets was hybridized to 32P-labeled probe DNA at 370C in4x NaCl/Cit (lx NaCl/Cit = 0.15 M NaCl/0.015 M sodiumcitrate, pH 7) containing 50% (vol/vol) formamide as de-scribed (25). After hybridization, the filters were washedtwice with 2x NaCl/Cit at room temperature and twice inO.1x NaCl/Cit containing 0.2% sodium dodecyl sulfate at650C, air-dried, and exposed to Kodak XRA-5 film.

Preparation of Labeled DNA Probe. The urokinase probe isa 637-base-pair EcoRI fragment from human cDNA clonesidentified from a cDNA library prepared from total RNA ofhuman fibroblasts transformed by simian virus 40 (8). Thisfragment contains the sequences encoding amino acids162-302 of human prourokinase and includes a 221-base-pairintervening sequence (8). The DNA probes were labeled with32Pby nick-translation (26) to specific activities of0.5-2 x 108cpm/0.2 ,ug ofDNA. DNA polymerase I was purchased fromBoehringer Mannheim; [32P]NTP was from Amersham.Chromosome Preparation. Metaphase chromosome

spreads were prepared from peripheral blood lymphocytes ofa normal (46, XY) male donor by using standard techniques.Air-dried slides were kept in the cold (40C) for at least 1 weekprior to their use in mapping studies.

In Situ Hybridization. In situ hybridization studies wereperformed by using modifications of the protocols of Can-nizzaro and Emanuel (27) and Yunis et al. (28). Air-driedmetaphase chromosome preparations on glass slides wereused 1-3 weeks after preparation. Slides were treated withRNase to remove any chromosomally bound RNA. Theslides were washed free of RNase and then dehydratedthrough an alcohol series. Chromosomal DNA was denaturedby immersing the slides in 2x NaCl/Cit containing 70%formamide at 70'C, followed by rapid transfer through analcohol series for dehydration.Probe DNA was labeled with 3H by nick-translation to a

specific activity of 4 x 107 cpm/tag according to the protocolof Lai et al. (29), and the DNA was separated fromunincorporated labeled nucleotides by chromatography on aSephadex column. Carrier DNA, salmon sperm, was addedat a 100Ox excess and DNA was ethanol-precipitated andresuspended in hybridization mixture [2x NaCl/Cit contain-

Abbreviation: GOT, glutamic-oxaloacetic transaminase (aspartateaminotransferase, EC 2.6.1.1).§Present address: Department of Pediatrics, The Milton S. HersheyMedical Center, The Pennsylvania State University, Hershey, PA17033.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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ing 50% formamide and 10% dextran sulfate (pH 7.0)]. ProbeDNA was denatured for 5 min at 70'C and quickly chilled onice. Probe DNA was added to the slides at a concentration of0.05 gg/ml. The slides were coverslipped, put in moistchambers, and hybridized for 18 hr at 370C. After hybridiza-tion, the slides were extensively washed at 390C to removenonspecifically bound labeled DNA and were dehydratedthrough an alcohol series. Slides were dipped in liquidnuclear track emulsion (Kodak NTB-2), stored in the dark,and developed at appropriate intervals. Slides were bandedby using a modified Wright's Giemsa protocol (27) andanalyzed under the light microscope.Metaphase spreads with good chromosome morphology

and few background grains were selected. The location ofspecific grains was determined using an idiogram from Yuniset al. (28).

RESULTSMapping of the Human Urokinase Gene on Chromosome 10.

A 637-base-pair-long urokinase cDNA fragment was used asprobe in Southern blot experiments. Fig. 1 A and B shows theresults ofhybridization of the probe with the Sst I and HindIIIdigests, respectively, ofDNA from (i) human-mouse somaticcell hybrids containing different complements of humanchromosomes and (ii) from the parental human and mousecells. A single positive hybridization band was detected incontrol human DNA (Fig. 1 A and B, lane 1), but no bandswere detected in the mouse DNA (Fig. 1 A and B, lane 16).As expected on the basis of the restriction map and sequenceof genomic DNA clones (34), bands of -7 kb and 10 kb wereobtained with Sst I and HindIII, respectively. The DNA ofthree of the 14 human-mouse hybrids was positive forhybridization (Fig. 1, lanes 4, 7, and 8). Digestion withBamHI endonuclease gave a 10.3-kb positive band in thesesame hybrids (not shown). The histogram in Fig. 2 wasconstructed from the analysis of these and other hybrids. Thecomplement of human chromosomes of the different hybridswas established by isoenzyme and cytogenetic analyses. Fig.

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FIG. 1. Southern blot analysis of Sst I-digested DNA (A) andHindIII-digested DNA (B) from human-mouse somatic cell hybridscontaining different complements ofhuman chromosomes. DNA (20,ug) was digested, run on 1% agarose gels, transferred onto nitrocel-lulose, and hybridized with nick-translated 32P-labeled urokinaseprobe. Lanes: 1, human DNA; 2-15, hybrid DNA; 16, mouse DNA.

Human ChromosomesHybrids 1 2 3 4 5 6 7 8 9 10111213141516171819202122 x Human

urokinaseDSK1B2A5 02DSK1B2A5 C20_PT47 M3

NufiPAFxBaWbI C557-37-IFI M21MUM2S5771-C105 +077-Bl0CIO_ ____77-810 CU25 *77-BIO Ct26 * +77-BIOCt2 _____ _77-B10 C30 __ __77-Bl0 C31 * _ __* *+

GMxLM 013GMxLM 0_ __GMxLMICUC275 S ______57-77F7DC7 __D206 53ipi+

FIG. 2. Histogram ofhuman chromosome complement containedin urokinase-positive hybrids from Fig. 1 and additional hybrids.Chromosome content was determined by isoenzyme and cytogeneticanalysis.

2 shows that the urokinase plasminogen activator genesegregates concordantly with human chromosome 10.

Subregional Mapping of the Urokinase Gene. To map theprecise region of chromosome 10 containing the urokinasegene, we used somatic cell hybrids between mouse cells IT22and human fibroblasts carrying a translocation of the distalthird ofthe long arm ofchromosome 10 to chromosome 17 (10pter-- 10q24 :: 17 q13 -- 17 qter; 17 pter-* 17 q13 :: 10 q24-* 10 qter) (30). These hybrids have been used to assign thegene for cytoplasmic glutamic-oxaloacetic transaminase(GOT; aspartate aminotransferase, EC 2.6.1.1) to the regionq24 -- qter of human chromosome 10 (31). As shown in Fig.3, three of the hybrids were positive (10.3-kb band) When theBamHI digests were hybridized with the urokinase probe.Two positive and two negative hybrids were then analyzed bystarch gel electrophoresis for expression of human GOT (notshown). Because (i) all of these hybrids lack chromosome 10and (ih) the GOT-positive hybrids bear the translocatedportion of the long arm of chromosome 10 on chromosome17, expression ofboth GOT and urokinase would indicate thelocalization of the urokinase gene to the distal two-thirds ofthe long arm ofchromosome 10. In fact, the same hybrids thatwere positive for human GOT were also positive forurokinase (Table 1).

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FIG. 3. Southern blot analysis of Bamffl-digested DNA frommouse (lane 10), human (lane 1) and mouse-human hybrids (lanes2-9) hybridized with nick-translated 32P-labeled urokinase probe.Size is shown in kb.

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Table 1. Segregation of human GOT and human urokinase in hybrids with thet(10;17) translocation

Human chromosome

t(10;17)

lOpter- 10q24-Clone no. 10 17 q24/17pl3-pter qter/17qter-pl3 Human GOT Urokinase

54-68 F1clone 6 - + - + + +

11 - + + + +

15 - + - - - -

54-68 F2clone5 - +

In Situ Hybridization. To confirm the localization of theurokinase gene to chromosome 10, we carried out in situhybridization using an 3H-labeled plasmid containing theurokinase cDNA fragment as probe. Metaphase chromosomepreparations from peripheral blood cultures of a normal male

were denatured and hybridized with the nick-translatedurokinase probe. After autoradiography, the chromosomeswere stained by the G-banding technique (27), and metaphasespreads were analyzed for grain localization (Fig. 4 Upper).One hundred metaphases were analyzed, and 172 grains were

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FIG. 4. Localization of the urokinase gene by in situ hybridization. (Upper) Metaphase spreads of chromosomes from cells of a normal male

donor after staining by G-banding technique and hybridization with 3H-labeled, nick-translated urokinase probe. The arrow indicates grain onchromosome 10. (Lower) Histogram of human autosomal grain distribution, showing localization of the urokinase gene to q24-qter ofchromosome 10 (based on analysis of 100 metaphase spreads); 17% of all grains were localized on the long arm of chromosome 10, and 83%

of these were detected on 10q22 -k qter. Bold vertical lines on the x axis divide the p (left) and q (right) arms.

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Proc. Natl. Acad. Sci. USA 82 (1985) 4451

identified over chromosomes. Of these, 30 (17.4%) werelocated on the long arm of chromosome 10. Approximately83% of the 10q grains were in the distal two-thirds of 10q, inthe region 10q22 -* qter, with most grains at 10q22 -) q24(Fig. 4 Lower). No other region of similar length showedsignificant hybridization with the urokinase probe. The nexthighest represented regions were 6q16 -) 6q22 and 7q21 --

7q31, with 8 grains each (4.6% of total). These 8 grains do notrepresent a significant deviation from the number expected ifthe grains were randomly distributed. However, the long armof chromosome 10 represents =3% of the haploid autosomelength. Our finding that >17% of the urokinase probehybridization is localized to this region is statistically signifi-cant (P < 0.01) and suggests that the urokinase gene islocalized on the q22 -- qter region of chromosome 10.

DISCUSSIONIncreasing evidence points to a direct role of plasminogenactivators in the determination ofthe malignant phenotype (1,9, 10, 17, 18). In particular, the urokinase form of plasmin-ogen activator has been shown to be required for themetastatic activity of the human Hep3 cell line when intro-duced in the chorion allantoid chamber of the chickenembryo (18). The immunohistochemical analysis ofurokinase in the Lewis lung carcinoma of the rat has shownthat the enzyme is localized only in those regions ofthe tumorthat are in contact with the normal surrounding cells-i.e., inthe invading cells (32). For these reasons it becomes impor-tant to understand the mechanisms of regulation of theexpression of the urokinase gene and to analyze the mecha-nisms that explain its deregulation in few specific cells. Thechromosomal localization of the urokinase gene is, of course,an important step in this direction, as it will allow (i) thediscovery of chromosomal abnormalities (if any) involvingthe urokinase gene and (ii) studies of urokinase gene expres-sion in somatic cells hybrids.The entire urokinase gene now has been isolated and

sequenced (34). An extensive analysis has been carried out tomeasure the number of urokinase genes in the humangenome. No evidence has been obtained for the presence ofmore than one gene or of pseudogenes (34). In the studyreported here, three restriction enzymes, HindIII, Sst I (Fig.1), and BamHI (Fig. 3), were used to map the humanurokinase gene on chromosome 10. All bands observed withthese three enzymes also have been found on recombinantphages carrying genomic urokinase clones (34). This ex-cludes the possibility of cross-hybridizing sequences interfer-ing with the mapping results.

Analysis of somatic cell hybrids between mouse cells andhuman fibroblasts carrying a t(10;17) chromosomal transloca-tion indicated that the urokinase gene is located on the distalthird of the long arm of human chromosome 10. In situhybridization of human metaphase chromosomes with theurokinase probe confirmed this finding. The chromosomallocation of tissue plasminogen activator is not as yet known,nor is it clear whether the locus of this or of urokinase isrearranged or translocated in human neoplastic diseases.These questions assume importance in the light of thepossible role of plasminogen activators in malignant trans-formation or tumor progression or both.The results of the analyses of somatic cell hybrids pre-

sented here, indicating that the gene for human urokinase islocated on human chromosome 10, contrast with those ofKucherlapati et al. (33), who instead reported the localizationof this gene on chromosome 6. However, no hybridization ofour urokinase probe with the DNA of hybrids Nu 9 andD2C16S3, which contain chromosome 6, was observed. Hy-brid Nu 9 contains only human chromosomes 6 and 7 (Fig. 2),and chromosome 6 is present in >90% of the cells of this

hybrid. Furthermore, of all hybrids positive for urokinase,none contained chromosome 6. We cannot offer any definiteexplanation of the discrepancy between the conclusionsdrawn from our work and the results of Kucherlapati et al.(33). The results ofthese authors were based on a rather smalldifference of migration of active urokinase on an electropho-retic gel in which the human enzyme migrated somewhatslower than the mouse enzyme. We have observed slightlydifferent migrations (i.e., corresponding to apparent Mrs of48,000 vs. 52,000) in urokinases immunoprecipitated from themedium of different human cell lines, which we have ascribedto a different degree of either glycosylation or NH2-terminaldegradation by proteases present in the medium (unpublishedresults). These kind of anomalies may have affected themapping results of Kucherlapati et al. (33).

We thank Marina Hoffman for editorial assistance, Jean Letofskyfor enzyme analysis, and Josephine Romano for expert technicalassistance. This work was supported by Grant CA 16685 from theNational Cancer Institute. F.B. and P.V. were supported by a grantfrom Progetto Finalizzato Ingegneria Genetica and from ProgettoFinalizzato Oncologia of Consiglio Nazionale delle Ricerche (Italy).

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