DNA AND CELL BIOLOGYVolume 13, Number 6, 1994Mary Ann Liebert, Inc., PublishersPp. 571-584

ßig-h3: A Transforming Growth Factor-ß-Responsive GeneEncoding a Secreted Protein That Inhibits Cell Attachment

In Vitro and Suppresses the Growth ofCHO Cells in Nude Mice

JOHN SKONIER,1 KELLY BENNETT,1 VICTORIA ROTHWELL,1 STEVE KOSOWSKI,1GREG PLOWMAN,1 PHIL WALLACE,1 SUSANNE EDELHOFF,2

CHRISTINE DISTECHE,2 MIKE NEUBAUER,1 HANS MARQUARDT,1JULIE RODGERS,1 and A.F. PURCHIO3

ABSTRACT

/3ig-h3 is a novel gene first discovered by differential screening of a cDNA library made from A549 humanlung adenocarcinoma cells treated with transforming growth factor-/31 (TGF-/31). It encodes a 683-amino-acid protein containing a secretory signal sequence and four homologous internal domains. Here we showthat treatment of several types of cells, including human melanoma cells, human mammary epithelial cells,human keratinocytes, and human fibroblasts, with TGF-/3 resulted in a significant increase in /3ig-h3 RNA.A portion of the /3ig-h3 coding sequence was expressed in bacteria, and antisera against the bacterially pro-duced protein was raised in rabbits. This antisera was used to demonstrate that several cell lines secreted a68-kD 0IG-H3 protein after treatment with TGF-/3. Transfection of /3IG-H3 expression plasmids into Chi-nese hamster ovary (CHO) cells led to a marked decrease in the ability of these cells to form tumors in nudemice. The 0IG-H3 protein was purified from media conditioned by recombinant CHO cells, characterized byimmunoblotting and protein sequencing and shown to function in an anti-adhesion assay in that it inhibitedthe attachment of A549, Hela, and WI-38 cells to plastic in serum-free media. Sequencing of cDNA clonesencoding murine /3ig-H3 indicated 90.6% conservation at the amino acid level between the murine and hu-man proteins. Finally, the /3ig-h3 gene was localized to human chromosome 5q31, a region frequently de-leted in preleukemic myelodysplasia and leukemia. The corresponding mouse /3ig-h3 gene was mapped tomouse chromosome 13 region B to Cl, which confirms a region of conservation on human chromosome 5and mouse chromosome 13. We suggest that this protein be named p68ß'g-h3.

INTRODUCTION Hanks et al, 1988; Madisen et al, 1988), TGF-/33(Derynck et al, 1988; Jakowlew et al, 1988a; TenDijke et

Transforming growth FACTOR-/3 (TGF-/3) refers to a at, 1988), TGF-/34 (Jakowlew et al, 1988b), TGF-05growing family of related dimeric proteins that regu- (Kondaiah et al, 1990), and the more distantly related

late the growth and differentiation of many cell types (Bar- Mullerian inhibitory substance (Cate et al., 1986), the in-nard et al, 1990; Massague, 1990; Roberts and Sporn, hibins (Mason et al, 1985), the bone morphogenetic pro-1990). Members of this family include TGF-/31 (Moses et teins (Wozney et al, 1988), and OP-1 (Özkaynak et al,al, 1981; Roberts et al, 1981; Derynck et al, 1985; Shar- 1990). Newly discovered members include OP-2 (Özkaynakpies et al, 1987), TGF-/32 (DeMartin et al, 1987; Ikeda et et al, 1992), GDF-1 (Lee, 1990), GDF-3 and GDF-9 (Mc-al, 1987; Marquardt et al, 1987; Seyedin et al, 1987; Pherron and Lee, 1993), and Nodal (Zhou et al, 1993).

'Bristol-Myers Squibb, Pharmaceutical Research Institute Seattle, WA 98121.department of Pathology, University of Washington, Seattle, WA 98195.3Advanced Tissue Sciences, La Jolla, CA 92037-1005.

571

572 SKONIER ET AL.

TGF-/3 was first characterized for its effects on cell pro-liferation. It both stimulated the anchorage-independentgrowth of rat kidney fibroblasts (Roberts et al, 1981) andinhibited the growth of monkey kidney cells (Tucker et al.,1984). Since then, it has been shown to have many diversebiological effects: it stimulates bone formation (Noda andCamilliere, 1989; Joyce et al, 1990; Mackie and Trechsel,1990; Marcelli et al, 1990; Beck et al, 1991), induces ratmuscle cells to produce cartilage-specific macromolecules(Seyedin et al, 1986), inhibits the growth of early hemato-poietic progenitor cells (Goey et al, 1989), T cells (Kehrl etal, 1986), B cells (Kasid et al, 1988), mouse keratinocytes(Coffey et al, 1988; Pietenpol et al, 1990), and several hu-man cancer cell ines (Roberts et al, 1985; Ranchalis et al,1987). It increases the synthesis and secretion of collagenand fibronectin (Ignotz and Massagué, 1986; Centrella etal, 1987), accelerates healing of incisional wounds (Mus-toe et al, 1987), suppresses casein synthesis in mouse

mammary expiants (Robinson et al, 1993), inhibits DNAsynthesis and phosphorylation of pRb in rat liver epithelialcells (Whitson and Itakura, 1992), stimulates the produc-tion of basic fibroblast growth factor (bFGF) bindingproteoglycans (Nugent and Edelman, 1992), modulatesphosphorylation of the epidermal growth factor (EGF) re-

ceptor and proliferation of epidermoid carcinoma cells(Goldkorn and Mendelsohn, 1992) and can lead to apopto-sis in uterine epithelial cells (Rotello et al, 1991), culturedhepatocytes, and regressing liver (Oberhammer et al,1992). It can mediate cardioprotection against reperfusioninjury (Lefer et al, 1990) by inhibiting neutrophil adher-ence to endothelium (Lefer et al, 1993), and it protectsagainst experimental autoimmune diseases in mice (Kuru-villaer al, 1991).

We have been interested in identifying and characteriz-ing novel genes induced by TGF-/3 (Brunner et al, 1991;Skonier et al., 1992) with the goal of studying the potentialrole of their encoded proteins in mediating some of thebiological effects of TGF-/3. We recently described a novelgene, /3ig-h3, which was transcriptionally induced byTGF-/3 after long-term (72 hr) treatment of A549 cells.cDNA sequence analysis predicted that /3ig-h3 encodes a683-amino-acid secreted protein that contains four regionsof internal homology and a carboxy-terminal Arg-Gly-Aspmotif (RGD motif; Skonier et al, 1992). In this report, wefurther demonstrate that treatment of several cell typeswith TGF-/3 results in an increase in /3ig-h3 RNA and secre-tion of a 68-kD /3IG-H3 protein (p680'g-h3). We also showthat transfection of /3ig-h3 into CHO cells markedly re-duces their ability to form tumors in nude mice and thatpurified, recombinant p680ig-h3 (rpóS/3'«113) can function asan antiadhesion protein in vitro. We have isolated the mu-rine cDNA encoding /3ig-h3 and shows a high degree of se-

quence identity (90.6%) between human and mouse. Fi-nally, by in situ hybridization, we localized the /3ig-h3 geneto human chromosome 5q31, a site often deleted in leuke-mias and myelodysplastic syndromes (Van Den Berghe etal, 1974; Le Beau et al., 1986; for review, see Carter et al.,1992). The mapping of /3ig-h3 to mouse chromosome 13region B to Cl confirmed a region of conservation betweenhuman chromosome 5q and mouse chromosome 13.

MATERIALS AND METHODSCell culture

A549 (human lung adenocarcinoma), A375 (human mel-anoma), HeLa, WI-38 (human lung fibroblasts), andHEPM (human embryonic palatal mesenchymal) cells were

grown in Dulbecco modified Eagle medium (DMEM) con-

taining 10% fetal calf serum (FCS). Human mammary epi-thelial cells (HMEC) and normal human epithelial kera-tinocytes (NHEK) were purchased from Clonetics (SanDiego, CA) and propagated in media supplied by the dis-tributor. Cells (60% confluent) were treated with 20 ng/mlrecombinant TGF-/31 (Gentry et al, 1988) for 48 hr incomplete medium.

RNA extraction and Northern blot analysisTotal RNA was extracted as described (Chomczynski

and Sacchi, 1987) and 20 pg was fractionated on a 1% aga-rose-formaldehyde gel (Lehrach et al, 1977), transferredto a nylon membrane (Hybond N, Amersham) and hybrid-ized to 32P-labeled probe as described (Madisen et al,1988). Filters were stained with méthylène blue to ensure

equal loading and transfer.

Antisera productionA portion of the /3ig-h3 coding region (comprising

amino acids 210-683) was expressed in Escherichia coli as a

Cro-fusion protein, purified as an insoluble aggregate as

described (Purchio et al, 1987) and fractionated by so-dium dodecyl sulfate-polyacrylamide gel electrophoresis(NaDodSO„-PAGE) (Laemmli, 1970). Bands containingthe fusion protein were emulsified in complete Freund'sadjuvant and injected subcutaneously into New Zealandwhite rabbits (5 sites, 20 pg of protein per site). Rabbitswere boosted every 3-4 weeks with fusion protein in in-complete Freund's adjuvant. Antibody production wasmonitored by immunoblotting (Burnette, 1981) using /3ig-h3 proteins produced in COS cells (Skonier et al, 1992).

ImmunoprecipitationCells were starved for 30 min in DMEM minus methio-

nine and cysteine plus 5% dialyzed FBS, and then labeledin the above media containing 200 pCUml 35S-labeled pro-tein labeling mix (New England Nuclear) for 4 hr. Super-natants were spun for 10 min at 500 g and incubated for 1hr with preimmune or anti-p680ig-h3 antiserum for 1 hr at4°C. Immunocomplexes were collected on ProteinA-Sepharose, washed four times with HO buffer (0.01 MTris pH 7.1 0.5% deoxycholate, 1.0% NP40, 0.1 MNaCl),boiled for 5 min in sample buffer, fractionated byNaDodSO„-PAGE (Laemmli, 1970), fluorographed, andsubjected to autoradiography.

Production of recombinant ßIG-H3The glutamine synthetase expression system (Celltech,

Berkshire, UK) was used to express p680>g-h3 in CHO cells

/3ig-h3: TGF-/3-RESPONSIVE GENE 573

(Cockett et al, 1990; Bebbington et al, 1992). The /3ig-h3coding region was cloned into the expression vectorpEE-14, which contains a cytomegalovirus (CMV) pro-moter (Celltech, Berkshire, UK) and transfected into CHOcells using calcium phosphate precipitation as instructed(Celltech, Berkshire, UK). Transfectants were selectedusing 25 fiM methionine sulfoxide (MSX) and individualclones were picked and expanded. Clones secretingrp680's-h3 were identified by immunoblotting of condi-tioned serum-free media. Positive clones used in this studywere designated CHO/H3cl.A13, CHO/H3cl.A19, andCHO/H3cl.A2g. Control CHO cells transfected withempty vector (pEE-14) and propagated in 25 ftM MSX asmass cultures or individual clones (identical results wereobtained with both) are designated as control CHO/-EE-14 cells.

Protein purification and sequencingSerum-free media conditioned by CHO/H3cl.A13 was

precipitated at 4CC for 20 hr with 50% ammonium sulfateand spun for 30 min at 30,000 x g. The pellet was dis-solved in phosphate-buffered saline (PBS) pH 7.5 andfractionated on a BioSil TSK-250 gel filtration column(BioRad, Richmond, CA) equilibrated in PBS. Fractionscontaining p680'g-h3 protein were identified by immuno-blotting, pooled, aliquoted, and stored at -70°C.

Proteins were recovered from NaDodS04-PAGE byelectroblotting onto a ProBlott membrane (Applied Bio-systems, Foster City, CA) using a mini-transblot electro-phoretic transfer cell (Bio-Rad Laboratories), as previouslydescribed (Matsudaira, 1987). The membrane was stainedwith Coomassie Brilliant blue, then destained and the68-kD band was excised for subsequent amino-terminal se-

quence analysis.Samples were sequenced in a pulsed-liquid phase protein

sequencer (model 476A, Applied Biosystems), equippedwith a vertical cross-flow reaction cartridge. The phenyl-thiohydantoin (pth) amino acid derivatives were analyzedby reversed-phase high-pressure Chromatograph (HPLC)(Marquardt et al, 1987). Data reduction and quantitationwere performed on a Macintosh Ilsi computer (AppleComputers, Inc.) and model 610A chromatogram analysissoftware (Applied Biosystems).

Isolation of mouse ßig-h3 sequencescDNA libraries were made from G8 (Hung et al, 1986)

cells and NIH-3T3 cells following the manufacturer's pro-tocol in the Stratagene ZAP cDNA Synthesis Kit. The G8library was screened with the human /3ig-h3 cDNA(Skonier et al, 1992) and one hybridizing clone was iso-lated. This clone contained an 800-bp insert consisting ofsequences homologous to the 3' untranslated region of/3ig-h3 and 240 bp of carboxy-terminal coding region. Thisfragment was used to screen the NIH-3T3 library andseven hybridizing clones were isolated. One clone (mh3-913) contained sequences homologous to the entire /3ig-h3coding region and both 5' and 3' untranslated regions (ac-cession number LI9932).

Adhesion assayAdhesion assays similar to those described to identify

proteins and their active domains involved in cell attach-ment were carried out on rp68#g-h3 (Ruoslahti et al, 1982;Graf et al, 1987; Dustin and Springer, 1989). Media con-

taining rp680'g-h3 was added to Costar six-well plates for 2hr at room temperature. The quantity of rp680'g"h3 addedis stated in the Results section for each appropriate experi-ment. A549, HeLa, and WI-38 cells were trypsinized,counted, and plated at 2 x 105 cells per well and thenplaced in an incubator with 5% C02, 37°C for 2 hr. Thewells were then washed two times with PBS. The cells re-

maining attached to the plate were trypsinized and countedin a Neubauer chamber.

Tumorigenesis in nude miceFemale nu/nu mice (6-8 weeks) were obtained from

Harlan Sprague-Dawley (Indianapolis, IN) and maintainedunder specific pathogen-free conditions. Mice were in-jected subcutaneously in the flank with either independentCHO/H3 clones or control CH3pEE-14 cells (3 x 107cells/mouse). Tumors were evaluated weekly and thetumor volumes calculated.

Chromosomal locationIn situ hybridization: The probe used for the in situ hy-

bridization mapping of the /3ig-h3 gene was a 6.5-kb sub-fragment of a human genomic clone of /3ig-h3. In situ hy-bridization to metaphase chromosomes from lymphocytesof a normal male donor was carried out using the probe la-beled with 3H to a specific activity of 3.5 x 107 cpm//tg asdescribed (Marth et al, 1986). The final probe concentra-tion was 0.04 fig/fd of hybridization mixture. The slideswere exposed for 2-3 weeks. Chromosomes were identifiedby Q banding.

The hybridization was repeated using fluorescence insitu hybridization (FISH). A cosmid clone correspondingto the human /3ig-h3 gene was labeled with biotin-14-dATPby nick translation (GIBCO BRL). The hybridization wascarried out following a modified protocol of Moyzis et al,1987). The hybridization signals were detected using a de-tection system from Vector. For the mapping of the mouse

/3ig-h3 gene, a cDNA probe containing an ~3-kb insert(pBSmh39.1) was labeled with biotin-11-dUTP by nicktranslation (GIBCO BRL). Mouse metaphase chromo-somes from spleen lymphocytes of a male C57BL/6Jmouse were obtained. A slightly modified protocol ofLemieux et al (1992) was followed for the hybridization.The hybridization signals were detected using a detectionsystem from Vector. Both human and mouse chromo-somes were banded using Hoechst 33258-actinomycin Dstaining and counterstained with propidium iodine. Thechromosomes and hybridization signals were visualized byfluorescence microscopy, using a dual band-pass filter(Omega or Chroma).

574 SKONIER ET AL.

RESULTSInduction of ßig-h3 RNA and protein by TGF-ß

We have previously shown that treatment of A549,H2981 (human lung adenocarcinoma), and HEPM cellswith TGF-/31 led to an increase in /3ig-h3 RNA synthesis,which was maximal at 48 hr (Skonier et al, 1992). Here weexamine /3ig-h3 induction in two addition established celllines and two primary cell lines. Cells were treated at 60%confluence for 48 hr with 20 ng/ml recombinant TGF-/31:RNA was extracted and hybridized to radiolabeled /3ig-h3probe. Figure 1 shows that treatment of HMEC (humanmammary epithelial cells), NHEK (normal human epithel-ial keratinocytes) cells, WI-38 (human lung fibroblasts),and A375 (human melanoma) cells with TGF-/3 resulted ina marked increase /3ig-h3 RNA. Oncostatin M, a growthregulator produced by differentiated histiocytic lymphomacells also inhibits the growth of A375 cells (Zarling et al,1986); however, treatment of A375 cells with oncostatin Mdid not lead to an increase in /3ig-h3 RNA (Fig. ID, lane3), suggesting that increased transcription of /3ig-h3 is nota generalized result of growth arrest.

Sequence analysis of cDNA clones predicts that /3ig-h3encodes a protein of 683 amino acids (p680>g-h3) which con-tains a secretory signal sequence (Skonier et al, 1992). Toexamine whether TGF-/31 treatment led to the secretion ofthe protein, we attempted to immunoprecipitate from con-ditioned media using antisera made against a bacteriallyexpressed protein. Four different cell lines were treatedwith TGF-/31 for 48 hr and then labeled with 35S-labeledprotein labeling mix; cell-free supernatants were immuno-precipitated and fractionated by NaDodS04-PAGE fol-lowed by autoradiography. Figure 2 shows that TGF-/31treatment of A375, A549, WI-38, and HEPM cells led tothe secretion of an approximately 68-kD (p68#g-h3) proteinimmunoprecipitated by the anti-p680¡g-h3 antisera (Fig. 2,

B

A375 A549

I -28S -28S

-18S

-28S

-18S

FIG. 1. Northern blot analysis of /3ig-h3 transcripts invarious cells after treatment with TGF-/3. Human mam-

mary epithelial cells (A), human keratinocytes (B), WI-38cells (human foreskin fibroblasts) (C), and A375 cells (hu-man melanoma cells) (D) were grown without (lane 1) orwith (lane 2) TGF-/3 (20 ng/ml) for 48 hr. Total RNA wasisolated and 20 pg was fractionated on an agarose-formal-dehyde gel, transferred to nylon filters, and hybridized to"P-labeled /3ig-h3 probe. Filters were washed and exposedfor autoradiography as described in Materials and Meth-ods. Lane 3 of panel D contains RNA from A375 cellstreated for 48 hr with oncostatin M (10 ng/ml).

X I

C/) M if) Md_ <o. d_ «az a z a

oc CQ.z a

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en mce en.z a

TGF-/3-

200-

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43-

29-3 4

WI-38

12 3 4

HEPMX

M C/lCQ. £E

TGF-/3-200-

97-68-

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FIG. 2. Immunoprecipitation of p680'g-h3 from mediacondition by various cell lines. A375 (human melanoma),A549 (human lung adenocarcinoma), WI-38 (human fore-skin fibroblast), and HEPM (human embryonic palatalmesenchymal) cells were left untreated (lanes 1 and 2) ortreated for 48 hr with TGF-/3 (lanes 3 and 4) and labeledfor 4 hr with 35S-labeled protein labeling mix. Conditionedmedia was collected and immunoprecipitated with preim-mune (NRS, lanes 1 and 3) or anti-p680'g-h3 (ap68ß''zh},lanes 2 and 4) antiserum. Samples were fractionated by re-

ducing NaDodSO„-PAGE (10% acrylamide), fluoro-graphed, and exposed for autoradiography.lane 4) but not by preimmune sera (Fig. 2, lane 3). Littlep680'g-h3 protein was detectable in these cells prior to TGF-/31 treatment (Fig. 2, lanes 1 and 2).

A549, A375, H2981, NHEK, and HMEC cells are

growth arrested by TGF-/3: WI-38 and HEPM cells are notinhibited by TGF-/3 and, in fact, are moderately growthstimulated (data not shown). Therefore, increased tran-scription of /3ig-h3 and secretion of p68/3IG-H3 protein isobserved in cells after TGF-/31 treatment regardless ofwhether TGF-/31 stimulates or arrests the growth of thecells.

Expression of ßig-h3 in CHO cells leads todecreased tumorgenicity in nude mice

To obtain sufficient amounts of p68#g-h3 protein for invitro analysis, we expressed the cDNA in CHO cells usingthe glutamine synthetase expression and amplification sys-tem (Celltech, Berkshire, UK) as described in Material andMethods. Immunoblot analysis of serum-free media condi-tioned by one of these clones (CHO/H3cl.A13) is shownin Fig. 3. When immunoblotting was performed after frac-

(3ig-h3: TGF-/3-RESPONSIVE GENE 575

B97-

68-

43-

29-1 1

FIG. 3. Analysis of recombinant p6S0's-h3 protein secreted by CHO cells. CHO cells were transfected with p680'g-h3 ex-

pression plasmids and clone CHO/H3cl.A13 was isolated as described in Materials and Methods. A. Media condi-tioned by CHO/H3cl.A13 cells (lanes 1 and 2) or control CHO/p33-14 cells (lanes 3 and 4) was fractionated by nonre-ducing NaDodS04-PAGE (10% acrylamide) and proteins were immunoblotted using anti-p680'g-h3 antiserum (lanes 2and 4) or preimmune serum (lanes 1 and 3). B. CHO/H3cl.A13 conditioned media was fractionated by reducingNaDodS04-PAGE (10% acrylamide) and proteins were immunoblotted using preimmune (lane 1) or anti-p680'g-h3 (lane2) antiserum. C. Control CHO-p33-14 (lane 1) or CHO/H3cl.A13 cells (lane 2) were labeled for 4 hr with "S-labeledprotein labeling mix in serum-free media and 20 ¡d of conditioned media was fractionated by nonreducing NaDodS04-PAGE (10% acrylamide). The gel was fluorographed and exposed for autoradiography. D. Media conditioned byCHO/H3cl.A13 cells was digested with neuraminidase (lane 1) or buffer only (lane 2), fractionated by reducingNaDodSO„-PAGE (7.5% acrylamide) and immunoblotted using anti-p680'g-h3 antiserum.

donation by NaDodS04-PAGE under nonreducing condi-tions, CHO/H3cl.A13 cells were found to secrete a majorimmunoreactive protein migrating with a molecular weightof about 68 kD (Fig. 3A, lane 2) that was not detected bypreimmune serum (Fig. 3A, lane 1). It was also not de-tected in CHO cells transfected with empty vector (Fig.3A, lanes 3 and 4). Analysis of total 35S-labeled proteinssecreted by CHO/H3cl.A13 cells (Fig. 3C, lane 2) showsthat rp680'g-h3 is one of the major proteins secreted; it isnot detected in control CHO cells (Fig. 3C, lane 1).

When immunoblotting is performed after fractionationby reducing NaDodS04-PAGE, three closely spacedrp68#g-h3-specific bands can be observed migrating at 68kD (Fig. 3B, lane 2) that are not detected in control CHOcells (Fig. 3B, lane 1). There are no predicted sites of TV-linked glycosylation in the /3ig-h3 cDNA sequence (Skonieret al, 1992) and rp680'g-h3 could not be labeled with [3H]-glucosamine, [3H]mannose, or ["Pjorthophosphate (datanot shown). Digestion with neuraminidase did not alter themigration of the rp68#g-h3-specific bands (Fig. 3D: notethat in this panel proteins were fractionated on a 7.5%polyacrylamide gel to maximize separation of the bands).In addition, one major amino-terminal sequence was ob-tained from purified recombinant p680'g-h3 (see below).Potentially, the heterogeneity could be due to carboxy-ter-minal processing, sulfation, or methylation.

Figure 4 shows the morphology and growth rate of the/3ig-h3-transfected CHO cells. A striking change in themorphology of the p68iJ'g-h3-expressing CHO cells was ob-served: whereas the control CHO/pEE-14 cells maintainedan elongated, spindle shape and unstructured packing (Fig.4A), CHO cells expressing rp68i*'g-h3 had a more cuboidalshape and organized packing (Fig. 4B). The growth rate oftwo independently isolated clones (CHO/H3cl.A13 and

CHO/H3cl.A2g) is shown in Fig. 4C; their growth was

slower than the control CHO/pEE-14 cells and theyreached a lower saturation density.

The tumorgenicity of the rp68#g-h3-expressing CHOcells was also investigated. Three independently selectedclones were injected subcutaneously into the backs of nudemice (3 x 107 cells per injection) and tumors were evalu-ated at 4 weeks. Table 1 shows that while the controlCHO/pEE-14 cells readily formed tumors, the p68#g-h3 ex-

pressing cells were greatly impaired in their ability to growin nude mice. The single tumor that grew from the CHO/H3cl.A13 cells remained small (3x3 mm) over 10 weeksof observation whereas the control CHO/pEE-14 cells usu-

ally produced tumor measuring 15 x 20 mm by 4 weeks.

Purification and sequencing of recombinantp68B'g-h3: Demonstration of antiadhesion activity

rp680ig-h3 was purified from serum-free media condi-tioned by CHO/H3cl.A13 cells by ammonium sulfate pre-cipitation and size chromatography on a TSK column asdescribed in Materials and Methods. rp680'g-h3 was moni-tored by immunoblotting and peak fractions were pooled.A sample of the purified protein was fractionated byNaDodS04-PAGE and stained with Coomassie blue (Fig.5, lane 1) or analyzed by immunoblotting (Fig. 5, lane 2): a

major 68-kD band is detected with minor bands migratingjust above and below the 68-kD band. The 68-kD bandwas subjected to amino-terminal sequence analysis afterelectroblotting from polyacrylamide gels. Automated Ed-man degradation of the 68-kD band was performed with27 pmoles. Unambiguous identification of PTH-deriva-tives of amino acids was possible up to residue 21. Theamino-terminal sequence of the 68-kD protein was identi-

576 SKONIER ET AL.

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144 192

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FIG. 4. Growth curve for p68#g-h3-producing CHO cellclones. A. Photomicrograph of control CHO/pEE-14cells (lOOx). B. Photomicrograph of CHO/H3cl.A13cells (100x). C. Control CHO/pEE-14, CHO/H3cl.A-13 (A13 in figure), and CHO/H3cl.A2 (A2 in figure) cellswere seeded at 5 x 105 cells in 100-mm dishes, grown forthe indicated times, trypsinized, and counted. Data pointsare the average of duplicate samples.

Table 1. Expression of p680'g-h3 Decreases theTUMORGENICITY OF CHO CELLS IN NUDE MlCE

Number of tumorsNumber of animals injected

Control CHO/pEE-14CHO/H3cl.A19Control CHO/pEE-14CHO/H3cl.A13Control CHO/pEE-14CHO/H3cl.A2g

8/100/108/101/107/100/10

Control CHO/pEE-14, clone A13, clone A19, and clone A2gcells were injected subcutaneously into nude mice (107 cells per in-jection). Tumors were evaluated at 4 weeks post injection.

NH2

p68/3¡q-h31 r Repeat 1 T Repeat 2 I Repeat 3 Repeat 4J-1-1-—-1-1-j—C00H23 139 274 411 537 642 683 a.a.number

RGD

B Coomassie Western200-

97-

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FIG. 5. Characterization of purified, recombinantp680'g-h3. A. Line diagram of p6$0'z-hi. The arrow showsthe signal sequence cleavage site at amino acid residue 23.The four internal repeats and the RGD motif at residue642 are indicated. B. p680ig-h3 was purified from mediaconditioned by CHO/H3cl.A13 cells as described in Mate-rials and Methods and a sample was fractionated by reduc-ing NaDodS04-PAGE. The gel was either stained withCoomassie blue (lane 1) or immunoblotted using anti-p68#'g-h3 antiserum (lane 2).

cal to the sequence predicted from /3ig-h3 cDNA (Table 2).The purified r/3ig-h3 was tested in an adhesion assay be-

cause it is a secreted protein with four regions of internalhomology and an RGD sequence, which are common fea-tures of proteins that modulate cell attachment (Piersch-bacher and Ruoslahti, 1986; Ruoslahti and Pierschbacher,1987; Elkins, 1990; Chang et al, 1993). rp680'g-h3 was

found to prevent the attachment of A549 cells to plastic.Individual wells of a 24-well tissue culture dish were incu-bated for 2 hr at 22°C with 7.5 pg of rp680'g-h3, BSA, or

serum-free medium from control CHO/pEE-14 cells puri-fied in a similar fashion as rp68#g-h3. A549 cells were thenadded in serum-free medium and allowed to attach for 2 hrat 37°C. The supernatants were removed and wells were

washed twice with PBS and photographed. Figure 6Ashows that in the presence of rp680>g-h3, the cells did notattach to the plate, but did attach when plated in the pres-ence of equivalent amounts of BSA or control CHO/pEE14 protein. Two-fold dilutions of p680'g-h3 demon-strated that the effect is concentration-dependent (Fig.6B). When A549 cells were plated in the presence of 7.5 pgof poSß'g-113, only 181 cells remained attached to the plate;with 1.8750 /.g of protein, 2,537 cells were adhered. In con-

trast, when A549 cells were plated in the presence of 7.5 pgof BSA, 15,481 cells remained on the plate. Similar resultswere obtained with HeLa cells, WI-38 cells, and CHO cells(data not shown). The antiadhesion activity is not a resultof cellular toxicity; rp680'g-h3, when tested in a number ofdifferent growth assays, never induced a growth inhibitoryfunction.

/3ig-h3: TGF-/MŒSPONSIVE GENE 577

Table 2. Verification of Predictedrp68<3'g-h3 Sequence

Position Residue Yield (pmoles)24 Gly 15.225 Pro 17.626 Ala 17.827 Lys 18.428 Ser 1.829 Pro 13.730 Tyr 16.031 Gin 11.432 Leu 11.233 Val 13.434 Leu 14.835 Gin 10.836 His 7.937 Ser 1.038 Arg 6.439 Leu 8.540 Arg 6.041 Gly 1.942 Arg 3.443 Gin 3.444 His 2.0

Chromosomal location of ßig-h3 to 5q31 in human

The /3ig-h3 gene was mapped on human metaphase chro-mosomes by in situ hybridization using a genomic probe. Atotal of 46 metaphase cells with autoradiographic grainswere examined. Of 98 sites of hybridization scored, 37(38%) were located on the distal portion of the long arm ofchromosome 5. The largest number of grains (31 grains) was

at bands q31. There was no significant hybridization on

other human chromosomes. This mapping was confirmedby using a cosmid clone for FISH. Of 50 human metaphasecells, all 50 (100%) showed signals on both chromatids of

B100,000

/3IG-H3 Adhesion Assay /3IG-H3 Control

0.937 1.875 3.75

Mg ¿3IG-H3FIG. 6. p680ig-h3 adhesion assay. A. PGS (300 ¡d) con-

taining 7.5 fig of purified rp680'g-h3 (left panel), 7.5 fig ofBSA (middle panel), or control pEE-14 media (right panel)was added to one well of a 24-well tissue culture dish andincubated for 2 hr at room temperature. At that time, 5 x105 A549 cells were added to each well and incubated at37°C for 2 hr in a tissue culture incubator. Wells werewashed with PBS and photographed. B. Dose-responsecurve for rp68#'g-h3. Assays were performed as described inA using A549 cells.

one or both chromosomes 5 at band q31 (Fig. 7A). Therewas a second site of hybridization in a small number of cells(5%) on chromosome 3 at band p21.

b)

131il i2b

11.21|

13|II

22 P231

33 r

35

>O50

Î13

FIG. 7. Assignment of /3IG-H3 to human chromosome 5 band q31 and of ßig-h3 to mouse chromosome 13 distal regionB or proximal Cl, as determined by FISH. A. Distribution of 50 signals of hybridization on a diagram of human chro-mosome 5. B. Distribution of 9 signals of hybridization on a diagram of mouse chromosome 13.

578 SKONIER ET AL.

Isolation, expression, and chromosomal location ofmouse homolog of ßig-h3

cDNA libraries were made from G8 (Hung et al, 1986)NIH-3T3 cells. The G8 library was first screened with thehuman /3ig-h3 cDNA and a hybridizing clone was isolated.This fragment had 90% sequence identity to the human 3'untranslated region and 248 bp of coding sequences. Thisclone was used to screen another cDNA library made forNIH-3T3 cells. The full-length clone (mh3-913) was iso-lated from this library. Comparison of the nucleotide se-

quences of mouse /3ig-h3 and human shows a high degreeof sequence identity both at the nucleotide and amino acidlevels (86% and 90.6%, respectively; Fig. 8). Northernblots of RNA from several mouse tissues were probed withthe mh3-913 insert to look more closely at the expressionof mouse /3ig-h3. Figure 9 shows that mouse /3ig-h3 is ex-

pressed in a variety of tissues. The highest level of expres-sion was in uterus; lower levels of expression were found inskeletal muscle, testes, thyroid, kidney, liver, heart, andstomach. Expression was also seen in mouse embryonic tis-sue starting at day 12. Expression was not found in brainor cerebellum. This is in agreement with a previous studyon /3ig-h3 expression in human tissue that also did not find/3ig-h3 message in human cerebellum. Mapping of the /3ig-h3 gene in mouse was done by FISH using a cDNA probe.Of 50 mouse metaphase cells, 9 (18%) showed signals on

one or both chromosomes 13 at distal band B or proximalband Cl (Fig. 7B). There was no significant hybridizationto other chromosomes.

DISCUSSION

The ability of TGF-/3 to regulate gene expression probablyplays a major role in contributing to the pleiotropic effectsof this protein, and it has been suggested that suppressionof c-myc expression plays a role in growth inhibition ofkeratinocytes by TGF-/3 (Pietenpol et al, 1990). We havebeen interested in gene regulation by TGF-/3 and have useddifferential hybridization to identify novel genes inducedby this growth factor (Brunner et al, 1991; Skonier et al,1992). Identifying and characterizing these genes and theirprotein products might shed light onto the mechanisms ofaction of TGF-/3. This approach has been successfully usedby Kallin et al to identify a growth arrest gene termed Til,a membrane protein related to a family of glycoproteinsexpressed in lymphocytes and tumor cells. Til mRNA wasinduced by TGF-/3 in proliferating CCL-64 cells and down-regulated in quiescent cells (Kallin et al, 1991).

We have previously identified a gene, /3ig-h3, which wasinduced approximately 10- to 20-fold in A549 cells aftergrowth arrest by TGF-/3: maximal induction was at 48 hr(Skonier et al, 1992). /3ig-h3 is predicted to encode a se-

creted protein of 683 amino acids containing four regionsof internal homology and a Arg-Gly-Asp (RGD) sequencenear the carboxyl terminus of the fourth repeat. In this re-

port, we show that /3ig-h3 RNA is induced in two addi-

tional cell lines (WI-38 and A375) and two primary celltypes (HMEC and NHEK) by TGF-/3. Using an antiseramade against a bacterially produced protein, we demon-strate that TGF-/3 treatment of several cell types results inthe increased secretion of an approximately 68-kD protein,p680ig-h3, which is not detected by preimmune sera (Fig. 2).A comparison of the murine and human p680'g-h3 aminoacid sequences reveals over 90% identity between the hu-man and murine sequences. This level of conservation,roughly 1.2 amino acid substitutions per amino acid siteper 10' years, is similar to hemoglobin chains and suggestsrigid structural and functional constrains are operatingupon the rate of amino acid substitution (Nei, 1987).

To begin to identify and characterize a biological ac-

tivity for p680'g-h3, we expressed the gene in CHO cells.Several independent clones expressing rp680>g-h3 were ob-tained, and we observed a marked change in the morphol-ogy of these cells. When compared to the parental CHOcells, the rp680'g-h3-expressing cells assumed a much more

cuboidal shape, grew slower, and reached a lower satura-tion density (Fig. 4). When injected into nude mice, theCHO clones expressing rp68ö'gh3 had a dramatically re-

duced ability to form tumors, suggesting that p680'g-h3 maybe involved in mediating negative growth signals. Otherproteins such as a5/31 fibronectin receptor have also beenshown to suppress the transformed phenotype of CHOcells (Giancotti and Ruoslahti, 1990). We have attemptedto transfect and express /3ig-h3 in various human tumorcell lines (A375, A549) using several promoters: we haveanalyzed over 50 individual neomycin-resistant coloniesand have not found any that express the gene. We are con-

tinuing in these efforts and are including some of the in-ducible promoters (Labow et al, 1990) in our constructs.

rp680'g-h3 was purified from media conditioned by re-combinant CHO cells and its identity was confirmed byprotein sequencing (Table 2). Since p68/3'g-h3 is a secretedprotein with four regions of internal homology and a RGDmotif, which are characteristics of proteins known to mod-ulate cell attachment, an adhesion assay was performed.The purified protein was shown to block the adhesion ofseveral cell types to plastic tissue culture dishes (Fig. 6). Inlight of this activity, we were surprised to find by carboxy-terminal sequencing of the recombinant /3ig-h3 protein thatthe RGD sequence was not present; this is most likely a re-

sult of carboxy-terminal processing. Therefore, the antiad-hesion activity is not mediated through the RGD sequence.However, it is of interest to note that a motif reminiscentof an active binding domain of laminin (PDSGR) is foundnear the end of the second region of homology of p680'g-h3(PDSAK). In addition, the Drosophila fasciclin I protein,which also has four regions of internal homology, has beenshown to mediate homophilic aggregation (Elkins et al,1990). Experiments are in progress to identify the requireddomains for the antiadhesion activity.

In this report, we show that a gene encoding a secretedprotein, póS^íg-h3 can result in decreased tumorgenicitywhen transfected in CHO cells; yet the purified protein didnot show any growth inhibitory activity (data not shown).While most of the well-characterized anti-oncogenes (p53,pRb, Wilms tumor suppressor gene) are thought to be

/3ig-h3: TGF-/3-RESPONSIVE GENE 579

AGCCACGAGCCTGCTTTCATCGTGCGTCCGCGCGTGCTCCAGCTCC_2J¡_i

-'

, Gly-

95Lys Ser Thr Val He Ser Tyr Glu Cys Cys Pro Gly Tyr Glu Lys Val Pro Gly Glu Lys Gly Cys Pro Ala AlaAAG TCG ACÁ GTC ATC AGT TAT CAG TGC TGT CCT GGA TAT GAA AAG GTC CCA GGA GAG AAA GGT TGC CCA GCA GCT 300

110 120Leu Pro Leu Ser Asn Leu Tyr Glu Thr Met Gly Val Val Gly Ser Thr Thr Thr Gln Leu Tyr Thr Asp Arg ThrCTT CCG CTC TCA AAT CTG TAT GAC ACC ATG GGA GTT CTG GGA TCG ACC ACC ACÁ CAG CTG TAT ACÁ GAC CGC ACÁ 375

13S r repeat 1 145Glu Lys Leu Arg Pro Glu Mec Glu Gly Pro Gly Ser Phe Thr He Phe Ala Pro Ser Asn Glu Ala Trp Ser SerGAA AAG CTG AGG CCT GAG ATG GAG GGA CCC GGA AGC TTC ACC ATC TTT GCT CCT AGC AAT GAG GCC TGC TCT TCC 4S0

160 L 170Leu Pro Ala Glu Val Leu Asp Ser Leu Val Ser Asn Val Asn He Glu Leu Leu Asn Ala Leu Arg Tyr His MetTTG CCT GCG GAA GTC CTG GAC TCC CTG GTC AGC AAC GTC AAC ATC GAA CTG CTC AAT GCT CTC CGC TAC CAC ATG 525

18$ 195Val Asp Arg Arg Val Leu Thr Asp Glu Leu Lys His Gly Met Thr Leu Thr Ser Met Tyr Gin Asn Ser Asn IleGTC GAC AGG CGG GTC CTG ACC CAT GAG CTC AAC CAC GGC ATG ACC CTC ACC TCC ATG TAC CAG AAT TCC AAC ATC 600

210 220Gin Ile His His Tyr Pro Asn Gly Ile Val Thr Val Asn Cys Ala Arg Leu Leu Lys Ala Asp His His Ala ThrCAG ATC CAT CAC TAT CCC AAT GGG ATT GTA ACT GTT AAC TGT GCC CGG CTG CTG AAG GCT GAC CAC CAT GCG ACC 675

23$ 245Asn Gly Val Val His Leu He Asp Lys Val He Ser Thr He Thr Asn Asn He Gin Gin He Ile Glu He GluAAC GGC GTG GTG CAT CTC ATT GAC AAG GTC ATT TCC ACC ATC ACC AAC AAC ATC CAC CAG ATC ATT GAA ATC GAG 750

260 270Asp Thr Phe Glu Thr Leu Arg Ala Ala Val Ala Ala Ser Gly Leu Asn Thr Val Leu Glu Gly Asp Gly Gin PheGAC ACC TTT GAG ACA CTT CGG GCC GCC GTG GCT GCA TCA GGA CTC AAT ACC GTG CTG GAG GGC GAC GGC CAG TTC 825

[repeat 2 28$ 295Thr Leu Leu Ala Pro Thr Asn Clu Ala Phe Glu Lys He Pro Ala Glu Thr Leu Asn Arg He Leu Gly Asp ProACA CTC TTG GCC CCA ACC AAC CAG GCC TTT GAG AAG ATC CCT GCC GAG ACC TTG AAC CGC ATC CTG GGT GAC CCA 900

310 320Glu Ala Leu Arg Asp Leu Leu Asn Asn His He Leu Lys Ser Ala Met Cys Ala Glu Ala He Val Ala Gly MetGAG GCA CTG AGA GAC CTG CTA AAC AAC CAC ATC CTG AAG TCA GCC ATG TGT GCT GAG GCC ATT GTA GCT GGA ATG 975

33$ 345Ser Met Glu Thr Leu Gly Gly Thr Thr Leu Glu Val Gly Cys Ser Gly Asp Lys Leu Thr He Asn Gly Lys AlaTCC ATG GAG ACC CTG GGG GGC ACC ACA CTG GAG GTG GGC TGC AGT GGG GAC AAG CTC ACC ATC AAC GGG AAG GCT 1050

360 370Val Ile Ser Asn Lys Asp He Leu Ala Thr Asn Gly Val He His Phe He Asp Glu Leu Leu He Pro Asp SerGTC ATC TCC AAC AAA GAC ATC CTG GCC ACC AAC GGT GTC ATT CAT TTC ATT GAT GAG CTG CTT ATC CCA GAT TCA 1125

385 395Ala Lys Thr Leu Leu Glu Leu Ala Gly Glu Ser Asp Val Ser Thr Ala He Asp He Leu Lys Gin Ala Gly LeuGCC AAG ACA CTG CTT GAG CTG GCT GGG GAA TCT GAC GTC TCC ACT GCC ATT GAC ATC CTC AAA CAA GCT GGC CTC 1200

410t- repeat 3 420Asp Thr His Leu Ser Gly Lys Glu Gin Leu Thr Phe Leu Ala Pro Leu Asn Ser Val Phe Lys Asp Gly Val ProGAT ACT CAT CTC TCT GGG AAA GAA CAG TTC ACC TTC CTG GCC CCC CTG AAT TCT GTG TTC AAA GAT GGT GTC CCT 1275

43$J- 445Arg He Asp Ala Gin Met Lys Thr Leu Leu Leu Asn His Met Val Lys Glu Gin Leu Ala Ser Lys Tyr Leu TyrCGC ATC GAC GCC CAG ATG AAG ACT TTG CTT CTG AAC CAC ATG GTC AAA GAA CAG TTG GCC TCC AAG TAT CTG TAC 1350

460 470Ser Gly Gln Thr Leu Asp Thr Leu Gly Gly Lys Lys Leu Arg Val Phe Val Tyr Arg Asn Ser Leu Cys He GluTCT GGA CAG ACA CTG GAC ACC CTG GGT GGC AAA AAG CTC CCA GTC TTT GTT TAT CGA AAT AGC CTC TGC ATT GAA 1425

465 495Asn Ser Cys He Ala Ala His Asp Lys Arg Gly Arg Phe Gly Thr Leu Phe Thr Met Asp Arg Met Leu Thr ProAAC AGC TGC ATT GCT GCC CAT GAT AAG AGC GGA CGG TTT GGG ACC CTG TTC ACC ATG GAC CGG ATG TTG ACA CCC 1500

510 520Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser AlaCCA ATG GGG ACA CTT ATG GAT GTC CTG AAG GGA GAC AAT CGT TTT AGC ATG CTG GTG GCC GCC ATC CAG TCT GCA 1575

53S y repeat 4 545Gly Leu Met Glu He Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu Ala Phe Gln Ala Met

. GGA CTC ATG GAG ATC CTC AAC CGC GAA GGG GTC TAC ACT GTT TTT GCT CCC ACC AAT GAA GCG TTC CAA GCC ATG 1650560 ± 570

Pro Pro Glu Glu Leu Asn Lys Leu Leu Ala Asn Ala Lys Glu Leu Thr Asn Ile Leu Lys Tyr His Ile Gly AspCCT CCA GAA GAA CTG AAC AAA CTC TTG GCA AAT GCC AAG GAA CTT ACC AAC ATC CTG AAG TAC CAC ATT GGT GAT 1725

585 595Glu Ile Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln Gly Asp Lys Leu Glu Val Ser

> GAA ATC CTG GTT AGC GGA GGC ATC GGG GCC CTC GTG CGG CTG AAG TCT CTC CAA GGG GAC AAA CTG GAA GTC AGC 1800610 620

Ser Lys Asn Asn Val Val Ser Val Asn Lys Glu Pro Va] Ala Glu Thr Asp He Met Ala Thr Asn Gly Val Val[ TCG AAA AAC AAT CTA GTG AGT GTC AAT AAG GAG CCT GTT GCC GAA ACC GAC ATC ATG GCC ACA AAC GGT GTG GTC 187$

63$ 645Tyr Ala Ile Asn Tlir Val Leu Gln Pro Pro Ala Asn Arg Pro Gln Glu Aro Glv Abp Glu Leu Ala Asp Ser Ala

' > TAT GCC ATC AAC ACT CTT CTC, CAG CCG CCA GCC AAC CGA CCA CAA CAA CGA GGA GAT GAC CTC GCA GAC TCT GCC 1950660 670

Leu Glu He Fhfe Lys Gln Ala Sei Ala Tyr Ser Aig Ala Ala Glr¡ Arg Ser Val Arg Leu Ala Pro Val Tyr Gln[ CTT GAA ATC TTC AAA CAC GCG TCA GCG TAT TCC ACG GCT GCC CAG AGG TCT CTG CGA CTT GCC CCT GTC TAT CAG 2025

Arg Leu Leu Glu Arg Met Lys His ••*

i CGG TTA CTG GAG AGG ATC AAG CAT TAGCAC^AAGACCGAGGAGGAGAGCCCTGCAGCAGCTTCCCGCCAGTTTCTCTCAGTTTGCCAAAGA 2116GACCATTGAATCTT'ITTGAAACCAAACAGCACACTTCAACATACATGGGCHGCACCATATTC 2215GGAGAAACGTTCTTTrTACCTTTGATCCCTCCAAACCGTCGTTGTTAACCCATTC^ 2 314AAGACCTCTCGAAAGCATCAATTTCCTGACTGTGCCAAGGCCTGATAAAGGCMCTACGGCATCT^ 2413TTCTCGAAAGCCTTGGCATGGTTCTGTAAAGCTCTTGTACCCCTÜGAGAAACGGCATCACTATAAGCTATGAGTTGAAC 2512TGTCTCCACACATGGTVTTC^AT^CTTCTATATTCCCCCTGCCCAC.GTA^ 2 611CATGGTGCATTTGTAATAATAAAACCAAAGAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAA 2673

ßigH3 MALFVRLLALALALALGPAATWGPAKSPYOLV[.OHSRLRGHOHGPNVCAVOKVIGTNRKYFTNCKOWYQRKICGKSTVISYECCPGYEKVPGEKGCPAA 100

ßlgM3 MALLMRLLTLAI^LSVGPAGTLAGPAKSPYOLVLOHSRLRGROHGPrWCAVOKVIGTNKKYFTIJCKOWORKICGKSTVISYECCPGYEKVPGEKGCPAA 100

ßigH3 LPLSNLYETIXVVGSTTTQLYTDRTEKLRPEMEGPGSFTIFAPSNEAWASLPAEVLDSLVSríVNIELLNALRYHMVGRRVLTDELKHGMTLTSMYQNSNI 2 00I I I I I I I t t : I I I I M I I I I I 1 I I I I I I I I ! I I I I I I I I I I I I I I I I 1

.

M ] 1 I I M I I I I I II I I 1 I I It I I I I I : I I I M I I I II ItI I I I I I I I I I IßlgM3 LPLSNLYETMGVVGSTTTOLyTDRTEKLRPEMEGPGSPTIFAPSNEAWSSLPAEVLDSLVSNVNIELLNALRYHMVDRRVLTDELKHGMTLTSMYONSNI 200

ßlgH3 QIHHYPr«IVTVNCARLLKADHHATr«VVHLIDKVISTITrmiÇX)IIEI^ 300I t I I I I I I II II I I I M I I I I I I I I I I I I I I I I M I I I I I I I I I I 11 1 I I I I M 1 I H I I I I I I 1 I I : I I I : I I : I I I I I I I M I I I I I

-

f II I I I I I I IßlgM3 QIHHYPNGIVTVNCARLLKADHHATNGWHLIDKVISTITWIQQIIEIEOT^ 300

ßigH3 EALKDLLMJHILKSAMCAEAIVAGLSVETLEGTTLE^GCSGCWLTIr^l^nSNKDILATNGVIHYIDELLIPOSAKTLFELAAESDVSTAIDLFROAGL 400I I I I I I I I I I I I I I I I I ! I I I I II : I : I I I : I I I I I I I I I I I

.

1 I I I I I I : I I I 11 I I I I I I ! I I : I I I I I I I 1 I II I I : II I : I i I I I I I I I : : : I I I I

ßlgM3 EÄLRDLLNNHILKSAMCAEAIVAGMSMF^LGGTTLE^GCSGDKLTIfWKAVISNKDILATNGVIHFIDELLIPDSAKTLLELAGESDVSTAIDlLKQAGL 400

ßlgH3 C^HLSGSERLTLLAPLNSVFKKÎTPPIimHTRNLLRNHIIKI^IASKYLYHGQTLETLGGKKLRVFVYRNSLCIENSCIAAHDKRGRYGTLrm 500:. I I I I

-

I.

I I : I I I I M I I I I I

-

I

.

I I I : :

-

I I I I :: I : I I I I I I I I I I I I : I I I I I I I I I I I I I M I I t I I I I I 1 I I ! I M I : I I I I I I I I : I IIplgM3 DTHLSGKEQLTFLAPI^SVFKDGVPRIDAQMKTLLLNHMVKEQLASKYLYSGC^^ 500

ßlgH3 PHGTVMDVLKGDrffiFSMLVAAIOSAGLTETl^REEWTVFAP^ 600I I I I I I I I I I I M I I I I M I I I I I I 1 I I

.

I I I I I M I I I I I M I I I.

f : I I I.

: I I : : I I I I.

I I M I I I I I I I I I I I I M I I I I I I I M I I 1 H I IßigM3 P>«T\mDVLKGDNRFSMLVAAIQSAGLMEILJ^REX}VYTVFAPTNEAFQ 600

ßlgH3 LlCNrAn/SVNKEPVAEPOIMATNGWHVITNVL^PPANRPiJERGDELADSALEIFKOASAFSRASORSVRLJkPVYOKLLERMKH 683I I I II I I I I I I I II

.

I I 1 1 I I I I I

- -

I

- -

I I I I I I I I 1 I I I I I I I 1 I I I I I I I I I I I I : I I I

.

I I 11 I I II I I 1:1 I I I I I IßlgM3 SKNNVVSVNKEPVAF^DIhWTNGV\^AINTVLQPPANRPQERGDELADSALElFKQASAYSRAAQRSVRLAPVYORLLERMKH 683

FIG. 8. Nucleotide and deduced amino acid sequence of murine ßig-h3 and comparison to human /3ig-h3. A. The sig-nal sequence is overlined and the RGD sequence underlined. Each of the four repeat sequences is shown in brackets(Skonier et ai, 1992). This nucleotide sequence has been submitted to GenBank and the assigned accession number isL19932. B. The deduced amino acid sequence for both mouse and human 0ig-h3 genes were compared. Using the sin-gle-letter code, identical residues are indicated by a vertical line, conservative changes by two dots, and less conservativechanges by one dot.

580 SKONIER ET AL.

1 2 3 4 5 6 7 8 9 10 11 12 13

fFIG. 9. Tissue expression of mouse /3ig-h3. Mouse tissueRNAs (lanes 1-9, 6 pg of poIy(A)*RNA; lanes 10-13, 30 /.gtotal RNA) were Northern blotted and probed with mouse/3ig-h3. Lane 1, Brain; lane 2, cerebellum; lane 3, heart;lane 4, kidney; lane 5, liver; lane 6, skeletal muscle; lane 7,testes; lane 8, thyroid; lanes 9 and 10, uterus; lane 11, 8-day mouse embryo; lane 12, 12-day mouse embryo; lane13, 16-day mouse embryo.

DNA-binding proteins and to exert their effect at the tran-scriptional level, others such as DCC do not. The DCCgene encodes a protein with sequence similarity to neuralcell adhesion molecules (Fearon et al, 1990). Other non-

DNA-binding proteins shown to have antiproliferative ac-

tivity when transfected into cells include the gap junctionprotein connexon (Eghbali et al, 1991), vimentin (Eiden etal, 1991), BTG1, a novel protein thought to be anchoredto the cell membrane (Rouault et al, 1992), gasl, an inte-gral membrane protein (Del Sal et al, 1992), vinculin (Fer-nandez et al, 1992), and a-actinin (Gluck et al, 1993).

Perhaps p680'g-h3 does not act as a typical growth inhibi-tor like TGF-/3 or interleukin-1 (IL-1): the fact thatp680>i-h:i has antiadhesion activity suggests that it may be a

component of the extracellular matrix (ECM), which mayexert its antiproliferative effects by binding to cell-surfacecomponents (integrins) in conjunction with other ECMmolecules that might be required for signal transduction,resulting in growth arrest. These accessory proteins maynot be available in tissue culture. We are presently at-tempting to identify p680>g-h3 cell-surface receptors as wellas performing immunolocalization of p680'g-h3 in varioustissues.

The chromosomal location of /3ig-h3 to 5q31 in humansis particularly interesting. The 5q31 deletion is the mostcommon karyotypic lesion found in all myelodysplasticsyndrome subtypes and human leukemias except chronicmyelomonocytic leukemia (CMML) (Van Den Bergh et al,1974; Le Beau et al, 1986; Pedersen and Jensen, 1991;Carter et al, 1992; Willman et al, 1993), and it has beensuggested that this region contains a tumor suppressorgene (Pedersen and Jensen, 1991). Deletions in this regionhave also been described in nasopharyngeal carcinoma(Kristensen et al, 1991), von Hippel-Lindau disease (Jor-dan et al, 1989), and bladder carcinoma (Barrios et al,1990). Recently interferon regulatory factor-1 (IRF-1) hasbeen mapped to the region 5q31 (Willman et al, 1993),and inactivating rearrangements of one alíele plus a dele-tion in the second alíele were identified in one case of acuteleukemia. These investigators went on to show that overex-

pressing IRF-1 reversed the transformed phenotype

(Harada et al, 1993). The fact that 0ig-h3 can also depresstumorgenicity makes investigating its possible mutations inpatients with acute monocytic leukemia (AML) and myelo-dysplastic syndrome (MDS) worthwhile; these experimentsare now in progress. The mapping of /3ig-h3 to mousechromosome 13B to Cl confirms a region of conservationbetween human chromosome 5q and mouse chromosome13 which includes the IL-9 gene (Mock et al, 1990).

What might the role of p680'g-h3 be in mediating some ofthe cellular effects of TGF-/3? One attractive hypothesis isthat upon induction by TGF-/3, it might participate inmediating some of the growth-inhibitory effects. TGF-/3induces /3ig-h3 in A549, A375, NHEK, HMEC, and H2981cells, all of which are growth arrested by TGF-/31. How-ever, p680'g-h3 is also induced by TGF-/31 in WI-38 andHEPM cells, two cell lines that are growth stimulated byTGF-/31. Although these results may seem contradictory atfirst, perhaps p680>g-h3 is pleiotropic with respect to its bio-logical effects and the particular response elicited byp68#'g-h3 depends on the population of cell-surface mole-cules available for it to interact with; these proteins maydiffer for mesenchymal and epithelial cells. It is interestingto note that the Ela oncogene of adenovirus, known for itstransforming activity, has also been shown to have anti-oncogenic activity (Frisch, 1991; Chinnadurai, 1992): theparticular response of a cell to a growth regulatory proteinmay depend on the context in which the protein is pre-sented. Experiments are in progress to identify either cell-surface or ECM components that interact with p68#g-h3 tostudy its mechanism of action further.

ACKNOWLEDGMENTS

This work was partially supported by grants from theMarch of Dimes Birth Defects Foundation (CD.), the Na-tional Institutes of Health (CD.), and the Deutsche For-schungsgemeinschaft, Germany (S.E.).

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Address reprint requests to:Dr. Kelly Bennett

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Received for publication September 18, 1993, and in revised formOctober 25, 1993.