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[CANCER RESEARCH 45, 5413-5416, November 1985]

DifferentialExpressionof TransformingGrowthFactor-«duringPrenatal

Developmentof the MouseDaniel R. Twardzik1

Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21701

ABSTRACT

Transforming growth factors (TGFs) are differentially expressed in the mouse during neonatal development. Highestlevels are seen early at Day 7, and lower levels, at Day 13. Bothsmall- and large-molecular-weight forms of TGF are found; theyshare many biochemical properties with rat TGF-«,including asimilar high-pressure liquid chromatography elution profile. Although the embryo-derived activities compete with epidermal

growth factor for binding to epidemial growth factor membranereceptors, they are immunologically distinct from epidermalgrowth factor. These embryonic polypeptides, however, docross-react in a competitive radioimmunoassay developed usinga synthetic peptide corresponding to the carboxy-17 amino acidsof rat TGF-a as the immunogen. The highly conserved TGF-a

family of peptides produced by some tumor cells may thereforerepresent derepressed forms of these embryonic growth factors.A functional role in neonatal development is proposed.

INTRODUCTION

TGFs2 are a family of low-molecular-weight, acid- and heat-stable, growth-regulatory peptides released into the viral culturemedium by virally transformed rodent (1-4) and certain humantumor cells (5-7). Functionally, two classes of TGFs (a and 0)

have been described, based on their interaction with the receptorfor EGF. TGF-a competes with EGF for binding to the EGFreceptor and activates a receptor-associated tyrosine kinase (8-10). TGF-a purified from medium conditioned by retroviral trans

formed rat embryo fibroblasts has been sequenced (11,12), andhuman and rat TGF-a has recently been cloned and expressedin bacteria (13,14). TGF-a is highly conserved between rodentand human species but shows only a 30% structural homologywith EGF. The anchorage-independent growth of NRK cellsassociated with TGF-a is strongly potentiated by TGF-/3. TGF-|8

has been isolated and partially purified from both normal tissues(15) and some tumor cells (16,17). It does not bind to the EGFreceptor and requires either EGF or TGF-a for stimulating the

clonogenic growth of NRK cells in soft agar (16).Nexo ef al. have reported that early mouse embryos (12 to 13

days) appear to have an excess of EGF receptor competingactivity as compared to the level of EGF detectable by radioimmunoassay (18). We (19) and others (20) have previously reported the isolation from normal mouse embryos of peptideswith properties characteristic of TGF-a, including stimulation ofanchorage-independent cell growth. In this study using a TGF-a

specific radioimmunoassay (21), we show that the TGF activities

1Present address: Oncogen, 3005 First Avenue, Seattle, WA 98121.2The abbreviations used are: TGF, transforming growth factor; TGF-a, trans

forming growth factor-«;EGF, epidermal growth factor; TGF-/3, transforming growthfactor-/?; HPLC, high-performance liquid chromatography; NRK, normal rat kidney;RIA, radioimmunoassay.

Received 4/3/85; revised 7/5/85; accepted 7/22/85.

we have previously described are indeed a member of the TGF-a family of growth factors and are differentially expressed in themouse during prenatal development.

MATERIALS AND METHODS

Mice. Hysterectomy-derived, barrier-maintained BALB/c mice were

obtained from the Frederick Cancer Research Facility animal productionfacility. Day of coital plug formation was taken as Day 0 of prenataldevelopment; average length of the fetus was also monitored (22). Wherepossible, beginning at Day 10 of prenatal development, the embryo wasdissected from the amnion, visceral yolk sac, and placenta. Conceptusesof similar ages from different litters were pooled and frozen at -70°C

prior to extraction. Acid:ethanol extraction was as previously described(23).

Chromatography. Gel filtration was performed on columns of Bio-GelP-30 (Bio-Rad, Richmond, CA) equilibrated in 1 M acetic acid. HPLC ofBio-Gel-purified embryonal TGF-a was as previously described (11).Separations were achieved utilizing a de-^Bondapak column (10-mm

particle size, 0.39 x 30 cm; Waters Associates). The mobile phase was0.05% trifluoroacetic acid, and the mobile-phase modifier was acetonitrile

containing 0.045% trifluoroacetic acid.Radioreceptor Assay. The binding of <25I-EGF(24) to its receptor on

monolayers of human epidermal carcinoma cells (A431) was modifiedfrom that described previously (25). Cells (1000/well) were fixed on 24-well plates (Linbro, Flow Laboratories) with 10% formalin in phosphate-buffered saline prior to assay. Under these assay conditions, 125I-EGF(1x 10'°cpm/nmol) saturates the binding assay at 2.5 to 3.0 nw; assays

were performed at 10% of the saturation value. TGF-a concentrations

are expressed as ng equivalents of EGF, the amount required to producean inhibition of 125I-EGFbinding equivalent to that produced by a known

amount of mouse submaxillary gland EGF (26).Radioimmunoassay. Each 100-i/l reaction contained the following:

20 HIM sodium phosphate (pH 7.4):200 mw CaCI:25 ITIM dithio-threitol:0.1% (w/v) bovine serum albumin:0.1% (w/v) NaN3:125l-peptide(2.5 x 104 cpm) corresponding to the 17 carboxy-terminal residues of

TGF-a (21):antiserum at a final dilution of 1:10,000. The reaction wasinitiated by the addition of antiserum and continued at 23°Cfor 90 min.

An equal volume of 10% formalin-fixed Staphylococcus aureus (Pansor-

bin; Calbiochem) was then added, and incubation was continued for anadditional 60 min at 23°C. The immunoadsorbent was removed bysedimentation, and the amount of bound 125l-peptide was measured. The

amount of bound peptide was corrected for nonspecific binding measured in the absence of antibody (less than 5% of the total).

RESULTS

BALB/c embryos at different days of prenatal developmentwere, when possible, dissected from extraembryonal membranes and placenta. Conceptuses were extracted with acidifiedethanol, and dilutions of solubilized embryonal polypeptides weretested for competing with 125I-EGFfor binding to EGF receptor-

rich human epidermoid carcinoma cells. As shown in Chart 1,two gestation-dependent peaks of EGF-competing activity areseen. Highest specific activities (>10 ng equivalents of EGF permg of protein) (27) are found early in prenatal development on

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TGF EXPRESSION DURING MURINE PRENATAL DEVELOPMENT

12-

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7 9 11 13 15 17 19Stageof PrenatalDevelopment

(days)Chart 1. Differential expression of EGF-competing activity during prenatal de-

vetopment in the mouse. Polypeptides were solubilized (acid:ethanol extraction)(19. 25) from pools of mouse embryos corresponding to days of gestation asindicated on the ordinate. Day of coital plug formation was assigned as Day 0 ofprenatal development. Dilutions of each extract were tested in duplicate forcompeting with 126I-EGFfor binding to formaldehyde-fixed human epidermal cancerous cells (A431) as previously described (1, 3). Quantitation was achieved bycomparing <2SI-EGFdisplaced from receptor by the test sample and a knownamount of cold EGF. Data are expressed as ng equivalents of EGF per mg ofLowry-determined protein (27).

Day 7. Levels appear to decline at Day 8 (<3 ng equivalents ofEGF per mg of protein) and remain low until Day 11; a lessdramatic burst of EGF-competing activity is seen from Day 12

to Day 15, with a maximum specific activity (2.5 ng equivalentsof EGF per mg of protein) attained at Day 13 of prenatal development.

Acid:ethanol-solubilized polypeptides from 7-day embryoswere chromatographed on Bio-Gel P-30 columns in 1 M aceticacid, and aliquot s of alternate fractions were assayed for EGF-

competing activity (Chart 2). Two peaks of activity are seeneluting with apparent molecular weights of 10,000 (Fractions 30to 34) and 20,000 (Fractions 18 to 21). The major M, 10,000embryonal EGF-competing activity élûtesfrom this column in thesame position as the low-molecular-weight form of TGF-a isolated from medium conditioned by feline sarcoma virus-transformed Fisher rat embryo cells. No detectable EGF-competing

activity is seen in those column fractions corresponding to theelution position of mouse submaxillary gland EGF. As previously

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1.0

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10 20 30Fraction

40 50

Chart 2. Bio-Gel P-30 chromatography of acid.ethanol-solubilizedpolypeptides(4.2 mg) from 7-day-okJembryos. Aliquots of alternate fractions (1.2 ml) werelyophilizedand tested for competing with 12SI-EGFfor binding to EGFreceptor withA431 cells as described in 'Materials and Methods." Column markers included

carbonic anhydrase (M, 29,000), RNase (M, 13.700). and insulin (M, 6.000). V„column, void volume.

U_O 3Lu -

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0.3

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10 20 30 40Fraction

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Charts. High-performance liquid chromatography of the M, 10,000 peak ofEGF-competing activity from gel filtration columns of solubilized polypeptidesderived from 7-day-old embryos. Peptides were eluted from a C,B-MBondapakcolumn with a linear gradient of 20 to 60% acetonitrile in 0.045% trifluoroacetícacid as described in 'Materials and Methods.* Aliquots of each fraction were

lyophilized and tested for EGF competition as described above. EGF equivalents(ng)were determined from a standard dilution curve using known amounts of coldmouse submaxillarygland EGF. EGFélûtesfrom this column at 35% acetonitrile.

described (19), two similar molecular weight forms of embryonalEGF-competing activity are also seen in 12- to 13-day-old em

bryos.The low-molecular-weight M, 10,000 EGF-competing activity

derived from 7-day-old conceptuses was further analyzed byhigh-performance liquid chromatography. As shown in Chart 3,a sharp peak of EGF-competing activity élûtesfrom a C,8-/tBondapak column at an acetonitrile concentration of 29 to 30%(Fractions 12 to 14) prior to the bulk of contaminating protein.The low-molecular-weight form of TGF-«isolated from retroviraltransformed rodent cells also élûtesfrom this column at this

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TGF EXPRESSION DURING MURINE PRENATAL DEVELOPMENT

Table 1Competitive radioiaimunoassay of embryonal EGF-receptor competing activities

Growth factors were tested in homologous radioimmunoassays for both TGF-«(21) and EGF (25) as previously described. Antigen (growth factors) Quantitätenwas based on the finding that EGF and rat TGF compete equivalently on a molarbasis for the EGF receptor. No reactivity where indicated included testing theantigen in excess of one order of magnitude than that required for 50% displacement in the homologous assay.

AntibodyAnti-EGF"

Anti-TGF-a"EGF

(ngEGF

equivalentsrequiredfor 50%

displacementin assay)80.20

No reactivityTGF-a

(ng EGFequivalents

requiredfor 50%

displacementinassay)6No

reactivity0.52Embryo-derived

M, 10,000activities0Day

7 Day13No

reactivity No reactivity1.2 2.0

8 EGF was purified from mouse submaxillary gland as described (26).b TGF-a was purified from medium conditioned by Snyder-Theilen feline sarcoma

virus-transformed Fisher rat embryo cells (4).c Acidiethanol-solubilized peptides from a peak of 12- to 13-day and 7-day-okJ

embryos, respectively, were chromatographed on Bio-Gel P-30 and P-10 columnsas described previously (19) and in 'Materials and Methods.' The Mr 10,000 species

from both age groups were used in this assay.d Antibody (IgG) against mouse EGF was prepared as described (25).8 Antibody was an IgG prepared against a synthetic peptide corresponding to

the carboxy-terminal 17 amino acid residues of rat TGF-a (21). Values required for50% displacement were determined from dilution curves constructed from duplicatepoints.

solvent concentration. Both the embryonal EGF-competing activity and rat TGF-a elute differently from mouse submaxillary gland

EGF (35% acetonitrile elution).Dilutions of the predominant low-molecular-weight (M, 10,000)

form of embryonal EGF-competing activity from both 7-day anda pool of 12- and 13-day embryos were analyzed in competitiveradioimmunoassays for both EGF and TGF-a. As shown in Table1, the M, 10,000 form of embryonal EGF-competing activity

derived from either Day 7 or Days 12 and 13 embryos and ratTGF-a (when tested at an order of magnitude higher concentration than half-maximal concentrations of EGF) do not competewith 125I-EGFfor binding to antibody prepared against EGF. ThisRIA is 5-fold more sensitive than the radioreceptor assay forEGF in which 1 ng of EGF is required for a 50% reduction inbinding to EGF receptor. Identical samples of the embryo-derived, low-molecular-weight, EGF-competing activities were alsotested in a competitive RIA for TGF-a. This assay was developedutilizing a synthetic peptide corresponding to the carboxy-17amino acid residues of rat TGF-a and, as shown in Table 1 andas previously reported (21), is specific for TGF-a and does not

detect EGF or urogastrone.With the antibody used in these studies, approximately 0.5 ng

of rat TGF-a is required for half-maximal displacement of 125I-

TGF carboxy terminal peptide from an IgG fraction preparedagainst the homologous synthetic peptide. When dilutions of thelow-molecular-weight embryonal EGF-competing activities were

tested in this assay, a 50% displacement of labeled peptide fromantibody was observed at 1.2 and 2.0 ng equivalent of EGF(value determined by radioreceptor assay), respectively, for theDay 7 and Days 12 and 13 embryonal forms of TGF-a.

DISCUSSION

In this study we show the differential expression of EGF-

competing activities during prenatal development in the mouse,and we present data which suggest that these activities, functionally related to EGF, are members of the TGF-a family of

growth factors. Supporting evidence includes: (a) the dominantform of embryonal EGF-competing activity does not competewith 125I-EGFfor binding to an antibody prepared against mousesubmaxillary gland EGF; they do, however, (ó)compete with 125I-TGF-a peptide for binding to an antibody made against a synthetic peptide corresponding to the carboxy-17 amino acids ofrat TGF-a. Embryonal murine TGF-a is, however, not as immu-nologically reactive as rat TGF-a derived from viral transformedrat fibroblasts. A plausible explanation is that the carboxy-terminal sequence of murine TGF-a (unknown) shares less structural homology with rat TGF-a (11) or that other polypeptidespresent in embryo preparations, such as as TGF-a bindingprotein, may compete for the dominant epitope on the antigen;(c) the embryo-derived growth factors elute from HPLC similarlyto TGF-a, differently than EGF. These studies, however, do notpreclude the possibility that EGF also plays a role in embryolog-ical development. Although we do not detect significant amountsof EGF RIA positive material in 12- to 13-day embryos, we havefound low levels of EGF-like material in older conceptuses.

Whether this is of maternal (blood derived) or fetal origin remainsto be determined.

I and others (1,3,4) have consistently observed two molecularweight forms of TGF-a in medium conditioned by tumor cells.The dominant, smaller form élûteswith Bio-Gel columns with an

apparent molecular weight of 10,000; however, primary sequence analyses (12) of the purified molecule demonstrate thatrat TGF-a peptide is composed of only 50 amino acid residues(Mr 6,000). The dominant form of embryonal TGF-a isolated inboth 7-day and 12- to 13-day embryos is the smaller M, 10,000

component and thus may also represent a 50 amino acid residuespecies. We have recently shown by immunoblotting analysis(21) that medium conditioned by retrovirus-transformed rodentcells also contains larger polypeptides (M, 24,000, 40,000, and42,000), which may represent TGF-a precursors at differentstages of processing. The M, 20,000 embryonal form of TGF-amay also represent a cleavage product of TGF-a, although the

low levels of the growth factor in embryo extracts precludedimmunological analysis.

Extraembryonal membranes and placenta were dissected fromthe embryo proper prior to extraction, beginning at Day 9 ofprenatal development. Because of the small size of the embryoproper, separation was not feasible on earlier conceptuses. Thus,we cannot rule out the possibility that TGF-a found in 7- to 8-

day embryos is of extraembryonal or placental origin. However,in an experiment specifically designed to test this hypothesis,attempts were made to partially disect extraembryonal membranes from 8- and 9-day-old embryos. No significant TGF-aradioimmunoassay-positive material was detected in ethanol ex

tracts derived from these preparations, thus suggesting a fetalorigin for TGF-a. In addition, the low specific activities of EGF-

competing material we previously described in human term placenta (28) suggest some maternal contribution to older mouseembryo preparations; however, these activities were not specifically defined as TGF-a or EGF.

Although we (19) and others (20) have previously reported theisolation of TGFs from mouse embryos, the criteria at that timefor establishing an activity as a member of the TGF-a family were

binding to the EGF receptor and stimulating the growth of normalrat kidney cells in a soft agar matrix (1, 9). Subsequently, it hasbeen shown by Assoian ef a/. (16) that anchorage-independentcell growth observed in partially purified TGF-a preparations was

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TGF EXPRESSION DURING MURINE PRENATAL DEVELOPMENT

a result of the interaction of a second class of factors (TGf-ß)and a ligand which binds to the EGF receptor, i.e., TGF-a or

EGF. Thus, a logical conclusion from this study is that thetransforming activity (reversible stimulation of anchorage-inde

pendent cell growth) we previously observed (19) in embryopreparations was a result of the synergistic interaction of embryonal TGF-a and factors functionally related to TGF-/3. Supplementing soft agar plates containing NRK cells and embryo poly-

peptides at concentrations which did not by themselves supportcolony formation with exogenous EGF or rat TGF-a demonstrated a peak of TGF-/3-like activity at Day 10 of prenataldevelopment.3 Thus, autocrine phenomena (29, 30) and most

likely paracrine are involved in the complex modulation of cellulargrowth (31) and behavior required in differentiation and development. More specifically, selected cell populations in the earlyembryo may release TGF-a (a potent mitogen) and thus preferentially expand concomitantly with (or without) TGF-/8 produc

tion. This could temporarily confer upon said population thepotential for anchorage-independent cell growth and could be ofadvantage if a unique cluster of cells is needed to invade neighboring tissues, as seen in organogeny.

The differential expression of cellular oncogenes (c-.'os) duringprenatal development has been reported (32). TGF-a derived

from tumor cells could represent an example of the derepressionof a fetal antigen associated with neoplastic transformation.Tumor-derived TGF-a is highly conserved among rats, mice, and

humans, thus suggesting an important functional role in prenataldevelopment. Beginning at about 7Vz days gestation, the mouseexhibits signs of organ differentiation; the neural groove, noto-

chord, and head process can be discerned. Days 12 and 13 arealso critical for brain maturation and neurodifferentiation (22).Recently, EGF cross-reactive material has been identified in rat

brain, and a neuromodulating function has been proposed (33).In that regard, elevated levels of TGF-a transcripts have beenfound in rat brain (14). Studies localizing TGF-a in embryos will

help elucidate the role of this embryonal form of EGF in prenataldevelopment and may also provide some insight into the relationship between growth factors and the complex process ofneoplastic transformation.

ACKNOWLEDGMENTS

I thank Don Fish and staff at the Frederick Cancer Research Facility forassistance in providing mouse embryos; Jane Ranchalis for excellent technicalsupport; and Lynda Taylor for help in the preparationof this manuscript.

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4. Twardzik, D. R., Todaro, G. J., Reynolds. F. H., Jr., and Stephenson, J. R.Similar transforming growth factors (TGFs)produced by cells transformed bydifferent isolates of feline sarcoma virus. Virology, 124: 201-207,1983.

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10. Reynolds,F. H., Jr., Todaro, G. J., Fryling, C., and Stephenson,J. R. Humantransforming growth factors (TGFs) induce tyrosine phosphorylation of EGFreceptors. Nature (Lond.),292: 259-262,1981.

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12. Marquardt, H., Hunkapiller, M. W., Hood, L. E., and Todaro, G. J. Rattransforming growth factor I: structure and relation to epidermalgrowth factor.Science(Wash. DC),223:1079-1082,1984.

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16. Anzano, M. A., Roberts, A. B., Meyers, C. A., Komoriya, A., Lamb, L. C.,Smith, J. M., and Sporn. M. B. Synergistic interaction of two classes oftransforming growth factors from murine sarcoma cells. Cancer Res., 42:4776-4778,1982.

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19. Twardzik, D. R., Ranchalis,J. R., and Todaro, G. J. Mouse embryos containtransforming growth factors related to those isolated from tumor cells. CancerRes., 42: 590-593,1982.

20. Proper, J. A., Bjomson, C. L., and Moses, H. L. Mouse embryos containpolypeptide growth factors capableof inducing a reversible neoplastic pheno-type in non-transformedcells in culture. J. Cell Physiol., 110:169-174,1980.

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1985;45:5413-5416. Cancer Res   Daniel R. Twardzik  Prenatal Development of the Mouse

duringαDifferential Expression of Transforming Growth Factor-

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