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[16] E x p r e s s i o n o f E p i d e r m a l G r o w t h F a c t o r P r e c u r s o r c D N A in A n i m a l C e l l s B y BARBARA MROCZKOWSKI

Introduction

Analysis of cDNA clones derived from mRNA transcripts encoding murine or human epidermal growth factor (EGF) has revealed that these peptides are synthesized as large precursor molecules comprised of 1217 and 1207 amino acid residues, respectively. 1'2 These unexpectedly large molecules contain a hydrophobic domain that may serve to anchor the precursor in the plasma membrane, thus making the EGF precursor structurally analogous to a receptor. In the kidney, where the EGF precursor does not undergo proteolytic processing, it is plausible that the precursor molecule functions as a receptor for a yet unidentified ligand. 3 Why EGF is encoded within a much larger membrane-bound precursor remains a matter of speculation. An interesting characteristic of the precursor molecule is that it contains eight regions of partial sequence similarity to mature EGF. Each repeat unit is comprised of approximately 40 amino acids and includes 6 cysteine residues spaced as in EGF. Whether these EGF-related sequences are ever processed to serve a biological function is not known. To gain a better understanding of the function of the EGF precursor and the factors resulting in its tissue-specific processing to EGF, we have used a bovine papillomavirus (BPV), mouse metallothionein I promoterbased expression vector, together with a human kidney cDNA encoding the EGF precursor, to produce high levels of this protein in mouse NIH 3T3 and C127 cells. The ability of BPV vectors to propagate extrachromosomally as multicopy plasmids, in conjunction with their ability to express foreign proteins efficiently (i.e., human insulin receptor, 4 human tissuetype plasminogen activatorS), has enabled us to establish stably transfected t A. Gray, T. J. Dull, and A. UUrich, Nature (London) 303, 722 (1983). 2 j. Scott, J. Urdea, M. Quiroga, R. Sanchez-Pescador, N. Fong, M. Selby, W. J. Rutter, and G. I. Bell, Science 221, 236 (1983). 3 L. B. Rail, J. Scott, G. I. Bell, R. J. Crawford, J. D. Penshow, H. D. Niall, and J. P. Coghlan, Nature (London) 313, 228 (1985). 4 j. Whittaker, A. K. Okamoto, R. Thys, G. I. Bell, D. J. Steiner, and C. A. Hofmann, Proc. Natl. Acad. Sci. U.S.A. 84, 5237 (1987). 5 V. B. Reddy, A. J. Garramone, H. Sasak, C.-M. Wei, P. Watkins, J. Galli, and N. Hsiung, DNA 6, 461 (1987).

METHODS IN ENZYMOLOGY, VOL. 198

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cell lines that express high levels of the hEGF precursor. This chapter presents specific protocols that our laboratory has used for the selection and establishment of stably transfected cell lines using BPV-derived shuttle vectors. Construction and Preparation of Bovine Papillomavirus Recombinant Expression Vectors Synthetic XhoI oligonucleotide linkers (New England Biolabs, Beverly, MA) are ligated to a 6.4-kilobase (kb) Sinai fragment containing the entire coding region and 3' and 5' flanking sequences of the human EGF precursor isolated from a hgtl0 adult human kidney cDNA library. 6 After XhoI digestion and removal of excess linkers on a low gelling temperature agarose gel (FMC BioProducts, Rockland, ME), the 6.4-kb fragment is ligated into the XhoI site of pBPV-MTH-Xho (kindly provided by D. Hamer, National Institutes of Health, Bethesda, MD). This eukaryotic shuttle vector is similar to that described by Pavlakis and Hamer 7 and contains the following structural elements: the 5.5-kb subgenomic fragment of BPV, the mouse metallothionein gene modified by replacement of the BglII site upstream of the initiator ATG by an XhoI site, and the origin of replication and ampicillin resistance gene of pML2. s The physical map of this construct, pBPV-MTH-hEGF, and the restriction map of the human prepro-EGF cDNA sequence are shown in Fig. 1. Vector DNAs used for transfection are prepared by a slight modification of the standard lysozyme-Triton method.

Plasmid Purification The quantities given are per 1 liter culture. 1. Harvest cells, spin 5000 rpm for 10-15 min at 4 °. 2. Resuspend in 10 ml sucrose solution [25% sucrose (w/v), 50 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0)]. 3. Add 2 ml of 5 mg/ml lysozyme freshly made up in 0.25 M Tris-Cl (pH 8.0) and 4 ml of 0.25 M EDTA (pH 8.0). Mix gently and incubate on ice for 10 min. 4. Add 16 ml of cold TLM [Triton lysis mix: 50 mM Tris-C1 (pH 8.0), 60 m M EDTA (pH 8.0), 0.1% Triton X-100]. Mix gently and incubate on ice for 15 min. 6 G. I. Bell, N. M. Fong, M. A. Wormsted, D. F. Caput, L. Ku, M. S. Urdea, L. B. Rail, and R. Sanchez-Pescador, Nucleic Acids Res. 14, 8427 (1986). 7 G. N. Pavlakis and D. H. Hamer, Proc. Natl. Acad. Sci. U,S.A. 80, 397 (1983). 8 M. Lusky and M. Botchan, Nature (London) 293, 79 (1981).

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EGF PRECURSORcDNA EXPRESSION

XhoI

177

XhoI

t

~

EcoRI

' MT-I

Eco

Barn HI pML

Sol I

BPV RESTRICTION MAP OF HUMAN EGF PRECURSOR COMPOSITE cDNA SEOUENCE

I

I

I

I

XMN1

STU1

I

I

SMA1

ECORI BCL1 NCO1

I

I

I

I

XMN1

BSTE2

PSTI

SCAI BAMHI

PVU2

I

I

I

II

I

AHA3

BALI

STU1 BSTXI

BALI

I

HGIE2 BSTXI

I I

BSI"XI

I

BSTE2

SPH1

I

I

II

PVU2 PVU2 BSM1

MST2

AVA3 B G L 1 APA1 SPH1 BALl HCIE2

I

APA1

SNAB1 PSTl SCA1

I

I

AHA3

I

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I

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I

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I

III

I

I

I

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I PVU2

I

I

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BALI BSTXI BSTX[

III

II

II

I

I

II

PST1

SSPI

AHA3 XMN1 SSP1 AHA3 AHA3 SSP1 STUI

HIND3 NDEI

II

Fic. 1. Expression vector pBPVMTMhEGF and restriction map of the human EGF precursor cDNA sequence. 5. Centrifuge the viscous lysate at 17,000 rpm for 30 min in a Beckman SW27 or SW28 rotor at 4 °. 6. To the cleared lysate from Step 5 add one-fourth initial volume of 30% P E G (polyethylene glycol, MW 6000-8000) made up in 1.5 M NaC1. Mix and incubate on ice for several hours or overnight. 7. Centrifuge at 10,000 rpm for 15 min at 4 °. Remove the supernatant and wash the pellet with 70% ethanol. 8. Dissolve the D N A in 2.1 ml of T E (10 m M Tris-Cl, p H 7.5, 1 m M EDTA). Add 2.3 g CsC12 and 0.15 ml of a 10 mg/ml ethidium bromide solution. 9. Centrifuge at 10,000 rpm for 1 hr at 20 ° to remove cell debris and film. Transfer the solution to a new tube.

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10. Supercoiled plasmid DNA is purified by density gradient centrifugation in a VTi 65 rotor (55,000 rpm, 12-15 hr at 20°). 11. Collect the lower, supercoiled band and extract with 2-propanol equilibrated with CsCl2-saturated TE. 12. Dialyze plasmid solution for 24 hr at 4 ° against four 4-liter volumes of TE. 13. Precipitate the plasmid DNA with 2 volumes of ice-cold 100% ethanol and 1/10 volume of 3 M sodium acetate (pH 6.0). Centrifuge at 10,000 rpm for 20 min at 4 ° and wash the pellet with 70% ice-cold ethanol prior to resuspension in TE. Cell Transfection Mouse N I H 3T3 cells or mouse C127 cells (mammary tumor cell line of an RIII mouse, ATCC CRL 1616) (2-4 × 105) are cotransfected with a calcium phosphate precipitate containing 5/zg of pBPV-MTH-hEGF and 0.5 tzg of pSV2Neo, 9 followed by osmotic shock with glycerol, r° Our protocol for transfection is based on the procedure published by Fordis and Howard. 11

Transfection On day 1, plate out cells at a density of 2 × 105 cells per T-25 Falcon flask. Prepare the DNA for precipitation and transfection as follows. Each T-25 flask is transfected with 5/zg of the BPV expression vector and 0.5 /.~g of pSV2Neo; we found this to be the optimum DNA concentration and ratio for NIH 3T3 and C127 recipient cells. Ethanol-precipitate the DNA from stock TE solutions by the addition of 1/10 volume of 3 M sodium acetate (pH 6.0) and 2 volumes of 100% ethanol, mix well, and place in dry ice-ethanol bath for 15 min, then collect the precipitate by centrifugation (13,000 g for 15 min) in a microcentrifuge at 4 °. Aspirate dry and rinse with 70% ethanol. Add 20/~1 of 70% ethanol to the precipitated DNA, mix gently, and evaporate in a Speed-Vac overnight to ensure that the DNA precipitate is dry. On day 2, prepare the following stock solutions. 10 × HEPES-buffered saline (10× HBS) contains 8.18% NaCI (w/v), 5.94% HEPES (w/v), and 0.2% NazHPO 4 • 7H20 (w/v). Prepare a 2 x HBS solution from the stock 10 × HBS and adjust the pH to 7.12 with 1 N NaOH (pH must be exact). The CaCI 2 solution is 2 M CaCI2.2HzO. Prepare 15% glycerol/HBS by 9 p. j. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327 (1981). J0 E. Frost and J. Williams, Virology 91, 39 (1978). H C. M. Fordis and B. H. Howard, this series, Vol. 151, p. 382.

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mixing 30 ml of 50% glycerol in water (w/v), 50 ml of 2 x HBS, and 20 ml doubly distilled water. Filter-sterilize the 2 x HBS, 2 M CaC1z, and the 15% glycerol/HBS solutions in a laminar flow hood.

Calcium Phosphate-DNA Coprecipitation Note that Steps 1-3 are performed in a laminar flow hood. The reaction volumes are given per single T-25 Falcon flask. 1. Resuspend the DNA in a 1 : 10 dilution of filter-sterilized TE (pH 7.5). For 5 ~g DNA resuspend in 50/~1 of a 1 : 10 dilution of TE, 15.5/~1 of 2 M CaClz, and 59.5/~l doubly distilled water. 2. Add the DNA solution dropwise to a tube containing 125 ~1 of 2 x HBS. During addition use a sterile cotton-plugged Pasteur pipette to direct a gentle stream of air in the tube to mix the components. The final concentration of DNA for the calcium phosphate coprecipitation step should be 20/~g DNA/ml. 3. Let the DNA incubate 30 rain at room temperature. 4. Wash the flask 2 times with serum-free Dulbecco's modified Eagle's medium (DMEM). 5. Add 5 ~g of the calcium phosphate-DNA precipitate to each T-25 flask containing 5.0 ml of DMEM. Return the flask immediately to the incubator to avoid pH changes in the medium. 6. Four hours later, osmotically shock the cells.* Wash the cells 2 times with DMEM. Overlay the cells with 3.0 ml of 15% glycerol/HBS. Incubate for 2 min at 37°. Wash 2 times with DMEM and feed with fresh complete medium. On day 4, split the transfected cells. A single T-25 flask is split into five large 100-ram tissue culture dishes containing selective medium.

Selection by Neomycin Resistance Transfected cells are fed twice a week with fresh medium containing the appropriate concentration of Geneticin. The selective medium for NIH 3T3 cells contains 600/.~g/ml of Geneticin (G418 sulfate, GIBCO, Grand Island, NY); the selective medium for C127 cells contains 1 mg/ml Geneticin. Geneticin-resistant colonies are readily observed after a 2-week period. Individual clones are isolated by removing the medium and placing a sterile cloning ring around the colony to be isolated. The colonies are * All solutions must be prewarmed to 37 °.

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then trypsinized ~2'j3 and subcultured directly into flat-bottomed 24-well plates (Linbro Flow Laboratories, McLean, VA).

Screening of Transformants Geneticin-resistant cells are subcultured into 12-well plates (Costar, Cambridge, MA), then tested for the expression of EGF precursor mRNA and protein by R N A - R N A hybridization and immunoblot analysis, respectively.

RNA Isolation and Blot Hybridization Analysis Cytoplasmic RNA is isolated from individual clones by detergent lysis and phenol/chloroform/isoamyl alcohol extractions. Briefly, each well is washed and scraped in 1.0 ml of calcium-free, magnesium-free phosphatebuffered saline. Cells are transferred to an Eppendorf tube and pelleted at 1500 rpm for I0 min. Cell pellets are resuspended in 500 ~1 of RNA lysis buffer [20 mM Tris-Cl pH (7.5), 0.15 M KCI, 5 mM MgCIz, and 0.5% Nonidet P-40 (NP-40)] and incubated on ice for 15 min. Lysates are spun in an Eppendorf microfuge (13,000 g) for 10 min, and the RNA is isolated from the supernatants by phenol/chloroform/isoamyl alcohol (25:24: 1) extractions followed by ethanol precipitation. Slot blots are performed using a minifold II slot-blot system (Schleicher and Schuell, Keene, NH) and Nytran membranes (Schleicher and Schuell). The total RNA from a single well is resuspended in 7.5 × SSC (1 × SSC is 0.15 M NaCI, 15 mM sodium citrate) containing 4.3 M formaldehyde and incubated at 80 ° for 10 min prior to spotting onto the nylon membrane. Blots are baked at 80 ° for 30 min under reduced pressure, then prehybridized for 3 hr at 55° in hybridization buffer {50% (v/v) formamide, 0.75 M NaC1, 0.15 M Tris-C1 (pH 8.0), 10 mM EDTA, 0.2 M sodium phosphate (pH 6.8), I × Denhardt's solution [0.02% bovine serum albumin (BSA), 0.02% (w/v) Ficoll, 0.02% (w/v) poly(vinylpyrrolidone)], 10% (w/v) dextran sulfate (MW 500,000), 0. I% sodium dodecyl sulfate (SDS)}. Hybridizations are carried out for 16 hr at 55 ° in fresh hybridization buffer containing [32p]CTP-labeled prepro-EGF RNA. The hybridization probe is a 1015-base pair (bp) EcoRI/BamHI fragment (Fig. 1) of preproEGF subcloned into the vector pGEM4 (Promega Biotec). High specific activity 32p-labeled probes of both sense and antisense strands are prepared 12 L. C. M. Reid, this series, Vol. 58, p. 152. 13 R. I. Freshney, "Culture of Animal Cells: A Manual of Basic Technique," Chap. 13, p. 129. Alan R. Liss, New York, 1983.

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by using SP6 and T7 RNA polymerases according to the manufacturer's protocol (Promega Biotec). Following hybridization, the blots are sequentially washed in a shaking water bath in a total volume of 40 ml for 1 hr at 68° in the following solutions14: Wash buffer I: 0.15 M Tris-Cl (pH 8.0), 0.75 M NaCI, 10 mM EDTA, 25 mM sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 0.1% SDS Wash buffer II: 30 mM Tris-C1 (pH 8.0), 0.15 M NaCI, 2.0 mM EDTA, 25 mM sodium phosphate (pH 6.5), 1 × Denhardt's, 0.1% sodium pyrophosphate Wash buffer III: 5 mM Tris-C1 (pH 8.0), 50 mM NaCI, 0.4 mM EDTA, 0.1% sodium pyrophosphate, 0.1% SDS The membranes are then covered with Saran plastic wrap and autoradiographed at - 7 0 °, using Kodak XAR-5 film and intensifying screens. Figure 2A shows a representative blot of RNA isolated from individual Geneticin-resistant clones immobilized onto Nytran membranes using the procedures outlined above. The slot blots are performed in duplicate and represent constitutive levels of prepro-EGF mRNA in the transfected cell lines. Figure 2B represents the relative steady-state levels of prepro-EGF mRNA in parental NIH 3T3 cells and in the transfected Geneticin-resistant cell lines hEGFI2, hEGFI 1, and hEGF19 in the presence or absence of 5 mM sodium butyrate. J5 We have observed that hybridization of the hEGF precursor RNA probe to mRNA isolated from various transfected NIH 3T3 or C127 cell lines results in at least a 10-fold increase in steadystate levels of mRNA after induction. Growth of transfected cells in the presence of heavy metals (i.e., zinc or cadmium) results in only a modest 2- to 3-fold increase in prepro-EGF mRNA. A major advantage of the use of sodium butyrate is that, unlike the heavy metals generally used to induce the metallothionein promoter, sodium butyrate is not toxic to the cells for incubation periods of up to 3 days. Detection of Recombinant Prepro-EGF

Immunoblot Analysis of Prepro-EGF in Transfected Cell Lines Polyclonal antibodies directed toward epitopes specific for the mature hEGF peptide are used to identify and quantitate relative levels of the hEGF precursor in transfected cell lines. Various aliquots of NP-40 lysates 14 D. DeLeon, K. H. Cox, L. M. Angerer, and R. C. Angerer, Dev. Biol. 100, 197 (1983). 15 B. W, Birren and H. R. Herschman, Nucleic Acids Res. 14, 853 (1986).

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Probe~ 861 bp anti-sense preproEGF A,

B. Induced

hEGF II h EGF 12 hEGF 18 hEGF32 hEGF28 hEGF25 hEGF 2 h EGF I h EGF 5 hEGF 30 hEGF 5 h EGF 16 h EGF 29 h EGF21 h EGF 6 h EGF 9

D

Cell

h EGF 12

+ --

h

EGF II

-t-

+

h EGF 19

NIH3T3

FIG. 2. Slot-blot analysis of prepro-EGF mRNA levels in transfected cells. (A) Analysis of total RNA isolated from duplicate cultures immobilized onto Nytran membranes and hybridized to a 32p-labeledantisense RNA probe. (B) Analysis of 5/~g of RNA isolated from control NIH 3T3 cells and three different transfected cell lines grown in the presence ( + ) or absence ( - ) of 5 mM butyric acid.

f r o m transfected N I H 3T3 cells induced for 12 hr with either butyric acid or sodium b u t y r a t e reveal the presence of a 170,000-dalton protein recognized b y a n t i - h E G F antiserum (Fig. 3). As o b s e r v e d with m R N A levels, the levels of p r e p r o - E G F protein are found to be consistently m u c h higher in cells e x p o s e d to butyric acid or sodium butyrate as o p p o s e d to ZnCI2 or CdCI2. The o p t i m u m sodium butyrate concentration for the induction of r e c o m b i n a n t p r e p r o - E G F in transfected N I H 3T3 or C127 cells is found to be 5 raM.

Immunoprecipitation of Prepro-EGFfrom Transfected Cells The synthesis of p r e p r o - E G F in transfected cell lines is examined by immunoprecipitating metabolically radiolabeled E G F precursor from solubilized cells. Briefly, transfected cells are grown to 70-80% confluency in 60-mm dishes prior to induction with 5 m M sodium butyrate for 8-12

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EGF PRECURSORcDNA EXPRESSION

Tnducer

183

J None IIButyric Sodium Acid IIButy rote 200,000

97,000

FIG. 3. Immunoblot analysis of the prepro-EGF content in transfected NIH 3T3 cells. Cells were incubated for 12 hr in the presence or absence of either 5 mM butyric acid or sodium butyrate. Aliquots (25 and 75/xl) of the NP-40 cellular lysates prepared from single 100-mm dishes were analyzed by Western blotting after SDS-polyacrylamide gel electrophoresis.

hr. The cells are washed 3 times with phosphate-buffered saline (PBS) and incubated at 37° for 30 rain in DMEM lacking cysteine and supplemented with 4 mM glutamine. The medium is then removed and replaced with fresh serum-free and cysteine-free DMEM to which [35S]cysteine (200/xCi/ ml, 940 Ci/mmol; NEN, Boston, MA) is added. After labeling, the cells are cooled to 4°, washed 3 times with PBS, harvested by scraping, and collected by centrifugation. Radioactively labeled cell pellets are solubilized by the direct addition of lysis buffer [20 mM Tris-Cl (pH 7.5), 0.15 M KCI, 5 mM MgCI 2, 0.5% Nonidet P-40]. Cells from each 60-ram dish are solubilized in 500/zl of lysis buffer. After a 10-rain incubation at room temperature, the lysate is clarified by centrifugation at 13,000 g for 8 min at 4°. Aliquots (100/xl) of the supernatant are immunoprecipitated with 5/zl of rabbit antiserum. Immunoprecipi-

184

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1

--

-I-

2

3

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FIG. 4. Immunoprecipitation of prepro-EGF from C!27 transfected mouse cells. Transfected C127 cells grown in 60-mm dishes were incubated in the absence (lane 2) or presence (lane 3) of 5 m M sodium butyrate for 12 hr prior to metabolic labeling with [35S]cysteine for 2 hr. Immunoprecipitates were separated electrophoretically on a SDS-polyacrylamide gel and examined by fluorography. Lane 1 represents I4C-labeled markers.

tates of mouse C127 transfected cell lysates incubated in the presence or absence of sodium butyrate indicate that the transfected cells synthesize a 150,000-160,000 dalton immunoreactive protein on induction with sodium butyrate (Fig. 4). As observed with transfected NIH 3T3 cells, no protein of 6.3 kDa, corresponding to mature EGF, is detected. Neither of the mouse cell lines expresses the appropriate proteolytic processing enzymes for production of mature EGF. The levels of expression of prepro-EGF in the stably transfected cell lines established in our laboratory remain constant for periods of up to 1 year in culture; on further passage a substantial decrease in the synthesis of recombinant protein is observed.

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ASSAYS FOR TRANSFORMING

G R O W T H F A C T O R ot

185

Summary This chapter outlines in detail the optimal conditions for the expression of human recombinant proteins in mouse cells using a bovine papillomavims-based mammalian expression vector. The procedures we have described were used to successfully express high levels of the human EGF precursor in our laboratory and the human insulin receptor in the laboratory of Whittaker. 4 Using this experimental approach we were able to demonstrate that expression of a cDNA for prepro-EGF produces a glycosylated membrane protein with biological activity. Acknowledgments I am grateful to Martha Reich for skilled technical assistance, Jonathan Whittaker for helpful discussions and valuable advice, Susan Heaver for secretarial assistance, and Ann M. Soderquist for critical reading of the manuscript.

[ 17] G e n e r a t i o n o f A n t i b o d i e s a n d A s s a y s for T r a n s f o r m i n g Growth Factor a By CATHERINE LUCAS, TIMOTHY S. BRINGMAN, a n d RIK DERYNCK

Introduction Two types of growth factors have been termed transforming growth factors (TGF) because they were discovered in an assay that evaluated their ability to elicit cellular transformation using an immortalized nonneoplastic fibroblast line. One of these, TGF-a, is a single-chain polypeptide structurally related to epidermal growth factor (EGF). Several TGF-a species are proteolytically derived from a transmembrane precursor form. The fully processed form is 50 amino acids long and lacks glycosylation, whereas the larger forms are glycosylated. All these forms and the transmembrane precursor are biologically active and interact with the same receptor as EGF. The best studied activity of TGF-a is its ability to stimulate DNA synthesis and mitosis of the many cell types that contain the corresponding cell surface receptors. 1-3TGF-fl is structurally unrelated t R. Derynck, Cell (Cambridge, Mass.) 54, 593 (1988). 2 R. Brachmann, P. B. Lindquist, M. Nagashima, W. Kohr, T. Lipari, M. Napier, and R. Derynck, Cell (Cambridge, Mass.) 56, 691 (1989). 3 S. T. Wong, L. F. Winchell, B. K. McCune, H. S. Earp, J. Teixido, J. Massagu6, B. Herman, and D. C. Lee, Cell (Cambridge, Mass.) 56, 495 (1989).

METHODS IN ENZYMOLOGY, VOL. 198

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.