[11]
TRANSFORMATION WITH SIGNALPEPTIDE-bFGF
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[11] Construction and Expression of Transforming Gene Resulting from Fusion of Basic Fibroblast Growth Factor
Gene with Signal Peptide Sequence B y SNEZNA ROGELJ, DAVID
STERN, and MICHAEL KLAGSBRUN
Introduction Basic fibroblast growth factor (bFGF) is a potent mitogen for cells of m e s o d e r m a l origin.l Basic F G F lacks a signal peptide and is not secreted by cells in culture. M a n y cell types, for example, endothelial cells, produce b F G F and h a v e b F G F receptors. Yet these cells a p p e a r normal and do not s e e m to undergo F G F - m e d i a t e d autocrine transformation. N I H 3T3 fibroblasts transfected with b F G F c D N A do not secrete b F G F ; they appear normal in culture and are not tumorigenic. 2'3 It has been suggested that lack of b F G F secretion prevents b F G F - r e c e p t o r interaction with the result that b F G F - p r o d u c i n g cells have a normal, nontransforming p h e n o t y p e . On the other hand, N I H 3T3 cells transfected with a construct of b F G F fused to a signal peptide sequence (spbFGF) are highly transformed morphologically, grow as nonadherent aggregates, and are highly tumorigenic and metastatic. 2-4 Thus, b F G F altered by the addition of a singal sequence is a transforming protein. Although s p b F G F transforms N I H 3T3 cells, there is no evidence of b F G F secretion, suggesting that transformation in these cells is the result of an internal autocrine loop. Overall Strategy The transforming b F G F expression v e c t o r p s p b F G F is constructed by fusing a sequence encoding the N-terminal 19 amino acid mouse h e a v y chain immunoglobulin signal sequence 5 u p s t r e a m to the 154 amino acid bovine brain b F G F sequence 6 (Fig. 1C). These sequences are placed under the transcriptional control of the Moloney leukemia virus long terminal i j. Folkman and M. Klagsbrun, Science 235, 442 (1987). 2 S. Rogelj, R. A. Weinberg, P. Fanning, and M. Klagsbrun, Nature (London) 331, 173 (1988). 3 S. Rogelj, R. A. Weinberg, P. Fanning, and M. Klagsbrun, J. Cell. Biochern. 39, 13 (1989). 4 A. Yayon and M. Klagsbrun, Proc. Natl. Acad. Sci. U.S.A. 87, 5346 (1990). 5 D. Y. Loh, A. L. M. Bothwell, M. E. White-Scharf, T. Imanishi-Kari, and D. Baltimore, Cell (Cambridge, Mass. ) 33, 85 (1983). 6 j. A. Abraham, A. Mergia, J. L. Whang, A. Tumolo, J. Friedman, K. A. Hjerrild, D. Gospodarowicz, and J. C. Fiddes, Science 233, 545 (1986).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
FIBROBLAST GROWTH FACTOR
[11] -
i
I
el
~1 ~S'UTICodinll Sequence [
~'UT
I
I!~5
1285
I
l
II0
71=6
[3' 1408
AmpR
pIIRori
' 5oo
"
U ,-
K W ' I IpWgl..q.
,82
1,01.,.
v
2so /
C
--/9MKCSWVIFFLMAVv515 I0
;:G~IT TL~oA ~24-
FIG. 1. (A) Restriction map of bovine brain basic FGF cDNA. (B) pspbFGF plasmid: The Moloney murine leukemia virus long terminal repeat (Mo-MuLV LTR) drives constitutive expression of the chimeric signal peptide-bFGF fusion protein. (C) Amino terminus of the predicted primary translation product of the signal peptide-basic FGF protein. The 19 amino acid mouse immunoglobulin heavy chain signal peptide is fused to the second amino acid (alanine) of bovine brain bFGF.
repeat (LTR) and are followed by RNA processing signals derived from the SV40 genome. Cloning of this functional composite gene is performed in a pUC 13-derived vector which contains the pBR322 origin of replication and the ampicillin resistance gene, thus allowing for growth of the mammalian vector in bacteria in the presence of ampicillin. The pspbFGF vector is introduced into NIH 3T3 fibroblasts by the calcium phosphate transfection procedure. Transforming potential is measured by the focus forming assay. Clonal cell lines are obtained by cotrans-
[11]
TRANSFORMATION WITH SIGNALPEPTIDE-bFGF
119
fecting the pspbFGF vector with the dominant selectable marker pSVneo. G418-resistant colonies are picked and expanded into cell lines which are assayed for the production of growth factor activity as measured by the ability of cell extracts and/or conditioned media to stimulate the incorporation of [3H]thymidine into BALB/c 3T3 DNA. Cell lines positive for the synthesis of growth factor activity are analyzed for specific bFGF expression by heparin affinity chromatography and by immunodetection (immunoprecipitation and Western blot) using specific anti-bFGF antibodies. 2,7 Materials A bovine bFGF cDNA clone was obtained from Dr. Judith Abraham (CalBio, Mountain View, CA). 6 pSV2-neo was obtained from Dr. Robert Weinberg (The Whitehead Institute for Biomedical Research, Cambridge, MA). Restriction enzymes, mung bean nuclease, and polymerases were obtained from New England BioLabs (Beverly, MA). G418 is obtained from GIBCO Laboratories (Grand Island, NY). Heparin-Sepharose is obtained from Pharmacia (Piscataway, N J). Construction of pspbFGF Expression Vectors A mammalian vector directing synthesis of a chimeric signal peptide-bFGF is constructed by replacing the epidermal growth factor (EGF) sequence for the bFGF sequence in the pUCDS3 vector. This vector was originally designed in collaboration with Dr. David L. Hare (Amgen Development Corp., Boulder, CO), for the expression and secretion of synthetic EGF fused to a signal peptide sequence in mammalian cells. 8 pUCDS3 consists of a Moloney murine leukemia virus long terminal repeat (MuLV LTR) that contains promoter/enhancer sequences, an SV40 polyadenylation site, sequences encoding a mouse immunoglobulin heavy chain signal peptide, 5 and chemically synthesized human EGF-encoding sequences. The signal peptide-encoding sequences are included so that the protein product is translocated to the endoplasmic reticulum (ER)/ Golgi compartment for subsequent secretion or expression on the cell surface, pUCDS3 was designed so that the coding sequences to be expressed are joined at the 5' end to a unique EcoRI site located just above 7 I. Vlodavsky, J. Folkman, R. Sullivan, R. Friedman, R. Ishai-Michaeli, J. Sasse, and M. Klagsbrun, Proc. Natl. Acad. Sci. U.S.A. 84, 2292 (1987). s D. F. Stern, D. L. Hare, M. A. Cecchini, and R. A. Weinberg, Science 235, 321 (1987).
120
FIBROBLAST GROWTH FACTOR
[1 1]
the signal peptidase cleavage site of the signal peptide and to a unique 3' SalI site located 5' to the polyadenylation site. The pUCSD3 vector was constructed as described previously.S Briefly, the signal peptide containing immunoglobulin heavy chain cDNA clone 17.2.25 is modified by oligonucleotide-directed mutagenesis to create an EcoRI site within the last two codons of the signal peptide sequence. This change in nucleotide sequence maintains the original amino acid sequence of the signal peptide. A 145-base pair (bp) DNA fragment encoding all of the signal peptide sequence, which includes a 79-bp intron, is excised from this modified 17.2.25 clone by cleavage at the newly created EcoRI site at the 3' end and at the AvaII site, 9 nucleotides 5' to the signal peptide initiating ATG codon. Using AvaII-pseudo-SalI and EcoRI-HindIII adapters, this fragment is ligated into a SalI- and HindIII-cleaved pFB 18RI vector to produce pUCDS2. The pFB18RI vector consists of a partial Sau3A fragment containing the Moloney leukemia virus LTR cloned into the BamHI site of pUC 13 that has had the single EcoRI site destroyed by cleaving with EcoRI restriction enzyme, filling-in with the Klenow fragment of DNA polymerase I, and religating with T4 DNA ligase. To provide for the RNA polyadenylation signal, the 155-bp HpaI-BarnHI fragment of the SV40 polyadenylation site was linked to synthetic HpaI-SalI and BarnHI-HindIII adapters and cloned into the SalI and HindIII restriction enzyme-digested pUC8 3' to the EGF gene. The EcoRI-HindIII fragment containing the EGF gene and the polyadenylation site was then cloned into the EcoRI- and HindIII-digested pUCDS2 to create pUCDS3. Basic FGF coding sequences are prepared as follows. The EcoRIflanked bFGF cDNA sequence is digested with NcoI restriction enzyme to remove all of the 5' end and some 350 bp of the 3'-end noncoding sequences (Fig. 1A). Cleavage with this enzyme yields a 1045-bp fragment with a 4-nucleotide overhang. The second, third, and fourth nucleotide of the overhang at the 5' end of the coding sequence specify for the proposed translation initiating ATG codon. The nucleotide overhang is removed by blunting with mung bean nuclease, and the fragment is ligated to 8-met EcoRI linkers and digested with the EcoRI enzyme to create EcoRI sticky ends. At the 5' end of the gene this removal of the NcoI overhang results in elimination of the sequences encoding for the initiating methionine, whereas the EcoRI site containing linker added to the blunted 5' end encodes for a serine which reconstructs the terminal amino acid of the signal sequence. This fragment is then further digested with a unique SspI restriction enzyme to remove an additional 439 bp from the 3'-end noncoding sequence and to create a 3' blunt end. The resulting 608-bp fragment contains 140 bp of the 3'-end noncoding sequence, a 4-nucleotide overhang and 2 bp from the EcoRI linker at the 5' end, and 462 bp of
[11]
TRANSFORMATION WITH SIGNAL P E P T I D E - b F G F
121
the coding sequence for the entire bFGF protein except the initiating methionine which has been removed. To construct an expression vector directing synthesis of a chimeric, immunoglobulin signal peptide containing bFGF protein (spbFGF), the 608-bp bFGF-encoding DNA fragment described above is cloned into the PUCDS3 vector from which the synthetic EGF sequences are removed by digestion with EcoRI and SalI restriction enzymes (Fig. 1B). The fusion joins the immunoglobulin signal peptide to alanine, which is the second amino acid residue of the putative open reading frame of the 18-kDa bFGF molecule. The amino terminus of the primary translation product of the chimeric spbFGF protein is illustrated in Fig. 1C. After translation, the signal peptide should be cleaved off in the endoplasmic reticulum, yielding an 18-kDa bFGF protein which differs from native bFGF only by lacking the first amino acid (Met), thus initiating with an alanine. Control vector pUCDS58 that expresses only the signal peptide sequence was constructed by cleavage of the pUCDS3 vector at the unique EcoRI site, filling-in with the large fragment of DNA polymerase I (Klenow fragment), and religating, resulting in a 4-nucleotide insertion. Transfection The transforming potential of the expression vectors is ascertained by introducing the plasmids into NIH 3T3 cells using the calcium phosphate transfection procedure of Graham and van der E b . 9 The NIH 3T3 cell line is chosen because it responds mitotically to bFGF treatment and is known to express bFGF receptors. A day prior to transfection, 100-mm dishes are seeded with 1 × l06 cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum, penicillin, and streptomycin. Milligram quantities of plasmid DNAs suitable for transfection are prepared by the alkaline lysis method, followed by a further purification on a CsCl gradient as detailed elsewhere, l0 Transfections are carried out using 0. l/~g of the dominant selectablemarked pSV2-neo and 0.8, 8.0, or 40/~g ofpspbFGF plasmid DNA. Carrier NIH 3T3 DNA is added to a final concentration of 75 ~g of DNA per 2 × ]06 cells (2 dishes). Positive control for cellular transformation is obtained using 0.8 t~g of pEJ6.6 plasmid DNA. Jl After a 4-hr exposure to calcium phosphate DNA precipitate, the ceils are glycerol-shocked for 3 min with 9 F. L. Graham and A. J. van der Eb, J. Virol. 52, 456 (1973). I0 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," p. 86. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. ii C. Shih and R. A. Weinberg, Cell (Cambridge, Mass.) 29, 161 0982).
122
FIBROBLASTGROWTHFACTOR
[11]
15% glycerol in phosphate-buffered saline, washed, fed with DMEM containing 10% calf serum, and incubated in 5% CO2 for further 24 hr. The transfected cells are then trypsinized and split in a ratio of 1 : 10 into 100mm dishes. One-half of the dishes are used for the focus forming assay 12 and the other half for the selection of G418-resistant monoclonal cell lines. 13
Focus Forming Assay For the focus forming assay, the cells are fed with DMEM containing 10% calf serum and antibiotics, refed every 5 days, and the foci counted 14, 21, and 28 days after seeding. The control plasmid pUCDS5 is unable to induce focus formation. In contrast, the pspbFGF plasmid induces foci visible to the naked eye within 10 days of transfection. These early foci have a characteristic spindly morphology but are present only at a low frequency of approximately 40 foci per 8 ~g of pspbFGF DNA per 106 cells. However, after an additional 2 weeks in culture, about 20 times more foci with the same characteristic morphology appear. The delayed foci do not arise through seeding of cells from the original foci, as the second wave of focus formation occurs even when the monolayer is overlayed with soft agar (0.6%, w/v) immediately after splitting of the transfected cells. When transfection is carried out using higher quantities of pspbFGF plasmid, fewer early and second-wave colonies are observed, suggesting that pspbFGF has a toxic effect on the transfected cells. For example, when 2 × 106 cells are transfected with 40/zg ofpspbFGF DNA, only 4-7 early colonies are obtained. The number of second-wave foci is still about 20 times the number of the original foci. Control Ha-ras oncogene, which induces focus formation at about 150 colonies per microgram of pEJ6.6 plasmid DNA per 106 cells, does not display such toxicity when larger quantities of plasmid DNA are used for transfection. The nature of this toxicity and delayed focus formation is not understood. Selection of Cell Lines Expressing Chimeric Fibroblast Growth Factor To obtain clonal cell lines synthesizing the chimeric signal pept i d e - b F G F proteins, transfected cells are placed into DMEM containing 10% calf serum supplemented with 0.5 mg/ml of the drug G418. The selective medium is changed every 3 days. Colonies of resistant cells are observed macroscopically after 10 to 14 days. Clonal cell lines are derived 12 H. Land, L. F. Parada, and R. A. Weinberg, Nature (London) 304, 596 (1982). J3 p. j. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327 (1982).
[11]
TRANSFORMATION WITH SIGNAL P E P T I D E - b F G F
123
from these colonies by placing glass cylinders around single colonies, trypsinizing the cells within the cylinder, and transferring the cells to 30-mm dishes. Upon confluency the clonal lines are expanded further. Again, the toxicity of pspbFGF clone is measurable. Addition of 8/zg instead of 0.8/xg of pspbFGF DNA to 0.1 /zg of transfected pSV2-neo DNA causes a 10-fold decrease in the surviving, G418-resistant colonies. A similar inhibitory effect by an oncogenic plasmid has been observed for the Abelson leukemia virus transforming gene. 14 The morphology of some of the G418-resistant pspbFGF-transfected clones is identical to the morphology of the foci seen in the focus forming assay and differs dramatically from the control pSV2-neo-transfected colonies. However, in about 90% of the G418-resistant, pspbFGF-transfected colonies, the spbFGF-induced morphological transformation is delayed. When these colonies are first isolated, they display the normal morphology and express barely detectable bFGF activity. However, during the first few rounds of passage, foci of stable transformed morphology arise in cultures grown from the original clonal cell line. When the cells from these foci of transformed cells are expanded into cell lines, they express higher levels of bFGF than the parental clonal line. This delayed focus formation in the pspbFGF-transfected, G418-resistant clonal cell lines is reproducible and is not caused by cross-contamination with the transformed cells since it occurs in clonal cell lines picked from plates that do not contain any transformed colonies.
Analysis of Basic Fibroblast Growth Factor Expression Basic FGF is measured by a combination of heparin affinity chromatography, bioassay on BALB/c 3T3 cells and/or endothelial cells, and immunoreactivity. These methods have been described in detail. 2'7'15 Briefly, samples are applied to columns of heparin-Sepharose or fast protein liquid chromatography (FPLC) TSK-heparin which are subsequently eluted with a gradient of NaC1. Basic FGF elutes at about 1.5-1.7 M NaC1. Fractions are analyzed for the ability to stimulate DNA synthesis (incorporation of tritiated thymidine) in confluent monolayers of BALB/c 3T3 cells and/or for the ability to stimulate the proliferation of endothelial cells. Active fractions are further analyzed in a Western blot for the ability to interact with anti-bFGF antibodies. Alternatively, cells are labeled with [35S]methionine and the bFGF detected by immunoprecipitation. Signal peptide14 S. F. Ziegler, C. A. Whitlock, S. P. Goff, A. Gifford, and O. N. Witte, Cell (Cambridge, Mass.) 27, 477 (1981). t5 R. Sullivan and M. Klagsbrun, J. Tissue Cult. Methods 10~ 125 (1986).
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FIBROBLAST GROWTH FACTOR
[12]
bFGF-transfected N I H 3T3 cells usually express about 0.8 units of b F G F per l 0 4 cells. It is thought that higher levels of expression are toxic to cells. Tumorigenicity Signal p e p t i d e - b F G F - t r a n s f o r m e d cells are highly tumorigenic in syngeneic m i c e ) Large tumors ( - 1 cm in diameter) appear within 2 weeks in 100% of the animals injected subcutaneously with 2 × 106 cells. The size of the tumors and their rate of growth are comparable to Ha-rastransfected N I H 3T3 cells. When the tumor tissue is cultured, G418resistant cell lines with the characteristic transformed morphology are obtained. These tumor-derived cell lines still express immunoprecipitable, heparin-binding, biologically active b F G F . 2'3 Acknowledgments The authors are grateful to Robert A. Weinberg in whose laboratory most of this work was done. We thank David L. Hare who collaborated in design and construction of pUCDS3. Financial support was provided in part by grants from the National Institutes of Health (EY05321 to R.A.W., CA37392 and CA45548 to M.K., and CA07813 to S.R.).
[12] I d e n t i f i c a t i o n a n d C h a r a c t e r i z a t i o n Factor-Related Transforming
of Fibroblast Growth G e n e hst-1
B y T E R U H I K O Y O S H I D A , KIYOSHI M I Y A G A W A , HIROMI SAKAMOTO,
TAKASHI S U G I M U R A , and MASAAKI TERADA
Introduction In contrast to many other classic peptide growth factors, the hst-1 transforming gene rather than its protein was identified first. ~The fact that the gene encodes a growth factor was then presumed from a deduced amino acid sequence 2 and from its remarkable sequence similarity to fibroblast growth factors (FGF). 3 Finally, growth factor activity was conI H. Sakamoto, M. Moil, M. Taira, T. Yoshida, S. Matsukawa, K. Shimizu, M. Sekiguchi, M. Terada, and T. Sugimura, Proc. Natl. Acad. Sci. U.S.A. 83, 3997 (1986). 2 M. Taira, T. Yoshida, K. Miyagawa, H. Sakamoto, M. Terada, and T. Sugimura, Proc. Natl. Acad. Sci. U.S.A, 84, 2980 (1987). 3 T. Yoshida, K. Miyagawa, H. Odagiri, H. Sakamoto, P. F. R. Little, M. Terada, and T. Sugimura, Proc. Natl. Acad. Sci. U.S.A. 84, 7305 (1987).
METHODS IN ENZYMOLOGY,VOL. 198
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any form reserved.
TRANSFORMATION WITH SIGNALPEPTIDE-bFGF
117
[11] Construction and Expression of Transforming Gene Resulting from Fusion of Basic Fibroblast Growth Factor
Gene with Signal Peptide Sequence B y SNEZNA ROGELJ, DAVID
STERN, and MICHAEL KLAGSBRUN
Introduction Basic fibroblast growth factor (bFGF) is a potent mitogen for cells of m e s o d e r m a l origin.l Basic F G F lacks a signal peptide and is not secreted by cells in culture. M a n y cell types, for example, endothelial cells, produce b F G F and h a v e b F G F receptors. Yet these cells a p p e a r normal and do not s e e m to undergo F G F - m e d i a t e d autocrine transformation. N I H 3T3 fibroblasts transfected with b F G F c D N A do not secrete b F G F ; they appear normal in culture and are not tumorigenic. 2'3 It has been suggested that lack of b F G F secretion prevents b F G F - r e c e p t o r interaction with the result that b F G F - p r o d u c i n g cells have a normal, nontransforming p h e n o t y p e . On the other hand, N I H 3T3 cells transfected with a construct of b F G F fused to a signal peptide sequence (spbFGF) are highly transformed morphologically, grow as nonadherent aggregates, and are highly tumorigenic and metastatic. 2-4 Thus, b F G F altered by the addition of a singal sequence is a transforming protein. Although s p b F G F transforms N I H 3T3 cells, there is no evidence of b F G F secretion, suggesting that transformation in these cells is the result of an internal autocrine loop. Overall Strategy The transforming b F G F expression v e c t o r p s p b F G F is constructed by fusing a sequence encoding the N-terminal 19 amino acid mouse h e a v y chain immunoglobulin signal sequence 5 u p s t r e a m to the 154 amino acid bovine brain b F G F sequence 6 (Fig. 1C). These sequences are placed under the transcriptional control of the Moloney leukemia virus long terminal i j. Folkman and M. Klagsbrun, Science 235, 442 (1987). 2 S. Rogelj, R. A. Weinberg, P. Fanning, and M. Klagsbrun, Nature (London) 331, 173 (1988). 3 S. Rogelj, R. A. Weinberg, P. Fanning, and M. Klagsbrun, J. Cell. Biochern. 39, 13 (1989). 4 A. Yayon and M. Klagsbrun, Proc. Natl. Acad. Sci. U.S.A. 87, 5346 (1990). 5 D. Y. Loh, A. L. M. Bothwell, M. E. White-Scharf, T. Imanishi-Kari, and D. Baltimore, Cell (Cambridge, Mass. ) 33, 85 (1983). 6 j. A. Abraham, A. Mergia, J. L. Whang, A. Tumolo, J. Friedman, K. A. Hjerrild, D. Gospodarowicz, and J. C. Fiddes, Science 233, 545 (1986).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
FIBROBLAST GROWTH FACTOR
[11] -
i
I
el
~1 ~S'UTICodinll Sequence [
~'UT
I
I!~5
1285
I
l
II0
71=6
[3' 1408
AmpR
pIIRori
' 5oo
"
U ,-
K W ' I IpWgl..q.
,82
1,01.,.
v
2so /
C
--/9MKCSWVIFFLMAVv515 I0
;:G~IT TL~oA ~24-
FIG. 1. (A) Restriction map of bovine brain basic FGF cDNA. (B) pspbFGF plasmid: The Moloney murine leukemia virus long terminal repeat (Mo-MuLV LTR) drives constitutive expression of the chimeric signal peptide-bFGF fusion protein. (C) Amino terminus of the predicted primary translation product of the signal peptide-basic FGF protein. The 19 amino acid mouse immunoglobulin heavy chain signal peptide is fused to the second amino acid (alanine) of bovine brain bFGF.
repeat (LTR) and are followed by RNA processing signals derived from the SV40 genome. Cloning of this functional composite gene is performed in a pUC 13-derived vector which contains the pBR322 origin of replication and the ampicillin resistance gene, thus allowing for growth of the mammalian vector in bacteria in the presence of ampicillin. The pspbFGF vector is introduced into NIH 3T3 fibroblasts by the calcium phosphate transfection procedure. Transforming potential is measured by the focus forming assay. Clonal cell lines are obtained by cotrans-
[11]
TRANSFORMATION WITH SIGNALPEPTIDE-bFGF
119
fecting the pspbFGF vector with the dominant selectable marker pSVneo. G418-resistant colonies are picked and expanded into cell lines which are assayed for the production of growth factor activity as measured by the ability of cell extracts and/or conditioned media to stimulate the incorporation of [3H]thymidine into BALB/c 3T3 DNA. Cell lines positive for the synthesis of growth factor activity are analyzed for specific bFGF expression by heparin affinity chromatography and by immunodetection (immunoprecipitation and Western blot) using specific anti-bFGF antibodies. 2,7 Materials A bovine bFGF cDNA clone was obtained from Dr. Judith Abraham (CalBio, Mountain View, CA). 6 pSV2-neo was obtained from Dr. Robert Weinberg (The Whitehead Institute for Biomedical Research, Cambridge, MA). Restriction enzymes, mung bean nuclease, and polymerases were obtained from New England BioLabs (Beverly, MA). G418 is obtained from GIBCO Laboratories (Grand Island, NY). Heparin-Sepharose is obtained from Pharmacia (Piscataway, N J). Construction of pspbFGF Expression Vectors A mammalian vector directing synthesis of a chimeric signal peptide-bFGF is constructed by replacing the epidermal growth factor (EGF) sequence for the bFGF sequence in the pUCDS3 vector. This vector was originally designed in collaboration with Dr. David L. Hare (Amgen Development Corp., Boulder, CO), for the expression and secretion of synthetic EGF fused to a signal peptide sequence in mammalian cells. 8 pUCDS3 consists of a Moloney murine leukemia virus long terminal repeat (MuLV LTR) that contains promoter/enhancer sequences, an SV40 polyadenylation site, sequences encoding a mouse immunoglobulin heavy chain signal peptide, 5 and chemically synthesized human EGF-encoding sequences. The signal peptide-encoding sequences are included so that the protein product is translocated to the endoplasmic reticulum (ER)/ Golgi compartment for subsequent secretion or expression on the cell surface, pUCDS3 was designed so that the coding sequences to be expressed are joined at the 5' end to a unique EcoRI site located just above 7 I. Vlodavsky, J. Folkman, R. Sullivan, R. Friedman, R. Ishai-Michaeli, J. Sasse, and M. Klagsbrun, Proc. Natl. Acad. Sci. U.S.A. 84, 2292 (1987). s D. F. Stern, D. L. Hare, M. A. Cecchini, and R. A. Weinberg, Science 235, 321 (1987).
120
FIBROBLAST GROWTH FACTOR
[1 1]
the signal peptidase cleavage site of the signal peptide and to a unique 3' SalI site located 5' to the polyadenylation site. The pUCSD3 vector was constructed as described previously.S Briefly, the signal peptide containing immunoglobulin heavy chain cDNA clone 17.2.25 is modified by oligonucleotide-directed mutagenesis to create an EcoRI site within the last two codons of the signal peptide sequence. This change in nucleotide sequence maintains the original amino acid sequence of the signal peptide. A 145-base pair (bp) DNA fragment encoding all of the signal peptide sequence, which includes a 79-bp intron, is excised from this modified 17.2.25 clone by cleavage at the newly created EcoRI site at the 3' end and at the AvaII site, 9 nucleotides 5' to the signal peptide initiating ATG codon. Using AvaII-pseudo-SalI and EcoRI-HindIII adapters, this fragment is ligated into a SalI- and HindIII-cleaved pFB 18RI vector to produce pUCDS2. The pFB18RI vector consists of a partial Sau3A fragment containing the Moloney leukemia virus LTR cloned into the BamHI site of pUC 13 that has had the single EcoRI site destroyed by cleaving with EcoRI restriction enzyme, filling-in with the Klenow fragment of DNA polymerase I, and religating with T4 DNA ligase. To provide for the RNA polyadenylation signal, the 155-bp HpaI-BarnHI fragment of the SV40 polyadenylation site was linked to synthetic HpaI-SalI and BarnHI-HindIII adapters and cloned into the SalI and HindIII restriction enzyme-digested pUC8 3' to the EGF gene. The EcoRI-HindIII fragment containing the EGF gene and the polyadenylation site was then cloned into the EcoRI- and HindIII-digested pUCDS2 to create pUCDS3. Basic FGF coding sequences are prepared as follows. The EcoRIflanked bFGF cDNA sequence is digested with NcoI restriction enzyme to remove all of the 5' end and some 350 bp of the 3'-end noncoding sequences (Fig. 1A). Cleavage with this enzyme yields a 1045-bp fragment with a 4-nucleotide overhang. The second, third, and fourth nucleotide of the overhang at the 5' end of the coding sequence specify for the proposed translation initiating ATG codon. The nucleotide overhang is removed by blunting with mung bean nuclease, and the fragment is ligated to 8-met EcoRI linkers and digested with the EcoRI enzyme to create EcoRI sticky ends. At the 5' end of the gene this removal of the NcoI overhang results in elimination of the sequences encoding for the initiating methionine, whereas the EcoRI site containing linker added to the blunted 5' end encodes for a serine which reconstructs the terminal amino acid of the signal sequence. This fragment is then further digested with a unique SspI restriction enzyme to remove an additional 439 bp from the 3'-end noncoding sequence and to create a 3' blunt end. The resulting 608-bp fragment contains 140 bp of the 3'-end noncoding sequence, a 4-nucleotide overhang and 2 bp from the EcoRI linker at the 5' end, and 462 bp of
[11]
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121
the coding sequence for the entire bFGF protein except the initiating methionine which has been removed. To construct an expression vector directing synthesis of a chimeric, immunoglobulin signal peptide containing bFGF protein (spbFGF), the 608-bp bFGF-encoding DNA fragment described above is cloned into the PUCDS3 vector from which the synthetic EGF sequences are removed by digestion with EcoRI and SalI restriction enzymes (Fig. 1B). The fusion joins the immunoglobulin signal peptide to alanine, which is the second amino acid residue of the putative open reading frame of the 18-kDa bFGF molecule. The amino terminus of the primary translation product of the chimeric spbFGF protein is illustrated in Fig. 1C. After translation, the signal peptide should be cleaved off in the endoplasmic reticulum, yielding an 18-kDa bFGF protein which differs from native bFGF only by lacking the first amino acid (Met), thus initiating with an alanine. Control vector pUCDS58 that expresses only the signal peptide sequence was constructed by cleavage of the pUCDS3 vector at the unique EcoRI site, filling-in with the large fragment of DNA polymerase I (Klenow fragment), and religating, resulting in a 4-nucleotide insertion. Transfection The transforming potential of the expression vectors is ascertained by introducing the plasmids into NIH 3T3 cells using the calcium phosphate transfection procedure of Graham and van der E b . 9 The NIH 3T3 cell line is chosen because it responds mitotically to bFGF treatment and is known to express bFGF receptors. A day prior to transfection, 100-mm dishes are seeded with 1 × l06 cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum, penicillin, and streptomycin. Milligram quantities of plasmid DNAs suitable for transfection are prepared by the alkaline lysis method, followed by a further purification on a CsCl gradient as detailed elsewhere, l0 Transfections are carried out using 0. l/~g of the dominant selectablemarked pSV2-neo and 0.8, 8.0, or 40/~g ofpspbFGF plasmid DNA. Carrier NIH 3T3 DNA is added to a final concentration of 75 ~g of DNA per 2 × ]06 cells (2 dishes). Positive control for cellular transformation is obtained using 0.8 t~g of pEJ6.6 plasmid DNA. Jl After a 4-hr exposure to calcium phosphate DNA precipitate, the ceils are glycerol-shocked for 3 min with 9 F. L. Graham and A. J. van der Eb, J. Virol. 52, 456 (1973). I0 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," p. 86. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. ii C. Shih and R. A. Weinberg, Cell (Cambridge, Mass.) 29, 161 0982).
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15% glycerol in phosphate-buffered saline, washed, fed with DMEM containing 10% calf serum, and incubated in 5% CO2 for further 24 hr. The transfected cells are then trypsinized and split in a ratio of 1 : 10 into 100mm dishes. One-half of the dishes are used for the focus forming assay 12 and the other half for the selection of G418-resistant monoclonal cell lines. 13
Focus Forming Assay For the focus forming assay, the cells are fed with DMEM containing 10% calf serum and antibiotics, refed every 5 days, and the foci counted 14, 21, and 28 days after seeding. The control plasmid pUCDS5 is unable to induce focus formation. In contrast, the pspbFGF plasmid induces foci visible to the naked eye within 10 days of transfection. These early foci have a characteristic spindly morphology but are present only at a low frequency of approximately 40 foci per 8 ~g of pspbFGF DNA per 106 cells. However, after an additional 2 weeks in culture, about 20 times more foci with the same characteristic morphology appear. The delayed foci do not arise through seeding of cells from the original foci, as the second wave of focus formation occurs even when the monolayer is overlayed with soft agar (0.6%, w/v) immediately after splitting of the transfected cells. When transfection is carried out using higher quantities of pspbFGF plasmid, fewer early and second-wave colonies are observed, suggesting that pspbFGF has a toxic effect on the transfected cells. For example, when 2 × 106 cells are transfected with 40/zg ofpspbFGF DNA, only 4-7 early colonies are obtained. The number of second-wave foci is still about 20 times the number of the original foci. Control Ha-ras oncogene, which induces focus formation at about 150 colonies per microgram of pEJ6.6 plasmid DNA per 106 cells, does not display such toxicity when larger quantities of plasmid DNA are used for transfection. The nature of this toxicity and delayed focus formation is not understood. Selection of Cell Lines Expressing Chimeric Fibroblast Growth Factor To obtain clonal cell lines synthesizing the chimeric signal pept i d e - b F G F proteins, transfected cells are placed into DMEM containing 10% calf serum supplemented with 0.5 mg/ml of the drug G418. The selective medium is changed every 3 days. Colonies of resistant cells are observed macroscopically after 10 to 14 days. Clonal cell lines are derived 12 H. Land, L. F. Parada, and R. A. Weinberg, Nature (London) 304, 596 (1982). J3 p. j. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327 (1982).
[11]
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from these colonies by placing glass cylinders around single colonies, trypsinizing the cells within the cylinder, and transferring the cells to 30-mm dishes. Upon confluency the clonal lines are expanded further. Again, the toxicity of pspbFGF clone is measurable. Addition of 8/zg instead of 0.8/xg of pspbFGF DNA to 0.1 /zg of transfected pSV2-neo DNA causes a 10-fold decrease in the surviving, G418-resistant colonies. A similar inhibitory effect by an oncogenic plasmid has been observed for the Abelson leukemia virus transforming gene. 14 The morphology of some of the G418-resistant pspbFGF-transfected clones is identical to the morphology of the foci seen in the focus forming assay and differs dramatically from the control pSV2-neo-transfected colonies. However, in about 90% of the G418-resistant, pspbFGF-transfected colonies, the spbFGF-induced morphological transformation is delayed. When these colonies are first isolated, they display the normal morphology and express barely detectable bFGF activity. However, during the first few rounds of passage, foci of stable transformed morphology arise in cultures grown from the original clonal cell line. When the cells from these foci of transformed cells are expanded into cell lines, they express higher levels of bFGF than the parental clonal line. This delayed focus formation in the pspbFGF-transfected, G418-resistant clonal cell lines is reproducible and is not caused by cross-contamination with the transformed cells since it occurs in clonal cell lines picked from plates that do not contain any transformed colonies.
Analysis of Basic Fibroblast Growth Factor Expression Basic FGF is measured by a combination of heparin affinity chromatography, bioassay on BALB/c 3T3 cells and/or endothelial cells, and immunoreactivity. These methods have been described in detail. 2'7'15 Briefly, samples are applied to columns of heparin-Sepharose or fast protein liquid chromatography (FPLC) TSK-heparin which are subsequently eluted with a gradient of NaC1. Basic FGF elutes at about 1.5-1.7 M NaC1. Fractions are analyzed for the ability to stimulate DNA synthesis (incorporation of tritiated thymidine) in confluent monolayers of BALB/c 3T3 cells and/or for the ability to stimulate the proliferation of endothelial cells. Active fractions are further analyzed in a Western blot for the ability to interact with anti-bFGF antibodies. Alternatively, cells are labeled with [35S]methionine and the bFGF detected by immunoprecipitation. Signal peptide14 S. F. Ziegler, C. A. Whitlock, S. P. Goff, A. Gifford, and O. N. Witte, Cell (Cambridge, Mass.) 27, 477 (1981). t5 R. Sullivan and M. Klagsbrun, J. Tissue Cult. Methods 10~ 125 (1986).
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bFGF-transfected N I H 3T3 cells usually express about 0.8 units of b F G F per l 0 4 cells. It is thought that higher levels of expression are toxic to cells. Tumorigenicity Signal p e p t i d e - b F G F - t r a n s f o r m e d cells are highly tumorigenic in syngeneic m i c e ) Large tumors ( - 1 cm in diameter) appear within 2 weeks in 100% of the animals injected subcutaneously with 2 × 106 cells. The size of the tumors and their rate of growth are comparable to Ha-rastransfected N I H 3T3 cells. When the tumor tissue is cultured, G418resistant cell lines with the characteristic transformed morphology are obtained. These tumor-derived cell lines still express immunoprecipitable, heparin-binding, biologically active b F G F . 2'3 Acknowledgments The authors are grateful to Robert A. Weinberg in whose laboratory most of this work was done. We thank David L. Hare who collaborated in design and construction of pUCDS3. Financial support was provided in part by grants from the National Institutes of Health (EY05321 to R.A.W., CA37392 and CA45548 to M.K., and CA07813 to S.R.).
[12] I d e n t i f i c a t i o n a n d C h a r a c t e r i z a t i o n Factor-Related Transforming
of Fibroblast Growth G e n e hst-1
B y T E R U H I K O Y O S H I D A , KIYOSHI M I Y A G A W A , HIROMI SAKAMOTO,
TAKASHI S U G I M U R A , and MASAAKI TERADA
Introduction In contrast to many other classic peptide growth factors, the hst-1 transforming gene rather than its protein was identified first. ~The fact that the gene encodes a growth factor was then presumed from a deduced amino acid sequence 2 and from its remarkable sequence similarity to fibroblast growth factors (FGF). 3 Finally, growth factor activity was conI H. Sakamoto, M. Moil, M. Taira, T. Yoshida, S. Matsukawa, K. Shimizu, M. Sekiguchi, M. Terada, and T. Sugimura, Proc. Natl. Acad. Sci. U.S.A. 83, 3997 (1986). 2 M. Taira, T. Yoshida, K. Miyagawa, H. Sakamoto, M. Terada, and T. Sugimura, Proc. Natl. Acad. Sci. U.S.A, 84, 2980 (1987). 3 T. Yoshida, K. Miyagawa, H. Odagiri, H. Sakamoto, P. F. R. Little, M. Terada, and T. Sugimura, Proc. Natl. Acad. Sci. U.S.A. 84, 7305 (1987).
METHODS IN ENZYMOLOGY,VOL. 198
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