InVitroCell.Dev.Biol.27A:89-96,January1991 © 1991TissueCuhureAssociation 0883-8364/91 $01.50+0.00
TRANSFORMED PHENOTYPE CONFERRED TO N I H / 3 T 3 CELLS BY ECTOPIC EXPRESSION OF HEPARIN-BINDING GROWTH FACTOR 1/ACIDIC FIBROBLAST GROWTH FACTOR PHANPIMOL BUNNAG, KAREN S. WADDELL, M. LEE VARBAN, AND ING-MING CHIU1 Department of lnternal Medicine and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210 (Accepted 2 November 1990)
SUMMARY Heparin-binding growth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen and angiogenic factor found in tissues such as brain, kidney and heart. The genomic and cDNA sequences indicate that HBGF-1 does not have a typical signal peptide sequence. HBGF-1 was shown to be localized to the extracellular matrix of cardiac myocytes, but the mechanism of secretion is not presently known. We have cloned the HBGF-1 cDNA which allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. A previous report showed that the truncated HBGF-1 confers partial transformed phenotype to the recipient fibroblasts. However, expression of full-length HBGF-1 has not been reported. The HBGF-1 coding sequence was cloned into the retroviral expression vector, SVX, and transfected into NIH/3T3 cells. Transfectants expressing full-length HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 is a unique band of approximately 20 kDa and can be detected in the cell homogenate but not in the conditioned medium. NIH/3T3 cells were conferred anchorage independence when HBGF-1 was provided exogenously. We showed the transformed cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfected cells expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus, NIH/3T3 cells acquired a full spectrum of transformed phenotype when full length HBGF-1 was expressed at high levels. Key words: aFGF; cellular transformation; NIH/3T3 cells; tumorigenesis; cDNA. predicted from the cDNA sequence (Chiu et al., 1990; Jaye et al., 1986). This was also confirmed by the amino acid sequence deduced from the genomic DNA sequence (Wang et al., 1989). These data suggest that HBGF-1 does not have a typical signal peptide. HBGF-1 may be localized to the extracellular matrix but the mechanism of secretion is not well understood (Weiner and Swain, 1989). The two smaller HBGF-1 proteins have deletions of the first 15 or 21 amino acid residues, respectively (Gimenez-Gallego et al., 1985). It is not clear whether these truncated forms represent proteolytic cleavage products in vivo or degradative products during the purification process. All three peptides have been shown to possess similar mitogenic activities when assayed using normal fibroblasts or endothelial cells (Burgess et al., 1986; Crabb et al., 1986; Gimenez-Gallego et at., 1985; Gospodarowicz, 1987). The study of biological factors that mediate cell growth is convergent with the study of oncogenes. This concept was strengthened by the report of identity between the structural gene for the B chain of platelet-derived growth factor and the c-sis proto-oncogene (Chiu et al., 1984). Subsequently, the receptors for epidermal growth factor and monocyte colony-stimulating factor have also been shown to be the proto-oncogene products of c-erbB (Downward et al., 1984) and c-fms (Sherr et al., 1985), respectively. A variety of growth factor genes, which have not been transduced by retroviruses, have been shown to become oncogenes upon activation (Chiu, 1989). These
INTRODUCTION Heparin-bindinggrowth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a polypeptide mitogen for a variety of mesenchymal and neuroectodermal cells (Folkman and Klagsbrun, 1987; Gospodarowicz, 1987) as well as prostatic epithelial cells (Crabb et al., 1986). It has been isolated from brain, kidney, and heart (Chiu et at., 1990). A variety of cell lines, including smooth muscle cells (Winkles et al., 1987), glioblastoma cells (Libermann et al., 1987; Wang et at., 1989) and two other human tumor cell lines (Lobb et at., 1986) have also been shown to express this mitogen. In both glioblastoma cells and smooth muscle cells, HBGF-1 serves as an autologous mitogen (Libermann et at., 1987; Winkles et at., 1987). HBGF-1 was also shown to be an angiogenic factor in vivo, and may be involved in pathological conditions, such as atherosclerosis, inflammation, diabetic retinopathy and cancer (Folkman and Klagsbrun, 1987). Three different forms of HBGF-1 have been isolated from different sources. The largest HBGF-1 protein (Burgess et al., 1986; Crabb et al., 1986) has a deletion of the first methionine residue as
t To whomrequests for reprints should be addressed at The Ohio State University,DavisMedicalResearch Center, Room $2052, 480 West Ninth Avenue, Columbus,OH 43210. 89
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BUNNAG ET AL.
gene products include granulocyte-macrophage colony stimulating factor (Lang et al., 1985), transforming growth factor a (Rosenthal et al., 1986), epidermal growth factor (Stern et al., 1987), and HBGF-2 (Blara et al., 1988; Neufeld et al., 1988; Rogelj et al., 1988; Sasada et al., 1988: Quarto et al., 1989). A truncated form of HBGF-1 has also been shown to partially transform NR6 cells (Jaye et al., 1988), a Swiss/3T3 mutant lacking epidermal growth factor receptor (Hung et al., 1986). Furthermore, at least three different oncogenes: int-2, hst/KS3 and FGF-5 have been shown to be members of the HBGF family (for a review, see Burgess and Maciag, 1989). However, expression of the full-length HBGF-1 was not possible (Jaye et al., 1987; 1988) and its transforming potential has yet to be manifested. The availability of the full-length cDNA clones of HBGF-1 (Chiu et al., 1990) allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. We have constructed the HBGF-1 coding sequence into the retroviral expression vector, SVX (Cepko et al., 1984). The expression construct was transfected into NIH/3T3 cells. Transfectants expressing HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 protein is a unique band of approximately 20 kDa and can he detected in the cell homogenate but not the conditioned medium. HBGF-1, when provided exogenously, conferred anchorage independence to NIH/ 3T3 cells. We showed that the transfected cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfectants expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus, a full spectrum of transformed phenotype is conferred to NIH/3T3 cells by ectopic expression of HBGF-1. These cell lines will be used to further characterize the mechanism of secretion and biochemical action of HBGF-1. MATERIALSAND METHODS
Plasmid DNA construction. All procedures used in cloning were essentially as described (Maniatis et al., 1982). The 355 bp BamHI-Bgl II fragment derived from pHBGF 1.1, comprised of the 3'-end of the protein-coding sequence (Chiu et al., 1990), was cloned into the unique BamHI site of SVX (Cepko et al., 1984). The 163 bp BamHI-BamH1 fragment derived from pHBGF 1.2, which contained the 5'-end of the coding region (Chiu et al., 1990), was then cloned into the unique BamHI site of the resultant plasmid and designated pSVX/HBGFI(+) (Fig. 1). Transfection. NIH/3T3 cells were transfected with expression vector DNA using the calcium phosphate precipitation method (Parker and Stark, 1979; Pulciani et al., 1982). To obtain more G418-resistant colonies, the expression vector was co-transfected with pSV2neo (Southern and Berg, 1987) in a 10:1 ratio in later experiments. The transfected cells were selected by culturing in medium containing 400 gg/ml of G418 (GIBCO). Genomic DNA was isolated from G418-resistant cell lines and analyzed by the method of Southern (Southern, 1975) using the 508 bp EcoRIBgllI fragment of HBGF-1 cDNA as a probe (Chiu et al., 1990). RNA was isolated using the guanidinium thiocyanate method (Chirgwin et al., 1979) and characterized by Northern hybridization (Maniatis et al., 1982). RNase protection analysis was performed as described using the KpnI transcript of pHBgl.0 template (Wang et al., 1989).
Cell density, focus formation and protein purification. Transfected cells (2 X 104 per well) were seeded in 6-well plates and cell numbers at confluency were determined after 14 days of culturing. For focus formation, transfected cells were mixed with NIH/3T3 cells in various ratios (1 × 105 per 10 cm plate) and incubated for three weeks and the number of foci was determined. For protein purification, cell homogenate from ten 10 cm plates was centrifuged at 10 000 ×g for 20 min and the supernatant or the medium conditioned from the transfectants was fractionated through a heparin-Sepharose CL-6B (Pharmacia) column as described (Winkles, et al., 1987). Fractions of 2.5 ml were collected and were monitored at 280 nm. Each fraction was stored at - 8 0 ° C for subsequent mitogenic assays. Mitogen assay. Growth factor activity was determined by measuring the incorporation of [3H]thymidine into the DNA of serumstarved Swiss/3T3 ceils. Ten/xl from each fraction of the heparinSepharose purified protein was added to the starved cells. Cells were labeled for 6 h with 1 gCi [3H]thymidine starting at 20 h after addition of mitogen. The labeled DNA was precipitated with trichloroacetic acid and the amount of incorporated [3H]thymidine was determined. The commercially available bovine HBGF-1 preparation (R&D Systems, Minneapolis) containing only the two truncated forms of the protein was used as a positive control. Western blotting analysis and protein labeling. HeparinSepharose purified fractions were concentrated in 10 mM sodium phosphate buffer, pH 7.2 using Centricon-3 membrane (Amicon). One tenth of the protein from each fraction was analyzed on a 15% SDS polyacrytamide gel (Laemmli, 1970) and transferred to a nitrocellulose filter. The filter was reacted first with 1:200 dilution of a rabbit anti bovine HBGF-1 antibody (generously provided by Tom Maciag, American Red Cross) then with 1:3000 dilution of goat antirabbit IgG conjugated with horseradish peroxidase (Bio-Rad). For protein labeling, transfected cells were grown to 80% confluency in a T150 flask and labeled with 1 mCi [3SS]methionine for 24 h. After incubation, both the conditioned media and cell lysates were purified using heparin-Sepharose chromatography and analyzed on a 15% SDS polyacrylamide gel. Anchorage independence and tumorigenicityassays. Anchorageindependent growth was assayed essentially as described (Rizzino, 1987). Cells (5 × 104 per well) were seeded in duplicate on 0.5% agar in the presence or absence of 5 ng/ml bovine HBGF-1 and 5 U/ml heparin. After incubation for 14 days, cell colonies were stained overnight with p-iodonitrotetrazolium violet (Sigma). Colony numbers from five different fields per well were determined using a Nikon Diaphot microscope. Capacity of transfected cell lines to induce tumor formation was also tested by injecting 1 × 106 cells subcutaneously into nude mice. NIH/3T3 cells were injected as a negative control. The nude mice were observed for tumor formation weekly for up to 3 months. RESULTS
HBGF-1 expression vector, pSVX/HBGF-I(+), was constructed by cloning the HBGF-1 cDNA coding sequence (Chiu et al., 1990) into the SVX vector (Cepko et al., 1984) which was derived from Moloney murine leukemia virus (Fig. 1). The viral long terminal repeat provided the promoter for both the HBGF-1 cDNA and G418 selection marker. To generate more G418-resistant colonies, the HBGF-1 expression vectors were co-transfected with pSV2neo (Southern and Berg, 1987). G418-resistant colonies were isolated
91
TRANSFORMING POTENTIAL OF HBGF-1/aFGF
BomH!
EcoRT B g / B m EcoRI
!
---H---
TRANSFORMED PHENOTYPE CONFERRED TO N I H / 3 T 3 CELLS BY ECTOPIC EXPRESSION OF HEPARIN-BINDING GROWTH FACTOR 1/ACIDIC FIBROBLAST GROWTH FACTOR PHANPIMOL BUNNAG, KAREN S. WADDELL, M. LEE VARBAN, AND ING-MING CHIU1 Department of lnternal Medicine and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210 (Accepted 2 November 1990)
SUMMARY Heparin-binding growth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen and angiogenic factor found in tissues such as brain, kidney and heart. The genomic and cDNA sequences indicate that HBGF-1 does not have a typical signal peptide sequence. HBGF-1 was shown to be localized to the extracellular matrix of cardiac myocytes, but the mechanism of secretion is not presently known. We have cloned the HBGF-1 cDNA which allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. A previous report showed that the truncated HBGF-1 confers partial transformed phenotype to the recipient fibroblasts. However, expression of full-length HBGF-1 has not been reported. The HBGF-1 coding sequence was cloned into the retroviral expression vector, SVX, and transfected into NIH/3T3 cells. Transfectants expressing full-length HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 is a unique band of approximately 20 kDa and can be detected in the cell homogenate but not in the conditioned medium. NIH/3T3 cells were conferred anchorage independence when HBGF-1 was provided exogenously. We showed the transformed cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfected cells expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus, NIH/3T3 cells acquired a full spectrum of transformed phenotype when full length HBGF-1 was expressed at high levels. Key words: aFGF; cellular transformation; NIH/3T3 cells; tumorigenesis; cDNA. predicted from the cDNA sequence (Chiu et al., 1990; Jaye et al., 1986). This was also confirmed by the amino acid sequence deduced from the genomic DNA sequence (Wang et al., 1989). These data suggest that HBGF-1 does not have a typical signal peptide. HBGF-1 may be localized to the extracellular matrix but the mechanism of secretion is not well understood (Weiner and Swain, 1989). The two smaller HBGF-1 proteins have deletions of the first 15 or 21 amino acid residues, respectively (Gimenez-Gallego et al., 1985). It is not clear whether these truncated forms represent proteolytic cleavage products in vivo or degradative products during the purification process. All three peptides have been shown to possess similar mitogenic activities when assayed using normal fibroblasts or endothelial cells (Burgess et al., 1986; Crabb et al., 1986; Gimenez-Gallego et at., 1985; Gospodarowicz, 1987). The study of biological factors that mediate cell growth is convergent with the study of oncogenes. This concept was strengthened by the report of identity between the structural gene for the B chain of platelet-derived growth factor and the c-sis proto-oncogene (Chiu et al., 1984). Subsequently, the receptors for epidermal growth factor and monocyte colony-stimulating factor have also been shown to be the proto-oncogene products of c-erbB (Downward et al., 1984) and c-fms (Sherr et al., 1985), respectively. A variety of growth factor genes, which have not been transduced by retroviruses, have been shown to become oncogenes upon activation (Chiu, 1989). These
INTRODUCTION Heparin-bindinggrowth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a polypeptide mitogen for a variety of mesenchymal and neuroectodermal cells (Folkman and Klagsbrun, 1987; Gospodarowicz, 1987) as well as prostatic epithelial cells (Crabb et al., 1986). It has been isolated from brain, kidney, and heart (Chiu et at., 1990). A variety of cell lines, including smooth muscle cells (Winkles et al., 1987), glioblastoma cells (Libermann et al., 1987; Wang et at., 1989) and two other human tumor cell lines (Lobb et at., 1986) have also been shown to express this mitogen. In both glioblastoma cells and smooth muscle cells, HBGF-1 serves as an autologous mitogen (Libermann et at., 1987; Winkles et at., 1987). HBGF-1 was also shown to be an angiogenic factor in vivo, and may be involved in pathological conditions, such as atherosclerosis, inflammation, diabetic retinopathy and cancer (Folkman and Klagsbrun, 1987). Three different forms of HBGF-1 have been isolated from different sources. The largest HBGF-1 protein (Burgess et al., 1986; Crabb et al., 1986) has a deletion of the first methionine residue as
t To whomrequests for reprints should be addressed at The Ohio State University,DavisMedicalResearch Center, Room $2052, 480 West Ninth Avenue, Columbus,OH 43210. 89
90
BUNNAG ET AL.
gene products include granulocyte-macrophage colony stimulating factor (Lang et al., 1985), transforming growth factor a (Rosenthal et al., 1986), epidermal growth factor (Stern et al., 1987), and HBGF-2 (Blara et al., 1988; Neufeld et al., 1988; Rogelj et al., 1988; Sasada et al., 1988: Quarto et al., 1989). A truncated form of HBGF-1 has also been shown to partially transform NR6 cells (Jaye et al., 1988), a Swiss/3T3 mutant lacking epidermal growth factor receptor (Hung et al., 1986). Furthermore, at least three different oncogenes: int-2, hst/KS3 and FGF-5 have been shown to be members of the HBGF family (for a review, see Burgess and Maciag, 1989). However, expression of the full-length HBGF-1 was not possible (Jaye et al., 1987; 1988) and its transforming potential has yet to be manifested. The availability of the full-length cDNA clones of HBGF-1 (Chiu et al., 1990) allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. We have constructed the HBGF-1 coding sequence into the retroviral expression vector, SVX (Cepko et al., 1984). The expression construct was transfected into NIH/3T3 cells. Transfectants expressing HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 protein is a unique band of approximately 20 kDa and can he detected in the cell homogenate but not the conditioned medium. HBGF-1, when provided exogenously, conferred anchorage independence to NIH/ 3T3 cells. We showed that the transfected cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfectants expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus, a full spectrum of transformed phenotype is conferred to NIH/3T3 cells by ectopic expression of HBGF-1. These cell lines will be used to further characterize the mechanism of secretion and biochemical action of HBGF-1. MATERIALSAND METHODS
Plasmid DNA construction. All procedures used in cloning were essentially as described (Maniatis et al., 1982). The 355 bp BamHI-Bgl II fragment derived from pHBGF 1.1, comprised of the 3'-end of the protein-coding sequence (Chiu et al., 1990), was cloned into the unique BamHI site of SVX (Cepko et al., 1984). The 163 bp BamHI-BamH1 fragment derived from pHBGF 1.2, which contained the 5'-end of the coding region (Chiu et al., 1990), was then cloned into the unique BamHI site of the resultant plasmid and designated pSVX/HBGFI(+) (Fig. 1). Transfection. NIH/3T3 cells were transfected with expression vector DNA using the calcium phosphate precipitation method (Parker and Stark, 1979; Pulciani et al., 1982). To obtain more G418-resistant colonies, the expression vector was co-transfected with pSV2neo (Southern and Berg, 1987) in a 10:1 ratio in later experiments. The transfected cells were selected by culturing in medium containing 400 gg/ml of G418 (GIBCO). Genomic DNA was isolated from G418-resistant cell lines and analyzed by the method of Southern (Southern, 1975) using the 508 bp EcoRIBgllI fragment of HBGF-1 cDNA as a probe (Chiu et al., 1990). RNA was isolated using the guanidinium thiocyanate method (Chirgwin et al., 1979) and characterized by Northern hybridization (Maniatis et al., 1982). RNase protection analysis was performed as described using the KpnI transcript of pHBgl.0 template (Wang et al., 1989).
Cell density, focus formation and protein purification. Transfected cells (2 X 104 per well) were seeded in 6-well plates and cell numbers at confluency were determined after 14 days of culturing. For focus formation, transfected cells were mixed with NIH/3T3 cells in various ratios (1 × 105 per 10 cm plate) and incubated for three weeks and the number of foci was determined. For protein purification, cell homogenate from ten 10 cm plates was centrifuged at 10 000 ×g for 20 min and the supernatant or the medium conditioned from the transfectants was fractionated through a heparin-Sepharose CL-6B (Pharmacia) column as described (Winkles, et al., 1987). Fractions of 2.5 ml were collected and were monitored at 280 nm. Each fraction was stored at - 8 0 ° C for subsequent mitogenic assays. Mitogen assay. Growth factor activity was determined by measuring the incorporation of [3H]thymidine into the DNA of serumstarved Swiss/3T3 ceils. Ten/xl from each fraction of the heparinSepharose purified protein was added to the starved cells. Cells were labeled for 6 h with 1 gCi [3H]thymidine starting at 20 h after addition of mitogen. The labeled DNA was precipitated with trichloroacetic acid and the amount of incorporated [3H]thymidine was determined. The commercially available bovine HBGF-1 preparation (R&D Systems, Minneapolis) containing only the two truncated forms of the protein was used as a positive control. Western blotting analysis and protein labeling. HeparinSepharose purified fractions were concentrated in 10 mM sodium phosphate buffer, pH 7.2 using Centricon-3 membrane (Amicon). One tenth of the protein from each fraction was analyzed on a 15% SDS polyacrytamide gel (Laemmli, 1970) and transferred to a nitrocellulose filter. The filter was reacted first with 1:200 dilution of a rabbit anti bovine HBGF-1 antibody (generously provided by Tom Maciag, American Red Cross) then with 1:3000 dilution of goat antirabbit IgG conjugated with horseradish peroxidase (Bio-Rad). For protein labeling, transfected cells were grown to 80% confluency in a T150 flask and labeled with 1 mCi [3SS]methionine for 24 h. After incubation, both the conditioned media and cell lysates were purified using heparin-Sepharose chromatography and analyzed on a 15% SDS polyacrylamide gel. Anchorage independence and tumorigenicityassays. Anchorageindependent growth was assayed essentially as described (Rizzino, 1987). Cells (5 × 104 per well) were seeded in duplicate on 0.5% agar in the presence or absence of 5 ng/ml bovine HBGF-1 and 5 U/ml heparin. After incubation for 14 days, cell colonies were stained overnight with p-iodonitrotetrazolium violet (Sigma). Colony numbers from five different fields per well were determined using a Nikon Diaphot microscope. Capacity of transfected cell lines to induce tumor formation was also tested by injecting 1 × 106 cells subcutaneously into nude mice. NIH/3T3 cells were injected as a negative control. The nude mice were observed for tumor formation weekly for up to 3 months. RESULTS
HBGF-1 expression vector, pSVX/HBGF-I(+), was constructed by cloning the HBGF-1 cDNA coding sequence (Chiu et al., 1990) into the SVX vector (Cepko et al., 1984) which was derived from Moloney murine leukemia virus (Fig. 1). The viral long terminal repeat provided the promoter for both the HBGF-1 cDNA and G418 selection marker. To generate more G418-resistant colonies, the HBGF-1 expression vectors were co-transfected with pSV2neo (Southern and Berg, 1987). G418-resistant colonies were isolated
91
TRANSFORMING POTENTIAL OF HBGF-1/aFGF
BomH!
EcoRT B g / B m EcoRI
!
---H---
