124

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.

[12]

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125

Met -Ser - G l y - P r o - G l y - T h r - A l a - A l a ~ ~ ~ ~ : ~ A•a-Pr•-Trp-A•a-G•y-Arg-G•y-G•y-A•a-A•a-A•a-Pr•-Thr-A•a-Pr•-Asn-G•y-Thr-Leu-G•uA•a-G•u-Leu-G•u-Arg-Arg-Trp-G•u-Ser-Leu-va•-A•a-Leu-ser-Leu-A•a-Arg-Leu-Pr•-va•Ala-Ala-Gln-Pro-Lys -Glu-Ala-Ala-Val-Gln-Ser-Gly-Ala-Gly-Asp-Tyr-Leu-Leu-Glyi le Lys -Arg-Leu-Arg-Arg-Leu-Tyr-Cys-Asn-Val -Gly- I le -Gly-Phe-His -Leu-Gln-Ala-Leu-P to_ Asp-GIy-Arg- I le-Gly-Gly-Ala-His-Ala-Asp-Thr -Arg-Asp-Ser-Leu-Leu-Glu-Leu-Ser-ProVal -Glu-Arg-Gly-Val -Val -Ser- I le-Phe-Gly-Val -Ala-Ser-Arg-Phe-Phe-Val -Ala-Met - Ser Ser-Lys -GIy-Lys -Leu-Tyr-Gly-Ser-Pro-Phe-Phe-Thr-Asp-Glu-Cys -Thr-Phe-Lys -Glu- i le Leu-Leu-Pro-Asn-Asn-Tyr-Asn-Ala-Tyr -Glu-Ser-Tyr-Lys -Tyr-Pro-Gly-Met -Phe- ile-AlaLeu-Ser-Lys -Asn-Gly-Lys-Thr-Lys -Lys -Gly-Asn-Arg-Val -Ser-Pro-Thr-Met -Lys -Val -Thr His-Phe-Leu-Pro-Arg-Leu

FIe. 1. Deduced amino acid sequence of the hst-1 protein (206 amino acids, 22 kDa). A hydrophobic core sequence of a putative signal peptide is shaded.

firmed by synthesizing a recombinant hst-1 protein. 4 In this chapter, we summarize these processes of identification and characterization of hst-1. High molecular weight DNA was extracted from 37 surgical specimens of gastric cancer and from 21 noncancerous portions of gastric mucosa. Three of the 58 samples, including one from noncancerous gastric tissues, showed transforming activity when transfected into NIH 3T3 cells. A genomic library was constructed from the DNA of a secondary transformant, T361-2nd-1, and Alu-containing portions were physically mapped. Three contiguous repetitive-free fragments were then identified to hybridize to the mRNA of the T361-2nd-1 transformant but not to the mRNA of the parental NIH 3T3 cells. One of the fragments was employed to screen a cDNA library of the T361-2nd-1 cells, and a novel transforming gene, which we designated hst (human stomach), was identified.l'2 Afterward the gene was renamed hst-1 when a close homolog of hst, termed hst-2, was cloned by cross-hybridization.5 The transforming activity of the hst-1 cDNA clone and various mutated derivatives was evaluated with an SV40-based eukaryotic expression vector. An open reading frame, termed ORF1, from nucleotides 239 to 856 of the cDNA was thus identified as encoding a transforming protein of 206 amino acids (Fig. 1). 2 All three independent NIH 3T3 foci induced by human gastric DNAs were found to be transformed by hst-1. Moreover, a number of subsequent reports have shown the presence of the "transforming" hst-1 gene in nongastric cancers, such as colon cancer, 6 hepatoma, 7'8 Kaposi sarcoma 9 4 K. Miyagawa, H. Sakamoto, T. Yoshida, Y. Yamashita, Y. Mitsui, M. Furusawa, S. Maeda, F. Takaku, T. Sugimura, and M. Terada, Oncogene 3, 383 (1988). 5 H. Sakamoto, T. Yoshida, M. Nakakuki, H. Odagiri, K. Miyagawa, T. Sugimura, and M. Terada, Biochem. Biophys. Res. Commun. 151, 965 (1988). 6 T. Koda, A. Sasaki, S. Matsushima, and M. Kakinuma, Jpn. J. CancerRes. 78, 325 (1987). 7 H. Nakagama, S. Ohnishi, M. Imawari, H. Hirai, F. Takaku, H. Sakamoto, M. Terada, M. Nagao, and T. Sugimura, Jpn. J. Cancer Res. 78, 651 (1987). 8 y . Yuasa and K. Sudo, Jpn. J. Cancer Res. 78, 1036 (1987). 9 p. Delli Bovi and C. Basilico, Proc. Natl. Acad. Sci. U.S.A. 84, 5660 (1987).

126

FIBROBLAST GROWTH FACTOR

[12]

(where hst was referred to as KS or K-fgf), melanoma, 1° and osteosarcoma. 1~ These reports indicate hst-1 is the most prevalent non-ras transforming gene. Identification of hst-1 Gene from Normal Genomic Libraries

Cloning o f hst-1 Gene from Cosmid Libraries Since the hst-1 cDNA clone was derived from an NIH 3T3 transformant, it was possible that the hst-1 amino acid sequence decoded from the ORF1 of the cDNA was a product of an artificial gene rearrangement during the transfection and not a genuine human protein. To address this question, an hst-1 genomic fragment was cloned from a normal human library. 5 Peripheral blood leukocytes were taken from a healthy male that had no previous or family history of serious illness. High molecular weight DNA was extracted, partially digested with Sau3AI, and fractionated by sucrose density gradient centrifugation. DNA fragments of 30-50 kilobase pairs (kbp) were ligated to the BamHI site of the cosmid vector LoristB jz and packaged in vitro. About 1 × 105 clones were screened with a probe corresponding to the ORF1 of the hst-1 cDNA under stringent conditions at 42° in a hybridization buffer containing 50% formamide. Two groups of cosmids were obtained, with two different patterns of hybridization to the ORF1 probe as shown in Fig. 2A; six of nine cosmids have three EcoRI fragments of 5.8, 2.8, and 0.8 kbp, whereas each of the remaining three clones has one 8.0-kbp EcoRI fragment hybridized to ORF1. These two groups of clones represented hst-1 and its close homolog hst-2, respectively. Partial physical maps are presented for these clones and for the clone L361-Hu3, which is an hst-1 clone derived from the original NIH 3T3 transformant T361-2nd-1 (Fig. 2B).

Transforming Potential o f Various hst-1 Genes All six hst-1 cosmids from the normal human library had transforming activity equivalent to that of L361-Hu3. A 4.0-kbp TaqI fragment, YT4.0, which contains a 1.3-kbp region upstream of TATA box (Fig. 2B) was fully transforming by itself. A mouse hst-1 fragment derived from the genome of NIH 3T3 cells showed a similar transforming activity. Although the cosmids containing hst-2 did not transform NIH 3T3 cells r0 j. Adelaide, M.-G. Mattei, I. Marics, F. Raybaud, J. Planche, O. De Lapeyriere, and D. Birnbaum, Oncogene 2, 413 (1988). IJ X. Zhan, A. Culpepper, M. Reddy, J. Loveless, and M. Goldfarb, Oncogene 1, 369 (1987). 12 S. H. Cross and P. F. R. Little, Gene 49, 9 (1986).

[12]

127

hst-1

B

A 1

2

3

TATA box

LYH-5

s,

kbp 8.0--,~ kbp

~.8-a 8.o_~ ~ 5.8 - -

2.8--1e

2.8--~

o.8-~-0.8 w

L361-Hu6

TATA box

El ISC I sc LYH-3 E

I

Sc H

t

scl ilIH_]..L~II I B

I E

J

1 kbp FIG. 2. (A) Southern blot analysis of EcoRl-digested DNAs hybridized with the ORF1 probe. Lane 1, human placental genomic DNA; lane 2, LYH-5 containing the hst-1 gene; lane 3, LYH-3 corresponding to the hst-2 gene. (B) Partial physical maps of LYH-5, L361-Hu6, and LYH-3. Boxes indicate exons of hst-I on LYH-5 and L361-Hu6, with the coding sequences (ORF1) stippled. The wavy line on the L361-Hu6 map indicates the rearrangement site in the genome of the NIH 3T3 transformant, T361-2nd-l. On LYH-3, a region which cross-hybridizes to ORF1 is hatched. B, BamHI; E, EcoR1; T, TaqI; Sc, SacI; SI, SalI; H, HindlII. The TaqI sites are indicated only on LYH-5.

morphologically in an ordinary focus assay, we found recently ]3 that they induce distinctive foci of transfected NIH 3T3 cells in a defined medium lacking platelet-derived growth factor (PDGF) and FGF. ]4 Another group of investigators cloned a gene, designated FGF6, which has transforming and tumorigenic activities.J5 The gene was identified by cross-hybridization with the hst-I gene, and a partial nucleotide sequence revealed that FGF6 is identical to hst-2.]6 Sequence analysis of the hst-1 genomic fragment showed that the coding sequence was completely identical to the ORF1 of the cDNA clone from the T361-2nd-1 transformant. 3 Thus, it was suggested that the mechanism of activation of the transforming potential of hst-1 is its transcriptional deregulation rather than a structural aberration. As described later, expression of hst-1 seems to be tightly suppressed in normal adult cells. One may speculate that such transcriptional silence is maint3 H. Sakamoto, unpublished results (1990). 14 X. Zhan and M. Goldfarb, Mol. Cell. Biol. 6, 3541 (1986). ~5 I. Marics, J. Adelaide, F. Raybaud, M.-G. Mattei, F. Coulier, J. Planche, O. de Lapeyriere, and D. Birnbaum, Oncogene 4, 353 (1989). i6 K. Naito and M. Terada, unpublished data (1989).

128

FIBROBLAST

Cys

1

HST1

79 V

I~-~

GROWTH

[12]

FACTOR

Cys •

171

206 a.a.

~i~i~iiiiii!ii~i[ ; 16

HST2/FGF6

~.

~ t 267

FGF5

I 155

bFGF

I 155

aFGF

[

~i~i~i] I ~ t

[ 194

KGF

INT2

I

~

~!~]

itl

[~i~iiiil

240

I

FIG. 3. Heparin-binding growth factor family. Amino acid sequences of currently known members of the family were aligned to that of the hst-1 protein. Gaps were introduced to achieve the best alignment. Hatched boxes indicate putative signal peptides, and two cysteine residues conserved among the family members are indicated by arrowheads. Stippled areas have significant sequence similarity with the hst-I protein as shown by percentage identity. Amino-terminal sequencing is not yet completed for the hst-2/FGF6 protein.

tained by gene methylation and/or by some flanking silencer sequence, both of which can be invalidated by transfection or gene cloning. This probably explains why hst-1 is so frequently found as a transforming gene in a variety of sources of human DNA.

Homology with Fibroblast Growth Factors and Related Molecules Upon realizing that the amino acid sequence decoded from ORF1 represents an authentic human hst-1 protein, we proceeded to search for homology among DNA and protein databases. As shown in Fig. 3, significant homology was noted with acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), the hst-2/FGF6 protein, the int-2 protein, FGF5, and keratinocyte growth factor (KGF). These constitute a potentially large and growing family of growth factors and oncogene products, the heparin-binding growth factor (HBGF) family. Fibroblast growth factors are potent and ubiquitous mitogens with

[12]

hst-I

129

diverse functions in a variety of target cells (reviewed in Ref. 17). They are known as angiogenic factors in vivo and in vitro. The factors also regulate the differentiation pathway of several cells, and bFGF acts as a mesoderm-inducing factor in Xenopus embryos, t8 int-2 was initially identified as one of the cellular genes activated transcriptionally by proviral insertion in mouse mammary tumor virus (MMTV)-induced breast cancers in mice. 19 Aside from these murine mammary tumors, expression of int-2 has been detected only in embryos and in some teratocarcinomas, z° FGF5 was identified by its ability to support growth of the transfected NIH 3T3 cells in a serum-free defined medium jl and was found to be expressed in neonatal brain. 21 KGF is a growth factor expressed in fetal and adult fibroblasts and is considered to be a mitogen specific for epithelial cells. 22

Synthesis of Recombinant hst-1 Protein Baculovirus and Silkworm Cells One of the potential advantages of the use of cells from multicellular organisms such as Bombyx mori (silkworm) over prokaryotes to synthesize human proteins is that we can expect posttranslational modifications to occur in a way more akin to human cells, such as glycosylation and recognition and cleavage of a signal peptide. Previous successes with the synthesis of a-interferon and interleukin 3 showed that biologically active materials were secreted into the culture medium of silkworm cells.Z3'24This also facilitates recovery and purification of the protein. Protein produced in Escherichia coli, on the other hand, is in many cases resistant to solubilization. The disadvantage of the baculovirus system is that the yield is lower and the procedure is more demanding than in the E. coli systems.

17 D.Gospodarowicz, this series, Vol. 147, p. 106. 18 j. M. W. Slack, B. G. Darlington, J. K. Heath, and S. F. Godsave, Nature (London) 326, 197 (1987). 19 G. Peters, S. Brookes, R. Smith, and C. Dickson, Cell (Cambridge, Mass.) 33, 369 (1983). 20 A. Jakobovits, G. M. Shackleford, H. E. Varmus, and G. R. Martin, Proc. Natl. Acad. Sci. U.S.A. 83, 7806 (1986). 21 X. Zhan, B. Bates, X. Hu, and M. Goldfarb, Mol. Cell. Biol. 8, 3487 (1988). 22 p. W. Finch, J. S. Rubin, T. Miki, D. Ron, and S. A. Aaronson, Science 245, 752 (1989). 23 S. Maeda, T. Kawai, M. Obinata, H. Fujiwara, T. Horiuchi, Y. Saeki, Y. Sato, and M. Furusawa, Nature (London) 315, 592 (1985). 24 A. Miajima, J. Schreurs, K. Otsu, A. Kondo, K. Arai, and S. Maeda, Gene 58, 273 (1987).

130

FIBROBLAST GROWTH FACTOR

[12]

Generation of Recombinant Baculovirus ORF1 with minimal flanking cDNA sequences (nucleotides 1 to 916 of the cDNA 2) is cloned into an EcoRI site of a baculovirus transfer vector, pBM030, z5 a gift from Dr. M. Furusawa (Daiichi Seiyaku Research Institute, Tokyo). The construct is placed the coding sequence of hst-1 under the strong promoter of a nuclear inclusion body polyhedrin gene of the B. mori nuclear polyhedrosis virus (BmNPV). 23 The vector is designed to express the insert as a nonfusion protein. Twenty-five micrograms of the plasmid is cotransfected with 5 /xg of BmNPV DNA into 5 × 105 of silkworm-derived BmN cells in a 25-cm 2 flask (Coming Glass Works, Corning, NY) by a standard calcium phosphate-mediated DNA transfection procedure. The viral DNA for cotransfection is prepared as described. 26 Recombinant virus harboring hst-1 is generated by homologous recombination between the transfer vector and the wild-type BmNPV genome in the transfected B m N cells. The recipient B m N cells were provided by Dr. S. Maeda (Tottori University, Japan) and are grown in TC-10 medium27 (Table I) supplemented with heat-inactivated 10% fetal calf serum (FCS, B6hringer, Mannheim, Germany) in a 27° air incubator with a tightly closed Corning flask cap. The medium is replaced every 4-5 days, and confluent cultures are transferred by gentle scraping with a silicon rubber policeman at a dilution ratio of 1 : 3. Freeze-thaw cycles of the BmN cells for storage are not recommended. The medium is replaced with 5 ml of fresh TC-10 on the next day of the transfection. A week later, the medium is harvested, and the recombinant virus in it is cloned by limiting dilution as follows: 200/zl of the 10 -5, 10 -6, and 10 -7 dilutions of the virus-containing medium are mixed with 100 to 200 fresh BmN cells and plated on a 96-well dish. Infected cells show a bizarre irregular shape after 1 week and are easily recognized among the round, smooth-surfaced uninfected cells under a phase-contrast microscope. In sharp contrast to cells infected with the wild-type virus, cells infected with the recombinant virus are identified by their lack of the characteristic massive nuclear inclusion body, polyhedrin, which has a different refractile index from normal cellular components. One more round of the limiting dilution is performed to obtain purified recombinant virus stocks, each derived from single clones. Ten to 20/xl of the purified 25 y . Marumoto, Y. Sato, H. Fujiwara, K. Sakano, Y. Saeki, M. Agata, M. Furusawa, and S. Maeda, J. Gen. Virol. 68, 2599 (1987). 26 S. Maeda, in "Invertebrate Cell System and Applications" (J. Mitsuhashi, ed.), p. 167. CRC Press, Boca Raton, Florida, 1989. 27 G. R. Gardiner and H. Stockdale, J. Invertebr. PathoL 25, 363 (1975).

[12]

131

hst-1

TABLE I PREPARATION OF TC-10 MEDIUMu Component

Amount

NaC1 KCI CaCI 2 • 2H20 MgCI 2 • 6H20 MgSO 4 • 7H20 Tryptose Glucose L-Glutamine

0.5 2.87 1.32 2.28 2.78 2.0 1.1 0.3

g g g g g g g g

Component

Amount

10 x Soluble amino acids b 10 x Insoluble amino acids C 1000 x Vitamins a NaH2PO4 • 2H20 NaHCO3 Kanamycin Distilled water

100 ml 100 ml 1 ml 0.89 g 0.35 g 60 mg to 900 ml

a This preparation yields 1000 ml of TC-10 medium (pH 6.30-6.35, 315 mOsm). Filter-sterilize over Millipak 20 (0.22 /zm, Millipore, Bedford, MA) and add 100 ml of heat-inactivated FCS before use. b 10 x Soluble amino acids (1000 ml): L-Arginine-HCl L-Aspartic acid L-Asparagine L-Alanine /3-Alanine L-Glutamic acid L-Glutamine Glycine L-Histidine

7.00 3.50 3.50 2.25 2.00 6.00 3.00 6.50 25.00

g g g g g g g g g

L-Isoleucine L-Leucine L-Lysine-HCl L-Methionine L-Proline L-Phenylalanine DL-Serine L-Threonine L-Valine

0.50 0.75 6.25 0.50 3.50 1.50 11.00 1.75 1.00

g g g g g g g g g

" 10x Insoluble amino acids (1000 ml): L-cysteine, 0.25 g; L-tryptophan,

1.00 g; L-tyrosine, 0.50 g. Dissolve in 500 ml of 40 m M HC1 by heating and then increase to 1000 ml with water. d 1000 x Vitamins (100 ml): Thiamin-HCl Riboflavin Calcium D-pantothenate Pyridoxine-HC1 p-Aminobenzoic acid

2.0 2.0 2.0 2.0 2.0

mg mg mg mg mg

Folic acid Nicotinic acid myo-Inositol

Biotin Choline chloride

2.0 2.0 2.0 1.0 20.0

mg mg mg mg mg

virus stock is then used to infect approximately 105 BmN cells in a Corning 75-cm 2 flask for the amplification of the virus stock. An aliquot of the amplified virus stock is used to infect a total of 2 x 108 BmN cells in 20 Corning 150-cm 2 flasks with 30 ml per flask of TC-10 medium with 10% FCS. The amount of the virus stock used for infection is titrated, so that the yield of the protein attains a maximum on the fourth day of infection. Then the medium is harvested, centrifuged at

132

FIBROBLAST GROWTH FACTOR

[12]

5,0

E

- 2.0

4.0,

- 1.5 t-.O *" 3.0 ."

"[ ¢"

2.0

-1.0

~s

..

.E E

=

- 0.5

1.o

0.0

i 5

t 10

I 15

i 20

i 25

0.0

30

Fraction number FIG. 4. Heparin affinity chromatography of the concentrated medium of B m N cells infected with the recombinant baculovirus. The dashed line represents the NaC1 gradient, and circles indicate the mitogenic activity of each fraction as determined by a [3H]thymidine incorporation assay with NIH 3T3 cells.

1000 rpm for 5 min to remove cells, and concentrated to around 60 ml by an Amicon (Danvers, MA) YM5 membrane.

Purification of Recombinant hst-1 Protein The concentrated medium is loaded onto an Affi-Gel heparin (Bio-Rad, Richmond, CA) column (1.6 × 5 cm), which is preequilibrated at 4° by a buffer consisting of 10 mM Tris-Cl (pH 7.0)/0.6 M NaCI and washed in the same buffer. The hst-1 protein is eluted with 60 ml of a 0.6-2.0 M linear gradient of NaC1 in 10 mM Tris-C1 (pH 7.0) at a flow rate of 30 ml/hr at 4 °. Two-milliliter fractions are collected and stored at - 70°. One microliter each of the fractions diluted 50-fold in phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA) is assayed by [3H]thymidine incorporation using NIH 3T3 cells (see below). Typically, the biologically active hst-1 fractions are eluted at 1.0-1.2 M NaCI (Fig. 4) and are concentrated to about 100/xl by a Centricon-10 apparatus (Amicon). The second step of the purification is reversed-phase high-performance liquid chromatography (HPLC). A 0.46 × 25 cm Vydac C4 column with

[12]

hst-1

133

5 tzl particle size and 300 A pore size (The Separation Group, Hesperia, CA) is equilibrated with 0.1% trifluoroacetic acid (TFA) and 30% acetonitrile on a Tosoh CCPM 8000 gradient liquid chromatography system. The sample is eluted by a 1.0 ml/min gradient of 30-60% acetonitrile in 0.1% TFA for 90 min at room temperature. Fractions of 1.0 ml are collected in siliconized Eppendorf tubes and stored at - 7 0 °. One-microliter aliquots of each fraction diluted 1 : 10 in PBS/0. I% BSA are analyzed in the [3H]thymidine incorporation assay. The HPLC-purified, biologically active hst-1 protein is about 18 kilodaltons (kDa) in size on SDS-PAGE. The final yield of the purified hst-1 protein is approximately 5/~g. Amino acid sequence analysis reveals cleavage of the N-terminal 58 amino acids containing the signal peptide. 28

Growth Factor Activity of Recombinant hst-1 Protein Stimulation of DNA Synthesis in N I H 3T3 Cells

The [3H]thymidine incorporation assay is performed essentially as described, 29 and the conditions for hst-1 are summarized here briefly. NIH 3T3 cells are cloned by limiting dilution to select those with good contact inhibition, well-aligned flat morphology, and high efficiency of transformation by transfected hst-1 expression vectors. The selected clones are expanded, stored in liquid nitrogen, and, once thawed, discarded after 1 month of culture. Approximately 1 x 104 cells in 0.5 ml of Dulbecco's modified Eagle's medium (DMEM) with 10% calf serum are incubated for 24 hr, then the medium is changed to DMEM with 0.5% calf serum and incubated for another 24-72 hr. As the pH of the HPLC eluent in acetonitrile/TFA is very low, test samples should be diluted at least 4-fold by PBS before addition to the medium. Cells are then incubated for 16 hr, and [3H]thymidine is added to final concentration of 18.5 kBq (0.5 ~Ci)/ ml. Eight hours later, the trichloroacetic acid-precipitable radioactivity is measured. As shown in Fig. 5, the purified hst-1 protein shows a potent mitogenic effect on NIH 3T3 cells, with half-maximal stimulation observed at 220 pg/ml in the absence of heparin and at 80 pg/ml in the presence of heparin. The potency in the presence of heparin is comparable to that of bovine pituitary bFGF (Takara-Shuzo, Kyoto, Japan). The morphological changes of N I H 3T3 cells in response to the hst-1 protein are characterized by a highly refractile, criss-cross appearance with long process formation. 28 K. Miyagawa, unpublished observations, 1989. 29 K. A. Thomas, this series, Vol. 147, p. 120.

134

FIBROBLAST

GROWTH

FACTOR

[12]

A

o

25_

X

E

~

0 Wi.thout heparin

20-

•

t-

.2 15_ O

O

u r-

10-

C

~ E

5-

N

0

0

I

I

I

10

100

I

1000

hst-1 protein (pg/ml) FIG. 5. hst-1 protein-induced stimulation of [3H]thymidineincorporation by NIH 3T3 cells. The mitogenic activity was measured in the absence and presence of 50 p.g/ml of heparin. The changes appear 12 hr after the addition of the protein, reached maximum after 24-48 hr and reverted after 72 hr.

Stimulation of H U V E Cell Growth H u m a n umbilical vein endothelial ( H U V E ) cells are isolated as described 3° and cultured in MCDB107 medium (Kyokuto, Tokyo, Japan) with 15% heat-inactivated fetal calf serum (FCS), 2.5 ng/ml of a F G F and 5 / x / m l of heparin (Sigma, St. Louis, MO) on fibronectin-coated flasks. H U V E cells are seeded onto 12-well plates at a density of 2 × 103 cells/ cm 2 in 2 ml o f medium per well. Twenty-four hours later, the medium is replaced by MCDB107 with 15% FCS and various concentrations of test samples. After 48 hr, the treatment is repeated once more, and cells are incubated for another 48 hr. The cell number in each well is determined with a Coulter particle counter after trypsinization. Half-maximal stimulation of H U V E cell growth is observed at about 30 pg/ml of hst-1 protein.

Anchorage-Independent Growth of N I H 3 T3 Cells A soft agar assay is done as described, 31 using 5 × 103 N I H 3T3 cells in the overlayer with 0.4% Bacto-agar (Difco, Detroit, MI) poured over the base layer of 0.9% agar, both in D M E M with 5% calf serum. The 30T. Imamura and Y. Mitsui, Exp. Cell Res. 172, 92 (1987). 31A. Rizzino, this series, Vol. 146, p. 341.

[12]

hst-1

135

number of colonies larger than 2000 / z m 2 is counted after 2 weeks of incubation at 37° in 5% CO2. The hst-1 protein induces anchorage-independent growth in a dose-dependent manner. An approximately 10-fold higher concentration of the protein is required to support soft agar colony formation of N I H 3T3 cells as compared to stimulation of DNA synthesis of the same cells. These data on the functions of the recombinant protein prove that hst-I actually encodes a heparin-binding growth factor, and, as for FGFs, angiogenic activity is also expected for the hst-1 protein. Recently, we observed a potent in vivo angiogenic activity of the recombinant hst-1 protein using the chorioallantoic membrane (CAM) assay and the rat cornea assay. 32

Expression of hst-1 Expression of hst-1 in Embryos and in Germ Cell Tumors

Northern blot analysis did not detect the hst-1 message in about 80 cancerous and noncancerous cells and tissues. 33 These samples included gastric cancers and Kaposi's sarcomas, in which types of cancers the transforming hst-1 gene was identified previously by transfection assays. To the best of our knowledge, the expression of hst-1 is confined to embryos and to some germ cell tumors (Fig. 6). The sizes of the hst-1 transcripts are 3.0 and 1.7 kilobases (kb) for human, and 3.0 kb for mouse. Differential Expression of hst-1 and int-2

The embryo-specific pattern of expression is also noted for int-2, 2° but hst-1 and int-2 are regulated in different ways. In vitro induction of differentiation of a mouse teratocarcinoma cell line, F9, is thought to simulate early stages of embryonic differentiation, z° F9 stem cells are grown on ungelatinized tissue culture dishes in DMEM supplemented with 15% heat-inactivated FCS. The cells should be transferred before they get to confluence and discarded 1 month after thawing out from liquid nitrogen storage. Retinoic acid (Sigma, type XX) and dibutyryl cyclic AMP (B6hringer) are added to final concentrations of 10 -7 and 10 -3 M , respectively, to 5 × 105 F9 cells carefully dispersed in a 100-mm dish (Falcon 3003) with 12 ml of the medium. The medium is renewed every 48 hr, and, after 5 32 T. Yoshida, unpublished observations, 1990. 33 T. Yoshida, M. Tsutsumi, H. Sakamoto, K. Miyagawa, S. Teshima, T. Sugimura, and M. Terada, Biochem. Biophys. Res. Commun. 155, 1324 (1988).

136

FIBROBLAST GROWTH FACTOR

A

[12]

B 1234

56 789101112

1 2 34

28S

28S

18S

18S

FIG. 6. (A) Northern blot analysis showing expression of hst-1 in human germ cell tumors. Lanes 1 to 9 contained RNA from surgical specimens of testicular germ cell tumors, lanes 10 and 11 RNA from noncancerous portions of testes, and lane 12 RNA from an immature teratoma cell line, NCC-IT. Three micrograms of poly(A) ÷ RNA was hybridized with the 3' one-third of the ORF1 probe. The positions of the 28 and 18 S rRNAs are indicated by arrowheads. (B) Expression of hst-1 in mouse embryos. Lane 1, day 11 embryos; lane 2, heads of day 14 embryos; lane 3, bodies of day 14 embryos with livers removed; lane 4, livers of day 14 embryos. Five micrograms of poly(A) ÷ RNAs was hybridized with probe M 1.8, a mouse genomic fragment of an hst-l-containing exon.

days, the cells are entirely differentiated to parietal endodermal cells, which are harvested for RNA extraction. 34 hst-1 is preferentially expressed in F9 stem cells and dramatically downregulated upon induction of parietal endodermal differentiation; in contrast, transcription of int-2 is very low in undifferentiated F9 cells, whereas it markedly increases after differentiation. We surmise that these related oncogenes are functionally coupled and convey distinct signals during embryogenesis. Chromosomal Localization of hst-1 Gone

Human hst-I and int-2 were mapped on the same chromosome band, 1lq13.3, 35 and cosmid mapping showed 36 that they are only 35 kbp apart, with the same transcriptional orientation (Fig. 7). This is also the case with the mouse genome, in which the two related oncogenes are within 17 34 T. Yoshida, H. Muramatsu, T. Muramatsu, H. Sakamoto, O. Katoh, T. Sugimura, and M. Terada, Biochem. Biophys. Res. Commun. 157, 618 (1988). 35 M. C. Yoshida, M. Wada, H. Satoh, T. Yoshida, H. Sakamoto, K. Miyagawa, J. Yokota, T. Koda, M. Kakinuma, T. Sugimura, and M. Terada, Proc. Natl. Acad. Sci. U.S.A. 85, 4861 (1988). 36 A. Wada, H. Sakamoto, O. Katoh, T. Yoshida, J. Yokota, P. F. R. Little, T. Sugimura, and M. Terada, Biochem. Biophys. Res. Commun. 157, 828 (1988).

[12]

137

hst-1

int-2 KB BN N K

5' ~

hst-1

BKB

BB

KB

Im

II

II

I I !

KB N K S

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,

I !

,c

35 kbp

>'

FIG. 7. Physical map of the human int-2-hst-I region at 1lq13.3. Exons are indicated by boxes. K, KpnI; B, BamHI; N, NotI; S, SalI.

kbp of o~e another on chromosome 7. 37 Their closeness on the genomes prompted the following two observations: first, hst-1 was also found to be activated transcriptionally in some MMTV-induced murine mammary tumors with or without int-2 activation. 37 Second, the genes are coamplifled in some human cancers. The incidence of coamplification is relatively high in breast c a n c e r s ( 1 2 - 2 2 % ) 38,39 and very high in esophageal cancers (47% in primary tumors and 100% in metastatic f o c i ) . 4° When the chromosomal localization of the hst-1 gene was determined, the committee on human gene nomenclature registered this gene officially as HSTF1 (heparin-binding secretory transforming factor 1). 35

Perspectives

The H B G F family is a large family, probably larger than currently known, encompassing numerous members with diverse functions and patterns of expression. Among the members of this family, hst-1 and int-2 are unique and especially interesting in that their expressions are tightly regulated, and specific roles in embryogenesis are expected. The receptors for HBGFs may also constitute a family, as genes homologous but not identical to the recently isolated bFGF receptor are being cloned in several 37 G. Peters, S. Brookes, R. Smith, M. Placzek, and C. Dickson, Proc. Natl. Acad. Sci. U.S.A. 86, 5678 (1989). 38 H. Tsuda, S. Hirohashi, Y. Shimosato, T. Hirota, S. Tsugane, H. Yamamoto, N. Miyajima, K. Toyoshima, T. Yamamoto, J. Yokota, T. Yoshida, H. Sakamoto, M. Terada, and T. Sugimura, Cancer Res. 49, 3104 (1989). 39 j. Adnane, P. Gaudray, M.-P. Simon, J. Simony-Lafontaine, P. Jeanteur, and C. Theillet, Oncogene 4, 1389 (1989). 4o T. Tsuda, E. Tahara, G. Kajiyama, H. Sakamoto, M. Terada, and T. Sugimura, Cancer Res. 49, 5505 (1989).

138

F I B R O B L A S T G R O W T H FACTOR

[13]

41,42 Evidently, one of the most exciting targets of the current biological research is analysis of the HBGF ligand-receptor systems, which may be involved in a number of important biological processes. laboratories

4I p. L. Lee, D. E. Johnson, L. S. Cousens, V. A. Fried, and L. T. Williams, Science 245, 57 (1989). 42 y . Hattori and M. Terada, unpublished results, 1990.

[13] P h o s p h o r y l a t i o n a n d I d e n t i f i c a t i o n o f P h o s p h o r y l a t e d F o r m s o f Basic F i b r o b l a s t G r o w t h F a c t o r B y JEAN-JACQUES F E I G E a n d A N D R E W BAIRD

Introduction Basic fibroblast growth factor (FGF) is a potent mitogen for endothelial cells and for a wide variety of mesoderm- and neuroectoderm-derived cells. 1-4 It has recently been suggested that the tight association (Kd --l0 nM) of basic FGF with the basement membrane regulates its bioavailability and controls its interaction with its high-affinity (Kd 10-20 pM) receptors. 5-7 It has thus been important to determine whether posttranslational changes in basic FGF participate in the regulation of its activity or modulate its extracellular and intracellular localization. Although many mechanisms might exist, we have studied the potential role of protein phosphorylation. Careful analysis of the primary structure of basic FGF revealed the presence of consensus sequences for the phosphorylation of basic FGF by both protein kinase C and cyclic AMP-dependent protein kinases (Table I). 8'9 Accordingly, we established that basic FGF is a substrate for these kinases and is synthesized as a phosphoprotein by bovine capillary endoJ W. H. Burgess and T. Maciag, Annu. Rev. Biochem. 58, 575 (1989). 2 D. Gospodarowicz, N. Ferrara, L. Schweigerer, and G. Neufeld, Endocr. Rev. 8, 95 (1987). 3 A. Baird and P. B6hlen, in "Handbook of Experimental Pharmacology" (M. B. Sporn and A. B. Roberts, eds.), p. 163. Academic Press, New York, 1990. 4 A. Baird, F. Esch, P. Morm~de, N. Ueno, N. Ling, P. B6hlen, S. Y. Ying, W. Wehrenberg, and R. Guillemin, Recent Prog. Horm. Res. 42, 143 (1986). 5 A. Baird and N. Ling, Biochem. Biophys. Res. Commun. 142, 428 (1987). 6 j. Folkman and M. Klagsbrun, Science 2.35, 442 (1987). 7 A. Baird and P. A. Waiicke, Br. Med. J. 45, 438 (1989). 8 j._j. Feige and A. Baird, Proc. Natl. Acad. Sci. U.S.A. 86, 3174 (1989). 9 j. j. Feige, J. D. Bradley, K. Fryburg, J. Farris, L. C. Cousens, P. Barr, and A. Baird, J. Cell Biol. 109, 3105 (1989).

METHODS IN ENZYMOLOGY, VOL. 198

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