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TUMOR CYTOTOXICFACTOR / HEPATOCYTEGROWTHFACTORFROMHUMANFIBROBLASTS: CLONINGOF ITS cDNA , PURIFICATIONAND CHARACTERIZATIONOF RECOMBINANTPROTEIN Nobuyuki Shima *, Masaya NagaoI , Fumiko Ogaki, Eisuke Tsuda, Akihiko Murakami, and Kanji Higashio Life Science Research I n s t i t u t e , Snow Brand Milk Products Co. Ltd, 519 Ishibashi-machi, Shimotsuga-gun, Tochigi 329-05, Japan IDepartment of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Received September 26, 1991 Summary: Two d i f f e r e n t forms of cDNA f o r F-TCF were isolated from cDNA l i b r a r y prepared with mRNAfrom human embryonic lung fibroblast, IMR-90 cells. One of them was completely identical to the cDNA for placenta type hepatocyte growth factor (HGF) and the other one was a variant cDNA for the HGF with a deletion of 15 base pairs in the coding region. The cDNAs were expressed in CHO cells and recombinant proteins were purified and characterized. The deleted form of recombinant F-TCF (rF-TCF) was s l i g h t l y lower in heparin a f f i n i t y than the intact form. Both rF-TCFs showed almost same dose-response curves for c y t o t o x i c i t y on Sarcoma 180 or Meth A sarcoma cells. Dose-response curves for the stimulation of DNA synthesis in rat hepatocytes were also almost same before reaching maximal a c t i v i t y at 12.5 ng/ml but s i g n i f i c a n t l y d i f f e r e n t at higher concentrations. The deleted form of rF-TCF maintained maximal a c t i v i t y in the dose range of 12.5 to 100 ng/ml, although the intact form decreased the a c t i v i t y dose-dependently at more than 25 ng/ml. This suggests that the deletion of five amino acids results in a conformational change which alters heparin binding and hepatocyte growth stimulating activities. ~ 1991Ac~demio Press. ~nc.
Fibroblasts play important roles in inflammation and repair of injured tissues not only producing matrix proteins but secreting inflammatory cytokines such as interleukin-1 (IL-I)(1), stimulating factors (5).
IL-6 (2),
IL-8 (3),
i n t e r f e r o n - B (4), and colony
We have also found that human embryonic lung fibroblast,
IMR-90 cells secrete a tumor cytotoxic factor (F-TCF) which has cytotoxic a c t i v i t y against several tumor cell lines (6,7). a large
Purified F-TCF is a heterodimer composed of
u-chain with Mw 52 to 56-kD and a small B-chain with Mw 30 to 34-kD, and
has structural s i m i l a r i t i e s to hepatocyte growth factor (HGF)(6). HGF was f i r s t
* To whom correspondence should be addressed. Abbreviations: FGF, fibroblast growth factor; F-TCF, fibroblast-derived tumor cytotoxic factor; HGF, hepatocyte growth factor; IL, interleukin; PCR, polymerase chain reaction. 0006-291X/91 $1.50 1151
Copyright © 1991 by Academic Press, Inc. All rights' of reproduction in any form reserved.
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p u r i f i e d and c h a r a c t e r i z e d as a mitogenic f a c t o r f o r a d u l t r a t hepatocytes(8-10) . Two d i f f e r e n t cDNAs f o r HGF were cloned from human placenta ( I i ) libraries
and deduced p r i m a r y s t r u c t u r e s were determined.
sequences, mismatch o f 14 amino acids was found.
Partial
and l i v e r (12) cDNA Between the primary amino a c i d sequence
analysis of F-TCF indicated t h a t t h i s p r o t e i n might be i d e n t i c a l to the HGF derived from placenta (6).
To f u r t h e r c l a r i f y
screened from IMR-90 cDNA l i b r a r y .
the i d e n t i t y of F-TCF and HGF, F-TCF cDNA was
In t h i s report, we describe the molecular cloning,
expression, p u r i f i c a t i o n and characterization of recombinant F-TCF.
MATERIALS AND METHODS Construction of cDNA l i b r a r y : The human embryonic lung f i b r o b l a s t , IMR-90 c e l l s were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented w i t h 10% FCS. Total RNAs were extracted by the guanidine thiocyanate/cesium c h l o r i d e gradient method (13) Poly(A) ÷ RNA was selected by o l i g o ( d T ) - c e l l u l o s e column chromatography (14). cDNA was synthesized with o l i g o ( d T ) p r i m e r (Pharmacia) as described (15). Double-stranded cDNAs with EcoRl adaptor (Pharmacia) were cloned i n ~ g t l O (Promega) and packaged in v i t r o with Gigapack Gold (Stratagene). Screening of the cDNA l i b r a r y : A mixture of 17-nucleotide long oligonucleotides (Fig. 1~ was predicted from the N-terminal amino acid sequence of F-TCF B - c h a i n (6). The o l i g o n u c l e o t i d e mixture was 5'end-labeled with 3zp and used as a h y b r i d i z a t i o n probe to screen the cDNA l i b r a r y . Plaque h y b r i d i z a t i o n was carried out as described (16). Polymerase chain reaction (PCR) was used as a method to obtain f u l l coding region of F-TCF cDNA. We chose i d e n t i c a l regions between placenta type and l i v e r type HGF cDNAs as primer sequences f o r PCR. 5'-non coding region (sence strand; p o s i t i o n s from -77 to -53) flanked by Sall r e c o g n i t i o n s i t e , 5'-GGTCGACTAGGCACTGACTCCGAACAGGATTC-3'and 3'-non coding region (antisense strand; positions from 2227 to 2203) flanked by Sphl r e c o g n i t i o n s i t e , 5'-GGCATGCACAGTTGTATTGGTGGGTGCTTCAG-3'were chemically synthesized and were allowed to react with the cDNA l i b r a r y . PCR was performed according to the method (17). Expression of F-TCF cDNA: F-TCF cDNA (BamHI-Sphl; 2.3-kb) was inserted i n t o pCDNAI ( I n v i t r o g e n ) under cytomegarovirus promoter. The constructed plasmid DNA (I0 # g ) and p S V 2 n e o ( l # g ) ( 1 8 ) were c o - t r a n s f e c t e d i n t o CHO (DXB11) c e l l s ( I x l O 6 c e l l s ) by the calcium phosphate method (19). A f t e r s e l e c t i o n w i t h G418, F-TCF high producing clones were screened by an ELISA employing anti-F-TCF monoclonal antibodies (20). The cloned c e l l s were grown in DMEM/HAM-F12(Gibco) with 10% FCS. A f t e r the c e l l s were grown to confluence, the medium was replaced with DMEM/HAM-F12 w i t h 5% FCS and the c e l l s were subsequently cultured f o r 7 days. P u r i f i c a t i o n and preparation of rF-TCF: rF-TCF was p u r i f i e d as previously described except f o r the f o l l o w i n g modifications. The conditioned medium of the transformant was a p p l i e d to heparin sepharose column (@2.5 X 8 cm, Pharmacia) at a flow rate of 6 ml/min. The column was washed w i t h e q u i l i b r a t i o n b u f f e r ( i 0 mM T r i s HCI, pH 7.5, c o n t a i n i n g 0.01% Tween 20 and 0.3 M NaCl) followed by e l u t i o n with 2 M NaCl in the e q u i l i b r a t i o n buffer. The eluate was dialyzed against PBS and was f u r t h e r p u r i f i e d by the combinations of Mono S and h e p a r i n - a f f i n i t y HPLCs as p r e v i o u s l y described (6). The amounts of the p u r i f i e d rF-TCF were determined by a p r o t e i n assay k i t (Bio-Rad) using BSA as a standard protein. B i o l o g i c a l assay: Sarcoma 180 and Meth A sarcoma c e l l s , which were obtained from National Cancer Center, were used as t a r g e t c e l l s f o r c y t o t o x i c assay as p r e v i o u s l y described (6,7). Adult r a t hepatocytes were isolated and prepared as described (21). E f f e c t of rF-TCF on DNA synthesis in r a t hepatocytes was determined by the published method (22).
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RESULTS Isolation and characterization,of cDNA clones:
5x105 plaques on Escherichia c o l i C600
high f r e q u e n c y l l s o g e n (Promega) were screened by h y b r i d i z a t i o n w i t h the oligonucleotide probe. We obtained one p o s i t i v e clone ( ~ - 1 3 ) insert (Fig. 1).
containing a 5.7-kb
The insert o f h - 1 3 contained an open reading frame which completely
matched with the corresponding region of the placenta type HGF cDNA (positions, 7722184).
N e x t we used PCR to obtain f u l l coding region of F-TCF cDNA.
A DNA band with
estimated size (2.3kb) was obtained from PCR reactant by electrophoresis with 1% agarose gel.
The DNA fragment was subcloned i n t o pUC18, and several clones were
analysed f o r DNA sequences. Two forms of cDNA f o r F-TCF were detected from PCR amplified DNA fragments (Fig. 1). One of them completely matched with the placenta type HGF cDNA and the other one was a variant of the HGF cDNA with a deletion of 15 base pairs in the coding region (positions, 483-497). P u r i f i c a t i o n of rF-TCFs:
On h e p a r i n - a f f i n i t y HPLC as a f i n a l p u r i f i c a t i o n step, the
intact and deleted forms of rF-TCF were eluted from the column at 1.31M and at 1.28
0
1
2
3
,
l
,
1//~
6.5 kb II--
2-13
3'-CAACATTTACCCTAAGG-5' G G G G G T C T T C A A VaIValAsnGlyIlePro
(1) (2)(3) (4) start-
(kd)
E I
5' - - I i
C)
AE ~
HE H K
I
\I/
a -chain A 483 497
~ 1468
t
-
-
-//--
3'
2184
ACT~TTTTGCCTTCGA~CT Se~rPheLeuProSerSelr
Q
94
--
67
--
43
--
31
-
21.5-14.4-
Fig. i , Schematic representation of F-TCF cDNAand mRNA. Relationship betweenthe cDNA clone, A -13 and the synthetic oligonucleotide mixture (below the cDNA clone) are shown above a diagramof the F-TCF mRNA. Non coding regions are representedby a line and the coding region is boxed. Black area shows the signal peptide. Open box indicates the coding regions for the a - and B -chain. Openarrowhead indicates the site of the 15 nucleotides deletion. The deleted sequence are shown below the open arrowhead. E, EcoRI; A, Ava m ; H, Hind • ; K, KpnI. Fig. 2, SDS-PAGEof purified rF-TCFs. Lane 1; the intact form of rF-TCF under non reducing conditions, Lane 2; the deleted form of rF-TCF under non reducing conditions, Lane 3; the intact form of rF-TCF under reducing conditions, Lane 4; the deleted form of rF-TCF under reducing conditions.
1153
a
B B'
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I00'
a
b
80'
//
60' o X 0
20"
.
1
.
.
2
.
4
.
;I
.
.
8 16 31 63 J25 250 500
.
.
.
.
.
1
2
.
.
4
.
.
.
.
.
.
8 16 31 63 125 250 500
rF-TCFs (ng/ml)
Fia. 3. Cytotoxic activity of intact or deleted form of rF-TCF against mousetumor cell lines. (a) Cytotoxicity on sarcoma 180 cells: 0 - - 0 ; the deleted form of rFTCF, 0 - - 0 ; the intact form of rF-TCF. (b) Cytotoxicity on Meth A sarcoma cells: mm--m ; the deleted form of rF-TCF, D - - D ; the intact form of rF-TCF. Results are presented as mean ± SD of t r i p l i c a t e cultures.
M NaCl, respectively.
For analysis of p u r i t y and molecular weight, the p u r i f i e d rF-
TCFs were subjected to SDS-PAGE and the gel was stained with s i l v e r .
Both purified
preparations showed two adjucent bands with 76 to 80-kD under non reducing conditions (Fig. 2).
These molecular weights coincided with those of natural F-TCF which we
previously reported (6).
Under reducing conditions, the preparations separated into
three bands with 52 to 56, 34 and 30-kD corresponding to ~ ,
B and
# ' - c h a i n s as
well as natural F-TCF (Fig. 2). Biological a c t i v i t i e s of rF-TCFs: Both rF-TCFs showed almost same dose-responses for c y t o t o x i c i t y on Sarcoma 180 or Meth A sarcoma cells (Fig. 3). which gave 50% c y t o t o x i c i t y
The amounts of rF-TCFs
on Sarcoma 180 and Meth A were 7 and 32 ng/ml,
r e s p e c t i v e l y . These were consistent with our previous r e s u l t s with natural F-TCF (Sarcoma 180 at 6 ng/ml and Meth A at 40 ng/ml )(7).
B o t h rF-TCFs showed growth
s t i m u l a t i n g a c t i v i t y f o r r a t hepatocytes as well as natural F-TCF. Dose-response curves f o r the two rF-TCFs were almost same before reaching maximal a c t i v i t y at 12.5 ng/ml but s i g n i f i c a n t l y d i f f e r e n t at more than 25 ng/ml (Fig. 4).
The intact form of
rF-TCF decreased the a c t i v i t y dose-dependently at more than 25 ng/ml. 1154
In contrast,
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20 I-
(zz~ X
10
5
1.6 3.1 6.3 12.5 25
50 100 200 500 rF-TCFs (n~/ml)
Fig. 4. E f f e c t of i n t a c t or d e l e t e d form of rF-TCF on DNA s y n t h e s i s in p r i m a r y cultured a d u l t r a t hepatocytes. Hepatocytes were c u l t u r e d with the i n t a c t form of rFTCE ( c l e a r bar) or the deleted form of rF-TCF (cross-hatched bar) f o r 24 hrs. Results are presented as mean + SD of t r i p l i c a t e c u l t u r e s . S t a t i s t i c a l s i g n i f i c a n c e was determined by Student's t - t e s t (* P < 0.01).
the deleted form maintained maximal a c t i v i t y in the dose range of 12.5 to 100 ng/ml and decreased the a c t i v i t y dose-dependentlyat more than 200 ng/ml.
DISCUSSION Two forms of cDNA encoding F-TCF were isolated from humanfibroblast cDNA library. When the cDNAs were introduced into pcDNAI vector and were expressed transiently in COS cells, authentic cytotoxic activities against Sarcoma 180 and Meth A sarcoma cells were detected in both conditioned media but not detected in the control medium from the cells transfected pcDNAI only (data not shown). This established that the cDNAs encoded F-TCF which were i d e n t i f i e d as a tumor cytotoxic factor. We previously reported that partial amino acid sequences for F-TCF are identical to placenta type HGF but different From l i v e r type HGF (6).
Indeed, one cDNA for F-TCF had a coding
sequence completely matched with placenta type HGF cDNA which has mismatch of 39 nucleotides compared to l i v e r type HGF cDNA. The other cDNAwas a variant of placenta type HGF cDNA with a deletion of 15 base pairs in the coding region.
Recently, the
same variant sequence was isolated from the cDNA l i b r a r i e s derived from human fibroblasts (23) and human leukocytes (24). 1155
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The d e l e t e d form of rF-TCF e l u t e d s l i g h t l y
earlier
than the i n t a c t form on the
h e p a r i n - a f f i n i t y HPLC. This indicates that the absence of f i v e amino acids (Phe-LeuPro-Ser-Ser) r e s u l t s in s t r u c t u r a l change t h a t a l t e r heparin-binding properties.
F-
TCF is s i m i l a r to f i b r o b l a s t growth f a c t o r (FGF) in c h a r a c t e r i s t i c of high a f f i n i t y f o r heparin.
Amino acid sequence, Phe-Leu-Pro is one of the conserved sequence in
a l l seven members of the FGF f a m i l y (25), and t h i s sequence is thought to c o n t r i b u t e to the h e p a r i n b i n d i n g a c t i v i t y
of b a s i c FGF ( 2 6 ) .
Primary sequence of F-TCF
indicates that the i n t a c t form of F-TCF has Phe-Leu-Pro sequence at the f i r s t
and the
second k r i n g l e s , r e s p e c t i v e l y , and the v a r i a n t F-TCF has only the second Phe-Leu-Pro sequence.
Thus, the second sequence might also contribute to the a f f i n i t y
in a d d i t i o n to the f i r s t
for heparin
sequence.
The comparison of s p e c i f i c a c t i v i t i e s of rF-TCFs revealed that the d e l e t i o n of f i v e amino acid residues did not a f f e c t c y t o t o x i c a c t i v i t y but affected hepatocyte growth s t i m u l a t i n g a c t i v i t y at higher concentrations.
These imply that d i f f e r e n t mechanisms
mediate the e f f e c t on growth i n h i b i t i o n and s t i m u l a t i o n in d i f f e r e n t type of c e l l s . In c o n t r a s t to the deleted form of rF-TCF, the i n t a c t form decreased the mitogenic activity
f o r r a t h e p a t o c y t e s at h i g h e r c o n c e n t r a t i o n s
(more than 25 n g / m l ) .
Recently, Strain et al. reported hepatocyte growth s t i m u l a t i n g a c t i v i t y of natural or recombinant HGF using a primary human hepatocyte bioassay (27).
They also i n d i c a t e
that both HGFs decrease the a c t i v i t y at higher concentrations (more than 5-10 ng/ml). Thus, m i t o g e n i c e f f e c t concentrations.
of HGF on h e p a t o c y t e s tends to decrease at the h i g h e r
In regard to HGF-heparin i n t e r a c t i o n ,
reported t h a t hepatocyte growth s t i m u l a t i n g a c t i v i t y
Zarnegar and Michalopoulus
of Hepatopoietin A, which is
found to be i d e n t i c a l to HGF (28), was i n h i b i t e d by heparin (10).
This suggests that
the binding of heparin to HGF may prevent the i n t e r a c t i o n between HGF and i t s receptor on hepatocytes.
Therefore, the d i f f e r e n c e of dose-response curves between the two
rF-TCFs may be caused by d i f f e r e n t a f f i n i t y cell
f o r heparin l i k e substances found on the
surface (29) and i n the e x t r a c e l l u l a r
m a t r i x (30), which may modulate F-TCF
a c t i v i t y in v i t r o . Recombinant expression of F-TCF cDNAs e s t a b l i s h e d t h a t they encoded the a c t i v e p r o t e i n s having both tumor c y t o t o x i c and hepatocyte growth s t i m u l a t i n g a c t i v i t i e s .
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Moreover, we confirmed that both rF-TCFs had migration inducing a c t i v i t y for human ovarian carcinoma, BG-1 and mouse melanotic melanoma, B-16 cells as well as natural FTCF (data not shown). Recent studies have also indicated that HGF or HGF like protein has multiple functions: growth stimulating a c t i v i t y for various type of cells such as endothelial c e l l s (7), keratinocytes (23), melanocytes (7,23), and renal tubular e p i t h e l i a l c e l l s (31); migration inducing a c t i v i t y f o r e p i t h e l i a l c e l l s (32), endothelial cells (33), and tumor cells (7); and differentiation inducing a c t i v i t y for HL-60 (7).
Moreover, Bottaro et al. has demonstrated that HGF receptor is a B -
subunit of the c-met proto-oncogene product, a membrane-spanning tyrosine kinase (34) These f i n d i n g s suggest that HGF is an e f f e c t o r molecule responsible f o r inflammation and repair and that fibroblasts may contribute to repair the injured tissues by secreting HGF.
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TUMOR CYTOTOXICFACTOR / HEPATOCYTEGROWTHFACTORFROMHUMANFIBROBLASTS: CLONINGOF ITS cDNA , PURIFICATIONAND CHARACTERIZATIONOF RECOMBINANTPROTEIN Nobuyuki Shima *, Masaya NagaoI , Fumiko Ogaki, Eisuke Tsuda, Akihiko Murakami, and Kanji Higashio Life Science Research I n s t i t u t e , Snow Brand Milk Products Co. Ltd, 519 Ishibashi-machi, Shimotsuga-gun, Tochigi 329-05, Japan IDepartment of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Received September 26, 1991 Summary: Two d i f f e r e n t forms of cDNA f o r F-TCF were isolated from cDNA l i b r a r y prepared with mRNAfrom human embryonic lung fibroblast, IMR-90 cells. One of them was completely identical to the cDNA for placenta type hepatocyte growth factor (HGF) and the other one was a variant cDNA for the HGF with a deletion of 15 base pairs in the coding region. The cDNAs were expressed in CHO cells and recombinant proteins were purified and characterized. The deleted form of recombinant F-TCF (rF-TCF) was s l i g h t l y lower in heparin a f f i n i t y than the intact form. Both rF-TCFs showed almost same dose-response curves for c y t o t o x i c i t y on Sarcoma 180 or Meth A sarcoma cells. Dose-response curves for the stimulation of DNA synthesis in rat hepatocytes were also almost same before reaching maximal a c t i v i t y at 12.5 ng/ml but s i g n i f i c a n t l y d i f f e r e n t at higher concentrations. The deleted form of rF-TCF maintained maximal a c t i v i t y in the dose range of 12.5 to 100 ng/ml, although the intact form decreased the a c t i v i t y dose-dependently at more than 25 ng/ml. This suggests that the deletion of five amino acids results in a conformational change which alters heparin binding and hepatocyte growth stimulating activities. ~ 1991Ac~demio Press. ~nc.
Fibroblasts play important roles in inflammation and repair of injured tissues not only producing matrix proteins but secreting inflammatory cytokines such as interleukin-1 (IL-I)(1), stimulating factors (5).
IL-6 (2),
IL-8 (3),
i n t e r f e r o n - B (4), and colony
We have also found that human embryonic lung fibroblast,
IMR-90 cells secrete a tumor cytotoxic factor (F-TCF) which has cytotoxic a c t i v i t y against several tumor cell lines (6,7). a large
Purified F-TCF is a heterodimer composed of
u-chain with Mw 52 to 56-kD and a small B-chain with Mw 30 to 34-kD, and
has structural s i m i l a r i t i e s to hepatocyte growth factor (HGF)(6). HGF was f i r s t
* To whom correspondence should be addressed. Abbreviations: FGF, fibroblast growth factor; F-TCF, fibroblast-derived tumor cytotoxic factor; HGF, hepatocyte growth factor; IL, interleukin; PCR, polymerase chain reaction. 0006-291X/91 $1.50 1151
Copyright © 1991 by Academic Press, Inc. All rights' of reproduction in any form reserved.
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p u r i f i e d and c h a r a c t e r i z e d as a mitogenic f a c t o r f o r a d u l t r a t hepatocytes(8-10) . Two d i f f e r e n t cDNAs f o r HGF were cloned from human placenta ( I i ) libraries
and deduced p r i m a r y s t r u c t u r e s were determined.
sequences, mismatch o f 14 amino acids was found.
Partial
and l i v e r (12) cDNA Between the primary amino a c i d sequence
analysis of F-TCF indicated t h a t t h i s p r o t e i n might be i d e n t i c a l to the HGF derived from placenta (6).
To f u r t h e r c l a r i f y
screened from IMR-90 cDNA l i b r a r y .
the i d e n t i t y of F-TCF and HGF, F-TCF cDNA was
In t h i s report, we describe the molecular cloning,
expression, p u r i f i c a t i o n and characterization of recombinant F-TCF.
MATERIALS AND METHODS Construction of cDNA l i b r a r y : The human embryonic lung f i b r o b l a s t , IMR-90 c e l l s were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented w i t h 10% FCS. Total RNAs were extracted by the guanidine thiocyanate/cesium c h l o r i d e gradient method (13) Poly(A) ÷ RNA was selected by o l i g o ( d T ) - c e l l u l o s e column chromatography (14). cDNA was synthesized with o l i g o ( d T ) p r i m e r (Pharmacia) as described (15). Double-stranded cDNAs with EcoRl adaptor (Pharmacia) were cloned i n ~ g t l O (Promega) and packaged in v i t r o with Gigapack Gold (Stratagene). Screening of the cDNA l i b r a r y : A mixture of 17-nucleotide long oligonucleotides (Fig. 1~ was predicted from the N-terminal amino acid sequence of F-TCF B - c h a i n (6). The o l i g o n u c l e o t i d e mixture was 5'end-labeled with 3zp and used as a h y b r i d i z a t i o n probe to screen the cDNA l i b r a r y . Plaque h y b r i d i z a t i o n was carried out as described (16). Polymerase chain reaction (PCR) was used as a method to obtain f u l l coding region of F-TCF cDNA. We chose i d e n t i c a l regions between placenta type and l i v e r type HGF cDNAs as primer sequences f o r PCR. 5'-non coding region (sence strand; p o s i t i o n s from -77 to -53) flanked by Sall r e c o g n i t i o n s i t e , 5'-GGTCGACTAGGCACTGACTCCGAACAGGATTC-3'and 3'-non coding region (antisense strand; positions from 2227 to 2203) flanked by Sphl r e c o g n i t i o n s i t e , 5'-GGCATGCACAGTTGTATTGGTGGGTGCTTCAG-3'were chemically synthesized and were allowed to react with the cDNA l i b r a r y . PCR was performed according to the method (17). Expression of F-TCF cDNA: F-TCF cDNA (BamHI-Sphl; 2.3-kb) was inserted i n t o pCDNAI ( I n v i t r o g e n ) under cytomegarovirus promoter. The constructed plasmid DNA (I0 # g ) and p S V 2 n e o ( l # g ) ( 1 8 ) were c o - t r a n s f e c t e d i n t o CHO (DXB11) c e l l s ( I x l O 6 c e l l s ) by the calcium phosphate method (19). A f t e r s e l e c t i o n w i t h G418, F-TCF high producing clones were screened by an ELISA employing anti-F-TCF monoclonal antibodies (20). The cloned c e l l s were grown in DMEM/HAM-F12(Gibco) with 10% FCS. A f t e r the c e l l s were grown to confluence, the medium was replaced with DMEM/HAM-F12 w i t h 5% FCS and the c e l l s were subsequently cultured f o r 7 days. P u r i f i c a t i o n and preparation of rF-TCF: rF-TCF was p u r i f i e d as previously described except f o r the f o l l o w i n g modifications. The conditioned medium of the transformant was a p p l i e d to heparin sepharose column (@2.5 X 8 cm, Pharmacia) at a flow rate of 6 ml/min. The column was washed w i t h e q u i l i b r a t i o n b u f f e r ( i 0 mM T r i s HCI, pH 7.5, c o n t a i n i n g 0.01% Tween 20 and 0.3 M NaCl) followed by e l u t i o n with 2 M NaCl in the e q u i l i b r a t i o n buffer. The eluate was dialyzed against PBS and was f u r t h e r p u r i f i e d by the combinations of Mono S and h e p a r i n - a f f i n i t y HPLCs as p r e v i o u s l y described (6). The amounts of the p u r i f i e d rF-TCF were determined by a p r o t e i n assay k i t (Bio-Rad) using BSA as a standard protein. B i o l o g i c a l assay: Sarcoma 180 and Meth A sarcoma c e l l s , which were obtained from National Cancer Center, were used as t a r g e t c e l l s f o r c y t o t o x i c assay as p r e v i o u s l y described (6,7). Adult r a t hepatocytes were isolated and prepared as described (21). E f f e c t of rF-TCF on DNA synthesis in r a t hepatocytes was determined by the published method (22).
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RESULTS Isolation and characterization,of cDNA clones:
5x105 plaques on Escherichia c o l i C600
high f r e q u e n c y l l s o g e n (Promega) were screened by h y b r i d i z a t i o n w i t h the oligonucleotide probe. We obtained one p o s i t i v e clone ( ~ - 1 3 ) insert (Fig. 1).
containing a 5.7-kb
The insert o f h - 1 3 contained an open reading frame which completely
matched with the corresponding region of the placenta type HGF cDNA (positions, 7722184).
N e x t we used PCR to obtain f u l l coding region of F-TCF cDNA.
A DNA band with
estimated size (2.3kb) was obtained from PCR reactant by electrophoresis with 1% agarose gel.
The DNA fragment was subcloned i n t o pUC18, and several clones were
analysed f o r DNA sequences. Two forms of cDNA f o r F-TCF were detected from PCR amplified DNA fragments (Fig. 1). One of them completely matched with the placenta type HGF cDNA and the other one was a variant of the HGF cDNA with a deletion of 15 base pairs in the coding region (positions, 483-497). P u r i f i c a t i o n of rF-TCFs:
On h e p a r i n - a f f i n i t y HPLC as a f i n a l p u r i f i c a t i o n step, the
intact and deleted forms of rF-TCF were eluted from the column at 1.31M and at 1.28
0
1
2
3
,
l
,
1//~
6.5 kb II--
2-13
3'-CAACATTTACCCTAAGG-5' G G G G G T C T T C A A VaIValAsnGlyIlePro
(1) (2)(3) (4) start-
(kd)
E I
5' - - I i
C)
AE ~
HE H K
I
\I/
a -chain A 483 497
~ 1468
t
-
-
-//--
3'
2184
ACT~TTTTGCCTTCGA~CT Se~rPheLeuProSerSelr
Q
94
--
67
--
43
--
31
-
21.5-14.4-
Fig. i , Schematic representation of F-TCF cDNAand mRNA. Relationship betweenthe cDNA clone, A -13 and the synthetic oligonucleotide mixture (below the cDNA clone) are shown above a diagramof the F-TCF mRNA. Non coding regions are representedby a line and the coding region is boxed. Black area shows the signal peptide. Open box indicates the coding regions for the a - and B -chain. Openarrowhead indicates the site of the 15 nucleotides deletion. The deleted sequence are shown below the open arrowhead. E, EcoRI; A, Ava m ; H, Hind • ; K, KpnI. Fig. 2, SDS-PAGEof purified rF-TCFs. Lane 1; the intact form of rF-TCF under non reducing conditions, Lane 2; the deleted form of rF-TCF under non reducing conditions, Lane 3; the intact form of rF-TCF under reducing conditions, Lane 4; the deleted form of rF-TCF under reducing conditions.
1153
a
B B'
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I00'
a
b
80'
//
60' o X 0
20"
.
1
.
.
2
.
4
.
;I
.
.
8 16 31 63 J25 250 500
.
.
.
.
.
1
2
.
.
4
.
.
.
.
.
.
8 16 31 63 125 250 500
rF-TCFs (ng/ml)
Fia. 3. Cytotoxic activity of intact or deleted form of rF-TCF against mousetumor cell lines. (a) Cytotoxicity on sarcoma 180 cells: 0 - - 0 ; the deleted form of rFTCF, 0 - - 0 ; the intact form of rF-TCF. (b) Cytotoxicity on Meth A sarcoma cells: mm--m ; the deleted form of rF-TCF, D - - D ; the intact form of rF-TCF. Results are presented as mean ± SD of t r i p l i c a t e cultures.
M NaCl, respectively.
For analysis of p u r i t y and molecular weight, the p u r i f i e d rF-
TCFs were subjected to SDS-PAGE and the gel was stained with s i l v e r .
Both purified
preparations showed two adjucent bands with 76 to 80-kD under non reducing conditions (Fig. 2).
These molecular weights coincided with those of natural F-TCF which we
previously reported (6).
Under reducing conditions, the preparations separated into
three bands with 52 to 56, 34 and 30-kD corresponding to ~ ,
B and
# ' - c h a i n s as
well as natural F-TCF (Fig. 2). Biological a c t i v i t i e s of rF-TCFs: Both rF-TCFs showed almost same dose-responses for c y t o t o x i c i t y on Sarcoma 180 or Meth A sarcoma cells (Fig. 3). which gave 50% c y t o t o x i c i t y
The amounts of rF-TCFs
on Sarcoma 180 and Meth A were 7 and 32 ng/ml,
r e s p e c t i v e l y . These were consistent with our previous r e s u l t s with natural F-TCF (Sarcoma 180 at 6 ng/ml and Meth A at 40 ng/ml )(7).
B o t h rF-TCFs showed growth
s t i m u l a t i n g a c t i v i t y f o r r a t hepatocytes as well as natural F-TCF. Dose-response curves f o r the two rF-TCFs were almost same before reaching maximal a c t i v i t y at 12.5 ng/ml but s i g n i f i c a n t l y d i f f e r e n t at more than 25 ng/ml (Fig. 4).
The intact form of
rF-TCF decreased the a c t i v i t y dose-dependently at more than 25 ng/ml. 1154
In contrast,
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20 I-
(zz~ X
10
5
1.6 3.1 6.3 12.5 25
50 100 200 500 rF-TCFs (n~/ml)
Fig. 4. E f f e c t of i n t a c t or d e l e t e d form of rF-TCF on DNA s y n t h e s i s in p r i m a r y cultured a d u l t r a t hepatocytes. Hepatocytes were c u l t u r e d with the i n t a c t form of rFTCE ( c l e a r bar) or the deleted form of rF-TCF (cross-hatched bar) f o r 24 hrs. Results are presented as mean + SD of t r i p l i c a t e c u l t u r e s . S t a t i s t i c a l s i g n i f i c a n c e was determined by Student's t - t e s t (* P < 0.01).
the deleted form maintained maximal a c t i v i t y in the dose range of 12.5 to 100 ng/ml and decreased the a c t i v i t y dose-dependentlyat more than 200 ng/ml.
DISCUSSION Two forms of cDNA encoding F-TCF were isolated from humanfibroblast cDNA library. When the cDNAs were introduced into pcDNAI vector and were expressed transiently in COS cells, authentic cytotoxic activities against Sarcoma 180 and Meth A sarcoma cells were detected in both conditioned media but not detected in the control medium from the cells transfected pcDNAI only (data not shown). This established that the cDNAs encoded F-TCF which were i d e n t i f i e d as a tumor cytotoxic factor. We previously reported that partial amino acid sequences for F-TCF are identical to placenta type HGF but different From l i v e r type HGF (6).
Indeed, one cDNA for F-TCF had a coding
sequence completely matched with placenta type HGF cDNA which has mismatch of 39 nucleotides compared to l i v e r type HGF cDNA. The other cDNAwas a variant of placenta type HGF cDNA with a deletion of 15 base pairs in the coding region.
Recently, the
same variant sequence was isolated from the cDNA l i b r a r i e s derived from human fibroblasts (23) and human leukocytes (24). 1155
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The d e l e t e d form of rF-TCF e l u t e d s l i g h t l y
earlier
than the i n t a c t form on the
h e p a r i n - a f f i n i t y HPLC. This indicates that the absence of f i v e amino acids (Phe-LeuPro-Ser-Ser) r e s u l t s in s t r u c t u r a l change t h a t a l t e r heparin-binding properties.
F-
TCF is s i m i l a r to f i b r o b l a s t growth f a c t o r (FGF) in c h a r a c t e r i s t i c of high a f f i n i t y f o r heparin.
Amino acid sequence, Phe-Leu-Pro is one of the conserved sequence in
a l l seven members of the FGF f a m i l y (25), and t h i s sequence is thought to c o n t r i b u t e to the h e p a r i n b i n d i n g a c t i v i t y
of b a s i c FGF ( 2 6 ) .
Primary sequence of F-TCF
indicates that the i n t a c t form of F-TCF has Phe-Leu-Pro sequence at the f i r s t
and the
second k r i n g l e s , r e s p e c t i v e l y , and the v a r i a n t F-TCF has only the second Phe-Leu-Pro sequence.
Thus, the second sequence might also contribute to the a f f i n i t y
in a d d i t i o n to the f i r s t
for heparin
sequence.
The comparison of s p e c i f i c a c t i v i t i e s of rF-TCFs revealed that the d e l e t i o n of f i v e amino acid residues did not a f f e c t c y t o t o x i c a c t i v i t y but affected hepatocyte growth s t i m u l a t i n g a c t i v i t y at higher concentrations.
These imply that d i f f e r e n t mechanisms
mediate the e f f e c t on growth i n h i b i t i o n and s t i m u l a t i o n in d i f f e r e n t type of c e l l s . In c o n t r a s t to the deleted form of rF-TCF, the i n t a c t form decreased the mitogenic activity
f o r r a t h e p a t o c y t e s at h i g h e r c o n c e n t r a t i o n s
(more than 25 n g / m l ) .
Recently, Strain et al. reported hepatocyte growth s t i m u l a t i n g a c t i v i t y of natural or recombinant HGF using a primary human hepatocyte bioassay (27).
They also i n d i c a t e
that both HGFs decrease the a c t i v i t y at higher concentrations (more than 5-10 ng/ml). Thus, m i t o g e n i c e f f e c t concentrations.
of HGF on h e p a t o c y t e s tends to decrease at the h i g h e r
In regard to HGF-heparin i n t e r a c t i o n ,
reported t h a t hepatocyte growth s t i m u l a t i n g a c t i v i t y
Zarnegar and Michalopoulus
of Hepatopoietin A, which is
found to be i d e n t i c a l to HGF (28), was i n h i b i t e d by heparin (10).
This suggests that
the binding of heparin to HGF may prevent the i n t e r a c t i o n between HGF and i t s receptor on hepatocytes.
Therefore, the d i f f e r e n c e of dose-response curves between the two
rF-TCFs may be caused by d i f f e r e n t a f f i n i t y cell
f o r heparin l i k e substances found on the
surface (29) and i n the e x t r a c e l l u l a r
m a t r i x (30), which may modulate F-TCF
a c t i v i t y in v i t r o . Recombinant expression of F-TCF cDNAs e s t a b l i s h e d t h a t they encoded the a c t i v e p r o t e i n s having both tumor c y t o t o x i c and hepatocyte growth s t i m u l a t i n g a c t i v i t i e s .
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Moreover, we confirmed that both rF-TCFs had migration inducing a c t i v i t y for human ovarian carcinoma, BG-1 and mouse melanotic melanoma, B-16 cells as well as natural FTCF (data not shown). Recent studies have also indicated that HGF or HGF like protein has multiple functions: growth stimulating a c t i v i t y for various type of cells such as endothelial c e l l s (7), keratinocytes (23), melanocytes (7,23), and renal tubular e p i t h e l i a l c e l l s (31); migration inducing a c t i v i t y f o r e p i t h e l i a l c e l l s (32), endothelial cells (33), and tumor cells (7); and differentiation inducing a c t i v i t y for HL-60 (7).
Moreover, Bottaro et al. has demonstrated that HGF receptor is a B -
subunit of the c-met proto-oncogene product, a membrane-spanning tyrosine kinase (34) These f i n d i n g s suggest that HGF is an e f f e c t o r molecule responsible f o r inflammation and repair and that fibroblasts may contribute to repair the injured tissues by secreting HGF.
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