JP3187113B2 - Cell growth factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an androgen-induced growth factor, a DNA encoding the factor, a plasmid having the DNA, a host cell having the plasmid, a method for producing the factor using the host cell, breast cancer. The present invention relates to a method for producing the factor using cells, a probe that hybridizes with the DNA, a method for detecting the DNA using the probe, and a cell growth agent having the factor.

[0002]

2. Description of the Related Art The self-proliferation mechanism of cancer cells is one of the central concepts of cancer cell proliferation, and various growth factors and growth factor receptors related to oncogene products have been reported. Is thought to form a self-propagating ring. Cancers arising from sex hormone target organs, such as breast and prostate cancers, are characterized by hormone-responsive proliferation. The earliest breast cancers require hormonal stimulation for their growth. Hormone-responsive growth of breast cancer cells has been attributed to growth factors secreted by hormone stimulation. Structural analysis and characterization of such hormone-induced growth factors are essential for elucidating the growth mechanism of hormone-responsive cancers.

The androgen-responsive mouse breast cancer cell, Shionogi carcinoma 115 (SC115), is widely recognized as a sex hormone-responsive cancer (Nonomura et al., Perspective
inAndrology (ed. Serino, M.) 431-438 (Raven, New
York, 1989)). Its proliferation is stimulated only by androgens. Androgen-dependent serum-free culture system (SC-3 cell line) obtained from SC115 tumor (Nakamura et al., J. Steroid Bi
ochem. 27, 459-464 (1987)) is a good model system for studying the molecular mechanisms by which sex hormones promote cancer cell growth. It has been suggested that androgen-dependent growth of SC-3 cells is mediated by heparin-binding growth factors in the self-proliferation mechanism (Nonomura et al., Supra; Nakamura et al., Supra; Nonomura et al., Cancer Res. 48 , 4904-4908 (198
8)).

[0004]

As described above, it has been suggested that androgen-dependent growth of SC-3 cells is mediated by heparin-binding growth factor in the self-proliferation mechanism. Not isolated or identified. Further, DNA encoding this factor has not been isolated or identified. If DNA encoding the factor can be obtained, the factor can be mass-produced using genetic engineering techniques. If a large amount of the factor can be obtained, an anticancer substance as an inhibitor of the factor can be searched. Further, if the DNA can be detected with a probe that hybridizes with the DNA, it is useful for cancer diagnosis. In addition, the factor itself is also expected as a cell growth reagent or drug due to the cell proliferation action of the factor.

[0005]

SUMMARY OF THE INVENTION The present invention is the first to report the cDNA cloning and functional expression of an androgen-induced self-growth factor obtained from androgen-dependent mouse breast cancer cells (SC-3 cells). is there. This androgen-induced growth fact (AIGF)
or) is a novel heparin-binding growth factor that is significantly induced by androgens and proliferates SC-3 cells in the absence of androgens.

The serum-free conditioned medium obtained from testosterone-stimulated SC-3 cells showed a remarkable growth promoting effect on SC-3 cells. The growth promoting activity was bound to heparin-Sepharose and eluted at 1.0-1.2 M sodium chloride concentration. After repeated heparin-Sepharose chromatography, the bioactive fraction was subjected to reverse phase HPLC (FIG. 1). The bioactive fraction obtained from reverse-phase HPLC was converted to SD under reducing and non-reducing conditions.
On S-PAGE, it contained two major proteins, 32 kDa and 28 kDa, respectively. Both the 32 kDa and 28 kDa proteins showed similar biological efficacy (FIG. 2). Both proteins are blocked at the N-terminus, but reversed-phase HPLC
Peptide mapping after digestion with lysyl endopeptidase revealed that most peptides from the two proteins were identical. The sequence of the peptide fragment (FIG. 3) was determined by reverse phase HPLC. From the sequence of the peptide in FIG.
^Was synthesized and cultured in the presence of 10 ^-8 M testosterone.
Polymerase-chain reaction was performed using random-primed single-stranded cDNA derived from total RNA of -3 cells.
240 bp DNA is obtained from one set of primers,
It contained various peptide sequences obtained by digesting AIGF with lysyl endopeptidase. This DNA fragment was used as an expression plasmid vector for mammalian cells-pcDL-SRα296 (Takebe
8, 466-472 (1988)) and oligo (dT) -primed double-stranded cDNA derived from polyadenylated RNA of testosterone-stimulated SC-3 cells.
It was used as a probe for screening of the cDNA library. 4 × 10 40 screens with ⁴ clones
Positive clones were obtained, of which 20 were infected with COS-7 cells. Eight clones were positive in the SC-3 cell proliferation test (FIG. 6). All bioactive clones are 1.0-1.
It contained a 2 kb insert.

The nucleotide sequence of one of these clones (pSC17) had an open reading frame of a putative 215 amino acid residues with the 3 'untranslated sequence. ATG
The start codon is located at nucleotide position 174, followed by a putative signal peptide for a hydrophobic residue downstream. The stop codon is at nucleotide position 819. This open reading frame encodes a sequence of 215 amino acids with one potential N-glycosylation site (FIG. 4). All peptides were included in this sequence except for peak 7, which was obtained by lysyl endopeptidase digestion (FIG. 4). Direct determination of the peptide sequence of AIGF failed to detect the N-terminus. von Heije's rule (G.
Based on Biochim. Et Biophys. Acta 947, 307-333 (1988)), the N-terminus of AIGF is assumed to be a glutamine residue at amino acid position 23, which is thought to cause N-terminal blocking by pyroglutamination. The mature protein has a molecular weight of about 22 kDa. However, when the nucleotide sequence of another clone, pSC15, was determined, 10 amino acids (residues 25-34, FIG. 4) of the pSC17 clone following the deduced signal sequence were replaced with 63 amino acid residues. (FIG. 4), suggesting the presence of a 28 kDa mature protein with another glycosylation site. The peptide sequence of peptide 7 is
It was found in pSC15 but not in pSC17 (FIG. 4).
These clones are differently spliced A
It appears to be a product from the IGF transcript. 3 on SDS-PAGE
The difference between the two major proteins at 2 kDa and 28 kDa and the difference between the theoretical value of the molecular weight and the value on SDS-PAGE was due to the glycosylation at the putative N-glycosylation position, as described above. This may be due to differences in transcript splicing. In fact, treatment with endoglycosidase F
The SDS-PAGE pattern of AIGF changed and the 32 kDa band became faint, while the intensity of the 28 kDa band increased and the 23 kDa band appeared. This is due to the glycosylation of 22 kDa and 28 kDa AIGF, resulting in 28 kDa and 32 kDa AIGF, respectively.
It suggests that GF is formed.

[0008] As a result of investigating the homology between AIGF and known proteins, it was found that AIGF shows homology to the FGF family. Overall, int-2, hst-1, FGF-5, F
Not only proteins encoded by GF-6 but also basic FGF
And 30-40% homology with KGF (Esch, F. et al., Pr.
oc. Natl. Acad. Sci. USA 82, 6507-6511 (1985);
Finch, PW et al., Science 245, 752-755 (1989); Dickso
n, C. et al., Nature 326, 833 (1987); Taira, M. et al., Pro.
c. Natl. Acad. Sci. USA 84, 2980-2984 (1987); Z
han, X. et al., Molec. Cell. Biol. 8, 3487-3495 (198
8); Marics, I. et al., Oncogene 4, 335-340 (1989)). FGF
Although the positions of the two cysteine residues are well conserved in the family, the cysteine residue at position 127 is only conserved in the AIGF sequence. Receptor binding site of basic FGF (Eriksson, AE et al., Proc. Natl. Acad. Sci. US
A. 88, 3441-3445 (1991)), amino acid residue 1 of mouse FGF
14-123 show only 44.4% homology with the predicted sequence of AIGF. AIGF signaling is transmitted by the basic FGF receptor (Nonomu
ra, N. et al., Cancer. Res. 50, 2316-2321 (1990)) or via a different receptor for AIGF itself. All proteins encoded by FGF-related oncogenes have an N-terminal signal peptide sequence. The presence of a signal peptide sequence is an important factor in determining whether a member of the FGF family is capable of transforming. CDNA encoding basic FGF having an immunoglobulin signal peptide sequence at the N-terminus
Can transform NIH 3T3 cells (Yayon,
A. et al., Proc. Natl. Acad. Sci. USA 87, 5346-5350.
(1990)). AIGF also has a signal sequence, which is SC-
This suggests a close link between AIGF and three-cell cancerous growth.

[0009] Androgen induction of AIGF was confirmed by Northern blot analysis. AIGF mRNA was not detected by Northern blot analysis in the absence of testosterone (FIG. 5). AIGF mRNA appeared 6 hours after stimulation with 10 ⁻⁸ M testosterone and increased significantly by 24 hours (FIG. 5).

The expression of AIGF cDNA in mammalian cells allows
It was revealed that IGF significantly promoted the growth of SC-3 cells in the absence of androgen (FIG. 6). In addition, AIGF
Changed the morphology of SC-3 cells, which was also found in SC-3 cells stimulated by testosterone (Tan
aka, A. et al., J. Steroid Biochem. Molec. Biol. 37,23-
29 (1990)). SC-3 cells stimulated with AIGF exhibited a fibroblastic appearance, in contrast to the epithelial appearance of SC-3 cells treated with conditioned medium of COS 7 cells transformed with the plasmid vector alone.

[0011] AIGF is a secreted heparin-binding growth factor having a signal peptide sequence and is significantly induced by androgens. AIGF promotes the growth of SC-3 cells in the absence of androgens and changes the morphology of SC-3 cells. These results conclude that hormone-dependent cancerous growth of SC-3 cells is mediated by AIGF secreted by the cancer cells themselves in response to hormonal stimulation.

[0012] The growth of breast and prostate cancer is affected by hormones. The identification of AIGF in the present invention reveals that hormone-induced self-proliferative factors play a central role in the growth of breast and prostate cancer and provide a rationale for hormonal treatment of such cancers .

As described above, first, the present invention provides a cell growth factor having an amino acid sequence from His at position 35 to Arg at position 215 in the amino acid sequence of SEQ ID NO: 1. The amino acid sequence is, as apparent from FIG.
It is common to pSC17 and pSC15 clones obtained in the present invention, and is considered to be an amino acid sequence necessary and sufficient for the growth factor of the present invention.

The present invention further relates to an amino acid sequence from Gln at position 23 to Arg at position 215 of the amino acid sequence of SEQ ID NO: 1 or 268 to Gln at position 23 of the amino acid sequence of SEQ ID NO: 2. A cell growth factor having an amino acid sequence up to the Arg position. The sequence from Met at position 1 to Ala at position 22, which is common to the amino acid sequences of SEQ ID NOs: 1 and 2, is assumed to be a signal sequence. Therefore, SEQ ID NOs: 1 and 2
A cell growth factor having an amino acid sequence obtained by removing a signal sequence from the amino acid sequence of the above is included in the present invention.

The present invention also relates to an amino acid sequence from Gly at position 2 to Arg at position 215 of the amino acid sequence of SEQ ID NO: 1 or 2 of the amino acid sequence of SEQ ID NO: 2.
A cell growth factor having an amino acid sequence from position Gly to position 268 Arg is provided.

The cell growth factor of the present invention has the following properties. (1) Induced by androgen. (2) Heparin binding. (3) Proliferate SC-3 cells in the absence of androgen. Of the above amino acid sequences, those in which one or more amino acid residues are added, removed, inserted, or substituted are included in the present invention as long as they have these properties.

Further, as is apparent from the analysis of the amino acid sequence, since the cell growth factor of the present invention has a glycosylation site, it is considered that one having a sugar chain is preferable.

The present invention also provides a DNA encoding the above-mentioned cell growth factor, a plasmid having the DNA and capable of expressing the cell growth factor, and a host cell having the plasmid. The host cells of the present invention include prokaryotic hosts such as Escherichia coli and Bacillus subtilis, yeast, COS cells, CHO cells, Hela cells, 3T
3 cells, 3T6 cells, Ltk ^- such as eukaryotic hosts such as cells, can be used normally all host cells used in the present transgenic art. Since the cell growth factor of the present invention is considered to have a sugar chain, the host is preferably a eukaryotic host. As the plasmid of the present invention, a normal expression plasmid / virus vector may be used depending on the host used. In the case of using a eukaryotic host, besides the above pcDL-SRα296, commercially available pSVK3, pBPV, pBP
MSG, pMSG-CAT, pSVL, pCH110, pCaMVCN (Pharmacia)
May be used.

The present invention provides a method for producing a cell growth factor, which comprises culturing the above host cell. Host cells capable of expressing the cell growth factor of the present invention can be cultured by a usual method depending on the properties of the host cells. In order to obtain the growth factor from the culture medium, affinity chromatography using heparin can be repeated as many times as necessary, and if necessary, can be isolated and purified by subjecting it to reverse phase HPLC.

Since the cell growth factor of the present invention can be stably supplied in large quantities at low cost, those produced by the above-mentioned production method are preferable, but breast cancer cells, preferably SC-3 cells stimulated with testosterone, are preferably used. The androgen-reactive mouse breast cancer cells can be cultured in a serum-free conditioned medium and obtained from the medium by the above-described isolation and purification method.

The present invention further relates to a DNA or RNA which hybridizes with the DNA or RNA encoding the cell growth factor.
Is specifically provided. The probe is a DNA probe labeled with a commonly used enzyme, radioisotope, or the like, and encodes the cell growth factor in normal Northern or Southern blot analysis.
Any DNA that specifically hybridizes with DNA or RNA and that can specifically detect the DNA or RNA can be used, and any of the DNA sequences described in SEQ ID NOS: 1 and 2 (or their complementary sequences) can be used. Some, especially Xho of pSC17 clone
Those having an I fragment are preferred.

Using this probe, DNA or RNA encoding the cell growth factor, or a gene highly homologous thereto can be detected by ordinary Northern or Southern blot analysis. .

The cell growth factor of the present invention is useful as a cell growth agent because it allowed SC-3 cells to grow even in the absence of androgen. In order to use the cell growth factor of the present invention as a cell growth agent, a stabilizer, a preservative, and the like used in the preparation of a general peptide reagent may be added.

Since the cell growth factor of the present invention proliferates cancer cells, it is useful for searching for a substance having an anticancer effect as an inhibitor of the present factor.

[0025]

EXAMPLES Purification of AIGF 4 L of conditioned medium obtained from serum-free culture of SC-3 cells stimulated with 10 ⁻⁸ M testosterone was concentrated 10-fold,
The sample was applied to a heparin-Sepharose column (gel layer volume; 5 ml) equilibrated with a 10 mM Tris-HCl buffer (pH 7.0) containing M NaCl, 0.1% CHAPS, 2 mM PMSF, and 500 μg / ml leupeptin. The column was washed with the above-mentioned equilibration buffer, and the adsorbed protein was eluted with 10 times the volume of the gel buffer containing 2 M NaCl. The eluted fraction was diluted with NaCl-free equilibration buffer to reduce the NaCl concentration to 0.6 M,
It was refractionated in the same manner using a Sepharose column (gel layer volume: 0.5 ml). The eluted fraction was applied to a 4.6 × 50 mm Cosmosil C4 reverse phase column, and developed with a concentration gradient of 0-60% acetonitrile in 0.1% TFA at an elution rate of 1 ml / min over 30 minutes ( (Fig. 1). [ ³ H] thymidine incorporation activity in SC-3 cells in each fraction was determined by the method of Yamanishi et al. (Yamanishi, H .;
And Cancer Res. 51, 3006-3010 (1991).
The biologically active fraction was lyophilized, subjected to a reducing (FIG. 2) and non-reducing condition on a 10-20% gradient acrylamide gel containing 0.1% SDS and silver-stained. The same sample of an adjacent lane developed under non-reducing conditions was divided into 38 equal fractions. 10 mM Tris-HCl containing 0.1% CHAPS (pH 7.
The protein was eluted by stirring overnight at 4 ° C in 5). The eluate was assayed for [ ³ H] thymidine incorporation activity (FIG. 2). The purified AIGF was denatured with 70% formic acid at room temperature for 2 hours and digested with 500 ng of lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.) in 50 mM Hepes buffer (pH 7.6) for 5 hours at 37 ° C. Digest the sample directly into a 3.9 × 150 mm μ-Bon
The sample was applied to a dasphere C18 reverse phase column (Fig. 3). The peptides were separated by a 0-60% acetonitrile linear gradient over 60 minutes at an elution rate of 1 ml / min, and different peaks were collected. Sequence analysis was performed on an ABI 477A protein sequencer (Applied Bios) using an online ABI 120A PTH analyzer.
ystems, Foster city, CA).

The structure SC-3 cells AIGF a (3 × 10 ^5/100 mm dish) were placed in MEM 10 ml of containing 2% fetal bovine serum and 10 ^-8 M testosterone treated with dextran-coated charcoal. Next day (day 0)
Was changed once. On the second day, nearly confluent SC-3 cells (approximately 4 × 10 ⁶ cells / dish) were collected, and total cellular RNA was extracted with acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski, P. et al., N.
Analyt. Biochem. 162, 156-159 (1987)). Randomly primed single-stranded cDNA is converted to reverse transcriptase (Sup
erscript, Bethesda Research Laboratories, Gaithers
burg, MD) using total RNA from SC-3 cells cultured in the presence of 10 ^-8 M testosterone. Oligonucleotide primers were synthesized according to the obtained amino acid sequence. Of the various primer sets, 5 '> ATHAAYGCIATG
GCIGARGAYGG <3 ′ (SEQ ID NO: 3) and 5 ′> GCCATRTACCA
When a set of ICCYTCRTA <3 ′ (SEQ ID NO: 4) (where H represents A or C or T, Y represents T or C, and R represents G or A), 1 is used in the polymerase chain reaction. Horn
A DNA band was obtained. Polymerase chain reaction includes single-stranded cDNA, synthetic oligonucleotides, Ampli
TaqTM recombinant Taq DNA polymerase (PERKIN-ELMER C
ETUS Instrument, Norwalk, CT), 94 ° C 90 seconds, 3
A reaction at 7 ° C. for 90 seconds and 72 ° C. for 180 seconds was repeated 30 times. The nucleotide sequence of the amplified cDNA was determined by the dideoxynucleotide chain termination method (Sanger, F. et al., Proc. Natl.
d. Sci. USA 74, 5463-5467 (1977)). A cDNA library was constructed from polyadenylated RNA of SC-3 cells cultured in the presence of 10 ^-8 M testosterone. Polyadenylated RNA was isolated using oligo (dT) latex. Oligo (dT) primed double-stranded cDNA is cDNA
The plasmid vector was synthesized using a synthesis system (Bethesda Research Laboratories) and the plasmid vector pB was synthesized using BstXI linker.
Bound to cDL-SRα296. Hybridization is
Performed at ³² ° C. in 1.5 × SSPE, 1.0% SDS, 0.5% Blotto at ³² ° C. using a ³² P-labeled probe obtained by polymerase chain reaction, and 50 ° C. in 2 × SSC, 0.1% SDS.
Then, the plate was washed with 0.1 × SSC and 1.0% SDS at 37 ° C. for 30 minutes. Twenty positive clones were used to infect COS 7 cells. The sequence of one of the bioactive clones (pSC17) was determined.

FIG. 4 shows the nucleotide sequence and the deduced amino acid sequence of AIGF. AIGF cDNA (pSC17 clone) encodes a protein of 215 amino acids. The underlined portion is the peptide sequence obtained by lysyl endopeptidase digestion (FIG. 3, Nos. 1 to 10). Two peptides are peaks in FIG.
It was included in 10. The shaded areas are the two primer areas used for the Polymerase Chain Reaction. Boxed portions are amino acid residues substituted between two different clones, pSC17 and pSC15. The potential for N-glycosylation is underlined.

[0028] The AIGF mRNA induction SC-3 cells (8 × 10 ^5/100 mm dish) by testosterone, and treated with dextran-coated charcoal in the absence of testosterone 2
The cells were placed in 10 ml of MEM containing% fetal calf serum. The next day, cells were washed with PBS and further cultured in MEM containing serum with or without ^10-8 M testosterone. Polyadenylated RNA was isolated at the times shown in FIG. 2 μg of polyadenylated RNA was electrophoresed in a 1% agarose gel containing 0.66 M formaldehyde and transferred onto a nylon membrane.
Hybridization is 1.5 × SSPE, 1.0% SDS,
Performed in a 0.5% Blotto at 65 ° C. for 18 hours. The blot is
2 × SSC, 0.1% SDS at 50 ° C for 30 minutes, followed by 0.1 × SS
C, washed with 1.0% SDS at 37 ° C for 30 minutes. Autoradiography was performed at -70 ° C for 48 hours.

[0029] FIG. 5, pSC17 black - the emission of XhoI fragment ³² P
The result of hybridization of the labeled AIGF cDNA probe and the blot is shown. In the figure, the transfer position of the ribosome RNA is shown on the left.

Expression of AIGF cDNA in Mammalian Cells COS 7 cells were treated with calcium phosphate precipitation to obtain 10 μg of cDNA.
Infected. Forty-eight hours after infection, media was harvested and assayed for [ ³ H] thymidine incorporation in SC-3 cells. The vector plasmid alone was used to infect COS 7 cells, and the conditioned medium was used as a control to assay for biological activity.

FIG. 6 shows a conditioned medium of COS 7 cells infected with a vector plasmid containing the AIGF cDNA (pSC17) (right) and SC-3 cells infected with the vector plasmid alone (left).
Fig. ³ shows the [ ³ H] thymidine uptake activity in cells.

[Sequence list]

SEQ ID NO: 1 Sequence length: 997 Sequence type: Nucleic acid sequence CGCGCGCGGC GAGCACGACA TTCCACCGGA CCCGCCGAGC CGCGTCGGGA TAGCCGCTGG 60 CCTCCCGCAC CCCGACCTCC CTCAGCCTCC GCACCTTCGG CTTGTCCCCC CGCGGCCTCC 120 AGTGGGGCCC GCTCGCCGCGCTGCGCCGCGCTGCGCCGCGTCGCCGCGTCGCCGCGCTGCGCCGCGCTGCGCCGGCTCGCCGGCT AGC TGC CTG CTG TTG CAC TTG CTG GTT 224 Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu Val 5 10 15 CTC TGC CTC CAA GCC CAG GTA ACT GTT CAG TCC TCA CCT AAT TTT ACA 272 Leu Cys Leu Gln Ala Gln Val Thr Val Gln Ser Ser Pro Asn Phe Thr 20 25 30 CAG CAT GTG AGG GAG CAG AGC CTG GTG ACG GAT CAG CTC AGC CGC CGC 320 Gln His Val Arg Glu Gln Ser Leu Val Thr Asp Gln Leu Ser Arg Arg 35 40 45 CTC ATC CGG ACC TAC CAG CTC TAC AGC CGC ACC AGC GGG AAG CAC GTG 368 Leu Ile Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His Val 50 55 60 65 CAG GTC CTG GCC AAC AAG CGC ATC AAC GCC ATG GCA GAA GAC GGA GAC 416 Gln Val Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Glu Asp Gly Asp 70 75 80 CCC TTC GCG AAG CTC ATT GTG GAG ACC GAT ACT TTT GGA AGC AGA GTC 464 Pro Phe Ala Lys Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg Val 85 90 95 CGA GTT CGC GGC GCA GAG ACA GGT CTC TAC ATC TGC ATG AAC AAG AAG 512 Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys Lys 100 105 110 GGG AAG CTA ATT GCC AAG AGC AAC GGC AAA GGC AAG GAC TGC GTA TTC 560 Gly Lys Leu Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val Phe 115 120 125 ACA GAG ATC GTG CTG GAG AAC AAC TAC ACG GCG CTG CAG AAC GCC AAG 608 Thr Glu Ile Val Leu Glu Asn Asn Tyr Thr Ala Leu Gln Asn Ala Lys 130 135 140 145 TAC GAG GGC TGG TAC ATG GCC TTT ACC CGC AAG GGC CGG CCC CGC AAG 656 Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg Lys 150 155 160 GGC TCC AAG ACG CGC CAG CAT CAG CGC GAG GTG CAC TTC ATG AAG CGC 704 Gly Ser Lys Thr Arg Gln His Gln Arg Glu Val His Phe Met Lys Arg 165 170 175 CTG CCG CGG GGC CAC CAC ACC ACC GAG CAG AGC CTG CGC TTC GAG TTC 752 Leu Pro Arg Gly His His Thr Thr Glu Gln Ser Leu Arg Phe Glu Phe 180 185 190 CTC AAC TAC CCG CCC TTC ACG CGC AGC CTG CGC GGC AGC CAG AGG ACT 800 Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg Thr 195 200 205 TGG GCC CCG GAG CCC CGA TAGGCGCTCG CCCAGCTCCT CCCCACCCAG Trp Ala Pro Glu Pro Arg 210 215 CCGGCCGAGG AATCCAGCGG GAGCTCGGCG GCACAGCAAA GGGGAGGGGC TGGGGAGCTG 908 CCTTCTAGTT GTGCATATTG TTTGCTGTTG GGTTTTTTTTTG TTTTTTGTTT TTTGTTTTTG 968 TTTTTTGTTT TTTAAACA997 AGA

SEQ ID NO: 2 Sequence length: 1178 Sequence type: Nucleic acid sequence TCCAGCAGCG GCTCAGAGGG GTTCGGCGCG CGCGGCGAGC ACGACATTCC ACCGGACCCG 60 CCGAGCCGCG TCGGGATAGC CGCTGGCCTC CCGCACCCCCG ACCTCCCTCA GCCTCCCCCGCC GCCTCGCCGCGCCCTCGCCCCCGCGCTCGTCCCCGCGCCCTCCCCCGCGCCCT AGC TGC CTG 232 Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu 1 5 10 CTG TTG CAC TTG CTG GTT CTC TGC CTC CAA GCC CAG GTA AGG AGC GCT 280 Leu Leu His Leu Leu Val Leu Cys Leu Gln Ala Gln Val Arg Ser Ala 15 20 25 GCG CAG AAG CGG GGG CCG GGC GCG GGG AAC CCA GCT GAC ACT CTC GGG 328 Gln Gly His Glu Asp Arg Pro Phe Gly Gln Gln Arg Ser Arg Ala Gly Lys 30 35 40 CAG GGA CAC GAG GAC CGA CCC TTC GGC CAG CGC AGC AGG GCT GGA AAG 376 Gln Gly His Glu Asp Arg Pro Phe Gly Gln Arg Ser Arg Ala Gly Lys 45 50 55 AAC TTT ACA AAT CCA GCC CCA AAC TAC CCC GAG GAG GGA TCT AAG GAA 424 Asn Phe Thr Asn Pro Ala Pro Asn Tyr Pro Glu Glu Gly Ser Lys Glu 60 65 70 75 CAG AGA GAC AGT GTC CTG CCT AAA GTC ACA CAG CGA CAT GTG AGG GAG 472 Gln Arg Asp Ser Val Leu Pro Lys Val Thr Gln Arg His Val Arg Glu 80 85 90 CAG AGC CTG GTG ACG GAT CAG CTC AGC CGC CGC CTC ATC CGG ACC TAC TAC 520 Gln Ser Leu Val Thr Asp Gln Leu Ser Arg Arg Leu Ile Arg Thr Tyr 95 100 105 CAG CTC TAC AGC CGC ACC AGC GGG AAG CAC GTG CAG GTC CTG GCC AAC 568 Gln Leu Tyr Ser Arg Thr Ser Gly Lys His Val Gln Val Leu Ala Asn 110 115 120 AAG CGC ATC AAC GCC ATG GCA GAA GAC GGA GAC CCC TTC GCG AAG CTC 616 Lys Arg Ile Asn Ala Met Ala Glu Asp Gly Asp Pro Phe Ala Lys Leu 125 130 135 ATT GTG GAG ACC GAT ACT TTT GGA AGC AGA GTC CGA GTT CGC GGC GCA 664 Ile Val Glu Thr Asp Thr Phe Gly Ser Arg Val Arg Val Arg Gly Ala 140 145 150 155 GAG ACA GGT CTC TAC ATC TGC ATG AAC AAG AAG GGG AAG CTA ATT GCC 712 Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys Lys Gly Lys Leu Ile Ala 160 165 170 AAG AGC AAC GGC AAA GGC AAG GAC TGC GTA TTC ACA GAG ATC GTG CTG 760 Lys Ser Asn Gly Lys Gly Lys Asp Cys Val Phe Thr Glu Ile Val Leu 175 180 185 GAG AAC AAC TAC ACG GCG CTG CAG AAC GCC AAG TAC GAG GGC TGG TAC 808 Glu Asn Asn Tyr Thr Ala Leu Gln Asn Ala Lys Tyr Glu Gly Trp Tyr 190 195 200 ATG GCC TTT ACC CGC AAG GGC CGG CCC CGC AAG GGC TCC AAG ACG CGC 856 Met Ala Phe Thr Arg Lys Gly Arg Pro Arg Lys Gly Ser Lys Thr Arg 205 210 215 CAG CAT CAG CGC GAG GTG CAC TTC ATG AAG CGC CTG CCG CGG GGC CAC 904 Gln His Gln Arg Glu Val His Phe Met Lys Arg Leu Pro Arg Gly His 220 225 230 235 CAC ACC ACC GAG CAG AGC CTG CGC TTC GAG TTC CTC AAC TAC CCG CCC 952 His Thr Thr Glu Gln Ser Leu Arg Phe Glu Phe Leu Asn Tyr Pro Pro 240 245 250 TTC ACG CGC AGC CTG CGC GGC AGC CAG AGG ACT TGG GCC CCG GAG CCC 1000 Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg Thr Trp Ala Pro Glu Pro 255 260 265 CGA TAGGCGCTCG CCCAGCTCCT CCCCACCCAG CCGGCCGAGG AATCCAGTTGGTT TTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGGTTGTT TTTAAACAAA 1173 AGAGA 1178

SEQ ID NO: 3 Sequence length: 23 Sequence type: nucleic acid sequence ATHAAYGCiA TGGCiGARGA YGG 23

SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid sequence GCCATRTACC AiCCYTCRTA

[Brief description of the drawings]

FIG. 1 is a diagram showing an elution profile of AIGF by reversed-phase HPLC.

FIG. 2 shows an SDS-PAGE profile of AIGF obtained by reverse-phase HPLC.

FIG. 3 shows peptide mapping after lysyl endopeptidase digestion of AIGF.

FIG. 4 shows the nucleotide sequence and deduced amino acid sequence of AIGF.

FIG. 5 is a diagram showing induction of AIGF mRNA by testosterone.

FIG. 6 shows the expression of AIGF cDNA.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ identification code FI (C12P 21/02 C12R 1:91) (58) Investigated field (Int.Cl. ⁷ , DB name) C07K 14/00-14 / 825 C12N 15/00-15/90 GenBank / EMBL / DDBJ / GeneSeq SwissProt / PIR / GeneSeq

Claims (12)

(57) [Claims] 1. The amino acid sequence of SEQ ID NO: 1
A cell growth factor comprising an amino acid sequence from His at position 35 to Arg at position 215. 2. The amino acid sequence of SEQ ID NO: 1
Amino acid sequence from Gln at position 23 to Arg at position 215 or 2 from Gln at position 23 in the amino acid sequence of SEQ ID NO: 2
The cell growth factor according to claim 1, comprising the amino acid sequence up to Arg at position 68. 3. The amino acid sequence of SEQ ID NO: 1
An amino acid sequence from Gly at position 2 to Arg at position 215 or the amino acid sequence of SEQ ID NO: 2 from position 2 Gly to 268
2. The cell growth factor according to claim 1, which comprises an amino acid sequence up to Arg. 4. The cell growth factor according to claim 1, which has the following properties. (1) Induced by androgen. (2) Heparin binding. (3) Proliferate SC-3 cells in the absence of androgen. 5. The cell growth factor according to claim 1, which has a sugar chain. 6. A DNA encoding the cell growth factor according to any one of claims 1 to 5. 7. The DNA according to claim 6, which has the DNA according to claim 6.
A plasmid capable of expressing the cell growth factor according to any one of the above. 8. A host cell having the plasmid according to claim 7. 9. The method for producing a cell growth factor according to claim 1, wherein the host cell according to claim 8 is cultured. 10. The cell growth factor according to claim 1, which is produced by the production method according to claim 9. 11. A method for producing a cell growth factor, comprising culturing a breast cancer cell and collecting the cell growth factor according to claim 1 from a culture medium. A cell growth agent having the cell growth factor according to any one of claims 1 to 5.