JPH05222096A - Cell growth factor - Google Patents

Abstract

PURPOSE:To obtain a cell growth factor having a specific amino acid sequence, useful for producing cell growth reagents, reagents for probing for carcinostatic substances, or cancer gene-diagnosing probes, by culture of breast cancer cells and then collecting a product from the resulting culture medium. CONSTITUTION:Breast cancer cells stimulated with testosterone are cultured in a serumless conditioned medium, and the resulting medium is concentrated and then injected into an immobilized heparin column. Thence, the protein adsorbed is eluted with a 2M NaCl-contg. buffer solution and the eluted fraction is diluted and put to reversed-phase column chromatography followed by elution and fractionation with 0.1% trifluoroacetic acid using 0-60% acetonitrile concentration gradient; the [H]thymidine intake activity in breast cancer cells in each fraction is determined, and fraction(s) having biological activity is collected and then put to SDS acrylamide gel electrophoresis to carry out fractionation, thus obtaining the objective cell growth factor having the amino acid sequence from the His at 35th site to the Arg at 215th site among an amino acid sequence of the formula.

Description

Detailed Description of the Invention

[0001]

The present invention relates to androgen-induced growth factor, DNA encoding the factor, plasmid containing the DNA, host cell containing the plasmid, method for producing the factor using the host cell, and 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 growth, and various growth factors and growth factor receptors related to oncogene products have been reported. It seems to form a self-propagating cycle. Cancers that originate from sex hormone target organs such as breast and prostate cancers are characterized by hormone-responsive growth. The earliest breast cancers require hormonal stimulation for their growth. Hormone-responsive growth of breast cancer cells has been mediated by growth factors secreted by hormone stimulation. Structural analysis and characterization of such hormone-induced growth factor are essential for elucidating the growth mechanism of hormone-responsive cancer.

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 growth 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 mechanism by which sex hormones promote the growth of cancer cells. It has been suggested that androgen-dependent proliferation of SC-3 cells is mediated by heparin-binding growth factors in the self-proliferation machinery (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, but this factor is still Not isolated / identified. In addition, the DNA coding for this factor has not been isolated or identified. If a DNA encoding the factor can be obtained, it becomes possible to mass-produce the factor using a genetic engineering technique. If a large amount of the factor can be obtained, an anticancer substance as an inhibitor of the factor can be searched for. Further, if the DNA can be detected by a probe that hybridizes with the DNA, it is useful for cancer diagnosis. In addition, due to the cell proliferation action of the factor, the factor itself is expected as a reagent for cell proliferation and a medicine.

[0005]

The present invention is the first to report the cDNA cloning and functional expression of 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, which is significantly induced by androgen and proliferates SC-3 cells even in the absence of androgen.

Serum-free conditioned medium obtained from testosterone-stimulated SC-3 cells showed a marked growth-promoting effect on SC-3 cells. The growth promoting activity was bound to heparin-Sepharose and eluted at a concentration of 1.0-1.2 M sodium chloride. After repeating heparin-Sepharose chromatography, the physiologically active fraction was subjected to reverse phase HPLC (Fig. 1). The bioactive fraction obtained from reverse-phase HPLC was analyzed by SD under reducing and non-reducing conditions.
It contained two major proteins of 32 and 28 kDa on S-PAGE, respectively. Both 32 kDa and 28 kDa proteins showed similar biological efficacy (Fig. 2). N-terminal of all proteins is blocked, but reversed-phase HPLC
Peptide mapping after digestion with lysyl endopeptidase in E. coli revealed that most peptides from the two proteins were identical. The peptide fragment (Figure 3) was sequenced by reverse phase HPLC. From the sequence of the peptide in FIG. 3, the putative condensation oligonucleotide primer
SC synthesized and cultured in the presence of 10 ^-8 M testosterone
Polymerase reaction was performed using random-primed single-stranded cDNA derived from total RNA of 3 cells.
240 bp of DNA was obtained from one primer set,
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
N. Molec. Cell. Biol. 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 the cDNA library. 40 by screening 4 x 10 ⁴ clones
Positive clones were obtained, 20 of which 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 estimated 215 amino acid residue open reading frame with a 3'untranslated sequence. ATG
The start codon is located at nucleotide position 174, followed downstream by a putative signal peptide of hydrophobic residues. 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 (Figure 4). All peptides except peak 7 obtained by digestion with lysyl endopeptidase were included in this sequence (FIG. 4). Direct determination of the AIGF peptide sequence 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 presumed to be a glutamine residue at amino acid position 23, which is thought to cause N-terminal block by pyroglutamation. , The molecular weight of the mature protein is about 22 kDa. However, when the nucleotide sequence of another clone, pSC15, was determined, 10 amino acids of pSC17 clone (residues 25-34, FIG. 4) following the putative 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
Found in pSC15 but not in pSC17 (Fig. 4).
These clones have differently spliced A
It appears to be a product from the IGF transcript. 3 on SDS-PAGE
The difference between the two major proteins of 2 and 28 kDa and the difference between the theoretical molecular weight and the value on SDS-PAGE is due to the glycosylation at the putative N-glycosylation position and above. This may be due to the difference in splicing of the transcript. In fact, with endoglycosidase F treatment
The SDS-PAGE pattern of AIGF was 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 the 22 and 28 kDa AIGF, resulting in 28 and 32 kDa AI, respectively.
It suggests that GF is formed.

[0008] As a result of investigating the homology between AIGF and a known protein, it was revealed that AIGF shows homology with the FGF family. As a whole, 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
The positions of the two cysteine residues are well conserved in the family, but 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 putative sequence of AIGF. AIGF signal transduction is based on the basic FGF receptor (Nonomu
ra, N. et al., Cancer. Res. 50, 2316-2321 (1990)) or via different receptors of AIGF itself. All proteins encoded by FGF-related oncogenes have an N-terminal signal peptide sequence. The presence of the signal peptide sequence is an important factor in determining whether a member of the FGF family has a transforming ability. CDNA encoding a 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-
It suggests a close relationship between 3-cell cancerous growth and AIGF.

The 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 at 6 hours and was significantly increased by 24 hours after stimulation with 10 ⁻⁸ M testosterone (FIG. 5).

Expression of the AIGF cDNA in mammalian cells results in A
It was revealed that IGF significantly promotes the growth of SC-3 cells in the absence of androgen (Fig. 6). In addition, AIGF
Alters 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)). AIGF-stimulated SC-3 cells exhibited a fibroblast-like appearance, contrasting with the epithelial-like appearance of SC-3 cells treated with conditioned medium of COS 7 cells transformed with the plasmid vector alone.

AIGF is a secretory heparin-binding growth factor having a signal peptide sequence, and is significantly induced by androgen. AIGF promotes the growth of SC-3 cells and changes the morphology of SC-3 cells in the absence of androgen. 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 hormone stimulation.

Breast and prostate cancer growth is hormone sensitive. The identification of AIGF in the present invention revealed that hormone-induced self-growth factors play a central role in the growth of breast and prostate cancer and provide a rationale for hormone treatment of such cancers. ..

From the above, the present invention firstly provides a cell growth factor having an amino acid sequence from His at the 35th position to Arg at the 215th position among the amino acid sequences set forth in SEQ ID NO: 1. The amino acid sequence is as shown in FIG.
It is common to pSC17 and pSC15 clones obtained in the present invention, and is considered to have an amino acid sequence necessary and sufficient for the growth factor of the present invention.

The present invention further includes an amino acid sequence from Gln at position 23 to Arg at position 215 in the amino acid sequence shown in SEQ ID NO: 1 or from Gln at position 238 in the amino acid sequence shown in SEQ ID NO: 2. A cell growth factor having an amino acid sequence up to Arg at the position is provided. The sequence from Met at position 1 to Ala at position 22 common to the amino acid sequences of SEQ ID NOs: 1 and 2 is presumed to be a signal sequence. Therefore, SEQ ID NOs: 1 and 2
The present invention includes a cell growth factor having an amino acid sequence obtained by removing the signal sequence from the amino acid sequence of.

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

The cell growth factor of the present invention has the following properties. (1) Induced by androgen. (2) It is heparin-binding. (3) SC-3 cells are grown in the absence of androgen. Among 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 clear from the analysis of the above amino acid sequence, the cell growth factor of the present invention has a glycosylation site, and therefore it is considered preferable that it has a sugar chain.

The present invention also provides a DNA encoding the above cell growth factor, a plasmid having the DNA and capable of expressing the cell growth factor, and a host cell having the plasmid. Examples of the host cell of the present invention include prokaryotic host cells such as Escherichia coli and Bacillus subtilis, yeast, COS cells, CHO cells, Hela cells, and 3T.
All host cells usually used in this technical field of gene recombination such as eukaryotic host cells such as 3 cells, 3T6 cells and Ltk ⁻ cells can be used. Since the cell growth factor of the present invention is considered to have a sugar chain, a eukaryotic host is preferable as the host. As the plasmid of the present invention, a usual expression plasmid / virus vector may be used depending on the host to be used, and when a eukaryotic host is used, in addition to pcDL-SRα296, commercially available pSVK3, pBPV, p
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. The host cell capable of expressing the cell growth factor of the present invention can be cultured by an ordinary method depending on the properties of the host cell. 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, subjected to reverse phase HPLC for isolation and purification.

The cell growth factor of the present invention is preferably produced by the above-mentioned production method from the viewpoint that it can be stably supplied in a large amount at low cost, but breast cancer cells, preferably testosterone-stimulated SC-3 cells, are used. It can also be obtained by culturing the androgen-reactive mouse breast cancer cells containing it in a serum-free conditioned medium and subjecting the medium to the isolation and purification method described above.

The present invention further comprises hybridizing with the DNA or RNA encoding the cell growth factor to obtain the DNA or RNA.
A probe capable of specifically detecting The probe is a DNA probe labeled with a commonly used enzyme, radioisotope, etc., and encodes the cell growth factor in a usual Northern or Southern blot analysis.
Any DNA can be used as long as it can specifically hybridize with DNA or RNA and can specifically detect the DNA or RNA, but it does not have the DNA sequence of SEQ ID NO: 1 or 2 (or its complementary sequence). Xho in some, especially pSC17 clones
Those having the I fragment are preferred.

Using this probe, it is possible to detect DNA or RNA encoding the cell growth factor, or a gene highly homologous thereto, by ordinary Northern or Southern blot analysis and the like. ..

The cell growth factor of the present invention proliferates SC-3 cells even in the absence of androgen, and is therefore useful as a cell growth agent. The cell growth agent may be added to the cell growth factor of the present invention with stabilizers, preservatives and the like which are usually used for formulation of peptide reagents.

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

[0025]

EXAMPLE 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 and
It was applied to a heparin-Sepharose column (gel layer volume; 5 ml) equilibrated with a 10 mM Tris-HCl buffer solution (pH 7.0) containing M NaCl, 0.1% CHAPS, 2 mM PMSF, and 500 μg / ml leupeptin. The column was washed with the above equilibration buffer, and the adsorbed protein was eluted with 10 times the equilibration buffer containing the gel layer volume containing 2 M NaCl. The eluate fraction was diluted with NaCl-free equilibration buffer to reduce the NaCl concentration to 0.6 M and heparin-
Re-fractionation was carried out in the same manner using a Sepharose column (gel layer volume; 0.5 ml). The elution fraction was applied to a 4.6 x 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 ( (Figure 1). [ ³ H] thymidine uptake activity in SC-3 cells of each fraction was determined by the method of Yamanishi et al. (Yamanishi, H.
, Cancer Res. 51, 3006-3010 (1991)).
The biologically active fractions were lyophilized, subjected to reducing (FIG. 2) and non-reducing conditions on a 10-20% gradient acrylamide gel containing 0.1% SDS and silver stained. The same sample of adjacent lanes 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 in 5) by stirring overnight at 4 ° C. The eluate was assayed for [ ³ H] thymidine incorporation activity (Figure 2). Purified AIGF was denatured with 70% formic acid for 2 hours at room temperature and digested with 500 ng of lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.) in 50 mM Hepes buffer (pH 7.6) at 37 ° C. for 5 hours. Digested sample directly on 3.9 × 150 mm μ-Bon
It was applied to a dasphere C18 reverse phase column (Figure 3). Peptides were separated with a linear gradient of 0-60% acetonitrile over 60 minutes at an elution rate of 1 ml / min, and different peaks were collected. Sequence analysis was performed using the ABI 477A Protein Sequencer (Applied Bios
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)
The medium was exchanged once. On the second day, SC-3 cells (approximately 4 × 10 ⁶ cells / dish) which had reached almost confluency were collected, and total cellular RNA was subjected to acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski, P. et al., N.
Analyt. Biochem. 162, 156-159 (1987)). Randomly primed single-stranded cDNA was labeled with reverse transcriptase (Sup
erscript, Bethesda Research Laboratories, Gaithers
Burg, MD) and synthesized from total RNA of SC-3 cells cultured in the presence of 10 ⁻⁸ M testosterone. The oligonucleotide primer was synthesized according to the obtained amino acid sequence. 5 '> ATHAAYGCIATG out of various primer sets
GCIGARGAYGG <3 '(SEQ ID NO: 3) and 5'> GCCATRTACCA
When using 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 in the polymerase chain reaction Horn
A DNA band was obtained. Polymerase-reactions are single-stranded cDNA, synthetic oligonucleotides, Ampli
TaqTM recombinant Taq DNA polymerase (PERKIN-ELMER C
ETUS Instrument, Norwalk, CT), 94 ° C for 90 seconds, 3
The 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. Aca
d. Sci. USA 74, 5463-5467 (1977)). The 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 a cDNA
It was synthesized by a synthesis system (Bethesda Research Laboratories), and plasmid vector p was constructed using BstXI linker.
It bound to cDL-SRα296. Hybridization is
Performed with ³² P-labeled probe obtained by polymerase chain reaction at 65 ° C. in 1.5 × SSPE, 1.0% SDS, 0.5% Blotto, 50 ° C. in 2 × SSC, 0.1% SDS.
It was washed for 0 minutes, followed by 0.1 × SSC, 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 of AIGF and the deduced amino acid sequence. AIGF cDNA (pSC17 clone) codes for a protein of 215 amino acids. The underlined portion is the peptide sequence obtained by digestion with lysyl endopeptidase (Fig. 3, Nos. 1 to 10). The two peptides are the peaks in Figure 3.
It was included in 10. The shaded parts are the two primer parts used for the polymerase chain reaction. Boxed portions are amino acid residues that are substituted between two different clones pSC17 and pSC15. Areas where N-glycosylation may occur are underlined in bold.

[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
It was placed in 10 ml of MEM containing% fetal bovine serum. The next day, cells were washed with PBS and further cultured in serum-containing MEM with or without 10 ⁻⁸ M testosterone. Polyadenylated RNA was isolated at the times indicated 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 x SSPE, 1.0% SDS,
It was carried out at 65 ° C. for 18 hours in 0.5% Blotto. The blot is
2 x SSC, 0.1% SDS at 50 ° C for 30 minutes, then 0.1 x SS
It was washed with C and 1.0% SDS at 37 ° C for 30 minutes. Autoradiography was performed at -70 ° C for 48 hours.

In FIG. 5, the XhoI fragment of pSC17 clone was digested with ³² P.
The result of having hybridized the labeled AIGF cDNA probe and a blot is shown. In the figure, the migration position of ribosomal RNA is shown on the left.

Expression of AIGF cDNA in mammalian cells COS 7 cells were treated with 10 μg of cDNA by the calcium phosphate precipitation method.
Infected. Forty-eight hours after infection, medium was harvested and assayed for [ ³ H] thymidine incorporation in SC-3 cells. COS 7 cells were infected with only the vector-plasmid, and the conditioned medium was also used as a control to assay the biological activity.

FIG. 6 shows SC-3 of conditioned medium (right) of COS 7 cells infected with a vector plasmid containing AIGF cDNA (pSC17) and SC-3 cells infected with only the vector plasmid (left).
³ shows [ 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 CTCAGCCTGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC 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 CCCAGCTC48 CCCCACCCAG 8GG Trp Ala Pro Glu Pro Arg 210 215 CCGGCCGAGG AATCCAGCGG GAGCTCGGCG GCACAGCAAA GGGGAGGGGC TGGGGAGCTG 908 CCTTCTAGTT GTGCATATTG TTTGCTGTTG GGTTTTTT TTTTTTGTTT TTTGTTTTTG 968 TTTTTTGTTTGATTGAAACAAA997

SEQ ID NO: 2 Sequence length: 1178 Sequence type: Nucleic acid sequence TCCAGCAGCG GCTCAGAGGG GTTCGGCGCG CGCGGCGAGC ACGACATTCC ACCGGACCCG 60 CCGAGCCG GCCCTGGCCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCCG 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 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 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 CCTTGTTGATTTGTTGATTTGTTGCTTGCTTCCCCCCCCCCTT 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 drawings]

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

FIG. 2 is a diagram showing an SDS-PAGE profile of AIGF obtained by reverse phase HPLC.

FIG. 3 is a diagram showing peptide mapping of AIGF after digestion with lysyl endopeptidase.

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

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

FIG. 6 shows the expression of AIGF cDNA.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location // (C12N 15/16 C12R 1:91) (C12P 21/02 C12R 1:91)

Claims (14)

[Claims] 1. Amino acid sequence of SEQ ID NO: 1
A cell growth factor having an amino acid sequence from His at position 35 to Arg at position 215. 2. Of the amino acid sequence set forth in SEQ ID NO: 1,
The amino acid sequence from Gln at the 23rd position to Arg at the 215th position or the amino acid sequence of SEQ ID NO: 2 from Gln at the 23rd position to 2
The cell growth factor according to claim 1, which has an amino acid sequence up to Arg at position 68. 3. Among the amino acid sequences set forth in SEQ ID NO: 1,
The amino acid sequence from Gly at position 2 to Arg at position 215 or the amino acid sequence of SEQ ID NO: 2 from Gly at position 2 to 268.
The cell growth factor according to claim 1, which has an amino acid sequence up to Arg at the position. 4. The cell growth factor according to claim 1, which has the following properties. (1) Induced by androgen. (2) It is heparin-binding. (3) SC-3 cells are grown 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 method according to claim 1, which comprises the DNA according to claim 6.
A plasmid capable of expressing the cell growth factor according to any one of 1. 8. A host cell containing the plasmid according to claim 7. 9. The method for producing a cell growth factor according to claim 1, which comprises culturing the host cell according to claim 8. 10. The cell growth factor according to any one of claims 1 to 5, which is produced by the production method according to claim 9. 11. A method for producing a cell growth factor, which comprises culturing a breast cancer cell and collecting the cell growth factor according to claim 1 from a culture medium. [Claim 12] A cell growth factor according to any one of claims 1 to 5, which is hybridized with DNA or RNA encoding the cell growth factor,
A probe capable of specifically detecting DNA or RNA. 13. A DN encoding the cell growth factor according to any one of claims 1 to 5, which uses the probe according to claim 12.
A or RNA detection method. 14. A cell growth agent comprising the cell growth factor according to claim 1.