CN1078494A - Protein compound, coding nucleotide sequence produces clone and its application - Google Patents

Abstract

The present invention relates to a proteinaceous composition, the group The compound can selectively inhibit the division of estrogen-sensitive tumor cells and/or are capable of exerting a cytotoxic effect on said tumor cells. Book Invention also relates to a kind of can be isolated in culture from the human lipoma isolated split cell line (this cell line produces said composition) and related Its conditioned medium (in which said composition is present) The invention also relates to various diagnostic and clinical applications of said products. use.

Description

The present invention relates to a proteinaceous composition capable of selectively inhibiting the division of estrogen-sensitive tumor cells and/or exerting a cytotoxic effect on said tumor cells. The present invention also relates to a cell line isolated from human liposarcoma capable of dividing in culture during which the cell line produces said composition and to said composition in a conditioned medium in which said composition is present. Cell line culture method.

The invention also relates to the diagnostic and clinical applications of said products.

A large number of cell lines originally isolated from human tumors capable of growing in vitro are known in the art. These cell lines are particularly useful in studying their physiological properties and in the production of specific factors.

To date, despite numerous attempts, no cell lines known to have a differentiated phenotype associated with adipose cells (or adipocytes) nor tumor isolates known to be of the liposarcoma class have been found in the prior art to the cell line. It is especially useful if these cells are derived from stromal tissue (the identity of which is currently poorly understood and which is thought to be controlling given some of the functions played by adjacent tissues). In fact, in vivo in the mammary gland in the presence of the hormone testosterone, adipocytes induce epithelial tissue degeneration [Kratochwill, K. Development and loss of androgen responsiveness in the emtryonic rudiment of thd mouse - 3 l mammary 6 gland. ], thereby exerting paracrine action, which may be mediated by one or more compounds released by said adipocytes [Kratochwill, K.1987, in cellular and Molecular Biology of Mammary Cancer book (D. Medina, W.K.dweel, G.Heppner, e.E.Anderson, eds.pp 1-8 Plenum, New York].

The studies described above specifically give the evidence needed for the in vitro isolation and growth of cell lines capable of performing the functions observed in vivo.

The present authors are the first to isolate and characterize a cell line capable of dividing in culture with a phenotype associated with the adipocyte phenotype. Surprisingly, the cell line produces at least one compound capable of selectively inhibiting the division of epithelial tumor cells derived from estrogen-sensitive epithelial (epithelila) tumors in a conditioned medium-based medium.

The term "phenotype associated with an adipocyte phenotype" refers to the manifestation of at least one differentiation characteristic of adipocytes, such as lipid synthesis. The term "epithelial tumor cells" refers to tumor cells derived from epithelial tumors, said cells having been isolated or still present on a tumor mass. The term "estrogen-sensitive epithelial neoplasm" refers to a neoplasm composed of cells bearing at least one hormone receptor belonging to the class of estrogen. The term "conditioned medium" refers to a medium in which the cells are cultured for at least 6 hours under temperature, temperature and pH conditions suitable for the growth of the cells. "Inhibiting cell division ability" refers to the ability of a compound to inhibit the growth of at least 50% of the cells in culture after at least 3 days of cultivation in said cell culture environment.

Thus, there is a clear need to identify and characterize compounds that are capable of selectively inhibiting tumor cell division derived from estrogen-sensitive epithelial tumors.

"heat shock" ("heat shock") P27 human protein [HSD27, has been described by Hickey E. etc. identification and sequencing, Nucl.Acids Res., 14,10 4127-4145 (1986)] has 199aa amino acid sequence, is identical with the protein sequence associated with estrogen-receptor and is called P29 [Coffer A.I. etc., Cancer Ressearch 45, 3686-3693, 1990].

Recently [Carper SW et al., Nucl.Acids Res.18, 6457, 1990] a cDNA clone obtained from lung cancer A549 cell line has been sequenced. The nucleotide sequence of the clone encodes a protein of 205 amino acids, of which the first 193 amino acids coincided exactly with those known for human HS _p 27. The insertion of two cytosine residues was detected corresponding to the codon Z encoding the sulfamate residue at position 194, which determined the shift of the reading frame to the next position at positions 6161-618 of the nucleotide sequence. At a stop codon Z, this insertion causes a modification of the C-terminal sequence of the HS _p 27 protein, differing by the last 5 amino acids (from position 195 to 199) and exhibiting an extension of 6 amino acids (from position 200 to 205) .

Of the prior art proteins described, there is no evidence that any of them have inhibitory activity against tumor cell division.

Among the compounds produced and secreted from the LSA cell line, the present inventors have identified and characterized a compound, whose composition is actually protein in nature, that selectively inhibits the division of estrogen-sensitive tumor epithelial cells , and can exert cytotoxic effects on these tumor cells. The inventors characterized the biochemical properties of said compound, called it P1LSA, and determined its amino acid sequence, demonstrating that it consists of 205 amino acids. The inventors surprisingly found that said amino acid sequence is identical to the human HS _p 27 protein sequence determined by Carper SW (supra) except for a single amino acid. The observed difference is determined by the substitution of guanine for cytosine at position 581 of the sequence encoding protein HS _p 27 at amino acid position 194 (arginine instead of proline).

Accordingly, the inventors have identified a novel compound with antitumor biological activity specific for estrogen-sensitive epithelial tumor cells.

The inventors identified the nucleotide sequence encoding the protein P1LSA and determined its amino acid sequence.

Accordingly, an object of the present invention is a compound, the composition of which is proteinaceous in nature, said compound capable of selectively inhibiting cell division of estrogen-sensitive epithelial tumor cells and/or capable of exerting Cytotoxic activity; said compound has a molecular weight in the range of 27 to 30 kilodaltons (KDa); said compound also contains a sequence from position 1 substantially homologous to the sequence of the corresponding human protein HS _p 27 (heat shock p27) The amino acid sequence from amino acid to amino acid 193, or a fragment related to the biologically active compound.

The term "substantially homologous amino acid sequences" refers to sequences that are 50% to 100% homologous within the scope of the present invention without causing a loss of biological activity of the protein. The term "biological activity" refers to the ability to selectively inhibit the division of estrogen-sensitive epithelial tumor cells (cytostatic activity) and/or exert cytotoxic effects on said tumor cells.

According to a preferred embodiment of the invention, said compound whose composition is substantially proteinaceous contains at its carboxyl terminus an amino acid sequence substantially homologous to the following sequence:

GluAlaAlaLysSerAspGluThrAlaAlaLys-NH2

Preferred said substantially proteinaceous compounds comprise the following amino acid sequence:

MetThrGluArgArgValProPheSerLeuLeuArgGlyProSer 15

TrpAspProPheArgAspTrpTyrProHisSerArgLeuPheAsp 30

GlnAlaPheGlyLeuProArgLeuProGluGluTrpSerGlnTrp 45

LeuGlyGlySerSerTrpProGlyTyrValArgProLeuProPro 60

AlaAlaIleGluSerProAlaValAlaAlaProAlaTyrSerArg 75

AlaLeuSerArgGlnLeuSerSerGlyValSerGluIleArgHis 90

ThrAlaAspArgTrpArgValSerLeuAspValAsnHisPheAla 105

ProAspGluLeuThrValLysThrLysAspGlyValValGluIle 120

ThrGlyLysHisGluGluArgGlnAspGluHisGlyTyrIleSer 135

ArgCysPheThrArgLysTyrThrLeuProProGlyValAspPro 150

ThrGlnValSerSerSerLeuSerProGluGlyThrLeuThrVal 165

GluAlaProMetProLysLeuAlaThrGlnSerAsnGluIleThr 180

IleProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195

AlaAlaLysSerAspGluThrAlaAlaLys 205.

According to a preferred embodiment of the present invention, said essentially proteinaceous compound is produced by LSA cells (DSM ACC.N.2029), preferably secreted by said cells in culture medium.

Another object of the present invention is as a pharmaceutical composition, preferably for the treatment of malignant tumors, more preferably for the treatment of estrogen-sensitive epithelial tumors. The composition contains the proteinaceous compound of the present invention or a physiologically acceptable salt thereof.

Another object of the present invention is the use according to the invention of said essentially proteinaceous compounds of the invention or their physiologically acceptable salts for the preparation of pharmaceutical compounds for antineoplastic therapy, preferably for the treatment of estrogen sensitivity Epithelial tumors.

Another object of the invention is a nucleic acid comprising a nucleotide sequence encoding a compound of the invention, the composition of which is proteinaceous in nature according to the invention. Preferably said nucleic acid comprises the following nucleotide sequence:

ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGC 45

TGGGACCCCTTCCGCGACTGGTACCCGCATAGCCGCCTCTTCGAC 90

CAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGG 135

TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180

GCCGCCATCGAGAGCCCCGCAGTGGCCGCGCCCGCCTACAGCCGC 225

GCGCTCAGCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCAC 270

ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCC 315

CCGGACGAGCTGACGGTCAAGACCAAGGATGGCGTGGTGGAGATC 360

ACCGGTAAGCACGAGGAGCGGCAGGACGAGCATGGCTACATCTCC 405

CGGTGCTTCACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCC 450

ACCCAAGTTTCTCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 495

GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540

ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585

GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618

Another object of the present invention is a plasmid or phage type vector containing the nucleotide sequence of the present invention.

Another object of the present invention is a mammalian cell line capable of dividing in vitro and having a phenotype associated with adipocytes, which also produces in its conditioned medium at least one tumor cell, preferably epithelial tumor cells, capable of selectively inhibiting the division of tumor cells, Compounds derived from division of estrogen-sensitive epithelial tumor cells are more preferred.

According to a preferred embodiment of the present invention, said cell line is isolated from liposarcoma (preferably derived from human), and this cell line is more preferably the LSA cell line deposited with DSM accession number 2029. It is emphasized again that the cell lines according to the invention produce compounds that are actually proteins with the above disclosed sequences.

Another object of the present invention is a medium conditioned for the growth of the cell lines disclosed and claimed herein.

Preferably, said conditioned medium comprises at least one compound capable of selectively inhibiting the division of tumor cells (preferably epithelial tumor cells, more preferably cells derived from estrogen-sensitive epithelial tumors), more preferably said compound is the protein disclosed in the present invention compound.

According to a preferred embodiment of the present invention, the medium is conditioned to grow LSA cell line (DSM ACC.N.2029).

Another object of the present invention is to use said conditioned medium to produce a pharmaceutical composition for the treatment of tumors, preferably epithelial tumors, more preferably estrogen-sensitive tumors.

Another object of the present invention is to purify, identify and characterize at least one compound capable of selectively inhibiting the division of tumor cells, preferably epithelial tumor cells, more preferably cells derived from estrogen-sensitive epithelial tumors, using said conditioned medium.

The present invention will be disclosed with some examples, which are only used to illustrate and illustrate the present invention, but not to limit the present invention. Attached here for reference are:

Accompanying drawing 1 represents the growth curve of LSA line under the two situations of having and having no serum;

Accompanying drawing 2 a shows the cytofluorescence analysis result of human normal thymocyte;

Accompanying drawing 2 b represents the cytofluorescence analysis result of LSA cell;

Figure 3 shows a bar graph of LSA-CM stimulation with 3[H]-thymidine incorporation in various cell lines. Among them, 1 is the control sample, 2 is the LSA-CM at 1/4 dilution, 3 is the LSA-CM at 1/2 dilution, 4 is the LSA-CM at undiluted time, 5 is the control sample, 6 is the undiluted LSA-CM, 7 is the control sample, 8 is the undiluted LSA-CM;

Accompanying drawing 4 shows the growth curve of MCF-7 cell line in two cases when there is and does not exist in LSA-CM;

Accompanying drawing 5 represents the growth curve of ZR-75-1 cell line under two kinds of situations when there is and does not exist in LSA-CM;

Accompanying drawing 6 shows the growth curve of MDAMB-231 cell line under two kinds of situations when having and not having LSA-CM to exist;

Accompanying drawing 7 represents the growth curve of NMNG cell line under two kinds of situations when there is no LSA-CM to exist;

Figure 8 shows growth curves of human cells obtained from ovarian (ovaric) carcinoma in the presence and absence of LSA-CM;

Figure 9 shows a bar graph of the growth of human cells obtained from ovarian cancer in the presence and absence of LSA-CM and/or purified P1 LSA.

Example 1

Isolation of LSA cell lines

A fragment of a mixed adipogenic-fibroblastic human liposarcoma was surgically removed from the leg of a 65-year-old adult woman in an aseptic manner.

Fragments were mechanically crushed with a chisel to obtain 1 mm fragments, and the resulting material was treated with the equivalent of 20 μ/ml collagenase (CLSP, Worthington, Regno Unito), 075 mg/ml trypsin (1/300, Inc. Pharmaceuticals), 2 The CTC solution of % chicken serum was treated at 37°C with constant shaking in Hank's medium (Soil) (GIBCO) without Ca ²⁺ and Mg ²⁺ ions and Ham F ₁₂ medium (Soil) containing 5% bovine serum. ) (GIBCO) for 4 hours to obtain a single-cell suspension.

Cell suspension was diluted in Ham F ₁₂ (GIBCO) medium supplemented with 5% bovine serum (GIBCO), penicillin (31 μg/ml), streptomycin (50 μg/ml) and amphotericin B (25 μg/ml) . Cells were plated on a 100mm diameter plate (Costar) at 200,000 cells/dish.

A cell clone capable of dividing in culture and having characteristics specific to adipocytes is obtained from the culture medium, said clone is called LSA, and is deposited in DSM with the accession number ACC 2029.

Figure 1 shows the growth curves of the LSA line, where the growth curves in the presence and absence of serum are evident. The LSA line exhibited 90% plating efficiency, with a doubling time of 50-52 hours in the presence of serum and 102 hours in the absence of serum, with medium changes every 72 hours.

Example 2

Characterization of LSA series

The DNA content of LSA cells was analyzed by a cytofluorometer (Partec Pas II flow-cytometer). Cells were trypsinized and fixed with 70% ethanol, and stained with a solution containing 5 μg/ml ethidium bromide, 12.5 μg/ml mithromycin and 15 _mg MgCl2 in 0.1 M Tris buffer (pH 7.5). The percentage of cells in each step of the Mitotic cycle was used by Flintoff, WE, Davidson, SV and Siminowitch, L. in "Isolation and partial Charcterization of Methotrexate-Resistent phenotype from Chinese hamster ovary cell" (Somatic cell Genet.2: 245-261 1976 ) and Ambesi-Impiom-bato, FS, Parks, LAM and Coon HG in "Culture of hormone-dependent functional epithelial cells from rat thyroid" (Proc. Natl. Acad. Sci. USA 77: 3455-3459, 1980) Obtained by the method disclosed in the article.

These values are compared with those obtained using normal human thymocytes as ploidy standards, as shown in Figure 2b. Taking the G ₁ -G ₀ peak of thymocytes as 1, under the same conditions, the G ₁ -G ₀ peak of LSA cells was 1.79, which means that the DNA inclusion is almost quadrupled. As shown in Figure 2a, the DNA content/cell was also almost double that obtained with thymocytes.

The cell distribution at various stages of the cell cycle of the LSA cell population in the logarithmic growth phase is shown in Table 1 below.

Table 1

stage cell percentage

G ₁ -G ₀ 59

S 27

G ₂ /M 14

The percentage of cells in the S phase indicates that the cell population is in an active growth phase.

Chromosome counts demonstrate between 80 and 90 chromosomes/cell.

When viewed under a light microscope, LSA cells appear as elongated bodies with abudant cytoplasm. Under the electron microscope, the nucleus is found to contain many nucleoli and organelles located in the perinuclear region. Cells are homologous to each other, mitochondria are relatively abundant, and cytoplasmic vesicles are constant in number and size. The most prominent feature is the presence of vacuolar regions within the cytoplasm that are electron reflective and typical of high lipid content bands, which were removed as a result of cell treatment formulated for observation under an electron microscope. In addition, there is also a well-developed Golgi apparatus (Golgi). To estimate lipid content, LSA cells were plated on Lab-Teck (NUNC) culture slants and grown at semi-confluence. After washing the cells, fix with 4% paraformaldehyde and 15% saturated picric acid in PBS (phosphate saline buffer) for 6 minutes, wash the cells twice with distilled water, then stain with Oil Red O solution for 5 minutes, and then wash with PBS Rinse twice and stain with 1% methylamine blue for 10 seconds.

If cells were grown in the presence of 5% bovine serum, lipids were dispersed into the cytoplasm, forming circular vesicles. Lipids were not formed in cells grown in serum-free medium, but were stimulated when cells were incubated with methylxanthine/desamethasone for 48 hours and with insulin for an additional 48 hours.

Example 3

Effect of LSA-conditioned media (SA-CM) on the in vitro growth of different cell lines

After LSA cells were grown to 80% confluence in 100mm dishes (Costar) containing _F12 medium containing 5% bovine serum, the cell monolayer was washed with PBS and cultured in serum-free _F12 medium. After 24 hours, the original medium was replaced with fresh medium, and the conditioned medium was collected every 6-24 hours for 30 days. The cells continued to grow, and after staining with Trypan Blue (Trypan Bluc), they appeared structurally and functionally alive when viewed under a contrast microscope.

Medium conditioned on LSA cells stimulates the growth of normal epithelial cells like CHO (CCL-61), FRTL-5 (CRL-1468) and NIH-3T3 (ECACC-87100803) grown for 24 hours in the absence of serum , as demonstrated by the ³ H-thymidine incorporation assay (see Figure 3). In contrast to the above results, medium conditioned on LSA cells showed an inhibitory effect on the growth of cytolytic activity when assayed in vitro with tumor cells containing estrogen receptors. The cells used are:

MCF-7 cells obtained from human breast carcinoma (Land, H. et al., Science 222:778 (1980), ECACC-86012803), which have estrogen receptors (Soule, H.O. et al., Natl. Cancer Inst. S1, 1409-1413 (1973)), androgen receptor (Horowity, K.B. et al., Steroids 26, 785-795 (1975)), insulin and triodothyronine receptor (MacGrath, C.M. Cancer Res., 43:1355-1360 (1983) );

ZK-75-1 (CRL-1504) cells obtained from human breast carcinoma, said cells being positive for the estrogen receptor.

MDAMB-231 (ATCC-HTB26) cells obtained from human breast carcinoma, which are negative for estrogen receptors.

NMMG (Burker, R.E. et al., Cancer Res., 38:3769-3773 (1978), CRL-1636) cells obtained from murine mammary epithelium which were slightly positive for estrogen receptors.

Ovarian cancer human cells obtained from women with peritoneal leaks due to ovarian cancer metastasis, et al. Said cell line is positive for the estrogen receptor.

The growth curves of the cell lines mentioned here were obtained by plating 100,000 cells/dish in 100 mm diameter in 10 ml DMEM (GIBCO) medium with 10% fetal calf serum (FCS, GIBCO). 2ml of LSA-CM was added to each dish.

Accompanying drawing 4 shows the growth curve of MCF-7 line, wherein the inhibitory effect of LSA-CM is obvious. In particular, after 4 days of treatment, removal of LSA-CM could not restore the proliferation ability of MCF-7 cells due to cytolysis.

Figure 5 shows the growth curves of the 7R-75-1 line in the presence and absence of LSA-CM, which caused a strong growth inhibition without any lysis.

Figures 6 and 7 show the growth curves of MDAMB-231 cells and normal epithelial NMMG cells in which no growth inhibition due to LSA-CM was evident, such cells do not exhibit estrogen receptors.

Figure 8 shows that LSA-CM conditioned medium has a cytotoxic effect on human ovarian cancer cells even after 24 hours of culture.

The effect of LSA-CM ^on3H -thymidine incorporation in different cell lines is listed in Table 2 below.

Table 2

Effect of LSA-CM on ³ H-thymidine incorporation in different cell lines

Cell Line No Effect Activation Inhibition

MCF 7 +

ZR-75-1 +

NMMG +

FRTL-S +

CHO +

NTH-3T3 +

Example 4

Effects of LSA-CM on Animals

Select 20 Balb/c fc 3H mice (Charles River) with obvious tumor masses infected by Bittner virus, which are suffering from estrogen-dependent breast cancer induced by the virus itself.

After daily intraperitoneal injection of 200μl LSA-CM for 15 days, growth inhibition and necrosis of tumor tissue were observed in 80% of the treated mice.

Example 5

Isolation of P1LSA protein from LSA-CM

LSA-CM medium was concentrated 100-fold, dialyzed against isotropic phosphate buffer at pH 7.4, and then gel-filtered on P6 resin (Pharmacia). The eluted fractions were tested for the ability to selectively inhibit the growth of MCF-7 cells. Only one component exhibited said biological activity. A sample of said fraction was separated by electrophoresis on a polyacrylamide gel under non-denaturing conditions and identified as an approximately 29 kDa protein designated P1LSA. The protein was electroeluted for the production of the polyclonal antibody described below and for sequence analysis.

The protein sequence is shown in Table 3 below.

As shown in Figure 9, the purified P1LSA protein has obvious cytotoxic effect on human ovarian cancer cells.

table 3

The amino acid sequence and corresponding coding sequence of P1LSA protein

ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGC 45

MetThrGluArgArgValProPheSerLeuLeuArgGlyProSer 15

TGGGACCCCTTCCGCGACTGGTACCCGCATAGCCGCCTCTTCGAC 90

TrpAspProPheArgAspTrpTyrProHisSerArgLeuPheAsp 30

CAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGG 135

GlnAlaPheGlyLeuProArgLeuProGluGluTrpSerGlnTrp 45

TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180

LeuGlyGlySerSerTrpProGlyTyrValArgProLeuProPro 60

GCCGCCATCGAGAGCCCCGCAGTGGCCGCGCCCGCCTACAGCCGC 225

AlaAlaIleGluSerProAlaValAlaAlaProAlaTyrSerArg 75

GCGCTCAGCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCAC 270

AlaLeuSerArgGlnLeuSerSerGlyValSerGluIleArgHis 90

ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCC 315

ThrAlaAspArgTrpArgValSerLeuAspValAsnHisPheAla 105

CCGGACGAGCTGACGGTCAAGACCAAGGATGGCGTGGTGGAGATC 360

ProAspGluLeuThrValLysThrLysAspGlyValValGluIle 120

ACCGGTAAGCACGAGGAGCGGCAGGACGAGCATGGCTACATCTCC 405

ThrGlyLysHisGluGluArgGlnAspGluHisGlyTyrIleSer 135

CGGTGCTTCACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCC 450

ArgCysPheThrArgLysTyrThrLeuProProGlyValAspPro 150

ACCCAAGTTTCTCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 495

ThrGlnValSerSerSerLeuSerProGluGlyThrLeuThrVal 165

GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540

GluAlaProMetProLysLeuAlaThrGlnSerAsnGluIleThr 180

ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585

IleProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195

GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618

AlaAlaLysSerAspGluThrAlaAlaLys 205

By comparison with the sequence stored in the database, it was proved that this sequence was identical with the corresponding human HSp27 protein described by Carper S.W. mentioned above, except for only one amino acid, the difference being the substitution of amino acid 194, said amino acid being Proline is replaced by arginine.

Example 6

Production of anti-P1LSA polyclonal antibody and inhibition of P1LSA protein activity

Dissolve 100 μg of P1LSA protein eluted from 10% polyacrylamide gel in 1 ml of PBS (phosphate saline buffer), and mix with 1 ml of Fisher’s complete adjuvant. The mixture was injected intramuscularly into the rabbits four times every 10 days. 30ml of immune serum was taken from the rabbit every other day for 20 days. Immunoglobulin (Ig) fractions were purified using Mab Trap G (Pharmacia) according to the manufacturer's instructions.

Immunoprecipitation and immunoblotting assays showed that immunoglobulins reacted in a specific manner with LSA-CM-derived P1LSA protein.

Pre-incubation of 100 μl LSA-CM with 10 μl 1 mg/ml Ig solution for 30 minutes at 37°C terminated the activity of inhibiting the division of tumor epithelial cells (MCF-7), and restored ³ H-thymus incorporation similar to untreated control samples And growth curves, it was proved that LSA-CM activity did not change after culturing with pre-immune serum.

These experimental data show that the P1LSA protein with the sequence disclosed in Table 3, isolated and purified according to the method disclosed in Example 5 above, actually plays a major role in the activity of inhibiting the division of epithelial cells mentioned herein.

Example 7

Nucleotide sequence encoding P1LSA protein

The nucleotide sequence encoding the P1LSA protein was determined from RNA poly A ⁺ purified from LSA cells according to standard methods, using the following primers in a PCR reaction:

Oligo 5': GGGAATTCTGAGCAGACGTCCAGAG

ECORI

Oligo 3': CGGAATTCCGGGCTAAGGCTTTACTTG

ECORI

The two sequences were deduced from the untranslated sequence of the HSp27 gene at the 5' and 3' positions, respectively.

The amplified product was directly cloned at the EcorI site of the vector pGEM4Z (Promega) and sequenced by the dideoxy method. The complete sequence of the encoded protein is shown in Table 3.

The present invention is disclosed in conjunction with its preferred versions, but it should be understood that modifications and changes made by those skilled in the art do not depart from the spirit and scope of the present invention.

Claims (28)

1, a kind ofly is essentially proteinic compound, it is characterized in that its can selectivity suppress the cell fission of the responsive epithelial tumor cell of oestrogenic hormon and/or can apply cytotoxic effect on said tumour cell; Its molecular weight ranges is between 27 to 30KDa, contains the aminoacid sequence of and people HSp27 (heat-shocked p27) protein homology basic from 1 amino acids to 193 amino acids; Or a kind of biological activity segment of said compound.

2, a kind of proteinic compound that is essentially as claimed in claim 1, said compound contains one and following amino acid sequences homologous aminoacid sequence at its C-terminal:

GluAlaAlaLysSerAspGluThrAlaAlaLys-NH ₂

3, a kind of proteinic compound that is essentially as claimed in claim 2, this compound contain a basic and following amino acid sequences homologous aminoacid sequence:

MetThrGluArgArgValProPheSerLeuLeuArgGlyProSer 15

TrpAspProPheArgAspTrpTyrProHisSerArgLeuPheAsp 30

GlnAlaPheGlyLeuProArgLeuProGluGluTrpSerGlnTrp 45

LeuGlyGlySerSerTrpProGlyTyrValArgProLeuProPro 60

AlaAlaIleGluSerProAlaValAlaAlaProAlaTyrSerArg 75

AlaLeuSerArgGlnLeuSerSerGlyValSerGluIleArgHis 90

ThrAlaAspArgTrpArgValSerLeuAspValAsnHisPheAla 105

ProAspGluLeuThrValLysThrLysAspGlyValValGluIle 120

ThrGlyLysHisGluGluArgGlnAspGluHisGlyTyrIleSer 135

ArgCysPheThrArgLysTyrThrLeuProProGlyValAspPro 150

ThrGlnValSerSerSerLeuSerProGluGlyThrLouThrVal 165

GluAlaProMetProLysLeuAlaThrGlnSerAsnGluIleThr 180

IleProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195

AlaAlaLysSerAspGluThrAlaAlaLys 205.

4, as among the claim 1-3 any one be essentially proteinic compound, it is produced by LSA cell (DSM ACC.N.2029), this compound is preferably secreted in substratum by said cell.

5, a kind of medicinal compositions, preferred antitumor usefulness comprises the proteinic compound of being essentially of one of above any claim or its physiologically acceptable salt.

6, composition as claimed in claim 5 is used for treating the estrogen-sensitive epithelial tumor.

7, a kind of purposes that is essentially acceptable salt on proteinic compound or its physiology of one of above claim is used to prepare the composition for the treatment of tumour, preferred therapeutic estrogen-sensitive epithelial tumor.

8, a kind of nucleic acid, it contains any one said nucleotide sequence that is essentially proteinic compound in the requirement of coding aforesaid right.

9, nucleic acid as claimed in claim 8, contain following nucleotide sequences:

ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGC 45

TGGGACCCCTTCCGCGACTGGTACCCGCATAGCCGCCTCTTCGAC 90

CAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGG 135

TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180

GCCGCCATCGAGAGCCCCGCAGTGGCCGCGCCCGCCTACAGCCGC 225

GCGCTCAGCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCAC 270

ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCC 315

CCGGACGAGCTGACGGTCAAGACCAAGGATGGCGTGGTGGAGATC 360

ACCGGTAAGCACGAGGAGCGGCACGACGAGCATCCCTACATCTCC 405

CGGTGCTTCACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCC 450

ACCCAAGTTTCCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 495

GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540

ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585

GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618

10, the plasmid or the phagotype carrier that contain one of claim 8 or 9 described nucleotide sequence.

11, a kind of Mammalia clone can be in external division, and has relevant phenotype with lipocyte, and it produces one of at least a claim 1-4 in its substratum or conditioned medium described, can selectivity suppresses the compound of tumour cell division.

12, as the Mammalia clone of claim 10, wherein said tumour cell comprises epithelial tumor cell.

13, as the Mammalia clone of claim 12, wherein said epithelial tumor cell comes from the estrogen-sensitive epithelial tumor.

14, as the Mammalia clone of one of claim 1-13, can have with two kinds of situations of serum free medium under external division.

15, as the Mammalia clone one of among the claim 11-14, wherein said clone is isolated from people's liposarcoma.

16, as the Mammalia clone of claim 15, wherein said clone is isolated from people's liposarcoma.

17, as the Mammalia clone of claim 16, wherein said clone is LSA system (DSM ACC.N.2029).

18, a kind of substratum is being condition according to the said cell line growth of one of claim 11-17.

19, according to the substratum of claim 18 applying condition, said substratum contains the compound that at least a energy selectivity suppresses tumour cell division.

20, according to the substratum of claim 19 applying condition, wherein said tumour cell is an epithelial tumor cell.

21, according to the substratum of claim 20 applying condition, wherein said epithelial tumor cell comes from the estrogen-sensitive epithelial tumor.

22, according to the substratum of claim 21 applying condition, wherein said clone is LSA clone (DSM ACC.N.2029).

23,, be used for the pharmaceutical composition of production for treating tumour according to the purposes of the conditioned medium one of among the claim 18-22.

24, according to the purposes of the conditioned medium of claim 23, wherein said tumour is an epithelial tumor.

25, according to the purposes of the conditioned medium of claim 24, wherein said epithelial tumor is an estrogen-sensitive.

26,, be used to purify, differentiate and characterize the compound that at least a energy selectivity suppresses tumour cell division according to the purposes of the conditioned medium among the claim 18-22.

27, according to the purposes of the conditioned medium of claim 26, wherein said cell comes from the estrogen-sensitive epithelial tumor.

28, according to the purposes of the conditioned medium of claim 27, wherein said cell comes from the estrogen-sensitive epithelial tumor.

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