WO1993018147A1

WO1993018147A1 - Protein compound capable of inhibiting tumoral growth

Info

Publication number: WO1993018147A1
Application number: PCT/IT1993/000020
Authority: WO
Inventors: Aldo Mancini
Original assignee: Istituto Nazionale Per Lo Studio E La Cura Dei Tumori Fondazione Giovanni Pascale
Priority date: 1992-03-09
Filing date: 1993-03-08
Publication date: 1993-09-16
Also published as: CN1078494A; FI944102A; EP0630404A1; CA2130089A1; KR950700412A; FI944102A0; HU9402317D0; HUT70972A

Abstract

This invention relates to a substantially proteinaceous composition which is capable of inhibiting selectively the division of estrogen-sensitive tumoral cells and/or of exerting a cytotoxic activity on such cells. This invention also relates to a cell line isolated from a human liposarcoma, capable of dividing in a culture, which produces said composition, as well as to its culture medium has been conditioned, wherein said composition is present. This invention also relates to the various uses of said products in diagnostic and clinical applications.

Description

PROTEIN COMPOUND CAPABLE OF INHIBITING TUMORAL GROWTH

This invention relates to a substantially proteic composition which is capable of inhibiting selectively the division of tumoral cells sensitive to estrogens and/or of exerting a cytotoxic activity on said cells. This invention also relates to a cell line isolated from a human liposarcoma capable of dividing in a culture, which line produces said composition, as well as to its culture conditioned medium in which said composition is present.

This invention is also concerned with the employments of said products in diagnostic and in clinical applications.

A large number of, cell lines capable of growing in vitro and originally isolated from human tumors are already known from the prior art. Such lines are particularly useful for the study of their biochemical and physiological properties, as well as for the production of specific factors.

In spite of the large number of attempts made up to the present time, no cell lines are known from the prior art which have a differentiated phenotype that can be associated to that of the adipose cells or adipocytes, and no lines are known which are isolated from tumors belonging to the class of liposarcomas. Such cells would be particularly useful as they derive from the stromal tissue, a tissue which at the present time has been very little characterized and for which a controlling role has been suggested as regards some of the functions performed by the adjacent tissues. Indeed, in vivo, in the mammary gland in the presence of the hormone testosterone, adipose cells induce the regression of epithelial tissue [Kratochwill, K. Development and loss of androgen responsiveness in the embryonic rudiment of the mouse mammary gland. Dev. Biol. 61: 358-365, 1977] so exerting paracrine action which possibly is mediated by one or more compounds released by said adipose cells [Kratochwill, K. 1987, in "Cellular and Molecular Biology of Mammary Cancer" (D. Medina, W. Kidwell, G. Heppner, e E. Anderson, eds.), pp. 1-8 Plenum, New York].

The investigations mentioned above put particularly into evidence the need for isolating and growing in vitro a cell line capable of performing the functions observed in vivo.

The Author of this invention has isolated and characterized for the first time a cell line which is capable of dividing in a culture, with a phenotype associable with that of adipose cells. Surprisingly, the cell line produces in the culture medium, denominated as conditioned medium, at least

one compound that is capable of inhibiting selectively the division of epithelial tumoral cells deriving from epithelial tumors which are sensitive to estrogens.

By the expression "phenotype associable to the phenotype of adipose cells" it is meant the manifestation of at least one differentiated character which is proper of adipose cells like the synthesis of lipids. By the expression "epithelial tumoral cells" it is meant tumoral cells derived from epithelial tumors, said cells being isolated or still present in the tumoral mass. By the expression "estrogen-sensitive epithelial tumors" it is meant tumors comprising

cells with at least one receptor for a hormone belonging to the class of estrogens. By the expression "conditioned medium" it is meant the culture medium in the presence of which the cells are incubated under temperature, humidity and pH conditions suitable for growing the same, for at least 6 hours. By the "capability for inhibiting cell division" it is meant the capability of a compound for inhibiting at least 50 % of the cell growth in a culture, after at least 3 days of incu bation in the culture soil of said cells.

Accordingly, there is evidently the need for identifying and characterizing the compound/s capable of inhibiting selectively the division of tumoral cells which are derived from estrogen-sensitive epithelial tumors.

In a paper published recently [Mendelsohn M.E. et al., Proc. Natl. Acad. Sci. USA, 88, 11212-11216, 1991] it is disclosed that the "heat shock" p27 human protein (HSp27), already identified and sequenced by Hickey E. et al., Nucl.

Acids Res., 14, 10 4127-4145 (1986), has the amino acid sequence of 199 aa. identical with that of a protein which is correlated to the estrogen-receptor and is called p29 [Coffer A.I. et al., Cancer Research 45, 3686-3693, 1990].

More recently [Carper S.W. et al., Nucl. Acids Res. 18, 6457, 1990], a cDNA clone obtained from the A549 cell line of human lung carcinoma has been sequenced. The nucleotide sequence of such clone codes for a protein of 205 amino acids in which the first 193 amino acids coincide with those of human HSp27 already known. In correspondence with the codon that codes for the amino acid residue in the position 194, the insertion of two cytosine residues has been detected, which determines a shift of the reading frame to the following stop codon in the positions 616-618 of the nucleotide sequence. The insertion thus causes a modification of the C-terminal sequence of the HSp27 protein that diverges for the last 5 amino acids (from the 195 position to the 199 position) and shows an extension of 6 more amino acids (from the position 200 to the position 205).

No inhibition activity of cell division of tumoral cells has been put into evidence in the prior art for any one of the proteins described.

Among the compounds produced and secreted from the LSA line the Author of this invention has identified and characterized a compound, whose composition is substantially protein aceous in nature, capable of inhibiting selectively the division of estrogen-sensitive tumoral epithelial cells and of exerting a cytotoxic activity on such cells. The Author has biochemically characterized said compound, which has been called p1LSA, and has determined its amino acid sequence, which has turned out to be made up of 205 amino acids. The Author has found surprisingly that said amino acid sequence is identical with the sequence of the human HSp27 protein already determined by Carper S.W. (ibid.) except for a single amino acid. The difference observed is in the amino acid 194 (arginine instead of proline) and is determined by a substitution of cytosine in the position 581 of the sequence that codes for the protein HSp27 with a guanine.

Accordingly, the Author has identified a new compound having an antineoplastic biological activity which is specific for estrogen-sensitive epithelial tumoral cells.

The Author has identified the nucleotide sequence that codes for the protein p1LSA and has determined its amino acid sequence.

Accordingly, it is an object of this invention a compound whose composition is substantially proteinaceous in nature, said compound being capable of inhibiting selectively the cellular division of estrogen-sensitive epithelial tumoral cells and/or of exerting a cytotoxic activity on said cells; said compound having a molecular weight in the range from 27 to 30 kDa; said compound also comprising an amino acid sequence from the amino acid 1 to the amino acid 193 which is substantially homologous to the corresponding sequence of human protein HSp27 (heat shock p27); or a fragment of said compound which is biologically active. By the expression "substantially homologous amino acid sequences" it is meant in the scope of this invention to designate sequences with homologies in the range from 50 % to 100 % which do not give rise to a loss of biological activity of the protein. By "biological activity" it is meant the capability of inhibiting selectively the division of estrogen sensitive epithelial tumoral cells (cytostatic activity) and/or of exerting a cytotoxic activity on said cells.

According to a preferred embodiment of this invention, said compound whose composition is substantially proteinaceous in nature comprises at its carboxyl end an amino acid sequence which is substantially homologous to the sequence:

GluAlaAlaLysSerAspGluThrAlaAlaLys-NH₂

Preferably said substantially proteinaceous compound comprises 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

GluAlaProMetProLysLevuAlaThrGlnSerAsnGluIleThr 180

IleProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195

AlaAlaLysSerAspGluThrAlaAlaLys 205.

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

It is a further object of this invention a composition for pharmacological uses, preferably for antineoplastic applications, and even more preferably for the treatment of estrogen-sensitive epithelial tumors, that comprises the substantially proteinaceous compound of this invention or salts thereof which are physiologically acceptable.

It is a further object of this invention the employment of said substantially proteinaceous compound according to the invention or of its salts which are physiologically acceptable for the preparation of pharmacological compounds for anti-neoplastic treatment, preferably for the treatment of estrogen-sensitive epithelial tumors.

It is an object of this invention a nucleic acid comprising a nucleotide sequence that codes for the compound whose composition is substantially proteinaceous

according to this invention, preferably said nucleic acid comprising the following nucleotide sequence:

ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGC 45

TGGGACCCCTTCCGCGACTGGTACCCGCATAGCCGCCTCTTCGAC 90

CAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGG 135

TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180

GCCGCCATCGAGAGCCCCGCAGTGGCCGCGCCCGCCTACAGCCGC 225

GCGCTCAGCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCAC 270

ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCC 315

CCGGACGAGCTGACGGTCAAGACCAAGGATGGCGTGGTGGAGATC 360

ACCGGTAAGCACGAGGAGCGGCAGGACGAGCATGGCTACATCTCC 405

CGGTGCTTCACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCC 450

ACCCAAGTTTCCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 495

GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540

ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585

GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618 A further object of this invention consists in plasmid or phage type vectors comprising the nucleotide sequences of this invention.

It is a further object of this invention a mammalian cell line capable of dividing in vitro, and having a phenotype associable to that of adipose cells, that produces also in its conditioned medium at least one compound capable of inhibiting selectively the division of tumoral cells, preferably epithelial tumoral cells, which even more preferably derive from estrogen-sensitive epithelial tumors.

According to a preferred embodiment of this invention, said cell line is isolated from a liposarcoma, preferably of human origin, and also more preferably such line is the LSA cell line deposited with the DSM with the accession number 2029. Again according to this invention, said cell line produces the substantially proteinaceous compound having the sequence disclosed above.

It is a further object of the invention a medium conditioned by the growth of cell lines herein disclosed and claimed.

Preferably said conditioned medium comprises at least one compound capable of inhibiting selectively the division of tumoral cells, preferably epithelial tumoral cells, and even more preferably cells deriving from estrogen-sensitive epithelial tumors, more preferably said compound being the protein compound disclosed according to this invention.

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

It is another object of this invention the employment of said conditioned medium for producing pharmaceutical compositions for treating tumors, preferably epithelial tumors, and even more preferably for the treatment of estrogen-sensitive epithelial tumors.

It is a further object of this invention the employment of said conditioned medium for the purification, identification and characterization of at least one compound capable of inhibiting selectively the division of tumoral cells, preferably of epithelial tumoral cells, and even more preferably of cells derived from estrogen-sensitive epithelial tumors.

This invention will be now disclosed by means of some examples that explain and illustrate the same but with no limitative purposes, and with reference to the enclosed figures wherein:

Figure 1 represents a growth curve of the LSA line both in the presence and in the absence of serum;

Figure 2a shows a cytofluorimetric analysis of human normal thymocytes;

Figure 2b shows a cytofluorimetric analysis of LSA cells:

Figure 3 shows an LSA-CM stimulation histogram of the incorporation of ³[H]-thymidine in various cell lines, where 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 undiluted, 5 is a control sample, 6 is the LSA-CM undiluted, 7 is the control sample, and 8 is the LSA-CM undiluted;

Figure 4 shows growth curves of the MCF-7 cell line, both in the presence and in the absence of LSA-CM:

Figure 5 represents growth curves of the ZR-75-1 cell line, both in the presence and in the absence of LSA-CM;

Figure 6 represents growth curves of the MDAMB-231 cell line, both in presence and in the absence of LSA-CM:

Figure 7 represeπts growth curves of the NMMG cell line in the absence and in the presence of LSA-CM;

Figure 8 represents growth curves of human cells from ovaric carcinoma both in the absence and in the presence of LSA-CM:

Figure 9 represents a histogram of human cell growth from ovaric carcinoma both in the absence and in the presence of LSA-CM and/or of purified p1LSA.

Example 1

Isolation of the LSA line

A fragment of human liposarcoma of mixed lipoblasticfibroblastic type is drawn in a sterile way from the leg of an adult woman, 65 years of age, by means of surgical techniques.

The fragment is disgregated mechanically by a chisel so as to obtain 1 mm fragments and then this material is treated with a CTC solution equivalent to 20 U/ml of collagenase

(CLSP, Worthington, . U.K. .), 0,75 mg/ml of trypsin (1/300, Inc. Pharmaceuticals), 2 % chicken serum, heat-inactivated and dialyzed (GIBCO) in Hank soil without Ca⁺⁺ and Mg⁺⁺ ions (GIBCO) and Ham F12 soil with 5 % bovine serum (GIBCO) for 4 hours at 37°C and under continuous stirring, in order to obtain a suspension of single cells.

The cell suspension is diluted with the Ham F12 (GIBCO) culture medium supplemented with 5 % bovine serum (GIBCO), penicillin (31 μg/ml), streptomycin (50 μg/ml) and fungizone

(2.5 μg/ml). The cells are plated at 200,000 cells/dish,

100 mm diameter (Costar).

A clone of cells which is capable of dividing in a culture and having the characteristics which are peculiar of the adipose cells has been obtained from ten dishes, said clone being called LSA and deposited with the DSM, accession number ACC 2029.

A growth curve of the LSA line is shown in Figure 1, in which the growth in the presence as well as in the absence of serum is evident. The LSA line shows a plating efficiency of 90 % and a duplication time of 50-52 hours in the presence of serum and of 102 hours in the absence of serum. The medium is changed every 72 hours.

Example 2

Characterization of the LSA line

The DNA content of LSA cells has been analyzed by means of a cytofluorometer (Partec Pas II flow-cytometer). The cell are trypsinized then fixed with 70 % ethanol and dyed with a solution containing 5 μg/ml of ethidium bromide, 12.5 μg/ml of mitramycin and 1.5 mg MgCl₂ in 0.1 M Tris buffer, pH 7.5. The cell percentage in the various steps of the mitotic cycle is obtained as disclosed by Flintoff, W.E., Davidson, S.V., and Siminowitch, L. "Isolation and partial characterization of Methotrexate-Resistent phenotype from Chinese hamster ovary cells" Somatic Cell Genet. 2:245-261, 1976; and Ambesi-Impiombato, F.S., Parks, L.A.M. and Coon H.G. Culture of hormonedependent functional epithelial ceils from rat thyroid. Proc. Natl. Acad. Sci. USA 77: 3455-3459, 1980. As shown in Figure 2b, the values are compared to those obtained with normal human thymocytes as ploidy standard. The value of 1 has been assigned to the G1-G0 peak of thymocytes. Under the same conditions the G1-G0 peak of the LSA cells has a value of 1.79, which is indicative of a DNA content which is almost tetraploid. Also the DNA content/cell is almost twice as much as that obtained with thymocytes as shown in Figure 2a.

The distribution of cells in the various steps of the cell cycle of an LSA cell population in the phase of logarithmic growth is shown in the following Table 1.

Table 1

Phase Cell percentage

G1/G0 59 S 27

G2/M 14

The presence of cells in the phase S confirms that the population is in the active growth phase.

The counting of the chromosomes has put into evidence a number of chromosomes/cell between 80 and 90.

The LSA cells as observed under an optical microscope look like elongated bodies with abudant cytoplasm. Under the electronic microscope, the cell nucleus contains many nucleoles and some organelles are present, localized in the perinuclear region. The cells are homogeneous to each other, the mitochondria are relatively abudant and the cytoplasmic vesicles are constant both in number and sizes. The most evident feature is the presence of vacuolar areas inside the cytoplasm, said areas being electron-reflecting and typical of zones containing high amounts of lipids, which is removed as a result of subjecting the cells to a treatment for their preparation for observation under the electron microscope. Moreover, a well developed Golgi apparatus is also present. In order to estimate the content of lipids, the LSA cells are plated on culture slides of the Lab-Teck (NUNC) and are grown by semi-confluence. The cells are washed and fixed for 6 minutes with 4 % paraformaldehyde and 15 % picric acid saturated in PBS (phosphate saline buffer). The cells are washed twice with distilled water, then they are dyed with an Oil Red 0 solution for 5 minutes, then they are washed twice with PBS and dyed with 1 % toluidine blue for 10 seconds.

If cells have been grown in the presence of 5 % bovine serum, lipids are dispersed throughout the cytoplasm, with round vesicles. The build up of lipids Is not present in cell grown in a medium without serum, but it is stimulated by incubating cells with methylxanthine/desamethasone for 48 hours and with insulin for a further period of 48 hours.

Example 3

Effects of conditioned medium by LSA (LSA-CM) cells on growth of different cell lines in vitro

LSA cells have been grown up to 80 % confluence on

100 mm plates (Costar) on F12 medium with 5% bovine serum. The cell monolayers are washed with PBS and incubated with F12 medium without serum. After 24 hours, the medium is replaced with fresh medium the conditioned medium is collected every 6-24 hours for the successive 30 days. The cells go on growing and they show structurally and functionally viable under phase-contrast microscope and after dying with Trypan Blue.

The medium conditioned by LSA cells is capable of stimulating 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 put into evidence by an ³H-thymidine incorporation test shown in Figure 3. Contrarily to the preceding results, the medium conditioned by LSA cells shows an inhibition effect of growth and a cytolytic activity when it is analyzed with in vitro tumoral cells containing receptors for estrogens. The cells employed are:

MCF-7 cells obtained from a human mammary carcinoma

(Land, H. et al., Science 222:778 (1980), ECACC-86012803) that possess receptors for estrogens (Soule, H.D. et al., Natl. Cancer Inst. 51, 1409-1413 (1973)), androgens (Horowitz, K.B. et al., Steroids 26, 785-795 (1975)), insulin and triodothyronine (MacGrath, CM. Cancer Res., 43: 1355-1360 (1983);

the ZR-75-1 (CRL-1504) cells obtained from a human mammary carcinoma, said cells being positive for estrogen receptors;

the MDAMB-231 (ATCC-HTB26) cells obtained froma a human mammary carcinoma, but that are negative for estrogen recep tors;

the NMMG (Burker, R.E. et al., Cancer Res., 38, 3769-3773 (1978), CRL-1636) cells, obtained from a mouse mammary epithelium, said cells being slightly positive for estrogen receptors;

a human cell line of ovaric carcinoma obtained from a woman suffering from peritoneal effusion due to ovaric carcinoma metastasis, said line being positive for estrogen receptor.

The growth curves of the cell lines mentioned herein have been obtained by plating 100,000 cells/dish of 100 mm diameter in 10 ml of DMEM (GIBCO) medium with 10 % bovine fetal serum (FCS, GIBCO). 2 ml of LSA-CM have been added to a set of dishes..

Figure 4 shows growth curves of the MCF-7 line in which the inhibition effect of LSA-CM is evident. In particular, after a 4 day treatment, the removal of LSA-CM cannot reestablish the proliferative capability of MCF-7 cells because of a cytolytic effect.

Figure 5 shows growth curves of the 7R-75-1 line in the presence and in the absence of LSA-CM. The presence of LSA-CM gives rise to a strong inhibition of growth without any cytolytic effect.

Figure 6 and 7 show the growth curves of MDAMB-231 cells and of normal epithelial NMMG cells, in which the absence of growth inhibition due to LSA-CM is evident. Such cells do not show estrogen receptors.

Figure 8 shows that the LSA-CM conditioned medium has a cytotoxic effect on the cells of human ovaric carcinoma, even after just 24 hours of incubation.

The LSA-CM effect on the incorporation of ³H-thymidine in different cell lines is shown in the following Table 2. Table 2

Effect of LSA-CM on ³H-thymidine incorporation

in different cell lines

Cell lines No effect Stimulation Inhibition

MCF7 +

ZR-75-1 +

NMMG +

FRTL-5 +

CHO +

NIH-3T3 +

Example 4

Effect of LSA-CM on animals

20 Balb/c fc3H mice (Charles River) which are affected by the Bittner virus are selected for the presence of an evident tumoral mass, an estrogen-dependent mammary carcinoma, which is induced by the virus itself.

After a peritumoral injection practised every day of 200μl of LSA-CM for 15 days, the inhibition of growth and the necrosis of the tumoral mass are observed in 80 % of the mice treated.

Example 5

Isolation of the p1LSA protein from LSA-CM

The LSA-CM medium is concentrated 100 x, then it is subjected to dialysis against a isotonic phosphate buffer

at pH 7.4 and then to gel filtration on P6 resin (Pharmacia). The fractions eluted are tested for their capability of inhibiting selectively the growth of MCF-7 cells. One only fraction shows said biological activity. A sample of said fraction is separated by electrophoresis on a polyacrylamide gel in non-denaturating conditions, identifying a protein of about 29 kDa which is called p1LSA. Said protein is electro eluted from the gel and employed for the production of polyclonal antibodies as disclosed in the following and for the sequence analysis.

The protein sequence is shown in the following Table 3. As shown in Figure 9, the purified p1LSA protein has an evident cytotoxic effect on the growth of human ovaric tumora cells.

Table 3

Amino acid sequence of the p 1LSA protein and

corresponding coding sequence

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 ACCCAAGTTTCCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 495

ThrGlnValSerSerSerLeuSerProGluGlyThrLeuThrVal 165

GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540

GluAlaProMetProLysLeuAlaThrGlnSerAsnGluIleThr 180

ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585

IleProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195

GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618

AlaAlaLysSerAspGluThrAlaAlaLys 205

From a comparison with the sequences deposited with data banks, the sequence turns out to be identical with that corresponding to the human HSp27 protein described by Carper S.W. mentioned above, except for just one amino acid. The difference consists in a substitution of the amino acid in the position 194, said amino acid turning out to be arginine instead of proline.

Example 6

Production of anti-p1LSA polyclonal anti- bodies, and inhibition of the activity of

the p1LSA protein

100 ug of p1LSA protein electroeluted from a 10 % polyacrylamide gel is dissolved in 1 ml of PBS (phosphate saline buffer) and mixed with 1 ml of a complete Freund adjuvant.

The mixture is injected through intramuscolar injection in a rabbit four times at 10 day intervals. 30 ml of immune serum are drawn from rabbits every other day for 20 days. The immunoglobulin (Ig) fraction is purified by the Mab Trap G (Pharmacia) according to the procedure recommended by the producer, Immunoprecipitation and i mmunob lt otting tests show that immunoglobulins react in a specific way with the p1LSA protein from LSA-CM. A pre-incubation of 100 μl of LSA-CM with 10 μl of an Ig solution at 1 mg/ml for 30 minutes at 37°C stops the activity of inhibition of tumoral epithelial cell (MCF-7) division, restoring an ³H-thymidine incorporation and growth curves similar to the untreated control sample. The LSA-CM activity turns out to be unaltered after incubation with preimmune serum.

Such experimental data show that the p1LSA protein isolated and purified according to the procedures disclosed in Example 5 and having the sequence disclosed in Table 3 is actually the active principle responsible for the activity of inhibition of epithelial cell division disclosed herein.

Example 7

Nucleotide sequence coding for the p1LSA

protein

The nucleotide sequence coding for the p1LSA protein is determined by purifying the RNA polyA⁺ from LSA cells following standard methods and employing the following primers for the PCR reaction: oligo 5' : CGGAATTCTGAGCAGACGTCCAGAG

EcoRI

oligo 3' : CGGAATTCCGGGCTAAGGCTTTACTTG

EcoRI The sequences are deduced respectively from the sequences at the positions 5' and 3' not translated of the gene that codes for the HSp27.

The amplification products are cloned directly in the EcoRI site of the vectors pGEM4z (Promega) and sequenced by means of the dideoxy method. The complete sequence of the coding portion is shown in the preceding Table 3.

This invention has been disclosed with reference to some preferred embodiments of the same but it is to be understood that modifications and/or changes can be introduced by those who are skilled in the art without departing from the spirit and scope of the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: 1st. Naz. per Studio e Cura dei Tumori Fond.

G. Pascale

(B) STREET: Via M. Semmola

(C) CITY: Naples

(E) COUNTRY: Italy

(F) POSTAL CODE (ZIP): 80131

(ii) TITLE OF INVENTION: Protein compound, coding nucleotide sequences, producing cell line and uses thereof

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: IT RM92/A000161

(B) FILING DATE: 09-MAR-1992

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: IT RM92/A000716

(B) FILING DATE: 30-SEP-1992

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 618 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(C) INDIVIDUAL ISOLATE: patient with liposarcoma

(F) TISSUE TYPE: Adipose tissue

(G) CELL TYPE: Adipocyte

(H) CELL LINE: LSA cell line deposited at DSM N. ACC2029

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA library from poly A+ RNA

(B) CLONE: p1LSA (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..618

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATG ACC GAG CGC CGC GTC CCC TTC TCG CTC CTG CGG GGC CCC AGC TGG 48 Met Thr Glu Arg Arg Val Pro Phe Ser Leu Leu Arg Gly Pro Ser Trp

1 5 10 15

GAC CCC TTC CGC GAC TGG TAC CCG CAT AGC CGC CTC TTC GAC CAG GCC 96 Asp Pro Phe Arg Asp Trp Tyr Pro His Ser Arg Leu Phe Asp Gln Ala

20 25 30

TTC GGG CTG CCC CGG CTG CCG GAG GAG TGG TCG CAG TGG TTA GGC GGC 144 Phe Gly Leu Pro Arg Leu Pro Glu Glu Trp Ser Gln Trp Leu Gly Gly

35 40 45

AGC AGC TGG CCA GGC TAC GTG CGC CCC CTG CCC CCC GCC GCC ATC GAG 192 Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala Ile Glu

50 55 60

AGC CCC GCA GTG GCC GCG CCC GCC TAC AGC CGC GCG CTC AGC CGG CAA 240 Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gln

65 70 75 80

CTC AGC AGC GGG GTC TCG GAG ATC CGG CAC ACT GCG GAC CGC TGG CGC 288 Leu Ser Ser Gly Val Ser Glu Ile Arg His Thr Ala Asp Arg Trp Arg

85 90 95

GTG TCC CTG GAT GTC AAC CAC TTC GCC CCG GAC GAG CTG ACG GTC AAG 336 Val Ser Leu Asp Val Asn His Phe Ala Pro Asp Glu Leu Thr Val Lys

100 105 110

ACC AAG GAT GGC GTG GTG GAG ATC ACC GGT AAG CAC GAG GAG CGG CAG 384 Thr Lys Asp Gly Val Val Glu Ile Thr Gly Lys His Glu Glu Arg Gln

115 120 125

GAC GAG CAT GGC TAC ATC TCC CGG TGC TTC ACG CGG AAA TAC ACG CTG 432 Asp Glu His Gly Tyr Ile Ser Arg Cys Phe Thr Arg Lys Tyr Thr Leu

130 135 140

CCC CCC GGT GTG GAC CCC ACC CAA GTT TCC TCC TCC CTG TCC CCT GAG 480 Pro Pro Gly Val Asp Pro Thr Gln Val Ser Ser Ser Leu Ser Pro Glu

145 150 155 160

GGC ACA CTG ACC GTG GAG GCC CCC ATG CCC AAG CTA GCC ACG CAG TCC 528 Gly Thr Leu Thr Val Glu Ala Pro Met Pro Lys Leu Ala Thr Gln Ser

165 170 175

AAC GAG ATC ACC ATC CCA GTC ACC TTC GAG TCG CGG GCC CAG CTT GGG 576 Asn Glu Ile Thr Ile Pro Val Thr Phe Glu Ser Arg Ala Gln Leu Gly

180 185 190

GGC CGA GAA GCT GCA AAA TCC GAT GAG ACT GCC GCC AAG TA 618

.Gly Arg Glu Ala Ala Lys Ser Asp Glu Thr Ala Ala Lys

195 200 205 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 205 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Thr Glu Arg Arg Val Pro Phe Ser Leu Leu Arg Gly Pro Ser Trp 1 5 10 15

Asp Pro Phe Arg Asp Trp Tyr Pro His Ser Arg Leu Phe Asp Gln Ala

20 25 30

Phe Gly Leu Pro Arg Leu Pro Glu Glu Trp Ser Gln Trp Leu Gly Gly

35 40 45

Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala Ile Glu 50 55 60

Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gln 65 70 75 80

Leu Ser Ser Gly Val Ser Glu Ile Arg His Thr Ala Asp Arg Trp Arg

85 90 95

Val Ser Leu Asp Val Asn His Phe Ala Pro Asp Glu Leu Thr Val Lys

100 105 110

Thr Lys Asp Gly Val Val Glu Ile Thr Gly Lys His Glu Glu Arg Gln

115 120 125

Asp Glu His Gly Tyr Ile Ser Arg Cys Phe Thr Arg Lys Tyr Thr Leu 130 135 140

Pro Pro Gly Val Asp Pro Thr Gln Val Ser Ser Ser Leu Ser Pro Glu 145 150 155 160

Gly Thr Leu Thr Val Glu Ala Pro Met Pro Lys Leu Ala Thr Gln Ser

165 170 175

Asn Glu Ile Thr Ile Pro Val Thr Phe Glu Ser Arg Ala Gln Leu Gly

180 185 190

Gly Arg Glu Ala Ala Lys Ser Asp Glu Thr Ala Ala Lys

195 200 205

Claims

C LA I MS

1. A substantially proteinaceous compound, characterize in that it is capable of inhibiting selectively the cellular division of estrogen-sensitive epithalial tumoral cells, and/or of exerting a cytotoxic activity on such cells; and characterized in that it has a molecular weight in the range between 27 and 30 kDa: it comprises an amino acid sequence from the amino acid 1 to the amino acid 193 which is substantially homologous to the corresponding sequence of human HSp27 (heat shock p27) protein; or a biologically active fragment of said compound.

2. A substantially proteinaceous compound according to claim 1, said compound comprising at its carboxyl end an amino acid sequence which is homologous to the sequence:

GluAlaAlaLysSerAspGluThrAlaAlaLys-NH₂.

3. A substantially proteinaceous compound according to claim 2, such compound comprising an amino acid sequence which is substantially homologous to the following amino acid sequence:

MetThrGluArgArgValProPheSerLeuLeuArgGlyProSer 15

TrpAspProPheArgAspTrpTyrProHisSerArgLeuPheAsp 30

GlnAlaPheGlyLeuProArgLeuProGluGluTrpSerGlnTrp 45

LeuGlyGlySerSerTrpProGlyTyrValArgProLeuProPro 60

AlaAlalleGluSerProAlaValAlaAlaProAlaTyrSerArg 75

AlaLeuSerArgGlnLeuSerSerGlyValSerGluIleArgHis 90

ThrAlaAspArgTrpArgValSerLeuAspValAsnHisPheAla 105

ProAspGluLeuThrValLysThrLysAspGlyValValGluIle 120

ThrGlyLysHisGluGluArgGlnAspGluHisGlyTyrIleSer 135

ArgCysPheThrArgLysTyrThrLeuProProGlyValAspPro 150

ThrGlnValSerSerSerLeuSerProGluGlyThrLeuThrVal 165 GluAlaProMetProLysLeuAlaThrGlnSerAsnGluIleThr 180

IleProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195 AlaAlaLysSerAspGluThrAlaAlaLys 205.

4. A substantially proteinaceous compound according to any one of the preceding claims, which is produced by LSA cells (DSM ACC. N. 2029), which compound is preferably secreted by said calls in the culture medium.

5. A composition for pharmacological use, preferably for use as anti-neoplastic, comprising the substantially proteinaceous compound according to any one of the preceding claims, or physiologically acceptable salts thereof.

6. A composition according to claim 5 to be employed for treatment of estrogen-sensitive epithelial tumors.

7. Use of the substantially proteinaceous compound according to any one of the preceding claims or of its salts physiologically acceptable, for the preparation of pharmacological compositions for antineoplastic treatment, preferably of estrogen-sensitive epithelial tumors.

8. A nucleic acid comprising a nucleotide sequence coding for said substantially proteinaceous compound according to any one of the preceding claims.

9. A nucleic acid according to claim 8 comprising the following nucleotide sequence:

ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGC 45

TGGGACCCCTTCCGCGACTGGTACCCGCATAGCCGCCTCTTCGAC 90

CAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGG 135

TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180

GCCGCCATCGAGAGCCCCGCAGTGGCCGCGCCCGCCTACAGCCGC 225

GCGCTCAGCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCAC 270

ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCC 315

CCGGACGAGCTGACGGTCAAGACCAAGGATGGCGTGGTGGAGATC 360

ACCGGTAAGCACGAGGAGCGGCAGGACGAGCATGGCTACATCTCC 405 CGGTGCTTCACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCC 450

ACCCAAGTTTCCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 495

GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540

ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585

GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618

10. Plasmid or phage vectors comprising the nucleotide sequences according to any one of the preceding claims 8 or 9.

11. A mammalian cell line capable of dividing in vitro, having a phenotype associable to that of adipose cells, which produces in its culture medium, or conditioned medium, at least one compound according to any one of the preceding claims 1-4 which is capable of inhibiting selectively the division of tumoral cells.

12. A mammalian cell line according to claim 11 wherein said tumoral cells comprise epthelial tumoral cells.

13. A mammalian cell line according to claim 12 wherein said epithelial tumoral cells derive from estrogen-sensitive epithelial tumors.

14. A mammalian cell line according to any one of the preceding claims 11-13 and capable of dividing in vitro both in the presence and in the absence of serum in the culture medium.

15. A mammalian cell line according to any one of the preceding claims 11-14 wherein said cell line is isolated from a liposarcoma.

15. A mammalian cell line according to claim 15 wherein said cell line is isolated from a human liposarcoma.

17. A mammalian cell line according to claim 15, wherein said cell line is the LSA line (DSM ACC. N. 2029).

18. A medium that has been conditioned by the growth of a cell line according to any one of the preceding claims 11-17

19. A medium that has been conditioned according to claim 18, said medium comprising at least one compound capable of inhibiting selectively the division of tumoral cells.

20. A medium that has been conditioned according to claim 19, wherein said tumoral cells are epithelial tumoral cells.

21. A medium that has been conditioned according to claim 20 wherein said epithelial tumoral cells derive from estrogensensitive epithelial tumors.

22. A medium that has been conditioned according to claim 21, wherein said cell line is the LSA line (DSM ACC.N. 2029).

23. Use of the conditioned medium

according to any one of the preceding claims 18-22 for the production of pharmaceutical compositions for treatment of tumors.

24. Use of the conditioned medium according to claim 23 wherein said tumors are epithelial tumors.

25. Use of the conditioned medium according to claim 24 wherein said epithelial tumors are estrogen-sensitive.

25. Use of the conditioned medium according to any one of the preceding claims 18-22 for the purification, identification and characterization of at least one compound which is capable of inhibiting selectively the division of tumoral cells.

27. Use of the conditioned medium according to claim 26, wherein said tumoral cells are epithelial cells.

28. Use of the conditioned medium according to claim 27 wherein said cells are derived from estrogen-sensitive epithelial tumors.