JP3850492B2 - Tenascin-inducing factor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a physiologically active substance that induces the production of tenascin involved in the growth and metastasis of cancer cells.
[0002]
[Prior art]
The epithelial cells are thought to play a leading role in the carcinogenesis of epithelial cells by the surrounding mesenchyme (sometimes called the stroma) that exists around the epithelial cells, especially the macromolecular group called the extracellular matrix. The extracellular matrix consists of collagen, laminin and type IV collagen that make up the basement membrane, and tenascin, a glycoprotein with molecular weights of 190 and 250 kDa (Chiquet-Ehrismann, R., et al., Cell, 47, pp .131-139, 1986) and the like.
[0003]
Tenascin exists as a hexamer in the surrounding mesenchyme, and is known to be transiently expressed during organ development, tissue regeneration, wound healing, cancer growth and invasion (Erickson, HP, Annu. Rev. Cell Biol., 5, pp. 71-92, 1989; Sakakura, T., Tumor Matrix Biology, pp. 101-129, CRC Press, 1995). The physiological activity of tenascin has been shown to be involved in cell behavior in the morphogenesis process and tissue repair process, such as cell adhesion and shedding, cell proliferation stimulation and inhibition, and hemagglutination activity. Tenascin is generally produced by fibroblasts, and it has been clarified that cancer cells derived from the epithelial system also produce tenascin, and tenascin production in cancer cells is derived from the surrounding mesenchyme of cancer cells. It has been suggested to be induced by sex factors (Hiraiwa, N., et al., J. Cell. Science, 104, pp. 289-296, 1993). Furthermore, transforming growth factor (TGFβ) and epidermal growth factor (EGF) are known as substances having tenascin-inducing activity (Chiquet-Ehrismann, R., et al., Cancer Res., 49, pp .4322-4325, 1989; Sakai, T., et al., J. Cell. Physiol., 165, pp. 18-29, 1995).
[0004]
[Problems to be solved by the invention]
As a result of studies on the role of tenascin in the proliferation and metastasis of cancer cells, the present inventors have used tenascin-deficient mice that the production of tenascin in cancer cells is regulated by an inducer derived from surrounding mesenchymal cells. (Kusakabe et al., Basic Breast Cancer Research, 5, 41-45, 1996). Moreover, it was proved that this inducer was expressed in a tenascin-dependent manner, that is, tenascin itself acted on the surrounding mesenchymal cells of cancer cells to promote the production of this inducer. Thus, the interaction between tenascin and the above inducer acts as a kind of amplification mechanism. However, since the inducer is produced in a very small amount, it has not yet been identified as a substance, and its physicochemical properties and physiological actions are almost unknown.
[0005]
Therefore, an object of the present invention is to separate and purify the above inducer to the extent that it can be used and identified as a substance, and to clarify its physicochemical properties and physiological actions.
[0006]
[Means for Solving the Problems]
Human squamous cell carcinoma-derived A431 cells rarely express tenascin in vitro, but express tenascin when transplanted into nude mice, and in vitro also express tenascin when the culture supernatant of mouse embryonic fibroblasts is added (Hiraiwa et al., J. Cell Science, 104, pp. 289-296, 1993). This result suggests that a factor that induces tenascin production is present in the culture supernatant of mouse fetal fibroblasts. The present inventor conducted further research and found that such activity was not observed in the culture supernatant of the mouse-derived cells knocked out of tenascin, and that the knockout-derived cells cultured in the culture dish coated with purified tenascin It was found that the culture supernatant has an activity to induce tenascin production. The inventors of the present invention further conducted intensive research to separate and purify this factor, and completed the present invention.
[0007]
That is, the present invention is expressed by cancer tissue mesenchymal cells, can induce tenascin production in cancer cells and / or cancer tissue mesenchymal cells, and the expression of the factor is induced by tenascin. Factors with the following physicochemical properties:
(1) Estimated molecular weight by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is about 12,000-16,000;
(2) The estimated molecular weight by gel filtration chromatography is about 4,500;
(3) substantially inactivated by boiling at 100 ° C and freeze-drying;
(4) No substantial loss of activity by heating, freezing, or thawing at 56 ° C for 30 minutes;
(5) No substantial loss of activity upon treatment with sodium dodecyl sulfate or 2-mercaptoethanol on SDS-PAGE, or with acetic acid or trifluoroacetic acid in reverse phase chromatography; and
(6) pI is about 7;
(7) substantially binds to concanavalin A (ConA) and the bound factor can be eluted by methylmannopyranoside;
(8) does not substantially bind to kidney bean lectin (PHA-E4), lotus bean lectin (Lotus), and lentil bean lectin (LCA); and
(9) does not substantially bind to heparin;
The factor specified by is provided.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
In order to isolate and purify the factor of the present invention, for example, murine fetal (14 days) skin cells are grown in DMEM containing 10% FCS, and then, for example, the following steps:
Step 1: Ultrafiltration between 50k and 6k;
Step 2: Dialysis against 20 mM Tris-HCl, pH 8.0 and purification by anion exchange chromatography, eg Q-Sepharose Fast Flow;
Step 3: Concanavalin A agarose adsorption and elution with methyl-α-D-mannopyranoside;
Step 4: Dialysis against 20 mM ammonium acetate, pH 6.0, and purification by cation exchange chromatography such as mono S HR 5/5; and Step 5: by a method including purification by reverse phase chromatography it can. However, the method for separating and purifying the factor of the present invention is not limited to the above method, and appropriate modifications and alterations may be added to the above method.
[0009]
The molecular weight of the factor of the present invention is estimated to be about 12,000-16,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and about 4,500 by gel filtration chromatography. In the gel filtration chromatography method, it is considered that the estimated molecular weight is smaller than the actual numerical value because the factor of the present invention has some interaction with the filler acrylamide gel. In the case of gel filtration chromatography, it is fractionated between cytochrome c (molecular weight 12,000) and cytidine (molecular weight 250). In addition, the factor of the present invention does not substantially lose activity even when treated with sodium dodecyl sulfate or 2-mercaptoethanol in SDS-PAGE, or treated with acetic acid or trifluoroacetic acid in reverse phase chromatography.
[0010]
The sugar chain present in the factor of the present invention is adsorbed to agarose bound with concanavalin A as an immobilized lectin, but is eluted by methylmannopyranoside, so that the sugar chain containing mannose or glucose The existence is clear. In addition, this sugar chain is characterized in that it does not substantially bind to other lectins such as kidney bean lectin (PHA-E4), lotus bean lectin (Lotus), and lentil lectin (LCA).
[0011]
The factor of the present invention is a factor expressed by a mesenchymal cell of a cancer tissue and acts on a cancer cell or a mesenchymal cell of a cancer cell and a cancer tissue, thereby causing the cancer cell and / or It has the effect of inducing tenascin production in mesenchymal cells of cancer tissue. The factor of the present invention is characterized by being expressed in a tenascin-dependent manner, and tenascin itself has an action of inducing the factor of the present invention. Therefore, expression of tenascin itself and the factor of the present invention is considered to be amplified by the interaction of these physiologically active substances.
[0012]
When measured under conditions where transforming growth factor (TGFβ) and epidermal growth factor (EGF) can confirm their tenascin-inducing activity, the factor of the present invention is insulin-like growth factor (IGF-II). The tenascin-inducing activity is almost the same. In addition, when A431 cells derived from human squamous cell carcinoma are stimulated with TGFα or EGF, a strong tyrosine phosphate is observed in the vicinity of a molecular weight of 180 kDa, but similar to other cytokines such as interleukin 1 (IL-1) and TNFα. Furthermore, the above-mentioned remarkable tyrosine phosphorylation action is not recognized in the factor of the present invention.
[0013]
The factor of the present invention has been confirmed to induce tenascin production on human squamous cell carcinoma-derived A431 cells, human breast cancer-derived MCF-7 cells, and mouse lung metastatic breast cancer-derived GLMT1 cells that do not produce tenascin in vitro. It is expected to have similar effects on various cancer cells. In addition, the factor of the present invention has been observed to promote the proliferation of human squamous cell carcinoma-derived A431 cells, and is considered to have a proliferation promoting effect on other cancer cells as well.
[0014]
Therefore, the factor of the present invention is a physiologically active substance expressed by mesenchymal cells of cancer tissue, and has the following biochemical properties:
(10) Expression of the factor of the present invention is tenascin-dependent;
(11) Acts on cancer cells or on mesenchymal cells of cancer cells and cancer tissue to induce tenascin production in cancer cells and / or cancer tissue mesenchymal cells;
(12) Tenascin-inducing activity similar to that of insulin-like growth factor (IGF-II): and
(13) substantially free of tyrosine phosphorylation;
Is a factor characterized by
[0015]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.
(1) Separation and purification of the factor of the present invention
(a) Materials Fetuses (within 20 animals) on day 14 were taken out from 1 to 2 uterus of pregnant mice, and organs were removed in physiological saline. The removed fetus was washed with PBS (−), finely crushed with a razor on a Teflon plate, and transferred to a 50 ml centrifuge tube. After washing twice with PBS (−), 20 ml of PBS (−) containing 0.05% trypsin and 0.53 mM EDTA was added to the sample, and the sample was treated at 37 ° C. with occasional stirring. After adding 20 ml of DMEM containing 10% FCS and centrifuging at 1,000 rpm for 5 minutes, the supernatant was discarded and 20 ml of DMEM containing 10% FCS was added. The mixture was stirred well, and when a large lump precipitated, the supernatant was seeded on 10 culture dishes having a diameter of 10 cm. After culturing at 37 ° C. and 5% CO ₂ for 1 to 2 days and growing sufficiently, the supernatant was collected and used as a material for separating and purifying the factor of the present invention. The cells were passaged 3 to 5 times larger, and the supernatant was collected 3 to 4 days after passage and used as a material in the same manner.
[0016]
The factor distribution of the present invention can be summarized as follows.
Mouse embryo (14 days) Skin cell (almost all fibroblasts) conditioned medium +++
(The activity of the factor of the present invention decreases after several passages)
Adult mouse skin cell preparation medium +
Wound adult mouse skin cell preparation medium ++
Tenascin knockout mouse fetal skin cell preparation medium-
Tenascin Knockout Mouse Fetal Skin Cell Preparation Medium (TN Coat) ++
[0017]
(b) The ultrafiltration culture supernatant was treated with an ultrafiltration module (Asahi Kasei, molecular weight cut off 50000), and the low molecular weight side was used as a purification material for the factor of the present invention. Since the low molecular weight side was not concentrated, when the amount was large, it was concentrated to about 3 liters with an ultrafiltration module having a molecular weight cut off of 6,000. This purified material was placed in a dialysis tube (Spectrapore No. 1, molecular weight cut off 6,000-8,000) and dialyzed against 20 mM Tris-HCl (pH 8.0) for 2 days and nights.
[0018]
(c) Anion-exchange chromatography Dialyzed sample was filtered through a 0.22 μm filter and then equilibrated with 20 mM Tris-HCl (pH 8.0). Q-Sepharose Fast Flow (Pharmacia Biotech, XK16 / 20 column) ) At a flow rate of 5 ml / min. Then, wash the column thoroughly with 20 mM Tris-HCl (pH 8.0) until the UV monitor value drops to the baseline, and further perform linear gradient elution with 0.3 M NaCl at a flow rate of 1 ml / min. 10 ml was fractionated per book.
[0019]
(d) Bioassay of factor of the present invention A431 cells derived from human squamous cell carcinoma are seeded in a 48-well plate at 1 × 10 ⁴ / well, and 5% CO ₂ in DMEM containing 10% FCS at 37 ° C. The cells were cultured in the presence for 24 hours. After the medium was replaced with a new medium, 1 μl of each chromatographic fraction was added and cultured in the same manner for 16 hours. Tenascin in this culture supernatant was detected by enzyme immunoassay or Western blotting. The outline of the enzyme immunoassay is as follows.
[0020]
Rat monoclonal anti-human tenascin antibody (clone 7-13: This antibody recognizes the FNIII 3-5 domain of the tenascin molecule, so it can recognize large and small tenascin molecules. P. Shrestha, et al., International Journal of Oncology , 8, pp.741-755, 1996) was diluted with 0.1 M carbonate buffer (pH 9.6) to 10 μg / ml. Dispense this diluted solution into a 96-well microtiter plate at 50 μl / well and leave it in a wet box for 1 hour at room temperature. Then, discard the diluted solution and add PBS containing 0.05% Tween 20 (- The wells were washed 3 times with Subsequently, PBS (−) containing 3% BSA and 0.1% NaN ₃ was dispensed to fill the well. After standing for 1 hour or longer, the solution in the well was discarded, and a sample for measuring tenascin was added to 50 μl / well. After allowing to stand at room temperature for 1 hour in a wet box, the reaction solution was discarded and washed 3 times with PBS (-) containing 0.05% Tween 20.
[0021]
Biotinylated 36-13-6 antibody diluted to 5 μg / ml in PBS (-) containing 3% BSA and 0.1% NaN ₃ in the well (this antibody recognizes only the large tenascin molecule because it recognizes the AD domain of the tenascin molecule P. Shrestha, et al., International Journal of Oncology, 8, pp.741-755, 1996) was added to 50 μl / well, and the mixture was allowed to stand at room temperature for 1 hour in a humidified box. The reaction solution was discarded, and the wells were washed three times with PBS (−) containing 0.05% Tween 20. Horseradish peroxidase-conjugated avidin diluted 1000-fold with PBS (-) containing 3% BSA was added to a concentration of 50 μl / well and left at room temperature for 1 hour in a humid box. The wells were washed 3 times with PBS (-) containing% Tween 20. In 0.1 M sodium acetate buffer (pH 6.0), 10 μl / 30 ml of 30% H ₂ O ₂ was added to 10 mg / 30 ml of orthophenylenediamine dihydrochloride and dispensed at 100 μl / well. The mixture was allowed to stand in the dark, and 2 N sulfuric acid was added to a concentration of 50 μl / well when the color development progressed sufficiently, and the absorbance at a wavelength of 492 nm was measured.
[0022]
(e) Reverse Phase Chromatography TFA was added to the Q-Sepharose fraction with confirmed tenascin-inducing activity to a final concentration of 0.1%. This fraction was applied to ProRPCHR5 / 10 (Pharmacia Biotech) equilibrated with H ₂ O containing 0.1% TFA at a flow rate of 1 ml / min, and when the absorbance at 280 nm was sufficiently lowered to the baseline, Linear gradient elution was performed with 60% acetonitrile containing 0.1% TFA, and fractions of 1 ml were fractionated, and tenascin-inducing activity was measured by the method described above.
[0023]
(f) Measurement of molecular weight by SDS-PAGE Using a glass tube measuring 5 mm in inner diameter and 12 cm in length, prepare a 15% acrylamide gel to a height of 10 cm and overlay the 3% acrylamide concentrated gel on top of each other. did. The sample treatment solution and the sample were mixed and allowed to stand at room temperature for 30 minutes, and then 50 to 100 μl of the reaction solution was applied to the gel and electrophoresed at a current of 5 mA per gel tube. When BPB reaches 1 cm from the end of the gel, finish the electrophoresis, remove the gel from the glass tube, slice the gel so that it is 5 mm apart, and add 100 μl PBS (-) to the test tube. Moved to. Extraction was performed overnight with shaking, and the tenascin-inducing activity of each extract was measured by the method described above. The estimated molecular weight of the factor of the present invention by this method was about 12,000-16,000. When tenasacin knockout GRS / A mice (Kusakabe et al., Breast Cancer Basic Research, 41-45, 1996) were subjected to the same procedure, tenascin-inducing activity was not detected after SDS-PAGE. This result indicates that the factor of the present invention is not expressed in cells lacking tenascin, and the factor of the present invention is expressed in a tenascin-dependent manner.
[0024]
(g) Measurement of molecular weight by gel filtration chromatography
The molecular weight of the factor of the present invention was measured using Superdex 75HR10 / 30 (Pharmacia Biotech). Bovine serum albumin (molecular weight: 67,000), soybean trypsin inhibitor (molecular weight: 20,100), cytochrome c (molecular weight: 12,400), and insulin (molecular weight: 5,800) were used as standard proteins, and regression was performed from the logarithmic value of these molecular weights and the elution position by the least square method. A straight line was calculated as a standard straight line. Tenascin-inducing activity was eluted at a molecular weight of around 4,500. In the molecular weight measurement by the gel filtration chromatography method, the apparent molecular weight may be reduced because the factor of the present invention has some interaction with the gel.
[0025]
(h) As a presumed lectin of the sugar chain by the immobilized lectin, binding to concanavalin A, kidney bean lectin (PHA-E4), lotus bean lectin (Lotus), and lentil lectin (LCA) was examined. The factor bound only to concanavalin A and the bound active fraction could be eluted by methylmannobiranosides.
[0026]
(i) Cytokine-like action Humans stimulated with the purification factor of the present invention, IL-1, IL-2, IL-3, IL-4, IL-6, TNFα, TGFα, TGFβ, PDGF, EGF, or IGF-II When a crude extract of squamous cell carcinoma-derived A431 cells was Western blotted with an anti-phosphotyrosine antibody (PY20, Tranduction Laboratories), TGFα and EGF showed strong tyrosine phosphorylation products at a molecular weight of about 180 k. And other cytokines showed no significant tyrosine phosphorylation compared to untreated ones. In addition, the tenascin-inducing activity of TGFβ and EGF, which have already been reported to be tenascin-inducing activity, was measured and some activity was observed. Under the same conditions, IGF-II is comparable to the factor of the present invention. showed that. For other cytokines, tenascin-inducing activity was not observed.
[0027]
(j) Other biological effects In vitro, human breast cancer MCF-7 that does not produce tenascin, mouse lung metastatic breast cancer GLMT1, and when the factor of the present invention was added in the same manner as (d) above, tenascin Production was observed. In addition, the factor of the present invention was found to significantly promote the proliferation of human squamous cell carcinoma-derived A431 cells. Furthermore, pulmonary metastatic breast cancer GLMT1 cells (which do not produce tenascin in vitro), which have the property of metastasizing to the lung after 1 to 1.5 months when transplanted subcutaneously in GR mice, form tumors even when transplanted into tenascin knockout GR mice However, metastasis to the lung hardly occurs, but after transplanting lung metastatic breast cancer GLMT1 cells to tenascin knockout GR mice, the factor of the present invention (Q fraction, 0.1 ml) was administered to normal GR mice. The tumor grew and metastasized to the lung (Fig. 1). In experiments using a culture system, it was also found that the factor of the present invention stimulates the proliferation of A431 cells as well as the induction of tenascin (FIG. 2).
[0028]
On the other hand, such activity was not observed in the culture supernatant of cells derived from mice knocked out of tenascin. In addition, when knockout-derived cells are cultured in a culture dish coated with tenascin purified from the culture supernatant of mouse embryonic fibroblasts, the activity of the factor that induces tenascin production is observed in the culture supernatant. became. This effect of tenascin was also observed in tenascin purified from the supernatant of human melanoma cell A375 cultured in GIT medium (Dainippon Pharmaceutical).
[0029]
【The invention's effect】
The factor of the present invention has a physiological effect that induces the expression of tenascin involved in the growth and metastasis of cancer cells. Therefore, the above-described factors provided by the present invention are useful for elucidating the mechanism of cancer cell proliferation and metastasis, and by measuring the expression level of the factors, the malignancy of cancer cells and the possibility of metastasis There is a possibility that it can be diagnosed accurately. Moreover, it is useful for the research of the inhibitor of this factor by which the usefulness as an anticancer agent is anticipated.
[Brief description of the drawings]
FIG. 1 shows the results of administration of the factor of the present invention (MTIF) in comparison with a non-administration group (MC) after transplantation of lung metastatic breast cancer GLMT1 cells to tenascin knockout GR male mice.
FIG. 2 shows the results of stimulation of A431 cell proliferation by the factor of the present invention in an experiment using a culture system. In the figure, TIF indicates the factor of the present invention, and TIF0.1, TIF0.01, and TIF0.001 are respectively 10 times, 100 times, and 1,000 times dilution of the Q fraction of the factor of the present invention. The results when used are shown.

Claims (1)

A method for producing a tenascin production inducer that is expressed by mesenchymal cells of cancer tissue and can induce tenascin production in cancer cells and / or mesenchymal cells of cancer tissue,
Using the culture supernatant obtained by culturing tenascin knockout mouse embryo-derived fibroblasts in a culture dish coated with tenascin purified from the culture supernatant of mouse embryo-derived fibroblasts, the following steps:
Step 1: Ultrafiltration between 50k and 6k;
Step 2: Dialysis against 20 mM Tris-HCl, pH 8.0, and purification by anion exchange chromatography;
Step 3: Concanavalin A Adsorption on agarose and elution with methyl-α-D-mannopyranoside;
Step 4:20 mM ammonium acetate, dialyzed against pH 6.0, and the cation exchange purification by chromatography; and Step 5: Purification by reverse phase chromatography seen including, and
The tenascin production inducer has the following physicochemical properties:
(1) SDS-estimated molecular weight by polyacrylamide gel electrophoresis (SDS-PAGE) is 12,000 to 16,000;
(2) the estimated molecular weight by gel filtration chromatography is 4,500 ;
(3) Inactivate by boiling at 100 ° C and freeze-drying;
(4) No loss of activity by heating, freezing or thawing at 56 ° C for 30 minutes;
(5) No activity is lost by treatment with sodium dodecyl sulfate or 2- mercaptoethanol on SDS-PAGE , or with acetic acid or trifluoroacetic acid in reverse phase chromatography; and
(6) pI is 7 ;
(7) binds to concanavalin A (ConA) and the bound factor can be eluted by methylmannopyranoside;
(8) does not bind to kidney bean lectin (PHA-E4) , lotus bean lectin (Lotus) , and lentil bean lectin (LCA) ; and
(9) Does not bind to heparin
A method that is a factor specified by

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