JPS61275221A - Protein - Google Patents

Abstract

NEW MATERIAL:A protein (molecular weight, 55,000-140,000 by two-dimensional electrophoresis; isoelectric point, 4.5-5.8). USE:Agent for the remedy and diagnosis of human and animal tumor, cancer and cerebroneurosis. PREPARATION:An animal cell (e.g. neuroblastoma N-18 strain of mouse- originated cell, leiomyoid cerebroma BC3H1 strain, pheochromocytoma PC12 strain as a rat-originated cell, human fatal brain Flow 3,000 strain, etc.) is cultured in proper culture liquid or serum, or is proliferated in an aminal body. The proliferated cells are collected in a mass and homogenized to extract the protein, and the proteins are separated from each other and purified by gel- filtration, ion-exchange chromatography, etc. The protein acts as an essential factor or promotion substance for the growth of tissue cell of cerebroneural system. The effect can be exhibited by applying the protein to a living body. It is also useful as a marker for various tumors.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a protein that can be used as a therapeutic or diagnostic agent for tumors, cancers, and cranial nervous system diseases in humans or other animals.

Conventional technology In the diagnosis of cancer, cancer-related antigens such as α-fetoprotein, carcinoembryonic antigen (cHA), and CA 19-9 are
It has been used as a T tumor marker. Furthermore, as various cancers develop and progress, it has been revealed that substances that are different from normal in the body ebb and flow, and efforts are being made to establish new cancer diagnostic methods using these as markers. ing.

From this perspective, the present inventor has conducted research on substances that appear only in the early stages of embryonic development in animals, and has conducted intensive research on proteins that appear and disappear over time during the development and differentiation of the mouse brain nervous system. found that certain proteins appear in brain embryos in a time-specific manner and are absent in adult normal animals. We believe that these proteins are specific to proliferating cells and can serve as markers for excessive proliferation, and have filed a patent application for their usefulness (Japanese Patent Application No. 58-206899).

Furthermore, as a result of examining patient's cranial nervous system tumor tissue obtained through brain surgery, the present inventor discovered that human cranial nervous system cells also contain specific proteins, just like mouse cranial nervous system cells. In view of its usefulness in the field of neurological diseases and cancer, a patent application was filed (patent application filed in 1986-1973).
550).

Problems to be Solved by the Invention The protein antigens related to the above patent application were extracted, separated, and extracted from the lung liver of mouse fetuses and the tissue cells of cranial nervous system tumors of patients.
These proteins must be purified, and it takes a lot of time and effort to industrially obtain them in large quantities, which is not economical. In particular, it is practically very difficult to collect a quantitative amount of tumor tissue from patients, and as described in the above-mentioned Japanese Patent Application No. 73550/1983, the small amount of tumor tissue obtained is planted in nude mice or hamsters and grown. It is necessary to take measures.

Means to Solve the Problems Therefore, the present inventors did not rely on animal brain and nervous system tissue obtained by surgery, but instead used brain and nervous system tumor cells derived from mice, rats, humans, etc. that had already been established as cell lines. They thought that if they collected a large amount of these, they might be able to discover fetal-specific or tumor-specific proteins that are not present in the brain neural tissue cells of normal mice, normal rats, and normal humans. Then, the present inventor cultivated the brain nervous system cells of various animals in culture vessels or collected the cells after growing them in the animal body, and extracted proteins specific to the brain nervous system tumor cells that do not exist in normal animal brain nervous tissue. The present invention was completed by separating, purifying, and identifying the substance.

The protein of the present invention exists in subcultured brain nerve tumor cells derived from various animals, has a molecular weight of 55,000 to 140,000 by two-dimensional electrophoresis, and an isoelectric point of 4.5 to 5.8. It is a protein characterized by the following.

The animal cells used by the present inventor include the following.

Neuroblastoma (Neurobl) is a mouse-derived cell.
astoma) N-18 strain, N-115 strain, N-
B41A3 strain, Neuro 2a strain, smooth muscle-like brain tumor (Smooth muscle-like brain tumor)
tumor) BC3H1 strain, rat-derived cells include Pheochromocytoma P
Cl3 strain, Glial cell tumor
mor) C6 strain, pituitary tumor (Pituitary
tumor) GH3% skeletal muscle myoblastoma (5kel
etal muscle myoblast > L
6 strains, and human-derived cells include IMR-32 strain and human fetal brain Flow3000 strain.

The present inventor cultured these cells in an incubator for an appropriate number of days using an appropriate culture medium and serum, or collected them in large quantities after growing them in an animal body, homogenized them using the following reagents, As a result of extracting proteins and analyzing them using two-dimensional electrophoresis, it was found that approximately 100 types of major proteins exist in cultured brain and nervous system tumor cells, and they are either undetectable or present only in small amounts in normal mouse and rat brains. figure,
It has been revealed that there are a total of eight or more types of proteins (NAK-1 to NAK-8, etc.) detected only in cultured cranial nervous system tumor cells, or in greater numbers, for each cranial nervous system tumor cell.

Separation and purification of this protein can be performed using known techniques. For example, when treating brain and nervous system tumor cells with Triton X-100
, homogenized with Tris/HCl buffer containing phenylmethylsulfonyl fluoride, extracted at low temperature, and then centrifuged at high speed. Alcohol such as ethanol was added to the resulting supernatant to precipitate the fraction. Each protein can be separated and purified by treatment with ion exchange chromatography and the like.

In addition, the physicochemical properties of each protein were confirmed by separating each protein in the crude homogenate fraction by two-dimensional electrophoresis on an SOS polyacrylamide gel and measuring their molecular weight and isoelectric point. . Table-1
summarizes the physical properties of each protein.

As can be seen from Table 1, the isoelectric point of each protein depends on its acidity, and some of these proteins are described in the above-mentioned Japanese Patent Application No. 58-206899 and Japanese Patent Application No. 73550-1987 filed earlier by the present inventor. Similar to the protein
It may be a sialoglycoprotein containing sialic acid. Generally, in the case of glycoproteins, molecular weight measurement by electrophoresis measures an apparent value. Furthermore, the molecular weights shown in Table 1 are considered to have an error of approximately ±2.000.

Some nervous system tumor cells contain nerve growth factor (nerv).
The presence of growth factor (NGF) induces differentiation, increases the number of neurites, and advances the processes. During this time, various proteins undergo changes. The present inventor examined the changes in the protein by changing the treatment time of NGF in pheochromocytoma PC12 cell lines, and determined whether it was not observed in normal rat brains and normal mouse brains as it was in cell lines not treated with NGP.
We have discovered proteins NAK-1 to NAK-8, which are present in very small amounts and are found in these cells. Among them N
The amount of AK-4 and NAK-5 proteins reached a maximum during the post-treatment period of NGF treatment.

The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.

Example 1 Rat adrenal medulla-derived pheochromocytoma (Pheochromocytoma)
cytoma) PC12 cell line (S, E, Pfeif
(obtained from Dr. Fer) on a 90 mm plastic dish (manufactured by Nunc) coated with collagen, and incubated at 37°C, pH 7, in a carbon dioxide gas incubator (5% CO2 + 95% air).
Cultured at 0 to 7.2. The culture medium is fetal bovine serum (FBS).
, 5%) and horse serum (HS, 10%).
ecco's modified Eagle's m
SDium (DME, 85%) was used. 1 cell
After reaching the stationary phase after 0 days of culture, the medium was removed and water-cooled Ca-Mg-free phosphate buffered saline (Dulb
Wash twice with ecco+P) 17°0), peel off the cells, collect in a centrifuge tube, and centrifuge in a refrigerated centrifuge (Hitachi) at 1,0
Cells of 3 x 10" per dish were collected by centrifugation at 00 rpm for 5 minutes. This was supplemented with 2% Triton X-100, 0.15
M NaCl, 10mM containing 50trg/mg phenylmethylsulfonyl fluoride (PMSF)
0.1 ml of hydrochloric acid buffer (PH7,6) was added, homogenized, and extracted at 4°C for 30 minutes. Next, high-speed centrifugation (40,000 rpm) was performed for 60 minutes, 10 times the volume of ethanol was added to the resulting supernatant, centrifugation was performed for 10 minutes (3,000 rpm), and the supernatant was removed. 9.5M urea and 2% NP-40 (manufactured by Hanisha) were added to the obtained precipitate.
, 2% ampholine (PH 3,5-10) (manufactured by Pharmacia), 5% mercaptoethanol, and 0.25% sodium dodecyl sulfate (hereinafter abbreviated as SOS) to prepare a solution. Next, this solution was subjected to two-dimensional electrophoresis to separate each protein and measure its molecular weight and isoelectric point. As a control protein, thyroglobulin (molecular weight 330,000
0>, lactoferrin (88,000), bovine serum albumin (67,000), ovalbumin (43,000), and aldolase (34,000)
) was used. A gel with the following formulation was used for first-dimensional electrophoresis. [Urea 2.76g, 30% acrylamide 0.67ml 1.10%NP-401ml Ampholine (
PH3,5-10) 0.25ml1.10% ammonia persulfate 10A11,5%TEMED 0.14ml1. water 0.91m1) (for 6 bottles). The sample was applied to this one-dimensional gel at 400v using 10mM H3PO4 as the anolyte and 20mM NaOH as the catholyte.
Electrophoresis was performed at 800V for 13 hours and then for 1 hour at 800V. Next, the gel after electrophoresis was soaked in SO3 sample buffer (10% glycerin, 5% β-mercaptoethanol, 2.3% SO
3,62,5mM) Lis-HCl buffer (PH6,8)
) and equilibrated with 7.5% acrylamide slab gel (25mM), 192mM glycine, 0.1%
SOS) Second-dimensional electrophoresis was performed for 4 hours at 120V. After each separated protein was stained with 0.05% Kumasi blue, 10% methyl alcohol, and 10% acetic acid for 1 hour, it was decolorized with 10% methyl alcohol and 10% acetic acid, and each protein spot was measured. NAK spots not found in normal rat brain and normal mouse brain
They were named NAK-1 or NAK-6. Furthermore, spots that exist in large amounts in this PC12 cell line, although only a small amount exists in normal rat brains, were named NAK-7 and NAK-8.

The results are shown in Table-1.

Example 2 The culture solution of rat pheochromocytoma PC12 cell line, which was started under the same conditions as in Example 1, was discarded after 2 days, and the culture was incubated at 2°5S N.
GF (V, Bocchini and P, V, An
geletti+ Proc, Nat, Acad, sc
10 ml of fresh culture solution containing 50 ng/ml of A.i, U, S, A., Dan (1969) 787-794) was added, and NGF treatment was performed (Tatsuro Koike
, Brain Research, 2 Birds (1983
) 293-303). After adding NGF, 1 day, 2 days, 3 days, and 5 days later, the culture medium of each dish with different NGF treatment times was discarded and washed twice with ice-cold phosphate buffered saline. In the same way as I
XIO" cells were collected. Next, the cells were treated in the same manner as in Example 1, and the homogenate was subjected to two-dimensional electrophoresis to separate and detect the extracted protein, and its properties were confirmed. Normal rat brain and NAK-1 or NAK-6 proteins, which are not found in normal mouse brain, were detected.

The amount of NAK-4 and NAK-5 proteins reached the maximum during the treatment period 3 days after NGF treatment. In addition, although there are only a few spots present in normal rat brains, spots present in large quantities in this PC12 cell line are seen in the same way as in the stationary phase PC cell line of Example 1, and the same < NAK-7 and NAK-8
(See Table 1).

Example 3 Rat-derived dahliacytoma C6 strain (derived from ATTC) was cultured in one dish for 7 days under the same conditions as in Example 1, except that the composition of the culture medium was 95% DME and 35% FB. Then, from the two plates, 1×10
8 cells were collected. Next, the proteins of this cell were extracted in the same manner as in Example 1, separated by two-dimensional electrophoresis, and protein spots that were not found in normal rat brain and normal mouse brain were detected, and NAK-1, NAK-2, NA
They were named K-3 and NAK-6. The amount of NAK-3 was higher than that in other cells. Furthermore, although only a small amount exists in the normal rat brain, a spot was found in which this cell was found in large quantities and was named NAK-7 (see Table 1).

Example 4 Rat-derived pituitary tumor GH3 cell lines (derived from ATTC) were cultured in three dishes under the same conditions as in Example 1 for 7 days.

And Example 1. 5 x 107 cells were collected in the same manner as described above. Next, the protein of this cell was extracted and separated in the same manner as in Example 1, and protein spots that were not found in normal rat brain and normal mouse brain were detected, and NAK-
1, NAK-2, NAK-3 and NAK-6. In addition, although only a small amount exists in normal rat brains, a large number of spots were observed in these cells and they were named NAK-7 (see Table 1).

Example 5 Rat-derived skeletal muscle myoblast If! The L6 strain (derived from ^TTC) was cultured in three dishes for 6 days under the same conditions as in Example 3. Then, in the same manner as in Example 1, 3X10? cells were collected. Next, the protein of this cell was extracted and separated in the same manner as in Example 1, and protein spots that were not found in normal rat brain and normal mouse brain were detected, and NAK-
1, NAK-2, NAK-3 and NAK-6. In addition, although only a small amount exists in normal rat brains, spots were found in these cells that were present in large quantities and were named NAK-7 and NAK-8 (see Table 1).

Example 6 Mouse-derived neuroblastoma N-18 cell lines were cultured in five dishes under the same conditions as in Example 3 for 7 days. And Example 1
4XIO" cells were collected in the same manner as in Example 1. Proteins from these cells were then extracted in the same manner as in Example 1, separated by two-dimensional electrophoresis, and protein spots not found in normal mouse brain were detected. , NAK-1, NAK-2
, NAK-3, NAK-6, NAK-7 and NAK-
8 (see Table 1). Even when the NAK-6 protein was analyzed by two-dimensional electrophoresis after treatment with neuraminidase, there was no change in the spot position.

Example 7 Mouse-derived neuroblastoma cancer cell N-115 cell line was used in Example 3
Five dishes were cultured for 7 days under the same conditions as above.

Then, 3×10 7 cells were collected in the same manner as in Example 1. Next, the proteins of this cell were extracted and separated in the same manner as in Example 1, and protein spots that were not found in normal mouse brain were detected.
They were named K-3, NAK-6, NAK-7 and NAK-8 (see Table 1). Even when the NAK-6 protein was analyzed by two-dimensional electrophoresis after treatment with neuraminidase, there was no change in the spot position.

Example 8 Mouse-derived smooth muscle-like brain tumor BC381 cell line (^TT
C), the composition of the culture medium was 90% DMB and FB51.
Three dishes were cultured for 14 days under the same conditions as in Example 1 except that the concentration was 0%. Then, in the same manner as in Example 1,
Next, the proteins of these cells were extracted and separated in the same manner as in Example 1, and protein spots that were not found in normal mouse and rat brains were detected. -2, NAK-3, and NAK-6. NAK-1 and NAK-2 were found in higher amounts than other cells.Also, although only a small amount exists in the normal rat brain, the present A large number of spots were observed in the cells and was named NAK-7 (Table-
(see 1).

These NAK-1 to NAK-8 proteins clarified by the present example are derived from the above-mentioned Japanese Patent Application No. 58-206899 and Japanese Patent Application No. 60-7355 previously filed by the present inventor.
Although they have similar molecular weights, they are different from the protein described in No. 0 in terms of isoelectric point, sensitivity to neuraminidase, etc., and are therefore considered to be different proteins.

Effect The protein of the present invention acts as an essential factor or promoter of the growth of tissue cells of the brain nervous system, and exhibits its effects when administered to a living body. This protein is also useful as a variety of tumor markers, and antibodies against these proteins are expected to be used in the diagnosis and treatment of cancer.

Claims (1)

[Claims] A protein present in animal-derived brain and nervous system tumor cells, having a molecular weight of 55,000 to 140,000 by two-dimensional electrophoresis, and an isoelectric point of 4.5 to 5.8.