JPH0892127A - Prophylactic and/or remedial agent for apoptosis-relating diseases, screening of apoptosis-controlling substances and diagnosis of diseases relating to apoptosis - Google Patents

Abstract

PURPOSE: To provide a prophylactic and/or remedial agent for diseases relating to apoptosis which controls the expression of G3PD (glyceraldehyde phosphate dehydrogenase), an important protein participating apoptosis. CONSTITUTION: Finding that protein G3PD known as a glycolytic enzyme participates the apoptosis, substances controlling the expression of the protein, G3PD, for example, 9-amino-1,2,3,4-tetrahydroacridine known as an antidementia and aurintricarboxylic acid known as an endonuclease inhibitor are used as active ingredients to prepare a prophylactic and/or remedial agent for diseases participating apoptosis. The polyclonal antibody and monocl-onal antibody prepared using G3PD as an antigen or antisense oligonucleotide synthesized from the DNA coding G3PD can be also used as a prophylactic and remedial agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a preventive and / or therapeutic agent for apoptosis-related diseases, a screening method for apoptosis-regulating substances, and a diagnostic method for apoptosis-related diseases. More specifically, it is characterized by regulating the expression of G3PD (also called glyceraldehyde-3-phosphate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase), which is a killer protein in neuronal cell apoptosis. And / or therapeutic agent for diseases associated with apoptotic apoptosis and protein G3PD
For preventing and / or treating a disease associated with apoptosis, which contains a substance that regulates the expression of spleen as an active ingredient, and a method for screening a substance that regulates apoptosis, which comprises using the protein G3PD
The present invention relates to a method for diagnosing a disease associated with apoptosis, which is characterized by using PD.

[0002]

BACKGROUND OF THE INVENTION Apoptosis or programmed cell death (planned cell death) is one of the cell death processes or modalities proposed by Kerr, Wyllie et al. (Brit.
J. Cancer, 26 , 239 (1972)). Apotosheath is seen during physiological ontogeny or the manifestation of diseases and drug effects, and is thought to occur based on the activation of the program inherent in individual cells. Distinct from some necrosis. Apoptosis differs from necrosis in that it involves RNA synthesis and protein synthesis, whereas necrosis does not.

There are various stimuli for inducing apoptosis, and the mechanisms thereof are diverse, but the morphological characteristics are common. The first observed morphological change is the formation of chromatin aggregates, which in most cases is accompanied by a DNA fragmentation reaction (Natu
Re, 284 , 555 (1980)). When chromatin is aggregated, cytoplasmic condensation occurs, and the cells themselves form cell fragments called apoptotic bodies, which are rapidly phagocytosed by surrounding cells and macrophages. It is said that this causes apoptosis.

The mechanisms of apoptosis include (1) lack of physiologically active substances [nerve growth factor (NGF), colony stimulating factor (CSF), etc.] and (2) apoptosis-inducing factor [tumor necrosis factor (TNF)]. And lymphotoxin, c-myc, p53 gene product, etc.] induce apoptosis (Cell, 69 , 119).
(1192) and Nature, 359 , 849 (1993)),
(3) Apoptosis is inhibited by an apoptosis inhibitor [bcl-2, etc.] (Nature, 359 , 552 (199
2) and Nature, 359 , 554 (1994)).

The reason why such a phenomenon of apoptosis has recently received attention is (1) that apoptosis plays an important role in the formation of an individual, (2) internally in vivo, In many cases, cell death due to external factors is due to apoptosis, and (3) in AIDS and other diseases, apoptosis is deeply involved in the decrease of somatic cells (lymphoid cells) that cause the disease.
(4) Various anti-cancer agents perform cancer cell destruction by apoptosis, and (5) What genes control apoptosis itself due to recent progress in genetic research, and how is information that leads to apoptosis? It can be mentioned that the knowledge of whether or not it is transmitted has been accumulated, and that there is a basic interest in cell biology.

[0006] To describe the relationship between apoptosis and disease in more detail, for example, when infected with HIV virus, the immune system is destroyed due to the death of lymphocytes and the resistance to infection is lost, but most of these lymphocytes are killed. According to apoptosis (Sience, 257 ,
217 (1992)). In autoimmune diseases and the like, it is known that the decrease of lymphoid cells is due to apoptosis. Further, it has been shown that the onset of encephalomyelitis, which is an autoimmune disease of the nervous system, is due to the death of spinal cells by apoptosis. Furthermore, diseases associated with neuronal death such as Alzheimer's disease and the relationship between learning and memory and apoptosis are also important. In relation to cancer, it has been revealed that the tumor suppressor gene p53 is involved in apoptosis of cells damaged by DNA, and the involvement of the tumor suppressor gene in apoptosis has been attracting attention. Furthermore, in relation to carcinogenesis, it is also known that many cells forming hyperplastic nodules as precancerous tissues die by apoptosis, and the remaining cells eventually change into cancer cells.

[0007]

2. Description of the Related Art Up to now, several kinds of polypeptides related to programmed cell death have been reported. Fas antigen is known as a typical one (Cell, 66 , 23
3 (1991)). Human Fas antigen is a polypeptide consisting of 335 amino acids, which has a signal peptide consisting of 16 hydrophobic amino acids at the N-terminal, and the mature protein has an extracellular region (157 amino acids) from the N-terminal side and a transmembrane membrane. It is presumed to have a structure such that it is divided into a region (17 amino acids) and a cytoplasmic region (145 amino acids). The Fas antigen is considered to function as a receptor for a factor (ligand) that induces cell death.

[0008]

OBJECT OF THE INVENTION The object of the present invention is to find a polypeptide deeply involved in programmed cell death in mammals and a DNA encoding the same, which is completely different from the known polypeptides represented by Fas antigen. . In the present invention, a protein deeply involved in programmed cell death was isolated and its amino acid sequence was determined. As a result, it was the same as the polypeptide of known glyceraldehyde phosphate dehydrogenase (hereinafter abbreviated as G3PD). This G3PD is known as a glycolytic enzyme as shown below, but it has never been known to be deeply involved in apoptosis. G3PD is
NAD ⁺ or N, which is one of the major enzymes that make up glycolysis
ADP ⁺ is used as a coenzyme to oxidatively phosphorylate glyceraldehyde-3-phosphate.

[0009]

[Chemical 1]

Further, it is a very important enzyme as a substrate-level phosphorylation enzyme which preserves the oxidation energy of the substrate as a phosphate bond. In order to isolate proteins that are deeply involved in programmed cell death, we present rat cerebellar granule cells as representative of mammalian cells that readily undergo apoptotic death in an in vitro system. (Hereinafter abbreviated as CGC) was selected. In such a central nervous system, it is known that almost half of the nerve cells die by apoptosis before being completely matured. The present inventors confirmed that, when CGC was actually cultured for 15 days, more than half of the cells were apoptotic. In addition, 9-amino-1,2,3,4-tetrahydroacridine (THA), which is known as an anti-dementia drug, or aurintricarboxylic acid (ATA), which is an inhibitor, was added on the 7th day and cultured for 15 days. ,
It was found that apoptosis is suppressed, and it was confirmed that quantitatively varying proteins were present when such a reagent was added.

When this protein was measured by SDS-PAGE analysis, when THA or ATA was added, the level of a protein with a molecular weight of 38 kD that was overexpressed (about twice as much) as cell death was suppressed. I found out that it was done. Therefore, the present inventors identified a 38 kD protein that is considered to be involved in this apoptosis. The cultured cells were purified by the blotting method and subjected to amino acid sequencing (see J. Bio. Chem., 262 , 10035). As a result, when the sequence from the N-terminal to the 23rd sequence was performed, it was completely the same as G3PD. Furthermore, in order to clarify the relationship between G3PD and apoptosis, antisense of G3PD was synthesized, and the antisense of G3PD was 7, 7, 11, and 1 in the culture of CGC.
Apoptosis was suppressed when added on day 3 to suppress overexpression of G3PD.

From the above, the 38 kD protein of the present invention was identified as G3PD. G3PD is known as a glycolytic enzyme, but it has never been known to be involved in apoptosis, and it was confirmed for the first time this time. It is quite difficult to predict the association between G3PD and apoptosis based on the action of glycolysis in apoptosis.

[0013]

The present invention relates to a polypeptide (hereinafter referred to as G3P) which is deeply involved in programmed cell death in mammals.
Call it D. ) Concerning. The G3PD of the present invention includes rat G3P whose action has been clarified by the present inventors.
G3 of other mammals having high homology with rat G3PD (meaning immunological equivalence such as cross-reactivity with antibody of rat G3PD) including D
PD, especially human G3PD.

The G3PD of the present invention includes modified G3PD in addition to natural G3PD. That is, natural G3P
One in which a part of the amino acid sequence of D is deleted (for example, a polypeptide consisting of only a mature protein portion lacking a signal peptide, or a polypeptide consisting of only a portion essential for activity expression in the mature protein), A part in which a part is substituted with another amino acid (for example, a part substituted with an amino acid having similar physical properties) and a part in which another amino acid is added or inserted are also included.

The G3PD of the present invention includes a DNA encoding the polypeptide, a vector comprising the DNA, a host cell transformed with the vector, an antibody of the polypeptide, and an antisense of the DNA encoding the polypeptide. Etc. are included. The DNA encoding G3PD of the present invention is chemically synthesized using its nucleotide sequence.
Alternatively, by chemically synthesizing a fragment of the base sequence and hybridizing it with a probe, the G of the present invention is obtained.
DNA encoding 3PD can be obtained. Furthermore, by introducing the vector DNA containing the present DNA into a suitable host and allowing it to grow, the desired amount of the DNA encoding the G3PD of the present invention can be obtained.

The method for obtaining the G3PD polypeptide of the present invention includes (1) a method of purifying and isolating from a living body or a cultured cell, (2) a method of synthesizing a peptide, or (3)
Examples of the method include a method of producing using a gene recombination technique, but the method described in (3) is industrially preferable.

The expression system (host-vector system) for producing a polypeptide using the gene recombination technique includes, for example, expression systems of bacteria, yeast, insect cells and mammalian cells. For example, in the case of expression in Escherichia coli, a start codon (ATG) is added to the 5 ′ end of the DNA encoding the mature protein portion, and the obtained DNA is used as an appropriate promoter (eg, trp promoter, la).
a vector that functions in E. coli (for example, pBR322, pUC18, pUC19, etc.) that is connected downstream of the c promoter, λPL promoter, T7 promoter, etc.
To prepare an expression vector. Next, E. coli transformed with this expression vector (for example, E. Coli DH
1, E. Coli JM109, E. Coli HB101 strain, etc.) can be cultured in an appropriate medium to obtain the desired polypeptide from the cells.

If a bacterial signal peptide (for example, pelB signal peptide) is used, the desired polypeptide can be secreted into the periplasm. Furthermore, it is also possible to produce a fusion protein with another polypeptide. When expressed in mammalian cells, for example, a DNA encoding the full-length G3PD is used as an appropriate vector (eg, retrovirus vector, papillomavirus vector, vaccinia virus vector, SV40).
Suitable promoter (eg, SV) in the system vector, etc.
40 promoter, LTR promoter, metallothionein promoter, etc.) to prepare an expression vector.

Next, an appropriate mammalian cell (eg, monkey COS-7 cell, Chinese hamster CHO cell, mouse L cell, etc.) is transformed with the obtained expression vector, and the transformant is cultured in an appropriate medium. By,
The desired polypeptide is secreted into the culture medium.
The polypeptide obtained as described above can be isolated and purified by a general biochemical method.

The G3PD-encoding DNA obtained by the present invention can be used as a probe to isolate the G3PD gene of animals other than rat including human. DNA or DNA encoding those genes
The fragment can detect the G3PD gene as a probe or a primer, and thus can be used for studying the relationship between the gene and a disease involved in apoptosis or for diagnosing the disease. In addition, those DNAs are expected to have great utility by ordinary gene recombination methods.
It can be used as an essential template when producing a D polypeptide, a polypeptide fragment or a derivative thereof.

The polypeptide thus produced,
The polypeptide fragment or modified polypeptide can be used as a prophylactic and / or therapeutic agent for diseases in which apoptosis is involved by regulating the expression thereof, for example, infectious diseases, decreased or enhanced immune function and brain function, or tumors. Be expected.

AI is a disease in which apoptosis is involved.
HIV, HTLV-1 related diseases such as DS, ARC (AIDS related diseases), adult T cell leukemia, hairy cell leukemia, myelopathy, respiratory disorders, arthritis, uveitis and virus related diseases such as hepatitis C , Cancer, collagen diseases such as systemic lupus erythematosus and rheumatoid arthritis, ulcerative colitis, Sjogren's syndrome, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, myasthenia gravis,
Autoimmune diseases such as insulin-dependent (type I) diabetes, myelodysplastic syndrome, periodic thrombocytopenia, aplastic anemia,
Idiopathic thrombocytopenia, various diseases associated with thrombocytopenia such as generalized intravascular coagulation, viral and drug-induced hepatitis such as C-type, A-type, B-type, F-type and liver disease of cirrhosis, Alzheimer's disease , Dementia such as Alzheimer-type senile dementia, cerebrovascular disorder, neurodegenerative disease, adult respiratory distress syndrome, infectious disease,
Examples include benign prostatic hyperplasia, uterine myoma, bronchial asthma, arteriosclerosis, various congenital malformations, nephritis, senile cataract, chronic fatigue syndrome, muscular dystrophy and peripheral neuropathy.

The substances that regulate the expression of the protein G3PD of the present invention are not only those substances which are currently confirmed to have the activity, but also those which are newly confirmed to have the activity in the future. Contains substances of In addition, the regulation here means suppression and enhancement. Currently, as substances that regulate the expression of the protein G3PD, for example, pentalenolactone, vinyl sulfone, nitric oxide, conindic acid (hepteridic acid), 4-chloroacetylpyridine, (S) -3-chlorolactaldehyde, acetylamide. , Dimethylaminopropionitrile, 2,5-hexanedione cyclohexamide (CHX), actinomycin-D (Act-D), aurintricarboxylic acid (ATA), 9-amino-1,2,3,4-tetrahydro Acridine (THA) and the like are known.

Furthermore, since a polyclonal antibody or a monoclonal antibody can be prepared by a conventional method using the polypeptide of the present invention or a fragment thereof as an antigen, the relationship between the polypeptide and a disease can be determined by quantifying the polypeptide using them. It can be used for research or diagnosis of diseases. Further, the monoclonal antibody can be used as a prophylactic and / or therapeutic agent in its intact form, in the form of a chimeric antibody with a human antibody, or in the form of being converted into a human form.

DNA encoding the polypeptide of the present invention
Not only serves as an important and essential template for producing the polypeptide of the present invention, which is expected to have great utility, but also stops the expression of the polypeptide by synthesizing an antisense oligonucleotide by a conventional method. It can also be used for prevention and / or treatment and the like.
Since the polypeptide of the present invention is involved in apoptosis, it can be used for screening of substances involved in apoptosis by measuring the expression of the polypeptide of the present invention.

[0026]

EXAMPLES The present invention will be described in more detail with reference to examples below, but these do not limit the scope of the present invention.

Example 1 Cultivation of cerebellar granule cells (CGC) CGC was performed by the method of Gallo et al. (J. Neurosci., 7 , 2203 (198).
7) See 8), and culture was performed from 8 day-old SD rats. That is, the cerebellum was cut into pieces of about 400 μm square.
Trypsin enzyme treatment was performed and the isolated cells were treated with poly-L-
It was sprinkled on a petri dish treated with lysine. Cell density is 1.7 ×
I made it 10 ⁶ . For long-term survival and maturation of CGC, the culture was performed in Eagle's medium containing 10% fetal calf serum (FCS), 2 mM glutamine, 50 μg / ml gentanuicin and 25 mM potassium chloride. To inhibit the growth of non-neuronal cells, cytosine arabinoside (10 μM) was added 20 hours after plating the cells on the dish. The culture medium was not changed during the experiment because the cells were necrotic by the excessive stimulation with glutamate by changing the culture solution.

Example 2: Analysis of amino acid sequence of 38 kD protein The analysis of N-terminal amino acid sequence of 38 kD protein was carried out according to the method of Matsudaira et al. (J. Bio.
Chem., 262 , 10035 (1987)). That is, the cells prepared by the method of Example 1 were cultured for 15 days, and the cells were collected using 50 mM Tris-HCl buffer (pH 7.4). The cell suspension was sonicated, and the resulting homogenate was centrifuged at 2 × 10 ⁵ g for 30 minutes. The suspension of the membrane fraction thus obtained and SDS
-1: 1 mixing with PAGE sample buffer to give a final protein concentration of 3 mg / ml. This sample 1
After heating 5 μl at 100 ° C. for 3 minutes, SDS-PAGE was performed under the following conditions.

Gel used: Tefco No. 01-003 SDS-
PAGE mini 12% (1.5 mm 15 well), electrophoresis tank: Tefco No. 03-001, electrophoresis conditions: 27 mA constant current, 1.5 hours

After completion of electrophoresis, the gel was separated from the stacking gel, immersed in a blotting buffer prepared with a solution of Tris (3.02 g) and glycine (14.4 g) in distilled water (1 liter), and shaken for 10 minutes. Applied Biosystems (Applie
ProBlott (registered trademark) manufactured by d Biosystem) was moistened with methanol and further immersed in a blotting buffer for 10 minutes before use. Blotting was performed at a constant current of 180 mA for 1 hour. After completion of the blotting, the membrane was washed by shaking with distilled water (10 minutes × 3). After washing, 0.1% Coomassie Brilliant Blue R in 1% acetic acid
Stained at -250 for 2-3 minutes. Further, it was decolorized with 50% methanol until a band became apparent. After washing with distilled water, the desired band was cut off. The cut out band was analyzed by ABI model 470A automated gas-phase protein sequencer / 120A PTH analyzer (ABI model 470A automated gas-phase protein seq.
uencer / 120A PTH analyzer) and the amino acid sequence of 23 cycles from the N-terminal was analyzed. The result of the amino acid sequence is shown in SEQ ID NO: 1.

When the homology search (homology search) of the amino acid sequence of SEQ ID NO: 1 was carried out, it was completely in agreement with the amino acid sequence of rat G3PD. The Xaa portion is cysteine in rat G3PD. This is something that cannot be detected by the usual Edman method. From the above, the 38 kD protein of the present invention was determined to be G3PD.

Example 3 Preparation of Antisense Oligonucleotides In order to regulate the expression of the rat G3PD gene, G3P
An antisense nucleotide of D was made. Further, in order to efficiently have an antisense effect, the vicinity of both 5'and 3'of the oligonucleotide was converted into an S-oligophosphothioate derivative. The gene sequence of G3PD is
Report by Yun Tso et al. (Nucleic Acids Resea
rch, 13 , 2485 (1985)). That is, antisense was prepared for a portion corresponding to A of 23 bp and C of 42 bp, which is a base sequence around ATG encoding methionine which is a protein translation initiation site. The detailed antisense nucleotides are shown below (SEQ ID NO: 2).

5'-GA ^* CCTTCACCCATCTT
The GTCTA ^* -3 '* part is the S-oligo conversion site.

The antisense nucleotide shown in SEQ ID NO: 2 is the Genosys Biot.
echnologies) method (J. Am.
Chem. Soc., 112 , 1254 (1990) and J. Org. Che
m., 55 , 4693 (1990)) was purchased from Kurabo Industries.

Example 4 Preparation of Sense Oligonucleotide A sense oligonucleotide was prepared as a negative antisense control. The detailed sense nucleotides are shown below (SEQ ID NO: 3).

5'-TA ^* GACAAGATGGGTGA
The AGGTC ^* -3 ′ * part is the S-oligo conversion site.

The sense nucleotide shown in SEQ ID NO: 3 was also manufactured by Kurano Co., Ltd., which was produced by the method of Iyer et al. At Genosys Biotechnologies as in Example 3.

Reference Example 1: Judgment of cell death by vital staining The cells prepared by the method of Example 1 were locked.
Wash with the solution of e) (twice with 2 ml), and 0.0008% Fluorescent diacetate (hereinafter abbreviated as FDA).
With 0.0002% propidium iodide (hereinafter abbreviated as PI) (J. Histochem. Cytochem., 33 , 7
7 (1985)). FDA decomposed the elastase of the cells, and the viable cells emitted yellowish green fluorescence. In the nuclei of dead cells, PI passes through the cell membrane and
It was bound to A and exhibited an orange-red color.

Example 5: Inhibition of apoptosis by rat G3PD antisense oligonucleotide The concentration of antisense oligonucleotide was Tris ED.
Adjusted to 1 mM with TA buffer. 5 μl of the thus prepared solution was added to 2.5 ml of the medium (prepared in Example 1) on the 7th, 9th, 11th and 13th days. This mixed medium was cultured until the 15th day. The number of dead cells was stained using the staining technique shown in Reference Example 1, and live and dead cells were identified by an epi-illumination fluorescence microscope and counted. The number of dead cells per (500 μm) ² (□) (dead cells / □) and the total number of cells (total cells / □) were measured (4 points per dish), and the average of 3 experiments Expressed as ± standard error. The survival rate was calculated by the following formula.

Survival rate (%) = (total number of cells per square-square
Dead cells per cell) / (total cells per square) x 100 The results are shown in Table 1.

Example 6 Inhibition of Apoptosis by Other Test Drugs As shown below, a test drug having a target concentration was added on a predetermined day and cultured in the same manner as in Example 5 for evaluation. The results are shown in Table 1. ATA: 7th day, THA: 7th day, rat G3PD sense oligonucleotide: 7th day, 9
Days 11, 11 and 13.

[0042]

[Table 1]

In Table 1, control means a control to which no test drug was added, and THA means 9-amino-1,2,3,3.
4-tetrahydroacridine, ATA represents aurintricarboxylic acid, AS represents rat G3PD
Represents an antisense oligonucleotide and S represents a rat G3PD sense oligonucleotide.

From Table 1, TH known as an anti-dementia drug
It was found that A and ATA, which is known as an endonuclease inhibitor, suppress cell death as compared with a control to which no drug is added. Also, rat G3P
It was found that the D antisense oligonucleotide completely suppressed cell death as compared with the sense oligonucleotide.

[0045]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 24 Sequence Type: Amino Acid Number of Chains: Single Strand Topology: Linear Sequence Type: Polypeptide Sequence Val Lys Val Gly Val Asn Gly Phe Gly Gly Arg Ile Gly Arg Leu Val 1 5 10 15 Thr Arg Ala Ala Phe Ser Xaa Asp 20

SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence GACCTTCACC ATCTTGTCTA

SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence TAGACAAGAT GGTGAAGGTC

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 31/435 ADV 31/70 ADY AED G01N 33/53 D

Claims (7)

[Claims] 1. Prevention and / or disease associated with apoptosis characterized by regulating the expression of the protein G3PD.
Or a therapeutic agent. 2. The diseases associated with apoptosis include viral infection, autoimmune disease, leukemia, HIV infection, cancer, cancer metastasis, cerebrovascular disorder, neurodegenerative disease, Alzheimer's disease, peripheral neuropathy or liver disorder. The preventive and / or therapeutic agent according to claim 1. 3. A preventive and / or therapeutic agent for a disease associated with apoptosis, which comprises a substance that regulates the expression of the protein G3PD as an active ingredient. 4. A preventive and / or therapeutic agent for an apoptosis-like disease involving an antisense nucleotide of protein G3PD. 5. A preventive and / or therapeutic agent for a disease associated with apoptosis, which comprises an antibody of the protein G3PD. 6. A method for screening a substance that regulates apoptosis, which comprises using the protein G3PD. 7. A method of diagnosing a disease associated with apoptosis, which comprises using the protein G3PD.